Allergic dermatitis is a common pruritic skin disease in dogs.　Dermatologic diseases mediated by allergic reactions in dogs include flea bite hypersensitivity, atopic dermatitis (AD), food hypersensitivity (FH), and contact hypersensitivity. Since inherited hypersensitive immune response is associated with the pathogenesis of AD and the number of predisposed pure breed dogs have been increasing as companion animals, AD is one of the most problematic diseases in small animal practice.　Moreover, recent studies revealed that FH could be diagnosed by elimination dietary trial and provocative challenge in many dogs sometimes concurrent with AD.　For these reasons, AD and FH are now accepted as clinically important diseases to be solved in veterinary medicine.

　Diagnosis of AD in dogs is based on its characteristic clinical features including pruritus, typical appearance and distribution of the skin lesion, recurrence of skin symptoms with secondary bacterial infections, familial history or breed predisposition, and frequent elevation of serum IgE specific to environmental allergens.　These clinical features are not specific to AD and can be also observed in dogs with FH.　Diagnosis of FH in dogs can be made only after the positive results in elimination dietary trial and provocative challenge because allergen-specific IgE and intradermal skin test (IDST) do not have sufficient sensitivity and specificity to food allergens.　For diagnosis of FH, highly efficient elimination diet is needed, however, there has been no elimination diet that can avoid both type 1 and type 4 hypersensitivity to food allergens.　Recently, a novel hypoallergenic diet for dogs was developed as a collaborative project between the University of Tokyo and a pet food company.　The diet has been manufactured from a mixture of amino acids and potato.　It is conceivable that amino acids do not induce either type 1 or type 4 hypersensitivity because of their small molecular weights unlike the proteins and peptides.　Moreover, it has been shown that potato induces FH in few dogs.　The study in Chapter 1 of the present thesis was carried out to examine the clinical efficacy of the newly developed hypoallergenic diet as an elimination diet to diagnose FH in dogs.

　Although dogs with AD are frequently shown to be hypersensitive to environmental allergens, especially house dust mites (HDM), it is usually difficult to eliminate HDM from the environment.　Therefore, treatments for AD in dogs have been carried out by administration of glucocorticoids and immunosuppressive drugs as well as allergen-specific immunotherapy (ASIT).　But, many dogs are often subject to adverse effects or reactions during the treatments with these therapies.　HDM avoidance measures in the environments have been carried out as a safe and effective treatment for controlling the symptoms in asthmatic human patients.　But, there has been no report on the efficacy of the HDM avoidance measures on the control of AD.　The study in Chapter 2 of this thesis was performed to know whether the HDM avoidance measures are effective to control the clinical symptoms and immune responses in dogs with AD that are sensitive to HDM.

　 In human patients with AD, complex immune responses are known to be associated with the pathogenesis to induce the skin lesion.　It has been shown that, in addition to immediate hypersensitivity, late phase reaction is also involved in the formation of the characteristic lesion of AD.　Moreover, predominance of Th2-skewed cytokines and chemokines has been demonstrated in the allergic skin lesions.　Recently, many of these immunologic abnormalites were shown in the skin lesions of dogs with AD.　Although a variety of immune cells are infiltrated conceivably to initiate and progress a series of immune reactions in the skin of humans and dogs with AD, many of the studies indicated significant roles of mast cells to develop the allergic reactions in the skin.　Mast cells are considered to play not only as effector cells in type 1 hypersensitivity but also as immunoregulatory cells in acquired and innate immunity.　Certain roles of mast cells in the late phase reaction have been also indicated in the experimental animal models.　To establish a fundamental research system on the role of mast cells in AD, the study in Chapter 3 of this thesis was carried out to culture and characterize mast cells derived from the skin of dogs in comparison to those from the bone marrow.

Chapter 1.　Usefulness of a new elimination diet composed of amino acids and potato for the diagnosis of canine food hypersensitivity

　Twenty dogs with suspected FH or FH concurrent with AD were enrolled in this study based on the history, onset of clinical signs at young age, non-seasonal pruritic chronic dermatitis, and rule-out of flea bite hypersensitivity, contact hypersensitivity and primary infectious skin diseases such as superficial pyoderma, mycotic skin disease and ectoparasitosis.　After satisfactory explanation to the owner, the elimination trial was carried out using the newly developed hypoallergenic diet (Aminoprotect care〓, Sankyo Lifetech, Co., Ltd. Japan) for 2 months.　During the period of elimination dietary trial, some dogs were treated using systemic antibiotics and anti-microbe shampoos for control of secondary skin infections, and received systemic glucocorticoid therapy for reducing their pruritus.　Before and after the dietary trial, the dogs' clinical symptoms were evaluated using the pruritus score, medication score of systemic glucocorticoid, and canine atopic dermatitis extent and severity index (CADESI). Fifteen of the 20 dogs could complete the elimination dietary trial.　The other 5 dogs were suspended because of diet unpalatability (3 dogs), progression of skin disease (1 dog) or diarrhea (1 dog).　Based on the comprehensive evaluation of the clinical scores, improvement of the clinical scores was seen in 10 of the 15 dogs that completed the trial.　Food provocation tests were then applied to 9 of the 10 improved dogs, and 6 dogs were confirmed to be hypersensitive to certain food allergens such as pork, beef and chicken from the recurrence of the clinical symptoms.　The results of the food elimination tests using the newly developed hypoallergenic diet and succeeding provocative challenge revealed that the diet was useful for the diagnosis of FH in dogs.

Chapter 2.　Effect of house dust mite avoidance measures on the clinical symptoms in dogs with atopic dermatitis

　For the study in this chapter, six dogs with AD sensitive to HDM were enrolled.　Clinical diagnosis with AD was based on Willemse's criteria.　All of the 6 dogs were shown to be sensitive to HDM by measurement of allergen-specific serum IgE and/or IDST.　HDM avoidance in which the decrease of a representative HDM allergen, Der 1, was performed by withdrawing conventional bedding and encasing furniture and bedding in covers allowing no mite penetration (Danizerock〓, Yamasei Corp., Tokyo, Japan).　The clinical symptoms of the dogs were quantitatively evaluated by recording CADESI, pruritus score, and glucocorticoid medication score before and after the HDM avoidance measures for approximately 20 weeks.　Der 1 concentration was measured by fluorescence sandwich ELISA using monoclonal antibodies directed to the allergen.　Seven to 23 weeks after the intervention, Der 1 concentration was shown to be significantly decreased on floors (from 4.73 to 0.39 μg/g dust, p=0.0306) and furnitures (from 9.78 to 0.17 μg/g dust, p=0.0082), but not on dogs' mats (from 1.32 to 0.09 μg/g dust, p=0.1425).　After the HDM avoidance measures, 4 of the 6 dogs showed overall improvement of their clinical symptoms.　Moreover, decrease of the level of serum IgE specific to HDM was shown in 4 of the 6 dogs after the intevention.　The results of this study indicate that decreasing the amount of HDM in the environment can have an efficacy on the control of clinical symptoms and hypersensitive immune reactions in dogs with AD.

Chapter 3.　Cultivation and characterization of skin-derived mast cells in comparison to the bone marrow-derived mast cells in the dog

　In this chapter, I carried out a study on the cultivation and characterization of skin-derived mast cells in comparison to the bone marrow-derived mast cells in the dog.　Skin biopsy samples from healthy dogs were enzymatically digested to obtain single cells.　After cultivation for 1 to 2 months in serum-free AIM-V medium supplemented with recombinant canine stem cell factor, the proliferating mononuclear cells were purified using anti-c-Kit antibody with magnetic cell sorting system.　Purity of the cells was assessed by co-expression of the surface c-Kit and Fc epsilon receptor 1 by using flow cytometory.　The cell morphology was examined by toluidine blue staining and transmission electron microscopy.　Immunocytochemistry for tryptase and enzymecytochemistry for chymase were employed to examine the protease phenotype.　A beta-hexosaminidase assay was performed to examine mast cell-specific degranulation induced by Ca(2+) ionophore, A23187, and anti-canine IgE antibodies.　These characteristics of skin-derived mast cells (skin MC) were compared with those of bone marrow-derived cultured mast cells (BMCMC).　The skin-derived culture showed the purity of mast cells as high as 91.7 ± 6.6 %.　These cells were 10 to 20 micrometers in diameter and contained a round or oval nucleus and metachromatic granules. Electromicroscopic examination revealed numerous electron-dense granules in the cytoplasm.　The cells were positive for both tryptase and chymase.　Release of beta-hexosaminidase induced by Ca(2+) ionophore and anti-canine IgE antibodies was seen in a dose-dependent manner.　In the morphological observation, the skin MC were larger than BMCMC and had longer cytoplasmic projections and larger numbers of cytoplasmic granules than BMCMC.　The amount of degranulation induced by anti-canine IgE antibodies was smaller in skin MC than BMCMC.　These results indicates the usefulness of the culture method employed in this study to obtain canine skin-derived mast cells with its typical characteristics that will function to develop allergic reactions in the skin.

　A series of the studies in this thesis were carried out for understanding the mechanisms to initiate and progress allergic dermatitis in dogs and for development of novel therapeutic strategies to the disease.　Nowadays, there is a rapid increase of the number of patients with allergic diseases in dogs as well as in humans.　The present studies provide basic and applied knowledge on the future development of novel therapies that will efficiently control the problematic allergic diseases in dogs and humans.

　本研究は、犬におけるアレルギー性皮膚炎の病態制御に関して、特に抗原回避による病態制御法の開発を目的として一連の研究を行ったものである。

第一章:犬の食物過敏症の診断におけるアミノ酸とジャガイモからなる新規除去食の有用性に関する研究

　アミノ酸とジャガイモからなる新規除去食(アミノプロテクトケア〓)が東京大学とペットフード企業の共同研究によって開発された。本章では、食物過敏症(Food hypersensitivity, FH)の診断におけるアミノプロテクトケア〓の除去食としての有用性を検討した。若齢時(多くは3歳齢未満)からの非季節性の痒みを伴う慢性皮膚疾患があり、ノミ過敏症および接触性過敏症の他、感染性(寄生虫性・真菌性・細菌性)皮膚疾患が除外され、FHまたはFHとアトピー性皮膚炎(Atopic dermatitis, AD)の両方を有する可能性がある20例を対象とした。飼い主の同意のもとで、アミノプロテクトケアによる2ヶ月間の除去食試験を行った。除去食試験前、中、後に、痒みスコアおよびプレドニゾロン(PDN)投与回数を記録するとともに、皮膚病変の程度を犬アトピー性皮膚炎重症度指数(CADESI)によって評価した。除去食試験を開始した20例のうち、15例では試験を完了することができたが、5例は途中で脱落した。その中止の理由としては、嗜好性の問題(3例)、皮膚症状の悪化(1例)、および下痢(1例)が挙げられた。試験前後の臨床スコアの変化を総合的に判断した結果、除去食試験を完了した15例のうち10例において症状が改善した。さらに、除去食試験有効例10例のうち9例において食物負荷試験を実施した。その結果、9例中6例において、豚肉、牛肉、鶏肉などの食物の投与によって皮膚症状が誘発されたことから、これら食物に対するFHが存在することが示された。アミノプロテクトケア〓を用いた除去食試験と食物負荷試験によってFHを診断できることが明らかとなり、そのFHの診断における臨床的有用性が示された。

第二章:HDMに感作された犬のアトピー性皮膚炎における防ダニ寝具を用いた環境改善効果に関する研究

　HDMはADにおける主要な感作抗原のひとつである。HDMに感作された犬のAD症例において防ダニ寝具(ダニゼロック〓)を用いた環境改善効果を臨床症状とHDM特異IgE値によって検討した。Willemseの提唱したAD の診断基準を満たし、かつ皮内反応および血清IgE検査においてHDM陽性の成犬6症例を対象とした。飼い主の同意のもと、家庭内の寝具を防ダニ寝具と交換し、20週以上にわたり観察した。治験開始前後に室内塵中のHDM抗原量(Der 1量)を定期的に測定し、環境改善前後における症例の臨床症状をCADESI、痒みのスコアおよびPDN投与回数によって評価した。6症例中4症例において室内塵中のDer 1量を人の喘息の感作閾値である2μg/g dust未満に維持することが可能であった。Der 1量を低値に維持することができた4症例のすべてにおいて臨床症状の緩和傾向を認め、さらに4症例中3症例においてHDM特異的IgE値が減少した。以上の結果から防ダニ寝具による室内環境改善がHDMに感作されたADの臨床症状のコントロールに有効であることが示唆された。

第三章:犬の皮膚肥満細胞培養系の確立とその骨髄肥満細胞との比較

　健常犬から皮膚肥満細胞培養系を確立し、その細胞の性状を骨髄由来肥満細胞と比較した。皮膚由来肥満細胞を採取するため、皮膚片を酵素処理し、細胞を単離後、犬リコンビナントStem cell factor (SCF)を添加した無血清AIMV培地を用いて4〜6週間培養した。肥満細胞の純化は抗マウスc-kit抗体を用いた磁気細胞分離システム(MACS〓)によって行った。純化した細胞は均一な単核円形浮遊細胞であり、豊富な異染性顆粒を有していた。フローサイトメトリーによる解析において、これら細胞はc-kitおよびFceRI陽性であり、純度は91.7±6.6%であった。免疫染色および酵素化学染色の結果、細胞質内顆粒はトリプターゼおよびキマーゼを含有し、MCTC型であることがわかった。皮膚肥満細胞は骨髄由来肥満細胞と比較して、トリプターゼおよびキマーゼをより多く含有していたが、細胞表面上のFceRIの発現量が低く、FceRIの架橋刺激による脱顆粒量も少ないことが示された。今回、確立した皮膚肥満細胞培養系はアレルギー性皮膚炎における免疫学的および薬理学的解析に有用であると考えられた。

　今回の一連の研究は、犬におけるアレルギー性皮膚炎の病態制御に関し、臨床的有用性を示した新たな知見を提供するものである。

　本申請論文を審査した結果、博士(獣医学)の学位を授与するに値すると判断した。