神経筋接合部疾患におけるイオンチャネルとレセプターの分子病態に関する免疫学的研究

金沢大学医学部(1)Lambert-Eaton筋無力症候群肺癌との合併頻度の高い本病は,骨格筋支配の末梢神経終末局在の電位依存性カルシウムチャネル(VGCC)を標的とする抗体が主役を演じ,チャネル機能障害の結果,神経終末からのアセチルコリン量子性遊離量が低下し発症する。分子免疫学的研究の結果,以下のことを明らかにした。(1)本病抗体の主たる抗原決定基はVGCCのP/Q型である。(2)P/Q型VGCC分子構造の中での抗体反応領域は,α1サブユニットのドメインIIとIVのS5-S6リンカー領域であった。(3)ドメインIIとIIIS5-S6リンカー領域合成ペプチドを抗原として,動物に疾患モデルを誘導することができた。(4)VGCC周辺機構を構成する蛋白質の一つで,カルシウム・センサーとしての役割を担うシナプトタグミンの,シナプス小胞開口中膜外に露呈する領域に照合して合成ペプチドは,これを抗原として動物を免疫すると,疾患モデルを昨出できた。(5)遺伝子操作で大腸菌に発現させたリコンビナント・シナプトタグミンを抗原として患者血清を検定すると,30%の症例で陽性,その50%は抗VGCC抗体陰性であったので,この蛋白質に対する免疫反応だけでも本病の一時的原因となりうる。(6)VGCCもシナプトタグミンも肺癌組織に発現,癌組織と神経系の免疫学的交叉反応が,発病の根底にあると示唆される。(2)重症筋無力症神経筋シナプスの後シナプス・アセチルコリン受容体に対する免疫反応が原因である本病で,特に胸腺腫合併例において,筋収縮疲労が本来のシナプス伝達疲労に加重していること,その原因が胸腺上皮細胞に発現するリアノジン受容体を認識し産生される抗体が筋の興奮収縮関連を阻害するにあることを明らかにした。また、自然発症胸腺腫ラット(Baffalo/Mna種)は,この病態の動物モデルであることを,胸腺の免疫組織化学,免疫学および電気生理学的指標で明らかにした。(1) Lambert-Eaton myasthenic syndrome (LEMS) LEMS,often ssociated with small cell lung carcinoma (SCLC), impairs the quantal release of acetylcholine (ACh) by antibodies against voltage-gated calcium channel (VGCC) in the motor nerve terminal. We focused attention on the P/Q-type VGCC,against which a majority of LEMS patients carry the specific antibody. This type of VGCC expresses in the motor nerve terminal and also in SCLC.In search of antigenic sites in the P/Q-type VGCC molecular structure, we synthesized peptides corresponding to the extracellular region (S5-S6 linker) of each of the four domains that form the alpha1A subunit of VGCC and tested their antigenicity. In LEMS patients\u27 sera, some were positive for anti-domain II,and the other were positive for anti-domain IV.Lewis rats immunized with domain II and III peptides, each being conjugated with KLH,showed such characteristic LEMS features as presence of antibodies to P/Q-type VGCC and reduced ACh quantal release. In addition, the possible role of synaptotagmin, a Ca^ sensor for exocytosis of synaptic vesicles taking place prior to ACh release, in the pathogenesis of LEMS was studied. The peptide corresponging to the extracellular region of synaptotagmin was found antigenic for the induction of an animal model of LEMS.A proportion of human LEMS antibodies reacted with the recombinant synaptotagmin in immunoblot.(2) Myasthenia gravis (MG) In MG in which muscle anti-ACh receptor antibodies play a crucial role, we focused attention on an additional impairment of excitation-contraction coupling in muscle, attributable to a defect caused by antibodies against ryanodine receptor (RyR). Many of MG patients with thymomas contained anti-RyR antibodies in serum ; these sera inhibited the calcium-induced release of calcium in response to caffeine in human muscle cell line. The Buffalo/Mna rat with spontaneous benign thymoma was shown as an animal model of impaired subcellular machineries in MG muscle as evidenced by RyR expressed in thymic epithelial cells, anti-RyR antibodies in serum and reduced contractile forces without abnormality in synaptic transmission and muscle membrane properties.研究課題/領域番号:07457154, 研究期間(年度):1995 – 1997出典：研究課題「神経筋接合部疾患におけるイオンチャネルとレセプターの分子病態に関する免疫学的研究」課題番号07457154（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-07457154/074571541997kenkyu_seika_hokoku_gaiyo/）を加工して作

合成アセチルコリン受容体立体構造と筋無力症病態解析、治療に関する分子免疫学的研究

金沢大学医学部アセチルコリン受容体(AchR)分子、立体構造のB、T細胞認識性の観点から研究し以下の成績を得た。1.AchR崩壊促進作用抗体の標的を分子構造中のα67-76、T細胞認識領域(ルイスラット)をα107〜116とした。2.両者単独、混合、人工的アミノ酸配列介左連結など6種の合成ペプチドを免疫原に選び、それぞれでラットを免疫、発病の状態を免疫学的、生理学的指標で評価した。3.疾患誘導に関わる抗原としてはα67〜76とα107-116の複合連結ペプチドが最も有意性が高く、一方、天然AchR蛋白質を抗原として免疫し発症した動物モデルでの血中抗体検定には、α-67-76とα107-116の間にβターン構造を強調すべく人工的アミノ酸配列(NPGG)を介在せしめた合成ペプチドが最も有用であった。2次元核磁気非鳴法(NMR)によるペプチド立体構造解析は、前者でαヘリックス、後者でβターン構造がつよく、免疫学的立体構造推定と一致した。4.動物モデルで疾患誘導性、抗体反応性が高かった合成ペプチドを含む6種の人工抗原のいずれも、ヒト重症筋無力症患者血中抗体検定用としては有違性が低かった。5.ヒトでの血液準化療法への応用に資すべく、α67〜76を骨格としてこれにつよいターン構造をとらせる人工的アミノ酸配列の連絡を行った。全12種のペプチドのうちCKGGLR-α67〜76-KC,KKC-α63〜77-C,KKC-α62〜77-C,KKC-α61〜77-Cのヒトおよびシビレエイ残基配列計8種のペプチドは、モノクローナル抗体(受容体崩壊促進作用)とつよく反応した。患者血中抗体との反応率は20%弱であった。6.アセチルコリンと受容体との結合を阻害する抗体の標的領域α183-200の合成ペプチドを吸着剤として、患者血液体外循環中に血二成分と反応させ、病原抗体の選択除去をはかる臨床応用(血液淨化療法)は、高い有効性を示した。Myasthenogenic regions in the acetylcholine receptor (AChR)alpha-subunit were studied in view of the conformation-dependent B-cell epitope expected at beta-turn and the MHC class II-restricted T-cell epitope expected at alpha-helix. Torpedo AChR alpha67-76 and alpha107-116 were synthesized as the main immunogenic region and the site specific for T-cell epitope in Lewis rat, respectively. Model peptides, synthesized by combining these natural sequence segments or by intervening the segment aligned as NPGG in natural sequence segments, were tested in terms of antigenic conformation. The model peptide, alpha107-116.alpha67-76.alpha107-116, was potently immunogenic in the induction of the animal model of myasthenia, accomplanied by the anti-peptide antibody cross-reactive with the native AChR.High antigenicity in antibody assays for vaious peptide- and native AChR-immunized rats was found when the model peptides, alpha107-116.alpha67-76 and/or alpha107-116. NPGG.aplha67-76, were used for measurement as antigens. Since none of these model peptides was reactive with antibody in human myasthenics, peptides were therefore designed to conforom to the antigen appropriate for binding with human myasthenic antibody. As the result, 4 model peptides, CKGGLR.alpha67-76.C,KKC.alpha63-77.C, KKC.alpha62-77, and KKCalpha 61-77-C, were potently reactive with monoclonal myasthenic antibody and useful for detecting antibody in human myasthenics.研究課題/領域番号:03454239, 研究期間(年度):1991 – 1993出典：研究課題「合成アセチルコリン受容体立体構造と筋無力症病態解析、治療に関する分子免疫学的研究」課題番号03454239（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-03454239/034542391993kenkyu_seika_hokoku_gaiyo/）を加工して作

アセチルコリン受容体と重症筋無力症の免疫、生化学および治療に関する研究

金沢大学医学部・附属病院・神経内科重症筋無力症病態解明と新しい治療法開拓のため、本病成立にかゝわる免疫反応の標的であるアセチルコリン受容体(AChR)を分子レベルから研究した。1.重症筋無力症発病機構の中で主役の一つを演ずるブロッキング抗体(AChRとAChとの結合阻事作用)のAChR分子構造上の標的を、一連のアミノ酸配列の中のα183ー200領域と特定、その合成ペプチドは高いACh結合能を有し、また本ペプチドを抗原としてラットを免疫すれば、疾患モデルを作出しうることを証明した。この領域に対する抗ペプチド抗体は、ヒト筋無力症患者血中にも証明できた。本領域の合成ペプチドを固定化した吸着剤と、体外楯環中の患者血液とを反応させ(処理後の血液は再び患者体内へ還流)、有意な臨床効果を得、分子レベルからの本病治療に資した。2.筋無力症発病機構の中でのもう一つの主役であるバインディング抗体(AChR崩壊促進、補体介右性細胞膜破壊作用)のAchR分子構造上の標的として、α67ー76,α70ー90,α125ー147を推定、それぞれの合成ペプチドを抗原としてラットを免疫、疾患モデルを作出し得、これを実証した。各領域に対する抗ペプチド抗体は、ヒト筋無力症血中にも証明でき、将来の免疫吸着療法の具体化に展望をひらいた。3.抗体産生ヘルパ-T細胞認識領域としてAChRαサブユニットのアミノ酸配列の中から、ヒトの場合19カ所を特定したが、抗体反応領域とは一致していなかった。4.重症筋無力症発症は、AChR分子構造の中で、βタ-ン構造をとるB細胞認識領域と、αヘリックス構造のT細胞認識領域の、両領域の免疫学的連係認識の上に成立するとの観点から、両者を種々のかたちで連結したり、人工的配列のアミノ酸を両者間に介在せしめたモデルペプチドを合成、より高い免疫原性、より強い抗体認識性を示す人工的な分子立体構造を特定して、特定の治療用人工抗原材製に資ずる情報を得た。Upon the availability of amino acid sequences and transmembrane topography of acetylcholine receptor (AChR) alpha-subunit, the research attempted to localize myasthenic domains on AChR, such as the sites recognized by the "blocking antibody" which prevents the binding of ACh with AChR and by the "binding antibody" which accelerates the degradation of AChR, by use of peptides synthesized referring to AChR molecular structure, resulting in the following : (1) The synthetic peptide, alpha183-200, was immunogenic in the induction of myasthenia in animals and antigenic in the detection of antibody in human myasthenic patients. (2) Myasthenic patients treated with plasmaperfusion by use of the synthetic peptide (alpha183-200)-bound adsorbent showed clinical improvement in association with the reduction of corresponding anti-peptide antibody and anti-native AChR blocking antibody in sera.(3) Synthetic peptides, alpha67-76, alpha70-90 and alpha125-147, were stimulatory to the induction of myasthenia in animals, and were useful to detect myasthenic antibody in humman myasthenic sera. (4) Nineteen segments in the molecular structure of ACha alpha-subunit were found to be T-cell epitopes, but they were not potent to stimulate B-cells. (5) Artificially formed peptides were synthesized by coupling natural AchR peptides and theoretical amino acid sequences based on the concept that the induction of myasthenia gravis depends on linked recognition of the B-cell epitope expected at beta-turn structure and the T-cell epitope expected at amphipathic alpha-helical structure.These conformationally modified AChR peptides were more immunogenic and antigenic than AChR peptides of natural sequences, and provided a provision for the antigenspecific therapy in myasthenia qravis.研究課題/領域番号:63480215, 研究期間(年度):1988 – 1990出典：研究課題「アセチルコリン受容体と重症筋無力症の免疫、生化学および治療に関する研究」課題番号63480215（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-63480215/634802151990kenkyu_seika_hokoku_gaiyo/）を加工して作

Randomised, double-blind, placebo-controlled study of tacrolimus in myasthenia gravis

Objectives: To evaluate the ability of tacrolimus to reduce the corticosteroid dose in patients with myasthenia gravis (MG) and the drug\u27s safety in a double-blind, placebo-controlled, parallel group study. Methods: Patients being treated with oral prednisolone at doses equivalent to 10-20 mg/day, and with stable symptoms, were randomised to tacrolimus or placebo in a 28-week double-blind study. The dose of corticosteroid was tapered with the procedures specified in the protocol. The primary efficacy endpoint was the mean daily prednisolone dose given in the last 12 weeks of the study. Results: Eighty patients received the study drug (40 patients in each group) and were included in the full analysis set. In the full analysis set, there was no significant difference in the primary efficacy endpoint between the two groups (p = 0.078). However, some secondary analyses suggested the steroid-sparing effect of tacrolimus. Tacrolimus was well tolerated, and no safety concerns were noted. Conclusions: This study suggests that tacrolimus has a potential advantage as a steroid-sparing agent in the treatment of MG patients. Copyright Article author (or their employer) 2011

Pseudo-Argyll Robertson pupil of patients with spinocerebellar ataxia type 1 (SCA1)

金沢大学保健管理センターA pseudo-Argyll Robertson pupil is a neurological sign indicating a normal near reflex but the absence of a light reflex (light-near dissociation), a lack of miosis, and pupil irregularity. It has been reported in patients with diabetes mellitus, multiple sclerosis, Wernicke\u27s encephalopathy, sarcoidosis, tumours, and haemorrhage.1 Although the appearance of pseudo-Argyll Robertson pupil is very similar to Holmes-Adie pupil, the first is distinguishable from the second by the location of lesions and pharmacological response. The responsible lesion in pseudo-Argyll Robertson pupil is in the central region, whereas that of Holmes-Adie pupil is peripheral. Dilute pilocarpine constricts the pupils of patients with Holmes-Adie pupil, but it is not effective in patients with pseudo-Argyll Robertson pupil. We present a patient with spinocerebellar ataxia type 1 (SCA1) and her asymptomatic younger brother who both exhibited pseudo-Argyll Robertson pupil

Hypocalcemic Action of a Pancreatic Factor and its Clinical Significance on the Myasthenic Patients

As reported in the previous short communication1), the authors have been engaged in isolating a protein anabolic factor from hog pancreas for the last eighteen years. The extraction procedure of fraction B reported herein, which is more effective against 15 myasthenic patients than any other drugs, has been established for the last five years

Synaptic Homeostasis and Its Immunological Disturbance in Neuromuscular Junction Disorders

In the neuromuscular junction, postsynaptic nicotinic acetylcholine receptor (nAChR) clustering, trans-synaptic communication and synaptic stabilization are modulated by the molecular mechanisms underlying synaptic plasticity. The synaptic functions are based presynaptically on the active zone architecture, synaptic vesicle proteins, Ca2+ channels and synaptic vesicle recycling. Postsynaptically, they are based on rapsyn-anchored nAChR clusters, localized sensitivity to ACh, and synaptic stabilization via linkage to the extracellular matrix so as to be precisely opposed to the nerve terminal. Focusing on neural agrin, Wnts, muscle-specific tyrosine kinase (a mediator of agrin and Wnts signalings and regulator of trans-synaptic communication), low-density lipoprotein receptor-related protein 4 (the receptor of agrin and Wnts and participant in retrograde signaling), laminin-network (including muscle-derived agrin), extracellular matrix proteins (participating in the synaptic stabilization) and presynaptic receptors (including muscarinic and adenosine receptors), we review the functional structures of the synapse by making reference to immunological pathogenecities in postsynaptic disease, myasthenia gravis. The synapse-related proteins including cortactin, coronin-6, caveolin-3, doublecortin, R-spondin 2, amyloid precursor family proteins, glia cell-derived neurotrophic factor and neurexins are also discussed in terms of their possible contribution to efficient synaptic transmission at the neuromuscular junction

Pathogenic Participation of MuSK-Biglycan Linkage Contributive to Synaptic Stability and Signalings in Myasthenia Gravis

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