Circulating lymphocytes and monocytes transcriptomic analysis of patients with type 2 diabetes mellitus, dyslipidemia and periodontitis.

Type 2 diabetes mellitus (T2DM), dyslipidemia and periodontitis are frequently associated pathologies; however, there are no studies showing the peripheral blood transcript profile of these combined diseases. Here we identified the differentially expressed genes (DEGs) of circulating lymphocytes and monocytes to reveal potential biomarkers that may be used as molecular targets for future diagnosis of each combination of these pathologies (compared to healthy patients) and give insights into the underlying molecular mechanisms of these diseases. Study participants (n = 150) were divided into groups: (H) systemically and periodontal healthy (control group); (P) with periodontitis, but systemically healthy; (DL-P) with dyslipidemia and periodontitis; (T2DMwell-DL-P) well-controlled type 2 diabetes mellitus with dyslipidemia and periodontitis; and (T2DMpoorly-DL-P) poorly-controlled type 2 diabetes mellitus with dyslipidemia and periodontitis. We preprocessed the microarray data using the Robust Multichip Average (RMA) strategy, followed by the RankProd method to identify candidates for DEGs. Furthermore, we performed functional enrichment analysis using Ingenuity Pathway Analysis and Gene Set Enrichment Analysis. DEGs were submitted to pairwise comparisons, and selected DEGs were validated by quantitative polymerase chain reaction. Validated DEGs verified from T2DMpoorly-DL-P versus H were: TGFB1I1, VNN1, HLADRB4 and CXCL8; T2DMwell-DL-P versus H: FN1, BPTF and PDE3B; DL-P versus H: DAB2, CD47 and HLADRB4; P versus H: IGHDL-P, ITGB2 and HLADRB4. In conclusion, we identified that circulating lymphocytes and monocytes of individuals simultaneously affected by T2DM, dyslipidemia and periodontitis, showed an altered molecular profile mainly associated to inflammatory response, immune cell trafficking, and infectious disease pathways. Altogether, these results shed light on novel potential targets for future diagnosis, monitoring or development of targeted therapies for patients sharing these conditions.

Systemic expression of genes related to inflammation and lipid metabolism in patients with dyslipidemia, type 2 diabetes mellitus and chronic periodontitis.

Inflammatory diseases, as periodontal disease (PD), has been associated with disturbance of lipid and glycemic metabolisms, as demonstrated by the increasing of PD patients with type 2 diabetes mellitus (T2D) and/or dyslipidemia comorbidities. We aimed to investigate the expression of inflammation and lipid metabolism genes, and correlations among clinical and biochemical characteristics in normoglycemic or T2D patients with dyslipidemia and PD, in comparison with healthy individuals. Five groups of 30 individuals each (150 patients) were formed based upon T2D, dyslipidemic and periodontal status. Blood analyses of lipid and glycemic profiles were carried out, and the gene expression was assessed by RT-qPCR. The systemic expression of IL6, TNFA and LEP genes were significantly higher in T2D, dyslipidemia and PD patients, while the PECAM1 gene showed the opposite. Higher RETN levels were found in patients with T2D independently of their glycemic control status. There were positive correlations between: TNFA, LEP and RETN with worse periodontal parameters; IL6, TNFA, ADIPOR1, LEP and RETN with waist-to-hip ratio; glycemic parameters with RETN; total cholesterol and triglycerides with LEP expression. We conclude that pro-inflammatory cytokines were related with worse lipid, glycemic and periodontal parameters, reinforcing that a hyper-inflammatory status connects systemic and oral inflammatory diseases.

Absolute quantification of Aggregatibacter actinomycetemcomitans in patients carrying haplotypes associated with susceptibility to chronic periodontitis: multifaceted evaluation with periodontitis covariants.

This study aimed to evaluate the association between haplotypes in the interleukin 8 (IL8) and IL4 genes previously associated to chronic periodontitis (CP) and the levels of Aggregatibacter actinomycetemcomitans (A.a.) in subgingival sites of patients with and without CP. Moreover, multifaceted evaluations were made to search associations among patients' genetic background with the A.a. levels and previous clinical/immunological/microbiological findings. Subgingival sites (n = 596) of 104 patients were divided into susceptible to CP by the IL8 haplotype ATC/TTC (IL8+); non-susceptible to CP by the IL8 AGT/TTC (IL8-); susceptible to CP by the IL4 TCI/CCI (IL4+); protection against CP by the IL4 TTD/CTI (IL4-). Subgingival biofilm samples from diseased and healthy sites of CP patients and from control sites of health patients were obtained for absolute quantification of A.a. by quantitative real-time polymerase chain reaction. For diseased sites, samples were collected before and 45 days after periodontal treatment. The IL4 but not the IL8 haplotypes were associated with levels of A.a. (in both periods). After periodontal treatment, higher levels of A.a. were found in subgingival sites of (IL4-) patients, and higher levels of IL-4 were associated with deeper probing pockets in these same patients. Significant correlations were found among genetic (patients carrying IL8 or IL4 haplotypes), microbiological and immunological data showing the interrelationship of different factors in the CP.

Elevated micronucleus frequency in patients with type 2 diabetes, dyslipidemia and periodontitis.

The over-production of reactive oxygen species (ROS) can cause oxidative damage to a large number of molecules, including DNA, and has been associated with the pathogenesis of several disorders, such as diabetes mellitus (DM), dyslipidemia and periodontitis (PD). We hypothesise that the presence of these diseases could proportionally increase the DNA damage. The aim of this study was to assess the micronucleus frequency (MNF), as a biomarker for DNA damage, in individuals with type 2 DM, dyslipidemia and PD. One hundred and fifty patients were divided into five groups based upon diabetic, dyslipidemic and periodontal status (Group 1 - poor controlled DM with dyslipidemia and PD; Group 2 - well-controlled DM with dyslipidemia and PD; Group 3 - without DM with dyslipidemia and PD; Group 4 - without DM, without dyslipidemia and with PD; and Group 5 - without DM, dyslipidemia and PD). Blood analyses were carried out for fasting plasma glucose, HbA1c and lipid profile. Periodontal examinations were performed, and venous blood was collected and processed for micronucleus (MN) assay. The frequency of micronuclei was evaluated by cell culture cytokinesis-block MN assay. The general characteristics of each group were described by the mean and standard deviation and the data were submitted to the Mann-Whitney, Kruskal-Wallis, Multiple Logistic Regression and Spearman tests. The Groups 1, 2 and 3 were similarly dyslipidemic presenting increased levels of total cholesterol, low density lipoprotein cholesterol and triglycerides. Periodontal tissue destruction and local inflammation were significantly more severe in diabetics, particularly in Group 1. Frequency of bi-nucleated cells with MN and MNF, as well as nucleoplasmic bridges, were significantly higher for poor controlled diabetics with dyslipidemia and PD in comparison with those systemically healthy, even after adjusting for age, and considering Bonferroni's correction. Elevated frequency of micronuclei was found in patients affected by type 2 diabetes, dyslipidemia and PD. This result suggests that these three pathologies occurring simultaneously promote an additional role to produce DNA impairment. In addition, the micronuclei assay was useful as a biomarker for DNA damage in individuals with chronic degenerative diseases.

Interleukin 4 haplotypes of susceptibility to chronic periodontitis are associated with IL-4 protein levels but not with clinical outcomes of periodontal therapy.

Different IL4 haplotypes were associated to susceptibility to/or protection against chronic periodontitis (CP). The aim of this study was to investigate if individuals carrying different haplotypes would present differences in clinical periodontal parameters and in the IL-4 levels at baseline, 45 and 90 days after non-surgical periodontal therapy. 62 patients were subdivided: genetically protected without CP (PH), genetically protected with CP (PCP), genetically susceptible with CP (SCP), genetically susceptible without CP (healthy) (SH). Clinical examination and gingival crevicular fluid (GCF) collection were performed for all patients, and IL-4 levels were measured by ELISA. At baseline, higher values for plaque index (PI, p = 0.013), gingival index (GI, p = 0.005) were observed for the SCP group in comparison to the PCP group but not after the completion of periodontal therapy. 45 and 90 days after the non-surgical therapy, PCP demonstrated significantly higher IL-4 levels than the SCP (p = 0.000002). Correlation analysis showed different results between clinical parameters and IL-4 production or GCF volume for groups with different genetic loads. The IL4 gene which was previously associated with susceptibility to CP was related with differences in the IL-4 protein levels in the GCF. However, independent of genetic carriage, individuals responded similarly to this therapy.

Pathogen levels and clinical response to periodontal treatment in patients with Interleukin 8 haplotypes.

The aim of this study was to investigate the effect of non-surgical treatment of periodontitis on the levels of periodontopathogens and clinical parameters in patients with different genetic backgrounds produced by polymorphisms in the Interleukin ( IL8) gene. Thirty patients grouped according to IL8 ATC/TTC or AGT/TTC haplotypes were submitted to non-surgical periodontal treatment. Levels of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola were determined in 240 subgingival plaque samples by qPCR. The association between IL8 haplotypes and the levels of periodontopathogens and clinical parameters was investigated by multilevel analysis accounting for the clustering of diseased sites analyzed within patients. It was observed that neither levels of periodontopathogens nor non-surgical treatment was associated with the IL8 haplotype. The clinical parameters after periodontal treatment were similar in diseased and healthy sites, independently of the IL8 haplotype. Nonetheless, in the same period, diseased sites of AGT/TTC patients harbored higher levels of P. gingivalis, T. denticola, T. forsythia, and red complex than those of ATC/TTC patients. However, the non-surgical periodontal therapy decreased the levels of these periodontopathogens and of the tested clinical parameters of diseased sites in both groups. Non-surgical therapy is equally effective in improving clinical parameters and decreasing the levels of periodontopathogens, independent of the genotype groups produced by the IL8 haplotype.

Haplotypes of susceptibility to chronic periodontitis in the Interleukin 8 gene do not influence protein level in the gingival crevicular fluid.

OBJECTIVE: Previously, we identified that the ATC/TTC haplotype formed by polymorphisms in the Interleukin-(IL)8 gene conferred susceptibility to chronic periodontitis (CP). The aim of the study was to investigate whether the IL8 haplotype ATC/TTC was associated with the volume of gingival crevicular fluid (GCF), the concentration of interleukin IL-8 in the GCF, as well as periodontal conditions in patients with CP in comparison to controls without CP. METHODS: Seventy-nine individuals (CP: n=41, controls: n=38) were grouped according to the presence (susceptible for CP) or absence (not susceptible for CP) of the IL8 ATC/TTC haplotype. After periodontal clinical evaluation, they were subdivided by the presence or absence of CP. GCF was collected from each patient and the IL-8 levels were determined by ELISA. The GCF volume of each subject was measured by means of a calibrated electronic device. Comparisons of means between carriers and non-carriers of the ATC/TTC haplotype were evaluated using the Mann-Whitney test. Linear regression and stepwise linear regression analysis were used to analyse the association of the GCF volume with potential covariates and their contribution for the phenotype. RESULTS: We did not find significant differences of both periodontal conditions and IL-8 concentration in the GCF of patients with the presence or absence of the IL8 ATC/TTC haplotype. However, the GCF volume was significantly higher amongst the patients affected by CP that are absent for the IL8 ATC/TTC haplotype. In addition, linear regression analysis showed a statistically significant association between GCF volume and CP, IL8 haplotype ATC/TTC and IL-8 concentration. CONCLUSIONS: The IL8 haplotype of susceptibility to CP was neither associated with IL-8 cytokine levels nor with clinical periodontal parameters. Also, CP, IL8 haplotype and IL-8 concentration showed a positive association with the GCF volume levels in the studied patients.

Haplotypes in the interleukin 8 gene and their association with chronic periodontitis susceptibility.

Interleukin-8 (IL-8), which is responsible for the migration and activation of neutrophils, is an important inflammatory mediator involved in the initiation and amplification of acute inflammatory reactions and chronic inflammatory processes. IL-8 plays an important role in periodontitis, an inflammatory disease characterized by the loss of connective tissue and alveolar bone. The aim of this study was to investigate whether the SNPs rs2227307 (+396) and rs2227306 (+781), and the haplotypes they formed together with the previously investigated rs4073 (-251), were associated with chronic periodontitis susceptibility. Clinical periodontal exams were performed and DNA samples were collected from 493 individuals (223 with periodontitis and 270 controls). Associations between SNPs, haplotypes, and subject phenotypes were analyzed using the χ(2) test followed by multivariate logistic regression modeling. We conclude that the +396TT genotype and the haplotypes ATC/TTC and AGT/TGC were significantly associated with chronic periodontitis susceptibility in Brazilians.