アラビアゴム重層遠心沈澱によるToxoplasma gondii感染マウス脳からのシストの分離法について

A multi-layer centrifugation technic with Gum arabic solution was examined with the object of purely separating cysts of Toxoplasma gondii from infected mouse brains. This paper outlines the preparations of infected mouse brain emulsions and of the Gum arabic solution having a certain specific gravity, and the experimental results on the recovery rate of cysts, the removal rate of brain tissue and the infectivity of cysts are also reported. The results followed multi-layer centrifugation with Gum arabic solution with a sp. gravity of 1.050 and 1.070 at 1,000 G for 15 min. were quite satisfactory.著者らはアラビアゴム重層法を用いてToxoplasma gondiiのBeverlev株感染マウスの脳からシストを分離する実験を行ない,以下の成績を得た.1.アラビアゴム重層法は,蔗糖重層法と比較して分離前の脳乳剤から高いシスト回収率と高度の脳組織除去率が得られた.2.比重,1.050および1.070のアラビアゴム分離液を用い,1,000G15分間の遠心沈澱によって満足すべき結果が得られた.3.1%脳乳剤をアラビアゴム重層法によって遠心沈澱し,平均92.3%のシスト回収率と約99%の脳組織除去率(比濁法により測定)を得た.4.本法によって分離されたシストはマウスに対する感染性に著明な変状が認められなかった.本法は無菌的操作も容易であり,分離術式も簡便であることから今後感染マウスの脳からシストを分離する実験に広く応用できるものと考えられる

マウス接種法および螢光抗体法による屠殺豚からのToxoplasma gondiiの検出について

Ninety five pigs suspicious of Toxoplasma-infection were selected from 18,867 ones killed at Isahaya City Slaughterhouse and used for the isolation of T. gondii with mouse inoculations of their hilar or hepatic lymph nodes and also for the microscopic detection of the parasite in the lymph nodes with direct fluorescent antibody technic. In the mouse inoculation method, Toxoplasma hemagglutination test was carried out with sera of mice killed 6 weeks after the inoculation of the lymph nodes into the mice. Further, T. gondii strains newly isolated were subinoculated into mice and hamsters to investigate their virulence. The isolation rate of T. gondii was 8/95 or 8.4%, while 33 of 95 (34.7%) were positive in hemagglutination test. Fluorescent antibody technic indicated a positive response in 19 of 95 pig lymph nodes (20.0%). Eight T. gondii strains were isolated and demonstrated a high virulence for mice and hamsters. In this paper, the methods used and the above mentioned results are stated in detail and discussed.長崎県諫早市立屠場に1966年12月より1967年3月までに搬入された豚18,867頭より,屠場獣医師の協力により選抜されたトキソプラズマ症の疑いある病変豚(肺水腫,肝壊死斑,腸充血など)95頭の肝または肺門リンパ腺の螢光抗体法(直接法)により原虫検出,マウス接種法による原虫分離,および接種マウスのHA抗体価を測定し,それらの成績を比較検討した.また分離された株の毒性についてもRH株と比較し検討した.1.マウス接種法によるトキソプラズマ原虫分離は95例中8例(原虫分離率8.4%)で,いずれも栄養型が検出された.分離8株中5株は継代初期においては,シスト型も検出された.2.マウス接種法によるHA抗体価陽性(256倍以上陽性)は95例中33(陽性率34.7%)であった.原虫分離8株はいずれもHA陽性であった.3.豚の肝または肺リンパ腺の割面スタンプ標本の,螢光抗体法(直接法)によるトキソプラズマ原虫の検出率は95例中19例(検出率20.0%)であった.この19例中4例からマウス接種法により原虫が分離できた.4.分離株のマウスおよびハムスターに対する毒性はRH株と同じ程度かやや弱毒であった

屠場の低温室内におけるToxoplasma gondiiの生存期間に関する研究

Examination was made of the survival period of Toxoplasma gondii in the low temperature rooms of a slaughterhouse. Materials for the examination were the mouse bodies themselves and the organs, such as the liver and brain, of mice infected with the RH or Beverley strain. These materials were stored in both of the refrigerating and the cold-storage rooms, then taken out of the rooms one after another at a short interval and examined on the existence of live Toxoplasma in them with the intraperitoneal inoculation into healthy mice. In the first experiment, it was revealed that the survival of the proliferative form of Toxoplasma in an infected mouse body was 8 days in the refrigerating room and 4 days in the cold-storage room, but a putrefactive sign was manifesting slowly in the mice stored more than 13 days in the refrigerating room and 6 days in the other. In the following experiment, the livers excised from RH-infected mice and the brains from Beverley-infected ones were stored only in the refrigerating room. It was recognized as the result that cysts were capable of survival for as long as 67 days and proliferative forms could survive for 11 days in the room. A control experiment was attempted on the resistance of T. gondii to -14℃ in a freezer and it was shown that both forms of this protozoa in the infected mouse organs could remain alive more than an hour but did not for 3 hours in a freezer of -14℃. Temperatures in both rooms were continuously measured by auto-recording thermometers. In the refrigerating room, it was 0.47℃ in average and the cold-storage room always had 3 to 4℃ higher temperature than the refrigerating room.Toxoplasma gondiiの低温に対する抵抗性についての報告は少なくない。しかし、これらの研究では、実験室内の冷蔵庫またはフリーザーなどの精確に調節された温度条件下に実施されたものである。屠畜肉やその内臓中に潜在する本原虫が、もっとも重要な感染源と見なされている現在では、屠場の低温室中で畜肉中の本原虫が、どれ程の期間生存しつづけるかが疫学上重要な意味を持つことはいうまでもない。事実、屠場の冷却室あるいは冷蔵室の温度は、頻回の扉の開閉や大量の温かい大動物肉塊の搬入などのため、相当な変動を受けることが予測され、実験室の冷蔵庫内温度条件とはかなり異なるものであると考えられた。　本研究は、以上の趣旨に沿って長崎市営屠場内の低温室を利用し、T. gondiiの増殖型および？子の生存期間を検討した。被検材料は、大動物肉塊や内臓を用いることができなかったので、RHおよびBeverley株感染マウスおよびその臓器を使用した。　第1実験では、PH株感染マウス自体を冷却室および冷蔵室に保存した。以後、毎日または隔日に保存マウス体を取り出し、その肝および脾乳剤を健常マウス腹膣内接種し、そのマウスからの原虫検出を試みた。接種ご30日以内に死亡したマウスは即時に、それ以上生存したものは屠殺して、その腹膣液および脳中の原虫の有無を顕微鏡下に検討した。その成績では、マウス体内のRH株増殖型は冷却室中で8日間、冷蔵室中で4日間生存することを認めた。しかし、保存マウスの腹膣内臓器の腐敗が冷却室では13日め以降から、また冷蔵室では6日めから認められたことから、その成績にはマウスの腐敗が影響を及ぼしていることが想像された。実験中の室温は、冷却室では平均0.9℃、最高2.5℃、最低-1.1℃、冷蔵室では平均4.2℃、最高低巾は5.1～3.2℃であった。　第2実験では、RH株感染マウス肝とBeverley株感染マウス脳を冷却室中に保存し、一定期間毎に取り出し、乳剤として健常マウスに接種した。本実験によりマウス肝中のRH株増殖型は11日間、マウスの脳中のBeverley株囊子は実に67日間冷却室で生存することが判明した。実験期間中の冷却室温度は平均0.47℃、最高低巾は3.2～-3.5℃におよんだ。対照実験として、-14℃のフリーザーの中で第2実験と同一材料を用いて検査したが、その結果、囊子、増殖型とも、1時間保存材料中に生存することを認めたが、3時間材料からは証明できなかった。　以上の実験で、屠場冷却室中で囊子は67日間、増殖型は11日間生存し、かつ、感染力を保有していることが判明したが、大動物肉塊や臓器中では、より長時間生存することが想定された

フィリピンの農山村、特にパラワン島におけるマラリアの疫学的調査

A field survey was undertaken on the malaria parasite rate and the spleen rate in some rural areas of the Philippines during the period from November, 1969, till March, 1971. The subject areas were Wawa, north from Manila; Sonlon, north from Davao; and Maruyogon, Mainit, Quezon and the Iwahig Penal Colony in Palawan Island. Persons subjected to the survey were residents, prisoners (colonists) in the penal colony and in some areas, primary school children. The spleen rate was examined by manual palpation in children under 12 years. Results obtained were summarized as follows: 1) The parasite rate was 2.5% (8/320) at Wawa, 0.5% (1/217) at Sonlon, 4.0% (16/401) at Maruyogon, 15.4% (10/65) at Mainit, 26.7% (8/30) in the native tribe and 5.7% (2/35) in settlers, and 7.1% (21/181) in school children at Quezon. 2) The spleen rate was 4.0% (7/177) at Maruyogon, 40.9% (18/44) at Mainit and 15.2% (34/224) at Quezon. 3) In the Iwahig Penal Colony, colonists in the Montible and Central Subcolonies were employed for the parasite examination. The parasite rate in the Montible was 12.1%(62/521). A significant difference in parasite rate was shown between the central district (6.7%) and the outskirts (16.3%) of the Montible Subcolony. Newcomers into the Orientation Unit of the Montible demonstrated 13.6% (9/66) in parasite detection and this fact would imply the high frequency of a new infection to colonists during the stay of the first 6 months. At the Mangahan (Agronomy) Unit of the Central Subcolony, 10.7%(3/28) in parasite rate was obtained. 4) The parasite species and the proportion observed in this survey were; P. falciparum 84.8%, P. vivax 13.9%, and P. malariae 1.2%.1969年11月より1971年3月の間に,フィリピンの農山村地域で,マラリア原虫率および脾腫率の野外調査を実施した.対象地はマニラ北方約60Kmのワワ,ミンダナオ島ダヴァオ北東約110Kmのソンロン,およびパラワン島のマルヨゴン,マイニット,ケソンおよびイワヒグ囚人部落で,対象人は一般住民,囚人部落では収容中の徒刑囚,またある地域では学童であった.脾腫率は12才以下の小人を対象に触診によって検査した.得られた成績を総括すると次の如くであった.1)原虫率は,ワワ2.5%(8/320),ソンロン0.5%(1/217),マルヨゴン4.0%(16/401),マイニット15.4%(10/65),たゞし,この地域の原住部族民では26.7%(8/30),移住者5.7%(2/35),ケソン中央小学校児童7.1%(21/181)であった.2)脾腫率は,マルヨゴン4.0%(7/177),マイニット40.9%(18/44),ケソン15.2%(34/224)の結果を得た.3)イワヒグ囚人部落ではモンテブレおよびセントラルの両サブコロニーの囚人を対象とした.モンテブレサブコロニーでは,12.1%(63/521)の原虫率をえたが,このサブコロニーの中央地区居住者の原虫率,6.7%と周辺地区居住者のそれ,16.3%との間には,明らかに有意差が認められた.同サブコロニーのオリエンテイション分団への新入囚人では,原虫検出率が13.6%(9/66)であった.この事は新入者の初期6ケ月間の滞在期間中におけるマラリア感染の頻度を物語るものと思われた.セントラルサブコロニーのマンガハン(耕作)分団での原虫率は10.7%(3/28)であった.4)検出した原虫種は,熱帯熱原虫がもっとも多く84.8%,三日熱原虫は13.9%,四日熱原虫は1.2%であった

Mutations in SERPINB7, Encoding a Member of the Serine Protease Inhibitor Superfamily, Cause Nagashima-type Palmoplantar Keratosis

“Nagashima-type” palmoplantar keratosis (NPPK) is an autosomal recessive nonsyndromic diffuse palmoplantar keratosis characterized by well-demarcated diffuse hyperkeratosis with redness, expanding on to the dorsal surfaces of the palms and feet and the Achilles tendon area. Hyperkeratosis in NPPK is mild and nonprogressive, differentiating NPPK clinically from Mal de Meleda. We performed whole-exome and/or Sanger sequencing analyses of 13 unrelated NPPK individuals and identified biallelic putative loss-of-function mutations in SERPINB7, which encodes a cytoplasmic member of the serine protease inhibitor superfamily. We identified a major causative mutation of c.796C>T (p.Arg266∗) as a founder mutation in Japanese and Chinese populations. SERPINB7 was specifically present in the cytoplasm of the stratum granulosum and the stratum corneum (SC) of the epidermis. All of the identified mutants are predicted to cause premature termination upstream of the reactive site, which inhibits the proteases, suggesting a complete loss of the protease inhibitory activity of SERPINB7 in NPPK skin. On exposure of NPPK lesional skin to water, we observed a whitish spongy change in the SC, suggesting enhanced water permeation into the SC due to overactivation of proteases and a resultant loss of integrity of the SC structure. These findings provide an important framework for developing pathogenesis-based therapies for NPPK

Effects of inorganic mercury and methylmercury on osteoclasts and osteoblasts in the scales of the marine teleost as a model system of bone

To evaluate the effects of inorganic mercury (InHg) and methylmercury (MeHg) on bone metabolism in a marine teleost, the activity of tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) as indicators of such activity in osteoclasts and osteoblasts, respectively, were examined in scales of nibbler fish (Girella punctata). We found several lines of scales with nearly the same TRAP and ALP activity levels. Using these scales, we evaluated the influence of InHg and MeHg. TRAP activity in the scales treated with InHg (10-5 and 10-4 M) and MeHg (10-6 to 10-4 M) during 6 hrs of incubation decreased significantly. In contrast, ALP activity decreased after exposure to InHg (10-5 and 10-4 M) and MeHg (10-6 to 10-4 M) for 18 and 36 hrs, although its activity did not change after 6 hrs of incubation. As in enzyme activity 6 hrs after incubation, mRNA expression of TRAP (osteoclastic marker) decreased significantly with InHg and MeHg treatment, while that of collagen (osteoblastic marker) did not change significantly. At 6 hrs after incubation, the mRNA expression of metallothionein, which is a metal-binding protein in osteoblasts, was significantly increased following treatment with InHg or MeHg, suggesting that it may be involved in the protection of osteoblasts against mercury exposure up to 6 hrs after incubation. To our knowledge, this is the first report of the effects of mercury on osteoclasts and osteoblasts using marine teleost scale as a model system of bone. © 2014 Zoological Society of Japan

Anti-invasive and antiangiogenic effects of MMI-166 on malignant glioma cells

<p>Abstract</p> <p>Background</p> <p>The constitutive overexpression of matrix metalloproteinases (MMPs) is frequently observed in malignant tumours. In particular, MMP-2 and MMP-9 have been reported to be closely associated with invasion and angiogenesis in malignant gliomas. Our study aimed to evaluate the antitumour effects of MMI-166 (Nalpha-[4-(2-Phenyl-2H- tetrazole-5-yl) phenyl sulfonyl]-D-tryptophan), a third generation MMP inhibitor, on three human glioma cell lines (T98G, U87MG, and ONS12) in vitro and in vivo.</p> <p>Methods</p> <p>The effects of MMI-166 on the gelatinolytic activity was analysed by gelatine zymography. The anti-invasive effect of MMI-166 was analysed by an in vitro invasion assay. An in vitro angiogenesis assay was also performed. In vitro growth inhibition of glioma cells by MMI-166 was determined by the MTT assay. The effect of MMI-166 on an orthotropic implantation model using athymic mice was also evaluated.</p> <p>Results</p> <p>Gelatine zymography revealed that MMP-2 and MMP-9 activities were suppressed by MMI-166. The invasion of glioma cells was suppressed by MMI-166. The angiogenesis assay showed that MMI-166 had a suppressive effect on glioma cell-induced angiogenesis. However, MMI-166 did not suppress glioma cell proliferation in the MTT assay. In vivo, MMI-166 suppressed tumour growth in athymic mice implanted orthotropically with T98G cells and showed an inhibitory effect on tumour-induced angiogenesis and tumour growth. This is the first report of the effect of a third generation MMP inhibitor on malignant glioma cells.</p> <p>Conclusions</p> <p>These results suggest that MMI-166 may have potentially suppressive effects on the invasion and angiogenesis of malignant gliomas.</p