マウスにおけるCyp2B誘導化合物による肝肥大及び肝発がんへのconstitutive androstane receptor(CAR)の関与に関する研究

　現代人は、環境や食事・水を介して様々な化学物質に暴露されている。これらの化学物質については、人体に与える影響のポテンシャルや暴露量との関係についてリスク評価が行われている。その一つの手法として、主にげっ歯類を用いた毒性試験が取り入れられており、ヒトへの有害性の推測に有用な情報をもたらしている。一方、生理機能や代謝反応には動物種差があることから、毒性試験の結果については実験動物における反応や影響の発現メカニズムを考慮した上でヒトへの外挿性を検討する必要がある。特に実験動物で発がん性が認められた化学物質の場合、ヒトへの外挿性の判断は有害性評価の最も重要なポイントとなる。　げっ歯類とヒトでの発がん性について種差が明らかとなっている化学物質の一例としてフェノバルビタール（PB）が挙げられる。PBは実験的にげっ歯類で肝肥大、肝発がんを起こすことが知られている。一方、ヒトにおいては疫学的研究結果から肝発がんに関与しないことが知られている。この種差については近年の研究の進展により徐々に明らかにされてきた。すなわち、げっ歯類においてPBは、cytochrome P450 2b（Cyp2b）等の代謝酵素誘導や肝細胞増殖を起こし、肝肥大・肝発がんを誘発するが、異物の解毒／代謝に関わる核内受容体であるconstitutive androstane receptor（CAR）をノックアウトしたマウス（CARKOマウス）では肝肥大／肝発がんが誘発されない。一方、ヒト由来のCARを発現させたマウスへの暴露では肝肥大は起こるものの、肝細胞増殖が起こらない。これらの知見の蓄積により、Cyp2b誘導化合物によるげっ歯類での肝発がんについて、CARの関与やその他PBの代謝といくつかの共通するkey eventを有する場合は、「ヒトへの外挿性なし」とするmode of action（MOA）の考え方が提唱され、広く受け入れられつつある。しかし、化学物質によるCAR活性化機序やその下流の薬物代謝酵素の誘導パスウェイ、あるいは細胞増殖に至る過程は完全には解明されておらず、様々なCyp2b誘導化合物がある中で、画一的な基準で肝発がん性のリスク評価を行ってしまうことには大きな懸念が残る。　そこで本研究では、げっ歯類でCyp2bを誘導して肝肥大・肝発がんを起こすピペロニルブトキシド（PBO）及びデカブロモジフェニルエーテル（DBDE）について、肝肥大及び肝発がんとCARとの関連性について調べ、これらCyp2b誘導化合物の肝発がん性のヒトへの外挿性を検討することを第一の目的とした。実験では、野生型及びCARKOマウスにPBO又はDBDEを混餌投与し、肝重量測定、病理組織学的検査、肝細胞増殖活性、及び肝薬物代謝酵素の発現量変化について検討するとともに、PB型の肝発がんに関わる変化（肝肥大、肝細胞増殖活性の亢進等）の比較を行い、外挿性について考察した。また、発がん性におけるCAR以外の核内受容体の関与についても調べることで、Cyp2b誘導化合物のMOA分析の妥当性を検討することを第二の目的とした。　第1章では、雄性の野生型マウス及びCARKOマウスにPBO又はDBDEを1週間、4週間、あるいは肝発がんイニシエーターであるジエチルニトロソアミン（DEN）単回投与後に27週間混餌投与し、肝肥大及び肝発がんにおけるCARの関与を調べた。その結果、PBOの5,000 ppm混餌投与では、野生型マウスで強くCyp2bを誘導しCARの活性化が示されたが、肝肥大は野生型マウスとCARKOマウスの両方で認められた。PBOの投与では遺伝子型に関わらずCyp3aの誘導もみられ、本剤による肝肥大にはCAR以外にpregnane X receptor（PXR）も関与している可能性が示された。肝細胞増殖活性は、野生型マウスではPBOとPBで、CARKOマウスではPBOのみで亢進していた。肝発がん性については、PBOはPBと同様に、野生型マウスで好酸性の増殖性病変を誘発し、CARKOマウスではこれが大幅に抑制されたことから、PBOによる肝発がんは主にCARに依存していることが明らかとなった。ただし、PBOはCARKOマウスにおいても陰性対照群よりも増殖性病変の発生頻度及び発生数が僅かながら高く、CAR以外にも酸化ストレスやPXRを介した肝臓での代謝変化も腫瘍発生に寄与している可能性も考えられた。DBDEの50,000 ppm混餌投与では、野生型マウスのみでCyp2bの誘導及び肝肥大が認められ、DBDEの肝肥大はCARに依存していることが明らかとなった。一方、DBDEによる肝発がん性については、野生型及びCARKOマウスの両方で好塩基性の増殖性病変を増加させ、Cyp1a1, Cyp1a2、Cyp1b1のmRNA発現レベルを上昇させたことから、肝発がん促進作用にはaryl hydrocarbon receptor（AhR）が関与している可能性が考えられた。　第2章では、マウスの肝腫瘍の発生頻度には性差があること、化合物によってはCARの感受性にも性差があることから、雌性の野生型マウス及びCARKOマウスにPBO又はDBDEを4週間、あるいはDEN単回投与後に27週間混餌投与し、第1章に準じた各種検索を実施し雌雄差を検討した。その結果、PBOによる肝肥大、肝発がんについて雌雄差は認められなかった。すなわち、雌マウスにおいても肝肥大にはCAR以外にPXRの関与が疑われた一方で、肝発がんについてはCARに依存して好酸性の増殖性病変が誘発された。DBDEについては、雄での肝肥大はCARに依存していたのに対し、雌ではCAR以外の経路が肝肥大に寄与する可能性が示された。また、雄性マウスでDBDE投与によりCARに依存せず増加した好塩基性の増殖性病変は、雌では野生型マウスで変異肝細胞巣の若干の増加がみられたのみであり、雌雄で感受性の違いがみられた。DENによるイニシエーション作用によって誘発される好塩基性の肝腫瘍は性ホルモンによる影響を受けることが知られており、本実験においてDBDEにより誘発される好塩基性の増殖性病変の感受性の雌雄差と一致するものであった。　第3章では、PBOによる肝肥大における投与用量と活性化される核内受容体の関連性について調べた。本章では5,000 ppm（高用量）に加え、中用量（1,000 ppm）、低用量（200 ppm）のPBO投与群を設定し、1週間の混餌投与を行い、CAR、PXR、peroxisome proliferator-activated receptor（PPAR）、及びAhRの各核内受容体の標的であるCyp2b、Cyp3a、Cyp4a、及びCyp1aの発現をmRNA及びタンパクレベルで解析した。その結果、高用量と異なり中用量では野生型マウスのみで肝肥大、Cyp2b10 mRNA発現レベルの増加、及び抗Cyp2b抗体に対する小葉中心性の陽性反応増強がみられたことから、中用量での肝肥大にはCARのみが寄与していることが明らかとなった。本章の実験結果から、PBOによる酵素誘導及び肝肥大では、投与量により関与する核内受容体が異なることが示された。以上の研究結果から、DBDEによる発がんについてはCAR以外の経路が示唆され、PBとは異なるタイプの肝発がんプロモーション作用を有すると考えられ、MOA分析によって「ヒトへの外挿性なし」との判断はできないと結論した。しかし、DBDEは好塩基性の腫瘍がもともと少ない雌マウスでは腫瘍を誘発しなかったこと、及び本研究でのDBDEは50,000 ppmと非常に高濃度であり、またDBDEの生体吸収性は非常に低いと言われていることから、実際のリスク管理においては、げっ歯類で誘発される腫瘍のタイプに応じた発生メカニズム考察や、ヒトでの暴露量も含めた考察が必要になるであろう。PBOについては、CAR活性化、好酸性の変異肝細胞巣／腺腫の誘発などのPBタイプのMOAのkey eventsを満たしていた。一方、PBOはCARKOマウスでも僅かながらに増殖性病変を増加させ、肝肥大にはPXRといった他の核内受容体の関与も示されたことから、CAR以外の経路も弱いながら肝発がん促進に寄与する可能性が示唆された。しかしながら、MOA分析結果のヒトへの外挿性については、key eventsの確認に加えてヒトと実験動物間での暴露量や代謝機能の違いを考慮に入れる必要があり、第3章では中用量のPBOによる肝肥大には、CARのみが関与していることを明らかにした。よって、中用量では増殖性病変の発生も完全にCARに依存していることが推察され、その場合は「ヒトへの外挿性なし」と判断できる可能性が高いと考えた。　化学物質のげっ歯類での肝発がん性について、ヒトへの外挿性を検討するために用いられるMOA分析は、化学物質を適切に管理しながら使用していくために非常に有用なツールである。しかし、通常実施されている野生型マウスやラットを用いた毒性試験では、CARに依存した肝肥大や肝発がんによって他の経路を介した作用がマスクされてしまう可能性があり、潜在的なリスクを見逃す恐れがある。本研究では、げっ歯類でCyp2bを誘導する非遺伝毒性の化学物質であるPBO及びDBDEについて、CARKOマウスを効果的に用いることで、肝肥大／肝発がんにおけるCAR及びその他の核内受容体の関与を明らかにし、げっ歯類での発がん性のヒトへの外挿性判断を行った。また、本研究で用いた化合物に限らず、Cyp2b誘導化合物の発がん性についてCARKOマウスを用いた実験手法をMOA分析と組み合わせることで、より正確で信頼性の高い評価を行うことができると結論した。There are many kinds of chemicals, which are naturally derived or artificially synthesized, in the atmosphere of modern life of humans. We are exposed to them by oral route as additives or residues in food or water, by respiration, or by cutaneous contact. To assess the effects of these chemicals to humans, a lot of experimental procedures have been undertaken. One of them is toxicity study with laboratory animals, usually with rodents such as rats and mice, and they provide useful information to speculate hazardous properties of the chemicals to humans. On the other hand, we have to consider the relevance of the toxic information obtained from animal studies to human reactions based on the mechanism of action, since each animal species have different physiological and metabological function. Especially in a case that carcinogenic potential was found in laboratory animals, the extrapolation to the humans would be the most important point in its risk assessment. One of the cases that species difference of carcinogenicity between rodents and humans has been clearly defined is phenobarbital (hereafter PB). PB is known to induce liver hypertrophy and liver tumor in rodents. In humans, it has been widely used as antiepileptic drug; however, comprehensive epidemiological researches revealed that hepatocarcinogenic property of PB in rodents is not relevant to humans. After late 1990’s, extensive researches using gene engineering have gradually revealed the mechanism of this species difference. PB induces liver hypertrophy accompanied by the induction of certain drug metabolic enzymes such as various cytochrome P450s (particularly Cyp2b), stimulation of cell proliferation activity, and inhibition of apoptosis, then long-term treatment with PB promotes liver tumorigenesis in rodents. However, in the mice lacking the gene of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor related to detoxification and metabolism of xenobiotics, PB does not induce Cyp2b, liver hypertrophy, or liver tumor; clearly indicating that CAR is essential for PB-inducible liver hypertrophy and hepatocarcinogenesis in mice. On the contrary, it was reported that the mice which express human CAR in their hepatocytes shows liver hypertrophy but no hepatocellular proliferation, suggesting that CAR is involved in the difference of susceptibility in the hepatocarcinogenic potential of PB between rodent and human. These researches for the species difference in the PB-inducible liver hypertrophy and hepatocarcinogenesis developed into a concept of the Mode of Action (MOA) analysis, which is used to assess the extrapolation of chemically induced hepatocarcinogenesis. In the MOA analysis, if rodents given a chemical show some key events like CAR activation, liver hypertrophy, hepatocellular proliferation, and so on, the hepatocarcinogenic potential of the chemical is considered “not human relevant.” However, there are various compounds that induce Cyp2b, and the mechanism of CAR activation by those chemical substances, the induction pathway of drug-metabolizing enzymes downstream thereof, or the process leading to cell proliferation has not been fully elucidated. Under such circumstances, there is a great concern that risk assessment of liver carcinogenicity will be conducted according to a uniform standard. Therefore, in this research, the involvement of CAR in liver hypertrophy and liver tumor induction were examined for piperonyl butoxide (PBO) and decabromodiphenyl ether (DBDE), which are known to induce Cyp2b and cause liver hypertrophy and tumor in rodents, and examined the extrapolation of their hepatocarcinogenic property to humans. In the experiments, wild-type and CAR knock-out (CARKO) mice were given PBO and DBDE in the diet and examined liver weight, histopathological examination, hepatocyte proliferative activity, and expression levels of hepatic drug-metabolizing enzymes, then the extrapolation of the hepatocarcinogenic property of PBO and DBDE to humans were discussed by MOA analysis. In addition, involvement of nuclear receptors other than CAR was investigated in the experiments in order to discuss the validity of the MOA analysis. In the Chapter 1, male wild-type and CARKO mice were given PBO and DBDE in the diet for 1 week, 4 weeks, or 27 weeks (after a single intraperitoneal administration of diethyl nitrosamine (DEN), which is a liver tumor initiator), and the involvement of CAR in liver hypertrophy and hepatocarcinogenesis was investigated. As a result, strong induction of Cyp2b, which indicates CAR activation, was observed in wild-type mice at 5000 ppm of PBO, but increase in liver weights and hepatocellular hypertrophy were observed in both wild-type and CARKO mice. Since PBO induced Cyp3a regardless of genotype, it was supposed that not only CAR but the pregnane X receptor (PXR, NR1I2) are involved in the liver hypertrophy caused by PBO. Hepatocellular proliferative activity was increased in PBO and PB groups in wild-type mice and in PBO group in CARKO mice. Regarding hepatocarcinogenesis, PBO induced eosinophilic proliferative lesions in wild-type mice as in PB, and those were considerably suppressed in CARKO mice, hence hepatocarcinogenesis by PBO is mainly dependent on CAR. In CARKO mice, however, PBO has a higher incidence of proliferative lesions than those in the negative control group, thus oxidative stress or the change in metabolic status in the liver via PXR may also contribute to tumorigenesis in addition to CAR. In DBDE treatment at 50000 ppm in the diet, induction of Cyp2b and liver hypertrophy were observed only in wild-type mice, and it was revealed that liver hypertrophy by DBDE is dependent on CAR. On the other hand, DBDE increased the number of basophilic proliferative lesions in both wild-type and CARKO mice, suggested that DBDE promotes liver carcinogenesis by a route independent on CAR. Since DBDE increased the mRNA expression levels of Cyp1a1, 1a2, and 1b1, the aryl hydrocarbon receptor (AhR) may be involved in the liver tumor promoting effect by DBDE. In the Chapter 2, sex difference in the relationship between liver hypertrophy/tumor induction and CAR activation was examined, since incidences of liver tumor in mice are generally different by sex and some chemicals were reported to have different effect on CAR activation between males and females. Female wild-type and CARKO mice were treated with PBO and DBDE for 4 weeks, or 27 weeks after single administration of DEN, then various examinations such as histopathology and mRNA expression levels were carried out in accordance with the Chapter 1. In consequence, there was no sex difference in liver hypertrophy and hepatocarcinogenesis induced by PBO. That is, PXR was suspected to be involved in liver hypertrophy in addition to CAR, while eosinophilic proliferative lesions were induced depending on CAR in female mice as well as in males. As for DBDE, liver hypertrophy in males was dependent on CAR, whereas in females a route other than CAR could contribute to liver hypertrophy. In addition, there was a sex difference in the promoting effect of basophilic proliferative lesions which were CAR-independently induced by DBDE in male mice but not obviously induced in female mice. The sex difference in susceptibility of basophilic proliferative lesions by DBDE is probably related to sex hormones considering the report which says that the induction of basophilic tumors by DEN initiation is altered by sex hormones. In Chapter 3, the relationship between dose levels of PBO and the involvement of nuclear receptors including CAR and others. In this experiment, in addition to 5000 ppm, male mice were treated with 1000 and 200 ppm of PBO in the diet for 1 week, and investigate the activation of CAR, PXR, PPAR and AhR by examine the target phase I liver metabolizing enzymes of each nuclear receptors, Cyp2b, Cyp3a, Cyp4a, and Cyp1a, respectively, in mRNA and protein levels. As a result, 5000 ppm of PBO induced liver hypertrophy regardless of CAR and the involvement of PXR was indicated by Cyp3a induction. On the contrary, 1000 ppm of PBO exhibited liver hypertrophy and Cyp2b induction only in wild-type mice; indicated that only CAR contributes at 1000 ppm of PBO. The result of this experiment revealed that the involvement of nuclear receptors alters by the dose levels of PBO.Based on the results from the experiments in the Chapter 1 to 3, liver tumor induction by DBDE has a pathway other than CAR and DBDE has a different mechanism of liver tumor promotion when compared to PB. Therefore, according to the MOA analysis, it cannot be said “not human relevant.” DBDE, however, did not induce basophilic-type tumors in female mice, thus more detailed disccusion depending on the type of tumor induced in rodents will be needed for the risk management of DBDE. Also, considering that the dose level of DBDE in this research was extremely high at 50000 ppm, and that it was reported that the orally administrated DBDE has very low absorbance to the body, it will be very important to discuss the exposure levels to humans in the risk management of the chemical.Meanwhile, the effects of PBO coincide with the key events in PB-type MOA such as Cyp2b induction, increase in hepatocellular proliferative activity, and induction of eosinophilic altered foci/adenomas. However, PBO slightly increased liver proliferative lesions even in CARKO mice and the involvement of PXR was suspected in the liver hypertrophy, suggesting the possibility that other pathways other than CAR contribute to the promotion of hepatocarcinogenesis. In the MOA analysis, it is necessary to take into consideration the key events as well as the differences on exposures or metabological function between laboratory animals and humans. The experiment in Chapter 3 revealed that only CAR is responsible for the liver hypertrophy induced by low dose of PBO. Hepatocarcinogenic property of PBO at low dose has not been investigated in this research, but it is supposed that the occurrence of liver tumors is also dependent on CAR at low dose, and in that case, it could be judged as “not human relevant”.The MOA analysis used to examine the extrapolation to humans for chemical hepatocarcinogenicity in rodents is a helpful tool for appropriate risk management of chemicals which can enrich our life. However, with traditional toxicity studies using wild-type mice or rats, there is a possibility that strong effect of CAR on the liver hypertrophy and tumor induction may cover the involvement of other pathway and that may make us misunderstand the potential risk of the chemical. In the present research, the effective use of CARKO mice shed light on the involvement of CAR and other nuclear receptors in liver hypertrophy and hepatocarcinogenesis. From the above, by combining experiments with CARKO mice and the MOA analysis, it is possible to conduct more accurate and highly reliable evaluation for hepatocarcinogenic property of Cyp2b-inducible chemicals.博士(獣医学)麻布大

A Case of Right Hepatic Artery Syndrome Diagnosed by Using SpyGlassDSTM System

We report the case of a 68-year-old woman who had abdominal pain and slightly elevated biliary enzymes. Magnetic resonance cholangiopancreatography detected biliary duct stenosis, while contrast-enhanced magnetic resonance imaging showed that the right hepatic artery transversed the extrahepatic bile duct at the level of bifurcation of the bile duct. We performed endoscopic retrograde cholangiopancreatography and peroral cholangioscopy with the SpyGlass DS? system. Then, mild extrinsic pulsatile compression of the bile duct was observed at stricture level with an intact bile duct epithelium. Therefore, she was diagnosed with right hepatic artery syndrome and underwent cholecystectomy. Six months later, her biliary enzyme level decreased, and the recurrence of pain gradually decreased

Improved brain MRI indices in the acute brain stem infarct sites treated with hydroxyl radical scavengers, Edaravone and hydrogen, as compared to Edaravone alone. A non-controlled study

<p>Abstract</p> <p>Background</p> <p>In acute stage of cerebral infarction, MRI indices (rDWI & rADC) deteriorate during the first 3-7 days after the ictus and then gradually normalize in approximately 10 days (pseudonormalization time), although the tissue is already infarcted. Since effective treatments improve these indices significantly and in less than the natural pseudonormalization time, a combined analysis of these changes provides an opportunity for objective evaluation on the effectiveness of various treatments for cerebral infarction. Hydroxyl radicals are highly destructive to the tissue and aggravate cerebral infarction. We treated brainstem infarction patients in acute stage with hydroxyl radical scavengers (Edaravone and hydrogen) by intravenous administration and evaluated the effects of the treatment by a serial observation and analysis of these MRI indices. The effects of the treatment were evaluated and compared in two groups, an Edaravone alone group and a combined group with Edaravone and hydrogen, in order to assess beneficial effects of addition of hydrogen.</p> <p>Methods</p> <p>The patients were divided in Edaravone only group (E group. 26 patients) and combined treatment group with Edaravone and hydrogen enriched saline (EH group. 8 patients). The extent of the initial hump of rDWI, the initial dip of rADC and pseudo-normalization time were determined in each patient serially and averages of these data were compared in these two groups and also with the natural course in the literatures.</p> <p>Results</p> <p>The initial hump of rDWI reached 2.0 in the E group which was better than 2.5 of the natural course but was not as good as 1.5 of the EH group. The initial dip of rADC was 0.6 in the E group which was close to the natural course but worse than 0.8 of the EH group. Pseudonormalization time of rDWI and rADC was 9 days only in EH group but longer in other groups. Addition of hydrogen caused no side effects.</p> <p>Conclusions</p> <p>Administration of hydroxyl radical scavengers in acute stage of brainstem infarction improved MRI indices against the natural course. The effects were more obvious and significant in the EH group. These findings may imply the need for more frequent daily administration of hydroxyl scavenger, or possible additional hydrogen effects on scavenger mechanisms.</p

Hydrogen(H2) treatment for acute erythymatous skin diseases. A report of 4 patients with safety data and a non-controlled feasibility study with H2 concentration measurement on two volunteers

BACKGROUND: We have treated 4 patients of acute erythematous skin diseases with fever and/or pain by H(2) enriched intravenous fluid. We also added data from two volunteers for assessing the mode of H(2) delivery to the skin for evaluation of feasibility of H(2) treatment for this type of skin diseases. METHODS: All of the four patients received intravenous administration of 500 ml of H2 enriched fluid in 30 min for more than 3 days except in one patient for only once. From two volunteers (one for intravenous H2 administration and the other for H2 inhalation), blood samples were withdrawn serially and air samples were collected from a heavy duty plastic bag covering a leg, before, during and after H2 administration. These samples were checked for H2 concentration immediately by gas chromatography. Multiple physiological parameters and blood chemistry data were collected also. RESULTS: Erythema of these 4 patients and associated symptoms improved significantly after the H2 treatment and did not recur. Administration of H2 did not change physiological parameters and did not cause deterioration of the blood chemistry. The H2 concentration in the blood from the volunteers rapidly increased with H2 inhalation and slowly decreased with cessation of H2 particularly in the venous blood, while H2 concentration of the air from the surface of the leg showed much slower changes even after H2 inhalation was discontinued, at least during the time of sample collection. CONCLUSION: An improvement in acute erythemtous skin diseases followed the administration of H2 enriched fluid without compromising the safety. The H2 delivery study of two volunteers suggested initial direct delivery and additional prolonged delivery possibly from a slowly desaturating reservoir in the skin to the surface

Immobilization-induced hypersensitivity associated with spinal cord sensitization during cast immobilization and after cast removal in rats

This study examined mechanical and thermal hypersensitivity in the rat hind paw during cast immobilization of the hind limbs for 4 or 8 weeks and following cast removal. Blood flow, skin temperature, and volume of the rat hind paw were assessed in order to determine peripheral circulation of the hind limbs. Sensitization was analyzed by measuring the expression of the calcitonin gene-related peptide (CGRP) in the spinal dorsal horn following cast immobilization. Two weeks post immobilization, mechanical and thermal sensitivities increased significantly in all rats; however, peripheral circulation was not affected by immobilization. Cast immobilization for 8 weeks induced more serious hypersensitivity compared to cast immobilization for 4 weeks. Moreover, CGRP expression in the deeper lamina layer of the spinal dorsal horn increased in the rats immobilized for 8 weeks but not in those immobilized for 4 weeks. These findings suggest that immobilization-induced hypersensitivity develops during the immobilization period without affecting peripheral circulation. Our results also highlight the possibility that prolonged immobilization induces central sensitization in the spinal cord.The final publication is available at link.springer.co

A basic study on molecular hydrogen (H2) inhalation in acute cerebral ischemia patients for safety check with physiological parameters and measurement of blood H2 level

BACKGROUND: In animal experiments, use of molecular hydrogen ( H(2)) has been regarded as quite safe and effective, showing benefits in multiple pathological conditions such as ischemia-reperfusion injury of the brain, heart, kidney and transplanted tissues, traumatic and surgical injury of the brain and spinal cord, inflammation of intestine and lung , degenerative striatonigral tissue and also in many other situations. However, since cerebral ischemia patients are in old age group, the safety information needs to be confirmed. For the feasibility of H(2) treatment in these patients, delivery of H(2) by inhalation method needs to be checked for consistency. METHODS: Hydrogen concentration (HC) in the arterial and venous blood was measured by gas chromatography on 3 patients, before, during and after 4% (case 1) and 3% (case2,3) H(2) gas inhalation with simultaneous monitoring of physiological parameters. For a consistency study, HC in the venous blood of 10 patients were obtained on multiple occasions at the end of 30-min H(2) inhalation treatment. RESULTS: The HC gradually reached a plateau level in 20 min after H(2) inhalation in the blood, which was equivalent to the level reported by animal experiments. The HC rapidly decreased to 10% of the plateau level in about 6 min and 18 min in arterial and venous blood, respectively after H(2) inhalation was discontinued. Physiological parameters on these 3 patients were essentially unchanged by use of hydrogen. The consistency study of 10 patients showed the HC at the end of 30-min inhalation treatment was quite variable but the inconsistency improved with more attention and encouragement. CONCLUSION: H(2) inhalation of at least 3% concentration for 30 min delivered enough HC, equivalent to the animal experiment levels, in the blood without compromising the safety. However, the consistency of H(2) delivery by inhalation needs to be improved

健常人におけるA1型とA2型のボツリヌス神経毒素の電気生理学的検査による比較

Botulinum neurotoxin type A1 (BoNTs/A1) and type B (BoNT/B) have been used for treating hyperactive muscle contractions. In the present study, we compared the effect of botulinum neurotoxin subtype A2 (6.5 mouse LD50 units A2 neurotoxin, A2NTX) and onabotulinumtoxinA (10 mouse LD50 units BoNT/A1 product) by measuring the compound muscle action potentials (CMAPs) before and after administration. In total, 8 healthy subjects were examined in the present study. A2NTX was injected into the extensor digitorum brevis (EDB) muscle, followed by onabotulinumtoxinA injection into the contralateral EDB muscle after 16 weeks. The CMAP amplitudes from the EDB, abductor hallucis (AH), and abductor digiti minimi pedis (ADM) muscles were measured after each BoNT injection on days 1, 3, 7, 14, 28, 56, 84, and 112 to assess the effect of the toxin. On day 14, both A2NTX and onabotulinumtoxinA produced an approximately 70% decline in EDB CMAP amplitude compared to the baseline values; significant reduction of the CMAP continued through day 112. The CMAP amplitudes from neighboring muscles (AH and ADM) remained intact throughout the study period, except for a slight but significant drop at day 28 after onabotulinumtoxinA injection compared to A2NTX. The current findings indicate that small doses (6.5 units and 10 units) of A2NTX and onabotulinumtoxinA have at least comparable onset and duration of action, although similar clinical effects were obtained with lower dose using A2NTX

Optimization of the proliferation and persistency of CAR T cells derived from human induced pluripotent stem cells

CARシグナルを補完する遺伝子改変により *iCAR-T細胞の固形がん治療効果が改善される. 京都大学プレスリリース. 2022-12-13.Genetic modifications boosting CAR signaling improve the therapeutic efficacy of iPSC-derived CAR-T cells against solid tumors. 京都大学プレスリリース. 2022-12-13.The effectiveness of chimaeric antigen receptor (CAR) T-cell immunotherapies against solid tumours relies on the accumulation, proliferation and persistency of T cells at the tumour site. Here we show that the proliferation of CD8αβ cytotoxic CAR T cells in solid tumours can be enhanced by deriving and expanding them from a single human induced-pluripotent-stem-cell clone bearing a CAR selected for efficient differentiation. We also show that the proliferation and persistency of the effector cells in the tumours can be further enhanced by genetically knocking out diacylglycerol kinase, which inhibits antigen-receptor signalling, and by transducing the cells with genes encoding for membrane-bound interleukin-15 (IL-15) and its receptor subunit IL-15Rα. In multiple tumour-bearing animal models, the engineered hiPSC-derived CAR T cells led to therapeutic outcomes similar to those of primary CD8 T cells bearing the same CAR. The optimization of effector CAR T cells derived from pluripotent stem cells may aid the development of long-lasting antigen-specific T-cell immunotherapies for the treatment of solid tumours