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.

Impact of fingolimod on CD4+ T cell subset and cytokine profile of relapsing remitting multiple sclerosis patients.

Fingolimod inhibits the egress of lymphocytes from lymphatic tissues and also directly affects their functions by modulation of the sphingosine-1-phosphate receptor 1 (S1P1). Our aim was to evaluate the impact of fingolimod on diverse CD4+ T cell subsets, and cytokines. Sixty-six relapsing remitting multiple sclerosis (RRMS) patients were treated with oral fingolimod (0.5 mg) for 6 months, and blood samples were collected at baseline, 3 months, and 6 months. Serum levels of seven cytokines and five chemokines were measured by multiplex immunoassay, and frequencies of peripheral blood mononuclear cell subsets were assessed by flow cytometry, and compared with those of 60 healthy controls. CCL2 (p = 0.039), and CCL5 (p = 0.001) levels were significantly higher in fingolimod-treated patients than healthy controls, whereas end-of-study serum levels of IL-6, IL-8, IL-17A, IL-22, IL-23, TNF-α, CXCL10, and CXCL13 were comparable to the baseline levels. Six months of fingolimod treatment reduced CD3+ T cell (mean ±â€¯standard deviation, 72.9% ±â€¯5.5 vs. 60.1% ±â€¯11.1, p < 0.001), CD4+ T cell (62.2% ±â€¯8.5 vs. 24.6% ±â€¯12.9, p < 0.001), CD4+CD25hi regulatory T cell (Treg) (3.4% ±â€¯1.3 vs. 2.0% ±â€¯1.4, p < 0.01), and CD19+ B cell (13.2% ±â€¯5.8 vs. 5.3% ±â€¯2.7, p < 0.001) frequencies, while CD8+ T cells (31.8% ±â€¯7.8 vs. 57.8% ±â€¯13.2, p < 0.001) were increased, and NK and NKT cells remained unchanged. The proportions of intracytoplasmic IL-4, IL-10, IFN-γ, and TNF-α-producing T cells were increased, whereas IL-17-producing cells remained relatively constant as measured by flow cytometry. Fingolimod appears to primarily diminish lymphocyte subsets involved in antigen presentation (CD19+ B and CD4+ T cells) rather than immune cells (CD8+ T, NK, and NKT cells) in charge of host defense against pathogens. In contrast, a relative increase is observed in pro- and anti-inflammatory cytokine-producing T helper subsets (IFN-γ, TNF-α, IL-4, and IL-10-producing CD4+ T cells), suggesting that effector T cells are suppressed to a lesser degree by S1P1 modulation.

CD133+ cells are associated with ADIPOCYTOKINES and endothelial dysfunction in hemodialysis patients.

BACKGROUND: Hemodialysis (HD) patients have increased risk of cardiovascular disease (CVD). Impaired stem cell health and adipocytokine metabolism may play important roles in the complex pathophysiological mechanisms of CVD in this patient population. We aimed to investigate the relationships between CD133+ cell counts, adipocytokines and parameters of endothelial dysfunction and atherosclerosis in HD patients. METHODS: In 58 chronic HD patients (male/female:28/30, mean age:58 ± 14 years), serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), leptin, adiponectin and resistin were measured by ELISA. Left ventricular mass index (LVMI), carotid intima-media thickness (CIMT), flow-mediated dilatation (FMD) of the brachial artery were measured. CD133+ cells were counted by flow cytometry (BD FACSCalibur-BD Bioscience,CA). RESULTS: CD133+ cell counts were inversely associated with FMD (r = -0.39, p = 0.007) and positively correlated with serum resistin (r = 0.45, p < 0.001) and serum TNF-α (r = 0.31, p = 0.02). Serum leptin levels were higher in high CD133 group compared to low CD133 group [32.37(12.74-72.29) vs 15.50(5.38-37.12)ng/mL, p = 0.03]. Serum leptin levels were correlated with TNF-α(r = 0.35, p = 0.009). Serum adiponectin levels were negatively correlated with serum leptin (r = -0.28, p = 0.03). Serum resistin levels were associated with TNF-α (r = 0.54, p < 0.001) and leptin (r = 0.29, p = 0.03). Serum IL-6 levels were significantly associated with LVMI (r = 0.31, p = 0.03). Serum IL-6 levels were significantly higher in patients with carotid plaque compared to patients without plaque [12.75(9.91-28.68) vs 8.27(5.97-14.04) pg/mL, p = 0.02]. In multiple linear regression analysis to determine the factors predicting LogFMD; dialysis vintage, LVMI and LogCD133+ cell counts were included as independent variables(R = 0.57, adjusted R-square = 0.27, p = 0.001). CD133+ cell count and LVMI were found to significantly predict FMD (p = 0.03 and p = 0.04 respectively). CONCLUSION: CD133+ cells were associated with inflammation and endothelial dysfunction in HD patients. Serum leptin, resistin and TNF-α levels were positively related to CD133+ cell count. Impaired regulation of undifferentiated stem cells and adipocytokines might contribute to endothelial dysfunction in HD patients.

ß-carotene treatment alters the cellular death process in oxidative stress-induced K562 cells.

Oxidizing agents (e.g., H2 O2 ) cause structural and functional disruptions of molecules by affecting lipids, proteins, and nucleic acids. As a result, cellular mechanisms related to disrupted macro molecules are affected and cell death is induced. Oxidative damage can be prevented at a certain point by antioxidants or the damage can be reversed. In this work, we studied the cellular response against oxidative stress induced by H2 O2 and antioxidant-oxidant (ß-carotene-H2 O2 ) interactions in terms of time, concentration, and treatment method (pre-, co-, and post) in K562 cells. We showed that co- or post-treatment with ß-carotene did not protect cells from the damage of oxidative stress furthermore co- and post-ß-carotene-treated oxidative stress induced cells showed similar results with only H2 O2 treated cells. However, ß-carotene pre-treatment prevented oxidative damage induced by H2 O2 at concentrations lower than 1,000 µM compared with only H2 O2 -treated and co- and post-ß-carotene-treated oxidative stress-induced cells in terms of studied cellular parameters (mitochondrial membrane potential [Δψm ], cell cycle and apoptosis). Prevention effect of ß-carotene pre-treatment was lost at concentrations higher than 1,000 µM H2 O2 (2-10 mM). These findings suggest that ß-carotene pre-treatment alters the effects of oxidative damage induced by H2 O2 and cell death processes in K562 cells.

K562 cells display different vulnerability to H2O2 induced oxidative stress in differing cell cycle phases.

Oxidative stress can be defined as the increase of oxidizing agents like reactive oxygen and nitrogen species, or the imbalance between the antioxidative defense mechanism and oxidants. Cell cycle checkpoint response can be defined as the arrest of the cell cycle functioning after damaging chemical exposure. This temporary arrest may be a period of time given to the cells to repair the DNA damage before entering the cycle again and completing mitosis. In order to determine the effects of oxidative stress on several cell cycle phases, human erytroleukemia cell line (K562) was synchronized with mimosine and genistein, and cell cycle analysis carried out. Synchronized cells were exposed to oxidative stress with hydrogen peroxide (H2O2) at several concentrations and different times. Changes on mitochondria membrane potential (ΔΨm) of K562 cells were analyzed in G1, S, and G2 /M using Rhodamine 123 (Rho 123). To determine apoptosis and necrosis, stressed cells were stained with Annexin V (AnnV) and propidium iodide (PI) for flow cytometry. Changes were observed in the ΔΨm of synchronized and asynchronized cells that were exposed to oxidative stress. Synchronized cells in S phase proved resistant to the effects of oxidative stress and synchronized cells at G2 /M phase were sensitive to the effects of H2O2 -induced oxidative stress at 500 µM and above.

Influence of conversion from calcineurin inhibitors to everolimus on fibrosis, inflammation, tubular damage and vascular function in renal transplant patients.

BACKGROUND: Conversion from calcineurin inhibitor (CNI) to mTOR inhibitors may reduce and even halt the progression of chronic allograft dysfunction (CAD) which is the most important cause of renal allograft loss. We aimed to investigate the effects of conversion from CNI to everolimus on parameters of fibrosis, inflammation, glomerulotubular damage and vascular functions in renal transplant recipients. METHODS: Fifteen stable renal transplant recipients who were under CNI treatment (male/female 13/2, mean age 41 ± 10 years) were enrolled and switched to everolimus. Serum and urinary transforming growth factor-ß (TGF-ß), urinary neutrophil gelatinase-associated lipocalin (NGAL) and monocyte chemoattractant protein-1 (MCP-1) were measured as markers of fibrosis, tubular damage and inflammation. As parameters of vascular functions, pulse wave velocity (PWV), augmentation index (AIx), serum asymmetric dimethyl-arginine and fibroblast growth factor-23 (FGF-23) were measured. All these measurements were repeated at the 3rd month of conversion. RESULTS: Estimated GFR (52 ± 7-57 ± 11 ml/min/l.73 m(2), p = 0.02) (was increased after conversion to everolimus. However, serum uric acid levels were significantly decreased (6.21 ± 1.21-5.50 ± 1.39 mg/dL, p = 0.01). Serum TGF-ß levels (8727 ± 2897-1943 ± 365 pg/mL, p = 0.03) and urinary NGAL levels (26 ± 10-12 ± 2 ng/mg creatinine, p = 0.05) were significantly decreased. However, urinary MCP-1, FGF-23, PWV and AIx did not change. Urinary TGF-ß was associated with urinary NGAL (r = 0.62, p = 0.01), urinary MCP-1 (r = 0.68, p = 0.005) and proteinuria (r = 0.50, p = 0.05). CONCLUSION: Conversion from CNI to everolimus resulted in significant decreases of serum TGF-ß and urinary NGAL which may represent less fibrosis and tubular damage. Association of urinary TGF-ß with NGAL and MCP-1 suggests that tubular damage, fibrosis and inflammation may act together for progression of CAD.

Relationship between lymphocytes, IL2 and the hormones E2, LH, PRG and FSH in menopausal and postmenopausal women.

PROBLEM In this baseline study, our aim is to show the relationship of parameters and gonad hormones in menopausal and postmenopausal women. METHOD Blood samples were taken from menopausal and postmenopausal women (12-14 months and ≥10 years, respectfully, since their last menstruation). Adolescents aged 13.7 ± 0.7 were used as controls. Hormones were measured by ELISA and percentages of CD45, CD4, CD8, CD3, CD19, IL-2, CD25 and HLA-DR were measured by flow cytometry. RESULTS Both groups showed an increase in the percentage of CD3, CD4 and CD8. Levels of CD19 were significantly lower in the postmenopausal group. However, changes in immunologic parameters during menopause were less marked than the hormonal changes observed in these groups. Most of the correlations LH × CD3 (-ve), LH × IL2R (-ve) and E2 × CD19 (+ve) suggesting how menopausal women with particularly high LH or low E2 levels may be affected. Only CD3 and HLA-DR correlated with the hormonal changes in the postmenopausal group. IL-2 levels were high in the menopausal group and low in the postmenopausal group; however, no correlation was observed. DISCUSSION Menopause is characterized by increased levels of IL-2, which has critical immune-modulatory effects. These changes may be related to the overall hormonal change process observed during menopause.