In vitro effects of Helicobacter pylori-induced infection in gastric epithelial AGS cells on microglia-mediated toxicity in neuroblastoma SH-SY5Y cells.

OBJECTIVE: To investigate the in vitro effects of H. pylori-conditioned medium (HCM) from gastric epithelial AGS cell cultures on microglia and neuronal cells. MATERIAL: H. pylori, human gastric epithelial AGS cells, microglia-like BV-2 cells and human neuroblastoma SH-SY5Y cells. TREATMENT: Treated AGS cells with H. pylori at ratios from 1:100 to 1:900 for 24 h. Cultured BV-2 cells and SH-SY5Y cells were treated with HCM from AGS cell cultures. METHODS: Cell viability was measured by a quantitative colorimetric assay with MTT. Nitric oxide (NO) was determined by using Griess reagent. IL-8 was measured by an enzyme-linked immunosorbent assay. Protein expressions were revealed by western blot analysis. RESULTS: H. pylori increased IL-8, NO, COX-2 and gp91(phox) in AGS cell cultures. When BV-2 cells were cocultured with AGS cells, HCM increased COX-2, gp91(phox), iNOS and NO of BV-2 cells. HCM also enhanced the degradation of I kappaB alpha in BV-2 cells. HCM up-regulated expression of nNOS, COX-2, and gp91(phox) of SH-SY5Y cells co-cultured with BV-2 cells. Particularly, the decrease of cell viability of SH-SY5Y induced by HCM was dependent on the presence of BV-2 cells. CONCLUSIONS: H. pylori-induced infection induces microglia-mediated inflammation and neurotoxicity. The present results suggest that microglia play a critical role in HCM-induced toxicity of neuronal SH-SY5Y cells.

Detection of genotypic clarithromycin-resistant Helicobacter pylori by string tests.

AIM: To evaluate the utility of the string test to detect genotypic clarithromycin-resistant Helicobacter pylori (H. pylori) by polymerase chain reaction (PCR)-restriction fragment length polymorphism. METHODS: Patients undergoing endoscopic examinations were enrolled in the present study. String tests were done on the next day of endoscopy. Segments of 23S rRNA were amplified from DNA obtained from string tests. PCR-restriction fragment length polymorphism was accomplished by restriction enzymes BbsI and BsaI recognizing the mutation site A to G at 2143 or at 2142 of 23S rRNA domain V, respectively. RESULTS: One hundred and thirty-four patients with H. pylori infection underwent string tests. To compare phenotypic resistance, 43 isolates were successfully cultured in 79 patients in whom 23S rRNA was successfully amplified. Of five patients with clarithromycin-resistant H. pylori, 23S rRNA of H. pylori isolates from four patients could be digested by BsaI. In 38 susceptible isolates, 23S rRNA of H. pylori isolates from 36 patients could not be digested by either BsaI or BbsI. The sensitivity and specificity of the string test to detect genotypic clarithromycin resistance were 66.7% and 97.3%, respectively. Positive and negative predictive values were 80% and 94.7%, respectively. CONCLUSION: String test with molecular analysis is a less invasive method to detect genotypic resistance before treatment. Further large-scale investigations are necessary to confirm our results.

Differentiation of virulence of Helicobacter pylori by matrix-assisted laser desorption/ionization mass spectrometry and multivariate analyses.

BACKGROUND: The ability to determine the virulence of a Helicobacter pylori strain would be helpful for predicting the development of gastrointestinal disease and suggesting medical treatment. METHODS: A protocol based on matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS) was established for the efficient detection of peptides and proteins in extracts of H. pylori cells. Two multivariate statistical methods-principal component analysis (PCA) and hierarchical clustering analysis-were used to analyze the resulting MALDI mass spectra of reference strains and clinical isolated/inoculated strains. RESULTS: Based on differences in their peptide and protein profiles, H. pylori strains having similar virulence genotypes were grouped together on the PCA score plot. Hierarchical cluster analysis revealed high conformity between the protein profiles and the respective virulence genotypes. The inoculated H. pylori strain, which was clustered in the same group with the high-virulence reference strains, also resulted in severe histopathological lesions in gerbils. CONCLUSIONS: MALDI-TOF MS combined with multivariate analyses shows the ability to rapidly differentiate H. pylori strains in terms of their virulence.

The study of interferences for diagnosing albuminuria by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry.

BACKGROUND: Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometric analysis of albumin (ALB) biomarkers is an alternative approach toward the rapid diagnosis of proteinuria for screening a large number of samples. The aim of this study is to reveal if interfering factors in urinary dipstick approach would affect the results of diagnosing albuminuria by MALDI-TOF MS. METHODS: The effects of various interfering chemicals on the diagnosis of albumin in urine were examined using both MALDI-TOF mass spectrometric and dipstick approaches. Semi-quantification of albumin was performed by using MALDI-TOF MS. RESULTS: Interferences from various drugs, detergents, vitamins and their metabolites, alkaline, and blood, which often cause false-positive and false-negative results in conventional urinary dipstick analysis, are avoided when using this MALDI-TOF MS approach. It was found that the intensity of +1 and +2 albumin ions varies with the albumin concentration. A log/log plot of the intensity ratio vs. albumin concentration is then used as a calibration curve for semi-quantifying the albumin in urines. CONCLUSIONS: MALDI-TOF MS is an effective approach toward avoiding interferences caused by various chemical compounds during the rapid diagnosis of albumin in urine. Semi-quantification of albuminuria is also achieved by this MALDI-TOF MS approach.

Lansoprazole-based sequential and concomitant therapy for the first-line Helicobacter pylori eradication.

OBJECTIVE: The aim of this prospective study was to compare the efficacy of the first-line lansoprazole-based sequential therapy and concomitant therapy (lansoprazole, amoxicillin, clarithromycin and metronidazole) for Helicobacter pylori (H. pylori) eradication. METHODS: A total of 169 patients with H. pylori infection were randomly assigned to either the sequential therapy group (n = 85) or the concomitant therapy group (n = 84). A follow-up endoscopy or urea breath test was examined at least 12 weeks after eradication. RESULTS: Comparable H. pylori eradication rate was observed in both the sequential therapy and concomitant therapy groups by either intention-to-treat analysis [sequential 80.0% (68/85) vs concomitant 88.1% (74/84); P = 0.27] or per protocol analysis [sequential, 85.3% (64/75) vs concomitant, 94.6% (70/74); P = 0.60]. Adverse effects were reported and good compliance was observed in both groups (P = 0.72). Although dual antibiotics resistance affected the therapeutic efficacy of sequential therapy (P = 0.03), not concomitant therapy (P = 0.74), it was not an independent factor for predicting the treatment outcome. CONCLUSION: First-line lansoprazole-based sequential and concomitant therapy were well-tolerated and comparable in terms of their H. pylori eradication rate.

Comparison between Single-Dose Esomeprazole- and Pantoprazole-Based Triple Therapy on the Effectiveness for Helicobacter pylori Eradication in Taiwanese Population.

Background and Study Aims. To compare the effectiveness of two regimens, single-dose esomeprazole- and pantoprazole-based triple therapy, for Helicobacter pylori (H. pylori) eradication. Patients and Methods. A total of 453 patients were enrolled for H. pylori eradication. They were randomly assigned to either EAC group (Esomeprazole 40 mg once daily, Amoxicillin 1 g twice daily, Clarithromycin 500 mg twice daily for 7 days) or PAC group (Pantoprazole 40 mg twice daily, Amoxicillin 1 g twice daily, Clarithromycin 500 mg twice daily for 7 days). Follow-up endoscopy or urea breath test was scheduled 12-16 weeks after the eradication to evaluate the therapeutic response. Results. Higher eradication rate in EAC group than PAC group was shown by intention-to-treat analysis (EAC 72% versus PAC 55%, P < 0.05) and per-protocol analysis (EAC 91% versus PAC 72%, P < 0.05). The incidence of adverse effects (EAC 19% versus PAC 17%, P = 0.712) and the compliance (EAC 87% versus PAC 91%, P = 0.083) were comparable between these 2 groups. Conclusions. Single-dose esomeprazole-based triple therapy is effective for H. pylori eradication.

Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to rapidly screen for albuminuria.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is used as an alternative method for the rapid diagnosis of albuminuria. This technique requires no further sample pretreatment than simply mixing the urine sample with a MALDI matrix and drying under ambient conditions. The resulting MALDI mass spectra reveal albumin ions having charges ranging from +1 to +5. The detection of albumin is possible using any of the three most common MALDI matrices - sinapinic acid (SA), 2,5-dihydroxybenzoic acid (2,5-DHB), or 4-hydroxy-alpha-cyanocinnamic acid (alpha-CHC). Using this analytical approach, the limit of detection for albumin in urine is 10(-6) M, approximately 5 to 10 times lower than that detectable through conventional chemical testing.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the detection of hemoglobins as the protein biomarkers for fecal occult blood.

In this study, we discover that hemoglobins (Hb), highly water-soluble globular proteins that are the most predominant proteins detected by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry in blood, can be used as protein biomarkers for fecal occult blood (FOB). Hemoglobins were extracted from the feces with pure water and separated from the solids in feces through centrifugation. Singly charged molecular ions of Hb-related alpha chains (theoretical MW: 15 126) and beta chains (theoretical MW: 15 867) were detected by MALDI-TOF operated in linear mode using 4-hydroxy-alpha-cyanocinnamic acid (alpha-CHC) as the matrix (with a volumetric ratio of 1:1). The detection limit of FOB using this method is estimated to be lower than 0.1 microg blood per mg of feces, which is approximately 10 to 100 times lower than that of the conventional chemical approaches. The foods and dietary supplements that commonly interfere with the conventional chemical assays of FOB - such as animal blood food products and tablets containing iron and vitamin C - do not interfere with the detection of Hb biomarkers during MALDI-TOF analysis.