カエルツボカビ（Batrachochytrium dendrobatidis）の起源およびツボカビ症の病理発生に関する研究

緒言　カエルツボカビ（Batrachochytrium dendrobatidis : Bd）は世界的な両生類の種類および個体数の減少に関与する新興病原体である。2006年、我々はアジアおよび国内で初めてツボカビ症を発見した。その後、全国調査や病性鑑定を行った結果、流行が起きている諸外国と異なる次の2点が明らかになった。①国内野生下両生類にBdによる大量死が認められない。②Bdのhaplotypeの多様性は、被害が大きい諸外国では低いが、日本は非常に高い。この状況は、Bdの被害が認められないアジア諸外国でも同様であった。このことから、Bdはアジアおよび日本を起源とし、世界中に拡散したという「アジア起源説」が提唱されている。しかし、アジア地域においてはBdが発見されて間も無く、感染率が低いため、未だ結論に至っていない。一方、ツボカビ症の病理発生機序には、皮膚ツボカビ症を発症した後、電解質異常をきたし、心不全によって死亡することが知られている。しかし、これは死因を特定したのみでその機序は不明である。カエルの皮膚はガス交換、電解質輸送、水吸収などを行っており、生命維持に重要な役割を担っていることから、ツボカビ症の病理発生機序を解明するためには、カエルに特徴的な生理学的機能の破綻を解明する必要がある。　本研究の目的は、アジア起源説を念頭に入れて、Bdの国内分布および遺伝子型解析からBdの由来を解明すること。そして、両生類の皮膚の生理学的特徴である皮膚に分布する水チャネルaquaporin（AQP）に注目して、皮膚ツボカビ症の病理発生機序を解明することである。第1章　ウシガエル幼生における口器ツボカビ症および検査法の確立　Bdの国内実態を的確に調べるために、後述する理由から、ウシガエル（Lithobates catesbeina）幼生の好適モニター動物としての検証を行った。ウシガエルは国内に広く分布しており、過去の調査によってBd感染率が高く、保菌するhaplotypeの種類も多いことが確認されている。また、幼生個体は採集が容易であり、かつ、Bdの感染部位である角質層が口器に限局していることから、口器のみを検査対象とすることができる。さらに本章では、的確にBdを検出する検査法についても検討した。　一生息池からウシガエル幼生59匹を採集し、これらの口器を病理検査（肉眼検査と組織検査）および口器から採取したswabを用いたPCR検査（nested PCR法）を行った。その結果、21匹の口器に肉眼的変化を認め、そのうち20匹にPCR検査または病理組織検査でBd感染を確認した。肉眼的変化がなかった38匹のうち17匹からPCR検査、病理組織検査のいずれか、または両方でBdを検出した。よって、62.7％の感染率であった。Bd感染による口器肉眼的変化の特徴は、嘴の①色素消失、②色素沈着層の菲薄化、③変形および④歯列の淡明化であった。　以上より、ウシガエル幼生はBdを高率に保菌し、目視によって容易に感染を確認できることから、Bdモニター動物として有効であると結論づけた。　ウシガエル幼生の口器のBdを的確に検出する検査法を確立するために、nested PCR法（swab、口器）および口器病理組織検査を比較した。2ヵ所の生息池から各60匹、合計120匹を採集した。1検体ですべての検査を実施できないため、目視検査によって口器異常を有する個体が同程度に含まれるように2群に分け、1群はswabおよび口器を材料とするnested PCR法、2群は口器病理組織検査を行った。各種検査の検出率は、swab nested PCR法（67%）、口器nested PCR法（65%）および口器病理組織検査（33%）の順に高かった。感度は口器よりもswabを材料とするnested PCR法の方が高かったが、統計学的に有意差を認めないこと、swabの採取は採取者の手技習熟度の影響を受けることから、口器を材料とするnested PCR法が推奨された。　以上より、ウシガエル幼生の口器を採材し、nested PCR法を行うことで、的確なBd感染状況を把握できると結論づけた。第2章　ウシガエル幼生におけるBd感染の季節変動の把握　Bd調査適期を決定するために、ウシガエル幼生のBd感染率の季節変動を検討した。2011年12月～2014年6月の31ヶ月間のうち、2012年12月を除く偶数月に長崎県の一生息池から15回にわたって、各回ウシガエル幼生を15匹または30匹、計345匹を採集した。これらの口器をnested PCR法に供し、感染率の推移を調べた。　その結果、日本におけるBdの感染率に明らかな季節変動があった。最も感染率が高かったのは4～6月で、平均79%であった。感染率が最も低かったのは8～10月で、感染を認めなかった。2月、12月は7～63%と、採集年によって感染率が異なっていた。　以上より、国内自然界におけるBd感染動態の年間サイクルは、冬～春にかけて増殖し、気温が発育至適温度を上回る夏季に大きく減少した後、晩秋～冬にかけて再び増殖すると結論づけた。従って、Bd国内実態調査の採集適期を2～6月とした。第3章　野生下ウシガエル幼生を対象としたBdの国内分布と遺伝子型　野生下ウシガエル幼生におけるBd感染率、保有haplotypeを解析し、Bd国内実態を調べた。2012年および2013年の各1～6月、26都府県44地域から835匹のウシガエル幼生を採集し、そのPCR産物からダイレクトシーケンス法によりBdのITS領域の塩基配列を決定し、既存のhaplotypeと比較、各haplotypeの国内分布を調べた。　その結果、19都県、29地域、266匹（感染率32%）に感染を認めた。検出地域におけるBd感染率は平均51%（3～100%）であった。266検体中68検体の塩基配列が決定され、これらは41種類のhaplotypeに型別された。そのうち、6 typeは既存のhaplotypeと一致し、その内訳は世界流行系統（A type：19、CW34 Clone N：1検体）、中国検出株（Bd50：1、Bd41：1 検体）、P type：2、Z type：2、検体であった。他35 typeは過去に報告のないものであった。なかでもA typeの高率かつ広域分布を確認した。国内、中国、アフリカ、ブラジル、オーストラリア、エクアドルおよびテキサス検出株の遺伝子データとともに、系統解析（近隣結合法）を行った。Clade形成に明瞭な地域差はなく、中国検出株と同じcladeを形成している検体も認められた。　以上より、Bdは日本およびアジアでは固有病原体（endemic）であり、長い歴史を経て宿主両生類と共に共進化している可能性があると考えた。第4章　Bd感染が皮膚水吸収に与える影響　ツボカビ症の病理発生を明らかにするために、次の実験を行った。Bd高感受性動物イエアメガエル（Litoria caerulea）感染群5匹、対照群5匹、計10匹を用いた。感染群には、培養したBd遊走子1×106、5,000または6,000個/mlの飼育水を1日浸漬させた後、7日間、通常飼育し、軽度ツボカビ症を発症させた。対照群は7日間、通常飼育した。これらのカエルを安楽死させた後、Ussing chamber改良装置を用いて大腿部と背部の皮膚の水透過性試験を行った。併せて、皮膚を病理組織学的に、血液を血清生化学的に調べた。Bd感染確認は全個体の皮膚を病理組織学的に観察した他、5匹（感染群：3匹、対照群：2匹）の皮膚のnested PCR検査を行った。　感染群5匹中、3匹にツボカビ症の特徴である高度の脱皮を認め、そのうち、1匹は食欲不振、元気消失を伴っていた。5匹中2匹に病理組織検査またはnested PCR法によりBd感染を認めた。水透過性試験において、大腿部皮膚の水透過性における水分調節機能の障害を確認した。すなわち、抗利尿ホルモン（arginine vasotocin: AVT）反応性に働くAQPa2S(AQP6vs)を介する水透過性が有意に減少し、AVT刺激の有無に関わらず水を透過させるAQP3を介する水透過性が有意に上昇した。背部皮膚には、感染群、対照群ともに水透過性を認めなかった。病理組織学的検索では、大腿部、背部ともに表皮角質層・表皮細胞層の肥厚および表皮細胞数の増数を認めたが、有意差は得られなかった。その他、表皮細胞の変性などを観察した。血清生化学検査では、血漿膠質浸透圧、Na、Mg、CaおよびClの減少を認めた。これらのうち、MgとCaの変化には有意差を認めたが、その他の成分の変化に有意差は認められなかった。　以上の結果より、ツボカビ症の病理発生機序において、Bd感染から心不全に至る経路の1つに皮膚水透過性を介する水分調節機能の障害が関与し、電解質バランスの恒常性を維持できなくなり、心不全に至ると結論づけた。感染群の一部にBd感染が確認できなかったこと、電解質成分の減少と病理組織変化が軽度であったことは、発症の程度が軽症であったことによるものと考えた。IntroductionChytrid fungs (Batrachochytrium dendrobatidis: Bd) is emerging pathogen that associated with global decline of amphibian species and individuals. We detected chytridiomycosis at first in Japan and in Asia in 2006. The Japanese field survey and pathological examination detected 2 differences with large scale damaged foreign countries; the amphibian mass die-off from Bd epidemic has not detected and diversity of Bd haplotype of Japan were higher than that of damaged foreign countries. These situations were similar in the Asian foreign countries where have not damaged by Bd. Thus, the hypothesis: Bd originated from Asia and Japan (Bd originated Asia hypothesis) has occurred, but this hypothesis has not concluded because Bd plevarence in Asia is very low and it had passed only short term after Bd detected in Asia. On the other hand, pathogenesis of chytridiomycosis has not yet been completely clarified because it is only observed cause of death known that infected frog dead by osmotic imbalance through loss of electrolytes and cardiac arrest after effected skin chytridiomycosis. To clarify pathogenesis of chytridiomycosis, disruption of amphibian unique physiological function by chytridiomycosis must be observed because the integument of frog is a site of regulated transport for water, ions (electrolytes) and respiratory gases. The aim of this study was to identify the origin of Bd by detecting distribution and genetic analysis of Bd in Japan with in mind that Bd originated Asia hypothesis. And to reveal pathogenesis of skin chytridiomycosis focused on the water channel, aquaporin (AQP) that amphibian unique physiological function.Chapter 1. Confirming of oral chytridiomycosis in bullfrog (Lithobates catesbeiana) larvae and establishment of Bd examination method We examined bullfrog (Lithobates catesbeiana) larvae (tadpole) usability as monitor of Bd field survey, since the reasons as follows. Bullfrog distributed over Japan widley, and it is known that this animal harbor Bd highly and its variety of haplotype is widely. We can capture bullfrog tadpole easily, and only examine the larval mouthparts that only distributed keratinized layer that area of Bd infection. Additionally, we established the examination method which can detect Bd infection exactly in this capter. Fifty nine wild-captured bullfrog tadpole mouthparts underwent macroscopic, histopathological examination and nested PCR. Macroscopic lesions were observed in 21 of 59 tadpoles, and its 20 tadpoles were detected Bd infection by nested PCR and/or histopathological examination. Seventeen in 38 tadpoles that were not observed macroscopic lesions, were detected Bd indection. Total prevalence was 62.7% in these tadpoles. The macroscopic lesion characters were depigmentation, thinning of pigmented layer and partial defect of jaw sheaths and whited tooth rows. We concluded that bullfrog tadpole is usable for Bd field survey because the animal is compromised host for Bd infection and its mouthparts legion is easily detected by macroscopic examination. To establish the usable examination method, we compared nested PCR (materials: swab and mouthparts) and histopathological examination of mouthparts. One hundred twenty wild-captured bullfrogs from 2 habitas were divided into 2 groups, each group were 60 individuals, because we unable to conduct all examination methods to same one tadpole. The group 1 was sampled mouthparts swab and dissected mouthparts, and conducted nested PCR. Another group were conducted histopathological examination. Detection rate of each examination was high in order of swab nested PCR (67%), mouthparts nested PCR (65%) and histopathological examination (33%). The mouthparts nested PCR was recommended as Bd survey, however, sensitivity of swab nested PCR was higher than that of mouthparts nested PCR, because the sampling of swab was effected by sampling techniques. We concluded that collecting bullfrog tadpole mouthparts and conduct nested PCR can detect Bd infection situation in Japanese field. Chapter 2. Seasonality of Bd infection of bullfrog tadpole To decide term of Bd field survey, we detected seasonality of Bd infection of bullfrog tadpole. We collected 15 or 30 tadpoles in even months without December 2012, and totally 345 tadpoles were collected in between December 2011 and June 2014. The prevalence rate at each collected month was detected by nested PCR of mouthparts of these tadpoles. The seasonality of Bd prevalence in Japanese bullfrog was revealed: the highest prevalence was April and June that average prevalence was 79%, the lowest prevalence was Augst and October that has not detected Bd infection. February and December detected Bd infection, however, the prevalence of these months were different in collected year that 7 to 63%. Bd infection dynamic cycle amoung the year is seems that Bd grows and diffuses in winter to next spring, and largely decreases in summer that temperature is higher than that of Bd growth optimal temperature, and grows in late autum to winter. We decided that sampling Bd survey conduct between February and June as described above.Chapter 3. Distribution and haplotype of Bd infected in bullfrog in Japanese field We surveyed the Bd situation in Japan such as prevalence and Bd haplotype that infected in wild-captued bullfrog in Japan. 2012 and 2013, we collected 835 tadpoles from 44 areas of 26 counties, between February and June in each year. We conducted nested PCR from these samples, and determined the ITS gene region sequence from Bd positive samples, and conducted phylogenetic analysis with Bd that has been detected in previous studies. Bd detected 266 individuals (32%) from 29 areas of 19 counties. The average prevalence was 51% (3 to 100%) in detected Bd areas. Sixty eight samples were detected sequence from 266 samples and were distinguished 41 haplotypes. Six types were already isolated haplotypes; Bd global pendemic lineage (A type: 19, CW34 Clone N: 1 sample), isolated in China (Bd50: 1, Bd41: 1 sample) and isolated in Japan (P type: 2, Z type: 2 samples). The other 35 types were newly identified haplotypes in this study. Interestingly, A type was detected highly and distributed widely in Japan. We conducted phylogenetic analysis by neighbor-joining method with Bd which isolated in previous study such as China, Africa, Brazil, Australia, Ecuador and Texas. Distinguishable areal difference was not observed in clade of phylogenetic tree, and partial clade was included with Chinese haplotypes. These result likely suggest that Bd is originated from Asia and Japan, and this pathogen seems to coevolved with host amphibians for long history.Chapter 4. The effect on skin water absorption by Bd infection To clarify the pathogenesis of chytridiomycosis, we performed skin water permeability test and histopathological examination of skin and biochemical test with animal experiment. Ten frogs, Litoria caerulea from commercial market were eradiated Bd because some were already infected, and randomly assigned to exposure and control groups (each group were 5 frogs). Infection group frogs were exposed Bd via shallow immersion in bath of diluted water containing 1 ×106, 5,000 or 6,000 zoospores per 1ml. After 24 h frogs were moved to fresh container and keep 6 days. Control group were immersed in diluted water for 24 h, and keep 6 days similarly. Thigh and dorsal skins were sampled and conducted water mermeability test using a modified Ussing chamber. Ventral and dorsal skins were conducted histopathological examination. And collected blood was conducted biochemical test. To obtain Bd infection, we examined skin of all frogs, and conducted nested PCR of 5 frog skins (infection group:3, control group: 2). Three of 5 frogs of infection group showed severe skin sloughing, and 1 frog was assossiated with anorexia and lethargy. Two of 5 frogs of infection group were obtained Bd infection by histopathological examination or nested PCR. Disruption of water regulatory function via thigh skin was observed: water absorption associated arginine vasotocin (AVT) reactive AQPa2s(AQP6vs) significantly decreased, however, water absorption associated with AQP3 that permeable with or without AVT stimulation significantly increased. Dorsal skin water permeability was not observed in all animals. Histologically, thicknning of stratum corneum and skin epidermal hyperplasia of both thigh and dorsal skin were observed in infected frogs, but not detected significant differences. The osomorality, Na, K and Cl level decreased slightly, and Mg and Ca level significantly decreased in biochemical test.博士(獣医学)麻布大

Observações preliminares sobre a toxicidade do ϒBHC e do cloreto de mercúrio à Artemia salina

Results of bio-assays using Artemia salina larvae in γBHC-HgCl2 mixture are presented. The results obtained are compared with those obtained using the two chemicals separately. The LC50 for γBHC was almost double that for HgCl2. The effect due to the primary dose of HgCl2 was observed for approximately 16% of the population (LC2.3) while for γBHC the population percentage was 2.3 (LC2.3). As a consequence, subsequent doses were more effective for HgCl2 than for γBHC. The effective dose 50% (ED50) is about the same for γBHC and for γBHC + HgCl2 mixture, though in higher doses BHC would be more effective, while in smaller doses the mixture would predominate.São apresentados os resultados de bioensaios com larvas de Artemia salinacolocadas em misturas de γBHC e HgCl2, a fim de se compararem os efeitos das mesmas com aqueles obtidos quando essas duas substancias atuam isoladamente. A CL50 para o yBHC foi quase que o dobro do valor para o HgCl2. O efeito da primeira dose foi observado para aproximadamente 16% da população (CL16), enquanto que, para o γBHC, o foi em 2,3% da população (CL2,3). Assim, as doses subseqüentes foram mais efetivas para o HgCl2 que para o γBHC. A dose efetiva 50% (ED50) ê mais ou menos igual para o γBHC e para o γBHC + HgCl2, mas em doses altas o YBHC seria mais efetivo, enquanto que em doses baixas a mistura e que o seria

琉球処分以降における転換期の唐手に関する一考察

The aim of this paper is to investigate how唐手(Toudi) was evaluated in society between 1879, when Okinawa Prefecture was established, and 1905 when Toudi was initially adopted as part of the gymnastics program at junior high schools in Okinawa. This attempt to clarify some aspects of Toudi after the Ryukyu Annexation is based on research using materials such as articles in Ryukyu Shinpo, the local newspaper, and educational magazines of that time. An examination of such documents reveals that the era was an important turning point for “Toudi” on its journey to becoming “Karate,” which is still written using the same characters, 唐手. Karate was, on one hand, condemned as “ one of the customsthat should be refined” under the assimilation educational policy of the Meiji Government. On the other hand, it was widely practised at local annual events and welcome parties for military personnel. Karate also found a place in school activities like farewell parties or sports meets of the Okinawa Prefecture Normal School

Fucoxanthin, a Marine Carotenoid Present in Brown Seaweeds and Diatoms: Metabolism and Bioactivities Relevant to Human Health

The marine carotenoid fucoxanthin can be found in marine brown seaweeds, the macroalgae, and diatoms, the microalgae, and has remarkable biological properties. Numerous studies have shown that fucoxanthin has considerable potential and promising applications in human health. In this article, we review the current available scientific literature regarding the metabolism, safety, and bioactivities of fucoxanthin, including its antioxidant, anti-inflammatory, anticancer, anti-obese, antidiabetic, antiangiogenic and antimalarial activities, and its protective effects on the liver, blood vessels of the brain, bones, skin, and eyes. Although some studies have shown the bioavailability of fucoxanthin in brown seaweeds to be low in humans, many studies have suggested that a dietary combination of fucoxanthin and edible oil or lipid could increase the absorption rate of fucoxanthin, and thus it might be a promising marine drug

ホゴシャ ノ コウドウ キロク シエン ノ タメ ノ LINE Bot ノ カイハツ

行動論的アプローチにおいて，支援者が対象児者の家族での行動に対して介入する際に，保護者が日常生活の中で当該行動の記録に基づいて助言することは重要である。しかしながら，非専門家である保護者が行動を記録する場合，記録行動の負担や維持が指摘されている。本研究の目的は，保護者の記録行動の負担の軽減と維持のために，スマートフォンのアプリケーションであるLINEを用いた記録行動を支援するLINE Botを開発することであった。LINE Botは，LINEの「お友達追加」の機能からQRコードを取得することで，スマートフォンやタブレットなどのデバイスで利用できる。記録者がLINEで入力した行動記録は，Googleスプレッドシートに自動で遷移する。また，通知機能や保護者の記録行動の即時強化として応答メッセージの機能を加えた。実装後，LINE Botの有効性と課題について考察した

Analysis of PD-L1 expression in non-small cell lung cancer microenvironment and its role as a potential predictive biomarker/

Objetivo: Analisar a literatura científica para a expressão de PD-L1 no infiltrado de células imunes de tumores do tipo CPCNP, além de seu potencial uso como biomarcador preditivo de desfechos clínicos e de resposta à imunoterapia com drogas anti PD-1 e anti PD-L1. Métodos: 5 bases de dados foram consultadas para buscas (PubMed, Web of Science, Scopus, Lilacs e Clinical Trials.gov.). Artigos foram incluídos se pertinentes, disponíveis através de acesso institucional e se escritos em Português, Inglês ou Espanhol. Não houve restrição na seleção quanto tipo de estudo ou ano de publicação. Resultados: 15 artigos foram selecionados. Foi observado relação entre o nível de expressão de PD-L1 e a presença de células dendríticas imaturas, além de expressão constitutiva da molécula em fibroblastos de pacientes com CPCNP. A expressão de PD-L1 nas células imunes infiltradas correlacionou-se com sobrevida aumentada e resposta tumoral melhor após terapia com atezolizumab, além de benefícios clínicos na terapia anti-PD-1. Outros artigos demonstram correlação significativa entre a expressão de PD-L1 em linfócitos T periféricos e desfechos clínicos. Discussão: Observações preliminares demonstraram que a expressão de PD-L1 nas células imunes estão relacionadas ao sucesso clínico da imunoterapia e ao microambiente imunossupressor visto no CPCNP.Objective: To analyse the recent findings regarding programmed-death ligand 1(PD-L1) expression on tumor infiltrating immune cells in NSCLC and its potential role as a predictive biomarker for clinical outcomes and for successful PD-1/PD-L1 blocking immunotherapy. Methods: 5 databases were accessed for search: PubMed, Web of Science, Scopus, Lilacs, and Clinical Trials.gov. Articles were selected if written in English, Portuguese or Spanish and if available via institutional access. Results: 15 articles were selected. PD-L1 expression was found to be related to the presence of immature DCs and had also constitutive expression on fibroblasts derived from NSCLC specimens. PD-L1 expression in tumor infiltrating immune cells was observed to be correlated with overall survival benefit and improved tumor response after atezolizumab therapy. A significant correlation between PD-L1 expression in peripheral T cells and clinical outcomes was also detected, besides the finding of significant correlation between an increased PD-L1 expression and clinical benefits in anti-PD-1 therapy. Discussion: Preliminary observations showed that PD-L1 expression in immune cells is related to an immunosuppressive milieu in NSCLC and to clinical benefits of immunotherapy