Ana Paula de Souza

Interleukin-8 Gene Promoter Polymorphism (rs4073) May Contribute to Chronic Periodontitis

BACKGROUND The proinflammatory chemokine interleukin (IL)-8 is important in the regulation of the inflammatory response. Analyses of the single nucleotide polymorphism (SNP) reference sequence (rs) 4073 showed that the A allele upregulated IL-8 levels after stimulation with lipopolysaccharides. We investigated the association of the SNP rs4073 with chronic periodontitis. METHODS Genotyping was performed by a standard polymerase chain reaction-restriction fragment length polymorphism assay in 289 genomic DNA samples of healthy control subjects and patients with chronic periodontitis; analyses were adjusted by multivariate logistic regression modeling. A real-time polymerase chain reaction performance was used to detect levels of the IL-8 mRNA. RESULTS The analysis pointed to a statistically significant association of chronic periodontitis with the heterozygous TA genotype (P = 0.001); the results showed an increase in the frequency of the A allele in the diseased group (36% in the control group versus 48% in the periodontitis group). The higher levels of the IL-8 mRNA were found in the periodontitis group, mainly in individuals who presented the TA genotype (P = 0.03). CONCLUSION The SNP rs4073 was associated with chronic periodontitis in non-smoker Brazilian subjects because the frequency of the A allele was higher in the disease group than in the control group, and the TA genotype was associated with increased levels of IL-8 mRNA transcripts.

DNA Methylation Status of the IL8 Gene Promoter in Aggressive Periodontitis

BACKGROUND Studies evaluating the methylation status of cytokine genes may have relevance for inflammatory diseases in which the expression of some cytokines is altered, such as periodontitis. This study observes the DNA methylation status in the interleukin-8 (IL8) gene promoter in cells of the oral epithelium of subjects affected by generalized aggressive periodontitis (AgP) and compares it to those of control subjects. METHODS Genomic DNA from epithelial oral cells of 37 generalized AgP patients and 37 controls were purified and modified by sodium bisulphite. Modified DNA was submitted by methylation-specific polymerase chain reaction, electrophoresed on 10% polyacrylamide gels, and stained. RESULTS Subjects who presented generalized AgP have a higher frequency of hypomethylation of the IL8 gene promoter in oral epithelium cells than that of controls (86.5% in the generalized AgP group versus 62% in the control group; P = 0.016; chi(2) test). CONCLUSIONS A marked hypomethylated status is found in the oral epithelial cells of subjects presenting with generalized AgP, compared to controls, in the promoter region of the IL8 gene. This hypomethylated status may reflect a generalized condition of oral epithelial cells, including gingival epithelium, because gingival epithelial cells were also collected during mouthwash use.

Inhibition of human gelatinases by metals released from dental amalgam.

The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in pathologic oral processes such as periodontal tissue destruction, root caries, tumor invasion and temporomandibular joint disorders. The aim of this study was to test the effect of metal ions released from dental amalgam on the major gingival gelatinolytic MMPs. Gingival human explants were cultured overnight in DMEM and the activity of secreted enzymes was analyzed by gelatin zymography in buffers conditioned with dispersed phase or concentional phase dental amalgams. The major enzymes present in conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation. The proteolytic activities of MMP-2 and MMP-9 were strongly inhibited by dispersed phase amalgams conditioned buffers. Inhibition of MMP-2 and MMP-9 activities was partly prevented by the addition of 1,10 phenanthroline, a divalent metal chelator, to the amalgam conditioned buffers. Dental amalgam conditioned buffer also inhibited the degradation of denatured type I collagen by purified MMP-2 on liquid phase assays. These findings suggest that the activity of oral tissue MMPs may be modulated by metal ions released from dental amalgam.

2-Hydroxyethyl methacrylate as an inhibitor of matrix metalloproteinase-2

This study evaluated the effect of different concentrations of 2-hydroxyethyl methacrylate (HEMA) on the inhibition of matrix metalloproteinase-2 (MMP-2) in vitro. Mouse gingival explants were cultured overnight in Dulbeccos modified Eagles minimal essential medium, following which the expression of secreted enzymes was analyzed by gelatin zymography and the effects of different amounts of HEMA on enzyme activity were investigated. The gelatinolytic proteinases present in the conditioned media were characterized as being matrix metalloproteinases (MMPs) by means of specific chemical inhibition. The MMPs present in the conditioned media were identified, using immunoprecipitation, as MMP-2. Three major bands were detected in the zymographic assays and were characterized, according to their respective molecular weights, into the following forms of MMP-2: zymogene (72 kDa), intermediate (66 kDa), and active (62 kDa). All forms of MMP-2 were inhibited by HEMA in a dose-dependent manner, implying that MMP-2 may be inhibited by HEMA in vivo.

TLR2 and TLR4 gene promoter methylation status during chronic periodontitis

AIM The objective of this study was to analyse the status of DNA methylation in the promoter region of the toll-like receptor (TLR)2 and TLR4 genes in gingival tissue samples from healthy subjects, smokers and non-smokers affected by chronic periodontitis. MATERIAL AND METHODS Genomic DNA and total RNA were purified from gingival tissue using the TRIZOL reagent protocol. Genomic DNA was then digested by methylation-sensitive restriction enzymes, amplified by polymerase chain reaction (PCR), electrophoresed on a 10% polyacrylamide gel and stained using SYBR Gold. Real-time PCR was also performed to verify the transcript levels. RESULTS The CpG dinucleotides analysed were observed to be unmethylated in the majority of DNA samples of the three groups and statistical differences were not found among groups (p>0.05). However, a trend towards methylation was observed in the TLR2 HhaI site in the samples of the periodontitis non-smoker groups. In fact, the analysis of all CpG sites together shows which complete methylation is observed in the shortest level in the samples of periodontitis non-smoker group. The analysis of transcript levels demonstrated no difference among groups (p>0.05). CONCLUSION The results demonstrated major unmethylation of the TLR4 gene promoter in all groups. However, the results for the TLR2 gene promoter are inconclusive; this gene was found as a mosaic of methylated and unmethylated DNA in the majority of samples of the three groups and we also observed a trend towards the DNA methylation of CpG sites recognized by the HhaI enzyme.

Association of matrix metalloproteinase gene polymorphism with temporomandibular joint degeneration.

Temporomandibular joint (TMJ) degeneration is a frequent cause of orofacial pain. Matrix metalloproteinases (MMPs) degrade extracellular matrix components and play an important role in TMJ degeneration. We investigated the frequency of the MMP1 1G/2G polymorphism (rs1799750), the MMP3 5A/6A polymorphism (rs3025058), and the MMP9 C/T polymorphism (rs3918242) in individuals with TMJ degeneration, in order to analyze the association of polymorphisms in these genes with TMJ degeneration. The population studied comprised 117 healthy controls and 115 individuals diagnosed with TMJ degeneration upon examination of magnetic resonance imaging (MRI) and computed tomography (CT) images. Genotypes were determined using PCR restriction fragment length polymorphism (RFLP). Logistic regression analyses revealed an association between the MMP1 2G/2G genotype and degeneration; in contrast, there was no association between either the MMP3 or the MMP9 genotype and degeneration. Our results may indicate a role for the MMP1 polymorphism in TMJ degeneration.

MMP3 and TIMP1 variants contribute to chronic periodontitis and may be implicated in disease progression

AIM Matrix metalloproteinases (MMPs) play a key role in the tissue destruction characteristic of chronic periodontitis. The purpose of this study was to investigate the association of MMP and TIMP polymorphisms with chronic periodontitis in two populations. MATERIAL AND METHODS A total of 34 polymorphisms spanning 12 MMP and 2 TIMP genes were genotyped in 401 individuals from Brazil (99 cases with chronic periodontitis and 302 controls), and 274 individuals from the US (70 cases and 204 controls). Individuals were considered cases if presenting at least three teeth exhibiting sites of clinical attachment loss ≥ 5 mm in two different quadrants. Controls were characterized by absence of clinical attachment loss and no sites with probing depth >3 mm. MMP3 and TIMP1 mRNA expression was evaluated in healthy and diseased periodontal tissues. RESULTS TIMP1 showed association with chronic periodontitis in the Brazilian population (for rs5906435, p = 0.0004), whereas MMP3 showed association in the US population (for rs679620, p = 0.0003; and rs650108, p = 0.002) and in the Brazilian population (for rs639752, p = 0.005). MMP3 and TIMP1 mRNA expression was significantly higher in diseased tissues when compared to control tissues. CONCLUSIONS Our results further support a role for variations in MMP3 in chronic periodontitis and report a novel association with TIMP1. These genes may be considered additional candidate genes for chronic periodontitis.

G-quadruplex formation enhances splicing efficiency of PAX9 intron 1

G-quadruplexes are secondary structures present in DNA and RNA molecules, which are formed by stacking of G-quartets (i.e., interaction of four guanines (G-tracts) bounded by Hoogsteen hydrogen bonding). Human PAX9 intron 1 has a putative G-quadruplex-forming region located near exon 1, which is present in all known sequenced placental mammals. Using circular dichroism (CD) analysis and CD melting, we showed that these sequences are able to form highly stable quadruplex structures. Due to the proximity of the quadruplex structure to exon–intron boundary, we used a validated double-reporter splicing assay and qPCR to analyze its role on splicing efficiency. The human quadruplex was shown to have a key role on splicing efficiency of PAX9 intron 1, as a mutation that abolished quadruplex formation decreased dramatically the splicing efficiency of human PAX9 intron 1. The less stable, rat quadruplex had a less efficient splicing when compared to human sequences. Additionally, the treatment with 360A, a strong ligand that stabilizes quadruplex structures, further increased splicing efficiency of human PAX9 intron 1. Altogether, these results provide evidences that G-quadruplex structures are involved in splicing efficiency of PAX9 intron 1.

Genetic analysis of the IL8 gene polymorphism (rs4073) in generalized aggressive periodontitis

OBJECTIVES Interleukin (IL)-8 is an important chemokine for regulation of the inflammatory response. A single nucleotide polymorphism (SNP) reference sequence (rs) 4073 in the IL8 gene has been shown to regulate IL-8 levels after stimulation with lipopolysaccharide. This study investigates the transmission pattern of the IL8 rs4073 risk allele A and its association with susceptibility to aggressive periodontitis (AgP) in families and in a case-control cohort of unrelated individuals from a Brazilian population. DESIGN Genotyping was performed by standard polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) in 13 nuclear families and 184 unrelated subjects. Statistical analysis was performed using the transmission disequilibrium test (TDT) for the family dataset and Chi-square test and multivariate logistic regression modelling for the case-control dataset. RESULTS TDT analyses did not detect evidence of over transmission of IL8 rs4073 alleles in affected and unaffected family members (allele T: 52%; allele A: 48%; p=0.2252). How expected, analyses of cases and unrelated controls showed a significant and inverse association of age with AgP; however, a lack of association between genotypes, ethnic groups and generalized AgP was observed. CONCLUSIONS The SNP (rs4073) was not associated with AgP in unrelated individuals and there is no evidence of over transmission of the alleles in families with AgP, from Brazilian individuals.

In situ study of the gelatinase activity in demineralized dentin from rat molar teeth

Matrix metalloproteinases (MMPs) in dentin are believed to participate in various physiological and pathological events in coronal dentin, but their exact source and location is not clear. The purpose of this study was to evaluate the activity of gelatinases in decalcified rat molars crowns by in situ zymography. Hemi-mandibles of five male Wistar rats were fixed in paraformaldehyde, decalcified in EDTA and glycerol solution and embedded in paraffin. Sections from the region of molar teeth were incubated with or without DQ gelatin in 50mM Tris-CaCl2 at 37°C for 2h and observed by means of confocal microscopy. Gelatinolytic activity was observed throughout the coronal dentin with varying intensities in different locations. High gelatinase activity was observed in the dentinal tubules, dentin-enamel junction (DEJ) and predentin, and it was weaker and less uniform in the intertubular dentin. This study shows that the location of gelatinase and relative activity can be detected by means of in situ zymography and confocal microcopy, and this methodology may provide a useful tool in studies on the role of gelatinases in tooth development, maturation and in pathological conditions.