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変形性膝関節症（以下，OA）に対する理学療法効果を検証するために適したモデルを検討することを目的に，5つのラットOAモデルを組織学的に比較した。Wistar系雄ラット10匹を用い，薬剤により化学的にOAを誘発するモデルと，膝関節の靭帯等の切除により外科的にOAを誘発する4つのモデル（内側側副靭帯切除，内側半月脛骨靭帯切除，内側半月板切除，前十字靭帯切除の各モデル）をそれぞれ2匹ずつ作成し，4週間後の膝関節軟骨の状態を比較した。その結果，化学的誘発モデルでは膝関節の内外側広範にわたる軟骨基質の染色性低下と軟骨細胞数の減少を認めた。外科的誘発モデルでは，内側側副靭帯切除モデル以外の全てのモデルで軟骨破壊所見を認め，内側半月板切除と前十字靭帯切除のモデルでは，部分的な軟骨下骨の露出まで至っていた。組織本来の修復力に依存する理学療法の介入効果を検証するためには，進行が緩徐な内側半月脛骨靭帯切除モデルが適している可能性が示唆された。

Metabolic syndrome （MS） is a worldwide healthcare issue and a dominant risk factor for the development of incurable diseases that affect the entire body. The hepatic manifestations of MS include nonalcoholic fatty liver disease （NAFLD） and its progressive variant nonalcoholic steatohepatitis （NASH）. NASH is known to extend into liver cirrhosis and hepatocellular carcinoma （HCC）. To determine the pathogenesis and effective treatment, an excellent animal model of NASH/HCC is required. We recently succeeded to develop two MS associated NASH mice model （TSOD mice and DIAR-MSG mice）. Their clinical course and pathological characters were quite similar to those of human MS-NASH patients. Interestingly, most of them developed HCC in aged, which pathological and functional characters were identical to those of human HCC. In addition, we established a novel mouse model of HCC based on type １ diabetes （DIAR-nSTZ mice） and reported its histopathological features. To compare these mice models from various aspects, we can highlight specific and useful characters of MS associated liver diseases including hepatocarcinogenesis.

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Highly active anti-retroviral therapy (HAART) is effective against human immunodeficiency virus type 1 ( HIV-1) infection, but several severe problems such as occurrence of multi-drug resistant (MDR) viruses are emerging after use of the currently available drugs. Thus, development of novel therapeutics with a different mechanism of action is urgently needed. A recombinant vesicular stomatitis virus (VSV) lacking its G gene ($ \Delta $G) and instead encoding human CD4 and CXCR4 is known to control CXCR4-tropic (X4) HIV-1 infection in culture. To test the value of such a recombiant virus more, we constructed VSV $ \Delta $ Gs expressing CD4 and CCR5 with or without modified dendritic cellspecific ICAM-3 grabbing nonintegrin (DC-SIGN) from rhesus macaques and examined if these viruses were effective against simian immunodeficiency virus (SIV) infection in culture. Our data demonstrated that the VSV $ \Delta $ Gs specifically infected and killed SIVinfected cells, resulting in long-term control of SIV infection in vitro. These results indicate that such VSVs have potential for novel anti-HIV-1 therapeutic candidates. In addition, to determine therapeutic value of anti-HIV-1 drug candidates, it is crucial to test them in animal models before clinical trials. Severe combined immunodeficiency (SCID) mice reconstituted with human peripheral blood mononuclear cells (PBMCs),termed hu-PBLSCID mice or humanized mice, have served as such a valuable small animal model. However, there was a limitation on use of this model because X4 HIV-1 does not efficiently infect these mice due to, at least in part, relatively low levels of expression of the CXCR4 coreceptor. To solve this problem, we generated human interleukin (IL)-4-transgenic huPBL-SCID mice that spontaneously synthesized human IL-4 which has been shown to enhance CXCR4 expression and promote X4 HIV-1 infection in vitro and investigated if X4 virus efficiently infected these mice. Our study showed that in vivo synthesis of human IL-4 augmented CXCR4 expression on human CD4+ T cells and led to productive X4 HIV-1 infection, and that the in vivo infection was definitely blocked by CXCR4 antagonist administration. These data indicate that human IL-4-transgenic hu-PBL-SCID mice can be a useful model for the X4 HIV-1 study and the testing of anti-X4 HIV-1 drug candidates.

A long-term treatment of clarithromycin (CAM), a 14-membered ring macrolide antibiotic, was shown to increase the median survival time for patients with unresectable non small-cell lung cancer. This study was addressed to determine the effects of CAM treatment on the expression of cytokine mRNA in spleen cells of mice bearing Lewis lung carcinoma (LLC). In mice which were not inoculated with LLC cells, there was no difference in the expression profile of cytokine mRNAs between the CAM-treated group and the non-CAM group. In mice bearing LLC, however, the expressions of mRNAs for IL -4, IL-12 p 40, and IFN-γ was increased, but that of IL-6 mRNA was decreased in the CAM -treated group, compared to the non-CAM group. Administration of anticancer agents, including CDDP and VDS, resulted in the suppresssion of cytokine mRNA expression. The combination therapy of CAM and these anticancer agents restored the expression level of mRNAs for IL-2, IL-4, IL-12, and IFN-γ up to the same level as observed in tumor-bearing mice treated with CAM alone, and also decreased IL-6 mRNA expression. When CAM administration was initiated 1 week after anticancer therapy, these changes for the expres- sion of cytokine mRNAs were more marked. These results suggest that CAM stimulates the well-balanced expansipn of helper T-cell subsets in tumor-bearing hosts recovering from immunosuppression caused by anticancer chemotherapy. Thus, CAM may be a promising adjuvant drug to anti-cancer chemotherapy, and treatment with this macrolide should be initiated after some interval but not immediately after basic cancer therapy.

To elucidate the cause of intractable asthma, guinea pigs were actively sensitized by intraperitoneal injection of ascaris suum as an antigen. We repeated the inhalation of aerosolized ascaris antigen eight times in the animal models which consisted of a non-treated group, a metopirone-treated group and a steroid-treated group. We investigated the course of asthmatic response by evaluating the ratio of expirations to inspirations after the inhalation challenge. The frequency of late asthmatic response (LAR) was increased by repeated inhalation of the antigen. There was no significant differnce in the frequency between non-treated group and steroid-treated group, but the metopirone-treated group showed strong LAR compared with the other two groups. The intensity of LAR was gradually increased and that of immediate asthmatic response (LAR) was decreased in the metopirone-treated group. Finally, we established a chronic asthma model in which LAR was strong and the asthma attack was prolonged over twenty-four hours. These results suggest that the model will be useful in clarifying the causes of intractable asthma in humans.

臭化メチルはヒトに中枢および末梢神経障害を惹起する。現在までの臭化メチル中毒動物の神経病理学的な研究にもかかわらず,臭化メチル中毒による多彩なヒトの神経障害は実験動物で十分に再現されていない。本研究では,1日6時間,週3日,3-8週間にわたり,500または290ppmの臭化メチルの繰り返し曝露を受けたラットの神経系病変を組織形態学的に系統的に検索し,未だ実験動物で再現されていない病変を明らかにすることを目的とした。その結果,10 -18日間の500ppmの臭化メチル曝露によってのみ,ヒトの脊髄後索病変に対応すると判断される頚髄薄束有髄線維の軸索変性およびヒトの大脳病変に対応すると判断される,1)尾状核-被殻の壊死病変,2)尾状核-被殻,視床および帯状皮質の神経細胞の萎縮およびニューロピルの染色性の低下が認められた。この実験により初めて明らかになった脊髄後索病変の作成は,繰り返し曝露が可能でかつ500ppm以上の濃度の臭化メチルの曝露によって達成されると推定される。, Methyl bromide exposure produces disorders of the central and peripheral nervous system. In the literature, there are many neuropathologic studies on the nervous system, especially on the central nervous system, of experimental animals intoxicated with methyl bromide presented. However, in our opinion, a variety of lesions of the nervous system described in the human methyl bromide intoxication have not been well reproduced in the experimental animals. In this study, male Wistar rats were subjected to a 6-hour exposure a day to methyl bromide at a concentration of 500 or 290 ppm 3 days a week for 3 to 8 weeks. The systematic neuropathologic studies of their central and peripheral nervous systems were carried out to reproduce the nervous system lesions in the human methyl bromide intoxication hitherto not produced in the experimental animals. Among the rats exposed to methyl bromide at a concentration of 500 ppm for 10 to 18 days, the axonal degeneration of myelinated fibers at the cervical level of the fasciculus gracilis, which probably corresponds to the degeneration of the spinal posterior column in humans, and the necrosis of the caudate-putamen and atrophy of neurons and pallor of the neuropile in caudate-putamen, thalamus and cingulate cortex, which probably correspond to the degenerative findings of the cerebrum in humans, were found. The rats exposed to methyl bromide at a concentration of 290 ppm for 8 weeks (24 days) did not show any noticeable abnormalities histologically. The results above suggest that the spinal cord lesion described for the first time is produced by the repeated exposure of rats to methyl bromide at a higher concentration than 500 ppm.

Mouse embryonal carcinoma cells have been studied extensively on cell differentiation, and are regarded as the stem cells of teratocarcinoma. However, it seems that the hypotheses on histogenesis of human germ cell tumors have followed the mouse tumor model too much. The characters of human embryonal carcinoma cells still remain unclear. Human embryonal carcinoma cells behave differently from mouse embryonal carcinoma cells in many aspects. Using human germ cell tum or cell lines in vivo and in nude mice, I successfully found that some of the human embryonal carcinoma, which were made a diagnosis in the customary way, belonged to undifferentited forms of yolk sac tumor correctly. A rat tumor model also supported my consideration. Moreover, I confirmed that yolk sac tumor cells had a potential of neoplastic metaplasia to the intestinal epithelium.

We developed a convenient animal experimental model to study the effects of passive smoking on urinary mutagenicity. Rats were individually placed into metabolic cages, and were exposed to a side-stream of cigarette smoke under specific conditions. Then 24-hour urine collections were performed. Mutagenicity was investigated by the Ames test using the Salmonella typhimurium strains TA98 and TA100. In strain TA98, the mutagenicity of urine was 5.4-fold and 5.3-fold increased with and without metabolic activation, respectively, by exposure to cigarette smoke, and that in strain TA100 was 4.7-fold and 4.4-fold increased. Using this animal model, the effect of passive smoking on urinary mutagenicity can be investigated conveniently. This model can be applied to a variety of experiments.

One of the reasons why many microbiologists and clinicians have been interested in Streptococcus milleri is that the streptococcus is not only part of the human commensals found in the mouth, nasopharynx, throat, gastrointestinal tract and genitourinary tract, but also has frequently been isolated from a wide variety of human infections, particulary from suppurative lesions. Another reason is that taxonomy and nomenclature of S milleri have been confused. Colman and Williams have claimed that S. milleri could be classified as one of viridans streptococci, whereas Facklam has not agreed the epithet S. milleri and proposed two approved names by lactose fermentation. Since then, many taxonomies and nomenclatures have been proposed by many researchers. Recently, S, milleri was genotypically classified into three distinct taxa, I.e. S. intermedius, S. constellatus, and S. anginosus, and this would be the last word on the taxonomy of S. milleri. Some S. milleri isolates caused dental caries, submandibular and subcutaneous abscess, and endocarditis in experimental animals, although the virulence factors of S. milleri have not been fully ascertained. Considering that S. milleri would be classified into the three approved species, the pathogenic mechanisms of the streptococci and possible associations between the approved species and specific infections will need to be studied in order to enhance the better understanding of the clinically significant streptococci.

1)ウイスター系ラットを実験動物に選び, エチレングリコール0.5%濃度, 飲水投与により, 尿中蓚酸排泄量は対照群の2～3倍に増加したが, 単独では結石形成はみなかった.2) 1α(OH)D3 0.5 μg隔日投与で, 尿中カルシウム排泄量は対照群の10～20倍に増加したが, 単独では結石形成はみなかった.3)エチレングリコールと活性型ビタミンDである1α(OH)D3を併用することにより, 各単独では結石形成が起こらない投与量で, 77.3%と高率に蓚酸カルシウム結石を発症することができた.4)エチレングリコール, 1α(OH)D3併用群では, 腎機能は対照群の約4分の1まで低下した, As calcium oxalate stones are the most important component in urolithiasis, an experimental model has to be designed to clarify the pathogenesis and aid in their prevention. Hyperoxaluria as well as hypercalciuria were produced in rats by administering ethylene glycol (0.5%, in drinking water administered ad libitum) and 1-alpha (OH) D3 (0.5 micrograms/rat given every other day), respectively, for three to four weeks. Neither drug alone produced stones efficiently as did the combination regimen of these two compounds. The occurrence of stones was 77.3%, and with only a moderate degree of renal functional impairment. Biochemical and histological data were obtained using this model.

Treatment with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) or N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) in the drinking water induces bladder cancer of rats or mice with high incidence. Induced bladder cancers of rats are mostly papillary, non-invasive type, whereas those of mice are non-papillary, invasive type. Histologically, most of BBN-induced bladder cancers are transitional cell carcinoma in both rats and mice. On the other hand, the incidences of squamous cell carcinoma increased in cases of mice, especially in mice treated with EHBN. Metastases and invasions of bladder cancers are more common in mice than in rats. There are strain and species differences in the bladder response to BBN and EHBN. In addition, a putative preneoplastic lesion, papillary or nodular hyperplasia of the epithelium is a good marker for early detection of bladder cancer development.

N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)を8週間投与するラット膀胱腫瘍実験モデルを用いて抗癌剤の効果判定を行った.1)抗癌剤単剤投与実験では, 5-FU, FT-207, CQ, VCRおよびCDDPの投与群において膀胱腫瘍の発生頻度が抑制され, ADM, MMC, CPM, NCSおよびBLM投与の各群では抑制されなかった.2)抗癌剤の経口投与実験において, 5-Fu関連物質は膀胱腫瘍発生の抑制がみられ, とくにUracil, 1-Hexylcarbamoyl-5-fluorouracil投与群では用量依存的に膀胱腫瘍発生の抑制がみられた, The chemotherapeutic agents were evaluated using the experimental urinary bladder tumor rat model induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Nine hundred and fourteen male rats received 0.05% BBN in drinking water for 8 weeks, and were divided into 35 groups to follow the regimens of chemotherapeutic agents. Thirty one groups received the agents after BBN treatment, and 4 groups were given the oral agents starting simultaneously with BBN treatment. All rats were killed at 20 weeks and incidence of the urinary bladder was examined histopathologically. The following 13 agents were evaluated; adriamycin (ADM), mitomycin-C (MMC), cyclophosphamide (CPM), 5-fluoro-uracil (5-Fu), N-(2-tetrahydrofuryl)-5-fluorouracil (FT-207), neocarcinostatine (NCS), carbazil quinone (CQ), bleomycin (BLM), vincristine (VCR) and cis-diammine-dichloroplatinum (CDDP) were dosed intraperitoneally, and N-(2-tetrahydrofuryl)-5-fluorouracil (FT-207), 1: 4 mixture of FT-207 and uracil (UFT) and 1-Hexylcarbamoyl-5-fluorouracil (HCFU) were dosed orally. Among these agents, 5-Fu, FT-207, CQ, VCR, CDDP, UFT and HCFU were effective in inhibiting the incidence of urinary bladder tumor induced by BBN. In conclusion, the experimental bladder tumor rat model induced by BBN seems to be useful in evaluating the effective chemotherapeutic agents for a superficial bladder cancer. The importance of the experimental animal model for the evaluation of chemotherapeutic agents is discussed.

Primary biliary cirrhosis (PBC) is supposed to be a disease entity in which autoimmune mechanisms may be operative, characterized by chronic nonsuppurative destructive cholangitis (CNSDC), cholestasis and epithelioid granuloma formation in its early stage. Thus far, although the pathogenesis of the disease remains largely unknown, there are a number of reports indicating hepatic lesions induced following graft-versus-host reaction (GVHR) in both human and experimental animals after the bone marrow transplantation. Recently, we demonstrated the bile duct lesions resembling CNSDC and the formation of epithelioid granuloma in the (B6xbm12) F1 mice with MHC class II-disparate GVHR, postinjection of spleen cells of parental B6 mice via the tail vein. By immunohistochemical examinations, both CD4^+ and CD8^+ T cells were found around the bile duct, and some of them were infiltrating among the bile duct epithelia on which the MHC class II antigen was aberrantly expressed. These findings indicate that MHC class II antigen might play an important role in the destruction of bile duct cells in PBC as well as in MHC class II-disparate GVHR.

研究科: 千葉大学大学院薬学研究院, 修了年:1991, 学位:千大院薬博乙第99号

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