Long-term Administration of Probiotics to Asymptomatic Pre-school Children for Either the Eradication or the Prevention of Helicobacter pylori Infection

Background:  The role of probiotics in the armamentarium remains to be defined. The aims of this study were to investigate whether the long-time administration of Lactobacillus gasseri OLL2716 (LG21) strain can eradicate H. pylori in asymptomatic pre-school children and/or prevent H. pylori infection.
Methods: A total of 440 children, from 5–7 years of age, attending a kindergarten in Thailand were screened by the Helicobacter pylori stool antigen (HpSA) test. Thereafter 132 H. pylori positive and 308 H. pylori negative children were recruited to eradication and randomized prevention arms, respectively. Children in the active and placebo treatment groups received Lactobacillus gasseri OLL2716 (LG21) containing cheese and ordinary cheese, respectively, for 12 months. Eradication was defined as reversion by HpSA at 12 months. Prevention was defined as persistently HpSA negative at 12 months.
Results:  Eighty-two of 132 H. pylori positive (62%) completed the eradication arm, of which 24 (29.3%) were negative at 12 months according to the HpSA test. In the randomized prevention arm, 123 of 156 (79%) and 99 of 122 (81%) completed active and placebo arms, respectively, of which 4.1% and 8.1%, respectively, were HpSA positive at 12 months based on a per-protocol analysis ( p  = .21).
Conclusion: Further trials are needed.     

The habitat disruption induces immune-suppression and oxidative stress in honey bees

The honey bee is a major insect used for pollination of many commercial crops worldwide. Although the use of honey bees for pollination can disrupt the habitat, the effects on their physiology have never been determined. Recently, honey bee colonies have often collapsed when introduced in greenhouses for pollination in Japan. Thus, suppressing colony collapses and maintaining the number of worker bees in the colonies is essential for successful long-term pollination in greenhouses and recycling of honey bee colonies. To understand the physiological states of honey bees used for long-term pollination in greenhouses, we characterized their gene expression profiles by microarray. We found that the greenhouse environment changes the gene expression profiles and induces immune-suppression and oxidative stress in honey bees. In fact, the increase of the number of Nosema microsporidia and protein carbonyl content was observed in honey bees during pollination in greenhouses. Thus, honey bee colonies are likely to collapse during pollination in greenhouses when heavily infested with pathogens. Degradation of honey bee habitat by changing the outside environment of the colony, during pollination services for example, imposes negative impacts on honey bees. Thus, worldwide use of honey bees for crop pollination in general could be one of reasons for the decline of managed honey bee colonies.     

Cleavage of Host Cytokeratin-6 by Lysine-Specific Gingipain Induces Gingival Inflammation in Periodontitis Patients

Background/PurposeLysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion.MethodsK6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay.ResultsWe identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359–378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt.ConclusionKgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease.     

Lipocalin 2 diminishes invasiveness and metastasis of Ras-transformed cells

Lipocalin 2, an iron-siderophore-binding protein, converts embryonic kidney mesenchyme to epithelia. We found that lipocalin 2 could also convert 4T1-Ras-transformed mesenchymal tumor cells to an epithelial phenotype, increase E-cadherin expression, and suppress cell invasiveness in vitro and tumor growth and lung metastases in vivo. The Ras-MAPK pathway mediated the epithelial to mesenchymal transition in part by increasing E-cadherin phosphorylation and degradation. Lipocalin 2 antagonized these effects at a point upstream of Raf activation. Lipocalin 2 action was enhanced by iron-siderophore. These data characterize lipocalin 2 as an epithelial inducer in Ras malignancy and a suppressor of metastasis.     

Trimeric structure of major outer membrane proteins homologous to OmpA in Porphyromonas gingivalis

The major outer membrane proteins Pgm6 (41 kDa) and Pgm7 (40 kDa) of Porphyromonas gingivalis ATCC 33277 are encoded by open reading frames pg0695 and pg0694, respectively, which form a single operon. Pgm6 and Pgm7 (Pgm6/7) have a high degree of similarity to Escherichia coli OmpA in the C-terminal region and are predicted to form eight-stranded beta-barrels in the N-terminal region. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pgm6/7 appear as bands with apparent molecular masses of 40 and 120 kDa, with and without a reducing agent, suggesting a monomer and trimer, respectively. To verify the predicted trimeric structure and function of Pgm6/7, we constructed three mutants with pg0695, pg0694, or both deleted. The double mutant produced no Pgm6/7. The single-deletion mutants appeared to contain less Pgm7 and Pgm6 and to form homotrimers that migrated slightly faster (115 kDa) and slower (130 kDa), respectively, than wild-type Pgm6/7 under nonreducing conditions. N-terminal amino acid sequencing and mass spectrometry analysis of partially digested Pgm6/7 detected only fragments from Pgm6 and Pgm7. Two-dimensional, diagonal electrophoresis and chemical cross-linking experiments with or without a reducing agent clearly showed that Pgm6/7 mainly form stable heterotrimers via intermolecular disulfide bonds. Furthermore, growth retardation and arrest of the three mutants and increased permeability of their outer membranes indicated that Pgm6/7 play an important role in outer membrane integrity. Based on results of liposome swelling experiments, these proteins are likely to function as a stabilizer of the cell wall rather than as a major porin in this organism.     

Production of 1,2-didocosahexaenoyl phosphatidylcholine by bonito muscle lysophosphatidylcholine/transacylase

1,2-Didocosahexaenoyl phosphatidylcholine (PC), which has highly unsaturated fatty acid at both sn-1 and sn-2 positions of glycerol, is a characteristic molecular species of bonito muscle. To examine the involvement of a de novo route in its synthesis, the molecular species of phosphatidic acid (PA) were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex, a novel phosphate-capture molecule. However, 1,2-didocosahexaenoyl species could not be detected. Next, 1,2-didocosahexaenoyl PC synthesis by the cytosolic lysophosphatidylcholine (LPC)/transacylase was examined using endogenous LPC from bonito muscle, in which the 2-docosahexaenoyl species is abundant. The LPC/transacylase synthesized 1,2-didocosahexaenoyl PC as the most abundant molecular species. For further characterization, the LPC/transacylase was purified to homogeneity from the 100,000 x g supernatant of bonito muscle. The isolated LPC/transacylase is a labile glycoprotein with molecular mass of 52 kDa including a 5-kDa sugar moiety. The LPC/transacylase showed a PC synthesis (transacylase activity) below and above the critical micelle concentration of substrate LPC, and fatty acid release (lysophospholipase activity) was always smaller than the transacylase activity, even with a monomeric substrate. These results suggest that the LPC/transacylase is responsible for the synthesis of 1,2-didocosahexaenoyl PC.     

A comparison of the peri-implant bone stress generated by the preload with screw tightening between ‘bonded’ and ‘contact’ model

A number of finite element analyses (FEAs) for the dental implant were performed without regard for preload and with all interfaces ‘fixed-bonded’. The purpose of this study was comparing the stress distributions between the conventional FEA model with all contacting interfaces ‘fixed-bonded’ (bonded model) and the model with the interfaces of the components in ‘contact’ with friction simulated as a preloaded implant (contact model). We further verified the accuracy of the result of the FEA using model experiment. In the contact model, the stress was more widely distributed than in the bonded model. From the model study, the preload induced by screw tightening generated strain at the peri-implant bone, even before the application of external force. As a result, the bonded model could not reproduce the mechanical phenomena, whereas the contact model is considered to be appropriate for analysing mechanical problems.