Antonín Fassmann

Promoter polymorphisms in the CD14 receptor gene and their potential association with the severity of chronic periodontitis

Periodontitis, a chronic inflammation of the tissues surrounding the teeth, is a common disease affecting all populations. The main aetiology remains a bacterial infection that leads to gingival inflammation, loss of alveolar bone, and tooth loss.1 Although the presence of pathogenic micro-organisms is required to trigger this process, the amplification and progression of the disease is believed to rely heavily on the production of host mediators in response to bacteria and/or their metabolic products.2 The CD14 molecule, described as the major endotoxin receptor, is one of the receptors which act on the recognition of lipopolysaccharides (LPS, endotoxin) and gram positive or mycobacterial cell wall components and thus can initiate the innate immune response to bacterial invasion.3–5 It is constitutively expressed primarily on the surface of monocytes, macrophages, neutrophils, and gingival fibroblasts (mCD14).6 In addition, a soluble form of CD14 (sCD14) is abundant in serum and is apparently derived both from the secretion of CD14 and from enzymatically cleaved glycosyl-phosphatidylinositol anchored mCD14.7 Besides the role of CD14 in the host defence, several other biological functions have been found. CD14 is involved in the phagocytosis of gram negative bacteria,8 LPS mediated bone resorption,9 and monocyte-endothelial cell interactions. Furthermore, changes in CD14 expression and serum sCD14 levels seem to be associated with a number of pathological states including periodontal diseases.10 CD14 production is genetically regulated. The gene for the CD14 receptor is on chromosome 5 (region q23-21), consists of ≈3900 bp organised in two exons, and encodes a protein of 375 amino acids.11 In the promoter region of the CD14 gene, a C to T transition was identified at position –159 upstream from the major transcription site, which is near to an SP1 binding site that has a major influence on the monocyte …

Interactions of Lymphotoxin alpha (TNF-beta), Angiotensin-Converting Enzyme (ACE) and Endothelin-1 (ET-1) Gene Polymorphisms in Adult Periodontitis

BACKGROUND Adult periodontitis is a complex multifactorial disease whose etiology is not well defined. To investigate whether the genes encoded within the HLA class III region may confer susceptibility to periodontitis, polymorphisms in the ET-1 and TNF-β genes were analyzed together with the I/D polymorphism of the ACE gene. METHODS We determined allele and genotype frequencies of the NcoI bi-allelic polymorphism of the TNF-β gene, the I/D (insertion/deletion) polymorphism of the ACE gene, and the TaqI polymorphism of the ET-1 gene in 63 Caucasian patients with adult periodontitis and 95 orally healthy controls. RESULTS We found a significant difference in a 3 locus combination of genotypes between patients and controls (P <0.05). In the next analyses, no significant differences were found in allele frequencies of single genes, but we did find a significant difference in the genotype distribution between cases and controls for TNF-β (P <0.03). Differences were also observed for 2 locus combinations of ACE and TNF-β genotypes (P <0.03), and the ET-1 and TNF-β (P <0.05) genes. Evidence of deviation from Hardy-Weinberg equilibrium was observed in the periodontitis group for TNF-β, with an absence of the B1 B1 homozygotes in patients. CONCLUSIONS This study is of an exploratory nature. Considering the number of significant results, however, at least a part of the observed associations may obviously be real and our findings suggest that interactions of the TNF-β, ET-1, and ACE genes may be involved in susceptibility to adult periodontitis. J Periodontol 2001;72:85-89.

Effects of activated and nonactivated platelet-rich plasma on proliferation of human osteoblasts in vitro.

PURPOSE The purpose of this study was to evaluate the effect of progressively increasing concentrations of activated and nonactivated platelet-rich plasma (PRP) on proliferation of human osteoblasts in vitro. MATERIALS AND METHODS Human osteoblasts (hFOB 1.19) obtained from the American Type Culture Collection (ATCC, Manassas, VA) were used in the experiment. PRP was obtained from a 28-year-old healthy male volunteer by means of a Haemonetics gradient density cell separator (Haemonetics, Munich, Germany). Human thrombin was used to activate PRP. Three independent experiments were conducted. Samples containing 10% (0.38x increase in platelet count), 25% (0.95x increase in platelet count), 50% (1.95x increase in platelet count), and 75% (2.86x increase in platelet count) of activated PRP and nonactivated PRP were prepared including controls. After culture periods of 24, 48, and 72 hours osteoblast proliferation was evaluated by counting the number of cells using a Multisizer 3 Coulter Counter (Beckman Coulter, Inc, Fullerton, CA). RESULTS After 24, 48, and 72 hours of incubation, the number of cells in the control group (without PRP) was higher than that of cells in samples containing activated or nonactivated PRP. Osteoblasts with 10% activated PRP (0.38x increase in platelet count) had the highest viability of all samples containing PRP. CONCLUSIONS Activated PRP resulted in higher proliferation of osteoblasts compared with nonactivated PRP at concentrations of 10% (0.38x increase in platelet count) and 25% (0.95x increase in platelet count) in culture. This study failed to show significant increases in proliferation of human osteoblasts treated with activated or nonactivated PRP compared with controls in vitro.

Association of Toll-like receptor 9 haplotypes with chronic periodontitis in Czech population.

AIM Toll-like receptors (TLRs) belong to the pattern recognition receptors family of signal molecules that recognize conserved microbial structures. The aim of this study was to analyse polymorphisms in the TLR genes and their association with chronic periodontitis (CP). MATERIAL AND METHODS Two polymorphisms (2408G/A, i.e. Arg753Gln and -16934A/T) in TLR-2 and three variants (-1486C/T, -1237C/T and+2848A/G) in the TLR-9 genes were studied in 222 patients with CP and 259 unrelated controls. All polymorphisms were detected using the polymerase chain reaction-restriction fragment length polymorphism methods. Subgingival bacterial colonization was investigated by the VariOr Dento test. RESULTS No significant differences were found in allele and genotype frequencies of all polymorphisms between patients and controls. Nevertheless, complex analysis revealed differences in TLR9 haplotype frequencies between both groups (p=0.001). Specifically, the haplotype T(-1486)/T(-1237)/A(2848) was significantly more frequent (9.6%versus 2.8%, p<0.000001) and the haplotype T(-1486)/T(-1237)/G (2848) of the TLR9 gene was less frequent (35.9%versus 43.3%, p=0.01) in patients than in controls. However, no significant relationships between periodontal pathogens, TLR polymorphisms and CP were found. CONCLUSIONS In conclusion, although no significant role of the TLR2 gene in periodontitis was found, our results indicate that TLR9 haplotypes may be associated with susceptibility to CP.

Plasminogen-activator-inhibitor-1 promoter polymorphism as a risk factor for adult periodontitis in non-smokers

Periodontal diseases belong to the most common chronic disorders affecting mankind. Smoking and impaired plasminogen activation with hypercoagulation and fibrinolysis inhibition have been proposed as having a role in predisposition to these diseases. We investigated relationships among adult periodontitis, smoking, and a variation in the deletion/insertion (4G/5G) promoter polymorphism of the plasminogen-activator-inhibitor-1 (PAI-1) gene in 304 Caucasian subjects. An association was detected between the deletion (4G) allele (and 4G/4G genotype) and periodontitis (P = 0.0022, Pcorr < 0.01; P = 0.014, Pcorr < 0.05). A stronger association occurred in non-smokers (P = 0.00021, Pcorr < 0.01; P = 0.0024, Pcorr < 0.05) where the presence of the PAI-1 gene 4G allele appears to be one of the risk factors for periodontitis.

Matrix metalloproteinase 8 (MMP8) gene polymorphisms in chronic periodontitis

OBJECTIVE Previous studies have suggested that some functional polymorphisms in the matrix metalloproteinase (MMPs) genes are associated with the risk of periodontal disease. However, to date no study has investigated MMP8 gene variants in relation to chronic periodontitis (CP). The aim of this study was to analyse polymorphisms in the MMP8 gene and their associations with microbial composition and clinical manifestation of CP. DESIGN A total of 619 unrelated Czech subjects were included in the present study. Two polymorphisms [-799C/T (rs11225395) and +17C/G (rs2155052)] in the MMP8 gene were studied in 341 patients with CP and 278 unrelated non-periodontitis controls. Both polymorphisms were detected using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. Subgingival bacterial colonisation (occurrence of bacteria in subgingival pockets and gingival sulci) was investigated by a commercial semiquantitative kit in selected subjects (N=169). RESULTS Our results showed no differences in the allele and genotype frequencies of the MMP8 -799C/T and +17C/G polymorphisms between patients with CP and controls (p>0.05). Nevertheless, the haplotype T(-799)/C(+17) was significantly more frequent in patients with CP than in controls [43.7% vs. 37.6%, p<0.05, OR=1.273 (95% CI: 1.013-1.601)]. Despite significant differences determined in the occurrence of periodontal bacteria between patients with CP and non-periodontitis controls (from p<0.000001 to p<0.05), no significant relationships between periodontal pathogens, MMP8 polymorphisms and CP were found (p>0.05). CONCLUSIONS Although none of the investigated SNPs in the MMP8 gene was individually associated with periodontitis, specific haplotype showed association with clinical manifestation of chronic periodontitis in a Czech population.

Haplotype analysis of interleukin-8 gene polymorphisms in chronic and aggressive periodontitis.

Objectives. Periodontitis is an inflammatory disease characterized by connective tissue loss and alveolar bone destruction. Interleukin-8 (IL8) is important in the regulation of the immune response. The aim of this study was to analyze four polymorphisms in the IL8 gene in relation to chronic (CP) and aggressive (AgP) periodontitis. Methods. A total of 492 unrelated subjects were included in this case-control association study. Genomic DNA of 278 patients with CP, 58 patients with AgP, and 156 controls were genotyped, using the 5′ nuclease TaqMan assay, for IL8 (rs4073, rs2227307, rs2227306, and rs2227532) gene polymorphisms. Subgingival bacterial colonization was investigated by the DNA-microarray detection kit in a subgroup of subjects (N = 247). Results. Allele and genotype frequencies of all investigated IL8 polymorphisms were not significantly different between the subjects with CP and/or AgP and controls (P > 0.05). Nevertheless, the A(−251)/T(+396)/T(+781) and T(−251)/G(+396)/C(+781) haplotypes were significantly less frequent in patients with CP (2.0% versus 5.1%, P < 0.02, OR = 0.34, 95% CI: 0.15–0.78, resp., 2.0% versus 4.5%, P < 0.05, OR = 0.41, 95% CI: 0.18–0.97) than in controls. Conclusions. Although none of the investigated SNPs in the IL8 gene was individually associated with periodontitis, some haplotypes can be protective against CP in the Czech population.

Interleukin-17A Gene Variability in Patients with Type 1 Diabetes Mellitus and Chronic Periodontitis: Its Correlation with IL-17 Levels and the Occurrence of Periodontopathic Bacteria

Interleukin-17 contributes to the pathogenesis of type 1 diabetes mellitus (T1DM) and chronic periodontitis (CP). We analyzed IL-17A −197A/G and IL-17F +7488C/T polymorphisms in T1DM and CP and determined their associations with IL-17 production and occurrence of periopathogens. Totally 154 controls, 125 T1DM, and 244 CP patients were genotyped using 5′ nuclease TaqMan® assays. Bacterial colonization was investigated by a DNA-microarray kit. Production of IL-17 after in vitro stimulation of mononuclear cells by mitogens and bacteria was examined by the Luminex system. Although no differences in the allele/genotype frequencies between patients with CP and T1DM + CP were found, the IL-17A −197 A allele increased the risk of T1DM (P < 0.05). Levels of HbA1c were significantly elevated in carriers of the A allele in T1DM patients (P < 0.05). Production of IL-17 by mononuclear cells of CP patients (unstimulated/stimulated by Porphyromonas gingivalis) was associated with IL-17A A allele (P < 0.05). IL-17A polymorphism increased the number of Tannerella forsythia and Treponema denticola in patients with CP and T1DM + CP, respectively (P < 0.05). IL-17A gene variability may influence control of T1DM and the “red complex” bacteria occurrence in patients with CP and T1DM + CP. Our findings demonstrated the functional relevance of the IL-17A polymorphism with higher IL-17 secretion in individuals with A allele.

Apolipoprotein E gene polymorphisms in relation to chronic periodontitis, periodontopathic bacteria, and lipid levels

OBJECTIVE Inflammatory periodontal diseases may be associated with common systemic conditions and, as recently described, alterations in lipid levels in the blood. The aim of this study was to determine the possible effects of apolipoprotein E (ApoE) genotypes on the lipid levels in healthy people and patients with chronic periodontitis (CP) in relation to periodontopathic bacteria. DESIGN This case-control study comprised 469 unrelated subjects. The genomic DNA of 294 patients with CP and 175 healthy/non-periodontitis controls were genotyped, using the real-time polymerase chain reaction (RT-PCR) method, for ApoE (rs429358 and rs7412) gene polymorphisms. Subgingival bacterial colonization was investigated by the DNA microarray using a periodontal pathogen detection kit and lipid levels were measured in a subgroup of subjects (N = 275). RESULTS There was no evidence for a significant association between ApoE gene polymorphisms and CP (P > 0.05). Patients with CP had increased levels of total cholesterol and low-density lipoprotein (LDL) compared to controls (P< 0.05); however, no significant difference was found for triglyceride and high-density lipoprotein (HDL) levels. ApoE gene variability influenced LDL levels marginally (P = 0.08) but it did not modify total cholesterol, triglyceride, and HDL levels or the occurrence of periodontal pathogens in subgingival pockets.(23) CONCLUSIONS: In the Czech population studied, ApoE genetic variations were not associated with susceptibility to CP or the presence of periodontopathic bacteria.

The Effect of IL-4 Gene Polymorphisms on Cytokine Production in Patients with Chronic Periodontitis and in Healthy Controls

Chronic periodontitis (CP) is an inflammatory disease of the teeth-supporting tissues in which genetic predisposition, dental plaque bacteria, and immune mechanisms all play important roles. The aim of this study was to evaluate the occurrence of IL-4 gene polymorphisms in chronic periodontitis and to investigate the association between polymorphisms and cytokines production after bacterial stimulation. Sixty-two subjects (47 CP patients and 15 healthy controls) with detected two polymorphisms in the IL-4 gene (-590C/T and intron 3 VNTR) were examined. Production of cytokines (IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-17, TNFα, INFγ, and VEGF) was studied after in vitro stimulation of isolated peripheral blood by mitogens (Pokeweed mitogen, Concanavalin A), dental plaque bacteria (Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, and Prevotella intermedia), and Heat Shock Protein (HSP) 70 by the Luminex multiplex cytokine analysis system. The results were correlated with IL-4 genotypes in patients with CP and healthy controls. The mononuclear cells isolated from peripheral blood of CP patients with selected IL-4 polymorphisms significantly altered the production of IFNγ, IL-10, IL-1β, IL-1α, TNFα, and IL-6 after stimulation by HSP 70 or selected bacteria (from P < 0.001 to P < 0.05). IL-4 gene polymorphisms may influence the function of mononuclear cells to produce not only interleukin-4 but also other cytokines, especially in patients with CP.