Clinical characteristics of patients with alcohol dependence comorbid with hypertension among regular drinkers: An internet-based, cross-sectional study in Japan.

While evidence suggests a strong association between alcohol and hypertension, little is known about the profile of patients with alcohol dependence comorbid with hypertension. This study aimed to clarify the clinical characteristics and health problems of this population through a web-based questionnaire survey using a research company's panel of adults in Japan. Of 20 000 regular drinkers, 176 on treatment for hypertension and with alcohol dependence (confirmed and/or an Alcohol Use Disorders Identification Test score ≥15 points) were included. Participants were asked about their health-related quality of life, work productivity, blood pressure (BP) control, receipt of brief interventions, and awareness of their alcohol dependence. Results were compared between the BP-controlled and BP-uncontrolled groups. The mean EQ-5D utility score was 0.838 in the entire population, and 0.786 vs. 0.892 in the groups (p < 0.0001). When 133 'employed' participants were compared, productivity loss was more apparent in the BP-uncontrolled group (presenteeism, 27.3% vs. 6.1%, p < 0.0001; absenteeism, 10.7% vs. 1.0%, p = 0.0003). The rate of dissatisfaction with BP control was 55.1% in the entire population (most [76.3%] of those dissatisfied considered alcohol a cause of inadequate BP control), ~78% in the uncontrolled group, and ~34% in the controlled group. Of those previously advised to reduce drinking or abstain from alcohol (60.2% of all participants), 63% (BP-uncontrolled group) and 55% (BP-controlled group) decreased their drinking. Though more than twice as many participants thought themselves to be alcohol-dependent in the BP-uncontrolled group than in the controlled group (41% vs. 15%), most (59% vs. 85%) showed no self-awareness of alcohol dependence. Patients with alcohol dependence comorbid with hypertension had impaired health status and reduced work productivity. They thought alcohol was the most common cause of inadequate BP control. Treatment beyond brief interventions is needed to enhance their awareness of alcohol dependence and their motivation to reduce drinking.

Long-term safety and efficacy of nalmefene in Japanese patients with alcohol dependence.

AIM: The safety and efficacy of nalmefene in Japanese patients with high or very high World Health Organization drinking risk level of alcohol dependence were assessed in a multicenter, randomized, double-blind, placebo-controlled, phase 3 (lead-in) study. Here, the long-term safety and efficacy of nalmefene in an open-label extension of the lead-in study are presented. METHODS: Patients who completed the 24-week lead-in study were eligible for the extension study, where they were treated with nalmefene 20 mg as needed for 24 weeks. The long-term safety and efficacy of nalmefene 20 mg during the total 48-week period were evaluated. Treatment-emergent adverse events during the study period were recorded and change from baseline in the number of heavy drinking days and total alcohol consumption were calculated. RESULTS: Overall, long-term nalmefene 20 mg was well tolerated; the main treatment-emergent adverse events reported in ≥5% of patients included nasopharyngitis (37.2%), nausea (36.5%), somnolence (21.2%), dizziness (16.8%), malaise (14.6%), and vomiting (12.4%). The number of heavy drinking days and total alcohol consumption decreased from baseline to 48 weeks (mixed model for repeated measures, least squares mean ± standard error, -15.09 ± 0.77 days/month and -53.20 ± 2.29 g/day, respectively) during the study. CONCLUSION: This long-term evaluation in Japanese patients with high or very high drinking risk levels of alcohol dependence indicated that nalmefene was safe, well tolerated, and efficacious.

OPC-167832, a Novel Carbostyril Derivative with Potent Antituberculosis Activity as a DprE1 Inhibitor.

There is an urgent need for new, potent antituberculosis (anti-TB) drugs with novel mechanisms of action that can be included in new regimens to shorten the treatment period for TB. After screening a library of carbostyrils, we optimized 3,4-dihydrocarbostyril derivatives and identified OPC-167832 as having potent antituberculosis activity. The MICs of the compound for Mycobacterium tuberculosis ranged from 0.00024 to 0.002 µg/ml. It had bactericidal activity against both growing and intracellular bacilli, and the frequency of spontaneous resistance for M. tuberculosis H37Rv was less than 1.91 × 10-7 It did not show antagonistic effects with other anti-TB agents in an in vitro checkerboard assay. Whole-genome and targeted sequencing of isolates resistant to OPC-167832 identified decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1), an essential enzyme for cell wall biosynthesis, as the target of the compound, and further studies demonstrated inhibition of DprE1 enzymatic activity by OPC-167832. In a mouse model of chronic TB, OPC-167832 showed potent bactericidal activities starting at a dose of 0.625 mg/kg of body weight. Further, it exhibited significant combination effects in 2-drug combinations with delamanid, bedaquiline, or levofloxacin. Finally, 3- or 4-drug regimens comprised of delamanid and OPC-167832 as the core along with bedaquiline, moxifloxacin, or linezolid showed efficacy in reducing the bacterial burden and preventing relapse superior to that of the standard treatment regimen. In summary, these results suggest that OPC-167832 is a novel and potent anti-TB agent, and regimens containing OPC-167832 and new or repurposed anti-TB drugs may have the potential to shorten the duration of treatment for TB.

Delamanid Kills Dormant Mycobacteria In Vitro and in a Guinea Pig Model of Tuberculosis.

Tuberculosis (TB) treatment is long and requires multiple drugs, likely due to various phenotypes of TB bacilli with variable drug susceptibilities. Drugs with broad activity are urgently needed. This study aimed to evaluate delamanid's activity against growing or dormant bacilli in vitro as well as in vivo Cultures of Mycobacterium bovis BCG Tokyo under aerobic and anaerobic conditions were used to study the activity of delamanid against growing and dormant bacilli, respectively. Delamanid exhibited significant bactericidal activity against replicating and dormant bacilli at or above concentrations of 0.016 and 0.4 mg/liter, respectively. To evaluate delamanid's antituberculosis activity in vivo, we used a guinea pig model of chronic TB infection in which the lung lesions were similar to those in human TB disease. In the guinea pig TB model, a daily dose of 100 mg delamanid/kg of body weight for 4 or 8 weeks demonstrated strong bactericidal activity against Mycobacterium tuberculosis Importantly, histological examination revealed that delamanid killed TB bacilli within hypoxic lesions of the lung. The combination regimens containing delamanid with rifampin and pyrazinamide or delamanid with levofloxacin, ethionamide, pyrazinamide, and amikacin were more effective than the standard regimen (rifampin, isoniazid, and pyrazinamide). Our data show that delamanid is effective in killing both growing and dormant bacilli in vitro and in the guinea pig TB model. Adding delamanid to current TB regimens may improve treatment outcomes, as demonstrated in recent clinical trials with pulmonary multidrug-resistant (MDR) TB patients. Delamanid may be an important drug for consideration in the construction of new regimens to shorten TB treatment duration.

Distinct propagation efficiencies of H5N1 influenza virus Thai isolates in newly established murine respiratory region-derived cell clones.

Inbred mice have been widely used for the study of influenza viruses as a mammalian model, while suitable cell lines derived from murine tissue have been limited. Here, we established several immortalized cell clones from respiratory regions of inbred mice (C57BL/6 and BALB/c) by transformation using simian virus 40 large T antigen expression vector. Twenty-five cell clones from C57/BL and BALB/c, designated as MRDC/C and MRDC/B series, respectively, showed different susceptibility to Thai isolates of influenza A virus H5N1. Two murine cell clones, C6 and B7 which were extensively studied expressed both SAα2,3 and SAα2,6 sialic acid receptors. Interestingly, the 6 Thai patient-derived H5N1 isolates examined showed varied virus propagation efficiency in murine cell clones, although there were only slight differences in their propagation in MDCK and A549 cell lines. The results indicate that the murine cell clones are useful for examining the propagation efficiency of H5N1 viruses in vitro.

GPI-anchorless human prion protein is secreted and glycosylated but lacks superoxide dismutase activity.

Prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that is thought to play a role in anti-oxidative stress. It remains controversial whether PrP elicits superoxide dismutase (SOD) activity itself or indirectly by activating cellular SOD. Our previous studies showed that soluble PrP produced by a baculovirus expression system did not exhibit any SOD activity in a marginally glycosylated form. In the present study, we developed a mammalian expression system for a truncated soluble form of human prion protein with the native signal peptide but without a GPI-anchor site, driven by the peptide chain elongation factor 1alpha promoter in stably transfected rabbit-kidney epithelial RK13 cells, to investigate the SOD activity of mammalian PrP. This recombinant product, denoted sPrP, is secreted in large quantities in medium and can be isolated in very high purity and yield (more than 1 mg sPrP per 2 litres medium). Characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tunicamycin treatment revealed that a fully glycosylated form of sPrP was secreted from the cells. SOD activity in cell lysate showed a decrease in sPrP-expressing RK13 cells and an increase in wild-type PrP-expressing RK13 cells compared to empty vector-transfected RK13 cells or parent cells. Purified or immunoprecipitated sPrP did not show any SOD activity. In conclusion, the GPI-anchor site, but not glycosylation, appears to be essential for the secretion of PrP. In addition, mammalian PrP itself does not act as a functional SOD enzyme.

A monoclonal antibody (1D12) defines novel distribution patterns of prion protein (PrP) as granules in nucleus.

A monoclonal antibody (mAb) panel to bovine prion protein (PrP) was studied by immunoblotting and immunohistochemistry for scrapie and bovine spongiform encephalopathy. A mAb panel recognized both normal (PrP(C)) and abnormal (PrP(Sc)) isoforms of PrP in murine, ovine and bovine brain tissues. Interestingly, an anti-bovine PrP mAb, 1D12, prepared by immunizing PrP gene-knockout mice with a synthetic polypeptides corresponding to codons 153-166 of the bovine PrP gene showed novel patterns of reactivity for prion-uninfected neuronal cells. An enzyme-linked immunosorbent assay-mapping of the mAb epitopes resulted in a reaction of monoclonal 1D12 to YEDRY and M corresponding to amino acids 156-160 and 165 of bovine PrP. Several patterns of bovine PrP(C) distribution in PrP-deficient neuronal cells (HpL3-4) transfected with bovine PrP were observed after different fixation methods. Stained cell surface was observed after formalin fixation by immunofluorescent assay of 1D12 with confocal microscopy, whereas granules in nucleus were stained after acetone fixation. No reactivity in the nucleus was observed to HpL3-4, or HpL3-4mPrP cells expressing mouse PrP. This is the first paper that has reported the detection of the PrP(C) at both cell surface and nuclei of prion-uninfected cell line.

Recent developments in prion disease research: diagnostic tools and in vitro cell culture models.

After prion infection, an abnormal isoform of prion protein (PrP(Sc)) converts the cellular isoform of prion protein (PrP(C)) into PrP(Sc). PrP(C)-to-PrP(Sc) conversion leads to PrP(Sc) accumulation and PrP(C) deficiency, contributing etiologically to induction of prion diseases. Presently, most of the diagnostic methods for prion diseases are dependent on PrP(Sc) detection. Highly sensitive/accurate specific detection of PrP(Sc) in many different samples is a prerequisite for attempts to develop reliable detection methods. Towards this goal, several methods have recently been developed to facilitate sensitive and precise detection of PrP(Sc), namely, protein misfolding cyclic amplification, conformation-dependent immunoassay, dissociation-enhanced lanthanide fluorescent immunoassay, capillary gel electrophoresis, fluorescence correlation spectroscopy, flow microbead immunoassay, etc. Additionally, functionally relevant prion-susceptible cell culture models that recognize the complexity of the mechanisms of prion infection have also been pursued, not only in relation to diagnosis, but also in relation to prion biology. Prion protein (PrP) gene-deficient neuronal cell lines that can clearly elucidate PrP(C) functions would contribute to understanding of the prion infection mechanism. In this review, we describe the trend in recent development of diagnostic methods and cell culture models for prion diseases and their potential applications in prion biology.

Novel single nucleotide polymorphisms in the specific protein 1 binding site of the bovine PRNP promoter in Japanese Black cattle: impairment of its promoter activity.

Susceptibility to transmissible spongiform encephalopathy and different alleles of the prion protein gene (PRNP) of humans and sheep are associated. A tentative association between PRNP promoter polymorphisms and bovine spongiform encephalopathy (BSE) susceptibility has been reported in German cattle, whereas none of the known polymorphisms within the bovine PRNP-coding sequence affect BSE susceptibility. In the present study, novel single nucleotide polymorphisms located in the 5'-flanking region of bovine PRNP affecting its expression were demonstrated in Japanese Black cattle. We sequenced exon 1, and the approximately 200-bp 5'-flanking region of the PRNP translation initiation site containing the proximal promoter of PRNP was harvested. We identified 7 single nucleotide polymorphisms: -184A-->G, -141T-->C, -85T-->G, -47C-->A, -6C-->T, +17C-->T and +43C-->T. Six segregated haplotypes in the population were cloned into luciferase-expressing plasmids, transfected into N2a cells, and their reporter activities were measured 48 h after transfection. Six haplotypes showed a decreased expression level including -6C-->T in specific protein 1 binding site (p < 0.05) or -141T-->C (p < 0.01) at 48 h compared with the wild-type haplotype. These results advocate that certain polymorphisms such as specific protein 1 binding site polymorphisms in the bovine PRNP promoter region in Japanese Black cattle could influence promoter activity, suggesting that breeding cattle with such substitutions may be a useful approach in reducing BSE risk.

Neurotoxic prion protein (PrP) fragment 106-126 requires the N-terminal half of the hydrophobic region of PrP in the PrP-deficient neuronal cell line.

The cytotoxicity of aged PrP(106-126) was examined using an immortalized prion protein (PrP) gene-deficient neuronal cell line. The N-terminal half of the hydrophobic region (HR) but not the octapeptide repeat (OR) of PrP was required for aged PrP(106-126) neurotoxicity, suggesting that neurotoxic signals of aged PrP(106-126) are mediated by this region.

Cell-autonomous PrP-Doppel interaction regulates apoptosis in PrP gene-deficient neuronal cells.

The Prnd-encoded prion protein (PrP)-like protein, Doppel (Dpl), is a homologue of Prnp-encoded PrP, and is N-glycosylated protein with glycosylphosphatidylinositol anchor like PrP. Recently, ectopic expressions of Prnp/Prnd chimeric mRNAs have been identified as the cause of late-onset ataxia observed in several lines of Prnp-knockout mice such as ZrchII, Ngsk, Rcm0, and Rikn mice. However, it remains unclear whether the toxic effect of Dpl expression is a cell-autonomous mechanism but rather reflect a systemic process of heterogeneous cell population in the brain. In this study, the cell-autonomous role of Dpl was estimated by investigating PrP-deficient cells (HpL3-4)-the SV40 large T-antigen immortalized and Rikn Prnp(-/-) mice-derived neuronal cell line expressing Prnp/Prnd chimeric mRNAs. The reverse transcription polymerase chain reaction revealed that serum deprivation did not increase Prnp/Prnd chimeric mRNAs, which in fact was translated into a small amount of Dpl in HpL3-4 cells, whereas serum deprivation induced apoptotic cell death of HpL3-4 cells. Dpl overexpression enhanced apoptotic cell death, whereas the toxic effect of Dpl on apoptotic cell death was neutralized by PrP expression. These results indicate that Dpl elicited dose-dependently toxic effects on PrP-deficient cells without affecting on PrP-expressing cells, suggesting that the PrP-Dpl interaction can regulate cell death in a cell-autonomous manner.

Cellular prion protein regulates intracellular hydrogen peroxide level and prevents copper-induced apoptosis.

The function of cellular prion protein (PrPC), which is a copper binding protein, remains unclear. To elucidate the mechanisms in which PrPC is involved in neuroprotection, we compared death signals in prion protein gene-deficient (Prnp-/-) primary cerebellar granular neurons (CGNs) to those with wild-type (WT) CGNs. When copper was exposed to these CGNs, ZrchI, and Rikn Prnp-/- CGNs were more sensitized and underwent apoptotic cell death more readily than WT CGNs. Furthermore, the level of intracellular hydrogen peroxide (H2O2) in WT CGNs increased by copper toxicity, whereas those in ZrchI and Rikn Prnp-/- CGNs did not. These results suggest that PrPC modulates the intracellular H2O2 level as a copper-binding protein to protect CGNs from apoptotic cell death possibly due to inhibiting a Fenton reaction.