犬の肺動脈狭窄症に対する非侵襲的治療法の確立

第一章　諸言　肺動脈狭窄症（以下PS）は、重度の場合生存期間短縮を引き起こすとされている。重度PS罹患犬の治療法には、カテーテルによるバルーン弁口拡大術（以下BV）や外科手術が挙げられるが、前者が治療の第一選択とされている。BVは術後に肺動脈弁逆流（以下PI）、三尖弁逆流（以下TR）、再狭窄などが合併症として挙げられている。獣医学領域においてもBV後の再狭窄は報告されているものの情報は限られており、その原因は不明であり、したがって対策がなされていないのが現状である。本研究の目的は、BVの合併症を把握し、対策および治療を提案することである。本研究ではまず臨床例の疫学的調査を実施し、BVの治療効果と合併症、中でも再狭窄率に注目し、再狭窄に関連する因子を探索することとした。さらに再狭窄弁を病理組織学的に検索し、正常弁や非再狭窄弁、BV未実施狭窄弁と比較検討することで再狭窄の発生機序について仮説を立てることとした。病理組織学的検索結果に基づき、犬の肺動脈弁内細胞を培養し薬物添加によって再狭窄の病態を再現することを試みた。薬物添加による培養細胞の再狭窄モデルに投薬を行うことで、再狭窄を抑制可能であるかを検討することとした。第二章　重度弁性肺動脈狭窄症症例に対するバルーン弁口拡大術の成績と合併症[目的] 本研究の目的は、犬の臨床例におけるBVの成功率、合併症の種類と発生数および発生時期、再狭窄に関連するとされる因子の探索である。[実験材料と方法] 本学に2006年9月から2014年5月までに来院し、重度PSと診断され、BVを実施した41例の犬を対象とした。術後の肺動脈右室圧較差（以下PG）の減少および合併症、再狭窄発生率を調査した。また手術時月齢、体重、術前PG、術後短期PG、BV回数、BVに使用したバルーン径・肺動脈径比、BV前β遮断薬およびBV前カンデサルタン投与の有無と再狭窄との関連を後方視的調査で行った。[結果] BV後短期成功率は95.1%（39/41）であった。成功と判断され長期間の追跡が可能であったのは22例であり、そのうち再狭窄が認められたのは6例であった。再狭窄の発覚時期は5.5ヵ月（1.5-68.2ヵ月）であった。成功例と再狭窄例における各因子を比較したところ、どの因子においても有意差は認められなかった。短期経過時にTR、PIが認められたのは12例と4例であったが、長期経過時には2例と3例に減少した。TR、PIの悪化が認められた症例で右心不全を呈した症例は認められなかった。[小括] 過去の報告同様にBVは成功率が高く効果的な治療法であった。再狭窄発生率は22例中6例で、再狭窄を呈するまでの経過は広範囲であった。再狭窄に関連する因子は、今回の検討では検出できなかった。BV実施後のTRやPIの悪化によって右心不全を呈した症例は認められなかった。過去の報告においても重症化は少ないことからも重度PSに対する治療法としてBVは合併症の少ないより安全な方法であると考えられた。第三章　バルーン弁口拡大術実施後再狭窄が認められた犬の病理学的調査[目的] これまでBV後の再狭窄の原因及びその発生機序の報告はなく、不明である。そこで本章では、BV後再狭窄と診断された1例について肺動脈弁の病理学的調査を実施することで、再狭窄の原因を探索することを目的とした。[実験材料と方法] BV実施後約1年半で再狭窄と診断され、再手術中に死亡した犬1頭の肺動脈弁と正常実験ビーグル犬3頭の肺動脈弁を採材し、特殊染色および免疫染色を用いた病理組織学的検査を実施した。[結果] 再狭窄弁は正常弁と比較して肉眼上での肥厚が認められた。再狭窄弁では正常弁と比較して酸性ムコ多糖類および膠原線維の蓄積、筋線維芽細胞の増加が認められた。[小括] 再狭窄弁では酸性ムコ多糖類および膠原線維が多く蓄積していたことから、これらにより肺動脈弁口部が狭小化していたことが明らかとなった。再狭窄弁では筋線維芽細胞の増加が認められた。筋線維芽細胞は細胞外基質（以下ECM）である酸性ムコ糖類と膠原線維を産生することから再狭窄には筋線維芽細胞の増加が関連している可能性が考えられた。第四章　バルーン弁口拡大術実施後短期および長期経過における肺動脈弁の病理学的変化の比較検討[目的] 本章ではBV実施後短期および長期における肺動脈弁の病理学的変化を検討することで、肺動脈弁に対する経時的なBVの影響および再狭窄の原因を病理組織学的に検索することを目的とした。[実験材料と方法] 自然発症PSに罹患した実験ビーグル犬2例にBVを実施し、短期および長期経過時に肺動脈弁を採材し、第三章同様に病理組織学的検査を実施した。BVを実施していないPS罹患犬1例を陽性コントロール、第三章で用いた正常犬を陰性コントロールとした。[結果] BV実施2例では再狭窄は認められなかった。BV実施2例短期経過時および陽性コントロールでは肉眼上で弁の肥厚が認められ、特殊染色検査では酸性ムコ多糖類、膠原線維の増加が認められた。長期経過では短期経過と比較して酸性ムコ多糖類のさらなる増加が認められた。膠原線維は短期と長期で比較して大きな差は認められなかった。短期で多く認められていた筋線維芽細胞は長期では減少していた[小括]本実験で用いた2例はBV実施後、長期経過時に肺動脈弁の肥厚は認められたものの、再狭窄を呈していないためBV成功例のモデルと考えられた。BV成功例は実施後、中長期的に細胞外基質による弁の肥厚を生じるが、筋線維芽細胞が不活化し、第三章で認められているような肺動脈弁口部を狭小化するほどのECMの過剰産生が停止していると考えられた。第五章　培養細胞を用いた再狭窄モデルの確立[目的] 前章より再狭窄は筋線維芽細胞によるECM過剰産生による肺動脈弁口部の狭小化によって起きるのではないかと考えられた。本実験では肺動脈弁内に存在する筋線維芽細胞を培養し、TGFβ-1を添加し細胞外基質の産生を促進させ、肺動脈再狭窄モデルを作製できると仮説を立てた。培養筋線維芽細胞にAngⅡ変換酵素阻害薬（以下ACEI）やARBを添加することでAT1受容体を介したTGFβ-１産生を抑制するかを検証することとした。[実験材料と方法] 実験ビーグル4頭から肺動脈弁を採材し、細胞を培養した。培養9日目に筋線維芽細胞を活性化するためにTGFβ-1（20ng/ml）を添加し、12日目に細胞数計測および培養液の採取を行った。培養液はヒアルロン酸（以下HA）およびアンジオテンシンⅡ（以下AngⅡ）の定量を実施した。定性は抗Vimentin抗体および抗α-smooth muscle actin（以下SMA）抗体を用い、蛍光免疫染色を行った。[結果] 培養細胞は全て筋線維芽細胞であった。TGFβ-1未添加群と添加群において細胞数およびHA濃度に有意差は認められなかった（P=1.00、P=0.56）。TGFβ-1添加群の4頭中3頭でHA濃度が多かった。AngⅡ濃度はTGFβ-1未添加群で有意に高値を示していた（P=0.04）。TGFβ-1添加群とACEIおよびARB添加群において細胞数、AngⅡ濃度およびHA濃度ではいずれの群でも有意差は認められなかった。[小括] TGFβ-1添加では細胞数において有意差が認められなかったがこれは過去の報告と一致していた。TGFβ-1添加群はHA産生において有意差は認められなかった。AngⅡはTGFβ-1未添加群で有意に上昇していたことからTGFβ-1添加によって筋線維芽細胞からのAngⅡ産生が抑制されるのではないかと考えられた。筋線維芽細胞はAngⅡの作用を受けてTGFβ-1を産生することからACEIとARBの投与が有効ではないかと考えられたが、本実験ではAngⅡとHA産生の抑制は認められなかった。第六章　総括　第二章の結果からBVの短期および長期成功率から示されているように今までの報告同様に重度PSに対して有効であることが再度示された。再狭窄はBV実施後中長期間で認められた。また医学領域においても再狭窄はBV実施後の長期経過中に多く認められていることから犬においても再狭窄率はBV後時間経過とともに上昇していくことが予想された。BV実施後のTRおよびPIの発生は他の論文でも報告されているが、いずれにおいても重症化をするのは珍しいとされていることから今回の結果と一致しており、今回のBVの結果は良好であったと考えられた。第三章において再狭窄はECMの産生過剰亢進による肺動脈弁口部の狭小化が原因と考えられた。再狭窄弁での筋線維芽細胞の増加がECMを過剰産生していることが考えられた。しかしながら第四章においてBV実施後長期経過した非再狭窄弁ではECMの増加が認められているにもかかわらず、筋線維芽細胞は減少していた。これらのことからBV実施後に筋線維芽細胞の活性化停止の有無が再狭窄発生と関連していると考えられた。第五章の結果ではTGFβ-1添加群とACEIやARB添加群ではHAとAngⅡの産生を抑制できなかったが、過去の報告ではECMの抑制が報告されていることから今回使用した添加濃度が低い可能性がある。そのため今後TGFβ-1とACEI、ARBの添加量は再検討する必要があると考えられた。これらの結果からBV実施の再狭窄はメカニカルストレスと筋線維芽細胞による細胞外基質の蓄積によって発生していると考えられた。再狭窄を予防するために細胞外基質産生を抑制する薬剤の検討が望まれる。Introduction Severe pulmonary valvular stenosis (PS) results in poor prognosis. The treatment options for dogs with severe PS include open chest surgery and balloon valvuloplasty (BV). BV has been recognized as the first-line treatment for severe PS in human as well as in dogs. Most common complications of BV are known as follows: restenosis of pulmonary valve, pulmonary valve insufficiency (PI) and tricuspid valve regurgitation (TR). Since there has been few reports in veterinary medicine focusing on restenosis after BV, the cause of restenosis has not been investigated yet. The aim of this study was to understand the prevalence of the complication in dogs after BV, to investigate the causes of the complications, and to propose prevention and treatment to avoid complications after BV. Study 1; The effectiveness and complications of BV in dogs with congenital PS[Aims] The aims of the first study were to reveal the prevalence of complications after BV, and to investigate the factors related to the complications, especially pulmonary restenosis.[Material and Method] Medical records of dogs referred to Azabu University Veterinary Teaching Hospital Cardiology Service from September 2006 to May 2014 were retrospectively reviewed. Forty-one dogs who underwent successful BV due to severe PS were included in the study. We investigated the effectiveness of BV using the reduction of the peak instantaneous pressure gradient across the pulmonary valve, the complications, especially the incidence of the restenosis. The following variables were investigated if it was related to restenosis: age (months) at the time of BV, weight (kg), PG prior to BV, PG at short-term follow-up, the number of balloon inflations, balloon annulus rate (BAR), concurrent congenital heart disease, atenolol administration prior to BV and candesartan administration prior to BV.[Results] The immediate success rate was 95.1% (39/41). Long-term follow-up was available for 22 out of 39 dogs. Restenosis was observed in 6 out of 22 dogs during long-term follow-up. The median time when restenosis was revealed was 5.5 months (ranging from 1.5 to 68.2 months) after BV. The significant difference was not observed between success group and restenosis group at each the factors. Although deterioration of TR severity was observed in 12 dogs at short-term follow-up, TR improved at long-term follow-up in 10 dogs. PI in 4 dogs worsened after BV in short-term follow-up, although the severity of PI in 2 dogs was improved during long-term follow-up. The right-sided heart failure due to TR and/or PI was not developed in any dogs during the observation period.[Discussion] It was confirmed that BV was effective procedure with high success rate in dogs, which was the same as the previous report. In the present study, 6 out of 22 dogs (27.3%) developed restenosis after successful BV at medium to long-term follow-up. Each the factors were not associated with incidence of restenosis in this study. The right-sided heart failure due to TR and/or PI was not developed in any dogs during the observation period. Exacerbation of TR and PI was observed in some dogs after BV; however, right-sided heart failure due to TR or PI was not observed in any of our dogs. Study 2; Histopathological and immunohistochemical findings in PS dogs with restenosis after BV[Aims] The cause of restenosis in dogs has never been described and no study has documented histopathological and immunohistochemical findings of the restenotic pulmonary valve in dogs with PS after BV. The aim of this study was to perform the pathological investigation of a pulmonary valve in one dog with PS diagnosed as the restenosis after BV.[Material and Method] A four-month-old female Chihuahua was presented to Azabu University due to severe PS with restricted ventricular septal defect. Although BV was successfully performed, restenosis was observed about 1.5 years after the procedure. Restenotic valve from this patient and three normal beagles were pathologically examined.[Results] The affected valve was markedly thickened due to the increased amount of the extracellular matrix (ECM), which was composed of acid mucopolysaccharide and collagen fibers. Immunohistochemical examination revealed prominently increased α smooth muscle actin (αSMA)-positive myofibroblasts in the affected valve. The affected valve was markedly thickened due to the increased amount of the ECM.[Discussion] It was suggested that thickening of the valve due to the increase of ECM would be associated with narrowing of the valve opening, resulting in restenosis. Immunohistochemical examination revealed prominently increased αSMA-positive myofibroblasts in the affected valve. Because these myofibroblasts produce acid mucopolysaccharide and collagen fibers, the increase in a myofibroblast could be related to the restenosis.Study 3; Pathological Changes of The Pulmonary Valve After Balloon Valvuloplasty in Two Dogs in Short-Term and Long-Term Follow-Up[Aims] Time course of pathological changes after BV was not known. The aim of this study was to reveal the histopathological changes of pulmonary valve in short-tem and long-term follow-up after BV.[Material and Method] BV was performed in two beagles with congenital PS. Pulmonary valve was obtained in short-term and long-term follow-up. Histopathological and immunohistochemical examinations were performed in pulmonary valves from short-term follow-up, long-term follow-up, negative controls and positive control (pulmonary valve from PS dog without BV).[Results] Two dogs with congenital PS after BV did not develop restenosis. Pulmonary valves of two dogs with congenital PS at short-term follow-up after BV and a positive control were markedly thickened. The amount of acid mucopolysaccharide and collagen fibers increased in pulmonary valves of two dogs with congenital PS. The amount of acid mucopolysaccharide was much increased in pulmonary valve at long-term follow-up, compared with that at short-term follow-up. The difference in collagen fibers between pulmonary valves at short-term and long-term follow-up was not observed. Number of myofibroblast was increased at short-term follow-up, but decreased at long-term follow-up.[Discussion] Although the thickening of the pulmonary valve was observed at long-term follow-up, two dogs with congenital PS after BV did not develop restenosis. Because the thickening of the valve was due to increased amount of ECM and myofibroblast was inactivated in long-term follow-up, it was supeculated that the increased ECM by myofibroblast had stopped at long-term follow-up.Study 4; An Establishment of The Culture Model of Restenotic Pulmonary Valve[Aims] We hypothesized that the cause of a restenosis was the narrowing of the valve opening induced by the overproduction of ECM by myofibroblast. The aims of this study was to establish the restenosis model using cultured myofibroblast from dogs’ pulmonary valve, and to investigate the possible medical approach to prevent restenosis after BV.[Material and Method] The pulmonary valve was obtained from four healthy beagles and the cell culture was performed. In order to activate myofibroblast, TGFbeta-1 (20 ng/ml) was added. Angiotensin converting enzyme inhibitor (ACEI) or angiotensinreceptor blocker (ARB) was added to cultured myofibrblast to evaluate whether or not they were effective to suppress ECM production. The number of cultured cells, the hyaluronic acid (HA) and the angiotensin II (AngII) of the medium were quantified on the day 12.[Results] All cultured cells were confirmed as myofibroblasts. Significant difference was not observed in the number of cell or HA concentration between control group (TGF beta-1 was not added) and TGF beta-1 added group, (P= 1.00, P= 0.56). AngII concentration in the control group was higher than that in added group (P= 0.04). Significant difference was not observed in the number of cells, AngII concentration or HA concentration between groups when ACEI or ARB was added.[Discussion] Cell culture from pulmonary valve was successfully established in this study. However, induction of increased HA was not achieved by adding TGF beta-1. ACEI or ARB did not suppress production of HA or AngII. DiscussionStudy 1 revealed that success rate of BV at the short-term and long-term follow-up were relatively high, which confirmed the effectiveness of this procedure in dogs. The restenosis was confirmed as one of the serious problem, and was observed during the medium to long-term follow-up after BV. Increased prevalence of restenosis over time was observed in human patients. The same tendency was also seen in dogs. The development of TR and PI after BV was although these complications did not get severe. In study 2, the cause of restenosis was speculated that the thickening of the valve that induced by the increasing amount of ECM. Increased number of myofibroblasts in restenotic valve produced the large amount of ECM. Increase of ECM at long-term follow-up after BV in spite of decreased number of myofibroblast was observed in the non-restenosis valve after BV. Therefore, it was considered that the restenosis development after BV was associated with whether myofibroblast was continuously activated or not. Mechanical stress would be one of the major causes of activation of myofibroblasts although further study was needed to confirm this hypothesis. Present study did not reveal the effectiveness of ACEI or ARB to prevent restenosis. Further study was warranted to investigate the possible medical approach.博士(獣医学)麻布大

Software Defined Media: Virtualization of Audio-Visual Services

Internet-native audio-visual services are witnessing rapid development. Among these services, object-based audio-visual services are gaining importance. In 2014, we established the Software Defined Media (SDM) consortium to target new research areas and markets involving object-based digital media and Internet-by-design audio-visual environments. In this paper, we introduce the SDM architecture that virtualizes networked audio-visual services along with the development of smart buildings and smart cities using Internet of Things (IoT) devices and smart building facilities. Moreover, we design the SDM architecture as a layered architecture to promote the development of innovative applications on the basis of rapid advancements in software-defined networking (SDN). Then, we implement a prototype system based on the architecture, present the system at an exhibition, and provide it as an SDM API to application developers at hackathons. Various types of applications are developed using the API at these events. An evaluation of SDM API access shows that the prototype SDM platform effectively provides 3D audio reproducibility and interactiveness for SDM applications.Comment: IEEE International Conference on Communications (ICC2017), Paris, France, 21-25 May 201

Effective treatment with mitotane for a canine case of presumed ectopic Cushing’s syndrome-related pheochromocytoma

Background: In humans, ectopic Cushing’s syndrome (ECS) is characterized by hypercortisolemia, which is caused by small lung carcinoma, bronchial carcinoids, and pheochromocytoma. In dogs, only a few cases of ECS associated with pheochromocytoma have been reported to date. Case Description: Herein, we describe a canine case of malignant pheochromocytoma that is presumed to be the cause of ECS. An 11-year-old, castrated, male Toy Poodle with hypercortisolemia was diagnosed with an adrenal tumor (AT) and treated with mitotane. Although repeated adrenocorticotropic hormone stimulation tests revealed improvement in the dog’s condition by mitotane treatment, its condition started declining 197 days post-diagnosis, and he died on day 280. The necropsy revealed the AT was a pheochromocytoma, not an adrenocortical tumor. However, because of no pathological change in the pituitary gland and the other adrenal gland, pheochromocytoma was presumed to be the cause of ECS. Conclusion: This is the first report that describes the effectiveness of mitotane against presumed ECS-related pheochromocytoma

Magnetized Fast Isochoric Laser Heating for Efficient Creation of Ultra-High-Energy-Density States

The quest for the inertial confinement fusion (ICF) ignition is a grand challenge, as exemplified by extraordinary large laser facilities. Fast isochoric heating of a pre-compressed plasma core with a high-intensity short-pulse laser is an attractive and alternative approach to create ultra-high-energy-density states like those found in ICF ignition sparks. This avoids the ignition quench caused by the hot spark mixing with the surrounding cold fuel, which is the crucial problem of the currently pursued ignition scheme. High-intensity lasers efficiently produce relativistic electron beams (REB). A part of the REB kinetic energy is deposited in the core, and then the heated region becomes the hot spark to trigger the ignition. However, only a small portion of the REB collides with the core because of its large divergence. Here we have demonstrated enhanced laser-to-core energy coupling with the magnetized fast isochoric heating. The method employs a kilo-tesla-level magnetic field that is applied to the transport region from the REB generation point to the core which results in guiding the REB along the magnetic field lines to the core. 7.7 $\pm$ 1.3 % of the maximum coupling was achieved even with a relatively small radial area density core ($\rho R$ $\sim$ 0.1 g/cm$^2$). The guided REB transport was clearly visualized in a pre-compressed core by using Cu-$K_\alpha$ imaging technique. A simplified model coupled with the comprehensive diagnostics yields 6.2\% of the coupling that agrees fairly with the measured coupling. This model also reveals that an ignition-scale areal density core ($\rho R$ $\sim$ 0.4 g/cm$^2$) leads to much higher laser-to-core coupling ($>$ 15%), this is much higher than that achieved by the current scheme

What Does Soil-Transmitted Helminth Elimination Look Like? Results From a Targeted Molecular Detection Survey in Japan

Background: Japan is one of the few countries believed to have eliminated soil-transmitted helminths (STHs). In 1949, the national prevalence of Ascaris lumbricoides was 62.9%, which decreased to 0.6% in 1973 due to improvements in infrastructure, socioeconomic status, and the implementation of national STH control measures. The Parasitosis Prevention Law ended in 1994 and population-level screening ceased in Japan; therefore, current transmission status of STH in Japan is not well characterized. Sporadic cases of STH infections continue to be reported, raising the possibility of a larger-scale recrudescence of STH infections. Given that traditional microscopic detection methods are not sensitive to low-intensity STH infections, we conducted targeted prevalence surveys using sensitive PCR-based assays to evaluate the current STH-transmission status and to describe epidemiological characteristics of areas of Japan believed to have achieved historical elimination of STHs. Methods: Stool samples were collected from 682 preschool- and school-aged children from six localities of Japan with previously high prevalence of STH. Caregivers of participants completed a questionnaire to ascertain access to water, sanitation and hygiene (WASH), and potential exposures to environmental contamination. For fecal testing, multi-parallel real-time PCR assays were used to detect infections of Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale and Trichuris trichiura. Results: Among the 682 children, no positive samples were identified, and participants reported high standards of WASH. Conclusions: To our knowledge, this is the first STH-surveillance study in Japan to use sensitive molecular techniques for STH detection. The results suggest that recrudescence of STH infections has not occurred, and that declines in prevalence have been sustained in the sampled areas. These findings suggest that reductions in prevalence below the elimination thresholds, suggestive of transmission interruption, are possible. Additionally, this study provides circumstantial evidence that multi-parallel real-time PCR methods are applicable for evaluating elimination status in areas where STH prevalence is extremely low.[Figure not available: see fulltext.

What does soil-transmitted helminth elimination look like? Results from a targeted molecular detection survey in Japan

Background: Japan is one of the few countries believed to have eliminated soil-transmitted helminths (STHs). In 1949, the national prevalence of Ascaris lumbricoides was 62.9%, which decreased to 0.6% in 1973 due to improvements in infrastructure, socioeconomic status, and the implementation of national STH control measures. The Parasitosis Prevention Law ended in 1994 and population-level screening ceased in Japan; therefore, current transmission status of STH in Japan is not well characterized. Sporadic cases of STH infections continue to be reported, raising the possibility of a larger-scale recrudescence of STH infections. Given that traditional microscopic detection methods are not sensitive to low-intensity STH infections, we conducted targeted prevalence surveys using sensitive PCR-based assays to evaluate the current STH-transmission status and to describe epidemiological characteristics of areas of Japan believed to have achieved historical elimination of STHs. Methods: Stool samples were collected from 682 preschool- and school-aged children from six localities of Japan with previously high prevalence of STH. Caregivers of participants completed a questionnaire to ascertain access to water, sanitation and hygiene (WASH), and potential exposures to environmental contamination. For fecal testing, multi-parallel real-time PCR assays were used to detect infections of Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale and Trichuris trichiura. Results: Among the 682 children, no positive samples were identified, and participants reported high standards of WASH. Conclusions: To our knowledge, this is the first STH-surveillance study in Japan to use sensitive molecular techniques for STH detection. The results suggest that recrudescence of STH infections has not occurred, and that declines in prevalence have been sustained in the sampled areas. These findings suggest that reductions in prevalence below the elimination thresholds, suggestive of transmission interruption, are possible. Additionally, this study provides circumstantial evidence that multi-parallel real-time PCR methods are applicable for evaluating elimination status in areas where STH prevalence is extremely low.[Figure not available: see fulltext.