Hiroshi Isogai

Specific inhibition of adherence of an oral strain of Bacteroides gingivalis 381 to epithelial cells by monoclonal antibodies against the bacterial fimbriae

Monoclonal antibodies against purified fimbriae from this organism blocked its adherence to buccal epithelial cells. Three clones of monoclonal antibodies against these fimbriae were selected for use. The isotype of the three was IgG1 kappa chain. The antibodies reacted with fimbriae or their partially dissociated oligomers, but not with their constituent monomers (43 K protein, fimbrilin) or with other B. gingivalis 381 components, in an enzyme-linked immunosorbent assay or by immuno-blotting. The antibodies agglutinated only B. gingivalis 381 cells and no other species of Bacteroides. The purified immunoglobulin G (IgG) antibodies inhibited bacterial adherence to the human buccal epithelial cells, but had no effect on bacterial haemagglutination to various animal and human erythrocytes. The papain-cleaved Fab fragment, which did not allow cell to cell cross-linking, also inhibited adherence of B. gingivalis 381 but did not interfere with haemagglutination. Thus the fimbriae of B. gingivalis 381 may be responsible for adherence to epithelial cells, which supports the notion that a different type of fimbria or a lectin-like protein may be acting as haemagglutinin in this bacterium.

Chemiluminescence of neutrophils from patients with Behçet's disease and its correlation with an increased proportion of uncommon serotypes of Streptococcus sanguis in the oral flora.

Zymosan-induced chemiluminescence was investigated in whole blood and in neutrophils: in both, the peak count was frequently elevated in Behçets disease, and was significantly higher than in healthy controls; similarly the peak time was shorter. There were more uncommon serotypes of Streptococcus sanguis in the oral flora of patients with Behçets disease. Common serotypes were present in the flora of healthy controls, but not in patients with the disease. The percentage of Strep. sanguis in the oral flora was significantly correlated with the level of chemiluminescence response. Thus infection with uncommon serotypes of Strep. sanguis may play a role in the aetiology of Behçets disease.

Colonization of Helicobacter pylori in the Gastric Mucosa of Mongolian Gerbils

Helicobacter pylori was orally inoculated into Mongolian gerbils. The organisms were able to colonize in the gastro‐mucosal layer of the gerbils, especially in those gerbils which had mucosal lesions caused by indomethacin treatment. The pathological changes developed by H. pylori infection were restricted to the stomachs, and only slightly inflammatory cells were observed.

Detection of Antibodies Against Borrelia burgdorferi in Patients With Uveitis

We determined the antibody response against Borrelia burgdorferi strains isolated from Japanese Ixodes ovatus and Ixodes persulcatus ticks by enzyme-linked immunosorbent assay and indirect immunofluorescence assay of serum specimens from 127 patients with uveitis. We examined samples of serum from Japanese patients with unclassified uveitis, iridocyclitis caused by herpes zoster virus, Behçets disease, Vogt-Koyanagi-Harada syndrome, sarcoidosis, or other conditions (sympathetic ophthalmia, Posner-Schlossman syndrome and acute anterior uveitis with ankylosing spondylitis). Serum from healthy individuals and patients with Lyme disease served as negative and positive control samples, respectively. Significantly higher antibody titers were demonstrated in patients with uveitis than in control subjects. Of 29 patients with unclassified uveitis, nine (31) had significantly increased antibody titers against B. burgdorferi strain H014 by ELISA testing. Five patients also showed higher IgG and IgM responses than in three control subjects with Lyme disease. All positive controls showed joint problems characteristic of rheumatoid arthritis. One of three patients had uveitis. The patients were diagnosed as having Lyme disease on the basis of their history and serologic tests. A positive antibody response was recognized in several patients with Behçets disease, Vogt-Koyanagi-Harada syndrome, sarcoidosis, and other conditions (acute anterior uveitis with ankylosing spondylitis), but not in control subjects.

Ceragenin CSA‐13 exhibits antimicrobial activity against cariogenic and periodontopathic bacteria

INTRODUCTION Ceragenin CSA-13 is a bile-acid-based mimic of endogenous antimicrobial peptides and shares a mechanism of action with many of these antimicrobial agents. Because CSA-13 is not peptide based, it is not a substrate for the proteases that are found in the oral cavity, which are capable of degrading antimicrobial peptides. Furthermore, the simplicity of the ceragenins makes them easier to prepare and purify than antimicrobial peptides. In this study, we examined the antimicrobial activities of CSA-13 against oral pathogens and found that this compound was bactericidal against all of the strains tested. METHODS The strains used were isolates of Streptococcus mutans and Porphyromonas species. Minimum inhibitory concentrations (MIC) were determined using agar dilution methods. In susceptibility testing, viable counts were determined after incubation with CSA-13. RESULTS CSA-13 was potent against all 23 strains tested with MICs of 1-8 microg/ml for S. mutans and 1-16 microg/ml for 24 strains of the genus Porphyromonas. The MIC(50) was 2 and the MIC(90) was 8 mug/ml for S. mutans. MIC ranges for protease-positive P. gingivalis and P. cangingivalis were 2-16 microg/ml, and 1-2 microg/ml for protease-negative P. circumdentaria. CSA-13 interacted with lipopolysaccharide-sensitized erythrocytes at a concentration of 5.0-20.0 microg/ml. CONCLUSION CSA-13 displays broad-spectrum activity against cariogenic and periodontopathic bacteria. CSA-13 was effective against protease-positive Porphyromonas. It was shown to bind to erythrocytes coated with lipopolysaccharide and lipoteichoic acid from diverse bacterial strains. These results suggest that CSA-13 may be useful for the prevention and treatment of oral microbial diseases.

In Vivo Synergy Between Green Tea Extract and Levofloxacin Against Enterohemorrhagic Escherichia coli O157 Infection

We studied the synergistic effects of Japanese green tea extract (JGTE) and levofloxacin (LVFX) against enterohemorrhagic Escherichia coli (EHEC) infection in a gnotobiotic mouse model. Mice fed on JGTE conferred a significant degree of protection against an oral challenge with EHEC. Complete elimination of the bacteria from the mice, was however, difficult. The combination of JGTE and LVFX increased the survival rate and reduced damage to target organs. Thus, dietary supplementation with JGTE improved the therapeutic effects of antibiotic treatment.

Biological activities of leptospiral lipopolysaccharide.

Lipopolysaccharide extracted with phenol-water from Leptospira interrogans serovar copenhageni strain Shibaura (L-LPS) showed various biological activities. In lethality for mice, L-LPS was active (LD 50, 3.4 mg/mouse) but about 12 times less potent than Escherichia coli LPS (E-LPS) per weight basis. L-LPS had pyrogenicity for rabbits, and the fever curves showed no evidence of the classical biphasic fever produced by E-LPS. In the bone marrow of mice, L-LPS caused hemorrhages and necrosis but less severe than those caused by E-LPS. Histopathologically, fresh hemorrhages were found in the intestine, spleen, lung and the other organs at 24 h after inoculation of L-LPS. Necrosis was also found in these organs and was particularly severe in mice inoculated with more than 2 mgL-LPS. Liver necrosis was found at 7th day after inoculation of L-LPS but not after inoculation of E-LPS. L-LPS had adjuvant activity just like E-LPS. L-LPS enhanced non-specific resistance to Salmonella infection and activated mouse peritoneal macrophages to kill these organisms. L-LPS was positive in limulus test just like E-LPS. These results demonstrated similarities of L-LPS and E-LPS. Some toxic effects of L-LPS were less than those of E-LPS, but some effects of L-LPS were more than those of E-LPS. L-LPS was antigenically active and the specificity was serogroup-associated. L-LPS was composed of carbohydrate (54%), lipid (12%), protein (5%). Arabinose, xylose and rhamnose were major sugars as detected by gas chromatography. 2-keto-deoxyoctanate (KDO) was not detectable.

Comparison of OspA Serotypes for Borrelia burgdorferi Sensu Lato from Japan, Europe and North America

Sixty‐one Borrelia burgdorferi sensu lato strains from various sources (ticks, human, and wild animals) in Japan and two strains from ticks in Far Eastern Russia were classified on the basis of reactivity with 16 monoclonal antibodies (mAb) to outer surface protein A (OspA) and by DNA‐DNA hybridization assay. Eleven OspA serotypes (J1 to J11) were recognized among the Japanese and the Far East Russian isolates (serotypes J1 to J9 were identified as B. garinii, serotype J10 was identified as B. afzelii, and serotype J11 corresponded to B. japonica), whereas 7 OspA serotypes for North American and European isolates previously reported (Bettina Wilske et al, J. Clin. Microbiol. 31:340‐350, 1993) were not observed except for OspA serotype 2 which showed identical reactivity with OspA serotype J10. This finding provides helpful information for understanding the geographical distribution of Lyme disease borrelia and the development of vaccine and diagnostic tests. In conclusion: 1. B. burgdorferi sensu stricto has not been observed in Japan, 2. Japanese B. afzelii isolates are closely related to those from Europe, 3. B. garinii isolates from Japan are highly heterogeneous and apparently different from European B. garinii isolates.

The gene for component-II of botulinum C2 toxin

The gene encoding component-II of the Clostridium botulinum C2 toxin was cloned from the chromosomal DNA of C. botulinum type C strain (C)-203U28, and the nucleotide sequence was determined. The gene (bc2h) encodes a protein with 721 amino acid residues and is located at 247 bp downstream of the gene for component-I. The N-terminal 16 amino acids were identical to those obtained by analysis of the purified component-II toxin. The ORF for bc2h had only 39% homology at the amino acid level with the C.perfringens iota-Ib protein and an ATP/GTP binding site which is present in the iota-Ib protein is missing from the protein encoded by bc2h. Both genes had a homologous region that predicts a transmembrane segment.

Protective Effect of Japanese Green Tea Extract on Gnotobiotic Mice Infected with an Escherichia coli O157:H7 Strain

We examined the effect of Japanese green tea extract (JGTE) on enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection in a gnotobiotic mouse model. Gnotobiotic mice inoculated with an EHEC strain developed neurologic and systemic symptoms, usually culminating in death. In contrast, none of mice receiving dietary JGTE showed clinical signs or death. This report describes the effect of JGTE, which includes the inhibition of bacterial growth in vivo. The Shiga‐like toxin (SLT) level in the feces of the JGTE diet group was significantly lower than that of the control group.