A hepatitis B virus-derived peptide combined with HBsAg exerts an anti-HBV effect in an HBV transgenic mouse model as a therapeutic vaccine.

Introduction: For complete or functional cure of hepatitis B virus (HBV) infection, application of immunotherapy is now being attempted. Recently, we reported that a 6-mer hepatitis B virus (HBV)-derived peptide, Poly6, exerts a strong anticancer effect in tumor-implanted mice through inducible nitric oxide synthase (iNOS)-producing DCs (Tip-DCs) in a type 1 interferon (IFN-I)-dependent manner, suggesting its potential as a vaccine adjuvant. Methods: In this study, we explored the potential of Poly6 in combination with HBsAg as a therapeutic vaccine against hepatitis B virus infection. We investigated the immunotherapeutic potential of Poly6 combined with HBsAg vaccination against hepatitis B virus infection in C57BL/6 mice or an HBV transgenic mouse model. Results: In C57BL/6 mice, Poly6 enhanced DC maturation and DC migration capacity in an IFN-I-dependent manner. Moreover, the addition of Poly6 to alum in combination with HBsAg also led to enhanced HBsAg-specific cell-mediated immune (CMI) responses, suggesting its potential as an adjuvant of HBsAg-based vaccines. In HBV transgenic mice, vaccination with Poly6 combined with HBsAg exerted a strong anti-HBV effect via induction of HBV-specific humoral and cell-mediated immune responses. In addition, it also induced HBV-specific effector memory T cells (TEM). Discussion: Our data indicated that vaccination with Poly6 in combination with HBsAg exerts an anti-HBV effect in HBV transgenic mice, which is mainly mediated by HBV-specific CMI and humoral immune responses via IFN-I-dependent DC activation, suggesting the feasibility of Poly6 as an adjuvant for an HBV therapeutic vaccine.

Locked nucleic acid real-time polymerase chain reaction method identifying two polymorphisms of hepatitis B virus genotype C2 infections, rt269L and rt269I.

BACKGROUND: The presence of two distinct hepatitis B virus (HBV) Pol RT polymorphisms, rt269L and rt269I, could contribute to the unique clinical or virological phenotype of HBV genotype C2. Therefore, a simple and sensitive method capable of identifying both types in chronic hepatitis B (CHB) patients infected with genotype C2 should be developed. AIM: To develop a novel simple and sensitive locked nucleic acid (LNA)-real time-polymerase chain reaction (RT-PCR) method capable of identifying two rt269 types in CHB genotype C2 patients. METHODS: We designed proper primer and probe sets for LNA-RT-PCR for the separation of rt269 types. Using synthesized DNAs of the wild type and variant forms, melting temperature analysis, detection sensitivity, and endpoint genotyping for LNA-RT-PCR were performed. The developed LNA-RT-PCR method was applied to a total of 94 CHB patients of genotype C2 for the identification of two rt269 polymorphisms, and these results were compared with those obtained by a direct sequencing protocol. RESULTS: The LNA-RT-PCR method could identify two rt269L and rt269I polymorphisms of three genotypes, two rt269L types ['L1' (WT) and 'L2'] and one rt269I type ('I') in single (63 samples, 72.4%) or mixed forms (24 samples, 27.6%) in 87 (92.6% sensitivity) of 94 samples from Korean CHB patients. When the results were compared with those obtained by the direct sequencing protocol, the LNA-RT-PCR method showed the same results in all but one of 87 positive detected samples (98.9% specificity). CONCLUSION: The newly developed LNA-RT-PCR method could identify two rt269 polymorphisms, rt269L and rt269I, in CHB patients with genotype C2 infections. This method could be effectively used for the understanding of disease progression in genotype C2 endemic areas.

rt269L-Type hepatitis B virus (HBV) in genotype C infection leads to improved mitochondrial dynamics via the PERK-eIF2α-ATF4 axis in an HBx protein-dependent manner.

BACKGROUND: In our previous report, the rt269I type versus the rt269L type in genotype C2 infection led to poor clinical outcomes and enhanced mitochondrial stress in infected hepatocytes. Here, we sought to investigate differences between the rt269L and rt269I types in mitochondrial functionality in hepatitis B virus (HBV) genotype C2 infection, mainly focusing on endoplasmic reticulum (ER) stress-mediated autophagy induction as an upstream signal. METHODS: Mitochondrial functionality, ER stress signaling, autophagy induction, and apoptotic cell death between rt269L-type and rt269I-type groups were investigated via in vitro and in vivo experiments. Serum samples were collected from 187 chronic hepatitis patients who visited Konkuk or Seoul National University Hospital. RESULTS: Our data revealed that genotype C rt269L versus rt269I infection led to improved mitochondrial dynamics and enhanced autophagic flux, mainly due to the activation of the PERK-eIF2α-ATF4 axis. Furthermore, we demonstrated that the traits found in genotype C rt269L infection were mainly due to increased stability of the HBx protein after deubiquitination. In addition, clinical data using patient sera from two independent Korean cohorts showed that, compared with rt269I, rt269L in infection led to lower 8-OHdG levels, further supporting its improved mitochondrial quality control. CONCLUSION: Our data showed that, compared with the rt269I type, the rt269L type, which presented exclusively in HBV genotype C infection, leads to improved mitochondrial dynamics or bioenergetics, mainly due to autophagy induction via activation of the PERK-eIF2α-ATF4 axis in an HBx protein-dependent manner. This suggests that HBx stability and cellular quality control in the rt269L type predominating in genotype C endemic areas could at least partly contribute to some distinctive traits of genotype C infection, such as higher infectivity or longer duration of the hepatitis B e antigen (HBeAg) positive stage.

Global prevalence and molecular characteristics of three clades within hepatitis B virus subgenotype C2: Predominance of the C2(3) clade in South Korea.

Hepatitis B Virus (HBV) genotypes reflect geographic, ethical or clinical traits and are currently divided into 10 genotypes (A-J). Of these, genotype C is mainly distributed in Asia, is the largest group and comprises more than seven subgenotypes (C1-C7). Subgenotype C2 is divided into three phylogenetically distinct clades, C2(1), C2(2), and C2(3), and is responsible for most genotype C infections in three East Asian nations, including China, Japan, and South Korea, which are major HBV endemic areas. However, despite the significance of subgenotype C2 with regard to clinical or epidemiologic aspects, its global distribution and molecular characteristics remain largely unknown. Here, we analyze the global prevalence and molecular characteristics between 3 clades within subgenotype C2 using 1,315 full genome sequences of HBV genotype C retrieved from public databases. Our data show that almost all HBV strains from South Korean patients infected with genotype C belong to clade C2(3) within subgenotype C2 [96.3%] but that HBV strains from Chinese or Japanese patients belong to diverse subgenotypes or clades within genotype C, suggesting clonal expansion of a specific HBV type, C2(3), among the Korean population. Our genome sequence analysis indicated a total of 21 signature sequences specific to the respective clades C2(1), C2(2), and C2(3). Of note, two types of four nonsynonymous C2(3) signature sequences, sV184A in HBsAg and xT36P in the X region, were detected in 78.9 and 82.9% of HBV C2(3) strains, respectively. In particular, HBV strains C2(3) versus C2(1) and C2(2) show a higher frequency of reverse transcriptase mutations related to nucleot(s)ide analog (NA) resistance, including rtM204I and rtL180M, suggesting an increased possibility of C2(3) infection in those with NA treatment failure. In conclusion, our data show that HBV subgenotype C2(3) is extremely prevalent in Korean patients with chronic HBV infection, which is distinct from two other East Asian nations, China and Japan, where diverse subgenotypes or clades within genotype C coexist. This epidemiologic trait might affect distinct virological and clinical traits in chronic HBV patients in Korea, where exclusively C2(3) infection is predominant.

Structural identification of the lipopolysaccharide-binding capability of a cupin-family protein from Helicobacter pylori.

We solved the crystal structure of a functionally uncharacterized protein, HP0902, from Helicobacter pylori. Its structure demonstrated an all-ß cupin fold that cannot bind metal ions due to the absence of a metal-binding histidine that is conserved in many metallo-cupins. In contrast, isothermal titration calorimetry and NMR titration demonstrated that HP0902 is able to bind bacterial endotoxin lipopolysaccharides (LPS) through its surface-exposed loops, where metal-binding sites are usually found in other metallo-cupins. This report constitutes the first identification of an LPS-interacting protein, both in the cupin family and in H. pylori. Furthermore, identification of the ability of HP0902 to bind LPS uncovers a putative role for this protein in H. pylori pathogenicity.

The effects of an electronic medical record on the completeness of documentation in the anesthesia record.

OBJECTIVES: The purpose of this study is to evaluate the completeness of anesthesia recording before and after the introduction of an electronic anesthesia record. METHODS: The study was conducted in a Korean teaching hospital where the EMR was implemented in October 2008. One hundred paper anesthesia records from July to September 2008 and 150 electronic anesthesia records during the same period in 2009 were randomly sampled. Thirty-four essential items were selected out of all the anesthesia items and grouped into automatically transferred items and manual entry items. 1, .5 and 0 points were given for each item of complete entry, incomplete entry and no entry respectively. The completeness of documentation was defined as the sum of the scores. The influencing factors on the completeness of documentation were evaluated in total and by the groups. RESULTS: The average completeness score of the electronic anesthesia records was 3.15% higher than that of the paper records. A multiple regression model showed the type of the anesthesia record was a significant factor on the completeness of anesthesia records in all items (ß=.98, p<.05) and automatically transferred items (ß=.56, p<.01). The type of the anesthesia records had no influence on the completeness in manual entry items. CONCLUSIONS: The completeness of an anesthesia record was improved after the implementation of the electronic anesthesia record. The reuse of the data from the EMR was the main contributor to the improved completeness.

Development of physicochemical nitrogen removal process for high strength industrial wastewater.

The biological nitrogen removal (BNR) process is the most common method for removing low quantities of ammonium from wastewater, but this method is not used for wastewater rich with ammonium. In this study, we have developed the physicochemical nitrogen removal process to remove the nitrogen content for a real industrial wastewater. The denitrification was accomplished by the physicochemical denitrification process using zinc powder and sulfamic acid in pH 2-4. The experimental parameters were examined by varying various reaction conditions such as pH, zinc feeding time, amount of sulfamic acid, and amount of nitrate concentration. For each experimental condition, the physicochemical denitrification process was determined by pH, amount of zinc powder and sulfamic acid, zinc feeding time and nitrate concentration.