Prominent transcriptomic changes in Mycobacterium intracellulare under acidic and oxidative stress.

BACKGROUND: Mycobacterium avium complex (MAC), including Mycobacterium intracellulare is a member of slow-growing mycobacteria and contributes to a substantial proportion of nontuberculous mycobacterial lung disease in humans affecting immunocompromised and elderly populations. Adaptation of pathogens in hostile environments is crucial in establishing infection and persistence within the host. However, the sophisticated cellular and molecular mechanisms of stress response in M. intracellulare still need to be fully explored. We aimed to elucidate the transcriptional response of M. intracellulare under acidic and oxidative stress conditions. RESULTS: At the transcriptome level, 80 genes were shown [FC] ≥ 2.0 and p < 0.05 under oxidative stress with 10 mM hydrogen peroxide. Specifically, 77 genes were upregulated, while 3 genes were downregulated. In functional analysis, oxidative stress conditions activate DNA replication, nucleotide excision repair, mismatch repair, homologous recombination, and tuberculosis pathways. Additionally, our results demonstrate that DNA replication and repair system genes, such as dnaB, dinG, urvB, uvrD2, and recA, are indispensable for resistance to oxidative stress. On the contrary, 878 genes were shown [FC] ≥ 2.0 and p < 0.05 under acidic stress with pH 4.5. Among these genes, 339 were upregulated, while 539 were downregulated. Functional analysis highlighted nitrogen and sulfur metabolism pathways as the primary responses to acidic stress. Our findings provide evidence of the critical role played by nitrogen and sulfur metabolism genes in the response to acidic stress, including narGHIJ, nirBD, narU, narK3, cysND, cysC, cysH, ferredoxin 1 and 2, and formate dehydrogenase. CONCLUSION: Our results suggest the activation of several pathways potentially critical for the survival of M. intracellulare under a hostile microenvironment within the host. This study indicates the importance of stress responses in M. intracellulare infection and identifies promising therapeutic targets.

Parabacteroides leei sp. nov., isolated from human blood.

An obligate anaerobic, Gram-negative, rod-shaped and non-spore-forming bacterium, designated as strain GYB001T, was isolated from the blood of a patient with a sigmoid colon perforation. Taxonomic characterization of the novel isolate was performed using a polyphasic approach. A phylogenetic analysis based on 16S rRNA gene and whole genome sequences revealed that GYB001T represented a member of the genus Parabacteroides, in the family Tannerellaceae. The closest species, based on 16S rRNA sequence, was Parabacteroides gordonii DSM 23371T with 97.4 % similarity. Average nucleotide identity and digital DNA-DNA hybridization values between strain GYB001T and P. gordonii DSM 23371T were 86.7 and 28.7% and between GYB001T and Parabacteroides faecis JCM 18682T were 86.6 and 27.7 %, respectively. The genome was 6.57 Mbp long with 43.3 mol% G+C content. Colonies on Brucella blood agar (BBA) were circular, convex, smooth, grey and small in size. Growth was observed on trypticase soy agar (TSA), TSA +5 % sheep blood and Euglena gracilis agar. Growth occurred at 18-42 °C on BBA in the presence of 0-3 % NaCl (w/v) and at pH 6.0-8.5. The major polar lipids were phosphatidylethanolamine and phospholipids. The major fatty acids in strain GYB001T were anteiso-C15 : 0 and iso-C17 : 0 3-OH, and the predominant respiratory quinones were menaquinone-10 (MK-10) and MK-9. The cell wall contained meso-diaminopimelic acid. Considering these phenotypic features and comparative genome analyses, we propose strain GYB001T as the type strain of Parabacteroides leei sp. nov. (=KCTC 25738T=KBN12P06525T=LMG 32797T).

Comparative genetic characterization of CMY-2-type beta-lactamase producing pathogenic Escherichia coli isolated from humans and pigs suffering from diarrhea in Korea.

BACKGROUND: Pathogenic Escherichia coli are an important cause of bacterial infections in both humans and pigs and many of antimicrobials are used for the treatment of E. coli infection. The objective of this study was to investigate the characteristics and relationship between humans and pigs regarding third-generation cephalosporin resistance and CMY-2-producing E. coli in Korea. RESULTS: All 103 third-generation cephalosporin-resistant E. coli isolates showed multidrug resistance. Also, except for ß-lactam/ß-lactamase inhibitor combinations, all antimicrobials resistant rates were higher in pigs than in humans. A total of 36 isolates (humans: five isolates; pigs: 31 isolates) were positive for the CMY-2-encoding genes and thirty-two (88.9%) isolates detected class 1 integrons with 10 different gene cassette arrangements, and only 1 isolate detected a class 2 integron. The most common virulence genes in pigs were LT (71.0%), F18 (51.6%), and STb (51.6%), while stx2 (80.0%) was the most frequently detected gene in humans. Stx2 gene was also detected in pigs (6.5%). Interestingly, 36 CMY-2-producing E. coli isolates showed a high diversity of sequence types (ST), and ST88 was present in E. coli from both pigs (11 isolates) and humans (one isolate). CONCLUSION: Our findings suggest that a critical need for comprehensive surveillance of third-generation cephalosporin resistance is necessary to preserve the usefulness of third-generation cephalosporins in both humans and pigs.

Fetuin as a potential serum biomarker to detect subclinical shedder of bovine paratuberculosis.

Paratuberculosis (PTB) is a chronic contagious granulomatous enteritis of wild and domestic ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). PTB causes considerable economic losses to the dairy industry through decreased milk production and premature culling. PTB-affected cattle undergo a subclinical stage without clinical signs and initiate fecal shedding of MAP into the environment. Current diagnostic tools have low sensitivity for the detection of subclinical PTB infection. Therefore, alternative diagnostic tools are required to improve the diagnostic sensitivity of subclinical PTB infection. In this study, we performed ELISA for three previously identified host biomarkers (fetuin, alpha-1-acid glycoprotein, and apolipoprotein) and analyzed their diagnostic performance with conventional PTB diagnostic methods. We observed that serum fetuin levels were significantly lowered in the subclinical shedder and clinical shedder groups than in the healthy control group, indicating its potential utility as a diagnostic biomarker for bovine PTB. Also, fetuin showed an excellent discriminatory power with an AUC = 0.949, a sensitivity of 92.6%, and a specificity of 94.4% for the detection of subclinical MAP infection. In conclusion, our results demonstrated that fetuin could be used as a diagnostic biomarker for enhancing the diagnostic sensitivity for the detection of subclinical MAP infections that are difficult to detect based on current diagnostic methods.

Endoplasmic Reticulum Stress and Impairment of Ribosome Biogenesis Mediate the Apoptosis Induced by Ocimum x africanum Essential Oil in a Human Gastric Cancer Cell Line.

Background and Objectives: Gastric cancer remains a major unmet clinical problem worldwide. Although conventional medical treatments are available, their curative effects are generally unsatisfactory. Consequently, it remains necessary to search natural products for potential alternatives in treating gastric cancer patients. Ocimum x africanum Lour. is a culinary herb that has been used in folk medicine for various diseases, but little is known regarding its anti-cancer activity against gastric cancer cells. In the current study, we focus on the anti-cancer mechanisms of O. x africanum essential oil (OAEO) in the AGS human gastric cancer cell line. Materials and Methods: After OAEO treatment, AGS cell viability was evaluated by MTT assay. Cell migration and apoptotic nuclear morphology were determined by wound-healing assay and DAPI staining, respectively. Gene expression levels of apoptosis-related genes were quantified by qRT-PCR. Differential protein expression was determined with an LC-MS/MS-based proteomics approach to identify the key proteins that may be important in the anti-cancer mechanisms of OAEO on AGS cells. The chemical constituents of OAEO were identified by GC-MS analysis. Results: We found OAEO to exhibit a potent growth-inhibiting effect on AGS cells, with an IC50 value of 42.73 µg/mL. After OAEO treatment for 24 h, AGS cell migration was significantly decreased relative to the untreated control. OAEO-treated AGS cells exhibited common features of apoptotic cell death, including cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. Apoptotic cell death was confirmed by qRT-PCR for apoptosis-related genes, revealing that OAEO decreased the expression of anti-apoptotic genes (BCL2 and BCL-xL) and activated pro-apoptotic genes and apoptotic caspase genes (TP53, BAX, CASP9, CASP12, and CASP3). Moreover, expression of CASP8 was not changed after treatment. Proteomic analysis revealed that OAEO may produce a signature effect on protein clusters relating to unfolded protein accumulation, thereby inducing severe ER stress and also impairing ribosome synthesis. STRING analysis revealed seven up-regulated and 11 down-regulated proteins, which were significantly associated with protein folding and ribosome biogenesis, respectively. Using GC-MS analysis, 6-methyl-5-hepten-2-one, citral, neral, and linalool were found to be the major chemical constituents in OAEO. Conclusions: Taken together, these results indicate that OAEO has a potential anti-proliferative effect on AGS cells. Our molecular findings show evidence supporting an important role of ER stress and ribosome biogenesis impairment in mediating the induction of cell death by OAEO through the mitochondrial-apoptotic pathway. This study, therefore, provides fundamental knowledge for future applications using OAEO as an alternative therapy in gastric cancer management.

Cohnella cholangitidis sp. nov., a novel species of the genus Cohnella isolated from a clinical specimen in Korea.

A Gram-positive, aerobic, rod-shaped bacterium, designated as strain 1605-214T, was isolated from the blood sample of a patient with cholangitis. Based on its 16S rRNA gene sequence, the strain 1605-214T belonged to the genus Cohnella and exhibited 97.9% sequence identity with Cohnella luojiensis DSM 24270T (GQ214052). DNA-DNA hybridization, digital DNA-DNA hybridization, and average nucleotide identity values between the two species were 23% ± 1.9, 21.1%, and 77.2%, respectively. The cellular fatty acids of strain 1605-214T were mainly comprised of anteiso-C15:0 (36.1%), iso-C16:0 (16.5%), and C16:0 (15.1%). The predominant quinone was menaquinone-7; predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and aminophospholipid-1. The cell wall peptidoglycan of strain 1605-214T contained meso-diaminopimelic acid. DNA G + C content of strain 1605-214T was 50.6 mol%. 5187 genes out of a total of 5413 (94.6%) were assigned putative functions using eggNOG v5.0. Based on genotypic characteristics and genomic sequence analysis results, strain 1605-214T was confirmed to represent a novel species of genus Cohnella, for which the name Cohnella cholangitidis sp. nov., was proposed.

Heterogeneity of Helicobacter pylori bab genotypes and their association with clinical outcomes in Korean gastroduodenal patients.

In this study, we aimed to investigate the prevalence of bab genes (babA, babB, babC) at their three loci (loci A, B, and C) in Helicobacter pylori strains from varied clinical manifestations of Korean gastroduodenal patients. The overall prevalence of H. pylori Korean strains positive for babA and babB was 91.1% and 92.2%, respectively, but all strains were negative for bab C. H. pylori strains with two loci occupied (loci A and B) were the most prevalent in Korean patients (85.6%), compared to one locus occupied (14.4%) (locus A or B). Twelve bab genotypes were detected, additionally, the distribution of three bab genotypes was significantly associated with different clinical outcomes among Korean patients. The genotypes babA/babB/- and babA/babA+babB/- were significantly associated with peptic ulcer disease (PUD) (63.3%) and gastritis (GT) (33.3%) patients, respectively. In addition, we found that the babA+babB/babA+babB/- genotype was significantly associated with gastric cancer (GC) (36.7%) as compared to GT (6.7%) or PUD (6.7%) (p<0.05) patients. This study provided evidence that the bab genotypes in H. pylori Korean strains were highly variable. Interestingly, three patterns of bab genotypes were significantly different among patients with different clinical outcomes in the population at high-risk for GC.

An In Vitro Anti-Cancer Activity of Ocimum tenuiflorum Essential Oil by Inducing Apoptosis in Human Gastric Cancer Cell Line.

Background and Objectives: The effects of Ocimum tenuiflorum essential oil (OTEO) against gastric cancer remain unknown and merit investigation. Materials and Methods: In the present study, the anti-cancer activity of OTEO was examined in a human gastric cancer cell line (AGS). After OTEO treatment, AGS cell viability was determined by an MTT assay, and inhibition of metastasis was determined by cell migration and invasion assays. The expression of apoptosis-related genes in treated AGS cells was determined by qRT-PCR. Results: OTEO significantly decreased AGS cell viability in a dose-dependent manner (IC50 163.42 µg/mL) and effectively inhibited cell migration and invasion. Morphological examination demonstrated that OTEO induced cell shrinkage, chromatin condensation, and fragmentation, which are considered typical morphologies of apoptotic cell death. Pro-apoptotic genes (TP53, BAX, and BAK) were significantly up-regulated, while anti-apoptotic genes (BCL-2 and BCL-xL) were significantly down-regulated after treatment with OTEO. In addition, significantly increased gene expression was detected for CASP8, CASP9, and CASP3 in AGS cells exposed to OTEO. GC-MS analysis demonstrated that the major compound of OTEO was caryophyllene (25.85%) and α-pinene (11.66%). Conclusions: This in vitro study demonstrates for the first time that OTEO has potential anti-gastric cancer activity and may induce apoptosis in AGS cells through extrinsic and intrinsic pathways.

Identification of Nontuberculous Mycobacteria in Patients with Pulmonary Diseases in Gyeongnam, Korea, Using Multiplex PCR and Multigene Sequence-Based Analysis.

BACKGROUND: Nontuberculous mycobacteria (NTM) are widely present in environments, such as soil and water, and have recently been recognized as important pathogenic bacteria. The incidence of NTM-related infections is steadily increasing. As the diagnosis and treatment of NTM infection should be distinguished from tuberculosis, and the treatment should be specific to the species of NTM acquired, accurate species identification is required. METHODS: In this study, two-step multiplex PCR (mPCR) and multigene sequence-based analysis were used to accurately identify NTM species in 320 clinical isolates from Gyeongsang National University Hospital (GNUH). In particular, major mycobacterial strains with a high isolation frequency as well as coinfections with multiple species were diagnosed through two-step mPCR. Multigene sequencing was performed to accurately identify other NTM species not detected by mPCR. Variable regions of the genes 16S rRNA, rpoB, hsp65, and 16S-23S rRNA internal transcribed spacer were included in the analysis. RESULTS: Two-step mPCR identified 234 (73.1%) cases of M. intracellulare, 26 (8.1%) cases of M. avium subsp. avium, and 13 (4.1%) cases of M. avium subsp. hominissuis infection. Additionally, 9 (2.8%) M. fortuitum, 9 (2.8%) M. massiliense, 2 (0.6%) M. abscessus, and 4 (1.2%) M. kansasii isolates were identified. Coinfection was identified in 7 (2.2%) samples. The sixteen samples not classified by two-step mPCR included 6 (1.9%) cases of M. chimaera, 4 (1.3%) M. gordonae, 1 (0.3%) M. colombiense, 1 (0.3%) M. mageritense, and 1 (0.3%) M. persicum identified by sequence analysis. CONCLUSIONS: The results of this study suggest a strategy for rapid detection and accurate identification of species using two-step mPCR and multigene sequence-based analysis. To the best of our knowledge, this study is the first to report the identification of NTM species isolated from patients in Gyeongnam/Korea.

Changes in Seroprevalence of Helicobacter pylori Infection over 20 Years in Jinju, Korea, from Newborns to the Elderly.

BACKGROUND: The objective of this study was to examine changes in the prevalence of cytotoxic-associated gene A (CagA) positive Helicobacter pylori infection in Jinju, Korea, over the last 20 years. METHODS: Three cross-sectional analyses were conducted concurrently. A total of 1,305 serum samples were collected from 1994-1995, 2004-2005, and 2014-2015, respectively. The presence of immunoglobulin (Ig) G, IgA, and IgM antibodies against H. pylori CagA protein was examined by western blotting. RESULTS: Overall, seropositivity for anti-CagA IgG antibody was significantly decreased from 63.2% to 42.5% over the last 20 years (P < 0.001). Anti-CagA IgG seropositivities in children and young adults aged 10-29 years decreased from 1994 (60.0%-85.0%) to 2015 (12.5%-28.9%). The age when plateau of increasing IgG seropositivity was reached in each study period shifted from the 15-19 year-old group in 1994-1995 (85.0%) to the 40-49 year-old group in 2014-2015 (82.5%). Overall seropositive rates of anti-CagA IgA and IgM antibodies did not change significantly either over the last 20 years. CONCLUSION: H. pylori infection rate in children and young adults declined over 20 years in Jinju, probably due to improved sanitation, housing, or economy.

Anti-Biofilm Effects of Synthetic Antimicrobial Peptides Against Drug-Resistant Pseudomonas aeruginosa and Staphylococcus aureus Planktonic Cells and Biofilm.

Biofilm-associated infections are difficult to manage or treat as biofilms or biofilm-embedded bacteria are difficult to eradicate. Antimicrobial peptides have gained increasing attention as a possible alternative to conventional drugs to combat drug-resistant microorganisms because they inhibit the growth of planktonic bacteria by disrupting the cytoplasmic membrane. The current study investigated the effects of synthetic peptides (PS1-2, PS1-5, and PS1-6) and conventional antibiotics on the growth, biofilm formation, and biofilm reduction of drug-resistant Pseudomonas aeruginosa and Staphylococcus aureus. The effects of PS1-2, PS1-5, and PS1-6 were also tested in vivo using a mouse model. All peptides inhibited planktonic cell growth and biofilm formation in a dose-dependent manner. They also reduced preformed biofilm masses by removing the carbohydrates, extracellular DNA, and lipids that comprised extracellular polymeric substances (EPSs) but did not affect proteins. In vivo, PS1-2 showed the greatest efficacy against preformed biofilms with no cytotoxicity. Our findings indicate that the PS1-2 peptide has potential as a next-generation therapeutic drug to overcome multidrug resistance and to regulate inflammatory response in biofilm-associated infections.

Cell-selectivity of tryptophan and tyrosine in amphiphilic α-helical antimicrobial peptides against drug-resistant bacteria.

The increasing emergence of drug-resistant bacteria creates a requirement for new antibiotics and various types of antibiotic materials such as proteins, peptides, polymers, and chemical compounds. Among these, antimicrobial peptides (AMPs) are considered to be promising antibiotic candidates for clinical treatments. In this study, we have designed a novel series of peptides with repeated sequences of minimum membrane-active motif, 'XWZX' basic sequence (X: lysine or arginine, Z: leucine, tyrosine, valine, or glycine), and an α-helical secondary structure. Some peptides displayed a potent antibacterial activity via membranolytic action and high therapeutic index (toxic dose/minimum inhibitory concentration) in vitro. Furthermore, in vivo experiments using bacterial ear-skin infection models verified that these peptides have the potential to be powerful and safe antibiotics. The present study provides a lead sequence for designing peptide antibiotics against bacterial membranes and information for cell-selectivity of hydrophobic amino acids with aromatic side chains such as Trp and Tyr.

ArmA and RmtB Were the Predominant 16S RMTase Genes Responsible for Aminoglycoside-resistant Isolates in Korea.

Pathogenic gram-negatives that produce 16S ribosomal RNA methyltransferases (16S RMTases) have already been distributed all over the world. To investigate the predominance of aminoglycoside resistance associated with 16S RMTases in Korea, we collected a total of 222 amikacin resistant Gram-negative clinical isolates from patient specimens between 1999 and 2015 from three hospital banks across Korea. ArmA and rmtB were the predominant 16S RMTase genes responsible for aminoglycoside-resistant isolates circulating in Korean community settings although only one rmtA-producing isolate was detected in 2006.

Helicobacter pylori Antigens Inducing Early Immune Response in Infants.

To identify the Helicobacter pylori antigens operating during early infection in sera from infected infants using proteomics and immunoblot analysis. Two-dimensional (2D) large and small gel electrophoresis was performed using H. pylori strain 51. We performed 2D immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) antibody immunoblotting using small gels on sera collected at the Gyeongsang National University Hospital from 4-11-month-old infants confirmed with H. pylori infection by pre-embedding immunoelectron microscopy. Immunoblot spots appearing to represent early infection markers in infant sera were compared to those of the large 2D gel for H. pylori strain 51. Corresponding spots were analyzed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS). The peptide fingerprints obtained were searched in the National Center for Biotechnology Information (NCBI) database. Eight infant patients were confirmed with H. pylori infection based on urease tests, histopathologic examinations, and pre-embedding immunoelectron microscopy. One infant showed a 2D IgM immunoblot pattern that seemed to represent early infection. Immunoblot spots were compared with those from whole-cell extracts of H. pylori strain 51 and 18 spots were excised, digested in gel, and analyzed by MALDI-TOF-MS. Of the 10 peptide fingerprints obtained, the H. pylori proteins flagellin A (FlaA), urease ß subunit (UreB), pyruvate ferredoxin oxidoreductase (POR), and translation elongation factor Ts (EF-Ts) were identified and appeared to be active during the early infection periods. These results might aid identification of serological markers for the serodiagnosis of early H. pylori infection in infants.

Dermabacter jinjuensis sp. nov., a novel species of the genus Dermabacter isolated from a clinical specimen.

A Gram-stain-positive, catalase-positive, facultatively anaerobic, non-motile, coryneform bacterium, designated strain 32T, was isolated from a closed pus sample from a patient having finger necrosis in Korea. Strain 32T was considered as representing a novel species according to its initial identification by matrix-assisted laser desorption/ionization-time-of-flight MS. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 32T belonged to the genus Dermabacter and was closely related to Dermabacter hominis DSM 7083T (=ATCC 49369T) (98.34 % similarity). Optimal growth was observed at 30-40 °C and pH 7. Growth occurred in the presence of 0-6 % (w/v) NaCl. Menaquinones MK-8, MK-7 and MK-9 were the major respiratory quinones. The major polar lipids were phosphatidylethanolamine, phosphatidylcholine, glycolipid and two unknown lipids. The major cellular fatty acids were anteiso-C17 : 0, anteiso-C15 : 0, iso-C16 : 0 and iso-C15 : 0. The DNA G+C content of strain 32T was 62.58 mol%, and the mean level of DNA-DNA relatedness between strain 32T and D. hominis ATCC 49369T was 49±1.6 %. Based on the phenotypic and genotypic characteristics, strain 32T is confirmed to represent a novel species of the genus Dermabacter, for which the name Dermabacter jinjuensis sp. nov. is proposed. The type strain is 32T (=NCCP 16133T=DSM 101003T).

Helicobacter pylori HP0425 Targets the Nucleus with DNase I-Like Activity.

BACKGROUND AND AIMS: Nuclear targeting of bacterial proteins has a significant impact on host cell pathology. Helicobacter pylori have many nuclear targeting proteins that translocate into the nucleus of host cells. H. pylori HP0425, annotated as hypothetical, has a nuclear localization signal (NLS) sequence, but its function has not been demonstrated. The aim of this experiment was to address the nuclear translocation of HP0425 and determine the effect of HP0425 pathology on host cells. MATERIALS AND METHODS: To investigate the nuclear localization of HP0425, it was expressed in AGS and MKN-1 cells as a GFP fusion protein (pEGFP-HP0425), and its localization was analyzed by confocal microscopy. Recombinant HP0425 (rHP0425) protein was overproduced as a GST fusion protein in Escherichia coli and purified by glutathione-affinity column chromatography. Purified rHP0425 was examined for cytotoxicity and DNase activity. RESULTS: The pEGFP-HP0425 fluorescence was expressed in the nucleus and cytosol fraction of cells, while it was localized in the cytoplasm in the negative control. This protein exhibited DNase activity under various conditions, with the highest DNase activity in the presence of manganese. In addition, the rHP0425 protein efficiently decreased cell viability in a concentration-dependent manner. CONCLUSIONS: These results suggest that HP0425 carrying a nuclear localization signal sequence translocates into the nucleus of host cells and degrades genomic DNA by DNase I-like enzymatic activity, which is a new pathogenic strategy of H. pylori in the host.

Correlations between the CagA Antigen and Serum Levels of Anti-Helicobacter pylori IgG and IgA in Children.

We tested correlations between anti-Helicobacter pylori IgG and IgA levels and the urease test, anti-CagA protein antibody, degree of gastritis, and age. In total, 509 children (0-15 years) were enrolled. Subjects were stratified as 0-4 years (n = 132), 5-9 years (n = 274), and 10-15 years (n = 103) and subjected to the urease test, histopathology, ELISA, and western blot using whole-cell lysates of H. pylori strain 51. The positivity rate in the urease test (P = 0.003), the degree of chronic gastritis (P = 0.021), and H. pylori infiltration (P < 0.001) increased with age. The median titer for anti-H. pylori IgG was 732.5 IU/mL at 0-4 years, 689.0 IU/mL at 5-9 years, and 966.0 IU/mL at 10-15 years (P < 0.001); the median titer for anti-H. pylori IgA was 61.0 IU/mL at 0-4 years, 63.5 IU/mL at 5-9 years, and 75.0 IU/mL at 10-15 years (P < 0.001). The CagA-positivity rate was 26.5% at 0-4 years, 36.5% at 5-9 years, and 46.6% at 10-15 years for IgG (P = 0.036), and 11.3% at 0-4 years, 18.6% at 5-9 years, and 23.3% at 10-15 years for IgA (P < 0.001). Anti-H. pylori IgG and IgA titers increased with the urease test grade, chronic gastritis degree, active gastritis, and H. pylori infiltration. Presence of CagA-positivity is well correlated with a high urease test grade and high anti-H. pylori IgG/IgA levels.

Immunohistochemical Expressions of MUC2, MUC5AC, and MUC6 in Normal, Helicobacter pylori Infected and Metaplastic Gastric Mucosa of Children and Adolescents.

OBJECTIVES: The aim of this study was to investigate expression of gastric mucins in children and adolescents and to assess their relations with age and Helicobacter pylori (H. pylori) infection. METHODS: Gastric biopsies were collected from 259 pediatric and adulthood patients with gastrointestinal symptoms among all of patients undergone gastroduodenoscopy from 1990 to 2004 at Gyeongsang National University hospital and assorted based on H. pylori infection, age, and intestinal metaplasia as follows; H. pylori infection before 5 years of age or not, H. pylori infection between 5 and 9 years of age or not, H. pylori infection between 10 and 14 years of age or not, H. pylori infection between 20 and 29 years of age or not and intestinal metaplasia between 21 and 35 years of age. Total 810 tissue slides from the subjects were examined regarding expressions of Mucin2 (MUC2), Mucin5AC (MUC5AC), and Mucin6 (MUC6) in nine groups using immunohistochemical stains. A semiquantitative approach was used to score the staining extent of tissue slide. RESULTS: Increased expressions of MUC2, MUC5AC, and MUC6 were noted in intestinal metaplasia compared with subjects infected with H. pylori between 20 and 29 years. Gastric expressions of MUC5AC were decreased in older than 5 years with H. pylori compared with in older than 5 years without H. pylori (p < .001). Expressions of MUC2 and MUC6 did not change significantly by H. pylori status. Some nuclear expressions of MUC2 and MUC6 were noted in children without intestinal metaplasia. CONCLUSIONS: MUC5AC might be affected by chronic H. pylori infection. In addition to biomarkers for intestinal metaplasia or prognostic factors for gastric cancer in adults, MUC2 and MUC6 in children might have an another role, based on ectopic gastric nuclear expressions of MUC2 and MUC6 in children without intestinal metaplasia.

Helicobacter pylori γ-glutamyl transpeptidase-induced Ca(2+) release via PLC-IP3 receptors in AGS cells.

In our previous study, γ-glutamyl transpeptidase (GGT) isolated from Helicobacter pylori induced apoptosis of AGS cells. Here, we investigate Ca(2+) effects on GGT-induced apoptosis. The GGT transiently and significantly increased intracellular Ca(2+) concentration ([Ca(2+)]i) in AGS cells in a dose-dependent manner (P < 0.05). The GGT-induced Ca(2+) increase resulted from Ca(2+) influx and release through the phospholipase C - inositol 1,4,5-trisphosphate (PLC-IP3) pathway. The GGT-induced apoptosis was significantly reduced by treatment with U73122 (a PLC inhibitor) and xestospongin (an IP3 receptor antagonist) (P < 0.05). These results indicate that GGT could induce apoptosis of AGS cells by high levels of [Ca(2+)]i.

Correlation between positive rate and number of biopsy samples on urease test in childhood Helicobacter pylori infection.

To identify the correlation between the number of gastric biopsy samples and the positive rate, we compared the results of urease test using one and three biopsy samples from each 255 children who underwent gastroduodenoscopy at Gyeongsang National University Hospital. The children were divided into three age groups: 0-4, 5-9, and 10-15 yr. The gastric endoscopic biopsies were subjected to the urease test. That is, one and three gastric antral biopsy samples were collected from the same child. The results of urease test were classified into three grades: Grade 0 (no change), 1 (6-24 hr), 2 (1-6 hr), and 3 (<1 hr). The positive rate of urease test was increased by the age with no respect to the number of gastric biopsy samples (one biopsy P = 0.001, three biopsy P < 0.001). The positive rate of the urease test was higher on three biopsy samples as compared with one biopsy sample (P < 0.001). The difference between one and three biopsy samples was higher in the children aged 0-9 yr. Our results indicate that the urease test might be a more accurate diagnostic modality when it is performed on three or more biopsy samples in children.