Antimicrobial Proteins in Intestine and Inflammatory Bowel Diseases

Mucosal surface of the intestinal tract is continuously exposed to a large number of microorganisms. To manage the substantial microbial exposure, epithelial surfaces produce a diverse arsenal of antimicrobial proteins (AMPs) that directly kill or inhibit the growth of microorganisms. Thus, AMPs are important components of innate immunity in the gut mucosa. They are frequently expressed in response to colonic inflammation and infection. Expression of many AMPs, including human β-defensin 2-4 and cathelicidin, is induced in response to invasion of pathogens or enteric microbiota into the mucosal barrier. In contrast, some AMPs, including human α-defensin 5-6 and human β-defensin 1, are constitutively expressed without microbial contact or invasion. In addition, specific AMPs are reported to be associated with inflammatory bowel disease (IBD) due to altered expression of AMPs or development of autoantibodies against AMPs. The advanced knowledge for AMPs expression in IBD can lead to its potential use as biomarkers for disease activity. Although the administration of exogenous AMPs as therapeutic strategies against IBD is still at an early stage of development, augmented induction of endogenous AMPs may be another interesting future research direction for the protective and therapeutic purposes. This review discusses new advances in our understanding of how intestinal AMPs protect against pathogens and contribute to pathophysiology of IBD

Immobilized Polydiacetylene Lipid Vesicles on Polydimethylsiloxane Micropillars as a Surfactin-Based Label-Free Bacterial Sensor Platform

Accurate detection and sensing of bacteria are becoming increasingly important not only in microbiology but in a variety of fields including medicine, food, public health, and environmental science. However, even new rapid methods are not convenient enough. Here, we describe a simple and efficient label-free bacterial detection system using the polydiacetylene (PDA) liposomes immobilized on the 3D polydimethylsiloxane (PDMS) micropillars. Our system utilizes the colorimetric response of amine functionalized PDA vesicles to surfactin, a bacterial cyclic lipopeptide commonly released by Gram-positive Bacillus species as an antibiotic. To improve the sensitivity of PDA vesicles to surfactin by increasing the number and surface area of immobilized vesicles, the PDA vesicles were immobilized on the micropillar structure to give a hierarchical 3D PDA vesicle structure. For the fabrication of the 3D micropillar structure, polydimethylsiloxane (PDMS) was used to overcome the limitations imposed by silicon-based fabrication. In contrast to the 2D-PDA-PDMS system, which could only hardly detect the presence of 500 μM surfactin, the 3D-PDA-PDMS system could efficiently detect the presence of 5 μM surfactin and the initial presence of 4 × 101 cells/ml of Bacillus subtilis NCIB3610, which actively produces surfactin. Furthermore, bacterial strains that are known to produce no surfactin, such as Staphylococcus aureus Newman, Escherichia coli DH5α, and Pseudomonas aeruginosa PA14 were not detected by our system suggesting that the 3D-PDA-PDMS system is highly specific to surfactin but not to other chemicals produced by bacteria. Taken together, our results suggest that the 3D-PDA-PDMS system can sensitively and selectively be used for the high throughput detection and screening of biotechnologically important surfactin-producing bacterial strains

Distribution of fluoroquinolone MICs in Helicobacter pylori strains from Korean patients

OBJECTIVES: The aim of this study was to assess the prevalence rate of primary fluoroquinolone resistance in Helicobacter pylori isolates from Korean patients over the past 16 years. METHODS: One hundred and thirty-five strains of H. pylori (34 strains in 1987, 36 in 1994 and 65 in 2003) were isolated from antral gastric mucosal biopsy specimens. The determination of MICs for the H. pylori isolates of ciprofloxacin, levofloxacin and moxifloxacin was examined by using the serial 2-fold agar dilution method. DNA sequences of the gyrA gene in fluoroquinolone-resistant strains were determined. RESULTS: The distribution of fluoroquinolone MICs (ciprofloxacin, levofloxacin and moxifloxacin) shifted to higher concentrations during 1987-2003. All of the levofloxacin- or moxifloxacin-resistant strains were resistant to ciprofloxacin. Sequence analysis in fluoroquinolone-resistant strains showed point mutation of the gyrA gene at A272G and G271A, indicating mutations of the codon Asp-91 in the fluoroquinolone-resistance-determining region of the DNA gyrase. CONCLUSIONS: These results suggest that resistance to fluoroquinolones has been increasing in the Korean population and the resistance is most likely mediated through point mutation in gyrA

α-Lipoic Acid Inhibits Helicobacter pylori-Induced Oncogene Expression and Hyperproliferation by Suppressing the Activation of NADPH Oxidase in Gastric Epithelial Cells

Hyperproliferation and oncogene expression are observed in the mucosa of Helicobacter pylori- (H. pylori-) infected patients with gastritis or adenocarcinoma. Expression of oncogenes such as β-catenin and c-myc is related to oxidative stress. α-Lipoic acid (α-LA), a naturally occurring thiol compound, acts as an antioxidant and has an anticancer effect. The aim of this study is to investigate the effect of α-LA on H. pylori-induced hyperproliferation and oncogene expression in gastric epithelial AGS cells by determining cell proliferation (viable cell numbers, thymidine incorporation), levels of reactive oxygen species (ROS), NADPH oxidase activation (enzyme activity, subcellular levels of NADPH oxidase subunits), activation of redox-sensitive transcription factors (NF-κB, AP-1), expression of oncogenes (β-catenin, c-myc), and nuclear localization of β-catenin. Furthermore, we examined whether NADPH oxidase mediates oncogene expression and hyperproliferation in H. pylori-infected AGS cells using treatment of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase. As a result, α-LA inhibited the activation of NADPH oxidase and, thus, reduced ROS production, resulting in inhibition on activation of NF-κB and AP-1, induction of oncogenes, nuclear translocation of β-catenin, and hyperproliferation in H. pylori-infected AGS cells. DPI inhibited H. pylori-induced activation of NF-κB and AP-1, oncogene expression and hyperproliferation by reducing ROS levels in AGS cells. In conclusion, we propose that inhibiting NADPH oxidase by α-LA could prevent oncogene expression and hyperproliferation occurring in H. pylori-infected gastric epithelial cells

Inhibition of proinflammatory cytokine expression by NF-kappaB (p65) antisense oligonucleotide in Helicobacter pylori-infected mice

BACKGROUND: Helicobacter pylori induces the expression of proinflammatory cytokines in vitro by activating nuclear factor-kappaB, a transcriptional regulator. However, it has not been clarified whether H. pylori-induced proinflammatory cytokines are also mediated through nuclear factor-kappaB in vivo. The aim of this study was to evaluate the role of nuclear factor-kappaB on the expressions of proinflammatory cytokines in H. pylori-infected mice. MATERIALS AND METHODS: We evaluated nuclear factor-kappaB (p65) activation in the H. pylori-infected gastric mucosa of mice by immunofluorescent staining using antip65 polyclonal antibody, and the expressions of proinflammatory cytokines with inhibition of nuclear factor-kappaB pathway by using phosphorothioate antisense and sense oligonucleotide against the nuclear factor-kappaB (p65). RESULTS: In the H. pylori-infected gastric mucosa of mice, immunofluorescent staining using antip65 polyclonal antibody showed nuclear factor-kappaB (p65) activation, which was particularly localized to epithelial cells. Tumor necrosis factor-alpha and interleukin-1beta concentrations in gastric mucosa by enzyme-linked immunosorbent assay (ELISA) were elevated in the infected group versus the uninfected group. Pretreatment with nuclear factor-kappaB (p65) antisense oligonucleotide inhibited the activation of nuclear factor-kappaB and the expressions of tumor necrosis factor-alpha and interleukin-1beta in H. pylori-infected gastric mucosa. Sense oligonucleotide did not influence on the expression of proinflammatory cytokines. CONCLUSIONS: H. pylori infection was found to activate the expressions of proinflammatory cytokines via nuclear factor-kappaB in vivo, and this may play an important role in the initiation of H. pylori-induced gastric inflammation

Up-regulation of inducible nitric oxide synthase and nitric oxide in Helicobacter pylori-infected human gastric epithelial cells: possible role of interferon-gamma in polarized nitric oxide secretion

BACKGROUND: Nitric oxide (NO) generated by nitric oxide synthase (NOS) is known to be an important modulator of the mucosal inflammatory response. In this study, we questioned whether Helicobacter pylori infection could up-regulate the epithelial cell inducible NOS (iNOS) gene expression and whether NO production could show polarity that can be regulated by immune mediators. MATERIALS AND METHODS: Human gastric epithelial cell lines were infected with H. pylori, and the iNOS mRNA expression was assessed by quantitative RT-PCR. NO production was assayed by determining nitrite/nitrate levels in culture supernatants. To determine the polarity of NO secretion by the H. pylori-infected epithelial cells, Caco-2 cells were cultured as polarized monolayers in transwell chambers, and NO production was measured. RESULTS: iNOS mRNA levels were significantly up-regulated in the cells infected with H. pylori, and expression of iNOS protein was confirmed by Western blot analysis. Increased NO production in the gastric epithelial cells was seen as early as 18 hours postinfection, and reached maximal levels by 24 hours postinfection. The specific MAP kinase inhibitors decreased H. pylori-induced iNOS and NO up-regulation. After H. pylori infection of polarized epithelial cells, NO was released predominantly into the apical compartment, and IL-8 was released predominantly into basolateral compartment. The addition of IFN-gamma to H. pylori-infected polarized epithelial cells showed a synergistically higher apical and basolateral NO release. CONCLUSION: These results suggest that apical NO production mediated by MAP kinase in H. pylori-infected gastric epithelial cells may influence the bacteria and basolateral production of NO and IL-8 may play a role in the tissue inflammation