Martina Kukletová

Lactobacillus spp. associated with early childhood caries

A group of 69 lactobacilli was isolated from caries lesions and root canals of early childhood caries (ECC) affected children treated in the Department of Pedodontics (Children’s Teaching Hospital, Brno, Czech Republic). Biochemical and physiological properties of all strains were characterized by API 50 CH kit and conventional tube tests. The rep-PCR fingerprinting with the (GTG)5 primer was used for genotypic grouping of the isolates. The (GTG)5-PCR fingerprinting grouped all analyzed strains into a few clusters in nearly full agreement with phenotype identification results and clarified the taxonomic position of 13 biochemically unidentified strains. In total, 20 strains of Lactobacillus fermentum, 17 L. rhamnosus, 14 L. casei/paracasei, 7 L. gasseri, 7 L. salivarius and 4 L. plantarum were identified. Mixtures of two or even three Lactobacillus spp. were isolated from a few root canal content samples. Results obtained by biotyping and (GTG)5-PCR were generally comparable except for L. gasseri strains that were not biochemically identified. The (GTG)5-PCR fingerprinting was shown to be quicker, easier to perform and more reliable than biotyping. Our results imply this molecular method as a good tool for screening and identification of Lactobacillus spp. inhabiting dental plaque.

Comparative evaluation of automated ribotyping and RAPD-PCR for typing of Lactobacillus spp. occurring in dental caries

A group of 67 Lactobacillus spp. strains containing Lactobacillus casei/paracasei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus salivarius species isolated from early childhood caries and identified to the species level in a previous study (Švec et al., Folia Microbiol 54:53–58, 2009) was characterized by automated ribotyping performed by the RiboPrinter® microbial characterization system and by randomly amplified polymorphic DNA fingerprinting (RAPD-PCR) with M13 primer to evaluate these techniques for characterization of lactobacilli associated with dental caries. Ribotyping revealed 55 riboprints among the analysed group. The automatic identification process performed by the RiboPrinter system identified 18 strains to the species level, however cluster analysis divided obtained ribotype patterns into individual clusters mostly corresponding to the species assignment of particular strains. RAPD-PCR fingerprints revealed by the individual Lactobacillus spp. showed higher variability than the ribotype patterns and the fingerprint profiles generated by the analysed species were distributed among one to four clusters. In conclusion, ribotyping is shown to be more convenient for the identification purposes while RAPD-PCR fingerprinting results indicate this method is a better tool for typing of Lactobacillus spp. strains occurring in dental caries.

Association of interleukin-6 (IL-6) haplotypes with plaque-induced gingivitis in children.

Objectives. The proinflammatory cytokine interleukin-6 (IL-6) is a key regulator of the host response to microbial infection and major modulator of extracellular matrix catabolism and bone resorption. The aim of this case-control study was to test differences between children with and without gingivitis in the distribution of IL-6 alleles at positions −174, −572, and −597 and their haplotypes. Material and methods. A total of 455 Caucasian children, aged 11 to 13 years, were enrolled in this study. According to gingival bleeding on probing indices, 183 were classified as healthy subjects and 272 as children with plaque-induced gingivitis. DNA for genetic analysis was obtained from buccal epithelial cells and PCR-RFLP methods were used for genotyping three selected IL-6 promoter polymorphisms. Results. Complex analysis revealed significant differences in haplotype frequencies between patients and healthy subjects (p<0.01). The CGA haplotype was significantly more frequent in children with gingivitis than in healthy subjects (41.5% versus 34.1%). In subanalyses, we found that IL-6 −174C allele was more frequent in patients (44.3%) than in healthy children (36.1%, p=0.016, Pcorr<0.05). Multivariate logistic regression analysis showed that allele C remained a risk factor for gingivitis in children (p=0.03) regardless of plaque or gender. However, the proportions of the IL-6 −597 and −572 genotypes were comparable between the two groups. Conclusions. Our results indicate that the three promoter polymorphisms in the IL-6 gene act in a cooperative fashion and suggest that IL-6 haplotypes could play a role in the pathogenesis of gingivitis in Caucasian children.

The association of MMP-9 and IL-18 gene promoter polymorphisms with gingivitis in adolescents.

OBJECTIVE Interleukin 18 (IL-18) is shown to be a proinflammatory cytokine that regulates the expression of matrix metalloproteinase 9 (MMP-9). The aim of this study was to test for differences between Czech adolescents with and without gingivitis in relation to MMP-9 and IL-18 polymorphisms. DESIGN A total of 298 Caucasian children, aged 11-13 years, were examined to assess gingival health. DNA for genetic analysis was obtained from buccal epithelial cells, and the MMP-9 -1562C/T and IL-18 -607A/C variants were identified with PCR-RFLP. RESULTS Gingivitis was present in 49.3% of the adolescents examined, the rest of the group was considered healthy. The IL-18 -607C and MMP-9 -1562T alleles were found in 58.9% and 8.3% of the healthy subjects, and in 62.2% and 15.0% of the patients with gingivitis, respectively. Although differences in allele frequencies were not significant for IL-18 variant, they were significant for MMP-9 polymorphism (p=0.01, p(corr) < 0.05). Furthermore, a highly significant association of the composite genotype (formed by the variants of the both genes) with gingivitis was found (p=0.004, p(corr) < 0.05). CONCLUSIONS The -1562 T allele of MMP-9 gene could have a role in gingivitis in adolescents. In addition, interaction of the MMP-9 and IL-18 genes could be considered a risk factor for the development of gingivitis in children.

GLUT2 and TAS1R2 Polymorphisms and Susceptibility to Dental Caries

Objective: Dental caries is one of the most frequent multifactorial diseases. Among the numerous factors influencing the risk of caries, genetics plays a substantial role, with heritability ranging from 40 to 60%. Gene variants affecting taste preference and glucose transport were recently associated with caries risk. The aim of this study was to analyze two common polymorphisms in the sweet taste receptor (TAS1R2) and glucose transporter (GLUT2) genes in children with dental caries and healthy controls in the Czech population. Methods: A total of 637 unrelated Caucasian children, aged 11-13 years, were included in this case-control study. One hundred and fifty-five subjects were caries-free (with decayed/missing/filled teeth, DMFT = 0) and 482 children were caries-affected (DMFT ≥ 1). The TAS1R2 (Ile191Val, rs35874116) and GLUT2 (Thr110Ile, rs5400) genotypes were determined using the 5′ nuclease TaqMan® assay for allelic discrimination. Results: Compared with subjects with the common Thr allele, carriers of the Ile allele of GLUT2 had significantly more frequently dental caries (p < 0.05, OR = 1.639, 95% CI: 1.089-2.466). Similarly, children with the Val allele for the TAS1R2 Ile191Val polymorphism were more frequently affected by caries than children who carried the Ile allele (p < 0.05, OR = 1.413, 95% CI: 1.014-1.969). In contrast, no significant associations between GLUT2 and/or TAS1R2 polymorphisms and fillings were found, but allele frequencies of the TAS1R2 variant were marginally significantly different between children with DMFT = 0 and DMFT ≥1 (p = 0.053, OR = 1.339, 95% CI: 0.996-1.799). However, no significant interaction between both genes and risk of dental caries was found. Conclusions: In conclusion, GLUT2 and TASR1 polymorphisms may influence the risk of caries in the Czech population.

Lack of Association between Lactotransferrin Polymorphism and Dental Caries

Objective: Dental caries is a complex, multifactorial disease and one of the most common illnesses worldwide. Its etiology is related to microbial, dietary and host factors. Recent evidence suggests a role of lactotransferrin (LTF) in caries. The purpose of this study was to determine the association between LTF gene polymorphism and dental caries. Methods: In this case-control study, 637 unrelated children, aged 11-13 years, were enrolled. The subjects were divided into two groups, i.e. caries-free (decayed/missing/filled teeth = 0) and caries-affected children (decayed/missing/filled teeth ≥ 1). The LTF rs1126478 (140A/G in exon 2, Lys/Arg) genotypes were determined by PCR with restriction analysis using the EarI enzyme. Results: Of 637 children, 155 (24.3%) were caries free. There were no statistically significant differences between caries levels and allele or genotype distributions in the total cohort. When the caries-affected group (n = 482) was stratified into low (decayed/missing/filled teeth = 1), moderate (2 ≤ decayed/missing/filled teeth ≤ 3) and high (decayed/missing/filled teeth ≥ 4) caries experience, allele and genotype frequencies were similar among all subgroups. Conclusions: The LTF 140A/G (exon 2, Lys/Arg) polymorphism was not associated with the susceptibility to or severity of dental caries in the Czech population.

ACE Insertion/Deletion Polymorphism Associated with Caries in Permanent but Not Primary Dentition in Czech Children

Objective: Dental caries is a multifactorial, infectious disease where genetic predisposition plays an important role. Insertion/deletion (I/D) polymorphism of angiotensin-converting enzyme (ACE) has very recently been associated with caries in Polish children. The aim of this study was to analyze ACE I/D polymorphism in a group of caries-free children versus subjects affected by dental caries in the Czech population. Materials and Methods: In this case-control study, 182 caries-free children (with decayed/missing/filled teeth, DMFT = 0), 561 subjects with dental caries (DMFT ≥1) aged 13-15 years and 220 children aged 2-6 years with early childhood caries (ECC, dmft ≥1) were included. Genotype determination of ACE I/D polymorphism in intron 16 was based on the TaqMan method. Results: Although no significant differences in the allele or genotype frequencies between the caries-free children and those affected by dental caries were observed, statistically significant differences between the children with DMFT = 0 and the subgroup of 179 patients with high caries experience (DMFT ≥4; p < 0.01 and p < 0.05, respectively) were detected. The comparison of DD versus II+ID genotype frequencies between the patients with DMFT ≥1 or DMFT ≥4 and healthy children also showed significant differences (31.5% or 35.6% vs. 23.6%, p < 0.05 or p < 0.01, respectively). A gender-based analysis identified a significant difference in the DD versus II+ID genotype frequencies only in girls (p < 0.05). In contrast, no significant association of ACE I/D polymorphism with ECC in young children was found (p > 0.05). Conclusions:ACE I/D polymorphism may be associated with caries in permanent but not primary dentition, especially in girls in the Czech population.

A histochemical study by light and electron microscopy of the distribution of dipeptidyl peptidase-IV activity in the human dental pulp.

This activity was demonstrated in blood vessels, fibroblast-like cells, odontoblasts and Schwann cells surrounding non-myelinated axons. It was present on both the luminal and abluminal plasma membrane of endothelial cells. The plasma membrane of fibroblast-like cells and their processes had patches of fine electron-dense end-product corresponding to DPP-IV activity, while the plasma membrane of odontoblasts was loaded with a consistent reaction product. DPP-IV activity in the non-myelin-forming Schwann cells was in the plasma membrane of the free surface in contact with the extracellular matrix as well as in the plasma membrane in close contact with the axolemma. The plasma membrane of Schwann cells producing myelin sheath had no positive reaction for DPP-IV. The DPP-IV activity in the plasma membrane of fibroblast-like cells and odontoblasts may be associated with fibronectin-mediated adhesion to collagen, whereas the membrane-bound DPP-IV activity in endothelium and non-myelin-forming Schwann cells may be involved in cleavage of neuropeptides of other biologically active peptides.

Vitamin D Receptor TaqI Gene Polymorphism and Dental Caries in Czech Children

Aim: We analyzed the VDR TaqI (rs731236) gene polymorphism in children with and those without dental caries. Methods: A total of 388 subjects, 153 caries-free (with decayed/missing/filled teeth [DMFT] = 0) and 235 children with dental caries (DMFT ≥1), were genotyped by the TaqMan method. Results: Although no significant differences in VDR TaqI allele and genotype frequencies between caries-free and caries-affected children were detected, a significant association between this polymorphism and gingivitis was found (p < 0.05). Conclusions: In contrast to previous studies from China and Turkey, the VDR TaqI gene variant cannot be used as a marker for identification of Czech children with increased dental caries risk.

Lack of Association between BMP2/DLX3 Gene Polymorphisms and Dental Caries in Primary and Permanent Dentitions

The aim of this study was to analyze the association between BMP2 (rs1884302) and DLX3 (rs2278163) gene polymorphisms and dental caries in primary and permanent dentitions. A total of 914 subjects were genotyped by the TaqMan methods: 176 caries-free children (with Decayed/Missing/Filled Teeth, DMFT = 0), 542 patients with dental caries in permanent dentition (DMFT ≥1), 83 caries-free children with primary teeth (with decayed/missing/filled teeth, dmft = 0), and 113 children with early childhood caries (ECC, dmft ≥1). There were no significant differences in allele/genotype frequencies between patients with caries in permanent dentition/ECC and caries-free children or between patients with very low (DMFT = 0-2), low (DMFT = 3-5), moderate (DMFT = 6-8), or high (DMFT ≥9) caries experience. Variability in BMP2 and DLX3 was not associated with caries in the Czech population.