Study of the molecular mechanism of influenza-associated encephalopathy (influenza-associated encephalopathy, mitochondria, adenosine triphosphate)
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Development of ATP extraction and quantification kit from peripheral blood of patients (adenosine triphosphate, multiple organ failure) (Measurements of adenylate nucleotides (i.e., ATP, ADP) are used extensively to monitor energy stasis in broad range of cell types and tissues. For applications involving mammalian and other animal tissue, ATP are commonly extracted with chaotropic reagents particularly trichloroacetic acid (TCA) and perchloric acid (PCA). Physical methods for disintegration (boiling, sonic disintegration) have also used. As expected, these methods are suitable for measurements of cellular ATP, however these methods are inappropriate for histionic ATP because of the inefficiency of nucleotides extraction. Consequently, we have developed an integrated kit that includes software programs measuring tissue or total blood ATP. As the first step, we have evaluated the clinically usefulness of this system for determining blood ATP levels in patients with acute disease such as influenza-associated encephalopathy. It was found that various patients with severe disease have decreased blood ATP levels. It can be anticipated that in the abovementioned disease states, ATP measurement will become a valuable clinical parameter of great potential clinical importance.)
Book / Paper
Academic Paper (Judged Full Paper):
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Dini Agriani Pasiana, Hironori Miyata, Junji Chida, Hideyuki Hara, Morikazu Imamura, Ryuichiro Atarashi and Suehiro Sakaguchi : Central residues in prion protein PrPC are crucial for its conversion into the pathogenic isoform, The Journal of Biological Chemistry, Vol.298, No.9, 102381, 2022.
Keiji Uchiyama, Hideyuki Hara, Junji Chida, Agriani Dini Pasiana, Morikazu Imamura, Tsuyoshi Mori, Hanae Takatsuki, Ryuichiro Atarashi and Suehiro Sakaguchi : Ethanolamine Is a New Anti-Prion Compound, International Journal of Molecular Sciences, Vol.22, No.21, 11742, 2021.
(Summary)
Prion diseases are a group of fatal neurodegenerative disorders caused by accumulation of proteinaceous infectious particles, or prions, which mainly consist of the abnormally folded, amyloidogenic prion protein, designated PrP. PrP is produced through conformational conversion of the cellular isoform of prion protein, PrP, in the brain. To date, no effective therapies for prion diseases have been developed. In this study, we incidentally noticed that mouse neuroblastoma N2a cells persistently infected with 22L scrapie prions, termed N2aC24L1-3 cells, reduced PrP levels when cultured in advanced Dulbecco's modified eagle medium (DMEM) but not in classic DMEM. PrP levels remained unchanged in prion-uninfected parent N2aC24 cells cultured in advanced DMEM. These results suggest that advanced DMEM may contain an anti-prion compound(s). We then successfully identified ethanolamine in advanced DMEM has an anti-prion activity. Ethanolamine reduced PrP levels in N2aC24L1-3 cells, but not PrP levels in N2aC24 cells. Also, oral administration of ethanolamine through drinking water delayed prion disease in mice intracerebrally inoculated with RML scrapie prions. These results suggest that ethanolamine could be a new anti-prion compound.
Hideyuki Hara, Junji Chida, Agriani Dini Pasiana, Keiji Uchiyama, Yutaka Kikuchi, Tomoko Naito, Yuichi Takahashi, Junji Yamamura, Hisashi Kuromatsu and Suehiro Sakaguchi : Vaporized Hydrogen Peroxide and Ozone Gas Synergistically Reduce Prion Infectivity on Stainless Steel Wire., International Journal of Molecular Sciences, Vol.22, No.6, 3268, 2021.
(Summary)
Prions are infectious agents causing prion diseases, which include Creutzfeldt-Jakob disease (CJD) in humans. Several cases have been reported to be transmitted through medical instruments that were used for preclinical CJD patients, raising public health concerns on iatrogenic transmissions of the disease. Since preclinical CJD patients are currently difficult to identify, medical instruments need to be adequately sterilized so as not to transmit the disease. In this study, we investigated the sterilizing activity of two oxidizing agents, ozone gas and vaporized hydrogen peroxide, against prions fixed on stainless steel wires using a mouse bioassay. Mice intracerebrally implanted with prion-contaminated stainless steel wires treated with ozone gas or vaporized hydrogen peroxide developed prion disease later than those implanted with control prion-contaminated stainless steel wires, indicating that ozone gas and vaporized hydrogen peroxide could reduce prion infectivity on wires. Incubation times were further elongated in mice implanted with prion-contaminated stainless steel wires treated with ozone gas-mixed vaporized hydrogen peroxide, indicating that ozone gas mixed with vaporized hydrogen peroxide reduces prions on these wires more potently than ozone gas or vaporized hydrogen peroxide. These results suggest that ozone gas mixed with vaporized hydrogen peroxide might be more useful for prion sterilization than ozone gas or vaporized hydrogen peroxide alone.
Keiji Uchiyama, Miyata Hironori, Yamaguchi Yoshitaka, Imamura Morikazu, Okazaki Mariya, Pasiana Dini Agriani, Junji Chida, Hideyuki Hara, Atarashi Ryuichiro, Watanabe Hitomi, Kondoh Gen and Suehiro Sakaguchi : Strain-Dependent Prion Infection in Mice Expressing Prion Protein with Deletion of Central Residues 91-106., International Journal of Molecular Sciences, Vol.21, No.19, 7260, 2020.
(Summary)
Conformational conversion of the cellular prion protein, PrP, into the abnormally folded isoform, PrP, is a key pathogenic event in prion diseases. However, the exact conversion mechanism remains largely unknown. Transgenic mice expressing PrP with a deletion of the central residues 91-106 were generated in the absence of endogenous PrP, designated Tg(PrP∆91-106)/ mice and intracerebrally inoculated with various prions. Tg(PrP∆91-106)/ mice were resistant to RML, 22L and FK-1 prions, neither producing PrP∆91-106 or prions in the brain nor developing disease after inoculation. However, they remained marginally susceptible to bovine spongiform encephalopathy (BSE) prions, developing disease after elongated incubation times and accumulating PrP∆91-106 and prions in the brain after inoculation with BSE prions. Recombinant PrP∆91-104 converted into PrP∆91-104 after incubation with BSE-PrP-prions but not with RML- and 22L-PrP-prions, in a protein misfolding cyclic amplification assay. However, digitonin and heparin stimulated the conversion of PrP∆91-104 into PrP∆91-104 even after incubation with RML- and 22L-PrP-prions. These results suggest that residues 91-106 or 91-104 of PrP are crucially involved in prion pathogenesis in a strain-dependent manner and may play a similar role to digitonin and heparin in the conversion of PrP into PrP.
Junji Chida, Hideyuki Hara, Keiji Uchiyama, Etsuhisa Takahashi, Hironori Miyata, Hidetaka Kosako, Yukiko Tomioka, Toshihiro Ito, Hiroyuki Horiuchi, Haruo Matsuda, Hiroshi Kido and Suehiro Sakaguchi : Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice., PLoS Pathogens, Vol.16, No.8, e1008823, 2020.
(Summary)
The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.
Suehiro Sakaguchi and Junji Chida : Prion Protein Is a Novel Modulator of Influenza: Its Potential Implications for Anti-Influenza Therapeutics., Current Issues in Molecular Biology, Vol.37, 21-32, 2019.
Nandita Rani Das, Hironori Miyata, Hideyuki Hara, Junji Chida, Keiji Uchiyama, Kentaro Masujin, Hitomi Watanabe, Gen Kondoh and Suehiro Sakaguchi : The N-Terminal Polybasic Region of Prion Protein Is Crucial in Prion Pathogenesis Independently of the Octapeptide Repeat Region, Molecular Neurobiology, Vol.57, 1203-1216, 2019.
(Summary)
Conformational conversion of the cellular isoform of prion protein, designated PrP, into the abnormally folded, amyloidogenic isoform, PrP, is an essential pathogenic event in prion diseases. However, the exact conversion mechanism remains largely unknown. Lines of evidence indicate that the N-terminal domain, which includes the N-terminal, positively charged polybasic region and the octapeptide repeat (OR) region, is important for PrP to convert into PrP after infection with prions. To further gain insights into the role of the polybasic region and the OR region in prion pathogenesis, we generated two different transgenic mice, designated Tg(PrP3K3A)/Prnp and Tg(PrP3K3A∆OR)/Prnp mice, which express PrP with lysine residues at codons 23, 24, and 27 in the polybasic region mutated with or without a deletion of the OR region on the Prnp background, respectively, and intracerebrally inoculated them with RML and 22L prions. We show that Tg(PrP3K3A)/Prnp mice were highly resistant to the prions, indicating that lysine residues at 23, 24, and 27 could be important for the polybasic region to support prion infection. Tg(PrP3K3A∆OR)/Prnp mice also had reduced susceptibility to RML and 22L prions equivalent to Tg(PrP3K3A)/Prnp mice. The pre-OR region, including the polybasic region, of PrP3K3A∆OR, but not PrP3K3A, was unusually converted to a protease-resistant structure during conversion to PrP3K3A∆OR. These results suggest that, while the OR region could affect the conformation of the polybasic region during conversion of PrP into PrP, the polybasic region could play a crucial role in prion pathogenesis independently of the OR region.
Jun Oda, Yukioka Tetsu, Kazunari Azuma, Takao Arai, Junji Chida and Hiroshi Kido : Endogenous genetic risk factor for serious heatstroke: the thermolabile phenotype of carnitine palmitoyltransferase II variant., Acute Medicine & Surgery, Vol.6, No.1, 25-29, 2018.
(Summary)
In serious heatstroke, elevated body temperature (>40°C) is considered the main cause of illness. Mitochondrial carnitine palmitoyltransferase II (CPT II) plays an important role in adenosine triphosphate (ATP) generation from long-chain fatty acids, and its thermolabile phenotype of polymorphisms leads to ATP production loss under high fever. Whether by heatstroke or influenza, high fever suppresses mitochondrial ATP production in patients with the thermolabile phenotype of polymorphisms. We investigated the relation between polymorphism and severity of heatstroke with a body temperature of over 40°C. We analyzed blood chemistry test results, Japanese Association for Acute Medicine Disseminated Intravascular Coagulation (JAAM DIC), Acute Physiologic and Chronic Health Evaluation II, and Sequential Organ Failure Assessment (SOFA) scores, and polymorphisms in 24 consecutive patients with severe heatstroke at two university hospitals. Eleven patients carried thermolabile CPT II variants (rs2229291; c.1055T G [p.Phe352Cys]) (F352C), and the genotype frequency was greater in heatstroke patients than in healthy volunteers. There was no significant difference in body temperature or blood chemistry data at emergency room arrival between patients with and without the CPT II variants. However, hospital days were longer and initial antithrombin activity was significantly lower in the variant group, suggesting a possible link with early phase vascular endothelial cell dysfunction. The JAAM DIC diagnostic criteria and SOFA scores were also higher in the group. There were no differences in the serum albumin, serum creatine kinase, and fibrin degradation product levels, and platelet counts. In addition to known risks (e.g., environmental temperature and old age), the CPT II polymorphism [F352C] can be a predisposing genetic risk factor for serious heatstroke with organ disfunction, and lower antithrombin activity.
Suehiro Sakaguchi and Junji Chida : Roles of Prion Protein in Virus Infections., DNA and Cell Biology, Vol.37, No.10, 808-811, 2018.
(Summary)
The normal cellular prion protein, designated PrP, is a membrane glycoprotein expressed most abundantly in brains, particularly by neurons, and to a lesser extent in non-neuronal tissues including lungs. Conformational conversion of PrP into the amyloidogenic isoform is a key pathogenic event in prion diseases. We recently found that PrP has a protective role against infection with influenza A viruses (IAVs) in mice by reducing reactive oxygen species in the lungs after infection with IAVs. The antioxidative activity of PrP is probably attributable to its function to activate antioxidative enzyme Cu/Zn-superoxide dismutase, or SOD1, through regulating Cu content in lungs infected with IAVs. Oxidative stress could play a pivotal role in the pathogenesis of a wide range of viral infections. Here, we introduce our and others' studies on the role of PrP in viral infections, and raise the attractive possibility that PrP might be a novel target molecule for development of antioxidative therapeutics against not only IAV infection but also other viral infections.
Junji Chida, Hideyuki Hara, Masashi Yano, Keiji Uchiyama, Rani Nandita Das, Etsuhisa Takahashi, Hironori Miyata, Yukiko Tomioka, Toshihiro Ito, Hiroshi Kido and Suehiro Sakaguchi : Prion protein protects mice from lethal infection with influenza A viruses., PLoS Pathogens, Vol.14, No.5, e1007049, 2018.
(Summary)
The cellular prion protein, designated PrPC, is a membrane glycoprotein expressed abundantly in brains and to a lesser extent in other tissues. Conformational conversion of PrPC into the amyloidogenic isoform is a key pathogenic event in prion diseases. However, the physiological functions of PrPC remain largely unknown, particularly in non-neuronal tissues. Here, we show that PrPC is expressed in lung epithelial cells, including alveolar type 1 and 2 cells and bronchiolar Clara cells. Compared with wild-type (WT) mice, PrPC-null mice (Prnp0/0) were highly susceptible to influenza A viruses (IAVs), with higher mortality. Infected Prnp0/0 lungs were severely injured, with higher inflammation and higher apoptosis of epithelial cells, and contained higher reactive oxygen species (ROS) than control WT lungs. Treatment with a ROS scavenger or an inhibitor of xanthine oxidase (XO), a major ROS-generating enzyme in IAV-infected lungs, rescued Prnp0/0 mice from the lethal infection with IAV. Moreover, Prnp0/0 mice transgenic for PrP with a deletion of the Cu-binding octapeptide repeat (OR) region, Tg(PrPOR)/Prnp0/0 mice, were also highly susceptible to IAV infection. These results indicate that PrPC has a protective role against lethal infection with IAVs through the Cu-binding OR region by reducing ROS in infected lungs. Cu content and the activity of anti-oxidant enzyme Cu/Zn-dependent superoxide dismutase, SOD1, were lower in Prnp0/0 and Tg(PrPOR)/Prnp0/0 lungs than in WT lungs. It is thus conceivable that PrPC functions to maintain Cu content and regulate SOD1 through the OR region in lungs, thereby reducing ROS in IAV-infected lungs and eventually protecting them from lethal infection with IAVs. Our current results highlight the role of PrPC in protection against IAV infection, and suggest that PrPC might be a novel target molecule for anti-influenza therapeutics.
Hideyuki Hara, Miyata Hironori, Das Rani Nandita, Junji Chida, Yoshimochi Tatenobu, Keiji Uchiyama, Watanabe Hitomi, Kondoh Gen, Yokoyama Takashi and Suehiro Sakaguchi : Prion Protein Devoid of the Octapeptide Repeat Region Delays BSE Pathogenesis in Mice., Journal of Virology, Vol.92, No.1, pii:e01368-17, 2018.
(Summary)
Conformational conversion of the cellular isoform of prion protein, PrP, into the abnormally folded, amyloidogenic isoform, PrP, is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals. We previously reported that the octapeptide repeat (OR) region could be dispensable for converting PrP into PrP after infection with RML prions. We demonstrated that mice transgenically expressing mouse PrP with deletion of the OR region on the PrP knockout background, designated Tg(PrPΔOR)/ mice, did not show reduced susceptibility to RML scrapie prions, with abundant accumulation of PrPΔOR in their brains. We show here that Tg(PrPΔOR)/ mice were highly resistant to BSE prions, developing the disease with markedly elongated incubation times after infection with BSE prions. The conversion of PrPΔOR into PrPΔOR was markedly delayed in their brains. These results suggest that the OR region may have a crucial role in the conversion of PrP into PrP after infection with BSE prions. However, Tg(PrPΔOR)/ mice remained susceptible to RML and 22L scrapie prions, developing the disease without elongated incubation times after infection with RML and 22L prions. PrPΔOR accumulated only slightly less in the brains of RML- or 22L-infected Tg(PrPΔOR)/ mice than PrP in control wild-type mice. Taken together, these results indicate that the OR region of PrP could play a differential role in the pathogenesis of BSE prions and RML or 22L scrapie prions. Structure-function relationship studies of PrP conformational conversion into PrP are worthwhile to understand the mechanism of the conversion of PrP into PrP We show here that, by inoculating Tg(PrPΔOR)/ mice with the three different strains of RML, 22L, and BSE prions, the OR region could play a differential role in the conversion of PrP into PrP after infection with RML or 22L scrapie prions and BSE prions. PrPΔOR was efficiently converted into PrPΔOR after infection with RML and 22L prions. However, the conversion of PrPΔOR into PrPΔOR was markedly delayed after infection with BSE prions. Further investigation into the role of the OR region in the conversion of PrP into PrP after infection with BSE prions might be helpful for understanding the pathogenesis of BSE prions.
Keiji Uchiyama, Mitsuru Tomita, Masashi Yano, Junji Chida, Hideyuki Hara, Nandita Rani Das, Anders Nykjaer and Suehiro Sakaguchi : Prions amplify through degradation of the VPS10P sorting receptor sortilin., PLoS Pathogens, Vol.13, No.6, e1006470, 2017.
(Summary)
Prion diseases are a group of fatal neurodegenerative disorders caused by prions, which consist mainly of the abnormally folded isoform of prion protein, PrPSc. A pivotal pathogenic event in prion disease is progressive accumulation of prions, or PrPSc, in brains through constitutive conformational conversion of the cellular prion protein, PrPC, into PrPSc. However, the cellular mechanism by which PrPSc is progressively accumulated in prion-infected neurons remains unknown. Here, we show that PrPSc is progressively accumulated in prion-infected cells through degradation of the VPS10P sorting receptor sortilin. We first show that sortilin interacts with PrPC and PrPSc and sorts them to lysosomes for degradation. Consistently, sortilin-knockdown increased PrPSc accumulation in prion-infected cells. In contrast, overexpression of sortilin reduced PrPSc accumulation in prion-infected cells. These results indicate that sortilin negatively regulates PrPSc accumulation in prion-infected cells. The negative role of sortilin in PrPSc accumulation was further confirmed in sortilin-knockout mice infected with prions. The infected mice had accelerated prion disease with early accumulation of PrPSc in their brains. Interestingly, sortilin was reduced in prion-infected cells and mouse brains. Treatment of prion-infected cells with lysosomal inhibitors, but not proteasomal inhibitors, increased the levels of sortilin. Moreover, sortilin was reduced following PrPSc becoming detectable in cells after infection with prions. These results indicate that PrPSc accumulation stimulates sortilin degradation in lysosomes. Taken together, these results show that PrPSc accumulation of itself could impair the sortilin-mediated sorting of PrPC and PrPSc to lysosomes for degradation by stimulating lysosomal degradation of sortilin, eventually leading to progressive accumulation of PrPSc in prion-infected cells.
Das Rani Nandita, Miyata Hironori, Hideyuki Hara, Keiji Uchiyama, Junji Chida, Masashi Yano, Watanabe Hitomi, Kondoh Gen and Suehiro Sakaguchi : Effects of prion protein devoid of the N-terminal residues 25-50 on prion pathogenesis in mice., Archives of Virology, Vol.162, No.7, 1867-1876, 2017.
(Summary)
The N-terminal polybasic region of the normal prion protein, PrP(C), which encompasses residues 23-31, is important for prion pathogenesis by affecting conversion of PrP(C) into the pathogenic isoform, PrP(Sc). We previously reported transgenic mice expressing PrP with residues 25-50 deleted in the PrP-null background, designated as Tg(PrPpreOR)/Prnp (0/0) mice. Here, we produced two new lines of Tg(PrPpreOR)/Prnp (0/0) mice, each expressing the mutant protein, PrPpreOR, 1.1 and 1.6 times more than PrP(C) in wild-type mice, and subsequently intracerebrally inoculated RML and 22L prions into them. The lower expresser showed slightly reduced susceptibility to RML prions but not to 22L prions. The higher expresser exhibited enhanced susceptibility to both prions. No prion transmission barrier was created in Tg(PrPpreOR)/Prnp (0/0) mice against full-length PrP(Sc). PrP(Sc)preOR accumulated in the brains of infected Tg(PrPpreOR)/Prnp (0/0) mice less than PrP(Sc) in control wild-type mice, although lower in RML-infected Tg(PrPpreOR)/Prnp (0/0) mice than in 22L-infected mice. Prion infectivity in infected Tg(PrPpreOR)/Prnp (0/0) mice was also lower than that in wild-type mice. These results indicate that deletion of residues 25-50 only slightly affects prion susceptibility, the conversion of PrP(C) into PrP(Sc), and prion infectivity in a strain-specific way. PrPpreOR retains residues 23-24 and lacks residues 25-31 in the polybasic region. It is thus conceivable that residues 23-24 rather than 25-31 are important for the polybasic region to support prion pathogenesis. However, other investigators have reported that residues 27-31 not 23-24 are important to support prion pathogenesis. Taken together, the polybasic region might support prion pathogenesis through multiple sites including residues 23-24 and 27-31.
Keiji Uchiyama, Miyata Hironori, Masashi Yano, Yoshitaka Yamaguti, Imamura Morikazu, Muramatsu Naomi, Das Rani Nandita, Junji Chida, Hideyuki Hara and Suehiro Sakaguchi : Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-terminal Residues 23-88 Restores Susceptibility to 22L Prions, But Not to RML Prions in PrP-Knockout Mice., PLoS ONE, Vol.9, No.10, e109737, 2014.
(Summary)
Prion infection induces conformational conversion of the normal prion protein PrPC, into the pathogenic isoform PrPSc, in prion diseases. It has been shown that PrP-knockout (Prnp0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM223-88)/Prnp 0/0 mice, neither developed the disease nor accumulated MHM2Sc23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM223-88)/Prnp 0/+ mice developed the disease with abundant accumulation of MHM2Sc23-88 in their brains. These results indicate that MHM223-88 itself might either lose or greatly reduce the converting capacity to MHM2Sc23-88, and that the co-expressing wild-type PrPC can stimulate the conversion of MHM223-88 to MHM2Sc23-88 in trans. In the present study, we confirmed that Tg(MHM223-88)/Prnp 0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM223-88)/Prnp 0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2Sc23-88 in their brains. We also found accelerated conversion of MHM223-88 into MHM2Sc23-88 in the brains of RML- and 22L-inoculated Tg(MHM223-88)/Prnp 0/+ mice. However, wild-type PrPSc accumulated less in the brains of these inoculated Tg(MHM223-88)/Prnp 0/+ mice, compared with RML- and 22L-inoculated Prnp 0/+ mice. These results show that MHM223-88 itself can convert into MHM2Sc23-88 without the help of the trans-acting PrPC, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrPC stimulates the conversion of MHM223-88 into MHM2Sc23-88, but to the contrary, the co-expressing MHM223-88 disturbs the conversion of wild-type PrPC into PrPSc.
Yamane Kazuhiro, Indalao L. Irene, Junji Chida, Yamamoto Yoshikazu, Hanawa Masaaki and Hiroshi Kido : Diisopropylamine dichloroacetate, a novel pyruvate dehydrogenase kinase 4 inhibitor, as a potential therapeutic agent for metabolic disorders and multiorgan failure in severe influenza, PLoS ONE, Vol.9, No.5, e98032, 2014.
(Summary)
Severe influenza is characterized by cytokine storm and multiorgan failure with metabolic energy disorders and vascular hyperpermeability. In the regulation of energy homeostasis, the pyruvate dehydrogenase (PDH) complex plays an important role by catalyzing oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic acid cycle and fatty acid synthesis, and thus its activity is linked to energy homeostasis. The present study tested the effects of diisopropylamine dichloroacetate (DADA), a new PDH kinase 4 (PDK4) inhibitor, in mice with severe influenza. Infection of mice with influenza A PR/8/34(H1N1) virus resulted in marked down-regulation of PDH activity and ATP level, with selective up-regulation of PDK4 in the skeletal muscles, heart, liver and lungs. Oral administration of DADA at 12-h intervals for 14 days starting immediately after infection significantly restored PDH activity and ATP level in various organs, and ameliorated disorders of glucose and lipid metabolism in the blood, together with marked improvement of survival and suppression of cytokine storm, trypsin up-regulation and viral replication. These results indicate that through PDK4 inhibition, DADA effectively suppresses the host metabolic disorder-cytokine cycle, which is closely linked to the influenza virus-cytokine-trypsin cycle, resulting in prevention of multiorgan failure in severe influenza.
Junji Chida, Rie Ono, Kazuhiko Yamane, Mineyoshi Hiyoshi, Masaji Nishimura, Mutsuo Onodera, Emiko Nakataki, Shichijo Koichi, Matsushita Masatomo and Hiroshi Kido : Blood lactate/ATP ratio, as an alarm index and real-time biomarker in critical illness, PLoS ONE, Vol.8, No.4, e60561, 2013.
(Summary)
The acute physiology, age and chronic health evaluation (APACHE) II score and other related scores have been used for evaluation of illness severity in the intensive care unit (ICU), but there is still a need for real-time and sensitive prognostic biomarkers. Recently, alarmins from damaged tissues have been reported as alarm-signaling molecules. Although ATP is a member of the alarmins and its depletion in tissues closely correlates with multiple-organ failure, blood ATP level has not been evaluated in critical illness. To identify real-time prognostic biomarker of critical illness, we measured blood ATP levels and the lactate/ATP ratio (ATP-lactate energy risk score, A-LES) in critically ill patients. Blood samples were collected from 42 consecutive critically ill ICU patients and 155 healthy subjects. The prognostic values of blood ATP levels and A-LES were compared with APACHE II score. The mean ATP level (SD) in healthy subjects was 0.62 (0.19) mM with no significant age or gender differences. The median ATP level in severely ill patients at ICU admission was significantly low at 0.31 mM (interquartile range 0.25 to 0.44) than the level in moderately ill patient at 0.56 mM (0.38 to 0.70) (P<0.01). Assessment with ATP was further corrected by lactate and expressed as A-LES. The median A-LES was 2.7 (2.1 to 3.3) in patients with satisfactory outcome at discharge but was significantly higher in non-survivors at 38.9 (21.0 to 67.9) (P<0.01). Receiver operating characteristic analysis indicated that measurement of blood ATP and A-LES at ICU admission are as useful as APACHE II score for prediction of mortality. Blood ATP levels and A-LES are sensitive prognostic biomarkers of mortality at ICU admission. In addition, A-LES provided further real-time evaluation score of illness severity during ICU stay particularly for critically ill patients with APACHE II scores of ≥20.0.
(Keyword)
adenosine triphosphate / Adolescent / Adult / Age Factors / Aged / Aged, 80 and over / Arteries / Biomarkers / Blood Chemical Analysis / children / Child, Preschool / Critical Illness / energy metabolism / Female / Hemoglobins / Humans / infant / Infant, Newborn / Intensive Care Units / Lactic Acid / Male / Middle Aged / Prognosis / Reference Values / risk / Time Factors / Veins / Young Adult
Etsuhisa Takahashi, Kosuke Kataoka, Irene L. Indalao, Keiko Konoha, Kazuyuki Fujii, Junji Chida, Dai Mizuno, Kohtaro Fujihashi and Hiroshi Kido : Oral clarithromycin enhances airway IgA immunity through induction of IgA class switching recombination and B-cell activating factor of the tumor necrosis factor family molecule on mucosal dendritic cells in mice infected with influenza A virus, Journal of Virology, Vol.86, No.20, 10924-1093, 2012.
(Summary)
We previously reported that the macrolide antibiotic clarithromycin (CAM) enhanced the mucosal immune response in pediatric influenza, particularly in children treated with the antiviral neuraminidase inhibitor oseltamivir (OSV) with low production of mucosal antiviral secretory IgA (S-IgA). The aims of the present study were to confirm the effects of CAM on S-IgA immune responses, by using influenza A virus (IAV) H1N1-infected mice treated with or without OSV, and to determine the molecular mechanisms responsible for the induction of mucosal IgA class switching recombination in IAV-infected CAM-treated mice. The anti-IAV S-IgA responses and expression levels of IgA class switching recombination-associated molecules were examined in bronchus-lymphoid tissues and spleens of infected mice. We also assessed neutralization activities of S-IgA against IAV. Data show that CAM enhanced anti-IAV S-IgA induction in the airway of infected mice and restored the attenuated antiviral S-IgA levels in OSV-treated mice to the levels in the vehicle-treated mice. The expression levels of B-cell-activating factor of the tumor necrosis factor family (BAFF) molecule on mucosal dendritic cells as well as those of activation-induced cytidine deaminase and Iμ-Cα transcripts on B cells were enhanced by CAM, compared with the levels without CAM treatment, but CAM had no effect on the expression of the BAFF receptor on B cells. Enhancement by CAM of neutralization activities of airway S-IgA against IAV in vitro and reinfected mice was observed. This study identifies that CAM enhances S-IgA production and neutralizing activities through the induction of IgA class switching recombination and upregulation of BAFF molecules in mucosal dendritic cells in IAV-infected mice.
Masaya Kubota, Junji Chida, Hideki Hoshino, Hiroshi Ozawa, Ayaka Koide, Hirohumi Kashii, Akiko Koyama, Yoko Mizuno, Ai Hoshino, Yamaguchi Miyoko, Dengbing Yao, Min Yao and Hiroshi Kido : Thermolabile CPT II variants and low blood ATP levels are closely related to severity of acute encephalopathy in Japanese children, Brain & Development, Vol.34, No.1, 20-27, 2012.
(Summary)
Despite the decrease in Reye syndrome after the discontinuation of aspirin, acute encephalopathy (non-Reye syndrome type) has been continually reported in Japan. Recent studies suggested that the thermolabile phenotype of carnitine palmitoyltransferase II (CPT II) variation [F352C] was closely related to the pathomechanism of influenza-associated encephalopathy (IAE) in Japanese, causing mitochondrial ATP utilization failure during periods of high fever, resulting in brain edema. So, we analyzed CPT II polymorphism and peripheral blood ATP levels as a signal of "energy crisis" in 12 and 10 patients with acute encephalopathy, respectively. Out of the 12 patients with acute encephalopathy, six showed thermolabile CPT II variants [F352C], and of these six, two patients died in spite of intensive care. In contrast, the remaining six patients with no thermolabile CPT II variant [F352C] showed a relatively mild clinical course. Blood ATP levels of the 10 patients in the acute phase of encephalopathy were significantly lower than those during the convalescent phase and also those of patients with febrile seizure status. Our data suggest that the thermolabile F352C CPT II variant, found only in Japanese, might be one of the predisposing factors to trigger the pathomechanism of acute encephalopathy in the Japanese population, and that it is causally related to the severity of disease. The decreased blood ATP level seems to reflect systemic mitochondrial dysfunction including the blood brain barrier during the acute phase of encephalopathy.
Hiroshi Kido, Yuushi Okumura, Etsuhisa Takahashi, Haiyan Pan, Siye Wang, Dengbing Yao, Min Yao, Junji Chida and Mihiro Yano : Role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol.1824, No.1, 186-194, 2012.
(Summary)
Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since the IVA genome does not have the processing protease for the viral hemagglutinin (HA) envelope glycoprotein precursors, entry of this virus into cells and infectious organ tropism of IAV are primarily determined by host cellular trypsin-type HA processing proteases. Several secretion-type HA processing proteases for seasonal IAV in the airway, and ubiquitously expressed furin and pro-protein convertases for highly pathogenic avian influenza (HPAI) virus, have been reported. Recently, other HA-processing proteases for seasonal IAV and HPAI have been identified in the membrane fraction. These proteases proteolytically activate viral multiplication at the time of viral entry and budding. In addition to the role of host cellular proteases in IAV pathogenicity, IAV infection results in marked upregulation of cellular trypsins and matrix metalloproteinase-9 in various organs and cells, particularly endothelial cells, through induced pro-inflammatory cytokines. These host cellular factors interact with each other as the influenza virus-cytokine-protease cycle, which is the major mechanism that induces vascular hyperpermeability and multiorgan failure in severe influenza. This mini-review discusses the roles of cellular proteases in the pathogenesis of IAV and highlights the molecular mechanisms of upregulation of trypsins as effective targets for the control of IAV infection. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.
(Keyword)
Animals / Antigen Presentation / Birds / Capillary Permeability / Host-Pathogen Interactions / Humans / Immune System / Influenza A virus / Influenza in Birds / Influenza, Human / Models, Biological / Multiple Organ Failure / Peptide Hydrolases
Min Yao, Dengbing Yao, Miyoko Yamaguchi, Junji Chida, Dengfu Yao and Hiroshi Kido : Bezafibrate upregulates carnitine palmitoyltransferase II expression and promotes mitochondrial energy crisis dissipation in fibroblasts of patients with influenza-associated encephalopathy, Molecular Genetics and Metabolism, Vol.104, No.3, 265-272, 2011.
(Summary)
Influenza-associated encephalopathy (IAE) is characterized by persistently high fever, febrile convulsions, severe brain edema and high mortality. We reported previously that a large proportion of patients with disabling or fatal IAE exhibit a thermolabile phenotype of compound variants for [1055T>G/F352C] and [1102G>A/V368I] of carnitine palmitoyltransferase II (CPT II) and mitochondrial energy crisis during high fever. In the present study, we studied the effect of bezafibrate, a hypolipidemic pan-agonist of peroxisome proliferator-activated receptor (PPAR), on CPT II expression and mitochondrial energy metabolism in fibroblasts of IAE patients and wild type (WT) fibroblasts from a healthy volunteer at 37°C and 41°C. Although heat stress markedly upregulated CPT II, CPT IA and PPAR-δ mRNA expression levels, CPT II activity, β-oxidation and ATP levels in WT and IAE fibroblasts at 41°C were paradoxically downregulated probably due to the thermal instability of the corresponding enzymes. Bezafibrate significantly enhanced the expression levels of the above mRNAs and cellular functions of these enzymes in fibroblasts at 37°C. Bezafibrate-induced increase in CPT II activity also tended to restore the downregulated ATP levels, though moderately, and improved mitochondrial membrane potential even at 41°C to the levels at 37°C in fibroblasts of IAE patients. L-carnitine, a substrate of CPT II, boosted the effects of bezafibrate on cellular ATP levels in WT and IAE fibroblasts, even in severe IAE fibroblasts with thermolabile compound variations of F352C+V368I at 37°C and 41°C. The results suggest the potential usefulness of bezafibrate for the treatment of IAE.
(Keyword)
Adenosine Triphosphate / Base Sequence / Bezafibrate / Blotting, Western / Brain Diseases, Metabolic / Carnitine / Carnitine O-Palmitoyltransferase / DNA Primers / Energy Metabolism / Fibroblasts / Gene Expression Regulation / Genomics / Hot Temperature / Humans / Influenza, Human / Japan / Membrane Potential, Mitochondrial / Microscopy, Fluorescence / Mitochondria / Molecular Sequence Data / Peroxisome Proliferator-Activated Receptors / RNA, Messenger / Real-Time Polymerase Chain Reaction / Sequence Analysis, DNA / Time Factors
Dengbing Yao, Min Yao, Miyoko Yamaguchi, Junji Chida and Hiroshi Kido : Characterization of compound missense mutation and deletion of carnitine palmitoyltransferase II in a patient with adenovirus-associated encephalopathy, The Journal of Medical Investigation : JMI, Vol.58, No.3,4, 210-218, 2011.
(Summary)
In mammals, carnitine palmitoyltransferase (CPT) system is a pivotal component of energy metabolism through mitochondrial fatty acid oxidation. The majority of patients with fatal or handicapped influenza-associated encephalopathy exhibit thermolabile compound homo/heterozygous mutations of CPT II. Compound CPT II mutations, [c.647A>G (p.Q216R)], [c.1102G>A (p.V368I)], [c.1939A>G (p.M647V)] and [c.745delG (p.G249EfsX16)], were found in a patient with adenovirus-associated encephalopathy and his family. The properties of these CPT II mutations were analyzed in COS-7 cells. CPT II mutations in the patient and his family were expressed in COS-7 cells and their molecular masses, enzyme activities, thermal instabilities and half-lives were analyzed. We identified two novel CPT II mutations in the patient, [c.647A>G (p.Q216R)] and [c.745delG (p.G249EfsX16)]. The CPT II Q216R mutation showed mild reduction of activity, thermal instability and short half-life but compound mutations with Q216R+V368I+M647V showed further enhancement of these disabilities, although mutations V368I and M647V had no such effects. CPT II mutation [c.745delG (p.G249EfsX16)] abolished enzyme activity and showed short half-life. The thermal instability and short half-life of the novel CPT II mutations, [c.647A>G (p.Q216R)] and [c.745delG (p.G249EfsX16)], could play important roles in energy crisis in the pathogenesis of virus-associated encephalopathy.
Junji Chida, Siye Wang, Pan Hai-Yan, Le Quang Trong and Hiroshi Kido : Influenza virus-cytokine-protease cycles are principal mechanisms of multi-organ failure in severe influenza and therapeutic approaches, Influenza and Other Respiratory Viruses, Vol.5, No.1, 281-286, 2011.
25.
Hai-Yan Pan, Hirotsugu Yamada, Junji Chida, Siye Wang, Mihiro Yano, Min Yao, Jianhua Zhu and Hiroshi Kido : Up-regulation of ectopic trypsins in the myocardium by influenza A virus infection triggers acute myocarditis, Cardiovascular Research, Vol.89, No.3, 595-603, 2010.
(Summary)
Influenza A virus (IAV) infection markedly up-regulates ectopic trypsins in various organs, viral envelope glycoprotein processing proteases, which are pre-requisites for virus entry and multiplication. We investigated the pathological roles of trypsin up-regulation in the progression of IAV-induced myocarditis, cytokine induction, and viral replication in the hearts, and also investigated the protective effects of trypsin inhibitor on cardiac dysfunction in vivo and selective knockdown of trypsin on IAV-induced cellular damage in cardiomyoblasts. The relationship of the expression among IAV RNA, trypsins, matrix metalloproteinase (MMP)-9, MMP-2, pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and tumour necrosis factor-α was analysed in mice hearts and cardiomyoblasts after IAV infection. The severity of myocarditis was most noticeable during Day 6-9 post-infection, along with peak expression of viral RNA, trypsins, particularly trypsin , MMPs, and cytokines. Cardiac ATP levels were the lowest at Day 9. Up-regulated trypsins, viral protein, and tissue-injured loci in the myocardium were closely localized. Trypsin inhibitor aprotinin treatment in vivo and selective trypsin - and trypsin -knockdown, particularly the latter, in H9c2 cardiomyoblasts significantly suppressed viral replication, up-regulation of MMPs, and production of active MMP-9 and cytokines, resulting in marked protection against cellular damage, ATP depletion, and apoptosis. IAV infection-induced cardiac dysfunction monitored by echocardiography was improved significantly by aprotinin treatment. IAV-induced trypsins, particularly trypsin , in the myocardium trigger acute viral myocarditis through stimulation of IAV replication, proMMP-9 activation, and cytokine induction. These results suggest that up-regulation of trypsins is one of the key host pathological findings in IAV-induced myocarditis.
Siye Wang, Le Quang Trong, Kurihara Naoki, Junji Chida, Youssouf Cisse, Mihiro Yano and Hiroshi Kido : Influenza Virus Cytokine Protease Cycle in the Pathogenesis of Vascular Hyperpermeability in Severe Influenza, The Journal of Infectious Diseases, Vol.202, No.7, 991-1001, 2010.
(Summary)
Severe influenza is characterized by cytokine storm and multiorgan failure with edema. The aim of this study was to define the impact of the cytokine storm on the pathogenesis of vascular hyperpermeability in severe influenza. Weanling mice were infected with influenza A WSN/33(H1N1) virus. The levels of proinflammatory cytokines, tumor necrosis factor (TNF) alpha, interleukin (IL) 6, IL-1beta, and trypsin were analyzed in the lung, brain, heart, and cultured human umbilical vein endothelial cells. The effects of transcriptional inhibitors on cytokine and trypsin expressions and viral replication were determined. Influenza A virus infection resulted in significant increases in TNF-alpha, IL-6, IL-1beta, viral hemagglutinin-processing protease trypsin levels, and viral replication with vascular hyperpermeability in lung and brain in the first 6 days of infection. Trypsin upregulation was suppressed by transcriptional inhibition of cytokines in vivo and by anti-cytokine antibodies in endothelial cells. Calcium mobilization and loss of tight junction constituent, zonula occludens-1, associated with cytokine- and trypsin-induced endothelial hyperpermeability were inhibited by a protease-activated receptor-2 antagonist and a trypsin inhibitor. The influenza virus-cytokine-protease cycle is one of the key mechanisms of vascular hyperpermeability in severe influenza.
Etsuhisa Takahashi, Kosuke Kataoka, Kazuyuki Fujii, Junji Chida, Dai Mizuno, Makoto Fukui, Hiro-O Ito, Kohtaro Fujihashi and Hiroshi Kido : Attenuation of inducible respiratory immune responses by oseltamivir treatment in mice infected with influenza A virus., Microbes and Infection, Vol.12, No.10, 778-783, 2010.
(Summary)
The antiviral neuraminidase inhibitor oseltamivir (OSV) is widely used to suppress viral replication in the treatment of influenza. Here, we report that OSV administration significantly suppressed respiratory mucosal secretory IgA responses with respect to antigen (Ag)-specific antibody (Ab) production and also the induction of Ag-specific IgA Ab-forming cells, but not systemic IgG responses, in weanling mice as a model of pediatric influenza. Neutralizing activities of the airway fluids in oral OSV-treated mice were significantly less than those of sham-treated mice. Our findings suggest the risk of re-infection in patients showing a low mucosal response following OSV treatment.
(Keyword)
Animals / Antibodies, Viral / Antiviral Agents / Female / Immunity, Mucosal / Immunoglobulin A / Immunoglobulin G / Immunosuppression / Immunosuppressive Agents / Influenza A virus / Mice / Mice, Inbred BALB C / Orthomyxoviridae Infections / Oseltamivir
Siye Wang, Le Quang Trong, Junji Chida, Youssouf Cisse, Mihiro Yano and Hiroshi Kido : Meckanisms of matrix metallo protease-9 wuregulation and tissue desfruction in rarious organs in influenza A virus infection, The Journal of Medical Investigation : JMI, Vol.57, No.1,2, 26-34, 2010.
(Summary)
Severe influenza is characterized clinicopathologically by multiple organ failure, although the relationship amongst virus and host factors that influence this morbid outcome and the underlying mechanisms of action remain unclear. The present study identified marked upregulation of matrix metalloproteinase (MMP)-9 and pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) in various organs after intranasal infection of influenza A WSN virus. MMP-9 and TNF-alpha were upregulated in the lung, the site of initial infection, as well as in the brain and heart. The infection-induced MMP-9 upregulation was inhibited by anti-TNF-alpha antibodies and by anti-oxidative reagents pyrrolidine dithiocarbamate and N-acetyl-L-cysteine, which inhibit activation of nuclear factor kappa B (NF-kappaB), as well as by nordihydroguaiaretic acid, which inhibits activation of activator protein 1 (AP-1). In addition, MMP-9 upregulation via TNF-alpha was also suppressed by inhibitors of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2 and p38, and partly by a c-Jun N-terminal kinase inhibitor. These results indicated that the influenza-induced MMP-9 upregulation in various organs is mediated through MAPK-NF-kappaB- and/or AP-1-dependent mechanisms. Strategies that neutralize TNF-alpha as well as inhibitors of MAPK-NF-kappa B- and/or AP-1-dependent pathways may be useful for suppressing the MMP-9 effect and thus preventing multiple organ failure in severe influenza.
Sawabuchi Takako, Suzuki Satoshi, Isase Kazuhiro, Ito Chika, Dai Mizuno, Todari Hajime, Watanabe Isamu, Talukder R. Sadiqur, Junji Chida and Hiroshi Kido : Boost of mucosal secretory immunoglobulin A response by clarithromycin in paediatric influenza, Respirology, Vol.14, No.8, 1173-1179, 2009.
(Summary)
The antiviral neuraminidase inhibitor oseltamivir (OSV) is used to treat influenza. The macrolide clarithromycin (CAM) is used to treat bacterial infections and has anti-inflammatory and immunomodulatory activities. This retrospective study investigated the immunomodulatory effects of CAM in children presenting with influenza A. The study recruited 40 children with acute influenza, and grouped them according to the treatment received: 5-day treatment with OSV (n = 14), CAM (n = 8), OSV + CAM (n = 12) and untreated (n = 6). The before and after treatment comparisons were made of the level of secretory IgA (sIgA) against influenza A virus (H3N2) and (H1N1), total sIgA, viral RNA copy numbers in nasopharyngeal aspirates and disease symptoms. Infection induced anti-viral mucosal sIgA in the nasopharyngeal aspirates of most patients of all treatment groups. Particularly prominent increases in the levels were found in the CAM and OSV + CAM groups. Low induction of anti-viral sIgA was observed in the OSV group, but the addition of CAM to OSV augmented sIgA production and restored local mucosal sIgA levels. The frequency of residual cough in the OSV + CAM group was significantly lower than in the other groups including the group treated with OSV. CAM boosted the nasopharyngeal mucosal immune response in children presenting with influenza A, even in those treated with OSV who had low production of mucosal anti-viral sIgA, and alleviated the symptoms of influenza.
Hiroshi Kido, Yuushi Okumura, Etsuhisa Takahashi, HY Pan, Siye Wang, Junji Chida, Le Trong Quang and Mihiro Yano : Host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses., Journal of Molecular and Genetic Medicine, Vol.3, No.1, 167-175, 2009.
(Summary)
Influenza A virus (IAV) is one of the most common infectious pathogens in humans and causes considerable morbidity and mortality. The recent spread of highly-pathogenic avian IAV H5N1 viruses has reinforced the importance of pandemic preparedness. In the pathogenesis of IAV infection, cellular proteases play critical roles in the process of viral entry into cells that subsequently leads to tissue damage in the infected organs. Since there are no processing protease for the viral membrane fusion glycoprotein hemagglutinin precursor (HA(0)) in IAV, entry of the virus into cells is determined primarily by the host cellular HA(0) processing proteases that proteolytically activate membrane fusion activity. HA(0) of seasonal human IAV has the consensus cleavage site motif Q(E)-T/X-R and is selectively processed by at least seven different trypsin-type processing proteases identified to-date in animal model experiments using mouse-adapted IAV or gene expression system in MDCK cells. As is the case for the highly pathogenic avian influenza (HPAI) A virus, endoproteolytic processing of the HA(0) occurs through ubiquitous cellular processing proteases, which selectively recognize the multi-basic consensus cleavage site motifs, such as R-X-K/R-R, and K-X-K/R-R. The cleavage enzymes for the R-X-K/R-R motif, but not K-X-K/R-R motif, have been reported to be furin and pro-protein convertase (PC)5/6 in the trans-Golgi network. Here we report new members of type II transmembrane serine proteases of the cell membrane, mosaic serine protease large form (MSPL) and its splice variant TMPRSS13, which recognize and cleave both R-X-K/R-R and K-X-K/R-R motifs without calcium. Furthermore, IAV infection significantly up-regulates a latent ectopic pancreatic trypsin, one of the potent HA processing proteases, and pro-matrix metalloprotease-9, in various organs. These proteases may synergistically damage the blood-brain barrier in the brain and basement membrane of blood vessels in various organs, resulting in severe edema and multiple organ failure. In this review, we discuss these proteases as new drug target molecules for IAV treatment acting by inhibition of IAV multiplication and prevention of multiple organ failure, other than anti-viral agents, viral neuraminidase inhibitors.
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 19565019
Dengbing Yao, Hiroshi Mizuguchi, Miyoko Yamaguchi, Hiroshi Yamada, Junji Chida, Koji Shikata and Hiroshi Kido : Thermal instability of compound variants of carnitine palmitoyltransferase II and impaired mitochondrial fuel utilization in influenza-associated encephalopathy., Human Mutation, Vol.29, No.5, 718-727, 2008.
(Summary)
Influenza-associated encephalopathy (IAE) is characterized by persistent high fever, febrile convulsions, severe brain edema, and high mortality in otherwise apparently healthy individuals. We have reported that a large proportion of patients suffering from disabling or fatal IAE, with transiently elevated serum acylcarnitine during high fever, exhibit a thermolabile phenotype of compound homo-/heterozygous variants of carnitine palmitoyltransferase II (CPT II, gene symbol CPT2). We characterized the enzymatic properties of five single and three compound CPT II variants in patients with IAE. The kinetic characteristics of WT and variant CPT IIs, expressed in COS-7 cells, indicated that the variants exert a dominant-negative effect on the homotetrameric protein of the enzyme. Among the variants, three compound variations found in patients with severe encephalopathy; [c.1055T>G (p.Phe352Cys); c.1102G>A (p.Val368Ile)], [c.1511C>T (p.Pro504Leu); c.1813G>C (p.Val605Leu)], and [c.1055T>G (p.Phe352Cys); c.1102G>A (p.Val368Ile); c.1813G>C (p.Val605Leu)], showed reduced activities, thermal instability, and short half-lives compared with the WT. Like other disease-causing mutant proteins, these variant proteins were poly-ubiquitinated and rapidly degraded by a lactacystin-sensitive proteasome pathway. COS-7 cells transfected with the compound variants had their fatty acid beta-oxidation decreased to 30-59% and intracellular ATP levels to 48-79%, and a marked reduction of mitochondrial membrane potential at 41 degrees C, compared with control cells transfected with WT at 37 degrees C. The unstable CPT II variants with decreased enzymatic activities may bring mitochondrial fuel utilization below the phenotypic threshold during high fever, and thus may play an important etiopathological role in the development of brain edema of IAE.
Junji Chida, Aiko Amagai, Masashi Tanaka and Yasuo Maeda : Establishment of a new method for precisely determining the functions of individual mitochondrial genes, using Dictyostelium cells., BMC Genetics, Vol.9, 25, 2008.
(Summary)
Disruption of mitochondrial genes may become a powerful tool for elucidating precisely the functions of individual mitochondrial genes. However, it is generally difficult to manipulate genetically mitochondrial genes, because 1) a mitochondrion is surrounded by inner and outer membranes, and 2) there are a large number of mtDNA copies in a single cell. This is the reason why we tried to establish a novel method for disrupting a certain mitochondrial gene (rps4), using Dictyostelium cells. Here, we have developed a new method for specifically disrupting a mitochondrial gene (rps4 ; ribosomal protein subunit S4), by a combination of homologous recombination and delivery of an appropriate restriction endonuclease (SfoI) into mitochondria. First, mitochondrially targeted SfoI whose expression is under control of the tetracycline (Tet)-regulated gene expression system was introduced into cells heteroplasmic with respect to the rps4 gene. Then, the heteroplasmic cells were produced by homologous recombination by use of the construct in which the unique SfoI site and the 5'-half of the rps4 coding region were deleted not to be digested by SfoI, and therefore their mitochondria have both the wild-type mtDNA and the mutant mtDNA with the disrupted rps4 gene. In response to removal of Tet from growth medium, SfoI was selectively delivered into mitochondria and digested only the wild-type mtDNA but not the mutated rps4. Thus one can gain rps4-null cells with only the mutated mtDNA, under the Tet-minus condition. The mitochondrial gene-disruption method presented here must be widely useful for precisely determining the functions of individual mitochondrial genes. This is the first report to demonstrate complete and specific mitochondrial gene disruption.
(Keyword)
mitochondria / mitochondrial DNA / Homologous Recombination
Junji Chida, Hitomi Yamaguchi, Aiko Amagai and Yasuo Maeda : The necessity of mitochondrial genome DNA for normal developmental of Dictyostelium cells., Journal of Cell Science, Vol.117, No.Pt 15, 3141-3152, 2004.
(Summary)
Most unexpectedly, there is now increasing evidence that mitochondria have novel and crucial functions in the regulatory machinery of the growth/differentiation transition, cell-type determination, cellular movement and pattern formation. Here we created rho delta cells with a reduced amount (about 1/4) of mitochondrial DNA (mtDNA) from Dictyostelium discoideum Ax-2 cells, by exposing Ax-2 cells to ca. 30 microg/ml of ethidium bromide (EtBr) in axenic growth medium. Importantly, the rho delta cells exhibited a series of fascinating behaviors: when they were starved, they showed a marked delay of differentiation and stopped their development at the slug stage, thus failing to construct fruiting bodies. Moreover, cell patterning and cell-type proportioning were found to be greatly modified in slugs (referred to as rho delta slugs) derived from rho delta cells. That is, prestalk differentiation was significantly enhanced in rho delta slugs, while prespore differentiation was markedly inhibited. In addition, the clear anterior prestalk/posterior prespore pattern was considerably disturbed in rho delta slugs, presumably because of incomplete sorting between the two types of differentiated cells. After the assay of phototaxis, rho delta slugs also exhibited highly disordered movement towards the light source. Taken together, these results suggest that mtDNA might have important multiple functions in a variety of cellular processes during Dictyostelium development.
(Keyword)
mitochondrial DNA / adenosine triphosphate / Dictyostelium discoideum
Kou-ichi Hosoya, Aiko Amagai, Junji Chida and Yasuo Maeda : Unique behavior and function of the mitochondrial ribosomal protein S4 (RPS4) in early Dictyostelium development., Zoological Science, Vol.20, No.12, 1455-1465, 2003.
(Summary)
Certain proteins encoded by mitochondrial DNA (mt-DNA), including mt-ribosomal protein S4 (rps4), appear to play important roles in the initiation of cell differentiation. Partial disruption of rps4 in Dictyostelium discoideum Ax-2 cells by means of homologous recombination greatly impairs the progression of differentiation, while the the rps4(OE) cells in which the rps4 mRNA was overexpressed in the extra-mitochondrial cytoplasm exhibit enhanced differentiation (Inazu et al., 1999). We have prepared a specific anti-RPS4 antibody and generated transformants (rps4(AS) cells) by antisense-mediated gene inactivation of rps4. Surprisingly, in the rps4(AS) cells the progress of differentiation was found to be markedly inhibited, suggesting that the antisense rps4 RNA synthesized in the extra-mitochondrial cytoplasm might be as effective as the partial disruption of rps4 gene. Immunostaining of the rps4(OE) cells with the anti-RPS4 antibody demonstrated that the RPS4 protein synthesized in the extra-mitochondrial cytoplasm is capable of moving to the nucleus, as predicted by PSORTII. Taken together with the results obtained using immunostained Ax-2 cells, we propose a possible pathway of RPS4 translocation coupled with differentiation.
(Keyword)
mitochondrial DNA / Ribosomal Protein / Dictyostelium discoideum
Junji Chida and Hiroshi Kido : Extraction and quantification of adenosine triphosphate in mammalian tissues and cells., Methods in Molecular Biology, Vol.1098, 21-32, 2014.
(Summary)
Adenosine 5'-triphosphate (ATP) is the "energy currency" of organisms and plays central roles in bioenergetics, whereby its level is used to evaluate cell viability, proliferation, death, and energy transmission. In this chapter, we describe an improved and efficient method for extraction of ATP from tissues and cells using phenol-based reagents. The chaotropic extraction reagents reported so far co-precipitate ATP with insoluble proteins during extraction and with salts during neutralization. In comparison, the phenol-based reagents extract ATP well without the risks of co-precipitation. The extracted ATP can be quantified by the luciferase assay or high-performance liquid chromatography.
Hiroshi Kido and Junji Chida : Pathogenesis of influenza-associated encephalopathy, --- CPT2 SNP as a phathogenetic risk factor ---, Journal of Clinical and Experimental Medicine, Vol.241, No.1, 23-28, 2012.
Hiroshi Kido, Dengbing Yao, Junji Chida, Youssouf Cisse and Yao Min : Pathogenesis of influenza-associated encephalopathy: disorder of fatty acid metabolism in mitochondria and increased membrane permeability in endothelial cells, The Medical Frontline, Vol.65, No.1, 52-60, 2010.
Junji Chida, Aiko Amagai and Yasuo Maeda : Validity of Dictyostelium mitochondrial DNA-less cells for elucidating the regulatory mechanisms of development., Journal of Plant Research, Vol.116, 149-150, 2003.
(Keyword)
mitochondrial DNA / Pattern Formation / Dictyostelium discoideum
Review, Commentary:
1.
Junji Chida, Hiroshi Kido and Suehiro Sakaguchi : 宿主因子を標的にした新たなインフルエンザ治療の試み, BIO Clinica, Vol.256, No.33, 52-55, Feb. 2018.
2.
Hiroshi Kido and Junji Chida : インフルエンザ脳症への新たな対応策は?, インフルエンザの最新知見 Q & A 2012, 171-172, May 2012.
3.
水野 葉子, 久保田 雅也, 柏井 洋文, 宮田 理英, 田沼 直之, 林 雅晴, Junji Chida, 山口 美代子, Yao Dengbing, Yao Min and Hiroshi Kido : Carnitine palmitoyltransferase II (CPT II) の熱不安定性遺伝子多型を持った急性脳症の1例, Japanese Journal of Pediatrics, Vol.65, No.5, 1057-1062, 2012.
Hiroshi Kido, Junji Chida, Min Yao and Siye Wang : Mechanisms of multi-organ failure in severe influemza, Nihon Rinsho. Japanese Journal of Clinical Medicine, Vol.68, No.8, 1565-1573, Aug. 2010.
(Summary)
Severe influenza is characterized by cytokine storm and multi-organ failure with edema. We found that the "influenza virus-cytokine-trypsin/MMP-9 cycle" in the endothelial cells is one of the key mechanisms of vascular hyperpermeability, the major pathogen of multi-organ failure. Upregulated TNF-alpha, IL-6 and IL-beta induce ectopic pancreatic trypsin and pro-MMP-9 in the endothelial cells and in various organs. Trypsin mediates the viral hemagglutinin processing, which is crucial for viral entry and multicycles of replication. In addition, trypsin is the most potent pro-MMP-9 convertase to form active MMP-9 and both proteases synergistically destruct matrix around blood vessels. In addition upregulated trypsin triggers through its receptor, PAR-2, the modification of cellular functions, such as increase in calcium mobilization and mitochondrial membrane permeability, suppression of ATP generation and loss of tight junction constituent, zonula occludens-1. High risk patients who have impaired mitochondrial fuel utilization will easy get into energy crisis, resulting in vascular hyperpermeability in severe influenza.
Hiroshi Kido, Dai Mizuno, Tunetomo Takei, Maki Nisino, Junji Chida and Youssouf Cisse : インフルエンザ経鼻ワクチン, Pediatrics of Japan, Vol.48, No.12, 1837-1844, Nov. 2007.
12.
Hiroshi Kido, Dengbing Yao, Le Trong Quang, Mariko Tsukane and Junji Chida : Analysis of SNPs and enzymatic disorder in the patients of influenza-associated encephalopathy., --- Disorder of fatty acid metabolism in mitochondria induced by high fever. ---, Nihon Rinsho. Japanese Journal of Clinical Medicine, Vol.64, No.10, 101-109, Oct. 2007.
Etsuhisa Takahashi, HY Pan, IL Indalao, Junji Chida and Hiroshi Kido : TRYPSIN KOCKDOWN AND TRYPSIN INHIBITOR ADMINISTRATION SUPPRESSED INFLUENZA VIRAL REPLICATION AND VIRUS-INDUCES MYOCARDITIS IN THE HEARTS AND CARDIOMYOBLAST CELLS, 7th General Meeting of the International Proteolisis, San Diego, Oct. 2011.
2.
Hiroshi Kido, Junji Chida, Siye Wang, Hai-Yan Pan and Min Yao : Influenza-Cytokine-Protease Cycle in the pathogenesis of vascular hyperpermeability in severe influenza and its possible treatments, BMB2010, Dec. 2010.
3.
Junji Chida, Siye Wang, Haiyan Pan, Min Yao, Dengbing Yao, Kazuhiko Yamane and Hiroshi Kido : Influenza virus-cytokine-protease cycle and mitochondrial ATP depletion are the principal mechanisms of multi-organ failure in severe influenza and therapeutic approaches, Options for the Control of Influenza VII, China, Hong Kong, Sep. 2010.
4.
Junji Chida, Siye Wang, Hai-Yan Pan, Min Yao, Dengbing Yao, Kazuhiko Yamane and Hiroshi Kido : Influenza virus-cytokine-protease cycle and mitochondrial ATP depletion are the principal mechanisms of multi-organ failure in severe influenza and therapeutic approaches., Oprions for the Control of Influenza, Hong Kong, Sep. 2010.
5.
Junji Chida, Siye Wang, Tadashi Enomoto, Yuushi Ohnishi and Hiroshi Kido : Up-regulations of ectopic pancreatic trypsin mRNAs by influenza virus infection and pathological role of them in multi-organ failure after virus infection., 5th International Proteolysis Society, Patras, Oct. 2007.
(Keyword)
influenza-associated encephalopathy / multiple organ failure / mitochondria
6.
Junji Chida, Masashi Tanaka, Aiko Amagai and Yasuo Maeda : A new approach to disrupt certain mitochondrial genes in Dictyostelium cells., International Conference on Mitochondria and Life 2005, Tokyo, Dec. 2005.
Junji Chida, Masashi Tanaka, Aiko Amagai and Yasuo Maeda : Unexpected and novel functions of mitochondria, revealed by Dictyostelium cells., International Conference on Mitochondria and Life 2005, Tokyo, Dec. 2005.
Junji Chida, Masashi Tanaka, Aiko Amagai and Yasuo Maeda : Establishment of a system for elucidating precisely the function of individual mitochondrial genes., 18th Annual International Dictyostelium Conference, Lyon, Aug. 2005.
Junji Chida, Hitomi Yamaguchi, Aiko Amagai and Yasuo Maeda : Contributions of mitochondrial DNA to cell differentiation and pattern formation in Dictyostelium development., 16th Annual International Dictyostelium Conference, Melbourne, Jul. 2003.
(Keyword)
mitochondrial DNA / adenosine triphosphate / Dictyostelium discoideum
10.
Chad Shaw, Nancy Van Driessche, Miroslava Ibarra, Sujata Sharma, Engi Okyay, Takahiro Morio, Mariko Katoh, Hideko Urushihara, Yoshimasa Tanaka, Junji Chida, Aiko Amagai, Yasuo Maeda, Dana Mahadeo, David Cotter, Adam Kusupa and Gad Shaulsky : Beyond development: transcriptional profiling of the Dictyostelium cell cycle, spore germination and de-differentiation., 15th Annual International Dictyostelium Conference, Sicilla, Sep. 2002.
Hideyuki Hara, Junji Chida and Suehiro Sakaguchi : Influenza virus infection triggers de novo generation of prions in neuronal cells, 第41回日本分子生物学会年会, Nov. 2018.
6.
Junji Chida, Hideyuki Hara and Suehiro Sakaguchi : Prion protein provides a protection against influenza A virus infection, 第66回日本ウイルス学会学術集会, Oct. 2018.
7.
Junji Chida, Hideyuki Hara and Suehiro Sakaguchi : Prion protein protects mice from lethal infection with Influenza A virues, 2017年度生命科学系学会合同年次大会 (ConBio2017) 第 40回日本分子生物学会年会/第90回日本 生化学会大会, Dec. 2017.
8.
Hideyuki Hara, Junji Chida and Suehiro Sakaguchi : Prion-infected neuroblastoma cells are resistant to influenza virus., 2017年度生命科学系学会合同年次大会 (ConBio2017) 第 40回日本分子生物学会年会/第90回日本 生化学会大会, Dec. 2017.
Rie Ono, Masaji Nishimura, Jun Oto, Hideaki Imanaka, Junji Chida and Hiroshi Kido : 重症患者の末梢血ATP乳酸値比, 日本麻酔科学会第58回学術集会，神戸, May 2011.
13.
Min Yao, Junji Chida, Hiyan Pan, Siye Wang and Hiroshi Kido : Effect of Bezafibrate on mitochondrial energy crisis in the fibroblast of severe influenza-associated encephalopathy patients, 第33回日本分子生物学会年会 第83回 日本生化学会大会合同大会,, Dec. 2010.
14.
Junji Chida, Min Yao, Dengbing Yao, Miyoko Yamaguchi and Hiroshi Kido : Pathogenesis of impaired systemic energy metabolism by severe influenza virus infection Analysis using mouse models of defect in mitochondrial -oxidation of long-chain fatty acids, 第33回日本分子生物学会年会 第83回 日本生化学会大会合同大会,, Dec. 2010.
15.
Hiroshi Kido, Junji Chida, Siye Wang, Hiyan Pan and Min Yao : Influenza-Cytokine-Protease Cycle in the pathogenesis of vascular hyper-permeability in severe influenza and its possible treatment, 第33回日本分子生物学会年会 第83回 日本生化学会大会合同大会,, Dec. 2010.
16.
Yao Min, Etsuhisa Takahashi, Pan Haiyan, Junji Chida and Hiroshi Kido : A Type II Transmembrane Serine Protease Serase-1B, a New Splice Variant of Polyserase-1/TMPRSS9, Plays a Role in Adipogenesis., 第83回日本生化学会大会, Dec. 2010.
17.
Hai-Yan Pan, Junji Chida and Hiroshi Kido : Up-regulation of ectopic trypsin in myocardium triggers acute myocarditis in severe influenza, Seikagaku, Dec. 2010.
18.
Min Yao, Junji Chida, Hai-Yan Pan, Siye Wang and Hiroshi Kido : Effect of Bezafibrate on mitochondrial energy crisis in the fibroblast of severe influenza-associated encephalopathy patients, Seikagaku, Dec. 2010.
19.
Junji Chida, Min Yao, Dengbing Yao, Miyoko Yamaguchi and Hiroshi Kido : Pathogenesis of impaired systemic energy metabolism by severe influenza virus infection Analysis using mouse models of defect in mitochondrial -oxidation of long-chain fatty acids-, Seikagaku, Dec. 2010.
20.
Hiroshi Kido, Junji Chida, Siye Wang, Hai-Yan Pan and Min Yao : Influenza-Cytokine-Protease Cycle in the pathogenesis of vascular hyper-permeability in severe influenza and its possible treatment., Seikagaku, Dec. 2010.
21.
Junji Chida, Siye Wang, Dengbing Yao, Min Yao, Miyoko Yamaguchi and Hiroshi Kido : インフルエンザ脳症の発症メカニズムの酵素学的な解析, 温熱生理研究会, Sep. 2010.
22.
Junji Chida : インフルエンザ脳症のリスク診断を可能にする DNA チップの開発と患者の重症度をモニターする末梢血の ATP 測定システムの開発, 第9回 次世代医療システム産業化フォーラム, Dec. 2008.
(Keyword)
influenza-associated encephalopathy / DNA 診断チップ / adenosine triphosphate
23.
Junji Chida, Tadashi Enomoto, Hiroki Arase and Hiroshi Kido : Molecular pathogenesis and multiplicity of influenza virus A/WSN/33 in plasminogen deficient mice., 第81回 日本生化学会大会, Dec. 2008.
Yuuji Onishi, Junji Chida, Youssouf Cisse and Hiroshi Kido : Molecular mechanism of the decrease in ATP levels by influenza virus infection., 第81回 日本生化学会大会, Dec. 2008.
Min Yao, Dengbing Yao, Miyoko Yamaguchi, Junji Chida and Hiroshi Kido : Effect of bezafibrate on mitochondrial energy crisis in the fibroblasts of severe influenza-associated encephalopathy patients., 第81回 日本生化学会大会, Dec. 2008.
Tadashi Enomoto, Hiroshi Yamada, Junji Chida, Siye Wang and Hiroshi Kido : Mechanism of augmentation of symptomatic of influenza virus infection by antipyretic, diclophenac., 第30回 日本分子生物学会，第80回 日本生化学会大会, Dec. 2007.
Junji Chida, Tadashi Enomoto, Yuushi Ohnishi, Mariam Nasreen and Hiroshi Kido : Down-regulation of mitochondrial related-genes by influenza virus infection and pathological role of them in multi-organ failure after virus infection., 第30回 日本分子生物学会年会，第80回 日本生化学会大会, Dec. 2007.
(Keyword)
influenza-associated encephalopathy / multiple organ failure / mitochondria
mitochondria / mitochondrial DNA / Dictyostelium discoideum
4.
Junji Chida, Hitomi Yamaguchi, Aiko Amagai and Yasuo Maeda : Mitochondrial genome DNA (mtDNA) is required for normal development of Dictyostelium discoideum cells., 第6回 細胞性粘菌研究会, Mar. 2004.
(Keyword)
mitochondria / mitochondrial DNA / Dictyostelium discoideum
5.
Takahiro Morio, Chad Shaw, Junji Chida, Nancy Van Driessche, Mariko Katoh, Miroslava Ibarra, Sujata Sharma, Engi Okyay, Aiko Amagai, Hideko Urushihara, Adam Kusupa, Yasuo Maeda and Gad Shaulsky : 細胞性粘菌の細胞周期における遺伝子発現プロファイルの解析, 第5回 細胞性粘菌研究会, Mar. 2003.
Studies of prion pathogenesis and non-prion pathogens for preventative and therapeutic development against prion diseases (Project/Area Number: 23H02798 )
Development of causative therapies for emerging infectious diseases targeting host inflammatory macrophages (Project/Area Number: 22K08600 )
Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice (Project/Area Number: 19K08930 )
Elucidation of the pathogenic mechanism and screening of therapeutic compounds in prion disease (Project/Area Number: 19H03548 )
Analysis of mechanism of a novel cancersuressor derived from microorganism (Project/Area Number: 16K15130 )
Prion protein protects mice from lethal infection with influenza A virus (Project/Area Number: 16K10029 )
Elucidation of the vesicular trafficking mechanism for prion propagation on the basis of our previous findings of the disturbed vesicular trafficking in prion disease (Project/Area Number: 26293212 )
Elucidation of molecular pathogenesis of influenza virus-associated encephalopathy using Prnp0/0 mice. (Project/Area Number: 25461596 )
New target and therapeutic approach to influenza virus-induced multiple organ failure (Project/Area Number: 23791180 )
Studies on real-time biomarker of illness severity inthe patients of critical illness and development of the diagnosis machine (Project/Area Number: 23659846 )
The analysis of host response determining the severe influenza and HIV infection. (Project/Area Number: 23591477 )
Elucidation of the molecular mechanisms of influenza-associated encephalopathy due to disorder of ATP generation in mitochondria (Project/Area Number: 21790992 )
Development of new therapeutics for a highly pathogenic influenza virus infection by inhibition of virus entry and multiplication (Project/Area Number: 21249061 )
Elucidation of the molecular mechanism of influenza-associated encephalopathy due to disorder of ATP generation in mitochondria (Project/Area Number: 19790724 )