Genetic Investigation of the Trail Mechanism in Diabetic and Non-diabetic Obese Patients.

Obesity is an important healthcare issue caused by abnormally increased adipose tissue because of energy-intake overcoming energy expenditure. Disturbances in the physiological function of adipose tissue mediate the development of diabetes. It is a metabolic disease that results from decreased insulin-levels and/or changes in the insulin action mechanism. Tumor Necrosis Factor-Associated Apoptosis-Inducing Ligand(TRAIL), which is a member of the Tumor Necrosis Factor(TNF)-family with an important role in adipose tissue biology, is included in many studies with its ability to induce apoptosis in cancer cells, but the number of human-studies conducted on the gene related to its protective-role against diabetes and obesity at this level is insufficient. Our study was carried out as a case and control and included three groups (80 diabetic obese, 80 non-diabetic obese, and 80 healthy individuals as the control group). The Real-Time-PZR(RT-qPZR), and DNA Sanger-Sequencing Methods were used for gene expression and gene squences. As a result of the analyses, TRAIL gene expression level was found to be higher in the controls than in the diabetic-obese and non-diabetic-obese group. This change in TRAIL gene expression suggests that TRAIL maybe a protective factor against diabetes. The presence of rs781673405, rs143353036, rs1244378045, rs767450259, rs759369504, rs750556128, and rs369143448 mutations, which was determined with the Sequencing-Method, was shown for the first time in the present study. In addition, it is the first study in which human TRAIL gene-expression and sequencing were performed together. We believe that these data will make an important contribution to the literature.

Effects of Blood Pressure Lowering With Different Antihypertensive Agents on Cognitive Function and Plasma Brain-derived Neurotrophic Factor Levels: A Comparative Study.

PURPOSE: Hypertension is a risk factor for cognitive impairment (CI). However, the specific effect of antihypertensive therapy on cognitive function is still controversial. We aimed to investigate the effect of antihypertensive agents targeting the renin-angiotensin system (RAS) on CI and brain-derived neurotropic factor (BDNF). METHODS: We included 62 patients who had been using the same antihypertensive agent for at least 3 months. Patients who had relevant conditions that could contribute to CI were excluded. After subjects were divided into 3 groups according to their current antihypertensive medication, the cognitive status of each patient was assessed by the mini-mental state examination (MMSE). BDNF and plasma renin activity were evaluated. RESULTS: There was a negative association between systolic blood pressure and MMSE independent of medication (rho = -0.251, P = 0.049). There was no significant correlation between MMSE and BDNF. The MMSE score was slightly higher in the non-RAS group, but the difference did not reach statistical significance (P = 0.09). There was also no significant difference in BDNF levels between study groups (P = 0.32). Mean plasma renin activity levels were significantly lower in the non-RAS group compared with the angiotensin converting enzyme inhibitor and angiotensin receptor blocker groups (P = 0.007). CONCLUSIONS: We suggest that the essential intervention for CI in hypertensive patients is appropriate for blood pressure control.

Could we use PD-1 and PD-L1 expression on lymphocytes and monocytes as predictive markers for prognosis of acute biliary pancreatitis?

PURPOSE: This study aimed to assess the significance of immunophenotyping and serum cytokines in predicting the clinical progression of acute biliary pancreatitis (ABP). MATERIALS AND METHODS: Cytokine levels, T-helper, cytotoxic T, natural killer (NK) cells, monocytes, HLA-DR, and PD-1, as well as PDL-1 immune checkpoints, were measured in ABP patients at the time of diagnosis and compared with results from healthy volunteers. The study also compared leukocyte counts, hematocrit, immunophenotyping results, cytokine statuses, and PD-1, PDL-1 expression between healthy volunteers and ABP subgroups categorized by pancreatitis severity. RESULTS: The study included 65 ABP patients and 20 healthy volunteers. Significant differences were observed between groups in hematocrit, leukocyte counts, total monocytes, lymphocytes, CD3+ total T cells, CD4+ Th cells, PD-1 expression on CD4+ and CD8+T lymphocytes, HLA-DR expression on CD14+ monocytes, NK cells, PD-L1 expression on CD14+ monocytes, classical and intermediate monocytes, as well as levels of IL-6, IL-8, IL-10, IL-18, and IL-33 cytokines. Moderate correlations were found with lymphocyte counts, PD-1+CD4+ cells, PD-L1+CD14+ cells, and strong correlations with HLA-DR+CD14+ cells. Hematocrit, CD3+ total T cells, NK cells, CD4+PD-1 + T cells, and CD8+PD-1 + T cells showed independent associations with the severity of ABP. Lymphocyte counts, CD14+HLA-DR+ cells, CD14+PD-L1+ cells, CD4+PD-1 + T cells, classical, and intermediate monocytes exhibited the highest Area Under the Curve rates in determining organ failure. CONCLUSIONS: Hematocrit, lymphocyte counts, CD14+HLA-DR+ cells, CD14+PD-L1+ cells, and intermediate monocytes emerged as parameters most closely associated with the severity and these parameters could be useful in predicting the severity of ABP.

The role of cytokines and T-bet, GATA3, ROR-γt, and FOXP3 transcription factors of T cell subsets in the natural clinical progression of Type 1 Diabetes.

Th cells play an important role in pathogenesis of type 1 diabetes (T1D). Peripheral blood mononuclear cells were isolated from peripheral blood samples from newly diagnosed (ND), 1-year (1YD), and 5-year T1D (5YD) patients (n:8 of each group), 8 healthy controls (HC), and cultured for 24 h under unstimulated (US) and stimulated conditions. Cell ratios of Th1, Th2, Th17, Treg, and intracellular levels of IFN-γ, TNF-α, IL-10, TGF-ß, IL-5, IL-13, IL-17, and IL-21 cytokines were evaluated using the flow cytometry. mRNA expressions of transcription factors T-bet, GATA3, ROR-γt, and FOXP3 of these cells were determined by real-time PCR. Reduced CD4+CD25high cell ratios were detected in ND. CD4+CD25high cells were found to be reduced in ND and 1YD compared to HC under IL-2-stimulated conditions. Intracellular IFN-γ and TNF-α levels were low in all patients under US and IL-12-stimulated conditions. IL-17A and IL-21 were found to be high in patients with IL-6-stimulated conditions. Expressions of IL-10 and TGF-ß have been observed to be reduced in patients. Th1/Th2, Th17/Treg, and Th1/Treg ratios were higher in patient groups. FOXP3 and GATA3 mRNA expressions were found to be low in patients, while RORγt and T-bet mRNA levels were higher than HC. Th1, Th17, and Treg cells and their cytokines have been shown to be associated with type 1 diabetes.

Hepatic insulin synthesis increases in rat models of diabetes mellitus type 1 and 2 differently.

Insulin-positive (+) cells (IPCs), detected in multiple organs, are of great interest as a probable alternative to ameliorate pancreatic beta-cells dysfunction and insulin deficiency in diabetes. Liver is a potential source of IPCs due to it common embryological origin with pancreas. We previously demonstrated the presence of IPCs in the liver of healthy and diabetic rats, but detailed description and analysis of the factors, which potentially can induced ectopic hepatic expression of insulin in type 1 (T1D) and type 2 diabetes (T2D), were not performed. In present study we evaluate mass of hepatic IPCs in the rat models of T1D and T2D and discuss factors, which may stimulate it generation: glycaemia, organ injury, involving of hepatic stem/progenitor cell compartment, expression of transcription factors and inflammation. Quantity of IPCs in the liver was up by 1.7-fold in rats with T1D and 10-fold in T2D compared to non-diabetic (ND) rats. We concluded that ectopic hepatic expression of insulin gene is activated by combined action of a number of factors, with inflammation playing a decision role.

Anticancer Properties of Fluorinated Aminophenylhydrazines on A549 Lung Carcinoma Cell Line.

BACKGROUND: Non-small cell lung cancer (NSCLC) is responsible for up to 85% of deaths associated with lung cancer. Chemotherapy is still an important treatment method on the treatment of inoperable cases. In this study, the anticancer properties of a series of Schiff bases were tested on the A549 cell line representing NSCLC. METHODS: Fluorinated Schiff bases (compounds 1-6) were synthesized based on 2-amino phenylhydrazines and benzaldehydes containing fluorine were used. The cytotoxic effects of the compounds on the A549 cell line were determined by colorimetric MTT assay and the antiproliferative effects of the compounds on the A549 cell line by the CFSE method. To demonstrate the development of apoptosis, cleaved caspase-3 expression in cells was tested using the immunofluorescence method. Morphological changes indicating apoptosis in cells were determined by histopathological staining methods (H & E, giemza, PAP). RESULTS: The strongest cytotoxic effect on A549 lung cancer cells was obtained with compound 6 (IC50: 0.64 µM) containing 5 fluorine atoms. The strongest antiproliferative effect on A549 cells was achieved with compound 5 (PI: 4.95) carrying 2 fluorine atoms. Apoptosis induction was effective in cell death. In addition to cleaved caspase-3 expression, chromatin condensation, marginalization, and apoptotic bodies were observed in the cells. CONCLUSION: Some of the compounds tested showed high cytotoxic and antiproliferative effects, indicating that these compounds could be potential chemotherapeutic agent candidates for lung cancer. The result of immunofluorescence and immunohistochemical analysis showing that the cytotoxic effects have been induced by apoptosis is an important advantage.

Combined evaluation of proliferation and apoptosis to calculate IC50 of VPA-induced PANC-1 cells and assessing its effect on the Wnt signaling pathway.

Pancreatic ductal adenocarcinoma (PDAC) is among the most deadly cancers. Since most patients develop resistance to conventional treatments, new approaches are in urgency. Valproic acid (VPA) was shown to induce apoptosis and reduce proliferation in PANC-1 cells. Wnt signaling pathway is known to be involved in apoptosis and PDAC onset. However, VPA-induced apoptosis and its impact on Wnt signaling in PDACs are not linked, yet. We aimed to calculate IC50 of VPA-induced PANC-1 cells by combined analyses of proliferation and apoptosis, while assessing its effect on Wnt signaling pathway. PANC-1 was induced with increased VPA doses and time points. Three independent proliferation and apoptosis assays were performed utilizing carboxyfluorescein succinimidyl ester and Annexin V/PI staining, respectively. Flow cytometry measurements were analyzed by CellQuest and NovoExpress. Taqman hydrolysis probes and SYBR Green PCR Mastermix were assessed in expression analyses of Wnt components utilizing 2-ΔΔCt method. Cell proliferation was inhibited by 50% at 2.5 mM VPA that evoked a significant apoptotic response. Among the screened Wnt components and target genes, only LEF1 exhibited significant four-fold upregulation at this concentration. In conclusion, cancer studies mostly utilize MTT or BrdU assays in estimating cell proliferation and calculating IC50 of drugs, which provided conflicting VPA dosages utilizing PANC-1 cells. Our novel combined approach enabled specific, accurate and reproducible IC50 calculation at single cell basis with no apparent effect on Wnt signaling components. Future studies are needed to clarify the role of LEF1 in this model.

Intraportal Transplantation of Pancreatic Islets in Mouse Model.

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation. This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 °C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Losartan may prevent the elevation of plasma glucose, corticosterone and catecholamine levels induced by chronic stress.

INTRODUCTION: Stress is a stimulus that activates the hypothalamic pituitary adrenal (HPA) axis and sympathetic nervous system (SNS). Increased activity of the SNS causes to increment or impairment in blood pressure, heart rate, body temperature and plasma glucose and adreno- corticotrophic hormone (ACTH) levels. Angiotensin II (Ang II), which is a product of the renin-angiotensin system (RAS), is an important factor affecting the activity of the SNS and responses to stress. We suggest that the blockade of Ang II may be worthwhile in the prevention and treatment of diabetes mellitus and cardiovascular diseases affected by stress. Therefore, we investigated the effects of immobilisation stress on blood glucose, norepinephrine (NE), epinephrine (E) and corticosterone levels and the effects of an Ang II receptor antagonist, losartan, on these parameters. MATERIALS AND METHODS: The rats were kept in small cylindrical cages for 60 min/day for 10 consecutive days to perform chronic immobilisation stress. Losartan (10 mg/kg) was given daily by gavage to Losartan (L) and Losartan + Chronic Stress (L+CS) groups. Control (C) and Chronic Stress (CS) groups received an equal volume of saline daily by gavage for 10 days. After the last stress regimen, blood samples were collected for plasma glucose, NE, E and corticosteroid measurements. RESULTS: Plasma glucose, NE, E and corticosterone levels in the CS Group increased significantly compared with the C group. In Group L+CS, the plasma glucose, NE, E and corticosterone levels decreased significantly vs. Group CS. In Group L there was no significant difference vs. Group C. CONCLUSION: It can be speculated that chronic blockade of RAS may decrease the excess sympathetic responses to stress in cardiovascular diseases and prevent the likely development of Type II diabetes mellitus.

Can immune parameters be used as predictors to distinguish between pulmonary multidrug-resistant and drug-sensitive tuberculosis?

INTRODUCTION: Despite the development and wide implementation of Directly Observed Therapy Strategies (DOTS), multidrug-resistant tuberculosis (MDR-TB) remains a serious global health threat. In this study, the role of host immune response in patients with MDR-TB is investigated and compared with that of patients with smear-positive drug-sensitive tuberculosis (SP-TB). MATERIAL AND METHODS: 27 patients with SP-TB, 20 patients with MDR-TB, and 20 healthy controls were included in the study. Immune parameters were determined by flow cytometry using monoclonal antibodies in order to compare the percentage values of these markers in the two study groups and the control group. RESULTS: The levels of lymphocyte subgroups in the gate of CD45(+)/CD14(-) lymphocyte: CD45(+), CD3(+), CD4(+), NK, CD3/HLA-DR, CD 95(+) cells were significantly lower; by contrast CD23(+), CD25(+), CD19(+), CD4(+)/CD8(+), HLA-DR cells were found to be lower, but not significantly so in patients with MDR-TB, compared to levels in patients in the SP-TB and control groups. Besides these findings, the levels of NKT cells and (γ)δ TCR(+) cells were significantly higher in the MDR-TB than in the healthy control and SP-TB group. CONCLUSIONS: The lower levels of CD3/ HLA-DR, CD4 (+), Fas (+), and NK, and the higher level of NKT together with (γ)δ T cells in patients with MDR-TB compared to those in SP-TB may indicate a profound immune suppression in MDR-TB patients and thereby may denote an accumulation in the bacterial load. Our findings may shed light on the pathogenesis and prognosis of MDR tuberculosis, and may point towards the use of flow cytometry findings as an aid to early diagnosis in MDR-TB patients.