Preliminary findings on the possible role of B-lymphocyte stimulator (BLyS) on diabetes-related periodontitis

Abstract The possible role of B-cell growth and differentiation-related cytokines on the pathogenesis of diabetes-related periodontitis has not been addressed so far. The aim of this study was to evaluate the effects of diabetes mellitus (DM) on the gene expression of proliferation-inducing ligand (APRIL) and B-lymphocyte stimulator (BLyS), two major cytokines associated to survival, differentiation and maturation of B cells in biopsies from gingival tissue with periodontitis. Gingival biopsies were obtained from subjects with periodontitis (n = 17), with periodontitis and DM (n = 19) as well as from periodontally and systemically healthy controls (n = 10). Gene expressions for APRIL, BLyS, RANKL, OPG, TRAP and DC-STAMP were evaluated using qPCR. The expressions APRIL, BLyS, RANKL, OPG, TRAP and DC-STAMP were all higher in both periodontitis groups when compared to the control group (p < 0.05). Furthermore, the expressions of BLyS, TRAP and RANKL were significantly higher in the subjects with periodontitis and DM when compared to those with periodontitis alone (p < 0.05). The mRNA levels of BLyS correlated positively with RANKL in the subjects with periodontitis and DM (p < 0.05). BLyS is overexpressed in periodontitis tissues of subjects with type 2 DM, suggesting a possible role of this cytokine on the pathogenesis DM-related periodontitis.

Cytokine profile and lymphocyte subsets in type 2 diabetes

Type 2 diabetes mellitus (T2D) is a metabolic disease with inflammation as an important pathogenic background. However, the pattern of immune cell subsets and the cytokine profile associated with development of T2D are unclear. The objective of this study was to evaluate different components of the immune system in T2D patients' peripheral blood by quantifying the frequency of lymphocyte subsets and intracellular pro- and anti-inflammatory cytokine production by T cells. Clinical data and blood samples were collected from 22 men (51.6±6.3 years old) with T2D and 20 nonsmoking men (49.4±7.6 years old) who were matched for age and sex as control subjects. Glycated hemoglobin, high-sensitivity C-reactive protein concentrations, and the lipid profile were measured by a commercially available automated system. Frequencies of lymphocyte subsets in peripheral blood and intracellular production of interleukin (IL)-4, IL-10, IL-17, tumor necrosis factor-α, and interferon-γ cytokines by CD3+ T cells were assessed by flow cytometry. No differences were observed in the frequency of CD19+ B cells, CD3+CD8+ and CD3+CD4+ T cells, CD16+56+ NK cells, and CD4+CD25+Foxp3+ T regulatory cells in patients with T2D compared with controls. The numbers of IL-10- and IL-17-producing CD3+ T cells were significantly higher in patients with T2D than in controls (P<0.05). The frequency of interferon-γ-producing CD3+ T cells was positively correlated with body mass index (r=0.59; P=0.01). In conclusion, this study shows increased numbers of circulating IL-10- and IL-17-producing CD3+ T cells in patients with T2D, suggesting that these cytokines are involved in the immune pathology of this disease.

Islet amyloid polypeptide is not a target antigen for CD8+ T-cells in type 2 diabetes

Type 2 diabetes [T2D] is a chronic metabolic disorder in which beta-cells are destroyed. The islet amyloid polypeptide [IAPP] produced by beta-cells has been reported to influence beta-cell destruction. To evaluate if IAPP can act as an autoantigen and therefore, to see if CD8+ T-cells specific for this protein might be present in T2D patients. Peripheral blood mononuclear cells [PBMC] were obtained from human leukocyte antigen [HLA]-A2+ T2D patients and non-diabetic healthy subjects. Cells were then screened for peptide recognition using ELISPOT assay for the presence of IFN-gamma producing CD8+ T-cells against two HLA Class I-restricted epitopes derived from IAPP [IAPP5-13 and IAPP9-17] and common viral antigenic minimal epitopes Flu MP 58-66, CMV495-503, EBV280-288 and HIV77-85 as controls. A total of 36.4% of patients and 56.2% of healthy subjects showed a response against IAPP5-13 peptide. No significant difference in response against this peptide was noted between the patients and the healthy donors. With respect to peptide IAPP9-17, although healthy subjects showed a higher mean number of spot forming cells than the patients, the difference was not significant; 36.4% of patients and 37.5% of controls responded to this peptide. The response of healthy subjects to the common viral peptides was stronger than that of the patients, though the result was not significant. It is unlikely that IAPP would be a target for CD8+ T-cells in diabetic patients; however, the trend observed toward a lower response of T2D patients against IAPP and common viral peptides may imply a decreased immune response in these patients

Role of Innate Immunity in the Pathogenesis of Type 1 and Type 2 Diabetes

The importance of innate immunity in host defense is becoming clear after discovery of innate immune receptors such as Toll-like receptor or Nod-like receptor. Innate immune system plays an important role in diverse pathological situations such as autoimmune diseases. Role of innate immunity in the pathogenesis of metabolic disorders such as type 2 diabetes, metabolic syndrome or atherosclerosis that has not been previously considered as inflammatory disorders, is also being appreciated. Here, the role of innate immunity in the development of type 1 diabetes, a classical organ-specific autoimmune disease, and type 2 diabetes will be discussed, focusing on the role of specific innate immune receptors involved in these disease processes.

Serological tests for the diagnosis of active tuberculosis: relevance for India.

Diagnostic tests for active tuberculosis (TB) based on the detection of antibodies (serological tests) have been commercially available for decades, although no international guidelines have recommended their use. An estimated 1.5 million serological TB tests, mainly enzyme-linked immunosorbent assays, are performed in India alone every year, mostly in the private sector. The cost of serological tests in India is conservatively estimated at US $15 million (825 million) per year. Findings from systematic reviews on the diagnostic accuracy of serological tests for both pulmonary and extra-pulmonary TB suggest that these tests are inaccurate and imprecise. A cost-effectiveness modelling study suggests that, if used as a replacement test for sputum microscopy, serology would increase costs to the Indian TB control sector approximately 4-fold and result in fewer disability-adjusted life years averted and more false-positive diagnoses. After considering all available evidence, the World Health Organization issued a strong recommendation against the use of currently available commercial serological tests for the diagnosis of TB disease. The expanding evidence base continues to demonstrate that the harms/risks of serological tests far outweigh the benefits. Greater engagement of the private sector is needed to discontinue the use of serological tests and to replace these tests with WHO-endorsed new diagnostics in India. The recent ban on import or sale of TB serological tests by the Indian health ministry is a welcome step in the right direction.

Immunostimulant, cerebroprotective & nootropic activities of Andrographis paniculata leaves extract in normal & type 2 diabetic rats.

Background & objectives: A large number of plants have been recognized to be effective in the treatment of diabetes mellitus. Persistent hyperglycaemia is associated with decreased function of immune system and cerebral ischaemia mainly due to increased oxidative stress and inflammatory response. Andrographis paniculata is a medicinal plant widely used in folk medicine for various purposes. In this study the effect of chronic administration (7 days) of methanolic extract of A. paniculata leaves was studied in rats with experimentally induced diabetes, nootropic and immunostimulant activities were evaluated. The effect of acute administration of methanolic extract of A. paniculata leaves was also studied for cerebroprotective activity. Methods: Type 2 diabetes was induced in rats by streptozotocin (STZ) (65 mg/kg) + nicotinamide (150 mg/kg). Various biochemical parameters were estimated using standard methods. Results: A significant (P<0.05) increase in cognitive function was observed in both normal and type 2 diabetic rats. Nootropic activity in terms of per cent reduction in latency period was more in type 2 diabetic rats. A significant increase in blood lymphocyte count, splenic lymphocyte count and peritoneal macrophage count was observed in both normal and type 2 diabetic rats. Immunostimulant activity was observed more in type 2 diabetic rats. The per cent decrease in cerebral infarction was more in type 2 diabetic rats when compared to normal rats. The per cent increase in superoxide dismutase (SOD) levels was more in type 2 diabetic rats. Interpretation & conclusions: The antioxidant activity of the methanolic extract of A. paniculata leaves was evident by decreased tissue malondialdehyde (MDA) levels and increased SOD levels. These properties may be responsible for the observed cerebroprotective activity. The methanolic leaf extract of A. paniculata showed significant immunostimulant, cerebroprotective and nootropic activities in normal and type 2 diabetic rats.

T-cell cytokine production and endothelial dysfunction in type 2 diabetic patients with nephropathy

This study assessed changes in serum levels of cytokines IFNgamma and IL-10 [biomarkers of inflammatory changes] and soluble biomarkers sICAM-1 and sE-selectin [biomarkers of endothelial dysfunction] in diabetic patients with and without nephropathy. IFNgamma and IL-10 were significantly elevated in patients with diabetic nephropathy [DN] and end-stage renal disease [ESRD] compared with controls and diabetic patients without DN. IFNgamma and IL-10 levels were significantly increased after haemodialysis. sICAM-1 and sE-selectin were significantly higher in diabetic, DN and ESRD groups compared with controls, and sICAM-1 but not sE-selectin was increased after haemodialysis

Reduced interleukin-17 expression of Burkholderia pseudomallei-infected peripheral blood mononuclear cells of diabetic patients.

Burkholderia pseudomallei is the causative agent of melioidosis. One of the main risk factors for B. pseudomallei infection in endemic areas is diabetes mellitus. The present study investigated IL-17 mRNA and protein expression by peripheral blood mononuclear cells in response to B. pseudomallei infection in 10 diabetic patients in comparison to 10 healthy blood donors. The IL-17 expression in diabetic patients was significantly lower (p < 0.05) than in the controls. However, IL-23 mRNA expression of the 2 groups was comparable. The present findings suggest that melioidosis affects T cell IL-17 production and that patients with diabetes mellitus have a defective IL-17 production in response to this type of infection.

Advanced glycosylated end products-mediated activation of polymorphonuclear neutrophils in diabetes mellitus and associated oxidative stress.

Two important consequences of hyperglycemia in diabetes are development of oxidative stress and formation of advanced glycation end products (AGE) which are known to be associated with diabetic complications. Relationship between AGE formation and development of oxidative stress (OS) is yet to be established. In the present study, the involvement of AGE in PMN-mediated ROS generation and the associated OS were investigated in type 2 diabetic mellitus (DM) patients. We assessed OS parameters (serum MDA, FRAP and GSH), PMN oxidative functions (respiratory burst and superoxide production) and total serum AGE in 90 subjects divided equally in three groups--control group, Group I consisting of type 2 diabetic patients without microvascular complications and Group II consisting of type 2 diabetic patients with microvascular complications. PMNs isolated from both groups (I and II) exhibited higher level of respiratory burst (RB) and produced increased amount of superoxide anion as compared to the controls. The increase was more pronounced in diabetes with complications, as compared to those without. Serum malondialdehyde (MDA) level was elevated, whereas glutathione (GSH) and ferric reducing ability of plasma (FRAP) levels were significantly reduced in diabetes as compared to the controls, suggesting the presence of oxidative stress in DM. A positive correlation between PMN oxidative function and OS parameters suggested the involvement of PMN in the development of OS in DM. Serum AGE level was also elevated in diabetic groups as compared to the controls. Further, the positive correlation between serum AGE level and PMN oxidative function suggested the involvement of AGE in increased RB and generation of reactive oxygen species (ROS) by resting diabetic PMN. The results of the study indicate that AGE-PMN interaction possibly upregulates NADPH oxidase, leading to enhanced ROS generation and thus contributes to the pathogenesis in diabetes.

Immunoglobulin concentration in gingival tissue of type 2 diabetic patients with periodontitis.

BACKGROUND: Diabetes mellitus is considered as a risk factor for the initiation and progression of periodontal disease. The diabetic patients often exhibit decreased immune response and increased susceptibility to infection. In the present study, a quantitative estimation of the gingival tissue immunoglobulin concentrations in diabetic and non diabetic subjects with periodontitis was assessed and compared with that of clinically healthy gingiva. METHOD: 40 gingival tissue samples obtained from 20 diabetic (Type 2) and 20 non-diabetic subjects were subjected to quantitative estimation of immunoglobulins G, A, and M. The data thus obtained were compared to the level of immunoglobulin found in clinically healthy gingiva. RESULTS: The IgG and IgA level in the tissues of both diabetic and non-diabetic subjects with periodontitis were found to be significantly higher than that of healthy subjects. The diabetic group also showed a significantly higher IgG and IgA levels compared to the non-diabetic group with periodontitis. CONCLUSION: These findings support the concept that the humoral immune response plays an important role in the pathogenesis of periodontal disease in diabetics. The significantly higher levels of immunoglobulin in the gingival tissues might be a protective mechanism against the increased bacterial challenge in diabetic subjects.

Traditional islet cell autoantibodies in diabetic patients with and without long-term complications

The aim of the present work was to demonstrate the presence of the traditional islet cell related autoantibodies in the diabetic patients with and without long term complications and to identify relevant predisposing markers of pre-clinical diabetic complications. There was a significant difference [P 0.001] between the percentage of islet cell autoantibodies [ICA], glutamic acid decarboxylase autoantibodies [GAD-Ab], and insulin autoantibodies [IAA] positive subjects in the diabetic groups and their matched control and potential groups. Type-1 diabetic groups had a higher percentage [P.0.05] of subjects positive for ICA, GAD-Ab, and IAA than Type-2 diabetic groups. The concentration of ICA in the studied population strongly correlated with the duration of the disease [r=0.705, p 0.05]. There was no significant difference [P>0.05] between the percentage of islet cell antigen-2 autoantibodies [IA2-Ab] positive subjects in the different groups of diabetic population and their control. In conclusion the traditional islet cell antibodies have a role in the detection and development of diabetes especially Type-1 rather than the long-term complications. Other more specific autoantibodies and immune responses, which were not studied, may have a role in the etiology and pre-clinical appearance of these chronic complications. KeyWords: Diabetes,Autoantibodies, Complications

Flow cytometry for the analysis of T cells expressing CD69 after stimulation with glutamic acid decarboxylase.

Type 1 diabetes mellitus is a T-cell mediated autoimmune disease in which the insulin-producing pancreatic beta cells are selectively destroyed. We recently found that the detection of cell-mediated immune response to glutamic acid decarboxylase (GAD) was more useful than the detection of specific autoantibodies for the diagnosis of type 1 diabetes mellitus. In this study, we established a flow cytometric analysis for the detection of activated T cells in whole venous blood, obtained from diabetic patients and normal controls after stimulation by GAD. Two millitiers of peripheral venous blood and 6 hours incubation time were used for performing the test. It was found that 33% (3/9) type 1 diabetic patients, 7.7% (1/13) type 2 diabetic patients and neither patients with fibrocalculous pancreatopathy nor normal controls had > or = 20% CD8+ T cells expressing CD69. The results suggest that flow cytometry may be a useful tool for the detection of surrogate markers of type 1 diabetes mellitus.

Chronic activation of the innate immune system may underlie the metabolic syndrome

CONTEXTO: The metabolic syndrome is characterized by a clustering, in free-living populations, of cardiovascular and diabetes risk factors generally linked to insulin resistance, obesity and central obesity. Consonant with the well-established inflammatory pathogenesis of atherosclerotic disease, the metabolic syndrome is now being investigated in relation to its inflammatory nature. OBJETIVO: We present cross-sectional findings demonstrating that markers of inflammation correlate with components of the metabolic syndrome, and prospective findings of the ARIC Study indicating that markers of inflammation and endothelial dysfunction predict the development of diabetes mellitus and weight gain in adults. We present biological evidence to suggest that chronic activation of the innate immune system may underlie the metabolic syndrome, characterizing the common soil for the causality of type 2 diabetes mellitus and cardiovascular disease. CONCLUSIONS: Better understanding of the role of the innate immune system in these diseases may lead to important advances in the prediction and management of diabetes and cardiovascular disease

Diabetes autoinmune del adulto en diabéticos tipo 2: frecuencia y características / LADA in type 2 diabetics: frequency and characteristics

Se realizó este trabajo para conocer frecuencia, características clínico-bioquímicas, inmunológicas y genéticas de la diabetes autoinmune en adultos (LADA) en 1 000 diabéticos tipo 2 con edades ü 35 años con distintos tiempos de duración de la diabetes. Se les determinó glucemia, anticuerpos antiislotes pancreáticos (ICA), anti-GAD65, anti-ICA512bdc/IA2, antimicrosomales tiroideos (AMT), antigástricos parietales (AGP), antinucleares (AN), microalbuminuria y péptido C en ayunas. Se encuestaron y se registraron algunas características clínicas. Se dividieron en 2 grupos según la presencia de ICA. Todos los diabéticos tipo 2 + para autoanticuerpos antiislotes (ICA y/o anti-GAD65) fueron identificados como LADA. Se detectó el 3,4 (por ciento) de diabético tipo 2 con ICA +, en los diabéticos tipo 2 ICA- el 22,0 (por ciento) presentó anticuerpos anti-GAD65. Se encontró que los diabéticos tipo 2 ICA+ eran más jóvenes, la duración de su diabetes era menor, presentaron menor IMC, disminución de los niveles de péptido C en ayunas, menos antecedentes familiares (padres) de DM2, valores menores en las tensiones arteriales diastólicas y sistólicas, mayor presencia de anticuerpos anti-GAD65, AMT y AGP en comparación con los diabéticos tipo 2 ICA-. Se observó que los diabéticos tipo 2 ICA+ (LADA) tienen características específicas que los asemejan a los diabéticos tipo 1, esto implicaría variaciones importantes en su tratamiento y evolución con respecto a los diabéticos tipo 2 ICA-. Se observó una baja frecuencia de ICA y alta de GAD en los diabéticos tipo 2 cubanos, las cuales fueron diferentes a la encontrada en poblaciones caucasianas. Los anticuerpos anti-GAD65 fueron superiores a los ICA para detectar los LADA. Las características clínicas e inmunológicas de estos pacientes muestran la lenta progresión de la destrucción autoinmune de las células b con implicaciones terapéuticas(AU)

This paper was aimed at knowing the frequency, clinico-biochemical, immunologic and genetic characteristics of autoimmune diabetes in adults (LADA) in 1 000 type 2 diabetic patients aged 35 or over with different times of duration of diabetes. Glycemia, anti-pancreatic islet cell antibodies (ICA), anti-GAD65 antibodies, anti-ICA512bdc/IA2 antibodies, anti-microsomal thyroid antibodies (AMT), anti-gastric parietal antibodies (AGP), antinuclear antibodies (AN), microalbuminuria and peptide C during fasting were determined. These patients were surveyed and some clinical characteristics were registered. They were divided into 2 groups according to the presence of ICA. All the type 2 + diabetics for anti-islet cell autoantibodies (ICA and/or antiGAD65) were identified as LADA. 3.4 percent of type 2 ICA + were detected. 22.0 percent of type 2 ICA - diabetics had anti-GAD65 antibodies. It was found that type 2 ICA + diabetics were younger, that their diabetes was shorter, that they had lower BMI, reduced levels of fasting peptide C, less DM2 history family (parents), lower values of diastolic and systolic arterial pressure, higher presence of anti-GAD65 antibodies, AMT and AGP in comparison with type 2 ICA - diabetics. It was observed that type 2 ICA+ diabetics (LADA) have specific characteristics that make them similar to type 1 diabetics, which would lead to important variations in their treatment and evolution as regards type 2 ICA - diabetics. Among the Cuban type 2 diabetics it was detected a low frequency of ICA and a high frequency of GAD, which were different to those found in the Caucasian populations. The anti-GAD65 antibodies were higher than ICA to detect LADA. The clinical and immunological characteristics of these patients show the slow progression of the autoimmune destruction of b-cells with therapeutic implications(AU)

Efecto inmunosupresor in vitro de las lipoproteínas de baja densidad / In vitro inhibition of lymphocyte proliferation by low density lipoproteins

Background: immune cells participate in the formation of atheromatous plate, however little is known about the effects of native or oxidatively modified lipoproteins on these cells. Aim: To study the effects of lipoproteins on in vitro mononuclear cell proliferation. Material and methods: peripheral blood mononuclear cells were obtained from 10 patients with type 2 diabetes mellitus (aged 52 ñ 9 years old with a disease duration of 8.2 ñ 5.7 years and a mean glycosilated hemoglobin of 9.3 ñ 2.2 percent) and 10 non diabetic healthy controls (aged 50.3 ñ 7.1 years old). These were stimulated with phytohemagglutinin (PHA) alone or in the presence of native LDLS, malondialdehyde modified LDLs or glycated LDLs. Proliferation was measured as 3H-thymidine incorporation and expressed as Stimulation Index (SI). Results: SI of patients and healthy subjects, after PHA stimulation were similar: (57.5 ñ 29.8 and 61.1 ñ 23.5) respectively LDLs did not induce proliferation in neither group. Native LDLs produced a 98 percent inhibition of PHA induced proliferation. Malondialdehyde modified and glycated LDLs caused a 50 percent inhibition. The suppressive effect was maintained when lipoproteins were incorporated to culture media 60 min prior or after PHA stimulation. Conclusions: Lipoproteins inhibit in vitro PHA induced peripheral blood mononuclear cell proliferation both in diabetic and in non diabetic subjects

Respuesta inmune contra lipoproteínas de baja densidad modificadas en pacientes con diabetes mellitus no-insulino dependiente / Immune response against modified low density lipoproteins in patients with non insulin dependent diabetes mellitus

LDLs obtainded from blood of healthy subjects, were glycated or altered with malondialbehyde and used as antigens. Serum autoantibodies against these LDLs were measured by ELISA in 22 patients with non insulin dependent diabetes mellitus aged 46 to 67 years old and 13 healthy controls aged 41 to 64 years old. Basal and LDL stimulated tumor necrosis factor production in vitro, by peripheral leukocytes of diabetics and controls was also measured. Results: The ratio of glycated LDL/native LDL antibodies was higher in diabetics than in controls (9.37 ñ 2.72 and 0.41 ñ 0.11 respectively p < 0.05) and the ratio of MDA modified LDL/native LDL antibodies was not significantly different (8.64 ñ 3.83 and 2.14 ñ 1.26 respectively, NS). Tumor necrosis or production by leukocytes was higher in diabetics than in controls in basal conditions (53.3 ñ 15.3 and 26.9 ñ 14.7 arbitrary units (a.u.) respectively), when stimulated withnative LDL (46.5 ñ 5 and 24.3 ñ 9.4 a.u. respectively), when stimulated with malondialdehyde modified LDL (50 ñ 16.2 and 24.4 ñ 7.7 a.u. respectively) or when stimulated with glycated LDL (38.3 ñ 8.8 and 14.4 ñ 7.5 a.u. respectively). Conclusions: Diabetic patients have an enhanced immune response against low density lipoproteins, factor that could contribute to the accelared atherogenesis of this disease

A study of cell-mediated immune response to pancreatic antigens in patients with fibrocalculous pancreatic diabetes.

In order to investigate whether there was any association between autoimmunity to pancreatic antigens with FCPD as well as IDDM, cell-mediated immune response to pancreatic antigens was studied by lymphoproliferation assay in 7 FCPD, 17 IDDM, 33 NIDDM patients and 102 normal controls. Optimal pancreatic antigen concentrations used were 100, 150 and 200 micrograms/ml. Positive results were considered for each concentration of antigens tested, at stimulation index (SI) > (mean +/- 2 SD) SI obtained from normal age-matched controls with the use of the corresponding concentration of antigen. The one who gave positive result with any of these optimal antigen concentrations was considered to be the responder to pancreatic antigens. With this criterion, the responders were found to be 3/7 (42.9%) FCPD, 6/17 (35.3%) IDDM and 6/33 (18.2%) NIDDM patients; while there were 11 of all 102 (10.8%) normal controls.