bluuebunny/crossref_metadata_2025 huggingface dataset:
The oral gram‐negative anaerobic microflora in young children: longitudinal changes from edentulous to dentate mouth
Authors: Könönen E. , Asikainen S. , Saarela M. , Karjalainen J. , Jousimies‐Somer H.
Published: December 2007

Eruption of primary teeth has a great influence on the oral environment by providing suitable niches for bacterial colonization. The composition of oral gram‐negative anaerobic microflora was investigated in 21 young children (mean age 32 months) with primary dentition. The bacterial findings of samples were compared with those of the same children collected at their edentulous infant period (mean age 3 months). During the primary dentition period, 2 samples were collected from each child: a sample with dental floss from gingival margin of 2 teeth and stimulated saliva pooled with a mucosal swab sample. Both samples were cultured aerobically and anaerobically using nonselective and selective media. Prevotell melaninogenica, nonpigmented Prevolella spp., Fusobacterium nucleatum group and Capnocytophaga spp. were found in all children at the older age, whereas they occurred in edentulous mouth in 76%, 62%, 67%. and 19%, respectively. The occurrence of Prevotella loescheii increased from 14% to 90%, Prevotella intermedia from 10% to 67%, Leptotrichia spp. from 24 to 71%, Campylobacter (Wolinella) spp. from 5 to 43% and Eikenella corrodens from 5 to 57%. Only the occurrence of Bacteroides gracilis and Veillonella spp. remained at about the same level. Species not isolated from the edentulous mouth, such as Prevotella denticola, Fusobacterium spp. other than the F. nucleatum group and Selenomonas spp. were now detected in 71%, 71% and 43% of the children. The stability of the colonizing P. melaninogenica strain(s) in the oral cavity was determined by using ribotyping; 1‐2 isolates per child from the edentulous infant period of 9 children and 3‐15 isolates per child from their primary dentition period were analyzed. The P. melaninogenica population proved to be intraindividually very heterogeneous, but one child had the same ribotype both in the infant and child period. Although a stable colonization seems to be possible already in the edentulous mouth, new species or strains colonizing oral surfaces create a multiform gram‐negative anaerobic microflora in young children with primary dentition.
Stress response of Porphyromonas gingivalis
Authors: Lu B. , McBride B. C.
Published: December 2007

The heat shock response of Porphyromonas gingivalis was examined by 1 ‐ and 2‐dimensional polyacrylamide gel electrophoresis after metabolic labeling with [14C]‐amino acids. When P. gingivalis cells were shifted from 37° C to 42° C, elevated synthesis of 4 proteins with the apparent molecular weight of 92, 80, 74, and 62 kDa was observed, and synthesis of a 50‐kDa protein decreased during heat shock. The 74‐ and 62‐kDa proteins were identified as homologs of Escherichia coli DnaK and GroEL respectively by Western immunoblotting. On 2‐dimen‐sional Western blots, 2 forms of DnaK and 4 forms of GroEL were identified due to their slightly different isolelectric points. dnaK and groEL gene homologs were identified in several P. gingivalis strains and some other black‐pigmented oral species. DnaK and GroEL homolog proteins were induced when P. gingivalis cells were challenged by ethanol. These proteins were not induced by oxidative stress or change in pH.
The role of the succinate pathway in sorbitol fermentation by oral Actinomyces viscosus and Actinomyces naeslundii
Authors: Takahashi N. , Kalfas S. , Yamada T.
Published: December 2007

The sorbitol fermentation by Actinomyces viscosus and Actinomyces naeslundii was studied with washed sorbitol‐grown cells. The fermentation was followed by titration of acids produced at pH 7.0 under anaerobic conditions. Metabolic end‐products and intracellular levels of NAD, NADH and glycolytic intermediates during the fermentation were also analyzed. Cell extracts were examined for certain enzyme activities. Bicarbonate was required for acid production from sorbitol and from a mixture of glucose and sorbitol. Malate and fumarate could also support the acid production of A. viscosus. The main end‐products were succinate and lactate but not ethanol. Cell extracts showed no activities of alcohol and aldehyde dehydrogenases, but they had activities of malate dehydrogenase and fumarate reductase. In the absence of bicarbonate, malate or fumarate, the intracellular NADH/NAD ratio increased and the levels of 3‐ and 2‐phospho‐glycerate and phosphoenolpyruvate decreased. The results indicate that oral sorbitol‐fermenting actinomyces lack the ethanol pathway that can contribute to NADH oxidation. To maintain intracellular redox balance during anaerobic sorbitol fermentation, these bacteria can oxidize surplus NADH through a succinate pathway.
Utilization of glutathione (l‐γ‐glutamyl‐l‐cysteinylglycine) by Fusobacterium nucleatum subspecies nucleatum
Authors: Carlsson J. , Larsen J. T. , Edlund M.‐B.
Published: December 2007

Although fusobacteria use amino acids and peptides as energy source, it is not known whether they are able to actively transport peptides into the cell. In the present study the tripeptide glutathione was used as a model substance to investigate peptide uptake in Fusobacterium nucleatum subsp. nucleatum. Cells harvested after 2 days of growth on blood agar or in their exponential growth phase in broth were suspended in buffer with glutathione, l‐cysteinylglycine and l‐cysteine. As a measure of cell uptake, the formation of hydrogen sulfide was followed. Cells from blood agar had a low capacity to form hydrogen sulfide from the tripeptide glutathione and the dipeptide l‐cysteinylglycine. However, hydrogen sulfide was formed from l‐cysteinylglycine, but not from glutathione or from l‐cysteine, by cells grown in broth in such a way that it strongly indicated an active transport of l‐cysteinylglycine with a Km of 18 μM. Hydrogen sulfide was efficiently formed from glutathione by cells grown in broth in the presence 1 mM glutathione. In these cells a glycylglycine‐dependent l‐γ‐glutamyl peptidase activity was induced. It is probable that the efficient utilization of glutathione for hydrogen sulfide formation mirrored the uptake of l‐cysteinylglycine after an l‐γ‐glutamyl peptidase had split l‐glutamate off from glutathione.
The gingival immune response to periodontal pathogens in juvenile periodontitis
Authors: Hall E. R. , Martin S. A. , Suzuki J. B. , Falkler W. A.
Published: December 2007

A gingival explant culture system was utilized to evaluate the reactivity of local immunoglobulins produced by juvenile periodontitis tissue. Gingival explant culture supernatant fluids were screened, via a standardized dot‐immunobinding assay, for antibodies reactive to: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus, Eikenella corrodens, Peptostreptococcus micros, Peptostreptococcus anaerobius, Capnocytophaga ochracea, Eubacterium nodatum and Fusobacterium nucleatum and one nonoral microorganism, Porphyromonas asaccharolytica. Of the 75 juvenile periodontitis supernatant fluids tested, the organisms that reacted with the highest numbers of supernatant fluids were E. nodatum (72%) and A. actinomycetemcomitans (49%). More juvenile periodontitis than healthy tissue samples showed supernatant fluid reactivity to P. intermedia, C. ochracea, E. nodatum and P. micros. No significant difference was observed between the juvenile periodontitis group supernatant fluids reactivity and the supernatant fluids of the other periodontal disease groups tested. Cluster analysis revealed the association, as determined by supernatant fluid reactivity, of P. micros and C. ochracea in the juvenile periodontitis group. The data from this investigation are consistent with a hypothesis of multiple possible etiologies of periodontal destruction in juvenile periodontitis and other forms of periodontal diseases.
Identification of linear antigenic sites on the Porphyromonas gingivalis 43‐kDa fimbrillin subunit
Authors: Brant E. E. , Sojar H. T. , Sharma A. , Bedi G. S. , Genco R. J. , De Nardin E.
Published: December 2007

The fimbrillin of Porphyromonas gingivalis is thought to be an important virulence factor that mediates adherence to host surfaces. The linear immunogenic and antigenic structure of P. gingivalis fimbrillin was investigated with synthetic pep‐tides corresponding to the amino acid sequence predicted from the cloned fimbrillin gene for P. gingivalis 2561. A series of continuous and overlapping pep‐tides corresponding to the entire sequence of P. gingivalis fimbrillin was used to immunize Wistar rats. The resulting polyclonal antibodies were used to test the antigenicity of the 43‐kDa fimbrillin protein by enzyme‐linked immunosorbent assay and Western blot analysis. All the peptides elicited specific antibodies directed to the corresponding peptides but differed in their ability to elicit antisera that cross‐reacted with either native or denatured fimbrillin. Antisera to various C‐terminal one‐third peptides were more reactive to the denatured monomeric form of fimbrillin by Western blot analysis. Antisera to peptide 99‐110 was by far the most reactive against the native form of the oligomeric fimbrillin as well as the partially denatured oligomeric form of fimbrillin. The results indicate that amino acid residues 99‐110 on the native fimbrillin protein are accessible to antibody binding and that the immunogen 99‐110, when conjugated to thyroglobulin, is able to mimic an epitope on the 43‐kDa fimbrillin.
Colonization of the oral cavity of mice by an unidentified streptococcus
Authors: Marcotte H. , Rodrigue L. , Coulombe C. , Goyette N. , Lavoie M. C.
Published: December 2007

While studying the oral bacterial biota of mice, we observed an unidentified streptococcus (TG) that eventually became the dominant species of the oral cavities of all other mice in our animal facility. We found that the strain is indigenous to Jackson Laboratory mice but is absent in animals from Charles River Laboratories. TG was also transmitted from artificially contaminated BALB/c mice to the oral cavities of 4 other mouse strains. Streptococcus sp. TG stimulated the secretory and systemic immune systems of artificially contaminated Charles River BALB/c mice but did not provoke clinical symptoms. The increase in antibody level to TG did not prevent its colonization and persistence in these mice. In mice from Jackson Laboratory, the secretory and systemic immune response to TG was significantly lower. In vitro, Streptococcus sp. TG inhibited murine oral lacto‐bacilli and staphylococci, probably due to the production of hydrogen peroxide.
Sulfate‐reducing bacteria in the periodontal pocket
Authors: van der Hoeven J. S. , van den Kieboom C. W. A. , Schaeken M. J. M.
Published: December 2007

This report is the first to describe the occurrence of sulfate‐reducing bacteria in the human mouth. Samples of subgingival dental plaque were examined for the presence of sulfate‐reducing bacteria. Using enrichment cultures, sulfate‐reducing bacteria were detected in 25 (58%) of 43 individuals, and in 39 (48%) of the 82 samples. Pure isolates of sulfate‐reducing bacteria, obtained from a limited number of enrichment cultures, belonged to the genera Desulfobacter and Desulfovibrio. These genera are also the predominant sulfate‐reducing bacteria in the human large intestine. The sulfate‐reducing bacteria use sulfate as terminal electron acceptor to oxidize low‐molecular‐weight organic compounds, mainly products of microbial fermentation such as acetate, lactate etc. The numbers of sulfate‐reducing bacteria in the mouth are assumed to be limited by sulfate. Potential sources of sulfate in the subgingival area include free sulfate in pocket fluid and glycosaminoglycans from periodontal tissues.
Localization of interleukin‐8 in human gingival tissues
Authors: Fitzgerald J. E. , Kreutzer D. L
Published: December 2007

Periodontitis and gingivitis are chronic inflammatory diseases of the periodontium and adjacent tissues. This site‐specific inflammation is characterized by a local infiltration of polymorphonuclear leukocytes and T lymphocytes. Interleukin‐8 is a low‐molecular‐weight cytokine that is thought to be responsible for the induction and maintenance of localized inflammation. We hypothesized that locally produced interleukin‐8 plays a central role in chronic inflammation of periodontitis by regulating the recruitment and activation of leukocytes in the gingival tissues. To test this hypothesis, we determined whether the interleukin‐8 antigen is present locally and is cell‐associated. Inflamed and control tissues were analyzed: 1) for the interleukin‐8 antigen; 2) by molecular weight; 3) for location; and 4) for the messenger RNA (mRNA) of interleukin‐8. The conclusions from these data were that: 1) interleukin‐8 antigen and mRNA was elevated in chronically inflamed gingiva; and 2) the major interleukin‐8 antigen was detected only in the epithelial cell layer. These results support that interleukin‐8 may play a crucial role in the recruitment and activation of neutrophils and T lymphocytes in periodontitis.
The cloning, expression and sequence analysis of a second Porphyromonas gingivalis gene that codes for a protein involved in hemagglutination
Authors: Progulske‐Fox A. , Tumwasorn S. , Lépine G. , Whitlock J. , Savett D. , Ferretti J. J. , Banas J. A.
Published: December 2007

Progulske‐Fox A, Tumwasorn S, Lepine G, Whitlock J, Savett D, Ferretti JJ, Banas JA. The cloning, expression and sequence analysis of a second Porphyromonas gingivalis gene that codes for a protein involved in hemagglutination.

It has been suggested that Porphyromonas gingivalis may possess more than one hemagglutinin. We have previously reported the cloning of a gene (hagA) that encodes a hemagglutinin. In this study we report the cloning, characterization, and sequencing of a second gene (hagB) that encodes a protein that also appears to be involved in hemagglutination. Antiserum to the clone (ST 7) was found to inhibit hemagglutination by P. gingivalis 381, and hemagglutinating inhibition activity of anti‐P. gingivalis antiserum was reduced by adsorption of the antiserum with cells of clone ST 7. Restriction mapping and Southern analysis indicates there is little or no DNA homology between this cloned 4.8‐kb Hind DNA fragment and a cloned hemagglutinin gene we have previously described. Minicell analysis of the cloned P. gingivalis chromosomal DNA fragment revealed that the major gene product is a 49‐kDa protein. Immunoaffinity chromotography using purified rabbit immunoglobulin G against the cloned protein resulted in the purification of a major reactive 49‐ to 50‐kDa protein from a P. gingivalis cell lysate. Nucleotide sequence analysis revealed the hagB open reading frame to be 1053 nucleotides in length with a mol% G+C of 59.9% coding for a protein of 350 residues with a calculated molecular weight of 39.375 kDa. This protein was also determined to be basic and hydrophilic and to contain a potential signal peptide. Comparison of both the nucleotide and derived amino acid sequences with computer‐based databases did not reveal any significant homologies between hagB and any other previously sequenced genes.
Distribution of biotypes and antimicrobial susceptibility of Actinobacillus actinomycetemcomitans
Authors: Avila‐Campos M. J. , Carvalho M. A. R. , Zelante F.
Published: December 2007

Eighty isolates of Actinobacillus actinomycetemcomitans from 30 Brazilian periodontitis patients were examined to determine the distribution of biotypes and in vitro antimicrobial susceptibility. Seventy‐seven percent of the isolates belonged to biotype X. All A. actinomycetemcomitans isolates were susceptible to cefoxitin, imipenem and tetracycline.
Feγ‐binding bacteria in periodontal lesions
Authors: Hillestad M. , Helgeland K. , Tolo K.
Published: December 2007

The proportion of bacteria exhibiting surface Feγ‐binding proteins was determined in periodontal pockets of 20 patients diagnosed with periodontal disease and in subgingival areas of 20 patients without periodontal lesions. Bacterial smears were examined by fluorescence microscopy based on DNA staining (Hoechst 33256) and staining of Feγ‐binding proteins by human biotin‐labelled Feγ and Texas red‐conjugated streptavidin. Feγ‐binding proteins were observed in all smears from the patients diagnosed with periodontitis, and in a majority of the smears high proportions of the bacteria were positive for Feγ‐binding proteins. In contrast, most smears from patients without periodontal lesions included low or undetectable proportions of bacteria with Feγ‐binding proteins.
Three subtypes of the tet(M) gene identified in bacterial isolates from periodontal pockets
Authors: Olsvik B. , Tenover F. C. , Olsen I. , Rasheed J. K.
Published: December 2007

The tet(M) genes were characterized from 84 isolates of 10 different bacterial species isolated from the periodontal pockets of 16 patients with periodontal disease. A 740 bp polymerase chain reaction product from the hypervariable region of the tet(M) structural gene was cleaved with the restriction enzymes AluI and HinfI. Three different restriction patterns were identified for each of the two enzymes. By DNA sequencing, using a direct solid‐phase automated sequencing method, the isolates could be grouped into 3 different clusters oftet(M) subtypes. The internal DNA homology within each subtype was 98‐100%; the homology between clusters was 89‐94%. Two different subtypes were identified in 9 of 10 bacterial species, and the remaining species had 3 different subtypes. One of the subtypes (M3) was seen mainly in the anaerobic isolates. This subtype was different from all earlier sequenced structural tet(M) genes present in the Genbank. Most patients had two different subtypes of tet(M), and a third subtype was seen in the 3 patients who exhibited the greatest variety of tetracycline‐resistant bacterial species. It appears that the presence of one subtype of the tet(M) gene within a patient or bacterial species does not prevent the acquisition of another subtype of the same gene. This study identified a new subtype of the tet(M) gene and grouped it into 3 distinct yet highly homologous genetic subtypes.
The inhibitory action of fatty acids on oral bacteria
Authors: Shapiro S.
Published: December 2007

Saturated and unsaturated fatty acids and fatty acid derivatives were examined for their growth‐inhibitory effects towards three selected oral bacteria: Porphyromonas gingivalis, Selenomonas artemidis, and Streptococcus sobrinus. Of the 45 compounds surveyed, only one, myristoleic acid, was inhibitory towards S. artemidis at a concentration <100 μg/ml. cis‐Hexadecenoic and cis‐octadecenoic acids were generally inhibitory towards P. gingivalis and S. sobrinus, but there was no correlation between the position of the double bond and the minimum inhibitory concentration. Supra‐minimum inhibitory concentrations of palmitoleic acid did not promote leakage of intracellular materials from either P. gingivalis or S. sobrinus, nor was l‐iso‐leucine uptake by S. sobrinus inhibited. Fatty acids and derivatives were also examined for prospective synergistic or antagonistic interactions with thymol vis‐à‐vis growth inhibition of the test strains. Laurie acid and myristic acid each behaved synergistically with thymol to inhibit the growth of at least one test strain, whereas cis‐10‐heptadecenoic acid and thymol were noticeably antagonistic towards the growth of S. sobrinus.
Development and evaluation of a selective and differential medium for the primary isolation ofPeptostreptococcus micros
Authors: Turng B.‐F. , Guthmiller J. M. , Minah G. E. , Falkler W. A.
Published: December 2007

Peptostreptococcusmicros, an anaerobic gram‐positive coccus, has been associated with periodontal and endodontic lesions, including those refractory to treatment, as well as many human polymicrobial infections in other body locations. A selective and differential medium for the primary isolation ofP. microswas developed and evaluated. Columbia CNA agar, a selective medium for gram‐positive cocci, was supplemented with glutathione and lead acetate (P. microsmedium: PMM).P. microshas a characteristic of rapidly utilizing the reduced form of glutathione to form hydrogen sulfide, which reacts with lead acetate producing a black precipitate in the medium. When grown on PMM,P. microscan be easily identified by its typical colonial morphology and the presence of a black precipitate directly under the colony. PMM was compared for the growth ofP. microswith phenylethyl alcohol agar (PEA) and Columbia base medium (CBM) with 80 strains ofP. microsand 30 strains of other gram‐positive cocci. AllP. microsisolates tested grew and showed the typical morphology ofP. microson PMM. Using colony counts on CBM as controls, there was an average 81.8% recovery in the number ofP. microscolonies on PMM, in contrast to an average 6.1 % recovery on PEA. Subgingival plaque and tongue samples from 12 adult periodontitis and 6 early‐onset periodontitis patients were cultured onto PMM for the isolation ofP. micros.P. microswas isolated on PMM and identified biochemically and enzymatically from both adult periodontitis and early‐onset periodontitis patients with higher percentages isolated from the diseased periodontal pockets of adult periodontitis patients; furthermore, this is the first isolation ofP. microsfrom tongue samples taken from periodontally diseased patients. This medium in cultural studies will further our understanding and assist future investigations ofP. microsinvolved in disease processes.
Biological and antigenic characterization of three BApNA‐hydrolyzing proteases from the culture supernatant of Porphyromonas gingivalis
Authors: Hinode D. , Masuda K. , Yoshioka M. , Hayashi H. , Nakamura R. , Grenier D. , Mayrand D.
Published: December 2007

Biological and antigenic distinction of 3 Nα‐benzoyl‐DL‐arginine p‐nitroanilide (BApNA)‐hydrolyzing proteases (Pase‐B, Pase‐C and Pase‐S) isolated from the culture supernatant of Porphyromonas gingivalis were determined. Immunoblotting analysis of these enzymes using a polyclonal antibody against Pase‐S, which is a soluble, clostripain‐like protease, revealed immunological distinction from Pase‐C, a vesicle‐associated thiol‐protease. Pase‐B, a vesicle‐associated clostripain‐like protease, reacted with the antibody and was also found to contain a considerable amount of carbohydrates in its structure, as compared with the others. Analysis of N‐terminal amino acids of Pase‐B provided a sequence not found in the SwissProt data bank or previously reported as N‐terminal sequences of proteases from P. gingivalis. Pase‐S, resembling Pase‐B in its hydro‐lytic specificity, cleaved only arginine residues of peptides and degraded type IV and denatured type I collagen. Pase‐C hydrolyzed Nα‐benzoyl‐DL‐lysine p‐nitro‐anilide and showed the strongest capacity of degrading native type I collagen. This enzyme was also the only one to possess hemagglutinating activity. Our findings suggest that Pase‐S from P. gingivalis is less active than Pase‐C and that the enzyme may be an isozyme of Pase‐B.
Prevotella intermedia fimbriae mediate hemagglutination
Authors: Leung K.P. , Fukushima H. , Nesbitt W. E. , Clark W. B.
Published: December 2007

Our earlier studies demonstrated that clinical strains of Prevotella intermedia, isolated from human periodontal pockets, possess various types of fimbria (surface appendages) as determined ultrastructurally. These bacteria have the ability to agglutinate selected mammalian erythrocytes. Hemagglutinating activity exhibited by these cells may be attributable to these surface structures. Strain 17, which posseses fimbriae of 8 nm in diameter and readily agglutinates human, monkey, sheep, rabbit, and mouse erythrocytes was selected to determine whether these fimbriae possessed the hemagglutinating activity. Fimbriae were mechanically sheared, concentrated by ammonium sulfate precipitation, solubilized in 10 mM Tris‐HCl buffer containing 0.5% deoxycholate and partly purified by ultracentrifugation in a 10–50% linear sucrose gradient. Isolated fimbriae banded at a density of 1.10–1.15 g/ml, appeared fairly uniform ultrastructurally, and possessed hemagglutinating activity. The hemagglutinating activity of P. intermedia whole cells and isolated fimbriae was reduced by treatment with proteases and eliminated by treatment with heat at 80°C for 10 min. The optimal pH for the hemagglutination was 7.0. In the process of hemagglutination, P. intermedia whole cells and isolated fimbriae bound to rabbit erythrocytes as observed by: (a) decrease in the hemagglutinating activity of bacterial whole cells and isolated fimbriae after incubation with rabbit erythrocytes, (b) increase in hemagglutinating activity of the red cells used for absorption and (c) the presence of P. intermedia whole cells and fimbriae on red cells after absorption as revealed by immunoelectron microscopy. Both the immune immunoglobulin G of the rabbit polyclonal anti‐fimbriae antibody and the immune immunoglobulin G and its Fab fragments of the murine monoclonal anti‐fimbriae antibody were effective inhibitors of hemagglutination mediated by isolated fimbriae. Immunogold labeling showed that the monoclonal antibody bound specifically to P. intermedia fimbriae. These results collectively suggest that the hemagglutination exhibited by P. intermedia may be attributable to the fimbriae or the fimbrial‐associated components.
Proteolytic artifacts in SDS‐PAGE analysis of selected periodontal pathogen:
Authors: Weidner M.‐F. , Grenier D. , Mayrand D.
Published: December 2007

The aim of the study was to examine whether proteolytic artifacts, which result in a loss and poor resolution of protein bands, occur during sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis analysis of cellular proteins from selected proteolytic (Porphyromonas gingivalis, Prevotella nigrescens and Treponema denticola) and non‐proteolytic (Fusobacterium nudeatum) bacteria. Conditions to limit or prevent proteolysis were also investigated. Bacterial cells were incubated in solubilizing buffer (SDS +β mercaptoethanol) at room temperature for various periods of time before boiling. A control assay consisted of trichloroacetic acid‐treated bacterial cells. Cellular proteins were separated by electrophoresis and stained with Coomassie blue. Proteolysis occurred very rapidly in the case of P. gingivalis (<30 s), whereas a longer incubation time (>1 h) was required to observe similar effects in P. nigrescens and T. denticola. No proteolysis was observed for F. nudeatum. In all cases, heat (100°C) and low pH (<4) treatments of bacterial cells could avoid production of proteolytic artifacts. Incorporation of specific protease inhibitors before solubilization of bacteria could also prevent proteolysis. More particularly, N‐α‐p‐tosyl‐l‐lysine chloromethyl ketone (TLCK), iodoacetamide and diisopropylfluorophosphate (50 mM) were highly efficient for P. gingivalis, P. nigrescens and T. denticola, respectively. When outer membranes of P. gingivalis were prepared in the presence of TLCK, numerous additional protein bands, not seen in the absence of TLCK, were detected. The present study suggests that specific protease inhibitors, effective in preventing proteolysis. should be identified and added during cell fractionation and protein purification procedures.
Identification of a repeated epitope recognized by human serum antibodies in a surface protein antigen of Streptococcus mutans
Authors: Senpuku H. , Nakai M. , Koga T. , Hanada N. , Nisizawa T.
Published: December 2007

This study determined the antigenic determinants of a 190‐kDa surface protein antigen of Streptococcus mutans that is involved in the initial attachment to the tooth surface. In 5 subjects, the reactivities of serum antibodies to 7 overlapping surface protein antigen fragments covering the entire antigen molecule and 19 sequential overlapping synthetic 19‐mer peptides covering the entire A‐region of the surface protein antigen were examined with enzyme‐linked immunosorbent assay (ELISA). The study showed that the A‐region of the antigen is strongly immunogenic in humans and contains several widely distributed epitopes. In addition, an amino acid sequence of the one of dominant epitopes in a certain subject was identified as LTAENTAI with ELISA inhibition assays using the relevant truncated peptides. This epitope was located both at the positions from L‐346 to 1–364 and L‐430 to E‐437 of the antigen molecule, and serum antibodies against the epitope were found in 3 of the 5 subjects.
Age‐related changes in salivary antibodies to commensal oral and gut biota
Authors: Percival R. S. , Marsh P. D. , Challacombe S. J.
Published: December 2007

The prevalence of mucosally derived infections appears to increase with age, suggesting dysfunction at the mucosal surfaces. The present investigation was undertaken to examine any age‐related changes in secretion rates and concentrations of secretory antibodies in whole and parotid saliva in a healthy adult population. A total of 116 subjects were subdivided into the following age groups: 20–39 years, 40–59 years, 60–79 years and 80 years and over. Specific immunoglobulin A (IgA), IgG and IgM antibodies in whole and parotid saliva to Streptococcus mutans (serotype c), Actinomyces viscosus NCTC 10951, and Escherichia coli NCTC 10418 were quantified by enzyme‐linked immunosorbent assay. IgA antibodies to all three organisms examined increased with age in both whole and parotid saliva, whereas IgG antibody levels to S. mutans in whole saliva were significantly decreased with age. IgG antibodies to E. coli in parotid saliva were reduced in older age groups. IgM antibody levels to S. mutans were reduced with age in both secretions, whereas IgM antibodies to A. viscosus were greatest in the oldest age groups. No significant changes with age were observed in salivary IgM antibody levels to E. coli. No significant reduction in the secretion rates of IgA antibodies were observed in parotid or whole saliva, whereas IgG and IgM antibody secretion rates to all three microorganisms were reduced in most age groups in both whole and parotid saliva. The results of this investigation have demonstrated age‐related changes with salivary antibodies, but, whereas salivary IgG and IgM antibodies showed decreases, salivary IgA levels generally increased with age. This suggests that the ability to form IgA antibody responses is not impaired with increased age, and that secretion rates and functional properties of antibodies may be as important as concentrations in protection against mucosal infective diseases.
Interleukin‐1 gene expression in macrophages induced by surface protein components of Porphyromonas gingivalis: role of tyrosine kinases in signal transduction
Authors: Saito A. , Sojar H. T. , Genco R. J.
Published: December 2007

We examined interleukin‐p (IL‐1β) mRNA expression and protein tyrosine kinase activities induced by surface protein components of Porphyromonas gingivalis to gain insights into signaling pathways leading to macrophage responses. Reverse transcriptase‐polymerase chain reaction analyses showed that native fknbriae, full‐length recombinant fimbrillin, and a lectin‐like 12‐kDa antigen of P. gingivalis induced rapid expression of mRNA encoding IL‐1β in BALB/c and lipopolysacharide‐hyporesponsive C3H/HeJ peritoneal macrophages. The antigens also specifically activated tyrosine kinase(s) in macrophages. The ability of the surface protein components to induce the cytokine mRNA accumulation was markedly abrogated by tyrosine kinase inhibitors. The findings reported here suggest that IL‐1β expression in macrophages is a functional consequence of tyrosine kinase activation by the P. gingivalis surface protein components.
Pathogenicity of Prevotella intermedia and Prevotella nigrescens isolates in a wound chamber model in rabbits
Authors: Hafström C. , Dahlén G.
Published: December 2007

The pathogenicity of 14 isolates identified as Prevotella intermedia or Prevotella nigrescens by serogrouping using monoclonal antibodies was compared in a tissue cage model in rabbits. Seven strains from periodontal abscesses, 5 strains from deep periodontal pockets and 2 strains from gingivitis were tested in the animal model comprising 6 Teflon tissue cages implanted on the back each of 34 rabbits. A total of 105–108 cells of P. intermedia or P. nigrescens strains were inoculated alone or together with either Actinobacillus actinomycetemcomitans or Streptococcus mitis. Five strains of Porphyromonas gingivalis were used as a reference. The infectivity was recorded as pus formation and log viable count in aspirated material for 3, 7 and 14 days. None of the Prevotella strains inoculated in monoculture survived more than 3 days, and they had no capacity to produce abscess. P. intermedia or P. nigrescens strains in combination with A. actinomycetemcomitans produced abscesses in 33–100% and with S. mitis in 42–100%. No difference in abscess formation or log viable count in samples after 14 days was recorded between serogroup I (P. intermedia) and serogroup II and III (P. nigrescens). The infectivity of P. intermedia or P. nigrescens strains did not differ whether they were isolated from periodontal abscess, periodontal pocket or gingivitis. P. intermedia and P. nigrescens strains produced abscesses in combination with a facultative anaerobic strain and appears to have a similar pathogenicity in the wound chamber model in rabbits.
Immunohistological study of lesions induced by Porphyromonas gingivalis in a murine model
Authors: Gemmell E. , Bird P. S. , Bowman J. J. D. , Xu L. , Polak B. , Walsh L. J. , Seymour G. J.
Published: December 2007

A previous study used a mouse model to demonstrate protection after challenge with Porphyromonas gingivalis ATCC 33277. In the present study, this same model was used to determine the phenotype of cells recruited into the lesions during the course of the protective immune response after immunization with this periodontal pathogen. BALB/c mice were immunized with 100 μg of P. gingivalis outer membrane antigens per mouse weekly for 3 weeks followed by challenge with live organisms 3 weeks after the final immunization. Hematoxylin and eosin‐stained sections showed inflammatory infiltrates in all lesions from control (immunized with adjuvant only) and immunized mice. The lesions developed central necrotic cores surrounded by neutrophils, phagocytic macrophages and lymphocytes. Neutrophils were the predominant cells in the lesions 1 day after challenge with significantly more in immunized than control mice. Acid phosphatase and nonspecific esterase‐positive macrophages were detected at day 4 and became the predominant cells in the healing lesions. CD4‐ and CDS‐positive T‐cells were present from day 1, and while numbers increased over time, there were no significant differences in control or immunized mice. When mice were depleted of CD4 or CDS cells prior to immunization with P. gingivalis, fewer neutrophils were found in the lesions 1 day after challenge compared with undepleted immunized mice. Acid phosphatase and nonspecific esterase‐positive macrophages were not affected by T‐cell depletion. The results suggest that the P. gingivalis‐induced lesion in immunized BALB/c mice is consistent with a strong innate immune response involving the recruitment of neutrophils in the first instance which may be under the control of T cells. This is followed by the infiltration of phagocytic macrophages which are involved in the healing process and do not appear to be regulated by T cells.
Detection of Bacteroides forsythus by immunomagnetic capture and a polymerase chain reaction‐DNA probe assay
Authors: Chandad F. , Guillot E. , Mouton C.
Published: December 2007

The aim of this study was to combine immunomagnetic capture and a polymerase chain reaction (PCR) followed by hybridization with a DNA probe for the detection of Bacteroides forsythus. Magnetic beads were coated with the immunoglobulin G fraction of an antiserum specific for B. forsythus. Aliquots were incubated with various concentrations of a suspension of B. forsythus or with a suspension containing 16 bacterial species, at a concentration of 1010 cells/ml, spiked with dilutions of B. forsythus. Beads with bound bacteria were boiled, and the target DNA in the supernatant was amplified to generate a 392‐bp PCR fragment specific for B. forsythus. The amplified product was detected by dot‐blot hybridization with a digoxigenin‐labeled 392‐bp probe. The detection limit was determined to be 10 cells/ml using immunocapture on a suspension of B. forsythus and 100 on spiked bacterial suspensions. Subgingival plaque samples were obtained from 39 Bolivian individuals with poor oral hygiene. Each sample was analyzed by the above procedure and by immunofluorescence. The overall prevalence of individuals harboring B. forsythus was 62% by immunofluorescence and 82% by PCR‐DNA probe assay. The immunocapture, PCR, DNA‐probe procedure should be useful for the detection of B. forsythus, particularly in false‐negative samples obtained by less sensitive techniques.
Immunochemical detection of CD14 on human gingival fibroblasts in vitro
Authors: Hiraoka T. , Izumi Y. , Sueda T.
Published: December 2007

The activation of monocytes and macrophages induced by lipopolysaccharide has been shown to contribute to the binding of lipopolysaccharide and lipopolysaccharide‐binding protein complex to the cell surface CD 14 molecule. To clarify the mechanism of the lipopolysaccharide‐induced modulation of the function of gingival fibroblasts, we investigated the effect of anti‐CD14 on interleukin 6 (IL‐6) production on human gingival fibroblasts in vitro. Immunochemical staining revealed weak positivity for CD14 on fibroblasts from healthy gingiva, while strong positivity for CD14 was found on fibroblasts from inflamed gingiva. Western blot profiles of the fibroblasts and monocytes showed a CD14‐positive reaction at 55 kDa. Fluorescein isothiocyanate–conjugated Escherichia coli lipopolysaccharide bound to fibroblasts more strongly in the presence of 10% fetal bovine serum than without serum. This binding, as well as IL‐6 production, was blocked by anti‐CD14 monoclonal antibody. The results showed that CD14 was present on human gingival fibroblasts, which suggests that lipopolysaccharide modulation of gingival fibroblast function depends on CD14.
Distribution of interleukin‐2 receptor α‐chain and cells expressing major histocompatibility complex class II antigen in chronic human periapical lesions
Authors: Márton I. J. , Dezsö B. , Radics T. , Kiss C.
Published: December 2007

In situ distribution of CD2+ T‐lymphocytes, CD4+ and CD8+ T‐cell subsets, CD14+ macrophages, interleukin‐2 receptor α‐chain (IL‐2Rα) and class II major histocompatibility complex antigen (major histocompatibility complex class II, HLA‐DR) expressing cells were determined in 14 chronic human periapical granulomas by imrnunohistochemical method using monoclonal antibodies. CD2+ lymphocytes were rather evenly distributed within the classical granulation tissue and comprised 55% of the mononuclear cells. Macrophages were distributed all over the periapical area, but their proportion was much less than that of T lymphocytes. Both small, lymphocyte‐like mononuclear cells and larger mononuclear cells resembling macrophages displayed mild to strong circumferential staining with the anti‐HLA‐DR antibody. The majority of lymphocytes expressed IL‐2Rα indicating the activated state of T cells within the lesion.
Functional analysis of pVT745, a plasmid from Actinobacillus actinomycetemcomitans
Authors: Novak K. R , Lee L. N. , LeBlanc D. J.
Published: December 2007

Plasmid pVT745 is a 25.1‐kb replicon isolated from Actinobacillus actinomycetemcomitans strain VT745. A previous report described the hybridization of pVT745 in 5 strain‐specific patterns to chromosomal DNA from 15 other A. actinomycetemcomitans strains. However, pVT745 does not share homology with the chromosome of the strain from which it was isolated, VT745. It was hypothesized that the shared areas of homology might represent insertion sequence elements and/or transposons possibly encoding resistance to one or more antibiotics. An antibiogram of strain VT745 demonstrated that this strain was uniformly susceptible to all antibiotics examined. Because insertion sequence elements and transposons are mobile genetic elements, a series of cell passaging experiments, followed by Southern hybridization was conducted in a attempt to detect transposition of pVT745 homologous DNA within the chromosomes of several A. actinomycetemcomitans strains. The results of these experiments suggested stability of the homologous DNA both within the chromosome and on the plasmid. It was also possible that pVT745 represented a lysogenic bacteriophage. Phage induction experiments were conducted under conditions that induced a previously described A. actinomycetemcomitans lysogenic phage, but no phage could be induced from strain VT745. Attempts to obtain isolates of VT745 cured of pVT745 were also unsuccessful.
The importance of fimbriae in the virulence and ecology of some oral bacteria
Authors: Hamada S. , Amano A. , Kimura S. , Nakagawa I. , Kawabata S. , Morisaki I.
Published: December 2007

Cumulative evidence indicates that bacterial adherence to mucosal and tooth surfaces as well as bacterial coaggregation are essential steps for colonization of various oral bacterial species. Bacterial fimbriae have been shown to play an important role in the interaction between bacteria and host cells or among bacterial cells. The properties of fimbriae from selected species of oral bacteria are discussed in terms of virulence traits and ecological significance. Among others, Porphyromonas gingivalis fimbriae have been most extensively studied. The fimbrial structure is composed of 41‐kDa nmbrillin proteins. DNA sequencing of the fimbrillin gene (fim A) from nine strains of P. gingivalis suggests intraspecies variation in the structure offimA, while retaining common immunochemical specificities. P. gingivalis fimbriae exhibit a wide variety of biological activities including immunogenicity, binding to various host proteins, stimulation of cytokine production and promotion of bone resorption, Actinobacillus actinomycetemcomitans also possesses fimbriae; however, little is known concerning their chemical, genetical, and biological properties. Fimbriae of Prevotella intermedia are shown to induce hemagglutination reaction, while those of Prevotella loescheii are found to cause coaggregation with other bacteria, i.e., Actinomyces viscosus and sanguis streptococci. Fimbriae from gram‐positive oral bacteria such as oral Actinomyces and sanguis streptococci are described. These fimbriae may participate in coaggregation, binding to saliva‐coated hydroxyapatite or glycoprotein of the surface layer of oral epithelial cells. Taken together, fimbriae are key components in cell‐to‐surface and cell‐to‐cell adherence of oral bacteria and pathogenesis of some oral and systemic diseases.
Antigenic components of Actinobacillus actinomycetemcomitans lipopolysaccharide recognized by sera from patients with localized juvenile periodontitis
Authors: Gu K. , Bainbridge B. , Darveau R. P. , Pagel R. C.
Published: December 2007

The dominant antigen of Actinobacillus actinomycetemcomitans recognized by high‐titer sera from patients with localized juvenile periodontitis is the serotype antigen located in the O‐side chains of lipopolysaccharide. Whether such sera contain antibodies reactive with other epitopes in lipopolysaccharide, as is the case for patients with rapidly progressive periodontitis, remains unknown. We prepared and characterized by gas liquid chromatography lipopolysaccharide, lipid A, core carbohydrate with no or few O‐side chains (core) and high‐molecular‐mass carbohydrate‐rich in O‐side chains (oligosaccharide) from A. actinomycetemcomitans ATCC 43718 (serotype b, Y4). Using enzyme‐linked immunosorbent assay (ELISA), sera from 36 patients with localized juvenile periodontitis were surveyed using whole‐cell sonicate as plate antigen. The seven highest titer sera were selected for further study. Specific IgG antibody binding was observed to intact lipopolysaccharide and to all the lipopolysaccharide fractions. The mean titers were highest for intact lipopolysaccharide (138.8 ELISA units), and lipid A (122 ELISA units), followed by the core fraction (81 ELISA units) and the oligosaccharide fraction (69.5 ELISA units). ELISA inhibition revealed that the core fraction at a concentration of 10 μg/test well inhibited antibody binding to A. actinomycetemcomitans lipopolysaccharide by a mean value of 56.7%. To further characterize antibody binding to the core fraction, ELISA inhibition was performed using as inhibitor the core carbohydrate fraction of the Re mutant of Salmonella minnesota, which is known to contain only alpha‐keto‐3‐deoxyoctonate residues and phosphate. This fraction at 10 μg/test well inhibited binding of antibodies from 6 of 7 test sera with a mean value of 49.2%. Thus, sera from patients with localized juvenile periodontitis contain antibodies that bind to the O‐side chains of lipopolysaccharide, as has been previously reported, but they also contain antibodies that bind to lipid A and to lipopolysaccharide core polysaccharide epitopes, specifically to alpha‐keto‐3‐deoxyoctonate moieties. The humoral immune response to A. actinomycetemcomitans in patients with localized juvenile periodontitis is more complex than previously reported and is very similar to that of patients with rapidly progressive periodontitis.
Variable serum immunoglobulin G immune response to genetically distinct Eikenella corrodens strains coexisting in the human oral cavity
Authors: Chen C. , Ashimoto A.
Published: December 2007

This study examined the variable serum immunoglobulin G (IgG) levels to genetically distinct autologous Eikenella corrodens strains by enzyme‐linked immunosorbent assay (ELISA). Twenty subjects, including 10 adult periodontitis patients, 5 juvenile periodontitis patients and 5 periodontally healthy subjects were examined. Each subject was colonized by 2–8 genetically distinct E. corrodens strains. The serum IgG levels to autologous E. corrodens within individuals were significantly different in 7 adult periodontitis patients, 4 juvenile periodontitis patients and a periodontally healthy subject. Poor correlation was found in diseased subjects between serum IgG levels to autologous strains and to reference strains ATCC 23834 or FDC 373. Four adult periodontitis patients and two juvenile periodontitis patients exhibited significant serum IgG levels to autologous E. corrodens strains (two standard deviations above the mean for periodontally healthy subjects); two of these six diseased subjects exhibited low serum IgG levels to reference strains and would have been classified as low immune responders if only reference strains had been used in ELISA. This study showed the importance of using autologous E. corrodens strains in the assessment of serum IgG immune responses to this organism.
Ribotype diversity of Actinomyces With similar intraoral tropism but different types of N‐acetyl‐β‐d‐galactosamine binding specificity
Authors: Hallberg K. , Holm C. , Hammarström K.‐J. , Kalfas S. , Strömberg N.
Published: December 2007

Sixty‐three isolates of Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus from three subjects clustered into 22 ribotypes. Unique ribotypes were found in the subjects and within individual tissue sites (bucca, tooth and tongue). A. odontolyticus ribotypes shared tongue‐specific binding properties, while those of genospecies 1 and 2 from buccal and tooth surfaces shared different types of N‐acetyl‐β‐d‐galactosamine binding specificity.