Sotirios Kalfas

The Cytolethal Distending Toxin Induces Receptor Activator of NF-κB Ligand Expression in Human Gingival Fibroblasts and Periodontal Ligament Cells

ABSTRACT Actinobacillus actinomycetemcomitans is associated with localized aggressive periodontitis, a disease characterized by rapid loss of the alveolar bone surrounding the teeth. Receptor activator of NF-κB Ligand (RANKL) and osteoprotegerin (OPG) are two molecules that regulate osteoclast formation and bone resorption. RANKL induces osteoclast differentiation and activation, whereas OPG blocks this process by acting as a decoy receptor for RANKL. The purpose of this study was to investigate the effect of A. actinomycetemcomitans on the expression of RANKL and OPG in human gingival fibroblasts and periodontal ligament cells. RANKL mRNA expression was induced in both cell types challenged by A. actinomycetemcomitans extract, whereas OPG mRNA expression remained unaffected. Cell surface RANKL protein was also induced by A. actinomycetemcomitans, whereas there was no change in OPG protein secretion. A cytolethal distending toxin (Cdt) gene-knockout strain of A. actinomycetemcomitans did not induce RANKL expression, in contrast to its wild-type strain. Purified Cdt from Haemophilus ducreyi alone, or in combination with extract from the A. actinomycetemcomitans cdt mutant strain, induced RANKL expression. Pretreatment of A. actinomycetemcomitans wild-type extract with Cdt antiserum abolished RANKL expression. In conclusion, A. actinomycetemcomitans induces RANKL expression in periodontal connective tissue cells. Cdt is crucial for this induction and may therefore be involved in the pathological bone resorption during the process of localized aggressive periodontitis.

Caspase 1 Involvement in Human Monocyte Lysis Induced by Actinobacillus actinomycetemcomitans Leukotoxin

ABSTRACT Actinobacillus actinomycetemcomitans, an oral bacterium implicated in the etiology of periodontal diseases, produces a leukotoxin that selectively lyses primate neutrophils and monocytes, the major populations of defense cells in the periodontium. Though lysis requires expression of the receptor lymphocyte function-associated molecule 1 (LFA-1) on the cell surface, not all LFA-1-expressing leukocyte populations are equally susceptible to the toxin. In this study, the susceptibility of human leukocytes to leukotoxin-induced lysis is compared to their expression of LFA-1 and the activity of caspase 1. Cytolysis was determined by the activity of lactate dehydrogenase released from peripheral human leukocytes after 1-h exposure to leukotoxin. Monocytes were lysed at leukotoxin concentrations of ≥5 ng/ml, while the corresponding values for neutrophils and lymphocytes were approximately 10 times greater. Similar LFA-1 expression was found in all susceptible cell populations irrespective of their degree of sensitivity to the toxin. Exposure of monocytes to leukotoxin increased their caspase 1 activity about fivefold within 10 to 20 min. Presence of the caspase 1 inhibitor Ac-YVAD-CMK significantly blocked the leukotoxin-induced lysis of monocytes only. At sublytic concentrations, leukotoxin induced no apoptotic activity in monocytes, as revealed by the lack of caspase 3 activation and DNA fragmentation. Monocytes are the most lysis-sensitive leukocytes for A. actinomycetemcomitans leukotoxin. Their lysis by this toxin depends on caspase 1 activation and proceeds through a process that differs from classical apoptosis.

Abundant Secretion of Bioactive Interleukin-1beta by Human Macrophages Induced by Actinobacillus actinomycetemcomitans Leukotoxin

ABSTRACT Actinobacillus actinomycetemcomitans produces a leukotoxin that selectively kills human leukocytes. Recently, we reported that macrophages are highly sensitive to leukotoxin and that their lysis involves activation of caspase 1. In this study, we show that leukotoxin also induces the production and release of proinflammatory cytokines from human macrophages. The macrophages were challenged with leukotoxin or lipopolysaccharide (LPS) from A. actinomycetemcomitans or LPS from Escherichia coli, and the production and secretion of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) were determined at the mRNA and protein levels by reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Leukotoxin (1 to 30 ng/ml) induced abundant production and secretion of IL-1β, while the effects on IL-6 and TNF-α production were limited. Leukotoxin (1 ng/ml) caused a 10-times-higher release of IL-1β than did LPS (100 ng/ml). The secreted IL-1β was mainly the bioactive 17-kDa protein. At higher concentrations (>30 ng/ml), leukotoxin caused secretion of mainly inactive cytokine, the 31-kDa pro-IL-1β. The presence of specific antibodies to IL-1β or of a caspase 1 inhibitor blocked the secretion and production of the cytokine. Supernatants of leukotoxin-challenged macrophages stimulated bone resorption when tested in a mouse calvarial model. The activity could be blocked by an IL-1 receptor antagonist or specific antibodies to IL-1β. We concluded that A. actinomycetemcomitans leukotoxin can trigger abundant production and secretion of bioactive IL-1β by human macrophages, which is mediated by activation of caspase 1.

FLUORIDE AND MUTANS STREPTOCOCCI LEVELS IN PLAQUE ON AGED RESTORATIONS OF RESIN-MODIFIED GLASS IONOMER CEMENT, COMPOMER AND RESIN COMPOSITE

The use of fluoride-releasing restoratives such as glass ionomer cements (GICs) has increased during the last decade. The antibacterial effect of released fluoride is thought to be a possible caries-preventive effect of these restorations. In this study fluoride concentrations in plaque on 1-year old resin-modified GIC, compomer and resin composite restorations were compared intraindividually and related to the occurrence of caries-associated bacteria. Plaque from class III restorations of the three restorative materials and from a proximal enamel surface in 18 individuals was analysed. Low fluoride levels were detected in all the samples, while the resin-modified GIC samples showed significantly higher amounts. The distribution of oral streptococci, mutans streptococci and lactobacilli did not differ significantly among the surfaces and did not correlate to the fluoride levels in the samples. A good correlation was found between the counts of mutans streptococci in saliva and their proportions in the plaque. The results indicate that the fluoride concentrations released in vivo from 1-year-old restoratives are not high enough to affect the plaque levels of the caries-associated bacteria mutans streptococci and lactobacilli.

Description of Mogibacterium pumilum gen. nov., sp. nov. and Mogibacterium vescum gen. nov., sp. nov., and reclassification of Eubacterium timidum (Holdeman et al. 1980) as Mogibacterium timidum gen. nov., comb, nov.

A new genus, Mogibacterium, is proposed for anaerobic, non-spore-forming, Gram-positive, rod-shaped bacteria which have been isolated from the periodontal pockets of adult human patients with periodontal disease and infected root canals. The novel isolates, strains D2-18T, BA11a-f and D5-2T, were inert in most of the conventional biochemical tests and phenotypically resemble asaccharolytic Eubacterium species. The protein profiles of whole cells on SDS-PAGE gels and Western immunoblotting reaction analysis distinguished these organisms from type strains belonging to the previously described Eubacterium species. The G + C content of the DNA is 45-46 mol% for Mogibacterium pumilum and 46 mol% for Mogibacterium vescum. The levels of DNA-DNA relatedness of these new species to other Eubacterium species, including Eubacterium limosum, Eubacterium brachy, Eubacterium lentum, Eubacterium nodatum, Eubacterium saphenum, and the more recently proposed Eubacterium minutum and Eubacterium exiguum (reclassified as Slackia exigua), are less than 2%. The DNA-DNA hybridization value between M. pumilum and M. vescum was 30%. Eubacterium timidum exhibited DNA homologies with Mogibacterium species which were low (17 and 18%) but clearly higher than with all the other Eubacterium species. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the closest phylogenetic neighbour of Mogibacterium species was E. timidum, and that these three species represent a novel lineage distinct from the previously described genera of Gram-positive, rod-shaped bacteria. On the basis of phenotypic characteristics and 16S rRNA gene sequence comparisons, it is also proposed that E. timidum is transferred to the genus Mogibacterium gen. nov. as Mogibacterium timidum gen. nov., comb. nov. (type strain ATCC 33093T).

Specific binding of lactoferrin to Escherichia coli isolated from human intestinal infections

The degrees of human lactoferrin (HLf) and bovine lactoferrin (BLf) binding in 169 Escherichia coli strains isolated from human intestinal infections, and in an additional 68 strains isolated from healthy individuals, were examined in a 125I‐labelled protein binding assay. The binding was expressed as a percentage calculated from the total labelled ligand added to bacteria. The HLf and BLf binding to E. coli was in the range 3.7 to 73.4% and 4.8 to 61.6%, respectively. Enterotoxigenic strains demonstrated a significantly higher HLf binding (median = 19%) than enteropathogenic, enteroinvasive, enterohaemorrhagic strains or normal intestinal E. coli isolates (medians 6 to 9). Enteropathogenic strains belonging to serotypes 044 and 0127 demonstrated significantly higher HLf binding compared to O26, O55, O111, O119 and O126. No significant differences in the degree of HLf or BLf binding were found between aerobactin‐producing and non‐producing strains. The interaction was further characterized in a high Lf‐binding EPEC strain, E34663 (serotype O127). The binding was stable in the pH range 4.0 to 7.5, did not dissociate in the presence of 2M NaCl or 2M urea, and reached saturation within two h. Unlabelled HLf and BLf displaced the 125I‐HLf binding to E34663 in a dose‐dependent manner. Apo‐ and iron‐saturated forms of Lf demonstrated similar binding to E34663. Among various unlabelled subepithelial matrix proteins and carbohydrates tested (in 104‐fold excess) only fibronectin and fibrinogen caused a moderate inhibition of 125I‐HLf binding. According to Scatchard plot analysis, 5,400 HLf‐binding sites/cell, with an affinity constant (Ka) of 1.4 × 10−7 M., were estimated in strain E34663. These data establish the presence of a specific Lf‐binding mechanism in E. coli.

Inhibitory effect of lactoferrin on the adhesion of Actinobacillus actinomycetemcotnitans and Prevotella intermedia to fibroblasts and epithelial cells

Adhesion of the periodontitis‐associated bacteria Actinobacillus actinomycetemcomituns and Prevotella intermedia to monolayers of fibroblasts, HEp‐2, KB and HeLa cells was quantified with radiolabeled bacteria. Bacterial adhesion was also examined microscopically with Giemsa‐stained nonradioactive preparations. The degree of bacterial adherence was dependent on the growth phase of the bacteria. Strains at the exponential phase adhered to a greater extent than those at the stationary phase of growth. Both human and bovine lactoferrins competitively inhibited the adhesion of A. actinomycetemcomitans and P. intermedia to all tested cell monolayers. The inhibitory effect was dose‐dependent in the concentration range 0.5–2500 μg/ml and not related to the bacterial growth phase. In the presence of lactoferrin, decreased association of bacteria with the cell monolayers was also found by microscopic examination of the preparations. The present findings indicate that lactoferrin may prevent the establishment of bacteria in periodontal tissues through adhesion‐counteracting mechanisms in addition to its bacteriostatic and bactericidal properties.

Salivary concentration of the antimicrobial peptide LL-37 in children

OBJECTIVE Antimicrobial peptides are important components of innate immunity, especially in the unique environment of the oral cavity. Lack of the human cathelicidin LL-37 has been implicated in severe periodontitis, whilst high salivary levels of LL-37 seem to increase caries resistance. Limited data exists about the concentration of LL-37 in saliva of young children. In this study, the salivary concentration of LL-37 was examined in relation to age, gender, type of dentition (primary, mixed or permanent) and caries experience of children. DESIGN Unstimulated whole saliva was collected from 49 systemically healthy and gingivitis free children aged 2-18 years old. Their caries activity was recorded. The salivary LL-37 concentration was determined by enzyme linked immunosorbent assay (ELISA). RESULTS LL-37 was detected in all saliva samples. Its concentration varied widely, with girls exhibiting higher peptide levels than boys. A positive correlation of the LL-37 concentration was observed with age. Children with primary dentition had significantly lower peptide concentration than those with mixed or permanent dentition. Significantly lower concentrations of LL-37 were also found in children with high caries activity, compared to caries free children or to children with low to moderate caries activity. CONCLUSIONS Our results reinforce the belief that LL-37 is an important molecule of immunity in the oral environment and it seems to play a protective role against caries.

Detection of bacterial interaction with lactoferrin by an enzyme-linked ligand binding assay (ELBA)

An enzyme-linked ligand binding assay (ELBA) was devised to measure the interaction between bacteria and human (H) or bovine (B) lactoferrin (Lf) linked to horseradish peroxidase. Reagents were calibrated for optimum colour development with o-phenylenediamine as chromophore and organisms that were either positive or negative in a radioisotope-labelled ligand binding assay (RLBA) with 125I-Lf. Good correlation of Lf binding (r = 0.89) was found between ELBA and RLBA with 169 randomly selected strains of Escherichia coli. A semi-quantitative scoring system for ELBA, corresponding to a similar system for RLBA, was established and shown to be valid for 517 strains from seven species of bacterial pathogens. ELBA was used to measure bacterial Lf binding-saturation and displacement kinetics and shown to be comparable with RLBA. ELBA may be a suitable method for examining the binding of Lf to bacteria without the need for radioactive isotopes.

Salivary Alpha-Amylase Activity and Salivary Flow Rate in Young Adults

The secretion of salivary alpha-amylase (sAA) is more associated with psychoneuroendocrinological response to stress than with the flow rate and age. The aim of this cross sectional study is to build an explanatory model based on patterns of relationship between age 20-39 in resting and stimulated saliva under no stressful condition in healthy volunteers. Both resting and stimulated saliva were collected from 40 subjects. The sAA values were log-transformed, the normality assumption was verified with the Shapiro-Wilk test and the reliability of the measurements was estimated by the Pearsons’ r correlation coefficient. The estimated model was based on the theory of the Linear Mixed Models. Significant mean changes were observed in flow rate and sAA activity between resting and stimulated saliva. The final model consists of two components, the first revealed a positive correlation between age and sAA while the second one revealed a negative correlation between the interaction of age × flow rate in its condition (resting or stimulated saliva), with sAA. Both flow rate and age influence sAA activity.