Aberrant localization of cornified cell envelop proteins related to abnormal keratinization in oral lichen planus

【緒言】口腔扁平苔癬（Oral lichen planus; OLP）はTリンパ球による上皮結合組織境界部（境界部）の傷害性病変である。病理組織学的には帯状リンパ球浸潤と基底層の液状変性に加え、しばしば角化亢進がみられる。しかし、角化異常に関する研究は乏しく、その発生機序は未だ不明である。角化には、周辺帯（Cornified cell envelope; CE）関連タンパク質が重要な役割を担っている。そこで我々は、OLPの角化異常の機序を明らかにするため、CE関連タンパク質の局在を免疫組織学的に検索し、上皮の傷害性変化や増殖性変化との関連を統計学的に解析した。【実験材料及び方法】両側頬粘膜の網状白斑型OLPの20例を実験材料とし、病理学的に著変のない頬粘膜5例を対照群とした。対照群の上皮厚径を基準に、OLP群の上皮を菲薄部と肥厚部に分類した。上皮の各層の厚径を計測し、基底層の細胞傷害の程度をG0からG3に分類して検討した。境界部の変化を確認するためにCollagen IV （COL4）、Keratin 19 （K19）、Desmoglein 1 （DSG1）、Ki-67に対する一次抗体を用い、角化異常の検討のためにCE関連タンパク質であるInvolucrin（IVL）、Transglutaminase 1（TGM1）、Transglutaminase 3（TGM3）に対する一次抗体を用いて免疫染色を行った。COL4、K19、DSG1、Ki-67は陽性率（陽性細胞数/一定領域の総細胞数）を、CE関連タンパク質は陰性率（下層部の陰性細胞層の厚径/上皮全層の厚径）を算出して解析した。【結果】OLP群の上皮では菲薄部と肥厚部が混在し、肥厚部より菲薄部で傷害の程度が有意に高かった。K19とCOL4陽性率は、対照群に比べOLP群で有意に低下したが、肥厚部と菲薄部の差はなかった。一方、DSG1陽性率は、対照群とOLP群間に有意差はないが、OLP群の肥厚部のDSG1陽性率は菲薄部よりも有意に高かった。Ki-67陽性率は対照群とOLP群間、肥厚部と菲薄部間でも有意差はなかった。IVLは対照群の有棘層下部から細胞質に陽性だが、OLP群では最下層から細胞膜に陽性であった。TGM1は対照群とOLP群の有棘層中央部から細胞膜に陽性だが、OLP群では下層部の細胞質にも陽性を示し、陰性率は対照群よりOLP群で有意に低かった。TGM3は対照群の有棘層下部から核と細胞質に陽性で、OLP群では有棘層中央部から細胞膜にも陽性を示した。陰性率は、対照群よりOLP群で高い傾向だった。TGM1とTGM3の陰性率は弱い負の相関関係を示し、OLP群の肥厚部のDSG1陽性率とTGM3陰性率の間で強い負の相関がみられた。【考察】OLP群ではK19とCOL4発現が減少し、境界部で傷害性変化が生じていることを確認できた。K19の減少は、上皮厚径、COL4およびKi-67と関連性はなく、対照群とOLP群間のみで有意差があり、基底細胞の形質の変化と考えられる。また対照群とOLP群ではKi-67とDSG1陽性率に差はなく、上皮性異形成症といえる明らかな所見はなかった。なお、OLP群の肥厚部でDSG1が有意に高値であった結果は、棘細胞症に伴う基底細胞の形質変化と考えられる。以上のように、実験に用いた試料は、研究対象として適切と考えられる。OLP群ではIVLの細胞膜移行像とともにTGM1が下層部の細胞質から広く発現していた。表皮と同様に、OLPでも基底細胞の細胞質内TGM1がIVLの膜移行を促して、IVLの表皮型の細胞内局在を示していると考えられる。興味深いことに、TGM3は本来細胞質に局在するが、OLPでは細胞膜に発現していた。このような報告はこれまでなく、TGM3の異常な膜発現もIVLの膜移行に関与する可能性がある。OLPではTGM1とTGM3は相補的な関係で分布していた。この関係は表皮のTGM1とTGM3の分布に類似している。また、OLPの上皮肥厚部でDSG1とTGM3の発現に強い相関があり、肥厚部の角化亢進にTGM3が関与する可能性がある。IVLの膜移行は周辺帯形成に必須である。正常粘膜と異なり、TGM1とTGM3の表皮型の上皮内発現が、IVLの細胞膜移行に関与すると考えられる。以上のように、TGM1やTGM3の異所性の局在が、OLPの角化亢進に重要な役割を担っている可能性が示された。2015博士（歯学）松本歯科大

Aberrant localization of cornified cell envelop proteins related to abnormal keratinization in oral lichen planus

松本歯科大学大学院歯学独立研究科博士（歯学）学位申請論

Ectopic transglutaminase 1 and 3 expression accelerating keratinization in oral lichen planus

Abstract Objective: Oral lichen planus (OLP) characterized by interface mucositis frequently shows hyper-keratinization. To clarify mechanisms of excess keratinization, we investigated key molecules for cornified cell envelope (CE). Methods: Involucrin (IVL), loricrin (LOR), transglutaminase 1 (TGase 1) and transglutaminase 3 (TGase 3) were immunohistochemically examined in 20 specimens of OLP; five specimens of buccal mucosa served as controls. Subsequently, the data were statistically analyzed. Results: IVL in OLP was localized in the cell membrane, in contrast to its localization in the cytoplasm in controls. No positive reaction indicative of LOR was noted in any specimens. Although the TGase 1 localization in controls was restricted to the upper three-quarters of the membrane, the localization in OLP was in both membrane and in the cytoplasm of full thickness mucosal layers. The TGase 3 localization pattern was dramatically altered from cytoplasmic to membranous in OLP. Conclusion: Our data suggest that aberrant TGase 1 and TGase 3 localization and distribution are closely related to hyper-keratinization in OLP. This is the first report of ectopic transglutaminase localization in OLP

Dental implant placement performed for two patients with maxillary sinus floor pneumatization

Asymmetry in the size and shape of the sinus is common. Placing dental implants with maxillary sinus floor pneumatization can be challenging. Implant placement is rarely difficult in the anterior maxilla due to insufficient bone volume below the maxillary sinus.The indications and methodology of implant treatment for patients are dependent on the planning of endosseous implant treatment in the esthetic zone with implant therapy and should be based on careful diagnosis.Knowledge and understanding of maxillary sinus floor pneumatization is important to achieve an accurate diagnosis and realize appropriate treatment and management

A case that has been diagnosed with glandular odontogenic cyst by histopathological examination

Glandular odontogenic cyst (GOC) is a rare odontogenic cyst, classified as a developmental odontogenic cyst by the WHO histological typing of odontogenic tumours in 1992. GOC generally occurs in the mandible and arises in each age. The clinical differential diagnosis of odontogenic tumor and cyst is important however, it is difficult to discriminate between them in clinical and radiographical findings. Thus, pathological searches become an essential step for the definite diagnosis. We report a case of GOC, that primary diagnosis was dentigerous cyst by biopsy, but the definite diagnosis was GOC after total extirpation of cyst. Because of a high recurrence rate of GOC, the patients should be followed for a long term

Stability of oral mucosal cell RNA stored in liquid–based cytology medium

We evaluated the stability of RNA stored in liquid–based cytology (LBC) medium for long periods. Specimens were taken from the five volunteers using LBC preparation methods. Experimental samples were preserved at one day, three days, seven days, one month, two months and three months in the medium at room temperature. The RNA was routinely extracted from each specimen, and amplified using human β–actin after reverse transcription method. In all cases, 186 bp β–actin bands were successfully detected until seventh days. However, the detection of RNA was impossible in some cases of one–month, two–month and three–month groups. These results suggest that the LBC method is superior in the preservation of RNA

A case of keratocystic odontogenic tumor occupying the maxillary sinus

Odontogenic keratocysts show a marked tendency toward proliferation or biological activity compared with jawbone cysts, being a disease highly　recurrent after extirpation, which has been characterized as a cystic tumor. The WHO tissue classification of odontogenic cysts revised in 2005 states that, of previous odontogenic keratocysts, those whose epithelium histologically becomes parakeratotic are keratocystic odontogenic tumors. We encountered a keratocystic odontogenic tumor that had extensively progressed in the right maxillary sinus of a 17–year–old boy. In diagnostic imaging, an opaque image containing an impacted tooth was detected, which seemed to invade the right maxillary sinus. Then, the tumor in the right maxillary sinus was resected with a radical operation under systemic anesthesia. Histopathological tests showed that the tumor was coated with stratified squamous epithelium presenting with parakeratosis, with no inflammation found in the fibrous connective tissues under the epithelium. Consequently, a keratocystic odontogenic tumor was diagnosed. The postoperative course was favorable, with no recurrence as of 1 year and 8 months after the operation

口腔粘膜上皮における角質化細胞エンベロープ形成へのトランスグルタミナーゼと基質たんぱく質の寄与

Cornified envelope formation is crucial for the final differentiation of keratinized epithelium.However,the mechanisms of cornified envelope formation in the oral epithelium remain unclear.The aim of this study was to clarify the differences in the distribution and expression of cornified envelope related proteins and genes between keratinized and non-keratinized oral epithelia.We immunohistochemically investigated the distribution patterns of transglutaminase1(TG1),transglutaminase3(TG3),and their substrate proteins involucrin(IVL),loricrin(LOR),and small pro[1]line rich proteins(SPRs),in 19 keratinized and 14 non-keratinized oral epithelium samples.TG1 and TG3 mRNA levels were investigated in both types of epithelium by real time reverse transcription polymerase chain reaction(RT-PCR)using paraf[1]fin-embedded specimens.Data were analyzed to identify factors involved in cornified envelope formation.We demonstrate that 11 localization patterns show statistically significant differences between keratinized and non-keratinized oral epithelia.These factors clearly drove the separation of the two groups during cluster analysis.TG1 mRNA levels in keratinized oral epithelium were significantly higher than those in non-keratinized oral epithelium.In conclusion,the characteristic distribution of trans[1]glutaminases and their substrates and the mRNA levels of TG1 can regulate cornified envelope formation in keratinized oral epithelium, together with the contribution of TG3 first reported in this paper2021博士（歯学）松本歯科大

Preventive Effects of Chlorogenic Acid on Alveolar Bone Loss in Ligature-Induced Periodontitis in Mice

Chlorogenic acid (CGA) is a polyphenol that is present in coffee beans, many vegetables, and fruits. Since CGA has been reported to exert antioxidant and anti-inflammatory effects, it is expected to protect against periodontitis. In the present study, we used a ligature-induced experimental periodontitis model and investigated the beneficial effects of CGA against alveolar bone resorption caused by experimental periodontitis. To examine the inhibitory effects of CGA on bone loss, a ligature was wrapped around the maxillary right second molar, and CGA was intraperitoneally injected once a day for 2 weeks. In another experiment to investigate the restorative effects of CGA on bone loss, a ligature was wrapped around the maxillary right second molar for 2 weeks, it was then removed, and CGA was intraperitoneally injected once a day for 2 weeks. At the end of the experiments, the maxillae were removed, and CT images were taken. Alveolar bone loss was measured as the distance from the cement–enamel junction to the alveolar crest. The statistical analysis was performed using GraphPad Prism6 (Dunn’s multiple comparison test). The results revealed that the ratio of the buccal alveolar bone loss (vs. the bone loss on the nonligated side) induced by ligation was significantly decreased by the administration of CGA (5 mg/kg) for 2 weeks. Moreover, the bone loss ratio on the buccal and palatal sides after 2 weeks of ligation was significantly decreased by the 2-week administration of CGA (5 mg/kg). The present results revealed that CGA exerted preventive effects against alveolar bone loss caused by experimental periodontitis

Phenotypic alteration of basal cells in oral lichen planus; switching keratin 19 and desmoglein 1 expression

In oral lichen planus, extracellular matrix and basal cells are damaged by T-lymphocytes. As a consequence, changes in expression of collagen fibers within the connective tissue and cytoskeletons of the epithelial tissue can be observed. With the goal of examining the characteristic changes undergone by basal cells as a consequence of T-lymphocytes damage in oral lichen planus, we investigated protein expression in the epithelial-connective junction. We selected 20 cases of oral lichen planus and 5 control samples of buccal mucosa. Subsequently, we divided the oral lichen planus cases into thin and thick parts based on the mean values of epithelial thickness from the control samples, and counted the positive rate of collagen IV, keratin 19, desmoglein 1, and Ki-67. Collagen IV immune-reactivity partially disappeared or thickened in oral lichen planus. The keratin 19 positive rate in oral lichen planus cases was significantly lower than in the controls. Desmoglein 1 positive rate of the thick part was significantly higher compared to the thin part of oral lichen planus. Thus, modifications in basal cells with both reduced keratin 19 expression and alterations of desmoglein 1 expression suggest that in oral lichen planus, as a consequence of cell injury or regeneration in the interface area, there is a disappearance of the “true basal cell nature”