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Suppression of the Excitability of Rat Nociceptive Primary Sensory Neurons Following Local Administration of the Phytochemical, Quercetin
Get PDFAlthough the modulatory effect of quercetin on voltage-gated Na, K, and Ca channels has been studied in vitro, the in vivo effect of quercetin on the excitability of nociceptive primary neurons remains to be determined. The aim of the present study was to examine whether acute local quercetin administration to rats attenuates the excitability of nociceptive trigeminal ganglion (TG) neurons in response to mechanical stimulation in vivo. Extracellular single unit recordings were made from TG neurons of anesthetized rats in response to orofacial non-noxious and noxious mechanical stimulation. The mean firing frequency of TG neurons in response to both non-noxious and noxious mechanical stimuli was dose-dependently inhibited by quercetin, and maximum inhibition of the discharge frequency of both non-noxious and noxious mechanical stimuli was seen within 10 min. The inhibitory effect of quercetin lasted for 15 minutes and was reversible. The mean magnitude of inhibition on TG neuronal discharge frequency with 10 mM quercetin was almost equal to that of the local anesthetic, 2% lidocaine. These results suggest that local injection of quercetin into the peripheral receptive field suppresses the excitability of nociceptive primary sensory neurons in the TG, possibly via inhibition of voltage-gated Na channels and opening voltage-gated K channels.journal articl
Studies on the regulation of hepcidin gene expression by activin B and BMP
Get PDF麻布大学博士(獣医学)Iron is essential nutrition for the organism, which supports protein function in transporting oxygen and maintaining cell living. Otherwise, iron overload in blood generates reactive oxygen species in tissues, following which causes disorder and tumorigenesis there. The hepcidin protein decreases iron and controls iron concentration in blood by inhibiting a transmembrane protein: ferroportin which exports iron from cells. The hepcidin gene expression is regulated by some factors, the BMP2/6 in basal expression and iron overload, activin B and interleukin in inflammation, and erythroferrone in erythropoiesis. In this study, we focused on and investigated two factors which are activin B and BMP2.
Chapter 1
Activin B, a homodimer of inhibin βB, belongs to the TGF-β family. In human and mouse, activin B is increased in inflammation and upregulates hepcidin gene expression. The upregulation of hepcidin expression in inflammation causes anemia which is called anemia of inflammatory disease. Anemia of inflammatory disease also often has occurred and has been considered an important disease in dogs. However, the function of canine activin B is largely unknown. In this chapter, we isolated the canine inhibin βB CDS and examined the signal transduction activity of canine activin B.
First, we tried to isolate the canine inhibin βB CDS by RT-PCR. In RT-PCR, we use a primer made from the genome sequence of the dingo, Canis lupus dingo. Inhibin βB mRNA sequences of human and dingo contain many GC-rich regions. We added 5% dimethyl sulfoxide (final concentration) to the RT-PCR mix and isolated the canine inhibin βB CDS. We deposited the canine inhibin βB CDS as LC630404 to DDBJ. We created an expression vector based on the canine inhibin βB CDS and call Ca InhβB (407). We transfected the Ca InhβB (407) vector to HEK293 cells and collected the conditioned medium. We detected inhibin βB in the conditioned medium by western blotting. In the conditioned medium from cells transfected with Ca InhβB (407), 12 kDa and 25 kDa bands and a 12 kDa band were detected in non-reduced and reduced conditions, respectively. The size of Inhibin βB is estimated as 12 kDa thereby this result indicates that the canine inhibin βB CDS: Ca InhβB (407) generated and released canine activin B.
Next, we examined the signal transduction activity of canine activin B. In human and mouse, it is known that activin B phosphorylates Smad2/3 and Smad1/5/8 via the ALK4/7 receptor and ALK2/3 receptor, respectively. We detected phosphorylated Smad3 and Smad1/5/8 by western blotting.in HepG2 cells added the conditioned medium from cells transfected with Ca InhβB (407). We also used A-83-01 and LDN-193189 as ALK4/7 and ALK2/3 inhibitors, respectively. The phosphorylation of Smad3 and Smad1/5/8 by the canine activin B was inhibited by A-83-01 and LDN-193189, respectively. Moreover, the phosphorylation of Smad1/5/8 by the canine, human and mouse activin B was inhibited by A-83-01, which is an inhibitor of ALK4/7.
In addition, we examined the level of transcriptional activity by the canine activin B by reporter assay. CACA-luc was used as a Smad2/3 response vector, and hepcidin-luc was used as a Smad1/5/8 response vector. The conditioned medium from cells transfected with Ca InhβB (407) upregulated CAGA-luc and hepcidin-luc activity. In reporter assay, we also use inhibitors, and the upregulation of transcriptional activity of CAGA-luc and hepcidin-luc were inhibited by A-83-01 and LDN-193189, respectively. Similar to the result of western blotting in Smad1/5/8, the upregulation of transcriptional activity of hepcidin-luc was also inhibited by A-83-01. These results were similar to using human and mouse inhibin βB vectors. These results suggest that the canine activin B, similar to human and mouse, phosphorylate Smad3 and Smad1/5/8, and upregulate TGF-β/activin and BMP signaling pathways via ALK4/7 and ALK1/5/8, respectively. But it is also newly suggested that the canine, human, and mouse activin B phosphorylate Smad1/5/8 and transmitted the BMP signaling pathway via ALK 4/7.
Chapter 2
BMP also belongs to the TGF-β family. In base expression and iron overload, BMP upregulates hepcidin gene expression via ALK2/3. Activated ALK2/3 phosphorylates Smad1/5/8 and phosphorylated Smad1/5/8 binds to the BMP response element (BMP-RE) on the hepcidin promoter and upregulates hepcidin gene transcription. In human and mouse livers, hepcidin is expressed normally. However, in HepG2 cells, which are derived from human liver blastoma, hepcidin is not expressed despite the presence of Smad1/5/8 and BMP-RE. In recent years, regulation of gene expression via BMP is regulated by epigenome in a gene, neurogenin1. In this chapter, we consider the association between hepcidin expression and epigenome through a comparison of HepG2 cells and hepatocytes.
First, we confirmed the hepcidin gene expression which is high in hepatocytes but low in HepG2 cells by RT-qPCR. With these cells, we examined the connection between the phosphorylated Smad1/5/8 and the DNA of hepcidin’s BMP-RE by chromatin immunoprecipitation qPCR. In hepatocytes, anti-phosphorylated Smad1/5/8 anti-body recovered DNA of hepcidin’s BMP-RE, but in HepG2 cells, it didn’t. These results suggested that in HepG2 cells hepcidin expression is low by phosphorylated Smad1/5/8 not connecting to hepcidin’s BMP-RE. Chromatin accessibility is one of the reasons why a transcription factor doesn’t connect to the gene promoter region. Next, we performed formaldehyde-assisted isolation of regulatory elements (FAIRE) to investigate chromatin accessibility. The DNA of hepcidin’s BMP-RE was recovered by FAIRE in hepatocytes but was not done in HepG2, which suggested, in HepG2 cells, the hepcidin’s BMP-RE was closed.
Next, we added 5-aza-2-deoxycytidine which inhibits DNA methylation and tends to open chromatin to HepG2 cells. We measured the mRNA level of hepcidin in the affected HepG2 cells. The mRNA level of hepcidin was upregulated by 5-aza-2-deoxycytidine. These results suggested that, in HepG2 cells, the DNA methylation on the hepcidin promoter closes the chromatin there and downregulates the hepcidin gene expression.
In chapter 1, we isolated the canine inhibin βB gene CDS and showed that canine activin B produced from this inhibin βB promotes activin/TGF-β and BMP signaling. In Chapter 2, we showed that DNA methylation and chromatin accessibility play important roles in hepcidin gene expression. These results will support the studies of hepcidin and anemia in dogs and epigenetics.生命にとって重要な血中鉄量の調節は、肝臓の実質細胞から産生されるペプチドホルモン、ヘプシジンにより行われている。ヘプシジンの発現量は血中鉄量と、細菌感染や腫瘍による炎症反応、および赤血球の新生の影響を受けて変化する。血中の鉄量増加時にはBone morphogenetic protein(BMP)が、炎症時にはアクチビンBやインターロイキン6が産生され、ヘプシジンの発現を促進する。造血の亢進時には赤芽球からBMPの作用を抑制するエリスロフェロンが産生され、ヘプシジン遺伝子の発現を抑制する。この内BMPとアクチビンBはTransforming growth factor-β(TGF-β)ファミリーに属するリガンドである。どちらのリガンドも細胞膜上の受容体であるActivin receptor-like kinases (ALK)2/3を介して細胞内へシグナルを伝達する。リガンドが結合したALK2/3受容体は転写因子であるSmad1/5/8をリン酸化により活性化させ、ヘプシジン遺伝子の発現を促進する。
TGF-βファミリーによるSmadを介した遺伝子発現制御は以前から調べられている。しかし、動物種差によるリガンドの機能の違いや、遺伝子の発現におけるエピジェネティックな制御に関する研究は十分とは言えない。本研究では、イヌのアクチビンBの遺伝子配列の単離とヘプシジン遺伝子を含む標的遺伝子発現制御、およびBMPによるヘプシジン遺伝子発現におけるエピジェネティックな制御に注目して解析を行った。
１．イヌインヒビンβB遺伝子の単離とその遺伝子産物アクチビンBによる
ヘプシジン遺伝子発現制御における細胞内情報伝達機構
アクチビンBはインヒビンβBサブユニットのホモ二量体である。アクチビンBによる遺伝子発現制御は、ヒトやマウスにおいて、ALK4/7受容体と細胞内伝達因子Smad2/3を介したアクチビン経路とALK2/3受容体と細胞内伝達因子Smad1/5/8を介したBMP経路の2つの経路を利用することが知られている。アクチビンBによるアクチビン経路は生殖、分化や発生において働く様々な遺伝子の発現制御で利用されているが、BMP経路は炎症時におけるヘプシジン遺伝子の発現促進での利用のみが報告されている。また、構造や機能がアクチビンBと類似するアクチビンAについては、その細胞内情報伝達と遺伝子発現制御についてよく調べられているが、動物種により機能が異なることが報告されている。そのため、アクチビンBにおいても制御する遺伝子やその情報伝達メカニズムだけでなく、動物種差も考慮する必要がある。しかし、イヌにおけるインヒビンβB遺伝子の正確な配列やその遺伝子産物の機能についての報告はほとんどない。本研究ではイヌインヒビンβB遺伝子のコーディング領域の塩基配列を同定し、その配列を基に作製した発現ベクターを用いてイヌアクチビンBによるシグナル伝達経路の評価を行うことで、イヌにおけるアクチビンBの基礎的な知見を得ることを目的とした。
イヌの亜種であるディンゴのインヒビンβBの推定遺伝子配列を参考にしたプライマーと、イヌ卵巣から作製したcDNAを用いてRT-PCRを行った。その結果、一般的なPCRの条件では増幅が見られなかったが、高GC配列への対策としてジメチルスルホキシドを添加したPCR条件では増幅が見られた。この配列を解析した結果、ヒトインヒビンβB遺伝子と同じ長さのCDSを含むイヌインヒビンβB遺伝子であることがわかり、この増幅産物からイヌのインヒビンβB遺伝子を単離することができた。このイヌインヒビンβB遺伝子の配列を基に作製した発現ベクターをヒト胎児腎臓由来細胞株HEK293細胞に導入し、イヌのインヒビンβB遺伝子産物を含む培養上清を回収した。この培養上清を用いて、イヌのアクチビンBのシグナル伝達経路の評価を行った。
ヒト肝ガン由来細胞株HepG2細胞に培養上清を添加したところ、アクチビン経路とBMP経路、それぞれの経路における活性化した情報伝達因子であるリン酸化Smad3及びリン酸化Smad1/5/8の増加が確認された。次に、アクチビン経路、もしくはBMP経路の活性化に応答するレポーターベクターを使用したレポーターアッセイ法を行った。HepG2細胞にそれぞれのレポーターベクターを導入した後に培養上清を加えたところ、どちらのレポーターベクターにおいても応答が見られた。アクチビンBが利用している受容体を確認するために、各種ALK受容体の阻害薬を用いてレポーターアッセイ法を行った。その結果、アクチビン経路の活性化にはALK4/7を、BMP経路の活性化にはALK2/3およびALK4/7を利用していることが明らかとなった。これらの結果はヒトやマウスのアクチビンBの場合でも見られたことから、動物種によるアクチビンBの作用機序に違いはないのかもしれない。BMP経路におけるALK4/7の利用はこれまで報告がなく、今後、アクチビンBによるヘプシジン遺伝子の発現機序を解明していく上で、この受容体を介した作用機序を明らかにしていく必要があるかもしれない。
1章では、イヌにおける機能的なインヒビンβB遺伝子を単離できたこと、イヌアクチビンBのシグナル伝達経路がヒトやマウスと同様であること、及びBMP経路においてはALK2/3だけでなくALK4/7受容体も介していることが示された。本研究はヘプシジン遺伝子発現制御に限らず、今後のイヌでのアクチビンBの作用機序の解明における基礎的な知見となりうると考えられる。
２．BMPによるヘプシジン遺伝子発現制御へのエピゲノムの影響
BMPは一般的に骨の形成促進や細胞の分化、発生に関わるサイトカインとして知られているが、ヘプシジン遺伝子の発現促進にも関与している。BMPによるヘプシジン遺伝子の発現促進は一般的なBMP応答遺伝子と同様に、受容体ALK2/3と細胞内情報伝達因子Smad1/5/8のリン酸化を介している。ALK2/3受容体によりリン酸化され活性化した転写因子Smad1/5/8が、ヘプシジン遺伝子上流のプロモーター領域に存在する2つのBMP応答領域に結合することでヘプシジン遺伝子の発現が促進される。ヘプシジン遺伝子は肝臓で発現しており、BMPによりその発現が促進されるが、ヒト肝ガン由来細胞株であるHepG2細胞ではリン酸化Smad1/5/8が存在しているにも関わらずヘプシジン遺伝子の発現は低いことが知られている。BMPによる発現促進において、エピゲノムの関与が疑われている。神経細胞への分化に関わるNeurog1遺伝子では、BMPによりリン酸化したSmadと遺伝子上流部の結合に、ゲノムDNAやDNAが巻き付いているヒストンタンパク質の修飾、DNAとヒストンの集合体であるクロマチンの構造といった、いわゆるエピゲノムが関係しているとの報告がある。本研究ではHepG2細胞におけるヘプシジン遺伝子の発現低下にエピゲノムによる発現制御が関与していると考え、ラット初代培養肝細胞とHepG2細胞において、ヘプシジン遺伝子の発現や遺伝子上流プロモーター領域におけるエピゲノムの比較を行った。これら実験を通して、正常な肝臓におけるヘプシジン遺伝子の発現とエピゲノムの関係を調査することを本研究の目的とした。
ラット初代培養肝細胞とHepG2細胞からRNAを抽出し、ヘプシジン遺伝子の発現量を定量したところ、過去の報告と一致して初代培養肝細胞では高い発現が見られ、HepG2では非常に低い発現しか見られないことを確認した。次に、リン酸化Smad1/5/8の結合状態を見るために抗リン酸化Smad1/5/8抗体でクロマチン免疫沈降（ChIP）法を行ったところ、初代培養肝細胞ではヘプシジン遺伝子上流プロモーター領域にリン酸化Smad1/5/8の結合が見られたのに対して、HepG2細胞では結合が見られなかった。DNAが巻き付いたヒストンの集まりであるクロマチンが凝集すると、転写因子が標的DNAに結合できなくなる。今回のHepG2細胞におけるリン酸化Smad1/5/8の結合不全にもクロマチンの凝集が関係している可能性を考え、開いたクロマチンを回収する方法であるFormaldehyde-Assisted Isolation of Regulatory Elements（FAIRE）法を行った。その結果、初代培養肝細胞ではヘプシジン遺伝子上流プロモーター領域のクロマチンは開いた構造を示したが、HepG2細胞では閉じた構造を示すことが分かった。
クロマチンの構造はリモデリング因子と呼ばれるタンパク質や、エピゲノムであるDNAとヒストンの修飾の影響を受けている。遺伝子の発現を抑制するエピゲノムの代表例としては、DNAのメチル化やヒストンの脱アセチル化、ヒストン3の27番目のリジンのメチル化が存在する。これらの修飾を行う酵素の阻害剤を添加した時のHepG2細胞におけるヘプシジン遺伝子の発現を定量した。その結果、DNAのメチル化を阻害する5-アザデオキシシチジンを加えたとき、ヘプシジン遺伝子の発現の増加が見られた。このことからヘプシジン遺伝子の上流部でのメチル化が、ヘプシジン遺伝子の発現に影響を与えている可能性が示された。
以上の結果から2章では、ヒトのヘプシジン遺伝子の発現においてDNAのメチル化と遺伝子上流部のクロマチンの凝集の関連が示唆された。今後はヘプシジン遺伝子の上流部におけるエピジェネティックな変化を起こす遺伝子の特定により、領域特異的なメチル化とクロマチン構造や遺伝子発現との関わりを明らかにする必要があると考える。doctoral thesi
Isolation, structural determination, and antiviral activities of metabolites from vanitaracin A-producing Talaromyces sp
Get PDFVanitaracin A, an anti-hepatitis B virus polyketide, has been previously isolated from Talaromyces sp. In the present study, we searched for novel compounds in the culture broth obtained from a vanitaracin A-producing fungus under various conditions. Three novel compounds (vanitaracin C, vanitaraphilone A, and 2-hydroxy-4(hydroxymethyl)-6-methylbenzaldehyde) were isolated, and their structures were determined using spectroscopic methods (1D/2D NMR and MS). In addition, the antiviral spectrum of vanitaracin A was examined by measuring its antiviral activities against rabies virus, Borna disease virus 1, and bovine leukemia virus. This compound exhibited antiviral activity against bovine leukemia virus, which is the causative agent of enzootic bovine leukosis. The anti-bovine leukemia virus effects of other compounds isolated from the vanitaracin A-producing fungus, namely, vanitaracins B and C, vanitaraphilone A, and 2-hydroxy-4-(hydroxymethyl)-6-methylbenzaldehyde, were also evaluated. Vanitaracin B, vanitaraphilone A and 2-hydroxy-4-(hydroxymethyl)-6methylbenzaldehyde were also found to exhibit activity against bovine leukemia virus. These findings reveal the broad-spectrum antiviral activity of the vanitaracin scaffold and suggest several candidates for the development of anti-bovine leukemia virus drugs.journal articl
Aberrant histone methylation in mouse early preimplantation embryos derived from round spermatid injection
Get PDFRound spermatid injection (ROSI) is the last resort and recourse for men with nonobstructive azoospermia to become biological fathers of their children. However, the ROSI-derived offspring rate is lower than intracytoplasmic sperm injection (ICSI) in mice (20% vs. 60%). This low success rate has hindered the spread of ROSI in ART (Assisted Reproductive Technology). However, the cause of the ROSI-zygote-derived low offspring rate is currently unknown. In the previous studies, we reported that H3K9me3 and H3K27me3 exhibited ectopic localizations in male pronuclei (mPN) of ROSI-zygotes, suggesting that the carried over histone to zygotes conveys epigenetic information. In this study, we analyzed other histone modifications to explore unknown abnormalities. H3K36me3 showed an increased methylation state compared to ICSI-derived embryos but not for H3K4me3. Abnormal H3K36me3 was corrected until 2-cell stage embryos, suggesting a long window of reprogramming ability in ROSI-embryos. Treatment with TSA of ROSI-zygotes, which was reported to be capable of correcting ectopic DNA methylation in ROSI-zygotes, caused abnormalities of H3K36me3 in male and female PN (fPN) of the zygotes. In contrast, round spermatid TSA treatment before ROSI, which was reported to improve the preimplantation development of ROSI-zygotes, showed beneficial effects without toxicity in fPN. Therefore, the results suggest that TSA has some negative effects, but overall, it is effective in the correction of epigenetic abnormalities in ROSI-zygotes. When attempting to correct epigenetic abnormalities, attention should be paid to epigenomes not only in male but also in female pronuclei.journal articl
Two cases of new-born puppies with transient pulmonary edema
Get PDFAcute pulmonary edema in puppies generally occurs due to congenital left-right shunts such as patent ductus arteriosus or large ventricular septal defects. Herein, we presented two cases of puppies with no apparent congenital cardiovascular disease. Case 1: A 12-day-old male Labrador Retriever, weighing 1.15 kg, was unable to suckle sufficiently from its dam and exhibited laboured breathing. Pulmonary edema was identified in all lung lobes by radiography, furthermore, echocardiography revealed significant enlargement of the left side of the heart. Pulmonary edema secondary to volume overload was suspected and furosemide was administered. The respiratory status was improved on the following day. Pimobendan was administered orally in addition to furosemide and both were withdrawn 6 weeks later when the heart size was normalized. Case 2: A 15-day-old female Standard Poodle, weighing 0.68 kg, was less active than other littermates and exhibited laboured breathing. Radiography revealed pulmonary edema in the right posterior lobe, dilatation of the caudal vena cava and ascites. Echocardiography revealed significant enlargement of the left atrium and ventricle perhaps owing to decreased left ventricular contractility. Furosemide and pimobendan were administered. One week later, appetite was improved and supraventricular tachycardia of 375 bpm was observed. Therefore, tachycardia-induced dilated cardiomyopathy was suspected which returned to sinus rhythm with diltiazem treatment, however, it was recurred. Upon sotalol monotherapy, a normal heart size was observed seven months later. In conclusion, we encountered two new-born puppies with transient pulmonary edema that were temporarily treated with pimobendan and furosemide.journal articl
Predicting an increased risk of severe clinical mastitis and economic loss using a threshold value of bovine leukemia virus proviral load
Get PDFOBJECTIVE
To establish a threshold value of bovine leukemia virus (BLV) proviral load (PVL) to identify increased risk of severe clinical mastitis, and to examine the prognosis and economic loss of clinical mastitis based on the newly established PVL cut-off value.
ANIMALS
97 lactating Holstein cows with clinical mastitis.
METHODS
Blood and milk samples were collected aseptically from each cow. Youden index was used for receiver-operating characteristic curve analysis with the severity rate of clinical mastitis as the dependent variable and PVL as an independent variable. PVL cut-off value was used as a criterion to compare the severity rate of clinical mastitis, percentage of cows with and without systemic treatments, number of treatments, cost of treatment, and prognosis.
RESULTS
PVL cut-off value was 17.8 copies/10 ng DNA for the dependent variable MILD vs SEVERE. The severity rate of clinical mastitis, percentage of cows given systemic treatments, and technical fees for medical treatment were significantly higher in the group above the PVL cut-off value than in the group below the PVL cut-off value and the negative group. Number of treatments was significantly higher in the group above the cut-off value than in the group below the cut-off value. There was no significant difference in prognosis after mastitis among the 3 groups.
CLINICAL RELEVANCE
These results suggested that PVL cut-off value of 17.8 copies/10 ng DNA was a useful threshold for increased economic losses in BLV-infected cows; it may also serve as a new standard value for the detection and culling of BLV-infected cows in Japan.journal articl
A thioacetamide-induced liver fibrosis model for pre-clinical studies in microminipig
Get PDFDrug-induced liver fibrosis models are used in normal and immunosuppressed small animals for transplantation and regenerative medicine to improve liver fibrosis. Although large animal models are needed for pre-clinical studies, they are yet to be established owing to drug sensitivity in animal species and difficulty in setting doses. In this study, we evaluated liver fibrosis by administering thioacetamide (TA) to normal microminipig and thymectomized microminipig; 3 times for 1 week (total duration: 8 weeks). The pigs treated with TA showed elevated blood cytokine levels and a continuous liver injury at 8 weeks. RNA-seq of the liver showed increased expression of fibrosis-related genes after TA treatment. Histopathological examination showed degenerative necrosis of hepatocytes around the central vein, and revealed fibrogenesis and hepatocyte proliferation. TA treatment caused CD3-positive T cells and macrophages scattered within the hepatic lobule to congregate near the center of the lobule and increased αSMA-positive cells. Thymectomized pigs showed liver fibrosis similar to that of normal pigs, although the clinical signs tended to be milder. This model is similar to pathogenesis of liver fibrosis reported in other animal models. Therefore, it is expected to contribute to research as a drug discovery and pre-clinical transplantation models.journal articl
Utility of In Vitro Cellular Models of Low-Dose Lipopolysaccharide in Elucidating the Mechanisms of Anti-Inflammatory and Wound-Healing-Promoting Effects of Lipopolysaccharide Administration In Vivo
Get PDFLipopolysaccharide (LPS) is a bacterial component that activates intracellular signaling pathways upon binding to the Toll-like receptor (TLR)-4/MD-2 complex. It is well known that LPS injected into animals and high-dose (100 ng/mL to 1 μg/mL) LPS treatment to innate immune cells induce an inflammatory response. In contrast, LPS is naturally present in the gastrointestinal tract, respiratory tract, and skin of humans and animals, and it has been shown that TLR-4-deficient animals cannot maintain their immune balance and gut homeostasis. LPS from commensal bacteria can help maintain homeostasis against mucosal stimulation in healthy individuals. Oral LPS administration has been shown to be effective in preventing allergic and lifestyle-related diseases. However, this effect was not observed after treatment with LPS at high doses. In mice, oral LPS administration resulted in the detection of LPS at a low concentration in the peritoneal fluid. Therefore, LPS administered at low and high doses have different effects. Moreover, the results of in vitro experiments using low-dose LPS may reflect the effects of oral LPS administration. This review summarizes the utility of in vitro models using cells stimulated with LPS at low concentrations (50 pg/mL to 50 ng/mL) in elucidating the mechanisms of oral LPS administration. Low-dose LPS administration has been demonstrated to suppress the upregulation of proinflammatory cytokines and promote wound healing, suggesting that LPS is a potential agent that can be used for the treatment and prevention of lifestyle-related diseases.journal articl
Serum amyloid A (SAA) concentration in cats with gastrointestinal lymphoma
Get PDFThe incidence of feline gastrointestinal (GI) lymphoma has recently increased. Serum amyloid A (SAA) levels are elevated in feline lymphoma. However, no reports have evaluated SAA concentrations and outcomes in feline GI lymphoma. This study aimed to evaluate the clinical utility of SAA and other factors in feline GI lymphoma to assess the outcomes with potential differences. The study included 39 client-owned cats diagnosed with GI lymphoma, which were divided into two groups: high- and low-grade lymphomas. Changes in SAA concentration, complete blood count (CBC), and biochemical profiles were analyzed at the time of initial presentation as well as on days 1, 28, and 56. Differences between the two groups were investigated. High-grade lymphoma was observed in 17 cats, whereas 22 cats showed low-grade lymphoma. SAA concentrations on the day of initial presentation were significantly higher in low-grade lymphoma than those in high-grade lymphoma (median, 12.4 µg/mL; range, 4.8–46.5 µg/mL vs. 3.8 µg/mL; 3.8–13.7 µg/mL; P=0.011). Elevated SAA concentration on day 56 in high-grade GI lymphoma was a poor prognostic factor. (Hazard Ratio=1.012, per 1 µg/mL increase; 95% confidence interval; 1.004–1.020, P=0.002). The SAA concentration on the day of initial presentation did not serve as a suitable prognostic factor and did not depend on the grade or stage of the lymphoma. However, continuous SAA concentration measurement may be useful for predicting the outcome of feline GI lymphoma.journal articl
Generation of a recombinant temperature-sensitive influenza D virus
Get PDFInfluenza D virus (IDV) is a causative agent of the bovine respiratory disease complex (BRDC), which is the most common and costly disease affecting the cattle industry. For developing a candidate vaccine virus against IDV, we sought to produce a temperature-sensitive strain, similar to the live attenuated, cold-adapted vaccine strain available against the influenza A virus (IAV). To this end, we produced a recombinant IDV (designated rD/OK-AL) strain by introducing mutations responsible for the adaptation of the IAV vaccine strain to cold conditions and conferring sensitivity to high temperatures into PB2 and PB1 proteins using reverse genetics. The rD/OK-AL strain grew efficiently at 33 °C but did not grow at 37 °C in the cell culture, indicating its high-temperature sensitivity. In mice, rD/OK-AL was attenuated following intranasal inoculation. It mediated the production of high levels of antibodies against IDV in the serum. When the rD/OK-AL-inoculated mice were challenged with the wild-type virus, the virus was not detected in respiratory organs after the challenge, indicating complete protection against IDV. These results imply that the rD/OK-AL might be a potential candidate for the development of live attenuated vaccines for IDV that can be used to control BRDC.journal articl