Identification of dihydroorotate dehydrogenase inhibitor, vidofludimus, as a potent and novel inhibitor for influenza virus.

Influenza A virus (IAV) infection causes respiratory disease. Recently, infection of IAV H5N1 among mammals are reported in farmed mink. Therefore, to discover antivirals against IAV, we screened a compound library by using the RNA-dependent RNA polymerase (RdRp) assay system derived from H5N1 IAV including a drug-resistant PA mutant (I38T) and a viral polymerase activity enhancing PB2 mutant (T271A). Upon screening, we found vidofludimus can be served as a potential inhibitor for IAV. Vidofludimus an orally active inhibitor for dihydroorotate dehydrogenase (DHODH), a key enzyme for the cellular de novo pyrimidine biosynthesis pathway. We found that vidofludimus exerted antiviral activity against wild-type and drug-resistant mutant IAV, with effective concentrations (EC50 ) of 2.10 and 2.11 µM, respectively. The anti-IAV activity of vidofludimus was canceled by the treatment of uridine or cytidine through pyrimidine salvage synthesis pathway, or orotic acid through pyrimidine de novo synthesis pathway. This indicated that the main target of vidofludimus is DHODH in IAV RdRp expressing cells. We also produced recombinant seasonal IAV H1N1 virion and influenza B virus (IBV) RdRp assay system and confirmed vidofludimus also carried highly antiviral activity against seasonal IAV and IBV. Vidofludimus is a candidate drug for the future threat of IAV H5N1 infection among humans as well as seasonal influenza virus infection.

Benralizumab in children with severe eosinophilic asthma: Pharmacokinetics and long-term safety (TATE study).

BACKGROUND: Benralizumab is an anti-interleukin-5 receptor α monoclonal antibody approved as an add-on maintenance treatment for patients with uncontrolled severe asthma. Prior Phase 3 studies have evaluated benralizumab in patients aged ≥12 years with severe uncontrolled asthma. The TATE study evaluated the pharmacokinetics (PK), pharmacodynamics (PD), and safety of benralizumab treatment in children. METHODS: TATE was an open-label, Phase 3 study of benralizumab in children aged 6-11 years from the United States and Japan (plus participants aged 12-14 years from Japan) with severe eosinophilic asthma. Participants received benralizumab 10/30 mg according to weight (<35/≥35 kg). Primary endpoints included maximum serum concentration (Cmax ), clearance, half-life (t1/2 ), and blood eosinophil count. Clearance and t1/2 were derived from a population PK (popPK) analysis. Safety and tolerability were also assessed. RESULTS: Twenty-eight children aged 6-11 years were included, with an additional two participants from Japan aged 12-14 years also included in the popPK analysis. Mean Cmax was 1901.2 and 3118.7 ng/mL in the 10 mg/<35 kg and 30 mg/≥35 kg groups, respectively. Clearance was 0.257, and mean t1/2 was 14.5 days. Near-complete depletion of blood eosinophils was shown across dose/weight groups. Exploratory efficacy analyses found numerical improvements in mean FEV1 , mean ACQ-IA, patient/clinician global impression of change, and exacerbation rates. Adverse events occurred in 22/28 (78.6%) of participants; none led to discontinuation/death. CONCLUSION: PK, PD, and safety data support long-term benralizumab in children with severe eosinophilic asthma, and were similar to findings in adolescents and adults. TRIAL REGISTRATION: ClinicalTrials.gov-ID: NCT04305405.

Development and validation of a new asthma questionnaire to help achieve a high level of control in school-age children and adolescents.

BACKGROUND: Maintaining good asthma control minimizes the risk of exacerbations and lung function decline and is a primary goal of asthma management. The Japanese Pediatric Asthma Guidelines (JPGL) employs different classification criteria for control status from other guidelines, stressing a higher level of control. Based on JPGL, we previously developed a caregiver-completed questionnaire for assessing and achieving best asthma control in preschoolers. In this study, we aimed to develop a questionnaire for school-age children and adolescents. METHODS: A working questionnaire comprising 14 items for patients and 34 items for caregivers was administered to 362 asthma patients aged 6-15 years and their caregivers. Separately, physicians filled out a questionnaire to determine JPGL-defined control. Logistic regression analysis was performed to construct a model to predict control levels using data from a randomly selected set of completed questionnaires from two-thirds of the subjects. Validation was performed using the remaining questionnaires. RESULTS: A set of 7 questions, encompassing self-assessed control status at the time of the visit and in the past month, and nocturnal/early morning asthma symptoms for patients and frequency of asthma symptoms, dyspnea, rescue beta-agonist use, and asthma hospitalization for caregivers, were selected and the 7-item model showed a good statistical fit with AIC of 110.5. The model has been named the Best Asthma Control Test for School Children and Adolescents (Best ACT-S). Best ACT-S scores differed significantly in the hypothetical direction among the groups of different JPGL-defined control levels, step-up/down treatment decisions, and presence/non-presence of exacerbations in the previous year. CONCLUSIONS: The Best ACT-S is a valid questionnaire for children/adolescents aiming for best asthma control.

[DEVELOPMENT AND VALIDATION OF QUALITY OF LIFE QUESTIONNAIRE FOR CHILDREN WITH FOOD ALLERGY REPORTED BY PARENTS].

OBJECTIVE: No questionnaire aimed at evaluating the quality of life (QOL) of children with food allergies has been developed in Japan. Therefore, this study was aimed at developing a Japanese version of a QOL questionnaire for children with food allergies to be responded to by their parents. METHODS: A 59-question primary questionnaire was developed for parents of children aged 0 to 15 years who had food allergies. Responses to the primary questionnaire were collected, and question items were reduced using factor analysis. Then, a secondary questionnaire consisting of nine questions was developed. The secondary questionnaire, the Food Allergy Quality of Life Questionnaire - Parent Form (FAQLQ-PF), and the parent-reported questionnaire on health-related quality of life in children and adolescents (KINDL) were administered to parents of children aged 3 to 15 years who had food allergies. RESULTS: Overall, 342 parents completed all questionnaires. The QOL scores of the secondary questionnaire were significantly correlated to those of the FAQLQ-PF (r=0.765) and weakly correlated to those of the KINDL (r=-0.358). In addition, QOL scores were significantly worse in patients with a history of anaphylaxis, who were prescribed an adrenaline auto-injector, and who were allergic to hen's eggs, cow milk, or wheat. CONCLUSION: We were able to develop a parent-reported validated Japanese version of the QOL questionnaire for children with food allergies.

[DEVELOPMENT AND VALIDATION OF QUALITY OF LIFE QUESTIONNAIRE FOR PARENT BURDEN OF CHILDREN WITH FOOD ALLERGY].

OBJECTIVE: Quality of life (QOL) questionnaires for parents of children with food allergies have been developed in the United States and Europe. However, no original Japanese QOL questionnaire has been developed till date. We aimed to develop an original questionnaire to evaluate the QOL in parents of children with food allergies in Japan. METHODS: We collected QOL-related questions from parents of children with food allergies aged 0-15 years, and created a primary questionnaire. Responses to the primary questionnaire were obtained from the parents again, and question items were reduced using factor analysis to create a secondary questionnaire comprising eight items. In addition to the secondary questionnaire, responses to the Food Allergy QOL Questionnaire-Parent Form (FAQLQ-PF) Japanese version, Parent reported Health-Related QOL in children and adolescents (KINDL) and Health-related QOL (SF-8) were obtained from parents to assess the validity of the secondary questionnaire. RESULTS: A total of 407 parents completed all questionnaires. The secondary questionnaire scores were positively correlated with those of FAQLQ-PF and weakly negatively correlated with the KINDL and SF-8 mental component summary scores. Parents of children with food allergies with ≥3 culprit foods or severe reactions to daily foods, a history of anaphylaxis, and those carrying adrenaline autoinjectors scored higher and had lower QOL. CONCLUSION: The developed original questionnaire is a valid QOL questionnaire for Parents of children with food allergies.

Novel Neplanocin A Derivatives as Selective Inhibitors of Hepatitis B Virus with a Unique Mechanism of Action.

Novel neplanocin A derivatives have been identified as potent and selective inhibitors of hepatitis B virus (HBV) replication in vitro. These include (1S,2R,5R)-5-(5-bromo-4-methyl-7H-pyrrolo[2,3-d]-pyrimidin-7-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (AR-II-04-26) and (1S,2R,5R)-5-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3-(hydroxylmethyl)cyclopent-3-ene-1,2-diol (MK-III-02-03). The 50% effective concentrations of AR-II-04-26 and MK-III-02-03 were 0.77 ± 0.23 and 0.83 ± 0.36 µM in HepG2.2.15.7 cells, respectively. These compounds reduced intracellular HBV RNA levels in HepG2.2.15.7 cells and infected primary human hepatocytes. Accordingly, they could reduce HBs and HBe antigen production in the culture supernatants, which was not observed with clinically approved anti-HBV nucleosides and nucleotides (reverse transcriptase inhibitors). The neplanocin A derivatives also inhibited HBV RNA derived from cccDNA. In addition, unlike neplanocin A itself, the compounds did not inhibit S-adenosyl-l-homocysteine hydrolase activity. Thus, it appears that the mechanism of action of AR-II-04-26 and MK-III-02-03 differs from that of the clinically approved anti-HBV agents. Although their exact mechanism (target molecule) remains to be elucidated, the novel neplanocin A derivatives are considered promising candidate drugs for inhibition of HBV replication.

Chromosome alignment-maintaining phosphoprotein CHAMP1 plays a role in cell survival through regulating Mcl-1 expression.

Antimitotic drugs such as vinca alkaloids and taxanes cause mitotic cell death after prolonged mitotic arrest. However, a fraction of cells escape from mitotic arrest by undergoing mitotic slippage, which is related to resistance to antimitotic drugs. Tipping the balance to mitotic cell death thus can be a way to overcome the drug resistance. Here we found that depletion of a mitotic regulator, CHAMP1 (chromosome alignment-maintaining phosphoprotein, CAMP), accelerates the timing of mitotic cell death after mitotic arrest. Live cell imaging revealed that CHAMP1-depleted cells died earlier than mock-treated cells in the presence of antimitotic drugs that resulted in the reduction of cells undergoing mitotic slippage. Depletion CHAMP1 reduces the expression of antiapoptotic Bcl-2 family proteins, especially Mcl-1. We found that CHAMP1 maintains Mcl-1 expression both at protein and mRNA levels independently of the cell cycle. At the protein level, CHAMP1 maintains Mcl-1 stability by suppressing proteasome-dependent degradation. Depletion of CHAMP1 reduces cell viability, and exhibits synergistic effects with antimitotic drugs. Our data suggest that CHAMP1 plays a role in the maintenance of Mcl-1 expression, implying that CHAMP1 can be a target to overcome the resistance to antimitotic drugs.

Correlation of hepatitis C virus-mediated endoplasmic reticulum stress with autophagic flux impairment and hepatocarcinogenesis.

Hepatitis C virus (HCV) infection has been known to use autophagy for its replication. However, the mechanisms by which HCV modulates autophagy remain controversial. We used HCV-Japanese fulminant hepatitis-1-infected Huh7 cells. HCV infection induced the accumulation of autophagosomes. Morphological analyses of monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP) tandem fluorescent-tagged LC3 transfection showed HCV infection impaired autophagic flux. Autophagosome-lysosome fusion assessed by transfection of mRFP- or GFP-LC3 and immunostaining of lysosomal-associated membrane protein 1 was inhibited by HCV infection. Decrease of HCV-induced endoplasmic reticulum (ER) stress by 4-phenylbutyric acid, a chemical chaperone, improved the HCV-mediated autophagic flux impairment. HCV infection-induced oxidative stress and subsequently DNA damage, but not apoptosis. Furthermore, HCV induced cytoprotective effects against the cellular stress by facilitating the formation of cytoplasmic inclusion bodies as shown by p62 expression and by modulating keratin protein expression and activated nuclear factor erythroid 2-related factor 2. HCV eradication by direct-acting antivirals improved autophagic flux, but DNA damage persisted. In conclusion, HCV-induced ER stress correlates with autophagic flux impairment. Decrease of ER stress is considered to be a promising therapeutic strategy for HCV-related chronic liver diseases. However, we should be aware that the risk of hepatocarcinogenesis remains even after HCV eradication.

Interaction of phospholipid scramblase 1 with the Epstein-Barr virus protein BZLF1 represses BZLF1-mediated lytic gene transcription.

Human phospholipid scramblase 1 (PLSCR1) is strongly expressed in response to interferon (IFN) treatment and viral infection, and PLSCR1 has been suggested to play an important role in IFN-dependent antiviral responses. In this study, we showed that the basal expression of PLSCR1 was significantly elevated in Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC). PLSCR1 was observed to directly interact with the EBV immediate-early transactivator BZLF1 in vitro and in vivo, and this interaction repressed the BZLF1-mediated transactivation of an EBV lytic BMRF1 promoter construct. In addition, PLSCR1 expression decreased the BZLF1-mediated up-regulation of lytic BMRF1 mRNA and protein expression in WT and PLSCR1-knockout EBV-infected NPC cells. Furthermore, we showed that PLSCR1 represses the interaction between BZLF1 and CREB-binding protein (CBP), which enhances the BZLF1-mediated transactivation of EBV lytic promoters. These results reveal for the first time that PLSCR1 specifically interacts with BZLF1 and negatively regulates its transcriptional regulatory activity by preventing the formation of the BZLF1-CBP complex. This interaction may contribute to the establishment of latent EBV infection in EBV-infected NPC cells.

Study of multiple genetic variations caused by persistent hepatitis C virus replication in long-term cell culture.

The most characteristic feature of the hepatitis C virus (HCV) genome in patients with chronic hepatitis C is its remarkable variability and diversity. To better understand this feature, we performed genetic analysis of HCV replicons recovered from two human hepatoma HuH-7-derived cell lines after 1, 3, 5, 7, and 9 years in culture: The cell lines 50-1 and sO harbored HCV 1B-1 and O strain-derived HCV replicons established in 2002 and 2003, respectively. The results revealed that genetic variations in both replicons accumulated in a time-dependent manner at a constant rate despite the maintenance of moderate diversity (less than 1.8% difference) between the clones and that the mutation rate in the 50-1 and sO replicons was 2.5 and 2.9 × 10-3 base substitutions/site/year, respectively. We found that the genetic distance of both replicons increased from 7.9% to 10.5% after 9 years in culture. In addition, we observed that the guanine + cytosine (GC) content of both replicon RNAs increased in a time-dependent manner, as observed in our previous studies. Finally, we demonstrated that the high sensitivity of both replicons to direct-acting antivirals was maintained even after 9 years in culture. Our results suggest that long-term cultured HCV replicon-harboring cells are a useful model for understanding the variability and diversity of the HCV genome and the drug sensitivity of HCV in patients with chronic hepatitis C.

Long-term safety and pharmacodynamics of mepolizumab in children with severe asthma with an eosinophilic phenotype.

BACKGROUND: Mepolizumab is approved for patients with severe asthma with an eosinophilic phenotype aged 12 or more (United States) or 6 or more (European Union) years, but its long-term use in children aged 6 to 11 years has not yet been assessed. OBJECTIVE: We sought to assess the long-term safety, efficacy, and pharmacodynamics of mepolizumab in children aged 6 to 11 years with severe asthma with an eosinophilic phenotype. METHODS: In this open-label, uncontrolled, repeat-dose extension study (NCT02377427), children aged 6 to 11 years with severe asthma with an eosinophilic phenotype (blood eosinophil counts ≥150 cells/µL at screening or ≥300 cells/µL in the previous year) received a body weight-dependent dose of subcutaneous mepolizumab of 40 mg (<40 kg) or 100 mg (≥40 kg) over 52 weeks. End points included the incidence of adverse events (AEs) and immunogenicity (primary), absolute blood eosinophil counts (cells per microliter; secondary), and annualized exacerbation rates and asthma control questionnaire/childhood asthma control test scores (exploratory). RESULTS: Over 52 weeks, 30 children received mepolizumab; 27 (90%) and 7 (23%) experienced on-treatment AEs and serious AEs, respectively. No serious AEs were treatment related. There were no fatal AEs. No specific patterns of AEs were evident, and no anti-drug antibody or neutralizing antibody responses were reported. Compared with baseline values, mepolizumab treatment reduced blood eosinophil counts and asthma exacerbations and improved asthma control across all treatment groups. CONCLUSION: Long-term safety, pharmacodynamic, and efficacy data from this study support a positive benefit-risk profile for mepolizumab in children with severe asthma with an eosinophilic phenotype and were similar to data in studies in adults and adolescents.

Breastfeeding and risk of food allergy: A nationwide birth cohort in Japan.

BACKGROUND: Although breastfeeding has been well-established as the preferred method for infant nutrition, its prophylactic effects on food allergy remain controversial. Infantile eczema has been linked to food allergy via percutaneous sensitization; however, this relationship has not been considered in previous studies. We aimed to uncover the prophylactic effects of breastfeeding on food allergy, focusing on eczema-mediated percutaneous sensitization. METHODS: This retrospective cohort study was based on 46,616 children from the Longitudinal Survey of Newborns in the 21st Century in Japan, begun in 2001. We classified participants into three groups based on infant feeding practices (exclusive breastfeeding, partial breastfeeding including only colostrum, and formula feeding only) and used information from at least one outpatient visit for food allergy during two observation periods (age 6-18 months and age 6-66 months) as health outcomes. We performed log-binomial regression analysis adjusted for potential confounders and stratified analysis according to infantile eczema status. RESULTS: Compared with formula feeding, partial breastfeeding including only colostrum reduced the risk of food allergy only in children with infantile eczema, (RR = 0.66, 95% CI: 0.46, 0.96 for age 6-66 months), whereas exclusive breastfeeding increased this risk in those without infantile eczema (RR = 2.41, 95% CI: 1.40, 4.15, age 6-66 months). The prophylactic effects of breastfeeding on food allergy in the infantile eczema group increased with shorter breastfeeding duration. CONCLUSIONS: Our results showed that breastfeeding, especially colostrum, had prophylactic effects on food allergy only among high-risk children with infantile eczema whereas prolonged breastfeeding increased the risk of food allergy.

Japanese guidelines for atopic dermatitis 2020.

Atopic dermatitis (AD) is a disease characterized by relapsing eczema with pruritus as a primary lesion, which is frequently encountered in clinical practice. Skin barrier dysfunction leads to enhanced skin irritability to non-specific stimuli and epicutaneous sensitization. In the lesion site, a further inflammation-related reduction in skin barrier function, enhanced irritability and scratching-related stimuli deteriorate eczema, leading to vicious cycle of inflammation. The current strategies to treat AD in Japan from the perspective of evidence-based medicine consist of three primary measures: (i) the use of topical corticosteroids and tacrolimus ointment as the main treatment for the inflammation; (ii) topical application of emollients to treat the cutaneous barrier dysfunction; and (iii) avoidance of apparent exacerbating factors, psychological counseling and advice about daily life. The guidelines present recommendations to review clinical research articles, evaluate the balance between the advantages and disadvantages of medical activities, and optimize medical activity-related patient outcomes with respect to several important points requiring decision-making in clinical practice.

The efficacy and safety of fluticasone/salmeterol compared to fluticasone in children younger than four years of age.

BACKGROUND: Fluticasone propionate 50 µg/salmeterol xinafoate 25 µg (FP/SAL) is widely used in adults and children with asthma, but there is sparse information on its use in very young children. METHODS: This was a randomized, double-blind, multicentre, controlled trial conducted in children aged 8 months to 4 years. During a 2-week run-in period, they all received FP twice daily. At randomization, they commenced FP/SAL or FP twice daily for 8 weeks. All were then given FP/SAL only, in a 16-week open-label study continuation. Medications were inhaled through an AeroChamber Plus with attached face mask. The primary end-point was mean change in total asthma symptom scores from baseline to the last 7 days of the double-blind period. Analyses were undertaken in all children randomized to treatment and who received at least one dose of study medication. RESULTS: Three hundred children were randomized 1:1 to receive FP/SAL or FP. Mean change from baseline in total asthma symptom scores was -3.97 for FP/SAL and -3.01 with FP. The between-group difference was not statistically significant (P = 0.21; 95% confidence interval: -2.47, 0.54). No new safety signals were seen with FP/SAL. CONCLUSION: This is the first randomized, double-blind study of this size to evaluate FP/SAL in very young children with asthma. FP/SAL did not show superior efficacy to FP; no clear add-on effect of SAL was demonstrated. No clinically significant differences in safety were noted with FP/SAL usage.

Dynamic feature of mitotic arrest deficient 2-like protein 2 (MAD2L2) and structural basis for its interaction with chromosome alignment-maintaining phosphoprotein (CAMP).

Mitotic arrest deficient 2-like protein 2 (MAD2L2), also termed MAD2B or REV7, is involved in multiple cellular functions including translesion DNA synthesis (TLS), signal transduction, transcription, and mitotic events. MAD2L2 interacts with chromosome alignment-maintaining phosphoprotein (CAMP), a kinetochore-microtubule attachment protein in mitotic cells, presumably through a novel "WK" motif in CAMP. Structures of MAD2L2 in complex with binding regions of the TLS proteins REV3 and REV1 have revealed that MAD2L2 has two faces for protein-protein interactions that are regulated by its C-terminal region; however, the mechanisms underlying the MAD2L2-CAMP interaction and the mitotic role of MAD2L2 remain unknown. Here we have determined the structures of human MAD2L2 in complex with a CAMP fragment in two crystal forms. The overall structure of the MAD2L2-CAMP complex in both crystal forms was essentially similar to that of the MAD2L2-REV3 complex. However, the residue interactions between MAD2L2 and CAMP were strikingly different from those in the MAD2L2-REV3 complex. Furthermore, structure-based interaction analyses revealed an unprecedented mechanism involving CAMP's WK motif. Surprisingly, in one of the crystal forms, the MAD2L2-CAMP complex formed a dimeric structure in which the C-terminal region of MAD2L2 was swapped and adopted an immature structure. The structure provides direct evidence for the dynamic nature of MAD2L2 structure, which in turn may have implications for the protein-protein interaction mechanism and the multiple functions of this protein. This work is the first structural study of MAD2L2 aside from its role in TLS and might pave the way to clarify MAD2L2's function in mitosis.

Activation of the Hypoxia Inducible Factor 1α Subunit Pathway in Steatotic Liver Contributes to Formation of Cholesterol Gallstones.

BACKGROUND & AIMS: Hypoxia-inducible factor 1α subunit (HIF1A) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with nonalcoholic fatty liver disease (NAFLD). NAFLD increases the risk for cholesterol gallstone disease by unclear mechanisms. We studied the relationship between HIF1A and gallstone formation associated with liver steatosis. METHODS: We performed studies with mice with inducible disruption of Hif1a in hepatocytes via a Cre adenoviral vector (inducible hepatocyte-selective HIF1A knockout [iH-HIFKO] mice), and mice without disruption of Hif1a (control mice). Mice were fed a diet rich in cholesterol and cholate for 1 or 2 weeks; gallbladders were collected and the number of gallstones was determined. Livers and biliary tissues were analyzed by histology, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunoblots. We measured concentrations of bile acid, cholesterol, and phospholipid in bile and rates of bile flow. Primary hepatocytes and cholangiocytes were isolated and analyzed. HIF1A was knocked down in Hepa1-6 cells with small interfering RNAs. Liver biopsy samples from patients with NAFLD, with or without gallstones, were analyzed by quantitative reverse-transcription polymerase chain reaction. RESULTS: Control mice fed a diet rich in cholesterol and cholate developed liver steatosis with hypoxia; levels of HIF1A protein were increased in hepatocytes around central veins and 90% of mice developed cholesterol gallstones. Only 20% of the iH-HIFKO mice developed cholesterol gallstones. In iH-HIFKO mice, the biliary lipid concentration was reduced by 36%, compared with control mice, and bile flow was increased by 35%. We observed increased water secretion from hepatocytes into bile canaliculi to mediate these effects, resulting in suppression of cholelithogenesis. Hepatic expression of aquaporin 8 (AQP8) protein was 1.5-fold higher in iH-HIFKO mice than in control mice. Under hypoxic conditions, cultured hepatocytes increased expression of Hif1a, Hmox1, and Vegfa messenger RNAs (mRNAs), and down-regulated expression of AQP8 mRNA and protein; AQP8 down-regulation was not observed in cells with knockdown of HIF1A. iH-HIFKO mice had reduced inflammation and mucin deposition in the gallbladder compared with control mice. Liver tissues from patients with NAFLD with gallstones had increased levels of HIF1A, HMOX1, and VEGFA mRNAs, compared with livers from patients with NAFLD without gallstones. CONCLUSIONS: In steatotic livers of mice, hypoxia up-regulates expression of HIF1A, which reduces expression of AQP8 and concentrates biliary lipids via suppression of water secretion from hepatocytes. This promotes cholesterol gallstone formation. Livers from patients with NAFLD and gallstones express higher levels of HIF1A than livers from patients with NAFLD without gallstones.

Dual effects of the Nrf2 inhibitor for inhibition of hepatitis C virus and hepatic cancer cells.

BACKGROUND: We previously showed that knockdown of nuclear factor E2-related factor 2 (Nrf2) resulted in suppression of hepatitis C virus (HCV) infection. In this study, whether brusatol, an Nrf2 inhibitor, has dual anti-HCV and anticancer effects was explored. METHODS: The anti-HCV effect of brusatol was investigated by analyzing HCV RNA and proteins in a hepatic cell line persistently-infected with HCV, HPI cells, and by analyzing HCV replication in a replicon-replicating hepatic cell line, OR6 cells. Then, dual anti-HCV and anticancer effects of brusatol and enhancement of the effects by the combination of brusatol with anticancer drugs including sorafenib, which has been reported to have the dual effects, were then investigated. RESULTS: Brusatol suppressed the persistent HCV infection at both the RNA and protein levels in association with a reduction in Nrf2 protein in the HPI cells. Analysis of the OR6 cells treated with brusatol indicated that brusatol inhibited HCV persistence by inhibiting HCV replication. Combination of brusatol with an anticancer drug not only enhanced the anticancer effect but also, in the case of the combination with sorafenib, strongly suppressed HCV infection. CONCLUSIONS: Brusatol has dual anti-HCV and anticancer effects and can enhance the comparable effects of sorafenib. There is therefore the potential for combination therapy of brusatol and sorafenib for HCV-related hepatocellular carcinoma.

Step Numbers and Hoehn-Yahr Stage after Six Years.

BACKGROUND: Freezing of gait (FOG) has been linked to increased numbers of steps taken while walking. We tested the hypothesis that an increased number of steps associated with FOG might predict the exacerbation of the severity of Parkinson's disease (PD). METHODS: We prospectively studied 26 patients. Clinical assessments were performed and balance was evaluated in 30 patients with Hoehn-Yahr stage III PD 6 years previously. Gait parameters were analyzed with the use of an originally designed, suddenly narrowed path. PD-related independent variables, balance investigation-related variables, and gait-independent-related variables were analyzed by multiple logistic regression analysis. RESULTS: The Hoehn-Yahr stage increased in 14 patients and was unchanged in 12 patients. The 36-item Short-Form Health Survey score (OR 1.079, p = 0.041, 95% CI 1.003-1.161) and the number of steps on the suddenly narrow path (OR 1.605, p = 0.047, 95% CI 1.006-2.56) were related to an increase in the Hoehn-Yahr stage. The number of steps was significantly higher on the suddenly narrowed path (11.3 ± 3.6) than on a straightly narrowed path (10.1 ± 3.2) at the time of final follow-up in the 26 patients (p < 0.001). CONCLUSIONS: An increased number of steps associated with FOG, which was elicited by the suddenly narrowed path, might be one predictor of an upgrade of stage in patients with Hoehn-Yahr stage III PD.

Early control treatment with montelukast in preschool children with asthma: A randomized controlled trial.

BACKGROUND: While Japanese guideline recommends initial control treatment for preschool children with asthma symptoms more than once a month, Western guidelines do not. To determine whether control treatment with montelukast was more effective than as-needed ß2-agonists in this population, we conducted a randomized controlled trial. METHODS: Eligible patients were children aged 1-5 years who had asthma symptoms more than once a month but less than once a week. Patients were randomly assigned in a 1:1 ratio to receive montelukast 4 mg daily for 48 weeks or as-needed ß2-agonists. The primary endpoint was the number of acute asthma exacerbations before starting step-up treatment with inhaled corticosteroids. This study is registered with the University Hospital Medical Information Network clinical trials registry, number UMIN000002219. RESULTS: From September 2009 to November 2012, 93 patients (47 in the montelukast group and 46 in the no-controller group) were enrolled into the study. All patients were included in the analysis. During the study, 13 patients (28%) in the montelukast group and 23 patients (50%) in the no-controller group had acute exacerbations with the mean numbers of 0.9 and 1.9/year, respectively (P = 0.027). In addition, 10 (21%) and 19 (41%) patients received step-up treatment, respectively. Cumulative incidence of step-up treatment was significantly lower in the montelukast group (hazard ratio 0.45, 95% confidence interval 0.21 to 0.92; P = 0.033). CONCLUSIONS: Montelukast is an effective control treatment for preschool children who had asthma symptoms more than once a month but less than once a week.

MIP-1α level in nasopharyngeal aspirates at the first wheezing episode predicts recurrent wheezing.

BACKGROUND: Respiratory virus-induced wheezing, such as that induced by respiratory syncytial virus (RSV) and human rhinovirus, is an important risk factor for recurrent wheezing and childhood asthma. However, no biomarkers for predicting recurrent wheezing have been identified. OBJECTIVE: We searched for predictors of recurrent wheezing using nasopharyngeal aspirates obtained from patients during the first wheezing episode who were hospitalized with an acute lower respiratory tract illness. METHODS: We enrolled 82 infants during the first wheezing episode (median age, 5.0 months) who were hospitalized for acute lower respiratory tract illness between August 2009 and June 2012 and followed these patients for 2.5 years. Nasopharyngeal aspirates and blood samples were obtained on the first day of hospitalization. Viral genomes were identified by using RT-PCR and sequencing. Levels of 33 cytokines, tryptase, IgE, anti-RSV IgE, and anti-RSV IgG were measured by using ELISAs or the Bio-Plex multiplex assay. Predictors of recurrent wheezing were examined by using a stepwise logistic regression model with backward elimination. RESULTS: Sixty percent of the patients experienced recurrent wheezing episodes. One or more viruses were detected in the nasopharynxes of 93% of the patients during the first wheezing episode. IFN-γ, IL-2, IL-9, MIP-1α, and MIP-1ß levels were significantly higher among patients with recurrent wheezing than among those without recurrent wheezing (P < .05 or .01). The stepwise model demonstrated that the MIP-1α level (odds ratio, 7.72; 95% CI, 1.50-39.77; P = .015) was the strongest independent predictor of the occurrence of recurrent wheezing. CONCLUSION: An increased MIP-1α level in nasopharyngeal aspirates from patients with acute respiratory symptoms during the first wheezing episode caused by viral infections might predict recurrent wheezing.