大学院医学系研究科（医学）

Cardiovascular diseases are the leading cause of mortality and disability worldwide. We have previously found that sphingosylphorsphorylcholine (SPC) is the key molecule leading to vasospasm. We have also identified the SPC/Src family protein tyrosine kinase Fyn/Rho-kinase (ROK) pathway as a novel signaling pathway for Ca^{2+}-sensitization of vascular smooth muscle (VSM) contraction. The present study aimed to investigate whether hesperetin can inhibit the SPC-induced contraction with little effect on 40 mM K^+-induced Ca^{2+}-dependent contraction and to elucidate the underlying mechanisms. Hesperetin significantly inhibited the SPC-induced contraction of porcine coronary artery smooth muscle strips with little effect on 40 mM K^+-induced contraction. Hesperetin blocked the SPC-induced translocation of Fyn and ROK from the cytosol to the membrane in human coronary artery smooth muscle cells (HCASMCs). SPC decreased the phosphorylation level of Fyn at Y531 in both VSMs and HCASMCs and increased the phosphorylation levels of Fyn at Y420, myosin phosphatase target subunit 1 (MYPT1) at T853 and myosin light chain (MLC) at S19 in both VSMs and HCASMCs, which were significantly suppressed by hesperetin. Our results indicate that hesperetin inhibits the SPC-induced contraction at least in part by suppressing the Fyn/ROK pathway, suggesting that hesperetin can be novel drug to prevent and treat vasospasm.

The hippocampal dentate gyrus has been identified to play a critical role in maintaining contextual memory in many mammalian species. To evaluate learning-induced synaptic plasticity of granule cells, we subjected male rats to an inhibitory avoidance (IA) task and prepared acute hippocampal slices. In the presence of 0.5 µM tetrodotoxin, we recorded miniature EPSCs in male rats experiencing four groups: untrained, IA-trained, unpaired, and walk-through. Compared with the untrained, IA-trained, unpaired, and walk-through groups, the unpaired group significantly enhanced mean mEPSC amplitudes, suggesting the experience-induced plasticity at AMPA receptor-mediated excitatory synapses. For inhibitory synapses, both unpaired and walk-through groups significantly decreased mean mIPSC amplitudes, showing the experience-induced reduction of postsynaptic GABA_A receptor-mediated currents. Unlike the plasticity at CA1 synapses, it was difficult to explain the learning-specific plasticity at the synapses. However, overall multivariate analysis using four variables of mE(I)PSC responses revealed experience-specific changes in the diversity, suggesting that the diversity of excitatory/inhibitory synapses onto granule cells differs among the past experience of animals include the learning. In comparison with CA1 pyramidal neurons, granule cells consistently showed greater amplitude and frequency of mE(I)PSCs. Fluctuation analysis further revealed that granule cells provide more postsynaptic AMPA receptor channels and greater single-channel current of GABA_A receptors of than CA1 pyramidal neurons. These findings show functional differences between two types of principal cells in the hippocampus.

前身に投与された治療薬が脳組織実質に到達するには、神経組織の血管により形成される血液脳関門を通過する必要がある。そのため、組織への損傷を最小限に抑えて血液脳関門を開くことができれば、難治性神経疾患の治療法開発に大きな進展をもたらすことが期待される。本研究では、血液脳関門を形成する血管内皮細胞に発現するBasiginに着目し、その内因性リガンドであるCyclophilin A (CypA) を用いて、血液脳関門機能を人為的に制御することを目的とした。マウス脳血管内皮細胞株を用いたin vivo解析により、CypAの投与がBasiginを介して血液脳関門機能を低下させること、それにより脳実質へ効率的に薬物を送達できることを示した。単層培養された血管内皮細胞において、CypAはタイト結合構成分子の一つであるClaudin-5を一過性かつ可逆的に細胞膜から消失させて、バリアー機能を低下させることを見出した。また、マウスへのCypAの単回静脈内投与では血液脳関門が一定期間開いた後、自発的に元の状態へ回復することが示され、そしてその限定された機関において、全身投与された水溶性薬物Doxorubicinが脳組織実質へ送達されることが明らかとなった。本研究の結果は、CypAの静脈内投与によって、脳実質への薬物送達を自在にコントロールできることを示しており、難治性神経疾患に対する治療法確立に向けた重要な成果であると考えられる。

【背景】近年、歯周炎や歯肉炎に関連する嫌気性グラム桿菌であるFusobacterium nucleatum (F. nucleatum) は大腸がんの発生や進行に関与することが報告されている。この菌の制御が大腸がんの予防につながる可能性があると考え、深紫外線発光ダイオード (DUV-LED) によるF. nucleatumの殺菌効果を検討した。【方法】DUV-LEDのF. nucleatumに対する殺菌効果を定性的、定量的に評価した。ピーク波長が265nmと280nmの2種類のDUV-LEDを使用した。F. nucleatumのDNAに対するダメージは、シクロブタンピリミジン二量体(CPD)とピリミジンピリミドン光生成物 (6-4PP) の生成で評価した。【結果】DUV-LEDでの265nmまたは280nmの波長を3分間照射したところ、コロニーの成長は観察されなかった。265nmのDUV-LED光照射下におけるF. nucleatumの生存率は10秒照射で0.0014%、20秒照射で0%に低下した。同様に、280nmのDUV-LED光照射では，10秒照射で0.00044%、20秒照射で0%に低下した。DUV-LEDから35mmの距離での放射照度は、265nmのLEDで0.265mW/cm^2、280nmのLEDでは0.415mW/cm^2であった。従って、致死量を示す放射エネルギーは265nm LEDは5.3mJ/cm^2、280nm LEDは8.3mJ/cm^2であった。265nmと280nmのDUV-LED光をF. nucleatumに照射した際のCPDと6-4PPの量はそれぞれ6.548ng/μg、1.333ng/μgであった。【結論】DUV-LED光は、F. nucleatumに対して、ピリミジン二量体を形成することにより殺菌効果を発揮した。

【背景】全層性治癒（Transmural healing healing; TH）は、クローン病の新たな治療標的として注目されているが、日本ではTHに関する臨床データはほとんどない。我々は、クローン病のモニタリング法として低被曝線量CTエンテログラフィ（CTE）を導入し、CTEによるTHの評価をレトロスペクティブに検討した。【方法】2009年 1月から2021年3月までに当院で低被曝線量CTEを施行したクローン病患者のうち、2週間以内に大腸内視鏡検査またはバルーン内視鏡検査を施行した122例を対象とした。放射線検査と内視鏡検査の結果は、それぞれ放射線科医と消化器内視鏡医が独立して検討した。CTEと内視鏡検査の診断の一致率を算出した。【結果】26名（21.3％）のクローン病患者がTHを達成し、カッパ係数は0.743と2人の放射線科医の間でかなりの一致が見られた。TH群と非TH群の比較では、クローン病活動指数（Crohn’s Disease Activity Index ; CDAI ）（P値 = 0.02)、内視鏡的治癒率（P値 ＜ 0.001）、血清アルブミン（P値 = 0.043）、血清C反応性蛋白（P値 = 0.018）に有意差が認められた。122名の患者のうち、69名（56.5％）はCTEの診断と内視鏡検査が一致し、22名（18.0％）はTHと内視鏡の両方の治癒を達成した。【結論】本研究は、日本における低被曝線量CTEによるクローン病のリアルワードデータを示すものである。本研究で用いたTHの基準はカッパ係数が高く、多くの施設で再現性を持って用いることができると考えられる。

背景：顕微鏡的多発血管炎 (MPA) は全身臓器が障害される疾患である。しかしながら、MPA患者における心臓超音波検査（心エコー図検査）における指標の特徴については不明な点が多い。 目的：本研究は、単施設後ろ向き研究で、ステロイド療法の新規導入または再導入後2週間以内に心エコーを行ったMPA患者15名を対象とし、その心エコー所見の特徴について検討することを研究の目的とした。30人の年齢・性別をマッチさせた心疾患のないコントロール群と比較検討した。 方法と結果：左室径、左室駆出率、拡張早期僧帽弁輪最大移動速度 (e’) に2群間で有意差は認めなかった。一方、MPA群では左房径、左房容積係数が有意に高く、左室流入血流速波形 (TMF) の早期拡張期充満速度(E波)と肺静脈流入拡張期血流速度 (D波) の高さ、三尖弁における収縮期右室-右房最大圧較差も有意に高く、TMFのE波の減速時間 (DCT) は短縮していた。血清CRPはTMFのE波高、E/A比およびDCTと相関が見られた。今回の研究では、MPA患者におけるe’の有意な低下がみられなかったことから、左室弛緩能の低下よりはむしろ左室スティフネスの上昇によって左室拡張機能低下が生じ、結果として左房拡大が生じている可能性が示唆された。 結語：急性期MPA患者では左室拡張機能低下によると考えられる左房拡大を生じていた。MPA患者、特に強い炎症反応を伴う患者では、心機能評価を行うことが重要であることが示唆された。

To investigate whether dantrolene (DAN), cardiac ryanodine receptor (RyR2) stabilizer, improves impaired diastolic function in an early pressure-overloaded hypertrophied heart, pressure-overload hypertrophy was induced by transverse aortic constriction (TAC) in mice. Wild-type (WT) mice were divided into four groups: sham-operated mice (Sham), sham-operated mice treated with DAN (DAN+Sham), TAC mice (TAC), and TAC mice treated with DAN (DAN+TAC). The mice were then followed up for 2 weeks. Left ventricular (LV) hypertrophy was induced in TAC, but not DAN+TAC mice, 2 weeks after TAC. There were no differences in LV fractional shortening among the four groups. Catheter tip micromanometer showed that the time constant of LV pressure decay, an index of diastolic function, was significantly prolonged in TAC but not in DAN+TAC mice. Diastolic function was significantly impaired in TAC, but not in DAN+TAC mice as determined by cell shortening and Ca^{2+} transients. An increase in diastolic Ca^{2+} leakage and a decrease in calmodulin (CaM) binding affinity to RyR2 were observed in TAC mice, while diastolic Ca^{2+} leakage improved in DAN+TAC mice. Thus, DAN prevented the progression of hypertrophy and improved the impairment of LV relaxation by inhibiting diastolic Ca^{2+} leakage through RyR2 and the dissociation of CaM from RyR2.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in the cardiac type 2 ryanodine receptor (RyR2). Using knock-in mouse (KI) model (R2474S/+), we previously reported that a single point mutation within the RyR2 sensitized the channel to agonists, primarily mediated by defective inter-domain interaction within the RyR2 and subsequent dissociation of calmodulin (CaM) from the RyR2. Here, we examined whether CPVT can be genetically rescued by enhancing the binding affinity of CaM to the RyR2. We first determined whether there was a possible amino-acid substitution within the CaM-binding domain in the RyR2 (3584-3603) that can enhance its binding affinity to CaM, and found that V3599K substitution showed the highest binding affinity of CaM to CaM-binding domain. Hence, we generated a heterozygous KI mouse model (V3599K/+) with a single amino acid substitution in the CaM-binding domain of the RyR2, and crossbred it with the heterozygous CPVT –associated R2474S/+ KI mouse to obtain a double heterozygous R2474S/V3599K KI mouse model. The CPVT phenotypes, bidirectional or polymorphic ventricular tachycardia, were inhibited in the R2474S/V3599K mice. Thus, enhancement of the CaM binding affinity of the RyR2 is essential to prevent CPVT-associated arrhythmogenesis.

Spontaneous rupture of hepatocellular carcinoma(HCC)is well known. However, rupture of HCC after transcatheter arterial chemoembolization(TACE)is a rare but potentially fatal complication. Herein, we report a case of ruptured HCC after TACE in a 73-year-old man with chronic liver injury(non-B and non-C).He had previously undergone subsegmental resection of S7 of the liver for HCC. One year later, multiple HCCs were detected. TACE was performed for the recurrent HCCs located on the surface of S2 of the liver. Epigastric pain and decreased blood pressure were observed after the treatment. Although his symptoms improved immediately, the patient gradually became anemic. Radiological examination after TACE revealed a hematoma around the S2 lesion. Therefore, we diagnosed rupture of HCC. We performed TAE and could control the bleeding. Few possible mechanisms of ruptured HCC after TACE include increasing pressure inside the tumor and capsular injury due to TACE. Consequently, HCC which has a high risk of rupture after TACE should be assured embolization and careful observation.

A 36-year-old man diagnosed with Charcot-Marie-Tooth(CMT)disease type 1B was treated in the department of neurology of this hospital. Due to a liver injury(AST 237IU/ml, ALT 496IU/ml)which occurred in November 200X, he was introduced to our department. As a result of scrutiny, we diagnosed him with chronic hepatitis C genotype 2a(HCV-RNA 5.4logIU/ml).He will be admitted to the hospital for Interferon(IFN)therapy in April next year. Although the mechanism is unknown, there are a few reports of peripheral neuropathy related to IFN therapy. Therefore, IFN therapy is generally unfavorable for patients with neuromuscular disorders. There are several IFN preparations used in Japan. IFN alfa associated peripheral neuropathy appears less than 0.1-5% of the time. While there is no report of peripheral neuropathy related to IFN beta, we performed IFN beta and Rivabirin combination therapy with a severe follow-up by a neurologist. We could accomplish treatment without the exacerbation of neurologic symptoms. He achieved sustained viral response. When IFN therapy was provided for chronic hepatitis B or chronic hepatitis C complicated with peripheral neuropathy, there is some possibility of performing antiviral therapy safely by using IFN beta.

Duodenal varices（DV）are the most common of ectopic varices. Although bleeding from DV is rare, it is difficult to control bleeding and sometimes fatal. We have encountered four clinical cases of ruptured DV. Case 1：A man in his 80s presented with a history of partial gastrectomy with Billroth-II reconstruction and LC due to chronic hepatitis C. We performed single-balloon endoscopy and injected 67% N-butyl-2-cyanoacrylate（NBCA）for DV on the afferent loop with red plug. Case 2：A woman in her 40s with primary biliary cirrhosis complained of tarry stool and anemia. We performed endoscopic injection sclerotherapy（EIS）with 67% NBCA for spurting bleeding point in duodenum. Case 3：A man in his 50s with liver cirrhosis（LC）due to chronic hepatitis B complained of tarry stool. We performed endoscopic variceal ligation（EVL）and balloon-occluded retrograde transvenous obliteration（B-RTO）for DV. Case 4：A man in his 60s with alcoholic LC complained of tarry stool. We performed EIS with 67% NBCA for DV. We added EVL, clipping, and argon plasma coagulation after EIS to control bleeding. To control bleeding was achieved in all cases.

Background The lipid-lowering therapy by statins may stabilize and reduce coronary plaques. We compare the effect of strong or moderate statins on the coronary plaque characteristics by using 64-slice multidetector computed tomography (MDCT). Methods and Results We analyzed 13 subjects with non-calcified coronary plaques(NCP)as determined by MDCT. Pitavastatin(PTV:2mg/day)or pravastatin(PRA:10mg/day)were randomly administered. MDCT were performed at 0, 6 and 12 months after lipid-lowering therapy. In PTV group(n=6),CT density of NCP increased by 35.6 ± 28.8 HU after 6 months(p=0.037)and by 30.6 ± 29.8 HU after 12 months(NS).NCP area was decreased by 35.9 ± 15.2 % after 6 months and by 41.0 ± 16.5 % after 12 months(both P=0.001).In PRA group(n=7),CT density of NCP did not significantly increased after 6 months(NS)and after 12 months(NS).NCP area was not significantly decreased at 6 months(NS),but decreased by 23.1 ± 16.7 % at 12 months(P=0.001). Conclusions Serial CT angiography revealed that the regression of NCP occurs rapidly by strong lipid lowering therapy compared to the moderate statin therapy.

Aim: To evaluate the efficacy of quantitative analysis for differentiating pancreatic ductal adenocarcinoma (PDAC) and inflammatory pancreatic masses (IPM) using time intensity curve (TIC) analysis based on contrast-enhanced endoscopic ultrasonography (CE-EUS). Methods: We reviewed 89 patients who had undergone CE-EUS for pancreatic solid lesions at our department between August 2012 and January 2016. CE-EUS images were recorded for 2 minutes after injection of the contrast agent. The diagnostic abilities of the enhanced patterns and TIC analysis were assessed. Results: The enhanced patterns of PDAC were mainly hypovascular and heterogeneous (66/77), while IPM were mainly isovascular and homogeneous (6/12). In PDAC, sensitivity was 77.9%, specificity 83.3%, and accuracy was 78.7%. In TIC analysis, the intensity reduction rate was significantly different at 10 and 30 seconds after peak intensity. After creating a cutoff value (49%) based on the receiver operating characteristic curve for the intensity reduction rate after 30 seconds, diagnosing PDAC with TIC analysis had a sensitivity of 67.5%, specificity of 100%, and accuracy of 71.9%. Combining enhanced pattern analysis with TIC analysis had a sensitivity of 90.9%, pecificity of 83.3%, and accuracy of 89.9%. Conclusion: Combining TIC analysis with CE-EUS improved diagnostic accuracy when differentiating between PDAC and IPM.

We performed this study to investigate whether serum cell-free DNA testing(liquid biopsy)of either methylated SEPT9 or methylated SST, which are possible biomarkers of HCC, in combination with AFP may improve clinical performance for the detection of HCC. Study subjects included 25 healthy controls, 15 patients with chronic liver disease without HCC, and 38 patients with HCC. Methylation level of serum cell-free SEPT9 and SST was measured by methylation-sensitive restriction enzymes followed by multiplex droplet digital polymerase chain reaction. According to receiver operating characteristic curve analysis to discriminate between non-HCC group(including control group and chronic liver disease without HCC group)and HCC group, area under the curve(AUC)was 0.73 by AFP and 0.73 by mSEPT9, whereas it was only 0.59 by mSST. Regarding combination tests, although negative predictive values were almost same between the AFP + mSEPT9 test and the AFP + mSST test(75.0% vs 72.2%),a positive predictive value of AFP + mSEPT9 test was higher than that of the AFP + mSST test(79.4% vs. 66.7%).In conclusions, methylated SEPT9 was a better companion marker of AFP for HCC screening than methylated SST.

Aberrant increases in protein phosphatase 1(PP1) activity have been shown to be associated with inefficient sarcoplasmic reticulum Ca^{2+} cycling, leading to cardiac dysfunction in the failing heart. In the present study, we investigated whether BNP promoter-inducible suppression of PP1β would ameliorate progression of pressure overload-induced heart failure in mice, a clinically relevant animal model. An Adeno-associated virus 9 (AAV9) vector encoding PP1βshRNA and a negative control (NC) shRNA driven by a brain natriuretic peptide (BNP) promoter with an emerald green fluorescent protein expression (EmGFP) cassette were used to test the hypothesis. AAV9 vectors (AAV9-BNP-EmGFP-PP1βshRNA and AAV9-BNP-EmGFP-NCshRNA) were introduced into the in vivo heart via the tail vein injection (4x10^{11} GC/mice) in 8-week-old C57BL6J mice, followed by transverse aortic constriction (TAC) 2 weeks after the AAV9 vector injection. Post TAC cardiac function was sequentially assessed every 2 week by echocardiography, followed by hemodynamic assessment at 1 month. AAV9-BNP-EmGFP-PP1βshRNA treatment suppressed myocardial PP1β expression by 15% compared with the NCshRNA group (p<0.001). The fractional shortening (%FS) of the left ventricle in the PP1βshRNA-treated group was significantly larger than the NCshRNA-treated group (21%±1.0% vs. 15%±0.01, p<0.01). The ratios of heart weight (HW) / body weight (BW) and lung weight (LW) / BW in the PP1βshRNA group were significantly smaller than those of the NCshRNA group (HW/BW: 9.20±0.49 vs. 10.6±0.45 mg/g

Background: Angiotensin II (AngII) increases reactive oxygen species (ROS) and induces glomerular sclerosis. Toll-like receptor 4 (TLR4)-mediated inflammation enhances the renal impairment in renal inflammatory diseases. The relationship between TLR4 and AngII-induced glomerular sclerosis is unknown.Methods: Mice lacking TLR4 function (Tlr4^{lps-d}) and wild-type (WT) mice were randomized into groups treated with AngII, norepinephrine (NE) or a sub-depressor dose of the AngII receptor blocker irbesartan along with AngII for 2 weeks. We then assessed the expressions of NADPH oxidase and monocyte chemoattractant protein-1 (MCP-1) and the inflammatory cell recruitment in the glomeruli. We also evaluated the mesangial matrix proliferation and ROS.Results: AngII and NE equally increased the systolic blood pressure compared to the control mice (p<0.05). In the WT mice treated with AngII, we observed glomerular sclerosis, an increase in NADPH oxidase, MCP-1 and the infiltration of macrophages as well as ROS content in the glomeruli compared to the control mice (p<0.05), whereas the Tlr4^{lps-d} mice showed little effects of AngII on these indices. In addition, the sub-depressor-dose irbesartan treatment reversed these changes. NE had little effects on these indices. Conclusions: TLR4 plays an important role in AngII-induced oxidative stress, inflammation and glomerular sclerosis through the AT1 receptor.

Aims: To clarify the usefulness of preservative-free ophthalmic preparations equipped with a filter. Methods: A total of 1,615 samples of in-use ophthalmic preparations were examined for microbial contamination. Results: Of 1,094 samples of preservative-containing ophthalmic preparations, 31 (2.8%) showed microbial contamination. Of 289 samples of preservative-free ophthalmic preparations without a filter, 6 (2.1%) were contaminated, consisting of 4 (13.8%) of 29 samples of hospital preparations and 2 (0.8%) of 260 samples of commercially available new quinolone antimicrobial agents. On the other hand, the microbial contamination rate in preservative-free ophthalmic preparations equipped with a filter was 0% (0 of 232 samples).The major contaminants detected in these preservative-containing ophthalmic preparations and preservative-free ophthalmic preparations without a filter were glucose-nonfermentative Gram-negative bacilli such as Pseudomonas fluorescens, Acinetobacter spp., and P. aeruginosa, coagulase (-) staphylococci, and Candida spp. The contaminant level was 10-99 colony forming units (CFU)/mL in 37.8% (14 of 37 samples), and 10^2 - 10^6 CFU/mL in 62.2% (23 of 37 samples). Conclusions: Preservative-free ophthalmic preparations equipped with a filter not only have zero risk of the oculotoxic effects of preservatives, but are also safe in terms of their lack of microbial contamination.

We investigated the relationship between myocardial oxidative stress and cardiac sympathetic hyperactivity in patients with takotsubo cardiomyopathy (TC) compared with acute anteroseptal myocardial infarction (AMI). Methods: In 10 TC patients and 10 AMI patients, electrocardiogram, echocardiography, cardiac catheterization were conducted, and plasma catecholamines and urinary (U) 8-hydroxy-2’-deoxyguanosine (8-OHdG) as a marker of oxidative DNA damage were taken for one week from onset. Results: On admission, the coronary sinus (CS) had significantly higher norepinephrine (NE) and 8-OHdG levels than the aortic root (Ao) and peripheral blood vessels. Circulating catecholamines in TC patients tended to be higher than those in AMI patients

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by stress- or exercise- induced ventricular tachycardia, frequently leading to sudden cardiac death. A considerable body of evidence accumulated over recent years suggests that mutation-linked cardiac ryanodine receptor (RyR2) defects cause Ca^2+ leak from sarcoplasmic reticulum, which triggers delayed afterdepolarization and leads to CPVT. However, the underlying mechanism, by which a single mutation in such a large molecule causes drastic effects on the channel function, remains elusive. Here we report that introduction of a human CPVT mutation S2246L into the mouse RyR2 induces aberrant activation of channel gating by forming abnormally tight domain-domain interaction between the S2246L mutable domain and the K201-binding domain. This produces more global conformational change in the RyR2: namely, an aberrant domain unzipping between the N-terminal (a.a. 1–600) domain and the central (a.a. 2000–2500) domain owing to the allosteric conformational coupling mechanism. Pharmacological correction of the defective inter-domain interactions can stop the aberrant Ca^2+ release and lethal arrhythmia. These results provide a new pathogenic mechanism of CPVT and a novel therapeutic strategy against CPVT