Petra Bořilová Linhartová

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.

Apolipoprotein E polymorphism is associated with both number of diseased vessels and extent of coronary artery disease in Czech patients with CAD

AIMS The impact of ApoE polymorphism on angiographic parameters was assessed in patients referred for coronary angiography. METHODS Elective coronary angiography was performed in 671 subjects (525 men, 146 women, mean age 60 ± 10 years) with symptoms of ischemic heart disease. The patients were divided into: no CAD group (smooth coronary vessels, n=83), one-vessel (n=155), two-vessel (n=170) and three-vessel disease (n=196). Patients with stenoses 0-50% were excluded. Within patients with CAD, we evaluated overall extent of CAD measured by the number of stenotic segments according to AHA (1 segment vs. 2-3 vs. ≥4), and the severity of the most serious stenosis (in percent). ApoE genotype was determined using real-time PCR. RESULTS The frequency of ε2/ε3 genotype (n=56) was lower in the three-vessel disease group compared to one-vessel disease (OR=0.25, P=0.0019), two-vessel disease (OR=0.31, P=0.0114) or no CAD group (OR=0.24, P=0.0057). Frequency of ε2/ε3 decreased with the number of affected segments (1 vs. ≥4: OR=0.35, P=0.0143). The ε3/ε4+ε4/ε4 genotypes (n=123) were more frequent in CAD patients altogether compared with no CAD group (OR=2.30, P=0.019), while no impact of the ε4 allele on angiographic parameters within the CAD patients was detected. In ε2/ε3 carriers with CAD, lower LDL-cholesterol, total cholesterol and lower use of lipid-lowering drugs were observed. CONCLUSIONS The results show predominantly focal form of CAD in patients with ε2/ε3 genotype. Lower LDL-cholesterol and total cholesterol may play the key role, although other contributing factors are discussed.

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.

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.

The NOD-like receptor (NLRP3) gene variability in patients with recurrent aphthous stomatitis

I diseases continue to be one of the biggest health problems in the world, affecting millions of people annually M. abscessus and other species of rapidly growing mycobacteria (RGM) are naturally resistant to antimicrobial compounds and disinfectants because they have an impermeable cell wall composed by peptideoglycan and mycolic acids. These RGM are responsible for various hospital outbreaks worldwide, causing lung infections in patients with cystic fibrosis, chronic lung disease (bronchiectasis, nodules and cavitations), post-surgical infections and skin and soft tissue infections in immunocompromised patients. The resistance of M. abscessus (Mabs) to the medications used in current therapy challenges the search for new treatment strategies. Previous studies on the search for new natural compounds with antimicrobial action highlighted the potential of Bixa orellana (urucum). The seeds of this plant are already used in folk medicine for treating heart disease, gastrointestinal problems and respiratory infections. In this study, we evaluated potential anti-inflammatory activities of hydroalcoholic (BoEH) and ethyl acetate (BoEA) extracts of B. orellana leaves, using a murine model of peritonitis induced by heat killed Mabs. C57BL/6 mice were orally treated with different concentrations of BoEH or BoEA. After one hour, peritonitis was induced by inoculation of 1x108 CFU of heat killed Mabs. BoEH and BoEA inhibited the migration of total leukocytes (Figure 1A-B), migration of polymorphonuclear cells (Figure 1C-D) and mononuclear cells (Figure 1E-F) into the peritoneum in the periods analyzed 4 and 24 hours after the induction of peritonitis. Our results suggest anti-inflammatory actions of the extracts tested, indicating this plant as natural source of compounds with potential for pharmacological and biotechnological applications.I is a member of the IL-17 family, and is known as CTLA8 in the mouse. It is produced by T lymphocytes and NK cells and has proinflammatory roles, inducing cytokine and chemokine production. However, its role in tumor biology remains controversial. We investigated the effects of locally produced IL-17A by transferring the gene, encoding it into mouse tumor cells including B16 melanoma, and MethA fibrosarcoma, either in a secretory or a membrane-bound form. Expression of the membranebound form on CT26 colon cancer cells dramatically enhanced their proliferation in in vitro. The enhanced growth was shown to be due to an increased rate of cell cycle progression. After synchronizing cells by adding and withdrawing colcemid, the rate of cell cycle progression in the cells expressing the membrane-bound form of IL-17A was much faster than that of the control cells. Both secretory and membrane-bound IL-17A induced the expression of Sca-1 on the cancer cells, which is commonly associated with aggressive phenotype of cancer cells. When tumor clones were grafted into syngeneic BALB/c mice, the tumor clones expressing the membranebound form IL-17A grew rapidly; those expressing the secretory form also grew faster than the wild type CT26 cells, but slower than the clones expressing the membrane-bound form. These results indicate that IL-17A promotes tumorigenicity, in part, by enhancing cell cycle progression. This finding should be considered in treating tumors and immune-related diseases.P which patients are at a higher risk for recurrent chronic rhinosinusitis with nasal polyps (CRSwNP) is one of the most challenging problems in clinical rhinology. A direct association between nasal polyp and eosinophil/neutrophil counts was reported. This study aimed to identify difference of eosinophils and neutrophils for formation of polyp by DNA methylation in CRS. We have previously shown that KRT 19, NR2F2, ADAMTS1, and ZNF222 levels are changed in nasal polyps (NPs) of patients with chronic rhinosinusitis (CRS) in patients. A study was performed from 30 patients with CRS with bilateral NP, examining the prognostic role of eosinophil and neutrophil levels. 30 patients with CRS were classified by the rate of eosinophils and neutrophils in tissue. The methylated genes detected by DNA methylation microarray were validated by methylation-specific polymerase chain reaction (PCR), bisulfite sequencing, and real-time PCR. DNA methylation microarray identified 43,674 CpG islands in 518 genes. Specific genes were found to have a hypermethylated signal, and some genes were significantly hypomethylated in the promoter region in eosinophils compared with neutrophils. Real-time PCR showed that the expression levels of genes were changed in eosinophils, when compared with neutrophils. We clearly demonstrated that the two subgroups of CRSwNP had characteristic differences in DNA methylation, which allows for pathophysiologically meaningful differentiations with likely therapeutic consequences. Further studies are needed to confirm the significance of these epigenetic factors in the mechanisms underlying NP formation.Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease where animal models are used to study its pathogenesis. We have developed a mouse model of an autoimmune disease that resembles human lupus by the injection of liposomes bearing non-bilayer phospholipid arrangements (NPA). We detected the presence of IgG antibodies against NPA in the serum of mice with lupus and patients with SLE. In this work, we determined by citofluorometry the presence of plasmacytoid dendritic cells (pDC) the main producer of type I interferons, and of NKT cells in the secondary lymphoid organs of mice 30 and 60 days after the injection of liposomes with or without NPA induced with 8 mM promazine. In both groups of mice injected with liposomes bearing NPA a significant increase of pDC cells (5-fold) was found which correlates with the high concentration of type I interferon previously detected in this mouse model of lupus and in patients with SLE. A significant increase of NKT (3-fold) was detected at 30 days, specifically in the NKT subpopulation CD4 + that is known to cooperate with B cells in response of lipid antigens; this increase suggests its probable involvement in adaptive immune responses, which lead to the production of anti-NPA IgG antibodies. The increase of pDC and NKT cells found by cytometry in secondary lymphoid organs in this work, suggests their involvement in the formation of anti-NPA IgG antibodies and the development of the disease resembling human lupus.Statement of the Problem: Recurrent aphthous stomatitis (RAS) is a multifactorial disease with an unclear etiopathogenesis, resulting from the interplay between genetic and environmental factors.1 As the dysregulation of the immune system can play a role in the RAS development2, single nucleotide polymorphisms (SNPs) in the genes for immune and inflammatory molecules were studied.3,4 The NOD-like receptor (NLRP3) gene, encoding the component of the inflammasome, has been proposed as one of the candidate genes for RAS.5 The aim of our study was to investigate three SNPs (rs4612666, rs10754558, rs3806265) in NLRP3 gene in patients with RAS and healthy controls in the Caucasian population Methodology: A total of 200 Czech subjects were enrolled in this case-control study. 143 healthy controls, 57 patients with RAS were genotyped by method based on polymerase chain reaction using 5′ nuclease TaqMan® assays. Clinical parameters such as complete blood count, levels of immunoglobulins including allergen-specific immunoglobulin E or presence of antibodies against cytomegalovirus, Epstein-Barr virus were determined in RAS patients. Findings: Although no significant differences in the NLRP3 (rs10754558, rs3806265) allele and genotype frequencies between patients with RAS and controls were observed, statistically significant differences in NLRP3 rs4612666 genotype frequencies between subjects with RAS and controls were found. Carriers of NLRP3 rs4612666 TT genotype had a higher risk of developing RAS in comparison to subjects with CT + CC genotypes (OR = 16.71, 95% CI = 1.96-142.14, P = 0.0024). No association between NLRP3 haplotypes and RAS was detected. Conclusion & Significance: In contrast to the previous study5, associations between NLRP3 (rs10754558, rs3806265) polymorphisms and RAS were not confirmed. However, we suggest that NLRP3 rs4612666 polymorphism can strongly influence the risk of developing RAS in the Czech population.Results: The prevalence of vitamin D insufficiency was 64% in 76 asthmatic and 62.5% in 446 atopic individuals; however there was no significant association between vitamin D and this outcomes. Negative correlation was found between vitamin D and specific IgE levels to Dermatophagoides pteronyssinus on atopic subjects (r=-0.11, p=0.04). Genetic variants in CYP2R1 gene, rs7935792 (C allele) (Beta 1.66; 95% CI 0.20-3.11) and rs7129781 (C allele) (Beta 1.55; 95% CI 0.07-2.96), were associated with vitamin D serum levels. In addition, the same variants had suggestive protection on asthma, but it was not significant (OR 0.74; 95% CI 0.39; 1.39; OR 0.73; 95% CI 0.38; 1.37, respectively). VDR variants rs7965397 (G allele) was positively associated with atopy (OR 1.43; 95% CI, 1.07-1.92); rs4328262 (G allele) (OR 1.44; 95% CI 1.09-1.90) and asthma rs2408876 (C allele) (OR 2.31; 95% CI; 1.18-4.53); rs2238317 (T allele) (OR 2.19; 95% CI 1.02-4.72).

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.

Polymorphisms in genes encoding purinoreceptor andosteoprotegerin and external apical root resorption in childrenafter orthodontic treatment

Orthodontically induced external apical root resoption (EARR) is a multifactorial inflammatory disease. Genes encoding proteins, which are involved in the modulation of inflammatory response and bone remodeling, are considered as the “candidate” for EARR. The aim of this study was to analyze possible associations of single nucleotide polymorphisms (SNPs) in the P2RX7 (encoding purinoreceptor) and TNFRSF11B (encoding osteoprotegerin) genes with EARR in Czech children after orthodontic treatment. A total, 99 orthodontically treated patients (69 healthy and 30 with EARR) were enrolled in this case-control study. Genotype determination of P2RX7 +489C/T (rs208294, Tyr155His) and +1068C/T (rs1718119, Thr348Ala), TNFRSF11B -163C/T (rs3102735) and TNFRSF11B +1181C/G (rs2073618, Lys3Asn) was based on polymerase chain reaction using 5 nuclease TaqMan® assays. While no significant differences were observed in allele or genotype frequencies of all four studied SNPs, specific combinations of variants in P2RX7 may be associated with lower/higher risk of EARR development (P<0.05). In addition, the length of treatment by orthodontic appliances positively correlated with the presence of EARR (P<0.05). Although the effect of P2RX7 SNPs themselves to the development EARR was not confirmed in the Czech population, haplotype analysis suggests, that variability in the P2RX7 gene, as well as the length of treatment, may be important factors contributing to the etiopathogenesis of post-orthodontic EARR.Results: 33.4% of the sample reported high stress. The study revealed significantly high stress in women with no or low income, chronic disease, sleep deprivation, no teeth brushing, irregular eating patterns, gestational diabetes, and no family support (P < 0.05). Self-reported oral health problems were significantly associated with high stress (P < 0.05). A multiple linear regression model shows no teeth brushing, chronic disease, sleep deprivation, gestational diabetes, and gingival redness predicted an increase in stress by (3.6, 2.4, 2.1, 1.4, and 1.4, respectively).TWAIN stands for “technology without an interesting name”. TWAIN is the interface standard for Windows and Mac that allows imaging hardware devices to communicate with imaging processing software. Prior to TWAIN, image acquisition devices all came with their own proprietary software, now nearly all imaging software is TWAIN compliant. If one wanted to work with a scanned image in a different application, one had to save the image to a disc first, then open the application of your choice and re-open the image there. Nearly all image processing software today is TWAIN compliant. If your software supports TWAIN, you will find an “Acquire” command in the menus or toolbar (though sometimes the command is hidden under an import in menu.

Immune mechanisms in periodontitis – effect of IL-4 gene polymorphisms on cytokine production

Introduction: Periodontitis is a chronic inflammatory disease of the teeth-supporting tissues in which genetic predisposition, dental plaque bacteria and immune mechanisms play important roles. The aim of this study was to assess the influence of IL-4 gene polymorphisms on cytokine production Methods: Cytokine production (IL-1alpha, IL-1beta, IL-4, IL-5, IL-6, IL-8 , IL-10, TNF–alpha, INF–gamma) was studied after in vitro stimulation of isolated peripheral blood mononuclear cells (PBMC) by mitogens and dental plaque bacteria (Aggregatibacter actinomycetemcomitans, Escherichia coli, Tannerrella forsythensis, Porphyromonas gingivalis and Heat Shock Protein 60). Cytokine production was detected by multiplex cytokine analysis system Luminex. The results were correlated with IL-4 genotypes in patients with chronic periodontitis (CP) and healthy controls. Results: We found that mononuclear cells isolated from peripheral blood of CP patients with investigated IL-4 polymorphisms (-589C/T, -33C/T, VNTR in intron 3) significantly changed production of IFN-gamma, IL-10, IL-1alpha and IL-1beta, TNF-alpha, IL-10, MCP-1, IL-6 after stimulation by all selected bacteria and HSP (p<0.05). Conclusion: In patients with CP, IL-4 polymorphisms may influence the function of mononuclear cells to produce not only IL-4 but also other cytokines. The results of this study support Seymour’s hypothesis (1999) about the role of TH2 produced cytokines such as IL-4 in genetic predisposition to chronic periodontitis.