Azabu University Science Information Repository / 麻布大学学術情報リポジトリ
Not a member yet
1842 research outputs found
Sort by
The Potential of Bemegride as an Activation Agent in Electroencephalography in Dogs
Get PDFThe present study investigated the potential of bemegride as a pharmacological activation agent that elicits epileptiform discharges (EDs) in interictal electroencephalogram (EEG) recordings in dogs. Four laboratory dogs with idiopathic epilepsy and four without epilepsy were included. The dogs were anesthetized using sevoflurane during EEG recordings. Bemegride was administered intravenously and repeatedly until EDs were enhanced or induced, or until the maximum dose (20 mg/kg) had been administered. Bemegride activated EDs in all dogs with epilepsy. These EDs predominantly occurred in each dog’s spontaneous irritative zones, which were identified without the administration of bemegride. EDs occurred after the administration of bemegride in 50% of dogs without epilepsy. The dose required for activation was significantly lower in dogs with epilepsy (median; 7.3 mg/kg) than in those without (median; 19.7 mg/kg) (p = 0.0294). The only suspected adverse effect associated with the administration of bemegride was vomiting in two dogs after awakening from anesthesia. There were no other adverse effects, including seizures. The present results demonstrated the potential of bemegride as a safe and effective pharmacological activation agent of EDs in anesthetized dogs with epilepsy and provided more options for the diagnosis and therapeutic planning of epilepsy, including presurgical evaluations, in dogs.journal articl
Effects of Inherent Lactic Acid Bacteria on Inhibition of Angiotensin I-Converting Enzyme and Antioxidant Activities in Dry-Cured Meat Products
Get PDFThe aim of this study was to investigate the inherent bacteria that contribute to expressing the angiotensin I-converting enzyme (ACE) inhibitory activity and the antioxidant activity of dry-cured meat products without a bacterial starter. Among the ten dry-cured meat product samples, Coppa and Milano salami exhibited high ACE inhibitory activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability, and oxygen radical absorbance capacity (ORAC). No consistent trend was observed in the pH values or the total peptide and imidazole dipeptide concentration of the products that exhibited high ACE inhibitory and antioxidant activities in the tested samples. To investigate the bacteria contributing to the ACE inhibitory and antioxidant activities of the product, 16S rRNA sequencing analysis, isolation, and identification of bacteria were performed using not only Coppa and Milano salami but also the Jamon Serrano and Parma prosciutto products that had low functional activities. Results suggest the Lactobacillales order, particularly the species Latilactobacillus sakei and Pediococcus pentosaceus, were the main inherent bacteria in Coppa and Milano salami, respectively, compared with the Jamon Serrano and Parma prosciutto products. Therefore, the inherent lactic acid bacteria in dry-cured meat products without bacterial starter is important for ACE inhibitory and antioxidant activities of the products.journal articl
Effective use of a growth-inhibiting sheet containing pyriproxyfen for cigarette beetle Lasioderma serricorne larvae: an implication from sheet shape and flour-food accumulation
Get PDFGrowth-inhibiting sheets containing pyriproxyfen have recently been developed to control the cigarette beetle Lasioderma serricorne. In this study, we evaluated effective use of a commercially available sheet through a rearing experiment in which the distribution of whole-wheat flour as a food and the shape of the sheet were manipulated. The results based on three replications indicated that the sheet effectiveness decreased by 0.51 times with each 1 cm increase in the flour depth on average, and that the effect was weakened by 0.58 times under conditions where the sheet shape did not match with the coverage of the flour. We also detected a 4.06-fold greater risk of allowing the individuals to develop from larvae into adults when the sheet shape was not matched compared to when it was matched. These findings suggest that an application covering the targeted area by the sheet without any gaps is essential to qualitatively and quantitatively prevent any development of the larvae.journal articl
A case report: Characteristic plain radiographic findings of a displaced abomasum in a heifer
Get PDFA 9-month-old Holstein heifer with a history of severely poor growth presented with diarrhoea. On physical examination, a metallic pinging sound was heard on the simultaneous percussion and auscultation of the left trunk. On the cranial abdominal radiography, the contour of a gas-filled balloon-like abomasum wall was delineated, which elevated to the dorsal abdomen. Radiopaque sand at the bottom of the abomasum had been pulled up caudodorsally by the gas-filled abomasum. After surgery, the gas-filled balloon-like appearance of the abomasum wall disappeared and the radiopaque sand was located in the normal position. To our knowledge, no reports on a displaced abomasum on plain radiographs are available. The radiographic findings described herein are characteristic imaging findings of a displaced abomasum. Abdominal radiography could be a new option as an auxiliary diagnostic approach for a displaced abomasum.journal articl
Human sapovirus propagation in human cell lines supplemented with bile acids
Get PDF麻布大学博士(学術)Sapoviruses are non-enveloped, single-stranded (+) RNA viruses belonging to the family Caliciviridae, similar to noroviruses. Many genotypes (likely equivalent to serotypes) of human sapovirus (HuSaV) exist; currently, at least 18 genotypes are known (GI.1-7, GII.1-8, GIV.1, and GV.1-2).
These viruses cause acute gastroenteritis in individuals of all age groups; HuSaV is known to cause infections, especially in infants and children. Human-to-human, food-borne suspected outbreaks due to HuSaV as well as human norovirus (HuNoV) have beenfrequently reported. However, these infectious diseases cannot be distinguished by
symptoms alone.
Cell-based viral infection and propagation systems for porcine sapovirus (PoSaV) have been established; however, such systems are lacking for HuSaVs, although they were discovered more than 40 years ago. HuNoV infection systems using conventional human cell lines, such as Caco-2 and BJAB, and human stem cells, such as intestinal enteroid cells, and fecal specimens as infection sources have been reported; however, their virus propagation efficiency is low, and virus stocks cannot be prepared. Therefore, it is important to construct an in vitro culture system that is convenient and that can facilitate the growth of various types of human gastroenteric viruses to establish infection control methods or to ensure food safety and risk profiling for public health.
In this study, we established an efficient HuSaV propagation system using conventional
human cell lines. The effect of bile acid supplementation on HuSaV propagation was also tested because PoSaV and most HuNoVs require bile acids for their propagation in cultured cells.
The summary of this research is as follows:
1. Bile acid requirement for HuSaV susceptibility in cultured human cells
Three cell lines (human testis tumor-derived NEC8 (JCRB0250), human duodenum
carcinoma-derived HuTu80 (HTB-40), and human ileocecal adenocarcinoma-derived
HCT-8 (CCL-244)) were used along with four bile acids (sodium cholic acid (CA), sodium deoxycholate (DCA), sodium glycocholate (GlyCA), and sodium glycochenodeoxycholate (GCDCA)), or bovine gall (Bile) for determining the maximum permissible concentrations of these bile acids based on cytotoxicity tests.
One day after inoculation with the HuSaV-positive fecal suspension (1 dpi), the monolayer cells were washed and replaced along with fresh culture medium, followed by culture for another 7 days. Viral RNA was extracted from the culture supernatant and detected via RT-PCR. The PCR signal was positive for HuSaV GI.1 in NEC8 and HuTu80 cells supplemented with GlyCA and GCDCA but was negative in HCT-8 cells, even in the presence of these bile acids. The PCR signal was positive for HuSaV GII.3 in HuTu80 cells supplemented with GlyCA, GCDCA, or bile. GlyCA was used in subsequent culture studies.
2. Efficient HuSaV propagation in human intestinal cells
To quantitatively investigate the HuSaV propagation efficiency, the kinetics of GI.1 HuSaV replication in the infected NEC8 and HuTu80 cell lines in the presence of GlyCA (0.5 mM for NEC81 and mM for HuTu80) were studied. The culture supernatants were collected on 1, 3, 5, and 7 dpi and 1, 3, 5, 7, 10, and 14 dpi from NEC8 and HuTu80 cells, respectively, and titrated using RT-qPCR. In NEC8, GI.1 HuSaV RNA remained at 2 log10 copies up to 7 dpi; it is difficult to maintain and culture NEC8 for more than 7 days. In HuTu80, GI.1 HuSaV RNA increased to 6.6 log10 copies on 3 dpi and reached 7.4 log10 copies on 5 dpi, which was maintained until 14 dpi. The propagation rate of GI.1 was 3.9 log10 higher than the initial value (1 dpi).
HuTu80 was selected for subsequent trials because GI.1 HuSaV propagated more efficiently in HuTu80 than in NEC8. In HuTu80, GII.3 HuSaV RNA titers gradually increased, and reached the peak of 7.7 log10 copies on 10 dpi, which was 3.6 log10 higher than the initial value (1 dpi), similar to GI.1 HuSaV. Furthermore, one GI.2 strain and two GII.3 strains were also propagated in HuTu80 cells. These results indicate that HuTu80 supports efficient HuSaV propagation of at least three genotypes.
3. Confirmation of intracellular replication, translation, and production of infective HuSaV progeny in HuTu80
The replication and translation of GI.1 and GII.3 HuSaVs in HuTu80 cells in the presence of GlyCA were confirmed using immunofluorescence (IF) by detection of double-stranded RNA (dsRNA; as a marker of the intermediate products during the replication of single-stranded RNA viruses), nonstructural protein NS1-2, and structural protein VP1. No signal was detected on 1 dpi, but all the target signals were confirmed on 5 and 10 dpi. The positive rate based on dsRNA (n = 3) was 5.6–8.2% (mean 7.6%) on 5 dpi and 10.4–14.3% (mean 12.3%) on 10 dpi for GI.1, and it was 6.4–8.0% (mean 6.5%) on 5 dpi and 7.1–11.1% (mean 8.7%) on 10 dpi for GII.3. A small proportion of HuTu80 cells was susceptible to HuSaVs. No cytopathic effect was observed in the infected cells. The viral RNA levels in the culture supernatants of both genotypes increased from 3–4 log10 copies / 100 μL on 1 dpi to 9 log10 copies / 100 μL on 5 dpi and later.
Virus-like particles (virions) in purified GI.1 and GII.3 HuSaV samples were visualized
using transmission electron microscopy. The average particle size of GI.1 and GII.3 (n = 15 each) was 44.1 ± 1.5 nm and 41.0 ± 1.9 nm, respectively. When the number of viral RNA copies in the culture supernatant before and after ultracentrifugation were compared, the recovery rate was 56.8% and 16.8% for GI.1 and GII.3, respectively.
The infectivities of progeny GI.1 and GII.3 HuSaVs harvested at 10 dpi were further
confirmed by passaging P0 (the virus from culturing the positive fecal sample) and P1
viruses (the passaged virus cultured in the P0 sample). Both P1 and P2 (the virus from the cultured P1 sample) cell culture supernatants exceeded 9 log10 copies / 100 μL at 10 dpi, and the VP1 detection ELISA revealed an increase in signal from P1 to P2. HuSaV successfully replicated, produced infectious progeny viruses, and was efficiently passaged in HuTu80 cells in the presence of GlyCA.
4. Minimum infectious dose and sensitivity of HuSaVs to heat and ultraviolet (UV)
treatments A series of ten-fold diluted GI.1 and GII.3 HuSaV-positive fecal suspensions was inoculated into HuTu80 cells. After removing the inoculum and washing the cells at 1 dpi, the culture supernatants were collected at 10 dpi, and the viral RNA of the culture supernatant was measured. The minimum infectious dose was > 2.3 log10 viral RNA copies for both GI.1 and GII.3 HuSaVs.
Furthermore, the attenuation of infection titers via heat and UV treatments was evaluated. GI.1 and GII.3 HuSaVs were heat-treated at 50 °C, 60 °C, and 70 °C for 10, 20, and 30 min or irradiated with UV light (wavelength 254 nm) on ice for 20, 40, and 60 min. HuSaV infection supernatant was collected as described above.
After heat treatment at 70 °C for 30 min, RNA levels of both the GI.1 and GII.3 HuSaVs
decreased to the RT-qPCR detection limit. However, after treatment at 50 °C for 30 min, GI.1 and GII.3 HuSaVs replicated at a level similar to that of a non-heat treated virus. The heat treatment sensitivity was not identical between GI.1 and GII.3 HuSaVs, and GII.3 HuSaV was more resistant than GI.1 HuSaV at 60 °C.
Regarding susceptibility to UV irradiation, both GI.1 and GII.3 HuSaV-diluted fecal
suspensions were insensitive to UV treatment, even at 5.4 J/ cm2. Along with these results, UV treatment was performed using passaged (P1) HuSaV stock mixed with feline calicivirus (FCV), and it was confirmed that the passaged GI.1 and GII.3 HuSaVs were sensitive to UV treatment at 1.8 J/cm2. Spiked FCV was almost inactivated under these conditions.
In this study, human testis-derived cells and two types of human intestinal-derived cells
(duodenum and ileocecal region) were selected for analyzing HuSaV susceptibility. This is because the PoSaV-Cowden strain grows in porcine testis cells in addition to porcine kidney cells (LLC-PK1) when supplemented with bile acids, and the PoSaV-Cowden strain infects the small intestine site in young pigs. Accordingly, HuSaV was grown in human testis tumor-derived cells (NEC8) and more efficiently in human duodenum carcinoma-derived cells (HuTu80) in the presence of a conjugated bile acid (i.e., GlyCA and GCDCA). In the human body, conjugated bile acids are the main bile acids in the duodenum. Therefore, it is physiologically consistent that conjugated bile acids are essential for HuSaV propagation in human duodenal cells and would be useful for analyzing infection mechanisms and developing HuSaV control methods.
In the kinetics study, despite the weak HuSaV VP1 signal, the HuSaV RNA signal reached a plateau at 5 dpi but the HuSaV VP1 signal substantially increased at 10 dpi (data not shown).
These time lags may correlate with HuSaV virion formation/release speed in the HuTu80 cell culture supernatant. Although these observations could also be attributed to the difference in assay sensitivity (i.e., RT-qPCR is more sensitive than ELISA), this
information was important to account for the difference in growth speed among various
genotypes and the virion collection timing.
Owing to lack of efficient cell culture systems, most studies on human gastroenteritis
viruses, including HuNoVs, have used viral RNA-positive specimens (feces) as resources; however, there are issues such as those related to finiteness, resource heterogeneity due to uncertain virion content, inaccuracy of results, and ethical restrictions in medical research.
Efficient in vitro replication and production of HuSaV using HuTu80 cells make it possible to confirm HuSaV infectivity in fecal specimens, and more importantly, could allowpreparatio n of a stock of infectious viruses by passage. Thus, the newly established system described in this study can help overcome these limitations and establish an efficient growth system for other human gastroenteritis viruses, including HuNoV.
HuSaV-RNA is detected throughout the year in symptomatic and asymptomatic individuals and in wastewater. HuSaV-RNA has also been found in edible bivalves such as oysters and clams. HuSaV distribution is identical to that of HuNoV, and both viruses are spreading globally. However, it is unclear whether detection of viral RNA can reflect the presence of the infectious virus, as current nucleic acid detection systems can only detect traces of viruses. The development of a conventional and efficient culture method for HuSaV using HuTu80 cells will allow analysis of viral dynamics and effective risk profiling from “infectivity.”
HuNoV is known to be inactivated upon heating at 60°C for 15 min. In contrast, HuSaV
was inactivated completely and was relatively stable even after 30 min of heating at 60°C.
These differences might reflect the differences between HuSaV and HuNoV or could be
partially responsible for lower propagation efficiency and shorter culture period for the
HuNoV culture system. Furthermore, the HuSaV-positive diluted stool sample and cultured HuSaV differed considerably in their UV irradiation susceptibility. Unknown fecal factor(s) could protect HuSaV from inactivation by UV irradiation. These findings are important for developing strategies for HuSaV control in public health settings.
Our findings could serve as reference for performing a wide range of studies, such as
detection and removal/inactivation of infectious HuSaV in clinical, food, and environmental resources for risk profiling; development of HuSaV infection control methods; identification of virus receptors and antiviral compounds; and analyses in human serum epidemiological studies such as those involving neutralization antibody retention surveys, among others.サポウイルス（sapovirus）は、カリシウイルス属に含まれるnon-envelope、単鎖（+）RNAウイルスである。ヒトサポウイルス（human sapovirus; HuSaV）は18種類の遺伝子型（GI.1～7、GII.1～8、GIV.1およびGV.1～2）を有し、ヒトノロウイルス（human norovirus; HuNoV）と同属で、かつ急性下痢症を引き起こすため、伝播・兆候・主訴などは集団下痢症事案においてノロウイルスと共に考慮すべき起因ウイルスとなる。実際には幼児や小児などでの集団発生例が多いが、成人での集団発生や食中毒事案の報告も相当数存在する。
一方、人工的な増殖方法の研究において、HuNoVではCaco-2細胞やB細胞のような汎用細胞株や、enteroidといった幹細胞により、一部の臨床株での培養報告はあるが、HuSaVでは発見から40年以上経過しても培養増殖に成功していない。また、これまでヒト急性下痢症の起因ウイルスにおいて、in vitroでの汎用性が高く、多様なgenotypeあるいは血清型の増殖を担保する方法もない。しかしながら、これらの手法を確立することは当該下痢症の発症解明、伝播様式解析、および感染制御手段の研究において非常に重要であり、同様の症状を呈するHuNoVへの本手法の将来的な転用・応用の可能性も高い。加えて公衆衛生学上はもちろん、食の安全性確保の観点からも、これまで不可能であったリスクプロファイリングなど実効性の高いオペレーションが可能となるため、非常に重要である。
今回、一部のHuNoVや唯一株化に成功した豚サポウイルス（porcine sapovirus; PoSaV）の報告において、培養時に胆汁酸要求性を示すことから、これに基づき各種胆汁酸適応濃度といくつかの細胞株との組み合わせからHuSaV増殖系確立の研究を開始した。またウイルスRNA検出系、既存抗血清を用いた抗原検出ELISAや蛍光抗体法による感染細胞の解析についても併せて検討した。本研究の概要は以下のとおりである。
1．HuSaV細胞培養における胆汁酸要求性
ヒト精巣腫瘍由来細胞NEC8（JCRB0250）、腸管癌細胞由来HuTu80（HTB-40）およびHCT-8（CCL-244）の3種類の細胞株を用いて、4種類の胆汁酸（コール酸ナトリウム（CA）、デオキシコール酸ナトリウム（DCA）、グリココール酸ナトリウム（GlyCA）、グリコケノデオキシコール酸ナトリウム（GCDCA））およびBileを細胞障害性試験に基づいた各最大許容濃度に従い添加した。
接種1日後（1 dpi）に洗浄および培養液交換を行い、7日間培養した。培養上清中のウイルスRNAを抽出し、RT-PCRにて検出した。HuSaV GI.1についてはNEC8、HuTu80ではGlyCAおよびGCDCAで明瞭な増幅シグナルが検出された。HCT-8ではいかなる培養条件でも不検出となった。この結果よりHuTu80を用いてHuSaV GII.3の培養検討を行ったところ、GlyCA、GCDCAおよびBileにて明瞭な増幅シグナルが検出された。NEC8およびHuTu80に共通してGlyCA含有条件でのシグナルが強いことから、以降の培養検討にはGlyCA（1 mM）を用いることとした。
2．培養細胞間におけるHuSaVの経時的増殖性比較
HuSaV GI.1について、NEC8およびHuTu80を用いて1 mM GlyCA含有下で各細胞をT25フラスコにて単層シート化したものに、陽性検体を7log10 copies of viral RNAを上限（20μL）として接種した。接種から一晩培養後に上清を取り除き、洗浄用培地にて2回洗浄して維持培地に交換してから経時的増殖性を比較検討した。NEC8については接種1、3、5および7日目まで、HuTu80については接種1、3、5、7、10および14日目まで、培養上清100μL中のウイルスRNAコピー数を定量した。NEC8では7日目まででウイルスRNAは2log10 copiesに止まった。加えてNEC8は7日を超える維持培養が困難なことから、これ以上の増殖は期待できなかった。一方HuTu80では培養3日後で6.6log10 copiesとなり、5日目には7.4log10 copiesに到達し、14日目まで維持された。
この結果からGI.1の増殖率は初期値（1dpi）から約3.9log10倍となり、これを踏まえて同様にHuSaV GII.3の経時的増殖性を確認したところ、GII.3はGI.1よりも若干緩やかに増幅するものの、10日目でピークとなる7.7log10 copiesに達し、初期値（1dpi）から約3.6log10倍でGI.1とほぼ同等となった。
さらにGI.2 1株およびGII.3 2株を用いたHuTu80での10日間培養における経時的増殖性検討においても明瞭な増殖性が認められた。以上の結果からHuTu80は少なくとも3つのgenotypeのHuSaV増殖をサポートすることが明らかになった。
3．HuTu80におけるHuSaVの細胞内動態とvirion、子孫ウイルス増幅の確認
HuTu80細胞におけるHuSaVの挙動に関して、dsRNA、非構造タンパクNS1-2およびVP1の経時的シグナルと動態について検討した。すなわち、細胞に感染させてから1、5、10日目に、各々の特異抗体を用いてIF法により確認した。1日目ではシグナル非検出であったが、5日目及び10日目で対象とするすべてのシグナルが確認された。dsRNAに基づく陽性率（n=3）は、GI.1において5日目で5.6-8.2%（平均7.6%）、10日目で10.4-14.3%（平均12.3%）であり、GII.3においては5日目で6.4-8.0%（平均6.5%）、10日目で7.1-11.1%（平均8.7%）となった。また、すべての感染細胞において細胞変性効果は認められなかった。加えて両genotypeでの各段階の培養上清中のウイルスRNAコピー数は3-4log10 copies/100μL（1日目）から5日目以降9log10 copies/100μLを超えた。
次に、精製HuSaVサンプルの透過電子顕微鏡観察によりウイルス様粒子（virion）を捕捉することができ、その平均粒径（各n=15）はGI.1で44.1±1.5 nm、GII.3で41.0±1.9 nmであった。各粒子の回収率について、培養上清の超遠心処理前後のRNAコピー数を比較したところ、GI.1で56.8%、GII.3で16.8%であった。そして陽性検体の培養で得られたウイルスサンプル（P0）を2世代まで各10日間の継代培養を繰り返し、各培養上清についてRNAコピー数とVP1検出ELISAシグナルを測定したところ、RNAコピー数はP1およびP2共に10日目で9log10 copies/100μLを超え、VP1検出ELISAではP1からP2にかけて共にシグナル増大が認められた。以上の事象から、HuSaVはGlyCA存在下でvirionが継代培養できていることが確認された。
4．HuSaV分離のためのウイルスRNA必要量と加熱および紫外線に対する感受性評価
HuSaV GI.1およびGII.3陽性検体の10倍段階希釈（5.3log10～1.3log10、2×1 RNA copies/100μL）を行い、各々をHuTu80細胞に接種して10日間後の培養上清ウイルスRNA量を測定したところ、共に2.3log10 copies/100μLを下回ると検出限界以下となることから、分離に要するRNA量は2.3log10 copies/100μL以上と考えた。
このことを踏まえ、陽性検体希釈サンプルを用いたドライバスによる加熱処理（50℃、60℃および70℃）と紫外線（波長254 nm）照射によるGI.1およびGII.3の経時的感染価減衰を評価したところ、両genotypeにて70℃･30分処理でRNAシグナルは検出限界以下となり、50℃･30分ではシグナル減衰は認められなかった。60℃処理ではgenotype間での相違が顕著で、GII.3で熱抵抗性が強いことが示された。また紫外線照射に対する感受性は、陽性検体では両genotypeともに5.4 J/cm2の照射でも感染価減衰は認められなかった。一方、両genotypeともP1培養上清を同試験に用いたところ、1.8 J/cm2の照射でRNAおよびVP1検出ELISAの両シグナルともに検出されず、照射サンプルに同時にスパイクしたFeline calicivirusも1.8 J/cm2の照射でわずかに検出されるにとどまった。
以上のように、まずヒト精巣由来細胞と2種類のヒト腸管由来細胞（十二指腸および回盲部）をHuSaV増殖の検討に使用した。その根拠はPoSaV-Cowden株の増殖がLLC-PK1細胞以外の精巣細胞で成立すること、幼豚でのin vivo実験で小腸部位に増殖が認められることによる。その結果、抱合型胆汁酸であるGlyCAあるいはGCDCAの存在がHuTu80細胞でのHuSaV増殖をサポートすることを確認した。ヒト体内における胆汁成分の合成・分泌および再吸収といった一連の代謝から、十二指腸付近の胆汁酸比率は抱合型が主体となることからも、ヒト体内の生理を反映した増殖を示唆していると考える。このことはヒトウイルス性胃腸炎のin vitroにおける病態生理が模倣され、その発症機構解析や制御手法開発などにも非常に有用である。
続いてHuTu80細胞によるHuSaV感染粒子（virion）の増殖が世界で初めて確認でき、GI.1およびGII.3では接種5-10日で培養上清中のRNAコピー数がピークとなる一方、IFの結果ではHuSaV感受性細胞は限局的であり、VP1検出ELISAの結果からも継代培養の繰り返しによるシグナル増加が示されたことからウイルスRNAとウイルス粒子作出にある種のtime-ragの発生が示唆された。このことはHuSaV株間での増殖速度やviron回収の検証において非常に重要な情報となった。
HuSaVのin vitro複製はウイルスRNAおよびVP1抗原共に非常に高いレベルに到達しており、継代培養による感染性ウイルスのストック作製が可能となった。これまでヒト胃腸炎ウイルスのほとんどが陽性検体（糞便）をリソースとして使用していることから、有限性、virion含量が不確定なためのリソース不均一性と研究結果の確度への影響、医学研究倫理の制約などから解放され、ヒトノロウイルス研究と比較しても非常に重要なブレークスルーとなった。さらに同様のstrategyによる汎用培養法への研究転換のきっかけともなり得るため、海外の関連研究者も注目している。
HuSaVはヒトノロウイルス同様に集団感染事例や食品媒介を疑うアウトブレイク報告がある一方で、その季節性や感染経路、最小感染量や発症者からの複数genotype検出の要因などが解明されていない。また今回の著者らの検出法により、近年生活排水から通年検出される実態が明らかになり、常に一定数の感染者あるいはウイルス排出者の存在が示唆されている。今回の研究成果により、これまでの遺伝子検出や遺伝子定量といった「ウイルスの痕跡トレース」のみならず、感染力を有するウイルスの存在や感染価を導くことが可能となるため、より精度の高いウイルス動態が解明されるとともに、リスクプロファイリングに基づく実効的な感染制御対策立案が可能となった。
さらに、加熱に対する感受性は60℃・30分で完全な不活化には至らず、60℃・15分で不活化されるヒトノロウイルスに対する対策では不十分であることが判明した。またUV照射についてはHuSaV陽性糞便希釈検体と、培養したHuSaVでは感受性に大きな相違が確認された。こうした結果も公衆衛生におけるHuSaV制御において、きわめて重要な要因となった。
本研究において、HuSaVの汎用培養法を発見したことから、感染経路解明やその対策立案への道が開けたとともに、本研究のさらなる発展による、抗体保有などのヒト血清疫学解析、HuTu80細胞を用いたウイルス受容体検索、抗ウイルス成分検索など幅広い研究展開が可能となった。doctoral thesi
Heart Rate Variability and Electroencephalogram as Predictors of the Therapeutic Outcome of Vagus Nerve Stimulation in Dogs with Epilepsy: A Preliminary Study
Get PDFThis preliminary study aimed to investigate the potential of heart rate variability (HRV) and electro-ecephalogram (EEG) as predictors of a therapeutic outcome of vagus nerve stimulation (VNS) therapy in dogs with epilepsy. We performed serial HRV and EEG analyses befor and until 6 months after VNS initiation and assessed temporal changes in HRV and EEG in four dogs that underwent VNS therapy. Two of the four dogs had reductions in seizure frequency and were determined to be responders, and the other two were non-responders. No differences in temporal changes in HRV and EEG were observed between responders and non-responders. However, HRV before VNS initiation showed that responders had higher parasympathetic activity than non-responders. EEG before VNS initiation revealed that both responders had their irritative zone in the temporal lobe. HRV and EEG assessments before VNS therapy probably helps predict its therapeutic outcome in dogs.P(論文)原著論文application/pdfORIGINAL ARTICLEdepartmental bulletin pape
Studies on the Development, Storage and Application of Equine Antivenom against Yamakagashi (Rhabdophis tigrinus)
Get PDF麻布大学博士(獣医学)Bites by Yamakagashi, Rhabdophis tigrinus are rare in Japan. Although there are serious cases of R. tigrinus bites, the incidence is extremely low. Thus, development of therapeutic drugs commercially has not progressed, and no therapeutic antivenom has been approved as a pharmaceutical drug. We conducted a study with the primary objective of experimentally manufacturing the Yamakagashi equine antivenom as a therapeutic drug. In the past, antivenom against R. tigrinus was experimentally manufactured using rabbits and goats as animals for immunization. It has been reported that these antivenom were used for treatment as an emergency measure and was found to be effective. However, this antivenom has already been used up and no longer available. In this study, we produced a new lot of Yamakagashi antivenom using horses for the first time as immunization animals. This new antivenom was stockpiled on the premise of extrajudicial use in clinical research from the viewpoint of emergency health risk management. This thesis consists of three chapters, in which the development, storage and application of the Equine antivenom against Yamakagashi (R. tigrinus) are described.
In Chapter 1, we described the new test-production of Yamakagashi equine antivenom. 11 g of Yamakagashi venom was extracted from about 500 heads of R. tigrinus, and two horses were immunized with the venom. Serum was collected after waiting for the antivenom titer to increase, and immunoglobulins were fractionated and purified by a method compliant with commercially available equine antivenom products. This was followed by lyophilization to produce 1,369 vials of "Freeze-dried Yamakagashi Antivenom, Equine (Lot 0001)" for therapeutic use in 2000. A purity test conducted as a quality control test of this product confirmed that only immunoglobulins (γ-globulin and T-globulin) were contained in the product. A potency test confirmed that one vial of the product neutralized about 13 mg of R. tigrinus venom. Quantitative testing of anticoagulant activity confirmed that each vial of the product could neutralize the activity of about 4 mg of R. tigrinus venom. Other general tests confirmed that this product is comparable in quality to other horse antivenom products on the market. This Yamakagashi equine antivenom can be an indispensable therapeutic drug to save the life of bite patients by R. tigrinus.
In Chapter 2, we described the quality retention of the "Freeze-dried Yamakagashi Antivenom, Equine (Lot 0001)" after a long period of time from the date of production. In 2021, twenty-one years has passed since the Yamakagashi equine antivenom (Lot 0001) has been manufactured. Therefore, quality control tests were conducted over time to confirm the effect of long-term storage on this product. As a result, no particular changes were observed in the physical and chemical tests (property test including dissolution time, insoluble foreign matter test, osmotic pressure ratio test, test of pH, test for protein content, endotoxin test, and sterility test) as of December 2020, as compared to the results immediately after test-manufacturing. Although the moisture content tend to increase after manufacturing, it was still much lower than the specified level, and thus conformed to the test. No changes were observed in test for freedom from abnormal toxicity and pyrogen test of the product after long-term storage. The potency test (anticoagulant activity) of the product also showed that the potency immediately after manufacture was sufficiently maintained in 2020. Based on the above results, we considered that Yamakagashi equine antivenom (Lot 0001) is still effective as a therapeutic drug even at 21 years after its production.
In Chapter 3, we summarized the cases in which the "Freeze-dried Yamakagashi Antivenom, Equine (Lot 0001)", as described in Chapter 1, was used in emergency situation in severe Yamakagashi bite patients. Since its test-production in 2000, the Yamakagashi equine antivenom has been used as an emergency drug in two serious cases of R. tigrinus bites in 2001 and 2011. Also, this product was used in two cases of severe Yamakagashi bite that occurred in 2017. As a result, these treatments with antivenom were successful in all cases, with no significant adverse reactions. As described above, it was confirmed that the Yamakagashi equine antivenom (Lot 0001) contributed to saving the patient's life. In the 2017 bite case, the antivenom was found to be remarkably effective, even though the product had already been test-manufactured 17 years ago. The 2017 therapeutic cases proved the retention of the quality stability after long-term storage of the product.
The conclusions of this study are as follows.
1) 1,369 vials of Yamakagashi equine antivenom (Lot 0001) were test-manufactured for the first time in Japan, using R. tigrinus venom as an antigen for immunization. This study is the first to demonstrate the production of Yamakagashi equine antivenom.
2) Yamakagashi equine antivenom (Lot 0001) was found to possess the same quality as other approved antitoxins for human use. In addition, one vial of the product was found to neutralize more than 4 mg of R. tigrinus venom.
3) Yamakagashi equine antivenom (Lot 0001) was found to generally retain its immediate post-production quality even in 2021, that is, 21 years after production. This equine antivenom could be stored for as long as 20 years was demonstrated for the first time.
4) Seventeen years after its test-production in 2000, the Yamakagashi equine antivenom (Lot 0001) was shown to be still effective in patients with severe cases of R. tigrinus bites, and thus demonstrating its life-saving efficacy.
As described above, the results of this study have contributed to the saving of lives and the development of medical care and snake antivenom therapy in Japan. The contribution of our study to science and society is deemed to be significant.【研究の背景】
日本国内での稀な毒蛇咬傷例として、やまかがしのそれがある。やまかがし咬傷には重篤例もあるが、発生が少ないことから治療薬の開発は進まず、医薬品として承認された治療用の抗毒素は存在しない。そこで今回、やまかがし咬傷に視点を当て、治療薬としてのやまかがしウマ抗毒素を試験的に製造することを目的として研究を実施した。やまかがし抗毒素は、過去には家兎および山羊を免疫用動物として作製されている。これらの製剤は、緊急措置の治療に用いられ、その有効性が認められていた。しかしながら、これらの抗毒素は既に枯渇し、現在は存在しない。今回、ウマを免疫用動物として初めて用いて、やまかがし抗毒素の試験的な製造を試みた。この新しい抗毒素は、緊急時の健康危機管理の観点から臨床研究の位置付けでの超法規的な使用を前提として備蓄されるものとなる。
【第1章】やまかがしウマ抗毒素の作製
やまかがし抗毒素のウマを用いた初めての試験製造について述べた。約500匹のやまかがしから、毒素11gを採取し、2頭のウマにやまかがし毒の免疫を行った。抗毒素価の上昇を待って血清を採集し、市販のウマ抗毒素製剤に準拠した方法で免疫グロブリン画分を精製した。その後、至適条件下で凍結乾燥させ、治療用の「乾燥やまかがしウマ抗毒素(Lot 0001)」1,369本を2000年に作製した。製剤の品質試験として実施した純度試験では、製剤中に免疫グロブリン(γ-グロブリンとT-グロブリン)のみが含まれることが確認された。力価試験では、製剤1本が約13mgのやまかがし毒を中和することが確認された。抗凝固活性の定量試験では、製剤1本中に約4mgのやまかがし毒を中和する活性を含むことが確認された。また、その他の一般性状試験では、当製剤が市販のその他のウマ抗毒素製剤と同等の品質であることが確かめられた。故に、このやまかがしウマ抗毒素は咬傷患者の救命に必須の治療薬になり得る。
【第2章】 やまかがしウマ抗毒素の安定性評価
作製日から長期経過した、やまかがしウマ抗毒素(Lot 0001)の品質保持について述べた。2021年にはやまかがしウマ抗毒素(Lot 0001)が、作製日から21年を経過する。このことから、長期間保存による製剤への影響を確認するため、経時的に品質試験を行った。その結果、性状確認試験、不溶性異物試験、浸透圧比試験、pH試験、たん白質含量試験、エンドトキシン試験および無菌試験については、製造直後の成績と比較して、2020年12月時点で変化を認めなかった。含湿度については、製造直後に比べて2020年では若干の上昇傾向がみられたが、上昇後でも規格内の低い値であった。製剤の異常毒性否定試験および発熱試験においても、長期間保存による変化は認められなかった。製剤の力価試験についても、製造直後の力価が2020年において保持されていた。以上の成績から、やまかがしウマ抗毒素(Lot 0001)は、製造から21年が経過した現時点でも製剤として有効であると考えた。
【第3章】 やまかがしウマ抗毒素の臨床評価事例
やまかがしウマ抗毒素(Lot 0001)が、緊急事態として発生したヒトやまかがし咬傷の重症例において使用された例ついて述べた。2000年の試験製造以降、2001年から2011年の間に2例の重篤なやまかがし咬傷患者に緊急避難的に当製剤が使用された。また、2017年に発生した2例のやまかがし重症患者に対しても当製剤が使用された。その結果、すべての症例で治療は成功し、顕著な副反応もみられなかった。以上のように、やまかがしウマ抗毒素(Lot 0001)が患者の救命に寄与したことが確認された。2017年の咬傷事例では、この時点で抗毒素は製造から既に17年が経過していたにもかかわらず、顕著な有効性が認められた。2017年の治療例は製剤の長期保存後の品質安定性の事実を支持する事例である。
本研究の結論は次のとおりである。
1）やまかがし毒を免疫用抗原として用いて、国内初の「やまかがしウマ抗毒素(Lot 0001)」の1,369本を試験製造した。本研究はやまかがしウマ抗毒素の作製について初めて示したものである。
2）やまかがしウマ抗毒素(Lot 0001)は、承認薬である他の抗毒素と同様に人用製剤としての品質を保有することが確認された。また、製剤1本は4mg以上のやまかがし毒を中和することが分かった。
3）やまかがしウマ抗毒素(Lot 0001)が、製造から21年を経過した2021年現在でも、概して、製造直後の品質を保持することが分かった。ウマ抗毒素が20年もの長期間保存出来る事実が本研究により初めて示された。
4）2000年の試験製造以降17年が経過したやまかがしウマ抗毒素(Lot 0001)が、やまかがし咬傷重篤患者に有効である事実が初めて示された。
以上のように、本研究の成果は、人命救助および日本の医療や蛇毒抗毒素療法の発展に貢献したものであり、本研究の社会的意義は大きいと考える。
本研究の一部は、以下の学術論文に公表済みである。
1． Morokuma, K., Kobori, N., Fukuda, T., Uchida, T., Sakai, A., Toriba, M., Ohkuma, K., Nakai, K., Kurata, T. and Takahashi, M.: Experimental manufacture of equine antivenom againt Yamakagashi (Rhabdophis tigrinus). Jpn. J. Infect. Dis., 64, 397-402, 2011.
(https://www.niid.go.jp/niid/JJID/64/397.pdf.)
2． Morokuma, K., Matsumura, T., Yamamoto, A., Sakai, A., Hifumi, T., Ato, M. and Takahashi, M.: Evaluation of the stability of Yamakagashi (Rhabdophis tigrinus) Equine Antivenom after 20 years storage. Tropical Biomedicine, 38, 111-118, 2021.
(https://secureservercdn.net/72.167.241.180/114.7f7.myftpupload.com/files/Vol38No2/tb-38-2-042-Morokuma-K.pdf)doctoral thesi
Evaluation of a D-Octaarginine-linked polymer as a transfection tool for transient and stable transgene expression in human and murine cell lines
Get PDFPoly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.journal articl