Localized scleroderma is an autoimmune disorder

金沢大学大学院医学系研究科Objectives. There have been many studies suggesting that localized scleroderma has a strong autoimmune background, although the lesions are usually limited to the skin and subcutaneous tissue. Here we summarize previous data on the autoimmunity of localized scleroderma, mostly published in the last two decades, because there has not been a review paper summarizing autoimmunity in this disorder. Methods. We classified the previous reports into three categories: antinuclear antibodies; cytokine and soluble receptors; and cell adhesion molecules and cell surface molecules. In each category, we introduce the important investigations. Results. High frequencies of antinuclear antibodies, detected by the indirect immunofluorescence method using cultured cells, are confirmed by many groups. The major autoantigens have been revealed to be histones. Recently, anti-topoisomerase II α antibody has been found to be detected highly frequently in localized scleroderma, while anti-topoisomerase I antibody, which is highly specific for systemic sclerosis, has not been detected in any case of localized scleroderma. In other studies, elevated serum cytokines and cell adhesion molecules suggest the immunoactivation of localized scleroderma. Conclusions. Many previous studies conclude that localized scleroderma involves autoimmune abnormalities and is one of the organ-specific autoimmune disorders targeting mainly skin, although the types of autoimmune abnormality are different from systemic sclerosis

Identification of Myocardial Damage in Systemic Sclerosis: A Nuclear Cardiology Approach

Myocardial involvement is an important prognostic factor in patients with systemic sclerosis, and early diagnosis and staging of the disease have been sought after. Since myocardial damage is characterized by connective tissue disease, including fibrosis and diffuse vascular lesions or microcirculation, nuclear myocardial perfusion imaging has been a promising option for evaluating myocardial damages in early stages. In addition to the conventional stress-rest perfusion imaging, the current use of quantitative electrocardiographic gated imaging has contributed to more precise evaluation of cardiac perfusion, ventricular wall motion, and diastolic function, all of which have enhanced diagnostic ability of evaluating myocardial dysfunction. Abnormal sympathetic imaging with Iodine-123 metaiodobenzylguanidine might be another option for identifying myocardial damage. This paper deals with approaches from nuclear cardiology to detect perfusion and functional abnormality as an early sign of myocardial involvement as well as possible prognostic values in patients with abnormal imaging results. The role of nuclear cardiology in the era of multiple imaging modalities is discussed

サイトカイン誘導制皮膚線維化マウスモデルにおけるコラーゲン合成亢進機構の解明

我々は世界に先駆けて新生マウス皮下にサイトカイン,特にTGF-β及びCTGFを連続注入することにより,サイトカイン誘導性皮膚線維化マウスモデルを確立した。このモデルにおいてはTGF-βが線維化を誘導し,CTGFは線維化を維持するというプロセスがin vivoにおいて確認されたが,両者の線維化誘導の役割の詳細は明らかにされていない。そこで平成17年度は,in vitroの皮膚線維芽細胞培養系を用いてその機序を明らかにすることを試みた。(1)TGF-βは単独でコラーゲン遺伝子を活性化する。(2)CTGFは単独ではコラーゲン遺伝子を活性化しない。(3)TGF-β単独でコラーゲン遺伝子は約1.5倍活性化されるのに対して,TGF-β及びCTGFを加えると約2倍活性化されるのに対して,TGF-β及びCTGFを加えると約2倍の活性がみられる。(4)TGF-β刺激を受けた皮膚線維芽細胞においては,c-mycの発現の低下がみられ,細胞増殖の低下もみられる。(5)TGF-β+CTGF刺激を受けた皮膚線維芽細胞においては,TGF-β単独より更に高度なc-mycの低下がみられた。(6)強皮症病変部由来線維芽細胞においては,逆にc-mycの上昇がみられ,TGF-β+CTGF刺激により,細胞上昇もみられた。以上の所見は,強皮症病変部由来皮膚線維芽細胞自身にサイトカイン,特にTGF-β,CTGF共存下における両者に対する異常反応を有することが本症の病因となりうることが示された。We established cytokine induced skin fibrosis model. We injected TGF-β first which induces skin fibrosis and CTGF next which maintain skin fibrosis. In this study, we tried to clarify the detailed mechanism of this fibrosis model using in vitro study culture systems.(1) TGF-β alone induced collagen m-RNA.(2) CTGF alone had no effect.(3) TGF-β plus CTGF induced collagen m-RNA more than TGF-β alone.(4) TGF-β suppressed c-myc-mRNA in normal fibroblasts.(5) In scleroderma fibroblasts c-myc-mRNA was enhanced by TGF-β plus CTGF stimulation.Thus, abnormal response to scleroderma fibroblast may contribute to skin fibrosis in scleroderma.研究課題/領域番号:16591093, 研究期間(年度):2004-2005出典：「サイトカイン誘導制皮膚線維化マウスモデルにおけるコラーゲン合成亢進機構の解明」研究成果報告書　課題番号16591093 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

結合組織成長因子(CTGF)の強皮症病変形成への関与

本研究は,トランスフォーミング成長因子(Transforming Growth Factor-β,TGF-β)皮下投与により誘導される新生マウスの皮膚線維化モデルを用いて,結合組織成長因子(Connective Tissue Growth Factor,CTGF)の役割とTGF-βとの相互作用について,その詳細を検討した。TGF-β単独投与により誘導される線維化は3日目をピークとして消失するが,TGF-βとCTGFを同時投与するか,あるいはTGF-β3日間投与後にCTGFを投与することにより不可逆的な線維化が形成されることが明らかにされた。以上の結果から,皮膚の線維化がTGF-βによって誘導され,CTGFによって維持されるという仮説が強く示唆された。そこで,このモデルを用いて抗CTGF中和抗体による線維化の抑制を試みたところ,一部抑制効果が確認され,さらにより中和活性の強い抗体のスクリーニングを試みている。またCTGFアンチセンスの作用についても検討する予定である。また,全身性強皮症患者の血清CTGF濃度を多数例で検討したところ,正常人に比較して有意な上昇がみられただけでなく,皮膚硬化や肺線維症などの疾患重症度と血清CTGF濃度との相関が認められ,線維化疾患の代表ともいえる全身性強皮症の病因にCTGFが関与していることが示された。今後我々が確立したTGF-βおよびCTGFによるマウス線維化モデルを用いて,全身性強皮症に対する治療薬のスクリーニングを行い,臨床応用に結びつけたい。In these studies, we investigated the role of CTGF, namely inter action with TGF-β in TGF-β induced skin fibrosis model of newborn mice.TGF-β solely induced fibrosis disappears with 3 day peak, although when CTGF was injected simultaneously or consecutively, combination of TGF-β and CTGF induces irreversible fibrosis.Above observations strongly support our hypothesis that TGF-β induces skin fibrosis and CTGF acts to maintain this fibrosis. In addition, we tried to block this fibrosis using anti- CTGF neutralizing antibody, we conformed partial suppression. We are going to screen more potent neutralizing antibody and antisense of DNA.We measured serum CTGF levels of systemic sclerosis using increased cases, we found elevated levels of CTGF the patients and the levels of CTGF was statistically correlated with severity of skin sclerosis and pulmonary fibrosis. Thus, we found strong correlation between CTGF and systemic sclerosis, which is a representative fibrotic disorder.In the future, we use our skin sclerosis mice model by TGF-β and CTGF to screen anti-scleroderma drugs.研究課題/領域番号:10670785, 研究期間(年度):1998-1999出典：「結合組織成長因子(CTGF)の強皮症病変形成への関与」研究成果報告書　課題番号10670785 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

DNA免疫法による抗CTGF抗体のサイトカイン誘導皮膚線維化マウスモデルの抑制

我々は形質細胞増殖因子β(TGF-β)と結合組織増殖因子(CTGF)という2つのサイトカインを新生マウス皮下に注入することによって確立した皮膚線維化モデルマウスにおける、抗CTGFマウスモノクロナル抗体による線維化の抑制を試みた。ヒトCTGFDNAを組み込んだベクターとジーンガンにてマウスに免疫するDNA免疫法により、CTGFmodule1〜4に対するモノクロナル抗体、17株の樹立に成功した。これら17株の抗CTGF中和活性についてスクリーニングを重ねたところ、抗CTGFmodule2に対する抗体の中和活性が最も強力であることが皮膚線維化モデルマウスにおいて示された。同抗体は、皮膚線維化モデルマウスに対して注入される前半のTGF-β及び後半のCTGFのいずれにおいても線維化を著明に抑制した。特に前半のTGF一β注入期においては、産生されたコラーゲンの蛋白量の50%、RT-PCRにおけるm-RNAの70%の抑制が得られた。以上のことは我々が得た抗ヒトCTGFモノクロナル抗体は、マウスの内因性のCTGFの活性をも抑制したことを意味する。今後は、皮膚以外においても、肝硬変モデル、腎硬化症モデル、肺線維症モデルなど他の線維化疾患の動物モデルにおいても、抗ヒトCTGFモノクロナル抗体の線維化抑制効果を検討するとともに、マウス抗ヒトCTGFモノクロナル抗体をヒト化し、各種ヒト線維化疾患、特に強皮症に対する新規治療薬確立への臨床応用の基盤を展開させたい。We established skin fibrosis animal model by subcutaneous injection of two cytokines, TGF-β and CTGF. We have conducted this project to inhibit this fibrosis model by anti-CTGF antibody.We succeeded in establishment of anti-human CTGF module 1, 2, 3 and 4 antibodies by DNA immunization method of human CTGF vector and gene gun. After the several screening studies, the anti-human CTGF module 2 antibody was revealed to be most potent of neutralizing activity.The anti-human CTGF antibody inhibited skin fibrosis dramatically, both in the early phase of TGF-β injection and the late phase of CTGF injection. In this experiment, this anti-human CTGF antibody inhibited mouse internal CTGF action.In the future, we are planning to create humanized anti-CTGF antibody for the clinical application.研究課題/領域番号:18591240, 研究期間(年度):2006-2007出典：「DNA免疫法による抗CTGF抗体のサイトカイン誘導皮膚線維化マウスモデルの抑制」研究成果報告書　課題番号18591240 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

Eisenhart Lift of 22--Dimensional Mechanics

The Eisenhart lift is a variant of geometrization of classical mechanics with $d$ degrees of freedom in which the equations of motion are embedded into the geodesic equations of a Brinkmann-type metric defined on $(d+2)$-dimensional spacetime of Lorentzian signature. In this work, the Eisenhart lift of $2$-dimensional mechanics on curved background is studied. The corresponding $4$-dimensional metric is governed by two scalar functions which are just the conformal factor and the potential of the original dynamical system. We derive a conformal symmetry and a corresponding quadratic integral, associated with the Eisenhart lift. The energy--momentum tensor is constructed which, along with the metric, provides a solution to the Einstein equations. Uplifts of $2$-dimensional superintegrable models are discussed with a particular emphasis on the issue of hidden symmetries. It is shown that for the $2$-dimensional Darboux--Koenigs metrics, only type I can result in Eisenhart lifts which satisfy the weak energy condition. However, some physically viable metrics with hidden symmetries are presented.Comment: 20 page

Decreased levels of regulatory B cells in patients with systemic sclerosis: Association with autoantibody production and disease activity

Objective. B cell abnormalities characterized by autoantibody production and polyclonal B cell activation play an important role in the pathogenesis of SSc. IL-10 producing regulatory B (Breg) cells also play an important role in the negative immune response. We identified a human Breg cell subset that was predominantly found within the CD24hiCD27+ B cell subpopulation. However, the role of Breg cells in SSc remains unknown. The aim of this study was to investigate the clinical association of Breg cells in SSc patients. Methods. Blood IL-10 producing Breg cell levels were determined by FACS in 35 SSc patients and 30 healthy subjects. In a follow-up study, we analysed six individual dcSSc patients before and after treatment. Results. The frequency of blood Breg cells was significantly lower in SSc patients than in healthy controls (P<0.0001). Similarly, the frequency of CD24hiCD27+ B cells was significantly lower in SSc patients than in healthy controls (P<0.0001). SSc patients with decreased Breg cell levels often had interstitial lung disease (P<0.05). Furthermore, Breg cell levels correlated negatively with the titre of anti-topo I antibody (Ab) and anticentromere Ab in SSc patients. For a follow-up study, Breg cell levels in dcSSc patients after treatment were found to be significantly increased compared with those before treatment (P<0.05), accompanied by decreased disease activity. Thus, Breg cell levels were inversely correlated with disease activity of SSc. Conclusion. These results suggest that decreased Breg cell levels may contribute to the development of SSc. © The Author 2015.Embargo Period 12 month

Expression of fractalkine and its receptor, CX3CR1, in atopic dermatitis: Possible contribution to skin inflammation

Background: Fractalkine (FKN) induces activation and adhesion of leukocytes expressing its receptor, CX3CR1. FKN is released from the cell surface through proteolytic cleavage as soluble FKN (sFKN). Objective: We sought to assess FKN and CX3CR1 expression in the skin, serum sFKN levels, and CX3CR1 expression on blood leukocytes in patients with atopic dermatitis (AD). Methods: FKN and CX3CR1 expression in the skin was examined immunohistochemically. mRNA expression of FKN, thymus and activation-regulated chemokine, and macrophage-derived chemokine in the skin was assessed by means of real-time RT-PCR. Serum sFKN levels were assessed by using ELISA. Blood leukocytes were stained for CX3CR1 by means of flow cytometric analysis. Results: FKN was strongly expressed on endothelial cells in skin lesions of patients with AD and psoriasis but not in normal skin. FKN mRNA levels in AD lesional skin increased to a similar extent to thymus and activation-regulated chemokine and macrophage-derived chemokine mRNA levels. CX3CR1-expressing cells in the affected skin of patients with AD or psoriasis increased compared with those in normal skin. Serum sFKN levels were increased in patients with AD but not in patients with psoriasis relative to levels in healthy control subjects. Serum sFKN levels were associated with the disease severity and decreased with the improvement of skin lesions in patients with AD. CX3CR1+ cell frequencies and CX3CR1 expression levels were decreased in CD8+ T cells, monocytes, and natural killer cells from patients with AD, but this was not observed in patients with psoriasis. Conclusions: These results suggest that through functions in both membrane-bound and soluble forms, FKN plays an important role in the trafficking of CX3CR1+ leukocytes during the inflammation caused by AD

Connective Tissue Growth Factor Gene Expression in Tissue Sections From Localized Scleroderma, Keloid, and Other Fibrotic Skin Disorders

Connective tissue growth factor (CTGF) is a novel peptide that exhibits platelet-derived growth factor-like activities and is produced by skin fibroblasts after activation with transforming growth factor-β. Coordinate expression of transforming growth factor-β followed by CTGF during wound repair suggests a cascade process for control of tissue regeneration. We recently reported a significant correlation between CTGF mRNA expression and histologic sclerosis in systemic sclerosis. To confirm the relation between CTGF and skin fibrosis, we investigated CTGF gene expression in tissue sections from patients with localized scleroderma, keloid, and other sclerotic skin disorders using nonradioactive in situ hybridization. In localized scleroderma, the fibroblasts with positive signals for CTGF mRNA were scattered throughout the sclerotic lesions with no preferential distribution around the inflammatory cells or perivascular regions, whereas the adjacent nonaffected dermis was negative for CTGF mRNA. In keloid tissue, the fibroblasts positive for CTGF mRNA were diffusely distributed, especially in the peripheral expanding lesions. In scar tissue, however, the fibroblasts in the fibrotic lesions showed partially positive signals for CTGF mRNA. In eosinophilic fasciitis, nodular fasciitis, and Dupuytren's contracture, CTGF mRNA was also expressed partially in the fibroblasts of the fibrotic lesions. Our findings reinforce a correlation between CTGF gene expression and skin sclerosis and support the hypothesis that transforming growth factor-β plays an important role in the pathogenesis of fibrosis, as it is the only inducer for CTGF identified to date