Haruhisa Hirata

Improvement of periodontal condition by probiotics with Lactobacillus salivarius WB21 : a randomized, double-blind, placebo-controlled study

AIM This randomized clinical study was designed to evaluate the effect of probiotic intervention using lactobacilli on the periodontal condition of volunteers without severe periodontitis. MATERIAL AND METHODS Freeze-dried Lactobacillus salivarius WB21 (WB21)-containing tablets or a placebo were given to volunteers in a double-blind randomized study. A total of 66 volunteers were finally enrolled and randomly assigned to receive tablets containing WB21 (6.7 x 10(8) CFU) with xylitol or xylitol alone (placebo) three times a day for 8 weeks. Periodontal clinical parameters and whole saliva samples were obtained at baseline (BL), 4 weeks, and the end of the interventional period (8 weeks). Salivary lactoferrin (Lf) levels were measured by enzyme-linked immunosorbent assay. Lactobacilli in saliva and plaque samples was detected by semi-quantitative RT-PCR using 16S rRNA primers. RESULTS Periodontal clinical parameters were improved in both groups after an 8-week intervention. Current smokers in the test group showed a significantly greater improvement of plaque index and probing pocket depth from BL when compared with those in the placebo group. Salivary Lf level was also significantly decreased in the test group smokers. CONCLUSION Our results indicate that probiotics could be useful in the improvement/maintenance of oral health in subjects at a high risk of periodontal disease.

Probiotic effects of orally administered Lactobacillus salivarius WB21‐containing tablets on periodontopathic bacteria: a double‐blinded, placebo‐controlled, randomized clinical trial

AIM This study was designed to evaluate whether the oral administration of lactobacilli could change the bacterial population in supra/subgingival plaque. MATERIAL AND METHODS Sixty-six healthy volunteers without severe periodontitis were randomized into two groups to receive lactobacilli or placebo for 8 weeks (8W): the test group (n=34) received 2.01 x 10(9) CFU/day of Lactobacillus salivarius WB21 and xylitol in tablets; the control group (n=32) received placebo with xylitol. Supra/subgingival plaque samples were collected at the baseline and after 4 weeks (4W) and 8W. The bacterial amounts in plaque samples were analysed by quantitative real-time polymerase chain reaction. RESULTS The numerical sum of five selected periodontopathic bacteria in the test group was decreased significantly in subgingival plaque at 4W [odds ratio (OR)=3.13, 95% confidence intervals (CI)=1.28-7.65, p=0.012]. Multivariate analysis showed that significantly higher odds were obtained for the reduction of Tannerella forsythia in subgingival plaque of the test group at both 4W (OR=6.69, 95% CI=2.51-17.9, p<0.001) and 8W (OR=3.67, 95% CI=1.45-9.26, p=0.006). CONCLUSION Oral administration of probiotic lactobacilli reduced the numerical sum of five selected periodontopathic bacteria and could contribute to the beneficial effects on periodontal conditions.

Production and Application of New Monoclonal Antibodies Specific for a Fecal Helicobacter pylori Antigen

ABSTRACT The aim of the present study was to establish monoclonal antibodies that could be used to produce a diagnostic test composed of one kind of monoclonal antibody recognizing a fecal Helicobacter pylori antigen. The need to develop such a test arose from disadvantages of the diagnostic test that uses a polyclonal antibody or plural kinds of monoclonal antibodies, such as the lower specificity for H. pylori antigen and the difficulty of reproduction with consistent quality. Mice were immunized with sonicated cells of the coccoid form of H. pylori, and fecal samples from H. pylori-positive subjects were screened by a direct sandwich enzyme immunoassay (EIA) for antibody production from 32 hybridoma clones. The three stable clones produced antibodies (21G2, 41A5, and 82B9) that reacted with the same soluble antigen. Gel filtration chromatography showed that the molecular masses of the cellular antigen and the fecal antigen were the same, 260 kDa. The antigen was labile in response to sodium dodecyl sulfate and heat treatments. A single-step direct sandwich EIA using a single monoclonal antibody, 21G2, was developed. The EIA could detect the antigen in 41 H. pylori clinical isolates and in fecal samples from seven H. pylori-positive subjects. Several kinds of Helicobacter species (Helicobacter felis, Helicobacter hepaticus, Helicobacter mustelae, and Helicobacter cinaedi) except H. pylori, major bacteria in feces (Campylobacter jejuni, Bacteroides vulgatus, Bifidobacterium breve, Bifidobacterium infantis, and Escherichia coli), and fecal samples from six H. pylori-negative subjects showed negative results. These results indicate that the new monoclonal antibodies and the new specific EIA would be useful as a noninvasive method of diagnosis of H. pylori infection.

Characterization and usefulness of stool antigen tests using a monoclonal antibody to Helicobacter pylori catalase

Background and Aim:  Two types of stool antigen tests have been used in the management of Helicobacter pylori infection. Testmate Pylori Antigen enzyme immunoassay (TPAg EIA) is a direct sandwich enzyme immunoassay (EIA) while Testmate Rapid Pylori Antigen (Rapid TPAg) is performed using immunochromatography. The aim of this study was to study the characterization and usefulness of these tests.

Catalase, a Specific Antigen in the Feces of Human Subjects Infected with Helicobacter pylori

ABSTRACT Recently, we reported the production of three new monoclonal antibodies with high specificity for a Helicobacter pylori antigen suitable for diagnosis of H. pylori infection. The aim of the present study was to identify the antigen recognized by these monoclonal antibodies concerning both H. pylori and the feces of human subjects infected with H. pylori. The cellular antigen was purified from an H. pylori cell extract by immunoaffinity column chromatography with the monoclonal antibody as a ligand. The amino-terminal amino acid sequences (eight residues) of the purified antigen and H. pylori catalase were the same. The molecular weights of native and subunit, specific catalase activity, and UV and visible spectra of the purified antigen were in good agreement with those of H. pylori catalase. The human fecal antigens were purified from two fecal samples of two H. pylori-positive subjects by ammonium sulfate precipitation, CM-Sephadex C50 chromatography, and the same immunoaffinity chromatography used for the H. pylori cellular antigen. The fecal antigens had catalase activity. The amino-terminal amino acid sequences (five residues) of the human fecal antigen and H. pylori catalase were the same. The monoclonal antibodies reacted with the native cellular antigen, but did not react with the denatured antigen, human catalase, and bovine catalase. The results show that the target antigen of the monoclonal antibodies is native H. pylori catalase and that the monoclonal antibodies are able to specifically detect the antigen, which exists in an intact form, retaining the catalase activity in human feces.

A new anti-MRSA antibiotic complex, WAP-8294A II. Structure characterization of minor components by ESI LCMS and MS/MS

The anti-MRSA antibiotic, WAP-8294A, was isolated from the fermentation broth of Lysobacter sp. The major component, WAP-8294A2, is composed of 1 mol of Gly, L-Leu, L-Glu, D-Asn, D-Trp, D-threo-β-hydroxyasparagine, N-Me-D-Phe and N-Me-L-Val, and 2 mol of L-Ser, D-Orn and D-3-hydroxy-7-Me-octanoic acid. The structure of the WAP-8294A2 was mainly determined as a cyclic depsipeptide by 2D NMR experiments. However, it was difficult to use the NMR experiment to determine the minor components, A1, A4 and Ax13, isolated in small amounts. In the present study, ESI MS/MS was applied to the structure elucidation of these minor components. The structures of these minor components were determined on the basis of the fragmentation pattern of the product ions of WAP-8294A2 in the ESI MS/MS. As a result, it was confirmed that A1 and A4 had the same amino acid sequence as A2, while A1 and A4 had the 3-OH-octanoic acid and 3-OH-8-Me-nonanoic acid, respectively, in the place of the 3-OH-7-Me-octanoic acid in A2. In the structure of Ax13, it was found that Gly of A2 was changed to β-Ala of Ax13.

Effects of orally administered Lactobacillus salivarius WB21 supplement on periodontal clinical parameters and microflora

The aim of this study was to evaluate whether the oral administration of lactobacilli could change the periodontal condition and microflora compared with placebo. Sixty-six healthy volunteers were randomized into two groups to receive lactobacilli or placebo for 8 weeks. The mean Plaque Index, Gingival Index, and bleeding on probing were significantly improved at 4 and 8 weeks in both groups, but showed the biggest change on smokers in the test group. Occurrence of Porphyromonas gingivalis and Treponema denticola in the subgingival plaque sample significantly decreased in the test group at 8 weeks. Oral administration of probiotic lactobacilli successfully reduced the prevalence of periodontopathic bacteria from the subgingival plaque, and possibly contributed to the beneficial effects on periodontal conditions.