Gastrokine 1 inhibits the carcinogenic potentials of Helicobacter pylori CagA.

Helicobacter pylori CagA directly injected by the bacterium into epithelial cells via a type IV secretion system, leads to cellular changes such as morphology, apoptosis, proliferation and cell motility, and stimulates gastric carcinogenesis. We investigated the effects of cytotoxin-associated gene A (CagA) and gastrokine 1 (GKN1) on cell proliferation, apoptosis, reactive oxygen species (ROS) production, epithelial-mesenchymal transition (EMT) and cell migration in CagA- or GKN1-transfected gastric epithelial cells and mucosal tissues from humans and mice infected with H.pylori. On the molecular level, H.pylori CagA induced increased cell proliferation, ROS production, antiapoptotic activity, cell migration and invasion. Moreover, CagA induced activation of NF-κB and PI3K/Akt signaling pathways and EMT-related proteins. In addition, H.pylori CagA reduced GKN1 gene copy number and expression in gastric cells and mucosal tissues of humans and mice. However, GKN1 overexpression successfully suppressed the carcinogenic effects of CagA through binding to CagA. These results suggest that GKN1 might be a target to inhibit the effects from H.pylori CagA.

In vitro and in vivo phototoxicity on gastric mucosa induced by methylene blue.

Background: Methylene blue (MB) is used endoscopically to demarcate tumors and as a photosensitizer in photodynamic therapy (PDT). However, there are few in vivo studies about its toxicity in healthy stomach tissue. We performed sequential in vitro and in vivo analyses of MB-induced phototoxicity. Methods: We performed in vitro experiments using the AGS human gastric cancer cell line treated with light-emitting diode (LED) irradiation (3.6 J/cm2) and MB. Cytotoxicity was evaluated using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. In vivo toxicity was evaluated in the stomach of beagles using the same dose of fiber-optic LED via gastroscopy, after spraying 0.1% and 0.5% MB solutions. Stomach tissue was also evaluated using the TUNEL assay. Results: In vitro, increased concentrations of MB led to higher TUNEL scores. However, cell viability was significantly lower after MB plus LED irradiation than after treatment with MB alone (P < 0.001). In vivo, the TUNEL score was highest immediately after treatment with 0.1% or 0.5% MB plus light irradiation, and the score was significantly higher in the LED illumination plus MB group than in the control group (P < 0.05). The elevated TUNEL score was maintained for 3 days in the MB plus light irradiation group but returned to normal levels on day 10. Conclusions: : Endoscopic light application with MB 0.5% concentration to the stomach may be regarded as a safe procedure despite some DNA injuries in the early period.

A Comparative Study of Hesperetin, Hesperidin and Hesperidin Glucoside: Antioxidant, Anti-Inflammatory, and Antibacterial Activities In Vitro.

The antioxidant, anti-inflammatory and antibacterial activities of hesperetin, hesperidin and hesperidin glucoside with different solubility were compared in vitro. Hesperetin was prepared by enzymatic hydrolysis from hesperidin, and hesperidin glucoside composed of hesperidin mono-glucoside was prepared from hesperidin through enzymatic transglycosylation. Solubility of the compounds was different: the partition coefficient (log P) was 2.85 ± 0.02 for hesperetin, 2.01 ± 0.02 for hesperidin, and -3.04 ± 0.03 for hesperidin glucoside. Hesperetin showed a higher effect than hesperidin and hesperidin glucoside on radical scavenging activity in antioxidant assays, while hesperidin and hesperidin glucoside showed similar activity. Cytotoxicity was low in the order of hesperidin glucoside, hesperidin, and hesperetin in murine macrophage RAW264.7 cells. Treatment of the cells with each compound reduced the levels of inflammatory mediators, nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Hesperetin was most effective at relatively low concentrations, however, hesperidin glucoside was also effective at higher concentration. Hesperetin showed higher antibacterial activity than hesperidin in both Gram-positive and -negative bacteria, and hesperidin glucoside showed similarly higher activity with hesperetin depending on the bacterial strain. In conclusion, hesperetin in the form of aglycone showed more potent biological activity than hesperidin and hesperidin glucoside. However, hesperidin glucoside, the highly soluble form, has been shown to increase the activity compared to poorly soluble hesperidin.

Differences of urease activity and expression of associated genes according to gastric topography.

BACKGROUND: We hypothesize that pH difference between acid-secreting corpus and non-secreting antrum might influence the activity of H. pylori's urease and/or related genes. We therefore measured urease activity and the expression of amiE whose encoded protein that hydrolyzes short-chain amides to produce ammonia. MATERIALS AND METHODS: Fifty-four patients were recruited into this study. Each gastroscopic biopsy specimen collected from the antrum and body of each patient was immediately used to measure urease activity using serial changes of urease activity (ammonia levels) during 60 minutes. Probe specific for amiE was labeled with a biotin nick-translation kit and was used to detect expression of these genes (mRNA) in fresh-frozen gastroscopic biopsy specimens using fluorescent in situ hybridization (FISH). RESULTS: Urease activity at 60 minutes from the gastric antrum and body of all patients infected with H. pylori was 399.5 ± 490.5 and 837.9 ± 1038.9 µg/dL, respectively (p = .004). Urease activity in the antrum was correlated with H. pylori density. Urease activity or H. pylori density in the antrum was significantly correlated with chronic active inflammation; in contrast, this correlation was not found in the gastric body. The expression level of amiE was 1.5 times higher (p < .05) in the gastric body compared with the antrum. CONCLUSION: Topographically, the urease activity in body was much higher than in antrum. The expression level of amiE was higher in the gastric body compared with the antrum.

A Comparative Study of Rutin and Rutin Glycoside: Antioxidant Activity, Anti-Inflammatory Effect, Effect on Platelet Aggregation and Blood Coagulation.

The effects of rutin and rutin glycoside with different solubility were compared on antioxidant activity and anti-inflammatory effects in vitro and the effects on platelet aggregation and blood coagulation in vitro and in vivo. Rutin glycoside (consisting of rutin mono-glucoside and rutin di-glucoside) was prepared via enzymatic transglycosylation from rutin. Rutin glycoside showed a higher effect than rutin on radical scavenging activity in antioxidant assays. Rutin showed a higher toxicity than rutin glycoside in murine macrophage RAW264.7 cells. They had similar effects on the levels of nitric oxide (NO), prostaglandin E (PGE) 2 and pro-inflammatory cytokines (such as tumor necrosis factor (TNF)-α, and interleukin (IL)-6) in the cells. Both rutin and rutin glycosides similarly reduced the rate of platelet aggregation compared to controls in vitro. They also similarly delayed prothrombin time (PT) and activated partial thromboplastin time (APTT) in an in vitro blood coagulation test. The effect of repeated administration of rutin and rutin glycoside was evaluated in vivo using SD rats. The platelet aggregation rate of rutin and the rutin glycoside administered group was significantly decreased compared to that of the control group. On the other hand, PT and APTT of rutin and rutin glycoside group were not significantly delayed in vivo blood coagulation test. In conclusion, rutin and rutin glycoside showed differences in antioxidant activities in vitro, while they were similar in the reduction of NO, PGE2, TNF-α and IL-6 in vitro. Rutin and rutin glycoside also showed similar platelet aggregation rates, and blood coagulation both in vitro and in vivo condition. Comparing in vitro and in vivo, rutin and rutin glycoside were effective on platelet aggregation both in vitro and in vivo, but only in vitro on blood coagulation.

Expression of the BabA adhesin during experimental infection with Helicobacter pylori.

The Helicobacter pylori babA gene encodes an outer membrane protein that mediates binding to fucosylated ABH antigens of the ABO blood group. We recently demonstrated that BabA expression is lost during experimental infection of rhesus macaques with H. pylori J166. We sought to test the generality of this observation by comparison of different H. pylori strains and different animal hosts. Challenge of macaques with H. pylori J99 yielded output strains that lost BabA expression, either by selection and then expansion of a subpopulation of J99 that had a single-base-pair mutation that encoded a stop codon or by gene conversion of babA with a duplicate copy of babB, a paralog of unknown function. Challenge of mice with H. pylori J166, which unlike J99, has 5' CT repeats in babA, resulted in loss of BabA expression due to phase variation. In the gerbil, Leb binding was lost by replacement of the babA gene that encoded Leb binding with a nonbinding allele that differed at six amino acid residues. Complementation experiments confirmed that change in these six amino acids of BabA was sufficient to eliminate binding to Leb and to gastric tissue. These results demonstrate that BabA expression in vivo is highly dynamic, and the findings implicate specific amino acid residues as critical for binding to fucosylated ABH antigens. We hypothesize that modification of BabA expression during H. pylori infection is a mechanism to adapt to changing conditions of inflammation and glycan expression at the epithelial surface.

In Vitro Bactericidal Effects of Photodynamic Therapy Combined with Four Tetracyclines against Clostridioides difficile KCTC5009 in Planktonic Cultures.

Surface disinfection in health-care facilities is critical to prevent dissemination of Clostridioides difficile (C. difficile). Tetracyclines (TCs) are broad-spectrum antibiotics that are associated with a low risk of development of C. difficile infection (CDI) and are used as photosensitizers (PS) in photodynamic therapy (PDT). We evaluated whether TCs may be useful environmental cleansing agents. We compared the in vitro ability to kill C. difficile of four TCs (TC, doxycycline, minocycline, and tigecycline) combined with PDT using ultraviolet A (UVA). We included chitosan, a cationic material, as a booster to increase the photodynamic bactericidal efficacy of TCs. PDT-induced bactericidal effects were assessed by the number of viable cells and the degree of DNA damage and membrane integrity. To avoid the intrinsic antibacterial activity of TCs at high concentrations, we used low concentrations of TCs (0.05 and 0.1 mg/mL). The bactericidal effect of treatment with chitosan plus PDT was over 100 times higher than that with PDT alone for each of the four TCs. DNA damage measured by ethidium bromide monoazide and real-time quantitative polymerase chain reaction was also greater for PDT plus chitosan treatment than for PDT alone or under control conditions: the threshold cycle (Ct) values for the control, PDT, and PDT plus chitosan were 14.67 ± 0.22, 20.46 ± 0.12, and 25.54 ± 0.17, respectively. All four TCs caused similar levels of severe cell membrane damage during PDT compared with control conditions. These data suggest that PDT combined with any of the four TCs plus chitosan might be an available tool to kill efficiently planktonic form of C. difficile.

Development of TaqMan probe-based real-time PCR method for erm(A),erm(B), and erm(C), rapid detection of macrolide-lincosamide-streptogramin B resistance genes, from clinical isolates.

To achieve more accurate and rapid detection of macrolidelincosamide- streptogramin B resistance genes, erm(A), erm(B), and erm(C), we developed a TaqMan probe-based real-time PCR (Q-PCR) method and compared it with conventional PCR (C-PCR), which is the most widely using erm gene identification method. The detection limit of Q-PCR was 5 fg of genomic DNA or 5-8 CFU of bacterial cells of Staphylococcus aureus. The utilization of Q-PCR might shorten the time to erm detection from 3-4 h to about 50 min. These data indicated that Q-PCR assay appears to be not only highly sensitive and specific, but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay will permit rapid and accurate identification of erm genes from clinical and other samples.

Comparison of high and low molecular weight chitosan as in-vitro boosting agent for photodynamic therapy against Helicobacter pylori using methylene blue and endoscopic light.

BACKGROUND: We reported in a previous study that photodynamic therapy (PDT) of Helicobacter pylori(H. pylori) could potentiate bactericidal effect by adding chitosan. As a next step, we compared the bactericidal effects of low molecular weight (LMW) combined with Photodynamic Therapy to high molecular weight (HMW) chitosan. METHOD: To perform PDT to kill H. pylori, we used endoscopic light as light source, methylene blue (MB) as a photosensitizer and chitosan (310-375, 50-190 kDa). We evaluated bacterial removal rate and its membrane damage by ethidium bromide monoazide PCR method (EMA q-PCR). 8-oxo-2'-dexoyguanosine by ELISA was measured for oxidative stress. RESULTS: At a chitosan concentration of ≤0.05%, the killing effect did not differ between the two molecular weights, and 100% bacterial removal rate was observed at a light energy ≥ 6.23 mJ/cm2 powers under 0.02% MB. After 15 min irradiation, LMW chitosan with high concentration of MB (0.004%) showed highest killing effects, which were consistent with the results of EMA q-PCR but not with the level of 8-OHdG. Bactericidal effects of LMW chitosan plus PDT using 0.002 and 0.004% MB for 15 min irradiation were significantly higher than those using HMW chitosan plus PDT. CONCLUSION: We found that PDT using methylene blue with LMW chitosan to kill H. pylori exerted greater bactericidal effects through bacterial membrane damage than PDT with HMW chitosan. These results suggest that it would be better to choose LMW chitosan to enhance the effect of PDT for clinical application, even at a very low concentration of PS.

Multiple genetic mutations caused by NKX6.3 depletion contribute to gastric tumorigenesis.

NKX family members are involved in a variety of developmental processes such as cell fate determination in the central nervous system, gastrointestinal tract, and pancreas. However, whether NKX6.3 contributes to gastric carcinogenesis remains unclear. The objective of this study was to examine roles of NKX6.3 depletion in mutagenesis and gastric carcinogenesis, focusing on its effects on genetic alterations and expression of genes. Our results revealed that NKX6.3 depletion induced multiple genetic mutations in coding regions, including high frequency of point mutations such as cytosine-to-thymine and guanine-to-adenine transitions caused by aberrant expression of AICDA/APOBEC family in human gastric epithelial cells. Interestingly, NKX6.3 downregulated AICDA/APOBEC family, NFκB, and CBFß genes by acting as a transcription factor while inhibiting deaminase activity in gastric epithelial cells. Functional relevance of NKX6.3 was validated in xenograft mice injected with NKX6.3 depleting cells. NKX6.3 depletion resulted in tumor formation and mutations of tumor-associated genes, including p53 and E-cadherin. Moreover, expression levels of NKX6.3 and its target genes were analyzed in tumors derived from mice implanted with NKX6.3 depleting cells and tissue samples of gastric cancer patients. Our results indicate that NKX6.3 depletion in gastric epithelial cells activates AICDA/APOBEC family, leading to accumulation of genetic mutations and eventually driving the development of gastric cancers.

Safety assessment of potential lactic acid bacteria Bifidobacterium longum SPM1205 isolated from healthy Koreans.

The safety assessment of Bifidobacterium longum SPM1205 isolated from healthy Koreans and this strain's inhibitory effects on fecal harmful enzymes of intestinal microflora were investigated. The overall safety of this strain was investigated during a feeding trial. Groups of SD rats were orally administered a test strain or commercial reference strain B. longum 1 x 10(9) CFU/kg body weight/day for four weeks. Throughout this time, their feed intake, water intake and live body weight were monitored. Fecal samples were periodically collected to test harmful enzyme activities of intestinal microflora. At the end of the four-week observation period, samples of blood, liver, spleen, kidney, and gut tissues were collected to determine for hematological parameters and histological differences. The results obtained in this experiment demonstrated that four weeks of consumption of this Bifidobacterium strain had no adverse effects on rat's general health status, blood biochemical parameters or histology. Therefore, it is likely to be safe for human use. Fecal harmful enzymes such as beta-glucosidase, beta-glucuronidase, tryptophanase and urease, were effectively inhibited during the administration of the B. longum SPM1205. These results suggested that this B. longum SPM 1205 could be used for humans as a probiotic strain.

NKX6.3 controls gastric differentiation and tumorigenesis.

NKX6.3 transcription factor is known to be an important regulator in gastric mucosal epithelial differentiation. The present study aimed to investigate whether NKX6.3 acts as an essential tumor suppressor in gastric carcinogenesis. Absent or reduced protein expression and decreased DNA copy number and mRNA transcript of the NKX6.3 gene were frequently observed in gastric cancers. Overexpression of NKX6.3 in AGSNKX6.3 and MKN1NKX6.3 cells markedly arrested cell proliferation by inhibiting cell cycle progression and induced apoptosis through both death receptor- and mitochondrial-pathways. In addition, stable NKX6.3 transfectants increased the expression of gastric differentiation markers, including SOX2 and Muc5ac, and decreased the expression of intestinal differentiation markers, CDX2 and Muc2. In ChIP-cloning and sequencing analyses, NKX6.3 coordinated a repertoire of target genes, some of which are clearly associated with cell cycle, differentiation and death. In particular, NKX6.3 transcriptional factor was found to bind specifically to the upstream sequences of GKN1, a gastric-specific tumor suppressor, and dramatically increase expression of the latter. Furthermore, there was a positive correlation between NKX6.3 and GKN1 expression in non-cancerous gastric mucosae. Thus, these data suggest that NKX6.3 may control the fate of gastric mucosal cells and function as a gastric tumor suppressor.

Antimicrobial resistance patterns of vancomycin-resistant Streptococcus equinus isolated from animal foods and epidemiological typing of resistant S. equinus by microbial uniprimer kit.

Raw milk samples, and cow and chicken intestines were tested to isolate vancomycin-resistant, gram-positive bacteria. From these samples, we isolated seven vancomycin-resistant Streptococcus equinus, two vancomycin-resistant viridans Streptococcus and two vancomycin-resistant Enterococcus faecium. The MICs of several antibiotics, including vancomycin, against these strains were tested. Seven isolates of S. equinus showed high level resistance to vancomycin and teicoplanin (>100 microg/mL). The cell wall thickness of these strains was compared with that of the sensitive strain by TEM and no differences were obserbed between these strains. We compared the strains of vancomycin-resistant Streptococcus equinus using PCR with Microbial Uniprimer Kit. We concluded that it is necessary to combine other methods in order to cluster and identify all isolates of S. equinus.

Two polymorphisms of interleukin-4 gene in Korean adult periodontitis.

Adult periodontitis is a multifactoral disease characterized by multiple genetic and environmental factors. In view of the importance of interleukin-4 (IL-4) gene as a genetic factor for adult periodontitis, we investigated the relationship between two polymorphisms (-590 C --> T polymorphism and 70 bp repeat polymorphism) of the human IL-4 gene and adult periodontitis in the Korean population. Genomic DNA was extracted from white blood cells of 32 adult periodontitis patients and 150 normal controls, respectively. There were no significant differences in the allele, genotype and haplotype distributions of two polymorphisms between normal controls and adult periodontitis group. Therefore, our results suggest that IL-4 gene locus contributes little to the interindividual susceptibility for adult periodontitis in Korean population.

Synergistic in vitro photodynamic antimicrobial activity of methylene blue and chitosan against Helicobacter pylori 26695.

BACKGROUND: Photodynamic therapy (PDT) is a method for killing cells (bacterial, fungal and cancer cells) or virus using photosensitizers (PS) and light of various wavelengths. In vitro PDT using endoscopic light against H. pylori was effective at a concentration of 0.2mg/mL of MB. The purpose of this study was to increase the effect of photodynamic modality against H. pylori by addition of chitosan to MB. METHODS: The bactericidal effect was measured by counting viable cells after PDT. The degree of damage to DNA was confirmed using alkaline gel electrophoresis. Cellular DNA damage was demonstrated by ethidium bromide monoazide-quantitative polymerase chain reaction (EMA-qPCR). RESULTS: In the groups treated with either 0.04 mg/mL MB alone or 0.02 mg/mL MB with endoscopic light for 15 min, viable cells were decreased approximately tenfold. The group treated with 0.04 mg/mL of MB with light, showed more effective bactericidal activity than 0.02 mg/mL of MB treatment. By 0.05% chitosan pre-treatment followed with 0.04 mg/mL of MB and light irradiation, viable cells were decreased 10(7)-fold. The DNA damage caused by PDT as demonstrated by alkaline gel electrophoresis was greater in the MB plus chitosan-treated group than in control and MB-treated groups. Cellular DNA damage demonstrated by EMA-qPCR was also greater in the group treated with MB plus chitosan than in the MB-treated group. CONCLUSION: The bactericidal effects with PDT using MB were increased with the concentration of photosensitizer and chitosan treatment, peculiarly before endoscopic light irradiation.

Neogenin as a receptor for early cell fate determination in preimplantation mouse embryos.

The first cell lineage determination in embryos takes place when two cell populations are set apart, each differentiating into the trophectoderm (TE) and inner cell mass (ICM), respectively. It is widely believed that position/polarity cues play a key role in triggering this differentiation, but it remains unclear how extracellular cues are transduced into cell fate determination. Here, we provide evidence that supports that neogenin is implicated in relaying extracellular cues into the first cell fate determination in preimplantation mouse embryos. A polarized and transient distribution of neogenin was manifested in early blastomeres. Neogenin up-regulation by its overexpression accelerated ICM development in the blastocyst concomitant with the activation of the ICM-specific transcription factors Oct3/4, Sox2, and Nanog while its depletion by small hairpin RNAs (shRNAs) caused a developmental abnormality of poorly endowed ICM accompanied by the deactivation of Oct3/4, Sox2, and Nanog. Treatment with netrin-1 among neogenin ligands further impaired both embryonic development and ICM formation while repulsive guidance molecule c (RGMc) led to opposite consequences, enhancing ICM formation. From this study, we propose a model whereby neogenin interprets its own expression level to control the first cell fate determination in response to extracellular cues.

The effect of Helicobacter pylori CagA on the HER-2 copy number and expression in gastric cancer.

We investigated whether Helicobacter pylori (H. pylori) CagA contributes to the DNA copy change and mRNA transcript expression of the HER-2 gene and, consequently, affects HER-2 protein expression to evaluate the significance of CagA and HER-2 amplification in gastric cancer. We used the AGS and MKN1 gastric cancer and HFE-145 immortalized non-neoplastic gastric mucosa cell lines. We also confirmed the effects of CagA on HER-2 expression in human gastric cancer tissues and gastric mucosal tissues of H. pylori infected C57BL/6 mice. Ectopic CagA expression in AGS, MKN1 and HFE-145 cells showed a significant increase in HER-2 gene copy number and expression. The gastric mucosae of H. pylori infected C57BL/6 mice also showed increased HER-2 DNA copy number and protein expression. In addition, CagA expression was detected in 17 (56.7%) of 30 gastric cancer tissues, and eight (47%) of them showed HER-2 DNA amplification of more than two-fold. In immunohistochemistry, HER-2 overexpression was detected in 12 (40%) of 30 gastric cancers and a positive correlation was observed among DNA copy number, the mRNA transcript, and protein expression of the HER-2 gene in gastric cancer (P<0.05). These results suggest that H. pylori CagA may induce overexpression of the HER-2 protein by increasing HER-2 DNA and mRNA copy number.

Point mutations in Helicobacter pylori's fur regulatory gene that alter resistance to metronidazole, a prodrug activated by chemical reduction.

BACKGROUND: Helicobacter pylori's Fur regulatory protein controls transcription of dozens of genes in response to iron availability, acidity and oxidative stress, and affects the vigor of infection and severity of disease. It is unusual among Fur family proteins in being active both when iron-loaded and iron-free. METHOLODOLGY/PRINCIPAL FINDINGS: We tested if H. pylori fur mutations could affect resistance to metronidazole (Mtz), an anti-H. pylori prodrug rendered bactericidal by chemical reduction. Point mutations were made by PCR in DNA containing fur and a downstream chloramphenicol resistance gene, and were placed in the H. pylori chromosome by transformation of a fur-deletion (Δfur) strain. Several substitutions affecting H. pylori Fur's ∼10 residue N terminal arm, which has no counterpart in prototype (E. coli-type) Fur proteins, increased Mtz resistance, as did mutations affecting the region between DNA binding and dimerization domains. Three types of mutations decreased resistance more than did Δfur: substitutions affecting the N-terminal arm; substitutions affecting the metal binding pocket; and nonsense mutations that resulted in a truncated Fur protein with no C-terminal dimerization domain. Most metal binding pocket mutations were obtained only in fur genes with additional inactivating mutations, and thus seemed deleterious or lethal because they. CONCLUSIONS/SIGNIFICANCE: These results establish that H. pylori Fur's distinctive N terminal arm is functional, and more generally illustrate that point mutations can confer informative phenotypes, distinct from those conferred by null mutations. We propose that fur mutations can affect Mtz susceptibility by altering the balance among Fur's several competing activities, and thereby the expression of genes that control cellular redox potential or elimination of bactericidal Mtz activation products. Further analyses of selected mutants should provide insights into Fur interactions with other cellular components, metabolic circuitry, and how H. pylori thrives in its special gastric niche.

In vitro photodynamic antimicrobial activity of methylene blue and endoscopic white light against Helicobacter pylori 26695.

Photodynamic therapy (PDT) is a method for inactivating cells (viral, bacterial and cancer cells) using photosensitizers (PS) and light of various wavelengths. Helicobacter pylori might be easily affected by light because it has few genes to repair light-induced DNA damage. In vitro PDT against H. pylori was conducted using endoscopic white light and methylene blue (MB) as the PS before application to in vivo study. The bactericidal effects were measured by counting viable cells after PDT. The degree of oxidative damage of DNA was confirmed using alkaline gel electrophoresis, real-time PCR (RT-PCR) and an assay of 8-hydroxy-2-deoxyguanosine (8-OHdG). In the control group, the number of viable cells was maintained constantly during the experiment. In the groups treated with either 0.2mg/mlMB alone or white light with 0.02mg/mlMB for 10min, bacteria decreased approximately a hundredfold. The killing effect increased proportionally to the PS concentration and the duration of irradiation. DNA damage by PDT proven by alkaline gel electrophoresis, RT-PCR and assay of 8-OHdG, was greater in PDT-treated groups than in control. PDT using MB and endoscopic white light showed effective bactericidal activity in vitro by oxidative DNA damage of H. pylori.

Surface properties of Helicobacter pylori urease complex are essential for persistence.

The enzymatic activity of Helicobacter pylori's urease neutralises stomach acidity, thereby promoting infection by this pathogen. Urease protein has also been found to interact with host cells in vitro, although this property's possible functional importance has not been studied in vivo. To test for a role of the urease surface in the host/pathogen interaction, surface exposed loops that display high thermal mobility were targeted for inframe insertion mutagenesis. H. pylori expressing urease with insertions at four of eight sites tested retained urease activity, which in three cases was at least as stable as was wild-type urease at pH 3. Bacteria expressing one of these four mutant ureases, however, failed to colonise mice for even two weeks, and a second had reduced bacterial titres after longer term (3 to 6 months) colonisation. These results indicate that a discrete surface of the urease complex is important for H. pylori persistence during gastric colonisation. We propose that this surface interacts directly with host components important for the host-pathogen interaction, immune modulation or other actions that underlie H. pylori persistence in its special gastric mucosal niche.