Expression Profile of Genes Potentially Associated with Adequate Glycemic Control in Patients with Type 2 Diabetes Mellitus.

Despite increasing research in type 2 diabetes mellitus (T2D), there are few studies showing the impact of the poor glycemic control on biological processes occurring in T2D. In order to identify potential genes related to poorly/well-controlled patients with T2D, our strategy of investigation included a primary screen by microarray (Human Genome U133) in a small group of individuals followed by an independent validation in a greater group using RT-qPCR. Ninety patients were divided as follows: poorly controlled T2D (G1), well-controlled T2D (G2), and normoglycemic individuals (G3). After using affy package in R, differentially expressed genes (DEGs) were prospected as candidate genes potentially relevant for the glycemic control in T2D patients. After validation by RT-qPCR, the obtained DEGs were as follows-G1 + G2 versus G3: HLA-DQA1, SOS1, and BRCA2; G2 versus G1: ENO2, VAMP2, CCND3, CEBPD, LGALS12, AGBL5, MAP2K5, and PPAP2B; G2 versus G3: HLA-DQB1, MCM4, and SEC13; and G1 versus G3: PPIC. This demonstrated a systemic exacerbation of the gene expression related to immune response in T2D patients. Moreover, genes related to lipid metabolisms and DNA replication/repair were influenced by the glycemic control. In conclusion, this study pointed out candidate genes potentially associated with adequate glycemic control in T2D patients, contributing to the knowledge of how the glycemic control could systemically influence gene expression.

Dyslipidemia rather than Type 2 Diabetes Mellitus or Chronic Periodontitis Affects the Systemic Expression of Pro- and Anti-Inflammatory Genes.

A high percentage of type 2 diabetes mellitus (T2D) patients are also affected by dyslipidemia and chronic periodontitis (CP), but no studies have determined the gene expression in patients that are simultaneously affected by all three diseases. We investigated the systemic expression of immune-related genes in T2D, dyslipidemia, and CP patients. One hundred and fifty patients were separated into five groups containing 30 individuals each: (G1) poorly controlled T2D with dyslipidemia and CP; (G2) well-controlled T2D with dyslipidemia and CP; (G3) normoglycemic individuals with dyslipidemia and CP; (G4) healthy individuals with CP; (G5) systemic and periodontally healthy individuals. Blood analyses of lipid and glycemic profiles were carried out. The expression of genes, including IL10, JAK1, STAT3, SOCS3, IP10, ICAM1, IFNA, IFNG, STAT1, and IRF1, was investigated by RT-qPCR. Patients with dyslipidemia demonstrated statistically higher expression of the IL10 and IFNA genes, while IFNG, IP10, IRF1, JAK1, and STAT3 were lower in comparison with nondyslipidemic patients. Anti-inflammatory genes, such as IL10, positively correlated with parameters of glucose, lipid, and periodontal profiles, while proinflammatory genes, such as IFNG, were negatively correlated with these parameters. We conclude that dyslipidemia appears to be the primary disease that is associated with gene expression of immune-related genes, while parameters of T2D and CP were correlated with the expression of these important immune genes.

Diabetes and increased lipid peroxidation are associated with systemic inflammation even in well-controlled patients.

BACKGROUND: The effect of the interaction between type 2 diabetes and dyslipidemia on inflammation and lipid peroxidation (LPO) has not been assessed. AIM: To investigate whether diabetes coupled with dyslipidemia alters oxidative metabolism leading to increased LPO products and inflammatory status. METHODS: 100 patients were divided into four groups based upon diabetic and dyslipidemic status: poorly controlled diabetes with dyslipidemia (DM-PC/D), well-controlled diabetes with dyslipidemia (DM-WC/D), normoglycemic individuals with dyslipidemia (NG/D), and normoglycemic individuals without dyslipidemia (NG/ND). Plasma was evaluated for an LPO product (MDA), antioxidant levels and inflammatory cytokines. RESULTS: Diabetics presented significantly higher levels of LPO (p<0.05) and the DM-PC/D had higher levels of proinflammatory cytokines and MDA in the plasma in comparison with normoglycemics (p<0.05). Interestingly IL1-ß, IL-6, and TNF-α in DM-WC/D were not statistically different from those in DM-PC/D. Normoglycemic individuals with dyslipidemia presented significantly increased levels of IL-6 and TNF-α when compared to normoglycemic without dyslipidemia (p<0.05). MDA levels were also positively correlated with the presence of DM complications (r=0.42, p<0.01). CONCLUSIONS: These findings show that dyslipidemia is associated with an increased inflammatory status, even in well-controlled diabetics and in normoglycemics. Our results suggest that lipid metabolism and peroxidation are important for the development of inflammation, which is elevated in several complications associated with diabetes.

Avaliação da expressão gênica e de lesões no DNA de índividuos portadores de diabetes mellitus tipo 2, dislipidemia e periodontite crônica / Gene expression and DNA damage in individuals with type 2 diabetes mellitus, dyslipidemia and chronic periodontitis

O objetivo deste estudo foi avaliar a expressão gênica de indivíduos portadores de diabetes mellitus tipo 2 (DM2, compensados e não compensados metabolicamente), dislipidemia e/ou periodontite crônica, e avaliar se tais alterações metabólicas apresentam efeito mutagênico. Cento e cinquenta pacientes, divididos em 5 grupos (grupo 1 - diabetes descompensado, com dislipidemia e com doença periodontal; grupo 2 - diabetes compensado, com dislipidemia e com doença periodontal; grupo 3 - sem diabetes, com dislipidemia e com doença periodontal; grupo 4 - sem diabetes, sem dislipidemia e com doença periodontal; e o grupo 5 - sem diabetes, sem dislipidemia e sem doença periodontal), foram avaliados quanto ao exame periodontal completo, exame físico e avaliação laboratorial da glicemia de jejum e perfil lipídico. De cada paciente foi coletado sangue para investigar a expressão gênica e as lesões no DNA. A avaliação da expressão gênica foi realizada por microarray e validada por RT-qPCR (Transcrição Reversa seguida de Reação em Cadeia da Polimerase em Tempo Real, ou quantitativo). As lesões no DNA foram avaliadas por meio do teste do micronúcleo. Os dados foram submetidos à análise bioinformática e estatística. Para verificar os resultados obtidos pelo microarray, os Grupos 1, 2 e 3 foram submetidos a comparações por pares. As análises de RT-qPCR confirmaram a expressão diferencial dos gene HLA-QA1, PDCD6, TRDV3, PPAP2B, HLA-DQB1, RIN3, VCAN, PPIC e SLC6A13. As frequências de micronúcleos foram significativamente mais elevadas em pacientes afetados por pelo menos uma das doenças sistêmicas, em comparação com aqueles sistemicamente saudáveis. Concluímos que foram identificados genes diferencialmente expressos em indivíduos portadores de DM2 também afetados por dislipidemia e periodontite crônica. Os resultados do micronúcleo confirmaram ser este teste útil como biomarcador para lesões no DNA, e demonstraram que as três patologias, ocorrendo simultaneamente, promoveram um papel adicional na produção de danos no DNA

The aim of this study was to evaluate the gene expression in individuals with type 2 diabetes (T2D, poor and well-controlled diabetics), dyslipidemia, and/or chronic periodontitis, and to assess whether these metabolic changes have mutagenic effect. One hundred and fifty patients were divided into 5 groups (group 1 ­ poor controlled diabetes with dyslipidemia and periodontal disease; group 2 ­ well-controlled diabetes with dyslipidemia and periodontal disease; group 3 ­ without diabetes with dyslipidemia and periodontal disease; group 4 ­ without diabetes, without dyslipidemia and with periodontal disease; and group 5 ­ without diabetes, dyslipidemia and periodontal disease), and were assessed a complete periodontal and physical examination, and laboratory evaluation of fasting glucose and lipid profile. From each patient, blood was collected to investigate gene expression and DNA damage. The gene expression was evaluated by microarray analysis and validated by RTqPCR (Polymerase Chain Reaction Real-Time, or quantitative). The DNA damage was evaluated using the micronucleus test. Data were subjected to statistical and bioinformatics analyses. To verify the results obtained by microarray, the Groups 1, 2 and 3 were submitted to pair comparisons. The RT-qPCR analysis confirmed the differential expression of HLA-QA1, PDCD6, TRDV3, PPAP2B, HLA-DQB1, RIN3, VCAN, PPIC and SLC6A13 genes. Significantly higher micronuclei frequencies were found in patients affected by any of the systemic diseases in comparison with the ones systemically healthy. We concluded that differentially expressed genes were identified in individuals with type 2 diabetes, dyslipidemia and chronic periodontitis. The results of the micronucleus confirmed that this test is useful as biomarker for DNA damage, and demonstrated that the three pathologies occurring simultaneously promoted an additional role in the production of DNA damage

Quantitation of malondialdehyde in gingival crevicular fluid by a high-performance liquid chromatography-based method.

Lipid peroxidation (LPO) has been associated with periodontal disease, and the evaluation of malondialdehyde (MDA) in the gingival crevicular fluid (GCF), an inflammatory exudate from the surrounding tissue of the periodontium, may be useful to clarify the role of LPO in the pathogenesis of periodontal disease. We describe the validation of a method to measure MDA in the GCF using high-performance liquid chromatography. MDA calibration curves were prepared with phosphate-buffered solution spiked with increasing known concentrations of MDA. Healthy and diseased GCF samples were collected from the same patient to avoid interindividual variability. MDA response was linear in the range measured, and excellent agreement was observed between added and detected concentrations of MDA. Samples' intra- and interday coefficients of variation were below 6.3% and 12.4%, respectively. The limit of quantitation (signal/noise=5) was 0.03 µM. When the validated method was applied to the GCF, excellent agreement was observed in the MDA quantitation from healthy and diseased sites, and diseased sites presented more MDA than healthy sites (P<0.05). In this study, a validated method for MDA quantitation in GCF was established with satisfactory sensitivity, precision, and accuracy.

Doença periodontal em pacientes com Diabetes Mellitus:influência de polimorfismos genéticos? / Periodontal disease in patients with Diabetes Mellitus:influence of genetic polymorphisms?

Nos últimos anos, evidenciou-se que, em muitas patologias, há fatores que não causam diretamente a doença, mas podem modificá-la, tornando o quadro clínico mais grave e a progressão mais rápida. Observa-se um número crescente de estudos investigando a relação entre Diabetes Mellitus (DM) e Doença Periodontal (DP), com alguns enfoques sobre a influência de fatores genéticos nesse processo. Existem polimorfismos genéticos cuja expressão está associada a fatores de risco. Portanto, para que as variantes genéticas afetem a severidade da doença ou aincidência de forma significativa, os polimorfismos genéticos devem agir cumulativamente ou pela interação com outros fatores, como a presença do diabetes. O objetivo deste estudo foi verificar, por meio de revisão sistemática da literatura, a influência de polimorfismos genéticos no perfil inflamatório da DP em pacientes com diabetes. Foram utilizadas as bases de dados BIREME e PubMed com os termos Periodontitis ou Periodontal disease, Polymorphism e Diabetes Mellitus. Realizando-se um refinamento na pesquisa bibliográfica, foram selecionados cinco referênciasque relacionavam DP crônica com DM e polimorfismos em genes de citocinas, especialmente Interleucina 1 (IL1) e IL6. Estudos associaram a presença de polimorfismos em pacientes portadores de diabetes à maior concentração de citocinas pró-inflamatórias no fluido gengival, quando comparados a pacientes sem diabetes. Conclui-se que, para confirmar essa associação, é necessária a realização de estudos longitudinais, investigando um maior número de genes para compreender melhor as relações causa-efeito entre polimorfismos genéticos, DM e DP.

Currently it is clear that there are several factors that can act as modifiers of diseases, without causing them directly, but having the potential to make these conditions to progress faster and more severe. There is a growing number of studies investigating the relationship between Diabetes Mellitus (DM) and Periodontal Disease (PD), includingsome studies focusing on the influence of genetic factors in this process. The aim of this study was to verify through a literature review, the influence of genetic polymorphisms in the development of PD in patients with DM. PubMed and BIREME were used as databases and the terms Periodontitis or Periodontal Disease, Polymorphism, Diabetes Mellitus were searched. After a refinement in the literature, five studies were selected and they were related to chronic PD with DM and polymorphisms in cytokine genes, especially interleukin 1 (IL1) e IL6. Polymorphismswere associated with a higher concentration of pro-inflammatory cytokines in the gingival crevicular fluid of diabetic patients when compared to non-diabetic. In conclusion, it is necessary to confirm this association with longitudinal studies that must investigate a larger number of cytokine genes in order to understand the cause-effectrelationship between genetic polymorphisms, DM and PD.

Avaliação clínica, imunológica e da resposta ao tratamento periodontal não-cirúrgico em indivíduos com e sem haplótipos de suscetibilidade genética à periodontite crônica no gene interleucina 8 / Clinical and immunological evaluation and the response to non-surgical periodontal therapy in the individuals with and without haplotypes of genetic susceptibility to chronic periodontitis in the Interleukin 8 gene

A Doença Periodontal (DP) tem caráter multifatorial, com a influência de fatores como a presença de microrganismos periodontopatogênicos, suscetibilidade genética do hospedeiro, reação do sistema imune, hábito de fumar e presença de doenças sistêmicas. O tecido periodontal inflamado produz várias citocinas, dentre elas a interleucina 8 (IL-8). Estudos recentes realizados por este grupo investigaram polimorfismos no gene IL8 em indivíduos com e sem periodontite crônica, onde foram observados indivíduos com 2 vezes mais predisposição genética à DP. O objetivo do presente estudo foi testar a hipótese de que a maior suscetibilidade genética à periodontite crônica dada pelo haplótipo ATC/TTC no gene IL8 seria acompanhada por diferenças nos índices clínicos periodontais, nos níveis da citocina IL-8 no fluido sulcular (FS) e na resposta ao tratamento periodontal não-cirúrgico. Foram selecionados 79 indivíduos divididos quanto à presença ou não do referido haplótipo no gene IL8, de forma que os indivíduos "suscetíveis" e "não suscetíveis" foram subdivididos quanto à presença ou ausência da periodontite crônica. Os indivíduos selecionados foram submetidos a exame clínico periodontal e coletas de FS antes e após 45 dias de finalizado o tratamento periodontal não-cirúrgico. A citocina IL-8 foi quantificada por meio de teste imunoenzimático (ELISA). Como resultado verificou-se que, comparando indivíduos suscetíveis e não-suscetíveis à DP, não houve diferença estatisticamente significante tanto nos índices clínicos periodontais quanto na resposta ao tratamento periodontal realizado, sendo que apenas o volume do FS, no período baseline, apresentou diferença significativa. Assim, a suscetibilidade genética à DP previamente observada nesses indivíduos não influenciou os índices clínicos periodontais avaliados, nem os níveis de IL-8 no FS e nem na forma como os mesmos responderam ao tratamento periodontal. Mais estudos investigando um número maior de genes são necessários para a melhor compreensão do envolvimento do fator genético na DP e sua interação com outros fatores, como o imunológico, dado o caráter multifatorial da DP

Periodontal disease is a multifactorial disease which is influenced by factors such as the presence of periodontopathogenic microorganisms, the reaction of the immune system, smoking, genetic susceptibility and the occurrence of systemic diseases. The inflamed periodontal tissue produces several cytokines like Interleukin 8 (IL-8). Recent studies carried out by our research group investigated polymorphisms in the IL8 gene in patients with and without periodontitis, where it was found individuals with a genetic predisposition to periodontitis. The purpose of this study was to test the hypothesis that the genetic susceptibility to chronic periodontitis given by ATC/TTC haplotype in IL8 gene would be linked to differences in the clinical periodontal parameters, IL-8 cytokine levels in the gingival crevicular fluid (GCF) and the response to non-surgical periodontal therapy. Seventy-nine individuals were selected and grouped according to the presence or not of the haplotype given in the IL8 gene, so that the "susceptible" and "nonsusceptible" individuals were subdivided by the presence or absence of chronic periodontitis. The selected individuals were submitted to periodontal clinical exam and the collection of GCF was done before and 45 days after the non-surgical periodontal therapy. The IL-8 cytokine was quantified through an immunoenzymatic assay (ELISA). As a result, it was verified that, comparing susceptible and non-susceptible individuals to chronic periodontitis, there wasn't any statistically significant difference as in clinical periodontal parameters as in response to non-surgical periodontal therapy. However, the volume of GCF, at baseline, presented a significant difference. Thus, the genetic susceptibility to chronic periodontitis previously observed in these individuals influenced neither in the clinical periodontal parameters, nor in IL-8 cytokine levels in the GCF. The way these individuals responded to non-surgical periodontal therapy was not also influenced by the genetic susceptibility previously observed. Therefore, further studies investigating a greater number of genes are needed to understand the involvement of the genetic factor in the periodontal disease and its interaction with other factors, such as the reaction of the immune system, given the multifactorial character of periodontal disease

Detection of the single nucleotide polymorphism (rs2227307) in the human interleukin 8 gene using a different PCR-RFLP assay / Detecção do polimorfismo de base única (rs2227307) no gene interleucina 8 humano usando PCR-RFLP

Interleukin 8 (IL-8) is a chemokine that acts as a potent chemoattractant for neutrophils. Single nucleotide polymorphisms (SNPs) in the human IL8 gene have been investigated in many disease association studies. We have developed a different PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment of Length Polymorphism) assay for genotyping the SNP (rs2227307) in the IL8 gene. This method was used for typing 147 white healthy Brazilian individuals, whose DNA was obtained from buccal epithelial cells and extracted with phenol: chloroform: isoamyl alcohol. Genomic DNA was amplified by PCR using a conventional thermal cycler. The PCR products (573 bp) were submitted to RFLP reactions. The RFLP fragments were analyzed in a 4% agarose gel stained with ethidium bromide. The genotype distribution observed in this study was consistent with the assumption of Hardy-Weinberg equilibrium and was similar (p=0.30) to those reported for other white populations in the SNP Database of the National Center for Biotechnology Information (NCBI). Because the PCR-RFLP method presented here was efficient, low cost, reproducible and convenient for laboratories with a limited level of technology worldwide, it should be useful for genotyping in case-control association or population genetic studies.

A Interleucina 8 é uma quimiocina com potente ação quimioatrativa para neutrófilos. Polimorfismos de base única (SNPs) no gene humano IL8 têm sido investigados em vários estudos de associação com doenças. Um método diferente de PCR-RFLP para genotipagem do SNP (rs2227307) do gene IL8 foi desenvolvido pelo nosso grupo. Esse método foi utilizado para genotipar 147 indivíduos brasileiros brancos saudáveis que tiveram seu DNA obtido de células da mucosa oral e extraído com fenol:clorofórmio:álcool isoamílico. DNA genômico foi amplificado por PCR usando um termociclador convencional, e a seguir os produtos da PCR (573 pb) foram submetidos a reações de RFLP. Os produtos de RFLP foram analisados em gel de agarose 4% impregnado com brometo de etídio. A distribuição do genótipo observado neste estudo foi consistente com o equilíbrio de Hardy-Weinberg e foi similar (p=0,30) ao reportado em outras populações brancas no banco de dados de SNPs do National Center for Biotechnology Information (NCBI). Este novo método de PCR-RFLP apresentado neste estudo foi eficiente, de baixo custo, reprodutível e conveniente para laboratórios com tecnologia limitada, podendo ser útil para utilização em estudos de genética de populações ou de associação com doenças (tipo caso-controle).