Salivary Outer Membrane Vesicles and DNA Methylation of Small Extracellular Vesicles as Biomarkers for Periodontal Status: A Pilot Study

Periodontitis is an inflammatory disease, associated with a microbial dysbiosis. Early detection using salivary small extracellular vesicles (sEVs) biomarkers may facilitate timely prevention. sEVs derived from different species (i.e., humans, bacteria) are expected to circulate in saliva. This pilot study recruited 22 participants (seven periodontal healthy, seven gingivitis and eight periodontitis) and salivary sEVs were isolated using the size-exclusion chromatography (SEC) method. The healthy, gingivitis and periodontitis groups were compared in terms of salivary sEVs in the CD9+ sEV subpopulation, Gram-negative bacteria-enriched lipopolysaccharide (LPS+) outer membrane vesicles (OMVs) and global DNA methylation pattern of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and N6-Methyladenosine (m6dA). It was found that LPS+ OMVs, global 5mC methylation and four periodontal pathogens (T. denticola, E. corrodens, P. gingivalis and F. nucleatum) that secreted OMVs were significantly increased in periodontitis sEVs compared to those from healthy groups. These differences were more pronounced in sEVs than the whole saliva and were more superior in distinguishing periodontitis than gingivitis, in comparison to healthy patients. Of note, global 5mC hypermethylation in salivary sEVs can distinguish periodontitis patients from both healthy controls and gingivitis patients with high sensitivity and specificity (AUC = 1). The research findings suggest that assessing global sEV methylation may be a useful biomarker for periodontitis.     

Regulation of Anti-Apoptotic SOD2 and BIRC3 in Periodontal Cells and Tissues

The aim of the study was to clarify whether orthodontic forces and periodontitis interact with respect to the anti-apoptotic molecules superoxide dismutase 2 (SOD2) and baculoviral IAP repeat-containing protein 3 (BIRC3). SOD2, BIRC3, and the apoptotic markers caspases 3 (CASP3) and 9 (CASP9) were analyzed in gingiva from periodontally healthy and periodontitis subjects by real-time PCR and immunohistochemistry. SOD2 and BIRC3 were also studied in gingiva from rats with experimental periodontitis and/or orthodontic tooth movement. Additionally, SOD2 and BIRC3 levels were examined in human periodontal fibroblasts incubated with Fusobacterium nucleatum and/or subjected to mechanical forces. Gingiva from periodontitis patients showed significantly higher SOD2, BIRC3, CASP3, and CASP9 levels than periodontally healthy gingiva. SOD2 and BIRC3 expressions were also significantly increased in the gingiva from rats with experimental periodontitis, but the upregulation of both molecules was significantly diminished in the concomitant presence of orthodontic tooth movement. In vitro, SOD2 and BIRC3 levels were significantly increased by F. nucleatum, but this stimulatory effect was also significantly inhibited by mechanical forces. Our study suggests that SOD2 and BIRC3 are produced in periodontal infection as a protective mechanism against exaggerated apoptosis. In the concomitant presence of orthodontic forces, this protective anti-apoptotic mechanism may get lost.     

Apoptosis Signaling Is Altered in CD4(+)CD25(+)FoxP3(+) T Regulatory Lymphocytes in Pre-Eclampsia

The aim of our study was to estimate the surface expressions of CD95 (APO-1/Fas) antigen and the intracellular expressions of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in CD4(+)CD25(+)FoxP3(+) T regulatory lymphocytes (Tregs) as well as the percentage of CD8(+)CD28(+) T cytotoxic cells in peripheral blood of patients with pre-eclampsia in comparison with healthy pregnant women in the third trimester of physiological pregnancy. Twenty-four women with pre-eclampsia and 20 normal third trimester pregnant women were included in the study. The lymphocytes were isolated from peripheral blood samples and labeled with monoclonal antibodies. The expressions of surface antigens and intracellular proteins were estimated using flow cytometry. The population of CD4(+)CD25(+)FoxP3(+) Treg cells was significantly lower in peripheral blood of patients with pre-eclampsia when compared to normal third trimester pregnant women. The percentages of CD4(+)CD25(+)FoxP3(+) Treg cells that express Bcl-2 protein were significantly lower in peripheral blood of patients with pre-eclampsia when compared to healthy pregnant women, whereas the percentages of CD4(+)CD25(+)FoxP3(+) Treg cells with the expressions of Bax protein did not differ in both groups. Moreover, the mean fluorescence intensity (MFI) of Bcl-2 protein in CD4(+)CD25(+)FoxP3(+) Treg cells was significantly lower and MFI of Bax protein significantly higher in pre-eclampsia when compared to the control group. The percentage of CD8(+)CD28(+) T cells did not differ in both studied groups but MFI of CD28 antigen on T CD8(+) cells was significantly higher in pre-eclampsia when compared to the control group. The obtained results suggest that the deficit of CD4(+)CD25(+)FoxP3(+) Treg lymphocytes which is observed in pre-eclampsia may be associated with altered apoptosis signaling in Tregs.     

Salivary Proteome Changes in Response to Acute Psychological Stress Due to an Oral Exam Simulation in University Students: Effect of an Olfactory Stimulus

The autonomic nervous system (ANS) plays a crucial role both in acute and chronic psychological stress eliciting changes in many local and systemic physiological and biochemical processes. Salivary secretion is also regulated by ANS. In this study, we explored salivary proteome changes produced in thirty-eight University students by a test stress, which simulated an oral exam. Students underwent a relaxation phase followed by the stress test during which an electrocardiogram was recorded. To evaluate the effect of an olfactory stimulus, half of the students were exposed to a pleasant odor diffused in the room throughout the whole session. Saliva samples were collected after the relaxation phase (T0) and the stress test (T1). State anxiety was also evaluated at T0 and T1. Salivary proteins were separated by two-dimensional electrophoresis, and patterns at different times were compared. Spots differentially expressed were trypsin digested and identified by mass spectrometry. Western blot analysis was used to validate proteomic results. Anxiety scores and heart rate changes indicated that the fake exam induced anxiety. Significant changes of α-amylase, polymeric immunoglobulin receptor (PIGR), and immunoglobulin α chain (IGHA) secretion were observed after the stress test was performed in the two conditions. Moreover, the presence of pleasant odor reduced the acute social stress affecting salivary proteome changes. Therefore, saliva proteomic analysis was a useful approach to evaluate the rapid responses associated to an acute stress test also highlighting known biomarkers.     

Thrombopoietin Contributes to Enhanced Platelet Activation in Patients with Type 1 Diabetes Mellitus

Atherosclerotic cardiovascular disease is the major cause of morbidity and mortality in patients with type 1 diabetes mellitus (T1DM). Enhanced platelet reactivity is considered a main determinant of the increased atherothrombotic risk of diabetic patients. Thrombopoietin (THPO), a humoral growth factor able to stimulate megakaryocyte proliferation and differentiation, also modulates the response of mature platelets by enhancing both activation and binding to leukocytes in response to different agonists. Increased THPO levels have been reported in different clinical conditions characterized by a generalized pro-thrombotic state, from acute coronary syndromes to sepsis/septic shock, and associated with elevated indices of platelet activation. To investigate the potential contribution of elevated THPO levels in platelet activation in T1DM patients, we studied 28 T1DM patients and 28 healthy subjects. We measured plasma levels of THPO, as well as platelet-leukocyte binding, P-selectin, and THPO receptor (THPOR) platelet expression. The priming activity of plasma from diabetic patients or healthy subjects on platelet–leukocyte binding and the role of THPO on this effect was also studied in vitro. T1DM patients had higher circulating THPO levels and increased platelet–monocyte and platelet–granulocyte binding, as well as platelet P-selectin expression, compared to healthy subjects, whereas platelet expression of THPOR did not differ between the two groups. THPO concentrations correlated with platelet–leukocyte binding, as well as with fasting glucose and Hb1Ac. In vitro, plasma from diabetic patients, but not from healthy subjects, primed platelet–leukocyte binding and platelet P-selectin expression. Blocking THPO biological activity using a specific inhibitor prevented the priming effect induced by plasma from diabetic patients. In conclusion, augmented THPO may enhance platelet activation in patients with T1DM, potentially participating in increasing atherosclerotic risk.     

Exosome Degeneration in Mesenchymal Stem Cells Derived from Patients with Type 1 Diabetes Mellitus

Type 1 diabetes mellitus is characterized by the destruction of pancreatic β-cells and requires the regeneration of these destroyed pancreatic β-cells for radical treatment. The degeneration of organelles in stem cells compromises stem cell quality; however, organelles in the mesenchymal stem cells of patients with type 1 diabetes mellitus have not been characterized previously. In this study, we use transmission electron microscopy to evaluate the degeneration of organelles in adipose-derived stem cells of patients with type 1 diabetes mellitus (T1DM ADSCs). Compared to adipose-derived stem cells from healthy humans, T1DM ADSCs degenerate differently, characterized by prominent enlarged spherical vesicles. The exosomes of T1DM ADSCs are found to be enlarged, reduced in number, and increased in the percentage of those positive for tetraspanin CD9. The findings of this study provide insight into the characteristics of stem cells in patients with type 1 diabetes mellitus.     

High Dietary Cholesterol Masks Type 2 Diabetes-Induced Osteopenia and Changes in Bone Microstructure in Rats

Type 2 diabetes mellitus (T2DM) often occurs concurrently with high blood cholesterol or dyslipidemia. Although T2DM has been hypothesized to impair bone microstructure, several investigations showed that, when compared to age-matched healthy individuals, T2DM patients had normal or relatively high bone mineral density (BMD). Since cholesterol and lipids profoundly affect the function of osteoblasts and osteoclasts, it might be cholesterol that obscured the changes in BMD and bone microstructure in T2DM. The present study, therefore, aimed to determine bone elongation, epiphyseal histology, and bone microstructure in non-obese T2DM Goto-Kakizaki rats treated with normal (GK-ND) and high cholesterol diet. We found that volumetric BMD was lower in GK-ND rats than the age-matched wild-type controls. In histomorphometric study of tibial metaphysis, T2DM evidently suppressed osteoblast function as indicated by decreases in osteoblast surface, mineral apposition rate, and bone formation rate in GK-ND rats. Meanwhile, the osteoclast surface and eroded surface were increased in GK-ND rats, thus suggesting an activation of bone resorption. T2DM also impaired bone elongation, presumably by retaining the chondrogenic precursor cells in the epiphyseal resting zone. Interestingly, several bone changes in GK rats (e.g., increased osteoclast surface) disappeared after high cholesterol treatment as compared to wild-type rats fed high cholesterol diet. In conclusion, high cholesterol diet was capable of masking the T2DM-induced osteopenia and changes in several histomorphometric parameters that indicated bone microstructural defect. Cholesterol thus explained, in part, why a decrease in BMD was not observed in T2DM, and hence delayed diagnosis of the T2DM-associated bone disease.     

Liver-Specific Overexpression of Prostasin Attenuates High-Fat Diet-Induced Metabolic Dysregulation in Mice

The liver has a most indispensable role in glucose and lipid metabolism where we see some of the most serious worldwide health problems. The serine protease prostasin (PRSS8) cleaves toll-like receptor 4 (TLR4) and regulates hepatic insulin sensitivity under PRSS8 knockout condition. However, liver substrate proteins of PRSS8 other than TLR4 and the effect to glucose and lipid metabolism remain unclarified with hepatic elevation of PRSS8 expression. Here we show that high-fat-diet-fed liver-specific PRSS8 transgenic mice improved glucose tolerance and hepatic steatosis independent of body weight. PRSS8 amplified extracellular signal-regulated kinase phosphorylation associated with matrix metalloproteinase 14 activation in vivo and in vitro. Moreover, in humans, serum PRSS8 levels reduced more in type 2 diabetes mellitus (T2DM) patients than healthy controls and were lower in T2DM patients with increased maximum carotid artery intima media thickness (>1.1 mm). These results identify the regulatory mechanisms of PRSS8 overexpression over glucose and lipid metabolism, as well as excessive hepatic fat storage.     

Repositioning the Role of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) on the TRAIL to the Development of Diabetes Mellitus: An Update of Experimental and Clinical Evidence

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF protein superfamily, represents a multifaceted cytokine with unique biological features including both proapoptotic and pro-survival effects in different cell types depending on receptor interactions and local stimuli. Beyond its extensively studied anti-tumor and immunomodulatory properties, a growing body of experimental and clinical evidence over the past two decades suggests a protective role of TRAIL in the development of type 1 (T1DM) and type 2 (T2DM) diabetes mellitus. This evidence can be briefly summarized by the following observations: (i) acceleration and exacerbation of T1DM and T2DM by TRAIL blockade or genetic deficiency in animal models, (ii) prevention and amelioration of T1DM and T2DM with recombinant TRAIL treatment or systemic TRAIL gene delivery in animal models, (iii) significantly reduced circulating soluble TRAIL levels in patients with T1DM and T2DM both at disease onset and in more advanced stages of diabetes-related complications such as cardiovascular disease and diabetic nephropathy, (iv) increase of serum TRAIL levels in diabetic patients after initiation of antidiabetic treatment and metabolic improvement. To explore the underlying mechanisms and provide mechanistic links between TRAIL and diabetes, a number of animal and in vitro studies have reported direct effects of TRAIL on several tissues involved in diabetes pathophysiology such as pancreatic islets, skeletal muscle, adipose tissue, liver, kidney, and immune and vascular cells. Residual controversy remains regarding the effects of TRAIL on adipose tissue homeostasis. Although the existing evidence is encouraging and paves the way for investigating TRAIL-related interventions in diabetic patients with cardiometabolic abnormalities, caution is warranted in the extrapolation of animal and in vitro data to the clinical setting, and further research in humans is imperative in order to uncover all aspects of the TRAIL-diabetes relationship and delineate its therapeutic implications in metabolic disease.     

Salivary Biomarkers in Patients with Sjögren’s Syndrome—A Systematic Review

Sjögren’s syndrome (SS) is a chronic autoimmune disease characterized by dry mouth and dry eyes, with lymphocytic infiltration of the exocrine glands. Saliva is becoming a useful tool to determine the clinical and pathological characteristics of SS because the collection method is easy and non-invasive. Since 1900, salivary proteomic analysis has been performed continuously using a variety of optimized analytical methods. Many studies have identified distinct characteristics of salivary proteins in patients with primary SS, and the changes were related to chronic inflammation and overproduction of immunoglobulins or downregulated secretory function. Several proteomic studies using whole or parotid saliva have evaluated whether several salivary proteins can be used to discriminate SS, including salivary β2-microglobulin, calprotectin, carbonic anhydrase VI, neutrophil gelatinase-associated lipocalin, sialic acid-binding immunoglobulin-like lectin-5, and tripartite motif-containing protein 29. In addition, salivary proinflammatory cytokine levels have been reported to be increased in patients with SS. Although these candidate salivary proteins have exhibited considerable differences in patients with SS, more data are needed to confirm their role as biomarkers. Moreover, the identification of salivary characteristics that can accurately reflect disease activity, predict treatment response and prognosis, and diagnose SS is anticipated.     

Detection of differential levels of proteins in the urine of patients with endometrial cancer: analysis using two-dimensional gel electrophoresis and o-glycan binding lectin

Cancers can cause some proteins to be aberrantly excreted or released in the urine, which can be used as biomarkers. To screen for potential biomarkers for endometrial cancer (ECa), the urinary proteins from patients who were newly diagnosed with early stage ECa and untreated controls were separated using two-dimensional gel electrophoresis (2-DE) and followed by image analysis. The altered levels of zinc alpha-2 glycoprotein, alpha 1-acid glycoprotein, and CD59 were detected in the patients compared to the controls. In addition, the urine of the ECa patients was also found to contain relatively lower levels of a fragment of nebulin when the 2-DE separated urinary proteins were probed using champedak galactose binding (CGB) lectin. The different levels of the nebulin fragment were further validated by subjecting the urinary protein samples to CGB lectin affinity chromatography and analysis of the bound fractions by LC-MS/MS. Our data is suggestive of the potential use of the differentially expressed urinary proteins as biomarkers for ECa although this requires further extensive validation on clinically representative populations.     

The Role of Circulating MicroRNA-126 (miR-126): A Novel Biomarker for Screening Prediabetes and Newly Diagnosed Type 2 Diabetes Mellitus

Recent studies suggested an association of endothelial microRNA-126 (miR-126) with type 2 diabetes mellitus (T2DM). In the current study, we examined whether circulating miR-126 is associated with T2DM and pre-diabetic syndrome. The study included 82 subjects with impaired glucose tolerance (IGT), 75 subjects with impaired fasting glucose (IFG), 160 patients with newly diagnosed T2DM, and 138 healthy individuals. Quantitative polymerase chain reaction (qPCR) was used to examine serum miR-126. Serum miR-126 was significantly lower in IGT/IFG subjects and T2DM patients than in healthy controls (p < 0.05). After six months of treatment (diet control and exercise in IGT/IFG subjects, insulin plus diet control and exercise in T2DM patients), serum miR-126 increased significantly (p < 0.05). An analysis based on serum miR-126 in the sample revealed a significantly higher odds ratio (OR) for the subjects with the lowest 1/3 of serum miR-126 for T2DM (OR: 3.500, 95% confidence interval: 1.901–6.445, p < 0.05) than subjects within the highest 1/3 of serum miR-126. Such an association was still apparent after adjusting for other major risk factors. The area under the curve (AUC) for the receiver-operating characteristic (ROC) analysis was 0.792 (95% confidence interval: 0.707–0.877, p < 0.001). These results encourage the use of serum miR-126 as a biomarker for pre-diabetes and diabetes mellitus, as well as therapeutic response.     

Early Signs of Atherogenic Features in the HDL Lipidomes of Normolipidemic Patients Newly Diagnosed with Type 2 Diabetes

Cardiovascular disease (CVD) is the major cause of death in patients with type-2 diabetes mellitus (T2DM), although the factors that accelerate atherosclerosis in these patients are poorly understood. The identification of the altered quantity and quality of lipoproteins, closely related to atherogenesis, is limited in routine to a pattern of high triglycerides and low HDL-cholesterol (HDL-C) and in research as dysfunctional HDLs. We used the emerging NMR-based lipidomic technology to investigate compositional features of the HDLs of healthy individuals with normal coronary arteries, drug-naïve; recently diagnosed T2DM patients with normal coronary arteries; and patients with recent acute coronary syndrome. Patients with T2DM and normal serum lipid profiles even at diagnosis presented significant lipid alterations in HDL, characterized by higher triglycerides, lysophosphatidylcholine and saturated fatty acids; and lower cholesterol, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, plasmalogens and polyunsaturated fatty acids, an atherogenic pattern that may be involved in the pathogenesis of atherosclerosis. These changes are qualitatively similar to those found, more profoundly, in normolipidemic patients with established Coronary Heart Disease (CHD). We also conclude that NMR-based lipidomics offer a novel holistic exploratory approach for identifying and quantifying lipid species in biological matrixes in physiological processes and disease states or in disease biomarker discovery.     

Role of Receptor Protein Tyrosine Phosphatases (RPTPs) in Insulin Signaling and Secretion

Changes in lifestyle in developed countries have triggered the prevalence of obesity and type 2 diabetes mellitus (T2DM) in the latest years. Consequently, these metabolic diseases associated to insulin resistance, and the morbidity associated with them, accounts for enormous costs for the health systems. The best way to face this problem is to identify potential therapeutic targets and/or early biomarkers to help in the treatment and in the early detection. In the insulin receptor signaling cascade, the activities of protein tyrosine kinases and phosphatases are coordinated, thus, protein tyrosine kinases amplify the insulin signaling response, whereas phosphatases are required for the regulation of the rate and duration of that response. The focus of this review is to summarize the impact of transmembrane receptor protein tyrosine phosphatase (RPTPs) in the insulin signaling cascade and secretion, and their implication in metabolic diseases such as obesity and T2DM.     

Activation of Peripheral Blood Mononuclear Cells and Leptin Secretion: New Potential Role of Interleukin-2 and High Mobility Group Box (HMGB)1

Activation of innate immunity and low-grade inflammation contributes to hyperglycemia and an onset of Type 2 Diabetes Mellitus (T2DM). Interleukin-2 (IL-2), leptin, High Mobility Group Box-1 (HMGB-1), and increased glucose concentrations are mediators of these processes also by modulating peripheral blood mononuclear cells (PBMCs) response. The aim of this study was to investigate if HMGB-1 and IL-2 turn on PBMCs and their leptin secretion. In isolated human PBMCs and their subpopulations from healthy individuals and naïve T2DM patients, leptin release, pro-inflammatory response and Toll-like Receptors (TLRs) activation was measured. After treatment with IL-2 and HMGB1, NK (Natural Killer) have the highest amount of leptin secretion, whilst NK-T have the maximal release in basal conditions. TLR4 (TAK242) and/or TLR2 (TLR2-IgA) inhibitors decreased leptin secretion after IL-2 and HMGB1 treatment. A further non-significant increase in leptin secretion was reported in PBMCs of naive T2DM patients in response to IL-2 and HMGB-1 stimulation. Finally, hyperglycemia or hyperinsulinemia might stimulate leptin secretion from PBMCs. The amount of leptin released from PBMCs after the different treatments was enough to stimulate the secretion of IL-1β from monocytes. Targeting leptin sera levels and secretion from PBMCs could represent a new therapeutic strategy to counteract metabolic diseases such as T2DM.