Role for the S-layer of Campylobacter rectus ATCC33238 in complement mediated killing and phagocytic killing by leukocytes from guinea pig and human peripheral blood.

OBJECTIVE: The role of the S-layer of Campylobacter rectus ATCC 33238 in complement-mediated killing and interaction with leukocytes of the intraperitoneal cavity from guinea pigs and human peripheral blood was studied in vitro. MATERIALS AND METHODS: Rabbit polyclonal anti-serum to whole C. rectus cells, a monoclonal antibody which recognizes 150 KDa S-layer protein antigen and a monoclonal antibody against lipopolysaccharide (LPS) were prepared. Sensitivities of C. rectus cells against complement mediated killing and phagocytic killing by peritoneal leukocytes of guinea pig and human peripheral leukocytes were examined in the presence or absence of a specific antibody. RESULTS: C. rectus ATCC 33238 cells were moderately sensitive to complement mediated killing in the presence of rabbit polyclonal antibody against whole cells, and slightly sensitive in the presence of monoclonal antibody against S-layer. Ingestion and phagocytic killing of C. rectus cells by leukocytes were enhanced by the rabbit anti-serum and monoclonal antibody against S-layer, but not by the monoclonal antibody against LPS, pre-immune rabbit serum or control ascites. Viability of leukocytes was dependent on the number of ingested C. rectus cells. CONCLUSIONS: The present study demonstrates that S-layer possessing C. rectus cells are resistant to complement mediated killing and phagocytic killing by leukocytes in the absence of specific antibody.

Effects of frequent mouthrinses with palatinose and xylitol on dental plaque.

The aim was to evaluate the effects of frequent mouthrinses with palatinose, xylitol and a mixture of palatinose and xylitol on plaque pH, plaque formation and cariogenic microorganisms. 15 subjects refrained from toothbrushing during 3 test periods and rinsed 15 x daily for 4 d with 10 ml of: (1) 50% palatinose, (2) 37.5% palatinose + 12.5% xylitol, or (3) 50% xylitol. A contrast period with no mouthrinses was also carried out. The 4 periods were carried out in a randomized order with a cross-over design. After the 4-day periods, 3 parameters were measured: (1) plaque pH during the first 30 min after a mouthrinse with palatinose, a mixture of palatinose and xylitol or xylitol alone, directly followed by a 2nd rinse with 10% sucrose; (2) number of mutans streptococci and lactobacilli in plaque and saliva; (3) plaque index. The most pronounced pH drop for the sugar substitutes was found when rinsing with 50% palatinose after the palatinose period, and the least pH drop with 50% xylitol after the xylitol period. The sucrose rinse gave similar pH fall after all 4 periods. The microbial data showed no differences between the 4 periods, but the mutans streptococcus counts in saliva decreased after the xylitol period in contrast to the 3 other periods. Regarding the plaque index, xylitol gave lower scores compared to the other 3 periods.

New directions for dental education in Japan.

To meet recent changes in the oral health sciences, general science, and social needs, the guidelines for the dental curriculum were revised by the Japanese Deans Meeting in 1994. This new program addresses the need to modify both undergraduate and postgraduate dental education in Japan. Lifelong education for dentists has been also modified recently. Reforming of dental education is pushed forward with the concept of continuity and flexibility. In this report, an overview of dental education and the most recent dental curriculum in Japan is briefly discussed.

Inhibitory effects of funoran on the adherence and colonization of mutans streptococci.

Funoran, a sulfated polysaccharide extracted from the seaweed Gloiopeltis furcata strongly inhibited the absorption of mutans streptococci to saliva-coated hydroxyapatite (S-HA), but enhanced that of Streptococcus sanguis ATCC 10556 and Streptococcus oralis ATCC 10557. Furthermore, funoran had a strong desorption activity against mutans streptococci preadsorbed to S-HA. In the presence of sucrose, the absorption of cells of Streptococcus sobrinus B13, Streptococcus mutans Ingbritt, and S. mutans MT8148R to S-HA was strongly inhibited by 0.01% funoran. The colonization of S. sobrinus 6715 inoculated on the molar teeth of experimental rats that were administered funoran was less frequent than that in a funoran-free group. The mean buccal and lingual, sulcal, and total caries scores of rat groups administered funoran were significantly lower than those of the funoran-free group.

Longitudinal study of caries, cariogenic bacteria and diet in children just before and after starting school.

Fifty 5-yr-old preschool children living in Akranes, a small community in West Iceland known to have a high caries prevalence, were investigated with respect to caries, salivary counts of mutans streptococci and lactobacilli, and consumption of cariogenic foods. Fifteen months later, after being in school for half a year, 43 of the 50 children were reexamined and investigated as before. Mean dmfs scores rose from 7.1 to 9.0, but the scores including initial caries rose from 9.7 to 15.3. Mutans streptococci were carried by 84% of children on both occasions with a mean count 2.1 and 3.6 x 10(5) cfu/ml. Lactobacillus carriage increased from 29 to 38% and the mean count from 5.1 to 13 x 10(3) cfu/ml at 6 yr. The frequency of consumption of sugar-containing foods increased from 4.2 to 5.2 intakes per day and between-meal snacks rose from 3.0 to 3.7 per day. Children classified as "misusing" sugar were 59% at 5 yr and 83% at 6 yr. The mean caries score at 6 yr for children "misusing" sugar was 10.7 but only 2.0 for those not misusing sugar. Thus the deterioration in dental health appears, in these children, to be associated with the increased consumption of sweets and other cariogenic between-meal snacks after starting school.

Polyclonal B cell activation, endotoxin tolerance, and limulus tests of endotoxin preparations of some periodontopathogens.

Potencies of polyclonal B-cell activation in C3H/HeN mice of Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis endotoxins were 0.36, 0.13 and 0.04, taking Salmonella abortusequi as 1.0. F. nucleatum and P. gingivalis endotoxins showed positive reactions in C3H/HeJ mice. Most activities in C3H/HeN other than that of F. nucleatum were suppressed by polymyxin B. In C3H/HeJ mice, similar inhibitions were only 60% for P. gingivalis and hardly observed with F. nucleatum. The resistances to polymyxin B could be due to protein in the endotoxins. A promoting effect of T cells added to B cells was observed only in the activity of F. nucleatum endotoxin in C3H/HeJ mice; there was no influence in other groups. Test endotoxins had nearly the same ability to produce colony stimulating factor as did references and could not produce the factor in tolerant mice. The clinical significance of tolerance is discussed. Regression lines of endotoxin doses and limulus activities of test endotoxins and Salmonella were parallel, either in specific or non-specific tests. The lines of two test groups were also parallel; values obtained by two tests were very close. These data indicate that the test endotoxins did not contain (1-3)-beta-D-glucan and elicited qualitatively similar limulus reactions to that of the reference, despite their different chemical natures. In conclusion, these test preparations had an endotoxicity similar to that of the reference and contribute to produce periodontitis through polyclonal B cell activation.

B-cell mitogenicity and IL-1 beta production of lipopolysaccharides from various Capnocytophaga strains.

To evaluate the etiological roles of Capnocytophaga species in the pathogenesis of periodontal disease, we examined the immunological activities of lipopolysaccharides (LPSs) from various Capnocytophaga strains. All LPSs from various Capnocytophaga species were mitogenic for BALB/c mouse spleen cells, although the responses were lower than those to reference LPSs from Escherichia coli or Salmonella typhimurium. LPSs of C. sputigena strains had polyclonal B cell activation and adjuvant activity and were comparable to reference LPSs. All LPSs from Capnocytophaga strains activated the interleukin-1 beta production from human peripheral monocytes, although the inducing activities of Capnocytophaga LPSs were lower than those of reference LPSs. It appears that LPSs from various Capnocytophaga strains activate certain immunological responses from lymphocytes and monocytes which may be important in the development and pathogenesis of periodontal disease.

Lipopolysaccharides from various Capnocytophaga strains possess potent Limulus amoebocyte lysate clotting activity.

Lipopolysaccharides (LPSs) were extracted by the hot phenol-water method from three ATCC strains (C. sputigena ATCC 33612, C. ochracea ATCC 33596, and C. gingivalis ATCC 33624) and three clinical isolates (C. sputigena TE-1, C. ochracea ONO-26, and C. gingivalis M-12). Endospecy was used to determine the Limulus amoebocyte lysate clotting activities. The activities of LPSs from Capnocytophaga strains were stronger than those of Escherichia coli, with the exception of the LPS from C. gingivalis M-12. Except for the LPS from C. sputigena TE-1, the SDS-PAGE analyses of these preparations showed slow-migrating and repeating ladder bands similar to the LPSs of E. coli and Salmonella typhimurium (wild type) and included both the core-lipid A region and various lengths of O-antigen. The LPS from C. sputigena TE-1 possessed fast-migrating bands and did not have an O-side chain.

Infection by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, and antibody responses at different ages in humans.

This study examined the serum IgG and IgM responses against Porphyromonas gingivalis and 3 serotypes of Actinobacillus actinomycetemcomitans, and the correlations of these responses with age and homologous infection. A total of 90 individuals were included in this study: 40 subjects with gingivitis, 40 periodontally healthy subjects, and 10 adult periodontitis subjects. The subjects in the gingivitis and periodontally healthy groups were divided into 4 stages based on their physiological age: early childhood, school age, puberty, and adult. In the gingivitis group, there was a positive correlation between increase in age and increase in serum IgG antibody levels against P. gingivalis until puberty. However, no statistically significant difference was found between the puberty stage and the adult stage. The average level of IgG antibodies against A. actinomycetemcomitans in the school age gingivitis group was significantly higher than that in the early childhood gingivitis group for all serotypes (p < 0.01). In serotype c, IgG antibody levels in the school age gingivitis group were significantly higher than in the early childhood gingivitis group or the adult gingivitis group (p < 0.01). With both P. gingivalis and A. actinomycetemcomitans, positive correlations between elevated IgG level and infections by these microorganisms were found in the puberty gingivitis and adult periodontitis groups.

Disinfectant effects of "Taisalite" and "Taifresh Ace" containing Irgasan DP300 against MRSA and HIV.

Sterilization and disinfection in dental clinics and laboratories are important in controlling disease vectors such as methicillin-resistant Staphylococcus aureus (MRSA) and human immunodeficiency virus (HIV). We determined the inactivation of disinfectant effects of "Taisalite" and "Taifresh Ace" containing Irgasan DP300 on MRSA, Streptococcus pyogenes, Candida albicans, and HIV. Exposure for 10 seconds to 50% or 5% Taisalite solution inactivated HIV completely. HIV was also completely inactivated by 10 minutes of exposure to 50% or 5% of Taifresh Ace solution. Taisalite possessed strong bactericidal activity against S. pyogenes and MRSA. C. albicans was resistant to 10 to 30 second exposures to Taisalite. MRSA and C. albicans were killed completely by exposure to 90% Taifresh Ace solution for 10 minutes. S. pyogenes was not highly sensitive to Taifresh Ace. The present study showed that Irgasan DP300 containing Taisalite is an excellent disinfectant against HIV and MRSA and that Taifresh Ace is a useful detergent against these micro-organisms.

Adherence and hydrophobicity of invasive and noninvasive strains of Porphyromonas gingivalis.

The adherence of Porphyromonas gingivalis to cell surfaces of periodontal tissues may play an important role in its pathogenicity. In this study, 12 strains of P. gingivalis, including both invasive and noninvasive strains, were investigated for adherence to gingival ligament components. The test strains of P. gingivalis adhered to collagen, fibronectin, and laminin to significantly different degrees. An overall positive correlation was noted between hydrophobicity and the number of cells that attached to collagen coated on hydroxyapatite beads. Invasive strains had low hydrophobicity and bound less to collagen than did noninvasive strains that possessed strong hydrophobic properties. 3H-fimbriae extracted from P. gingivalis were found to attach to collagen-coated hydroxyapatite. The fimbriae extracted from noninvasive strains bound strongly to collagen, whereas invasive strains' fimbriae with low hydrophobicity bound weakly to collagen or saliva-coated hydroxyapatite. These data suggest that 1) fimbriae play an important role in colonization through their hydrophobic activity; 2) fimbriae of noninvasive strains are associated with the major adhesin for attachment to gingival tissue, whereas fimbriae of invasive strains are weakly involved in adherence; and 3) there is no correlation between colonization and the invasiveness of P. gingivalis.

Significance of serum antibody against surface antigens of Actinobacillus actinomycetemcomitans in patients with adult periodontitis.

This study was undertaken to examine the prevalence of Actinobacillus actinomycetemcomitans, its serotype distribution and the serum immune responses against its surface antigens in 41 Japanese patients with adult periodontitis. The dominant A. actinomycetemcomitans serotype isolated was serotype c. Immunoblot analysis of 3 serotypes of A. actinomycetemcomitans-sonicated antigens and the patient sera revealed that the reactivities with serotype c were the most frequent and that heat-stable surface serotype-specific antigen appeared to be immunodominant. Elevated serum immunoglobulin G titers to extracted lipopolysaccharide and fimbriae antigen of A. actinomycetemcomitans were noted for the patient sera by enzyme-linked immunosorbent assay. High serum immunoglobulin G titers to the fimbriae antigen detected in patients without cultivable A. actinomycetemcomitans suggested the possibility that the elicited antibody to the antigen played a role in eliminating A. actinomycetemcomitans from the periodontal lesions.

The relationship between polysaccharide antigen and interleukin-1 beta producing activity in Porphyromonas gingivalis.

Strains of Porphyromonas gingivalis were serologically classified into three groups (a, b, and c) by immunodiffusion test using the autoclave extracted antigens. All tested strains had proteolytic activity, but the activity differed from strain to strain, regardless of serogroup. It was found that serogroup a strains had more collagenolytic activity than did serogroup b (p < 0.05), but that the three groups have no remarkable differences in trypsin activity. All autoclave extracted serogroup-specific antigens induced the release of interleukin-1 beta (IL-1 beta) from human peripheral monocytes. Serogroup-specific antigen of an invasive strain designated 16-1 (serogroup b) induced the highest release of IL-1 beta among all samples. The results of immunoblotting tests and IL-1 beta production indicated that there are serogroup-specific polysaccharide other than lipopolysaccharide in P. gingivalis.

Cloning and expression of the aspartate carbamoyltransferase gene from Treponema denticola.

Treponema denticola seems to play a central role in the etiology of human periodontal disease. We have cloned an antigenic protein-coding sequence from T. denticola ATCC 33520. The protein-coding region was found to be a 3-kbp HindIII-HindIII fragment. The open reading frame consists of 1,426 bp and codes for a protein with an M(r) of 54,919. The deduced amino acid sequence showed 33.8% homology with that of the aspartate carbamoyltransferase of Escherichia coli. The gene products showed aspartate carbamoyltransferase activity.

A sensitive enzymatic method (SK-013) for detection and quantification of specific periodontopathogens.

Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola have been found to predominate in periodontal pockets of patients with adult periodontitis. These microorganisms hydrolyze the synthetic peptide N-benzoyl-DL-arginine-2-naphthylamide (BANA). In this study, we developed an enzymatic method, designated SK-013, to detect the existence of these microorganisms in subgingival plaque bacteria. This enzymatic method was based on the observation of the hydrolysis of N-carbobenzoxy-glycyl-glycyl-arginyl-3,5-dibromo-4-hydroxyaniline (N-CBz-Gly-Gly-Arg-DBHA) and made more sensitive by adding an enhancing system. The SK-013 was specifically positive for P. gingivalis, B. forsythus, T. denticola, and some strains of Capnocytophaga species, but was not specific for any of the other bacterial strains tested. This SK-013 system may be valuable for detection and quantification of periodontal disease-associated bacteria in subgingival plaque and thus for diagnosis of periodontal infections.

Corroding characteristics of Eikenella corrodens.

Fourteen strains of Eikenella corrodens isolated from human oral cavity were studied to determine corroding characteristics. Nine out of the 14 strains produced corroding colonies under anaerobic culture condition. One of them produced corrosion even in an aerobic culture. No morphological differences in surface structures were observed between corroding and non-corroding strains of E. corrodens by transmission electron microscopy. The morphology of corroding colonies of E. corrodens was then examined by scanning electron microscopy. The surface of the corroding-colony center was smoothly convex. A boundary line was clear between the smooth center and the surrounding corrosion region. Double or triple frill-like structures surrounded the center portion with small convexities. Spreading bacterial masses were observed in the outer portion of the colony. Morphological observations of the corroding colony edge indicated that a surface translocation termed "twitching motility" or "gliding motility" occurs in the outer portion and plays a role in its colonization of periodontal regions.

[Subgingival microflora in children of early childhood, school age and circumpuberty. The proportion and frequency of gram-negative bacteria in periodontally healthy and gingivitis groups].

The present study characterized the microbial profiles of the gingival sulci in children. Subgingival samples from 36 gingivitis lesions of 18 patients and 36 sites in 18 healthy persons were examined. The tested individuals were divided into three stages according to physiological maturation i.e. early childhood, school age, circumpuberty. Puberty was confirmed through examination of wrist radiographs. Using continuous anaerobic techniques, samples were dispersed, diluted and then inoculated on selective and nonselective media and cultured under the condition appropriate gaseous phase respectively. Isolates were identified microbiologically and counted. All values were evaluated statistically. The samples were simultaneously examined by dark-field microscopy. Changes in the proportions and the frequency of periodontophathic bacteria were distinct in different stages of physiological maturation. Black-pigmented Bacteroides species were commonly found in gingivitis lesions. Bacteroides intermedius was frequently detected in the subgingival samples from children with gingivitis. In all stages, the proportion of black-pigmented Bacteroides and B. intermedius in the gingivitis groups were found to be significantly higher than that of the healthy groups. Statistical analysis revealed that levels of B. intermedius increased in circumpuberty stage compared with the 2 younger stages. Black-pigmented Bacteroides and B. intermedius were closely related to GI, PlI in 3 stages. Bacteroides gingivalis was found only in two gingivitis sites of a circumpubertal child with gingivitis. Actinobacillus actinomycetemcomitans was detected in 4 out of 12 sites in the school age group, and in 6 out of 12 sites in the circumpuberty group with gingivitis respectively. In the circumpuberty group, the proportion of A. actinomycetemcomitans in the gingivitis group was significantly higher than that of the healthy group, and A. actinomycetemcomitans was closely related to GI, PlI, CI, PPD. Eikenella corrodens was found to be associated with gingivitis in the school age and circumpuberty groups. No correlation was found in the detection of Fusobacterium nucleatum in 3 stages. Microscopic examination showed that the proportion of rods, fusiforms, filaments, motile rods, spirochetes in the gingivitis groups was significantly higher than that of the healthy groups, while the proportion of cocci in gingivitis groups was significantly lower than that of the healthy groups in 3 stages. Spirochetes were closely related to GI, PlI, PPD in all stages.

Clinical, microbiological, and immunological studies following initial preparation in adult periodontitis.

This study investigated the clinical, microbiological and immunological changes induced in adult periodontitis by initial preparation. Microflora in periodontal lesions was mainly examined by cultural methods, and serum IgG antibody levels were measured by an enzyme linked immunosorbent assay. Black-pigmented gram negative anaerobes, especially Porphyromonas (Bacteroides) gingivalis, was predominant in periodontal pockets with adult destructive periodontitis. Significantly elevated serum IgG antibody levels to P. gingivalis was found in high frequency, and these serum IgG antibody levels correlated with the presence of P. gingivalis in periodontal pockets, not with other bacteria tested. Total-CFU was significantly reduced and clinical conditions were improved after supragingival plaque control, but the microflora themselves showed few changes. Although reduction of total-CFU and changes in microflora were observed following scaling and root planing, it was found to be difficult to completely eliminate periodontopathic bacteria from periodontal pockets. Although serum IgG antibody levels to P. gingivalis was significantly reduced following initial preparation, the levels were still elevated from those of the controls. It was found that the presence of P. gingivalis was correlated with clinical conditions, including bleeding on probing and residual pocket depth following initial preparation.

Clinical, microbiological and immunological studies on recurrent periodontal disease.

The present study was performed to evaluate the clinical, microbiological and immunological aspects in the early stages of recurrent periodontal disease. After clinical monitoring of pockets with recent evidence of disease recurrence, microbiological samples for cultural analysis, serum and gingival crevicular fluid (GCF) samples were taken for IgG antibody analysis from 14 sites, 7 in 6 recurrent periodontitis patients and 7 in 7 periodontally healthy control subjects. IgG responses of serum antibody to 8 gram-negative bacterial strains were compared with those of GCF sampled from the recurrent site. The results clearly demonstrated the predominance of Bacteroides gingivalis in most subgingival plaque samples during the early stages of disease recurrence; the mean proportions of B. gingivalis were significantly different from those of the healthy sites (p less than 0.05). 4 out of 6 serum samples showed the elevated antibody responses to B. gingivalis 381; and this was closely correlated to homologous infection by this micro-organism in recurrent sites. Elevated serum antibody responses were also noted to Eikenella corrodens 1073 and Actinobacillus actinomycetemcomitans Y4. However, no relationship with homologous infection was found for A. actinomycetemcomitans. 3 out of 6 GCF samples had greater antibody titers than the serum, suggesting local antibody synthesis by the gingival cells in the recurrent pockets. The present study showed that B. gingivalis might play an important role in the pathogenesis of disease recurrence in its early stages.