type 2 diabetes mellitus ( t2 dm ) is the most common endocrine disorder and its incidence 
 is increasing worldwide . this condition is a serious public health concern due to the 
 need for lifelong care , premature death and the fact that it remains incurable . insulin resistance , whereby target tissues do 
 not respond to this hormone , is a characteristic of the disease 's first phase , when 
 there is often a corresponding hyperinsulinemia . this resistance comes together with inflammatory processes in the 
 development of diabetes chronic complications . among these complications , periodontal diseases ( pd ) are very 
 common and immune modulating factors are necessary for pathogen clearance , but also 
 contribute to host tissues damage , as those seen in pd . clues to the involvement of inflammation in diabetes date back to more than a century 
 ago . proinflammatory molecules as tumor necroses factor ( tnf)- , leptin , interleukin 
 ( il)-6 , resistin , monocyte chemoattractant protein-1 ( mcp-1 ) and visfatin , among others , 
 are expressed at high levels in activated macrophages and/or other cells . proinflammatory cytokines such as tnf- and il-1 activate jnk and ikk/nf-b through 
 classical receptor - mediated mechanisms which are also activated by pattern recognition 
 receptors , bound to substances as lipopolissacharide ( lps ) from gram negative bacteria . these include the toll like receptors ( tlrs ) and the receptor for advanced glycation end 
 products ( rage ) . prolonged hyperglycemia and the accompanying production of excess 
 amounts of advanced glycation end products ( ages ) can activate nf-b . jnk promotes 
 insulin resistance through the phosphorylation of serine residues in insulin receptor 
 signaling ( irs)-1 that negatively regulates normal signaling through the insulin 
 receptor / irs-1 axis . nf-b induces insulin resistance by promoting the expression of 
 numerous target genes as those for tnf- , il-6 , il-8 , mcp-1 , mip-1 , mip-2 , resistin , 
 icam-1 , vcam-1 . the inflammation associated with pd , characterized by elevated pro - inflammatory 
 cytokines , innate immune receptor expression , and cellular infiltrate is exacerbated in 
 patients with t2 dm where tlr-4 and rages play a significant role and contribute to 
 induce responses by oral epithelial cells . the local production of cytokines in response to periodontal bacteria and their products 
 results in higher serum concentrations of pro - inflammatory biomarkers and a poor glycemic control in t2 dm 
 patients is associated with a loss of mucosal barrier integrity and accumulation of 
 innate immune receptor ligands resulting in an exacerbation of ongoing 
 inflammation . thus , the adverse effect of periodontitis in t2 dm may be explained by an increase in 
 systemic inflammation , which contributes to insulin resistance . the interrelation between more severe pd and dm is established and there are 
 evidences on the importance of cytokine analysis in t2 dm evolution and therapy blockade . as chronic or recurring inflammation contributes to an aberrant continuation of acute 
 phase response and may also lead to further diabetes complications , such as micro and 
 macroangiopathy and impaired healing , it is suggested that periodontal 
 disease with increased inflammatory response at local and systemic levels may collaborate to insulin resistance 
 present on t2 dm pathogenesis . however , scientific evidence on the effects of chronic 
 periodontitis on diabetes mellitus remains inadequate and inconclusive . furthermore , whether periodontal therapy 
 may help to control serum levels of inflammatory cytokines still remains 
 controversial . while some 
 studies showed an effective improvement in periodontal parameters , circulating 
 inflammatory markers and glycemic control after periodontal treatment in patients with 
 t2 dm , others have shown that 
 these responses were inconsistent across individuals and not sustainable over 
 time . the elevated levels of systemic inflammatory mediators , such as il-6 , in obesity or 
 metabolic syndrome enhance the host response to periodontal pathogens , hence increasing 
 the chance of periodontal destruction . il-6 is a multifunctional cytokine produced by a 
 variety of cells including macrophages , neutrophils , and endothelial cells . the double 
 edge effects ( i.e. , pro- and anti - inflammatory ) of this molecule entail complexity in 
 investigating its role and , until now , no evidence from animal or human studies supports 
 the hypothetical model in which metabolic syndrome - induced il-6 increases the risk of 
 destructive periodontal disease . interleukin 8 ( il-8 ) is a chemokine important for recruiting neutrophils during healing 
 and its levels were shown to be tightly linked to increased susceptibility to 
 periodontitis . monocyte chemoattractant protein 1 ( mcp-1 ) is also thought to play an important role in 
 inflammatory processes . it has been implicated as a key factor in recruitment and 
 activation of peripheral blood leukocytes in atherosclerotic lesions and adipose tissue . elevated levels of circulating mcp-1 have been found in patients with t2 dm , and 
 experimental data have shown that this protein increases macrophage infiltration , 
 inflammation and insulin resistance in transgenic mice . some studies have evaluated 
 markers such as interleukin-6 ( il-6 ) , tumor necrosis factor - a ( tnf- ) , c - reactive 
 protein ( crp ) , and il-1 and related them to insulin resistance and glycemic control ; however , their results are still 
 conflicting . based on the evidences that il-6 , il-8 and mcp-1 are important key markers of immune 
 response , which have been considered as critical mediators of initiation , progression 
 and/or suppression of chronic periodontitis , and that no previous study had analyzed 
 this combination of mediators in groups with and without diabetes and periodontal 
 disease , this study aimed to evaluate serum levels of inflammatory markers , il-8 , il-6 
 and mcp-1 , in type 2 diabetic patients in the presence of chronic periodontitis . this study was conducted between january 2012 and march 2013 in full accordance with the 
 helsinki declaration of 1975 , as revised in 2000 . the study protocol and the informed 
 consent form were reviewed and approved by the biomedical sciences institute ethics 
 committee ( # 011/cep ) from the university of so paulo ( so paulo , brazil ) . t2 dm 
 individuals were recruited from the diabetes center at the federal university of so 
 paulo . non - diabetic patients were recruited from the school of dentistry , university of 
 so paulo . all 
 volunteers received full mouth periodontal clinical examination performed at six 
 sites per tooth ( excluding third molars ) from a calibrated examiner 
 ( hpca ) . the 
 presence of supragingival biofilm was recorded as plaque index ( pl ) , marginal gingival 
 bleeding was recorded as gingival bleeding ( gb ) ; bleeding on probing ( bp ) , probing depth 
 ( pd ) and attachment level ( al ) were also evaluated . the probe used was north caroline 
 ( unc-15 , hu - friedy , chicago , il , usa ) . pl , gb and bp were recorded as absent 
 ( 0 ) or present ( 1 ) . inclusion criteria were : age 35 years , confirmed diagnosis of type 
 2 diabetes over 3 years ( for individuals with t2 dm ) , generalized moderate to severe 
 chronic periodontitis ( american academy of periodontology , 1998 ) ( 30% of sites with pd 
 > 4 mm and bleeding on probing ) and 15 remaining teeth . pregnant women , smokers , 
 patients with body mass index ( bmi ) > 40 kg / m or who received periodontal 
 therapy , antibiotic or antiseptic therapy 6 months prior to the study were excluded . blood samples were obtained and glycated hemoglobin ( hba1c ) levels were determined for 
 all individuals at the clinical laboratory of the university of so paulo hospital . approximately 5 ml of blood were collected by venipuncture in untreated tubes ( bd 
 vacutainer rapid serum tube , becton dickinson co. , so paulo , sp , brazil ) . serum was 
 obtained by centrifugation at 2,500 g for 2 minutes , aliquoted and 
 stored at -80c until further analysis . quantitative measurement of il-6 , il-8 and mcp-1 
 was assayed by enzyme - linked immunosorbent assay ( human standard elisa development kit , 
 peprotech inc . , rocky hill , ct , usa ) and read out using a micro plate reader ( model 680 , 
 bio - rad laboratories inc . serum from each patient was tested in 
 triplicate , and a calibration curve was added to each plate . patients with t2 dm and periodontitis 
 were divided in two groups , as adequate glycemic control when hba1c < 8.0% ( dma+p , n = 
 10 ) and inadequate glycemic control when hba1c 8.0% ( dmi + p , n=10 ) . the other 3 groups 
 were composed of t2 dm individuals without periodontitis ( dm , n = 10 ) , non - diabetic with 
 periodontitis ( p , n=6 ) and non - diabetic without periodontitis ( h , n=6 ) . periodontal clinical data were analyzed using appropriate statistical software ( spss 
 version 17.0 for windows , spss inc . , data were analyzed by non - parametric statistical methods since data were 
 not normally distributed . tests were based on median values with variability 
 measures ( 25% and 75% quartiles ) . bop data , percentages of positive sites were obtained 
 per patient and , thereafter , median values were calculated for the groups . 
 for pd and al , measured by millimeters , percent frequency was first obtained per patient 
 and , thereafter , as a median value for the group . data from elisa were analyzed by kruskal - wallis and dunn 's multiple comparison test 
 ( prism 5.0 project , graph pad software inc . , la jolla , ca , usa ) . values were obtained 
 per patient and a median value was calculated for each group . a total of 546 patients were assessed for eligibility , and only 42 fulfilled the 
 inclusion criteria ( figure 1 ) . flow diagram of patients included in the study demographic and hba1c data from each group are described in table 1 . demographic characteristics and glycated level ( meanstandard deviation ) for 
 participants in each group dm = type 2 diabetes mellitus ; p = periodontitis ; bmi = body mass index . groups : h 
 ( dm - p- ) , p ( dm - p+ ) , dm ( dm+p- ) , dma+p ( dm+with adequate glycemic control p+ ) , 
 dmi+p ( dm+with inadequate glycemic control p+ ) 
 table 2 provides periodontal clinical parameters 
 for each group with periodontitis : dmi+p , dma+p and p. there were significant 
 differences between diabetic ( dmi+p and dma+p ) and non - diabetic ( p ) for pd ( p=0.04 ) and 
 al ( p = 0.01 ) values indicating a more severe disease among the diabetic patients . intergroup clinical 
 parameters comparison for dmi + p and dma+p showed no significant differences ( data not 
 shown ) . medians and quartiles ( 25 - 75% ) of clinical parameters for the dma+p , dmi+p and p 
 groups statistically significance difference between dma+p;dmi+p and p 
 ( kruskall - wallis test and tukey test ; =5% ) . gb= gingival bleeding index ; 
 pl = visible plaque index ; bop = bleeding on probing ; pd = probing depth ; 
 al = attachment level there were no differences between groups for il-6 ( p=0.6351 ) , il-8 ( p=0.9460 ) and mcp-1 
 ( p=0.2987 ) sera leveis ( figure 2 ) . il-6 ( a ) , il-8 ( b ) and mcp-1 ( c ) serum levels in patients without periodontitis 
 and t2 dm ( h ) , patients with periodontitis and without t2 dm ( p ) , patients with t2 dm 
 and without periodontitis ( dm ) , patients with t2 dm with adequate glycemic control 
 and periodontitis ( dma+p ) and patients with t2 dm with inadequate glycemic control 
 and periodontitis ( dmi+p ) . mcp-1=monocyte chemoattractant protein-1 ; 
 il-6=interleukin 6 ; il-8=interleukin 8 ; t2dm = type 2 diabetes mellitus . the present study evaluated clinical and inflammatory parameters among groups with and 
 without t2 dm and/or periodontitis . t2 dm patients presented significantly higher pd and al values when compared to group p , 
 without diabetes and with periodontitis . this result is consistent with a number of 
 cross - sectional and longitudinal studies which had shown that the increased severity of pd in 
 t2 dm patients is due to an interorgan crosstalk under inflammatory conditions . the positive correlation between glycemic control and severity of periodontal disease 
 has been reported in the literature . thus , we had expected to find a significant relationship between 
 hba1c levels ( representing adequate - hba1c < 8.0% - and inadequate - hba1c 8.0% - 
 glycemic control ) and periodontitis severity ( in terms of increased pd and bop ) . however , the periodontal clinical parameters data did not differ according to the 
 diabetes control , corresponding to dma+p and dmi+p groups , respectively . we can 
 speculate that , in order to observe correlation , we would need to consider a stricter 
 glycemic control of individuals with t2 dm , that is , hba1c between 6.5 and 7.0% , which 
 would hamper even more the group selection . besides , hba1c refers to glycemic control in 
 the last 2 or 3 months and we do not know how the chronic glycemic control in these 
 individuals was . we also have to point out that hyperglycemia is only one factor of the 
 multifactorial process of periodontal disease . the role of 
 locally produced il-6 in the periodontitis pathogenesis has been demonstrated , since high levels of this cytokine were detected in symptomatic large 
 lesions rather than in asymptomatic small lesions . this cytokine is overexpressed in sites with pd mainly in 
 patients without diabetes and patients with well - controlled t2 dm when compared with 
 patients with poorly controlled diabetes . the effect of periodontal treatment on il-6 levels showed 
 conflicting data , yielding a tendency toward a decrease or increase in 
 serum levels in t2 dm patients . furthermore , a controversial study suggested an increase or no change in serum il-6 
 levels in obesity and metabolic syndrome . it should be noticed that t2 dm individuals evaluated in the present 
 study were under treatment with insulin , which may have induced low levels of 
 inflammatory biomarkers such as il-6 , counteracting the high levels induced 
 by periodontal inflammation . in the present study , there were no significant differences in the sera levels of the 
 studied inflammatory markers , mcp-1 and il-8 , among the groups , although previous data 
 demonstrated that both were overexpressed in the gingival tissue with chronic 
 periodontitis . a search in the 
 literature revealed no study in humans regarding t2 dm and/ or periodontitis and mcp-1 
 serum or gingival fluid levels . in rats experimental models , mcp-1 gingival crevicular 
 fluid concentrations did not significantly differ between groups with diabetes and 
 periodontitis and its levels were higher than in control healthy group . high levels of il-8 were shown in the gingival fluid of subjects with periodontal 
 disease . however , subjects with t2 dm presented significantly lower levels of il-8 in the 
 gcf when compared to healthy ones and there was no correlation between il-8 gingival 
 levels and hba1c level . furthermore , il-8 levels in serum 
 tended to decrease after periodontal therapy in t2 dm patients . due to the use of restrictive inclusion and exclusion criteria , in an attempt to 
 minimize the occurrence of confounding factors , however , it is interesting to consider that some studies evaluated only 
 specific dental sites through gingival crevicular fluid analysis , whereas the serum 
 analysis provides a more valuable analysis of the inflammatory markers in the subject . 
 to the best of our knowledge , there have been no studies evaluating these serum markers 
 under all different conditions analyzed in this study . nevertheless , this study confirms the higher severity of periodontal disease in 
 individuals with diabetes , highlighting the need for special oral healthcare for these 
 patients . this study pointed out the great variability of the serum inflammatory 
 biomarkers under all systemic and periodontal conditions studied . we can suggest that 
 follow - up studies with stricter glycemic control and larger samples should be conducted 
 in order to investigate the cumulative influence of periodontal conditions on serum 
 inflammatory biomarkers in individuals with t2 dm . although periodontitis was more severe in patients with diabetes , the serum levels of 
 the investigated inflammatory markers were not different among the periodontitis 
 patients .