Gene polymorphisms and serum levels of mannose-binding lectin in Czech patients with recurrent aphthous stomatitis: A case-control study.

BACKGROUND: Recurrent aphthous stomatitis is one of the most prevalent oral mucosal immunological diseases. A recent case-control study in the Egyptian population suggested that single nucleotide polymorphism Gly54Asp (rs1800450) of the mannose-binding lectin 2 gene might affect the mannose-binding lectin serum level and recurrent aphthous stomatitis development. The aim of this study was to determine the distribution of six functional mannose-binding lectin 2 gene polymorphisms and analyse their role in recurrent aphthous stomatitis susceptibility in the Czech population. METHODS: The study included 227 subjects; 137 healthy people and 90 patients with recurrent aphthous stomatitis. Six mannose-binding lectin 2 gene polymorphisms (rs11003125, rs7096206, rs7095891, rs5030737, rs1800450, rs1800451) were analysed by the SNaPshot assay method, mannose-binding lectin serum levels were determined by enzyme-linked immunosorbent assay (ELISA) method in a subgroup of subjects (N = 87). RESULTS: No significant differences in mean of mannose-binding lectin serum levels between healthy controls and patients with recurrent aphthous stomatitis were observed (383 ng/ml ± 249 standard deviation (SD) vs. 316 ng/ml ± 177 SD in remission phase vs. 343 ng/ml ± 254 SD in active phase; p > 0.05), also the allele and genotype frequencies of the studied mannose-binding lectin 2 polymorphisms did not differ significantly between the two groups (p > 0.05, odds ratio (OR): 0.75-1.23). Moreover, the distribution of mannose-binding lectin 2 haplotypes and haplogenotypes was similar in the healthy subjects and patients with recurrent aphthous stomatitis (p > 0.05, OR: 0.75-1.23). CONCLUSIONS: This study did not confirm the previously reported association of the mannose-binding lectin 2 Gly54Asp gene variant and low mannose-binding lectin serum level as the risk factors for susceptibility to recurrent aphthous stomatitis. In addition, no significant relationships between mannose-binding lectin 2 functional haplotypes or haplogenotypes and recurrent aphthous stomatitis were observed.

Association of the angiotensin I converting enzyme (ACE) gene polymorphisms with recurrent aphthous stomatitis in the Czech population: case-control study.

BACKGROUND: Recurrent aphthous stomatitis (RAS) is multifactorial disease with unclear etiopathogenesis. The aim of this study was to determine distribution of the angiotensin I converting enzyme (ACE) gene polymorphisms and their influence on RAS susceptibility in Czech population. METHODS: The study included 230 subjects (143 healthy controls and 87 patients with RAS) with anamnestic, clinical and laboratory data. Five ACE gene polymorphisms (rs4291/rs4305/rs4311/rs4331/rs1799752 = ACE I/D) were determined by TaqMan technique. RESULTS: The allele and genotype distributions of the studied ACE I/D polymorphisms were not significantly different between subjects with/without RAS (Pcorr > 0.05). However, carriers of II genotype were less frequent in the RAS group (OR = 0.48, 95% CI = 0.21-1.12, P = 0.059). Stratified analysis by sex demonstrated lower frequency of II genotype in women (OR = 0.33, 95% CI = 0.09-1.17, P < 0.035, Pcorr > 0.05, respectively) than in men with RAS (P > 0.05). Moreover, the frequency of AGTGD haplotype was significantly increased in RAS patients (OR = 13.74, 95% CI = 1.70-110.79, P = 0.0012, Pcorr < 0.05). In subanalysis, TGD haplotype was significantly more frequent in RAS patients (P < 0.00001) and CGI haplotype was less frequent in RAS patients (P < 0.01), especially in women (P = 0.016, Pcorr > 0.05). CONCLUSIONS: Our study indicates that while the AGTGD and TGD haplotypes are associated with increased risk of RAS development, CGI haplotype might be one of protective factors against RAS susceptibility in Czech population.

Gene variability in matrix metalloproteinases in patients with recurrent aphthous stomatitis.

BACKGROUND: The development of recurrent aphthous stomatitis (RAS), inflammatory disease of oral mucosa, is influenced by both environmental and genetic factors. The aim of this study was to investigate polymorphisms located in seven genes coding different types of matrix metalloproteinases (MMPs)-collagenases (MMP1, MMP8, and MMP13), gelatinases (MMP2 and MMP9), stromelysin (MMP3), and membrane-type metalloproteinase (MMP16) in patients with RAS and healthy controls. METHODS: Totally, 223 subjects were included in this case-control study and their detailed anamnestic, clinical, and laboratory parameters were recorded. Seventy-seven patients with RAS and 146 controls were genotyped for seventeen polymorphisms in the MMPs genes using the real-time polymerase chain reaction (PCR) or PCR with restriction analysis. RESULTS: Allele, genotype, and haplotype frequencies of the studied polymorphisms between RAS patients and controls were similar, except for allele distributions of MMP1 rs1144393, MMP9 rs3918242, and MMP16 rs10429371, which were different between patients with RAS and healthy controls (P = .023, P = .049 and P = .025, all Pcorr  > 0.05, respectively). Moreover, the comparison of genotype frequencies (TT vs CC + CT) of the MMP16 rs10429371 variant showed a marginally significant difference between RAS patients and controls (P = .05, Pcorr  > 0.05, OR = 1.68, 95% CI = 0.95-2.98). CONCLUSIONS: No significant relationship between investigated polymorphisms in seven MMPs genes and RAS development in the Czech population was observed in this study.

Interleukin Gene Variability and Periodontal Bacteria in Patients with Generalized Aggressive Form of Periodontitis.

Host genetic predispositions to dysregulated immune response can influence the development of the aggressive form of periodontitis (AgP) through susceptibility to oral dysbiosis and subsequent host-microbe interaction. This case-control study aimed to perform a multilocus analysis of functional variants in selected interleukin (IL) genes in patients with the generalized form of AgP in a homogenous population. Twelve polymorphisms in IL-1 gene cluster, IL-6 and its receptor, IL-10, IL-17A, and IL-18 were determined in 91 AgP patients and 210 controls. Analysis of seven selected periodontal bacteria in subgingival sulci/pockets was performed with a commercial DNA-microarray kit in a subgroup of 76 individuals. The pilot in vitro study included stimulation of peripheral blood monocytes (PBMC) from 20 individuals with periodontal bacteria and measurement of IL-10 levels using the Luminex method. Only the unctional polymorphism IL­10-1087 A/G (rs1800896) and specific IL-10 haplotypes were associated with the development of the disease (P < 0.05, Pcorr > 0.05). Four bacterial species occurred more frequently in AgP than in controls (P < 0.01, Pcorr < 0.05). Elevated IL-10 levels were found in AgP patients, carriers of IL­10-1087GG genotype, and PBMCs stimulated by periodontal bacteria (P < 0.05, Pcorr > 0.05). We therefore conclude that a combination of genetic predisposition to the altered expression of IL-10 and the presence of specific periodontal bacteria may contribute to Th1/Th2 balance disruption and AgP development.

Association of the NOD-like receptor 3 (NLRP3) gene variability with recurrent aphthous stomatitis in the Czech population.

BACKGROUND: Recurrent aphthous stomatitis (RAS) is a multifactorial disease with unclear etiopathogenesis in which disturbance of immunological processes may be involved. The aim of our study was to investigate three single nucleotide polymorphisms (SNPs) rs3806265, rs4612666, rs10754558 in NOD-like receptor 3 (NLRP3), the gene encoding the component of inflammasome, in patients with RAS and healthy controls in the Czech population. METHODS: A total of 207 subjects were included in this case-control study. Sixty-four patients with RAS and 143 healthy controls were genotyped by a method based on polymerase chain reaction using 5' nuclease TaqMan® assays. Detailed anamnestic, clinical, and laboratory data were obtained from all subjects. RESULTS: The allele and genotype frequencies of NLRP3 polymorphisms (rs10754558 and rs3806265) between both groups were similar. However, statistically significant differences in NLRP3 rs4612666 genotypes between the patients with RAS and controls were found; carriers of the TT genotype had a higher risk of developing RAS than subjects with the CT+CC genotypes (OR = 14.69, 95%CI = 1.73-124.72, P = .004, Pcorr < .05). No associations between NLRP3 haplotypes and RAS were observed. CONCLUSIONS: Our study indicates that the NLRP3 rs4612666 polymorphism may be involved in the development of RAS in the Czech population.

Recurrent aphthous stomatitis and gene variability in selected interleukins: a case-control study.

Genetic factors, especially those related to immune system functioning, have been intensively studied to determine their role in the development of recurrent aphthous stomatitis (RAS). The aim of the present study was to analyze gene variability in interleukin (IL)2, IL4 (and its receptor α, IL4Rα), IL10, and IL13, which were selected based on literature review and/or their functional relevance, in Czech patients with RAS and in healthy controls. In total, 252 subjects (178 controls and 74 patients with RAS) were enrolled in this case-control study, and their detailed anamnestic, clinical, and laboratory data were obtained. Nine polymorphisms in the genes encoding interleukins were determined using PCR techniques. There were no significant differences in allele or genotype frequencies of the IL2, IL4, IL4Rα, IL10, and IL13 polymorphisms rs2069762/rs2069763, rs2243250/rs79071878, rs1801275, rs1800896, and rs1800925, respectively, between controls and patients with RAS. The minority alleles rs1800871 and rs1800872, which encode variants of IL10, were associated with a statistically significantly higher risk of RAS, as confirmed by the results of genotype and haplotype analyses. We suggest that variability in the IL10 gene may play an important role in the development of RAS in the Czech population.

Association study of interleukin-1 family, interleukin-6, and its receptor gene polymorphisms in patients with recurrent aphthous stomatitis.

BACKGROUND: Recurrent aphthous stomatitis (RAS) is one of the most common oral chronic ulcerative disease in which proinflammatory cytokines such as interleukin-1 (IL-1) and interleukin-6 (IL-6) are thought to play an important role. The aim of this study was to investigate the possible association between polymorphisms in the IL-1 cytokine family, IL-6 or its receptor and RAS in the Czech population. METHODS: A total of 248 subjects, 184 healthy controls, and 64 patients with RAS were genotyped for IL-1A-889C>T, IL-1B-511C>T, IL-1B+3953C>T, IL-1RN86 bp variable number of tandem repeats (VNTRs) in intron 2, IL-6-597G>A, IL-6-572G>C, IL-6-174G>C, and IL-6R+48992A>C by polymerase chain reaction (PCR) methods. RESULTS: No significant differences between investigated polymorphisms in healthy subjects and patients with RAS were detected (P>.05). In addition, complex analysis also revealed similar IL-1 or IL-6 haplotype frequencies between both groups (P>.05). CONCLUSIONS: In conclusion, IL-1 and IL-6 or its receptor gene variants cannot be used as markers for identification of Czech patients with increased risk of recurrent aphthous stomatitis.

Effect of Blood Component Coatings of Enosseal Implants on Proliferation and Synthetic Activity of Human Osteoblasts and Cytokine Production of Peripheral Blood Mononuclear Cells.

The study monitored in vitro early response of connective tissue cells and immunocompetent cells to enosseal implant materials coated by different blood components (serum, activated plasma, and plasma/platelets) to evaluate human osteoblast proliferation and synthetic activity and inflammatory response presented as a cytokine profile of peripheral blood mononuclear cells (PBMCs) under conditions imitating the situation upon implantation. The cells were cultivated on coated Ti-plasma-sprayed (Ti-PS), Ti-etched (Ti-Etch), Ti-hydroxyapatite (Ti-HA), and ZrO2 surfaces. The plasma/platelets coating supported osteoblast proliferation only on osteoconductive Ti-HA and Ti-Etch whereas activated plasma enhanced proliferation on all surfaces. Differentiation (BAP) and IL-8 production remained unchanged or decreased irrespective of the coating and surface; only the serum and plasma/platelets-coated ZrO2 exhibited higher BAP and IL-8 expression. RANKL production increased on serum and activated plasma coatings. PBMCs produced especially cytokines playing role in inflammatory phase of wound healing, that is, IL-6, GRO-α, GRO, ENA-78, IL-8, GM-CSF, EGF, and MCP-1. Cytokine profiles were comparable for all tested surfaces; only ENA-78, IL-8, GM-CSF, and MCP-1 expression depended on materials and coatings. The activated plasma coating led to uniformed surfaces and represented a favorable treatment especially for bioinert Ti-PS and ZrO2 whereas all coatings had no distinctive effect on bioactive Ti-HA and Ti-Etch.

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.

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-1ß, 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-1ß, 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.

Immune markers in oral discomfort patients before and after elimination of oral galvanism.

BACKGROUND: An enhanced release of metals in the mouth due to galvanic cell formation is considered to be one of the causes of oral discomfort. The aim of this study was to investigate the influence of galvanic cell on salivary immune defense factors. MATERIAL AND METHODS: The levels of IgA1, IgA2, sIgA, lysozyme and antiIgA/HSP60 were evaluated in representative samples from 159 patients with galvanism, from 177 patients without galvanism and in two control groups. All the participants underwent personal history taking, clinical examination, galvanic currents measurement and saliva collection. RESULTS: Electro active dental materials were removed in 30 patients. There was a significant increase in IgA2 level, a significant decrease in antiIgA/HSP60 levels and an increase in IgA1, sIgA and lysozyme levels found after the removal of electro active restorations. Morphological symptoms disappeared in 70% of the treated patients. CONCLUSION: The study confirmed that pathologic galvanic phenomena influences the immune defense reactions in the oral cavity and thus may cause the symptoms of oral discomfort. A measurement of the galvanism and a subsequent removal of electro active restorations should become a common therapeutic procedure in the patients with oral discomfort.

Serotonin transporter gene (SLC6A4) variability in patients with recurrent aphthous stomatitis.

OBJECTIVE: The aim of this study was to investigate five polymorphisms in the SLC6A4 gene in patients with recurrent aphthous stomatitis (RAS) and healthy controls. DESIGN: Totally, 239 subjects were enrolled in this case-control study: 86 patients with RAS and 153 healthy individuals were genotyped for serotonin transporter length polymorphic region (5-HTTLPR) polymorphism, variable number tandem repeat (STin2) and single nucleotide polymorphisms (rs25531, rs3813034, rs1042173) in the SLC6A4 gene by polymerase chain reaction with/without restriction analysis. RESULTS: No significant differences in the allele or genotype frequencies in all studied polymorphisms between RAS patients and healthy controls (P > 0.05) were detected. However, the haplotype analysis detected a higher frequency of LA12 (HTTLPR, rs25531, STin2) haplotype in RAS patients in comparison with healthy controls (P < 0.05, OR = 1.63, 95 % CI = 1.07-2.49). CONCLUSIONS: Our study indicates a possible relationship between SLC6A4 and susceptibility to RAS in the Czech population.

Antimutagenic effect of phenethyl isothiocyanate.

Using the Ames bacterial mutagenicity test, the comet assay, and an in vivo micronucleus test, we investigated the effect of the chemoprotective substance phenethyl isothiocyanate (PEITC) on the mutagenic activity of indirect-acting mutagens and carcinogens aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and direct-acting mutagen and carcinogen N-nitroso-N-methylurea (MNU). In the Ames test, the antimutagenic activity of PEITC was studied in the concentration range 0.3-300 microg/plate. PEITC at concentrations of 0.3, 3 and 30 microg/plate reduced dose-dependently mutagenicity of AFB1 and IQ in both S. typhimurium TA98 and TA100 strains. In the case of the direct mutagen MNU, the antimutagenic effect of PEITC was detected only at concentration of 30 microg/plate in the strain TA100. The PEITC concentration 300 microg/plate was toxic in the Ames test. The 24 h pre-treatment of HepG2 cells with PEITC at concentration 0.15 microg/ml resulted in a significant decrease of DNA breaks induced by MNU at concentrations 0.25 and 0.5 mM. Although a trend towards reduced strand break level were determined also at PEITC concentrations 0.035 and 0.07 microg/ml it did not reach the statistical significance. No effect, however, of PEITC on IQ-induced DNA breaks was observed. Chemopreventive effect of PEITC was revealed also in vivo. Pretreatment of mice with PEITC concentrations of 25 and 12.5 mg/kg b.w. administered to mice in three daily doses resulted in reduction of micronucleus formation in mice exposed to all three mutagens under study, with statistically significant effect at concentration of 25 mg/kg. Results of this study indicate that the strong PEITC antimutagenic properties may have an important role in the prevention of carcinogenesis and other chronic degenerative diseases that share some common pathogenetic mechanisms.

Interleukin-1 Gene Variability and Plasma Levels in Czech Patients with Chronic Periodontitis and Diabetes Mellitus.

Recent studies have suggested a bidirectional relationship between chronic periodontitis (CP) and diabetes mellitus (DM). Immunoregulatory factors such as cytokines play an important role in etiopathogenesis of both diseases. The aim of this study was to analyze variability in interleukin-1 (IL-1) gene cluster and IL-1ß plasma levels in patients with CP, DM, and a combination of both diseases. A total of 1016 individuals participating in this case-control study-225 healthy controls, 264 patients with CP, 132 with type 1 diabetes (T1DM), and 395 patients with type 2 diabetes (T2DM)-were genotyped using methods based on polymerase chain reaction for IL-1 gene polymorphisms (IL-1A (-889C/T, rs1800587), IL-1B (+3953C/T, rs1143634), and IL-1RN (gene for IL-1 receptor antagonist, IL-1RA, 86 bp tandem repeats in intron 2)). Levels of IL-1ß were measured by Luminex methods in subgroups of controls, CP, T1DM + CP, and T2DM + CP subjects. Although no significant associations were found in the genotype and allele frequencies of IL-1A (-889C/T), significant differences in the allele frequencies of IL-1B (+3953C/T) were observed between controls and CP patients (P < 0.05). In T1DM patients, IL-1RN ∗ S "short" allele and IL-1RN 12 genotype were significantly less frequent than those in controls (P < 0.01). In haplotype analysis, TTL haplotype decreased the risk of CP development (P < 0.01), whereas CCS and CTL haplotypes (P < 0.01 and P < 0.05) were associated with T1DM. Although IL-1ß levels were measured significantly higher in mononuclear cells after stimulation by mitogens, HSP70, or selected periodontal bacteria than in unstimulated cells, IL-1 genotypes did not correlate with circulating IL-1ß levels. In the Czech population, significant associations between the IL-1B polymorphism with CP and the IL-1RN variant with T1DM were found. Haplotype analysis suggests that variability in IL-1 gene cluster may be one of the factors in the CP and T1DM pathogenesis, although single variants of these polymorphisms are not substantial for protein production.

Oral Microbiota Composition and Antimicrobial Antibody Response in Patients with Recurrent Aphthous Stomatitis.

Recurrent aphthous stomatitis (RAS) is the most common disease of the oral mucosa, and it has been recently associated with bacterial and fungal dysbiosis. To study this link further, we investigated microbial shifts during RAS manifestation at an ulcer site, in its surroundings, and at an unaffected site, compared with healed mucosa in RAS patients and healthy controls. We sampled microbes from five distinct sites in the oral cavity. The one site with the most pronounced differences in microbial alpha and beta diversity between RAS patients and healthy controls was the lower labial mucosa. Detailed analysis of this particular oral site revealed strict association of the genus Selenomonas with healed mucosa of RAS patients, whereas the class Clostridia and genera Lachnoanaerobaculum, Cardiobacterium, Leptotrichia, and Fusobacterium were associated with the presence of an active ulcer. Furthermore, active ulcers were dominated by Malassezia, which were negatively correlated with Streptococcus and Haemophilus and positively correlated with Porphyromonas species. In addition, RAS patients showed increased serum levels of IgG against Mogibacterium timidum compared with healthy controls. Our study demonstrates that the composition of bacteria and fungi colonizing healthy oral mucosa is changed in active RAS ulcers, and that this alteration persists to some extent even after the ulcer is healed.

Metallic pigmentation of human teeth and gingiva: morphological and immunological aspects.

The composition of metallic pigmentations in gingiva and dental roots was determined by means of transmission electron microscopy with energy dispersive x-ray microanalysis. The systemic immune response to the metals found in the oral cavity was evaluated in 10 patients by using a modified lymphocyte proliferation test. Immunological results were compared with a group of five controls without metallic materials and pigmentation. Dense particles of various shapes and sizes, as well as of diverse extracellular and intracellular localization patterns, were detected in the pigmented lamina propria gingivae. Metallic deposits consisted predominantly of silver accompanied by selenium or sulfur or both. Besides, Ag, Au, Cr, Ni, Fe, Hg, Cu, and Ti were identified in dentinal tubules of teeth reconstructed with dental alloys. Nine patients with metallic pigmentations had a positive lymphocyte proliferative response to one or more metals present in their own metal reconstructions. Results of this study thus indicated that dental alloys-by virtue of their corrosion process-might pose a significant risk to immunologically susceptible patients.

Correction: Enhanced Mitogenic Activity of Recombinant Human Vascular Endothelial Growth Factor VEGF121 Expressed in E. coli Origami B (DE3) with Molecular Chaperones.

[This corrects the article DOI: 10.1371/journal.pone.0163697.].

Removal of dental amalgam decreases anti-TPO and anti-Tg autoantibodies in patients with autoimmune thyroiditis.

OBJECTIVES: The impact of dental amalgam removal on the levels of anti-thyroid peroxidase (anti-TPO) and anti-thyroglobulin (anti-Tg) antibodies was studied in patients with autoimmune thyroiditis (AT) with and without mercury allergy. METHODS: Thirty-nine patients with AT were tested by an optimized lymphocyte proliferation test MELISA for allergy (hypersensitivity) to inorganic mercury. Patients were divided into two groups: Group I (n = 12) with no hypersensitivity to mercury and Group II (n = 27) with hypersensitivity to mercury. Amalgam fillings were removed from the oral cavities of 15 patients with hypersensitivity to mercury (Group IIA) and left in place in the remaining 12 patients (Group IIB). The laboratory markers of AT, anti-TPO and anti-Tg autoantibodies, were determined in all groups at the beginning of the study and six months later. RESULTS: Compared to levels at the beginning of the study, only patients with mercury hypersensitivity who underwent amalgam replacement (Group IIA) showed a significant decrease in the levels of both anti-Tg (p=0.001) and anti-TPO (p=0.0007) autoantibodies. The levels of autoantibodies in patients with or without mercury hypersensitivity (Group I and Group IIB) who did not replace amalgam did not change. CONCLUSION: Removal of mercury-containing dental amalgam in patients with mercury hypersensitivity may contribute to successful treatment of autoimmune thyroiditis.

In vivo effects of dental casting alloys.

OBJECTIVE: Corrosion products of different metallic alloys used in prosthetic dentistry often cause the development of a bluish-grey pigmentation of gingiva and oral mucosa. The aim of this study was to determine the content of metals in metallic pigmentations and evaluate the immune response to metals found in the oral cavity. MATERIAL AND METHODS: The local tissue reactions were investigated clinically by electron microscopy and by energy dispersive x-ray microanalysis. An extensive anamnesis of the patients was recorded as well as earlier contacts with health care institutions. The immunological response to metallic components of dental alloys was evaluated in 34 patients by MELISA, a modified test for lymphocyte proliferation. In addition, cytokines in culture media were determined in 10 persons by the Human Inflammation Antibody Array. RESULTS: Dense particles containing metals were found in the matrix among collagen fibrils and in close proximity of the lamina basalis of the gingival epithelium. Particles were also localized intracellularly in fibroblasts, macrophages, and endothelial cells. Metallic depositions consisted predominantly of silver accompanied by selenium and sulphur. Twenty five out of 34 patients revealed high lymphocyte reactivity (positive MELISA test) to one or more metal components of dental restorations. A correlation between the positivity in MELISA test and number of dental alloys in the oral cavity was also found. Twenty MELISA positive patients suffered from serious health problems (various allergies, autoimmune diseases, Parkinson's syndrome etc.). Nickel and inorganic mercury were the most common sensitizers in vitro. The cytokine assay revealed that mercury chloride activated predominantly TH2 lymphocytes, while nickel chloride activated mainly TH1 lymphocytes. CONCLUSIONS: Metallic pigmentations in the oral cavity demonstrate a corrosion process and may pose a risk in immunologically susceptible patients.

Enhanced Mitogenic Activity of Recombinant Human Vascular Endothelial Growth Factor VEGF121 Expressed in E. coli Origami B (DE3) with Molecular Chaperones.

We describe the production of a highly-active mutant VEGF variant, α2-PI1-8-VEGF121, which contains a substrate sequence for factor XIIIa at the aminoterminus designed for incorporation into a fibrin gel. The α2-PI1-8-VEGF121 gene was synthesized, cloned into a pET-32a(+) vector and expressed in Escherichia coli Origami B (DE3) host cells. To increase the protein folding and the solubility, the resulting thioredoxin-α2-PI1-8-VEGF121 fusion protein was co-expressed with recombinant molecular chaperones GroES/EL encoded by independent plasmid pGro7. The fusion protein was purified from the soluble fraction of cytoplasmic proteins using affinity chromatography. After cleavage of the thioredoxin fusion part with thrombin, the target protein was purified by a second round of affinity chromatography. The yield of purified α2-PI1-8-VEGF121 was 1.4 mg per liter of the cell culture. The α2-PI1-8-VEGF121 expressed in this work increased the proliferation of endothelial cells 3.9-8.7 times in comparison with commercially-available recombinant VEGF121. This very high mitogenic activity may be caused by co-expression of the growth factor with molecular chaperones not previously used in VEGF production. At the same time, α2-PI1-8-VEGF121 did not elicit considerable inflammatory activation of human endothelial HUVEC cells and human monocyte-like THP-1 cells.