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.

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.

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.

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.

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.

Identification of Autoantigens in Pediatric Gastric Juices.

Purpose: This study aimed to investigate the presence of autoantigens in the gastric juices of children. Methods: Gastric juice and serum samples were obtained from 53 children <15 years of age who underwent gastric endoscopy. Among these, 8, 22, and 23 participants were in the age groups 0-5, 6-10, and 11-15 years, respectively. These samples were analyzed using two-dimensional electrophoresis (2-DE), immunoblot analysis, and matrix-assisted laser desorption ionization-time of-flight mass spectrometry. Furthermore, we reviewed the histopathological findings and urease test results and compared them with the results of 2-DE and immunoblot analysis. Results: There were no statistically significant differences in urease test positivity, grades of chronic gastritis, active gastritis, or Helicobacter pylori infiltration of the antrum and body among the three age groups. Three distinct patterns of gastric juice were observed on 2-DE. Pattern I was the most common, and pattern III was not observed below the age of 5 years. Histopathological findings were significantly different among active gastritis (p=0.037) and H. pylori infiltration (p=0.060) in the gastric body. The immunoblots showed large spots at an approximate pH of 3-4 and molecular weights of 31-45 kDa. These distinct, large positive spots were identified as gastric lipase and pepsin A and C. Conclusion: Three enzymes, which are normally secreted under acidic conditions were identified as autoantigens. Further investigation of the pathophysiology and function of autoantigens in the stomach is required.

Relationship between headache and mucosal mast cells in pediatric Helicobacter pylori-negative functional dyspepsia.

BACKGROUND: Although many patients with functional dyspepsia experience headache concurrently with dyspeptic symptoms, studies suggesting mechanisms underlying this phenomenon are limited. Herein, we explore the relationship between gastrointestinal inflammatory cells and presence of headache associated with dyspeptic symptoms in children with HELICOBACTER PYLORI -negative functional dyspepsia. METHODS: Fifty-six patients with H. PYLORI -negative functional dyspepsia underwent upper endoscopy with biopsy to investigate recurrent epigastric pain or discomfort. Patients were divided into two groups according to self-reported presence of headache associated with dyspeptic symptoms. Inflammatory cells including mast cells, and enteroendocrine cells in the gastroduodenal mucosa were evaluated. Associations between headache presence and cellular changes in the gastroduodenal mucosa were examined. RESULTS: Headache was not associated with the grade of lymphocytes, neutrophil infiltration, or enteroendocrine cell density in the gastroduedenal mucosa. However, headache was significantly associated with high mast cell density in the body (27.81 ± 8.71 vs. 20.30 ± 8.16, P < 0.01) and duodenum (23.16 ± 10.40 vs. 14.84 ± 5.88, P < 0.01). CONCLUSIONS: Presence of headache associated with dyspeptic symptoms is strongly related to mucosal mast cell density in pediatric patients with H. PYLORI -negative functional dyspepsia. Thus, our results may help clinicians understand and treat headache during dyspeptic symptoms in such pediatric patients.

Anthocyanins from black soybean inhibit Helicobacter pylori-induced inflammation in human gastric epithelial AGS cells.

Infection with Helicobacter pylori leads to gastritis, peptic ulcers and gastric cancer. Moreover, when the gastric mucosa is exposed to H. pylori, gastric mucosal inflammatory cytokine interleukin-8 (Il-8) and reactive oxygen species increase. Anthocyanins have anti-oxidative, antibacterial and anti-inflammatory properties. However, the effect of anthocyanins in H. pylori-infected cells is not yet clear. In this study, therefore, the effect of anthocyanins on H. pylori-infected human gastric epithelial cells was examined. AGS cells were pretreated with anthocyanins for 24 hrs followed by H. pylori 26695 infection for up to 24 hrs. Cell viability and ROS production were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 2',7'-dichlorofluorescein diacetate assay, respectively. Western blot analyses and RT-PCR were performed to assess gene and protein expression, respectively. IL-8 secretion in AGS cells was measured by ELISA. It was found that anthocyanins decrease H. pylori-induced ROS enhancement. Anthocyanins also inhibited phosphorylation of mitogen-activated protein kinases, translocation of nuclear factor-kappa B and Iκßα degradation. Furthermore anthocyanins inhibited H. pylori-induced inducible nitric oxide synthases and cyclooxygenase-2 mRNA expression and inhibited IL-8 production by 45.8%. Based on the above findings, anthocyanins might have an anti-inflammatory effect in H. pylori-infected gastric epithelial cells.

Changing pattern of antibiotic resistance of Helicobacter pylori in children during 20 years in Jinju, South Korea.

BACKGROUND: The antimicrobial resistance capability of Helicobacter pylori is one of the critical factors in the failure to treat this pathogen. The purpose of this study was to investigate the changing pattern of primary antibiotic resistance rates in children in the southern central part of South Korea from 1990 to 2009. METHODS: H. pylori strains were isolated from children who had undergone upper endoscopy at Gyeongsang National University Hospital, including 58 children from 1990-1994 and 33 children from 2005-2009. The susceptibility of H. pylori strains to erythromycin, clarithromycin, azithromycin, amoxicillin, tetracycline, metronidazole, furazolidone, levofloxacin, ciprofloxacin, moxifloxacin, and rifabutin was tested using the serial twofold agar dilution method. RESULTS: The resistance rate to erythromycin increased significantly from 13.8% in 1990-1994 to 33.3% in 2005-2009 (P = 0.032). Clarithromycin resistance increased from 6.9% to 18.2%. Metronidazole resistance decreased from 32.8% to 27.3%. The minimum inhibitory concentration of azithromycin and erythromycin showed definite shifts to higher concentrations in 2005-2009 compared with the strains sampled in 1990-1994 (P = 0.021 and P = 0.025, respectively). The frequency of both macrolide- and metronidazole-resistant strains was 13.8% in 1990-1994 and 15.2% in 2005-2009. No associations were detected between multidrug-resistant strains and the two study periods. CONCLUSIONS: The antibiotic resistance rates of H. pylori in Jinju had a different pattern to other regions. The antibiotic resistance rates of H. pylori showed geographic variation, and local data should be provided as a guideline for treating H. pylori infection.

Changes in anti-group a rotavirus antibody seroprevalence and levels in the Western Gyeongnam province of Korea over 16 years.

To observe how anti-group A rotavirus antibody seropositivity rates and levels have changed in the western region of Gyeongnam Province, 2,030 serum samples collected at four collection periods (1989-1990, 1994-1995, 1999-2000, and 2004-2005) were tested by Enzyme-Linked Immunosorbent Assay for IgG, and IgA antibodies reacting to recombinant VP6 protein. The seroprevalences exhibit no regular patterns over a 16-yr period. For all four collection periods, the anti-rVP6 IgG levels rose steadily during the first 5 months of life, after which they remained high. However, the 2-9 yr and 10-39 yr groups had significantly higher IgG levels in 1999-2000 and 2004-2005, respectively, than in the other collection periods. The 1-5 mo, 40- ≥ 60 yr, and 4-29 yr groups had significantly higher IgA levels in 1989-1990, 1999-2000, and 2004-2005, respectively. The 4 yr (25.0%), 5-9 yr (18.8%), 10-14 yr (41.1%), 20-29 yr (35.0%), and 30-39 yr (20.0%) groups in 2004-2005 had significant higher IgA seropositivity rate compared to the other three collection periods. These observations suggest that in the western region of Gyeongnam Province since the late 1990s, rotavirus reinfection has occurred more frequently than previously, with all ages being at risk.

A novel repeat sequence-based PCR (rep-PCR) using specific repeat sequences of Mycobacterium intracellulare as a DNA fingerprinting.

Repetitive sequence-based PCR (rep-PCR) is a potential epidemiological technique that can provide high-throughput genotype fingerprints of heterogeneous Mycobacterium strains rapidly. Previously published rep-PCR primers, which are based on nucleotide sequences of Gram-negative bacteria may have low specificity for mycobacteria. Moreover, it was difficult to ensure the continuity of the study after the commercial rep-PCR kit was discontinued. Here, we designed a novel rep-PCR for Mycobacterium intracellulare, a major cause of nontuberculous mycobacterial pulmonary disease with frequent recurrence. We screened the 7,645 repeat sequences for 200 fragments from the genome of M. intracellulare ATCC 13950 in silico, finally generating five primers with more than 90% identity for a total of 226 loci in the genome. The five primers could make different band patterns depending on the genome of three different M. intracellulare strains using an in silico test. The novel rep-PCR with the five primers was conducted using 34 bacterial samples of 7 species containing 25 M. intracellulare clinical isolates, compared with previous published rep-PCRs. This shows distinguished patterns depending on species and blotting assay for 6 species implied the sequence specificity of the five primers. The Designed rep-PCR had a 95-98% of similarity value in the reproducibility test and showed 7 groups of fingerprints in M. intracellulare strains. Designed rep-PCR had a correlation value of 0.814 with VNTR, reference epidemiological method. This study provides a promising genotype fingerprinting method for tracing the recurrence of heterogeneous M. intracellulare.

Effects of Helicobacter pylori γ-glutamyltranspeptidase on apoptosis and inflammation in human biliary cells.

BACKGROUND: Several studies have reported the presence of H. pylori in individuals with hepatobiliary diseases, but in vitro and in vivo studies are still needed. Here, we determined the effects of H. pylori γ-glutamyltranspeptidase (GGT) on the induction of apoptosis and IL-8 production in a human cholangiocarcinoma cell line (KKU-100 cells). METHODS: Cell viability and DNA synthesis were examined by MTT and BrdU assays, respectively. RT-PCR and western blot analysis were performed to assess gene and protein expression, respectively. IL-8 secretion in KKU-100 cells was measured by ELISA. RESULTS: Exposure to the H. pylori ggt (+) strain decreased KKU-100 cell survival and DNA synthesis when compared with cells exposed to the H. pylori ggt mutant strain. Treatment with recombinant H. pylori GGT (rHP-GGT) dramatically decreased cell survival and DNA synthesis, and stimulated apoptosis; these features corresponded to an increased level of iNOS gene expression in KKU-100 cells treated with rHP-GGT. RT-PCR and western blot analyses revealed that rHP-GGT treatment enhanced the expression of pro-apoptotic molecules (Bax, Caspase-9, and Caspase-3) and down-regulated the expression of anti-apoptotic molecules (Bcl-2 and Bcl-xL). The extrinsic-mediated apoptosis molecules, including Fas and activated Caspase-8, were not expressed after treatment with rHP-GGT. Furthermore, rHP-GGT significantly stimulated IL-8 secretion in KKU-100 cells. CONCLUSION: Our data indicate that H. pylori GGT might be involved in the development of cancer in hepatobiliary cells by altering cell kinetics and promoting inflammation.