Esin Aktas

Relationship between CD107a expression and cytotoxic activity

NK cells play important roles in innate immunity against tumors and infections of the host. Studies show that CD107a (LAMP-1) may be a marker for degranulation of NK and activated CD8+ T cells. In our study, the relationship between the expression of CD107a, cytokine secretion and cytotoxic activity in CD56+ NK, CD8+ T cells and lymphocytes has been determined after various stimuli. Effector cells from PBMCs of healthy subjects were isolated and K562 cell line was used as target of cytotoxicity. IL-2 stimulation resulted in a significant increase of CD107a expression in CD56+ NK, CD8+ T cells and lymphocytes. Increased expression of CD107a after IL-2 stimulation of NK cells was parallel to the increase of cytotoxicity. Our results suggest that CD107a expression may be a sensitive marker for the cytotoxic activity determination.

Regulatory NK Cells Suppress Antigen-Specific T Cell Responses

The immune system has a variety of regulatory/suppressive processes, which are decisive for the development of a healthy or an allergic immune response to allergens. NK1 and NK2 subsets have been demonstrated to display counterregulatory and provocative roles in immune responses, similar to Th1 and Th2 cells. T regulatory cells suppressing both Th1 and Th2 responses have been the focus of intensive research during the last decade. In this study, we aimed to investigate regulatory NK cells in humans, by characterization of NK cell subsets according to their IL-10 secretion property. Freshly purified IL-10-secreting NK cells expressed up to 40-fold increase in IL-10, but not in the FoxP3 and TGF-β mRNAs. PHA and IL-2 stimulation as well as vitamin D3/dexamethasone and anti-CD2/CD16 mAbs are demonstrated to induce IL-10 expression in NK cells. The effect of IL-10+ NK cells on Ag-specific T cell proliferation has been examined in bee venom major allergen, phospholipase A2- and purified protein derivative of Mycobecterium bovis-induced T cell proliferation. IL-10+ NK cells significantly suppressed both allergen/Ag-induced T cell proliferation and secretion of IL-13 and IFN-γ, particularly due to secreted IL-10 as demonstrated by blocking of the IL-10 receptor. These results demonstrate that a distinct small fraction of NK cells display regulatory functions in humans.

Human NK1 and NK2 subsets determined by purification of IFN-γ-secreting and IFN-γ-nonsecreting NK cells

The in vivo existence of human NK cell subsets similar to Th1 and Th2 cells was demonstrated in freshly isolated IFN‐γ‐secreting and IFN‐γ‐nonsecreting NK cells. The IFN‐γ‐secreting NK subset showed a typical cytokine pattern with predominant expression of IFN‐γ, but almost no IL‐4, IL‐5 and IL‐13. In contrast, the IFN‐γ‐nonsecreting NK subset was composed ofIL‐4, IL‐5 and IL‐13‐producing NK cells. Short‐time stimulation or 2 weeks of in vitro differentiation of NK cells led to distinct patterns of cytokine production similar to freshly‐purified IFN‐γ (+) or IFN‐γ (–) NK cell subsets. NK cells stimulated with IL‐12 produced increased levels of IFN‐γ and decreased levels of IL‐4. In contrast, stimulation of NK cells with IL‐4 inhibited IFN‐γ, but increased IL‐13 production. Freshly‐purified IFN‐γ (+) and IFN‐γ (–) or in vitro differentiated NK1 and NK2 subsets showed similar cytotoxicity to K562 cells. These results demonstrate that circulating NK cells retain effector subsets in humans with distinct cytokine profiles and may display different inflammatory properties.

Different natural killer (NK) receptor expression and immunoglobulin E (IgE) regulation by NK1 and NK2 cells

Many studies concerning the role of T cells and cytokines in allergy have been performed, but little is known about the role of natural killer (NK) cells. Accordingly, the expression of co‐stimulatory, inhibitory and apoptosis receptors, cytokine profiles and their effect on immunoglobulin isotypes were investigated in polyallergic atopic dermatitis (AD) patients with hyper immunoglobulin E (IgE) and healthy individuals. AD patients showed significantly decreased peripheral blood NK cells compared to healthy individuals. Freshly isolated NK cells of polyallergic patients spontaneously released higher amounts of interleukin (IL)‐4, IL‐5, IL‐13 and interferon (IFN)‐γ compared to healthy individuals. NK cells were differentiated to NK1 cells by IL‐12 and neutralizing anti‐IL‐4 monoclonal antibodies (mAb), and to NK2 cells by IL‐4 and neutralizing anti‐IL‐12 mAb. Following IL‐12 stimulation, NK cells produced increased levels of IFN‐γ and decreased IL‐4. In contrast, stimulation of NK cells with IL‐4 inhibited IFN‐γ, but increased IL‐13, production. The effect of NK cell subsets on IgE regulation was examined in co‐cultures of in vitro differentiated  NK  cells  with  peripheral  blood  mononuclear  cells  (PBMC) or B cells. NK1 cells significantly inhibited IL‐4‐ and soluble CD40‐ligand‐stimulated IgE production; however, NK2 cells did not have any effect. The inhibitory effect of NK1 cells on IgE production was blocked by neutralization of IFN‐γ. Except for CD40, NK cell subsets showed different expression of killer‐inhibitory receptors and co‐stimulatory molecules between the polyallergic and healthy subjects. These results indicate that human NK cells show differences in numbers, surface receptor and cytokine phenotypes and functional properties in AD.

An ultrasensitive tumor enriched flow-cytometric assay for detection of isolated tumor cells in bone marrow of patients with breast cancer

BACKGROUND An ultrasensitive tumor enriched flow-cytometric assay was used to determine its feasibility in detection of isolated tumor cells (ITC) in bone marrow (BM) of patients with breast cancer. METHODS Epithelial cells were removed by magnetic microbeads conjugated with an anti-cytokeratin 7/8 monoclonal antibody to enrich tumor cells in BM samples. A specific gate for MCF-7 breast cancer cells (gate(MCF-7 cells)) was also taken into consideration in addition to a gate including all enriched BM cells (gate(enriched BM cells)) in flow-cytometric analysis to enhance the specificity of the method. RESULTS Nineteen patients with stage I/II were evaluated. Ten patients (53%) were found to have cytokeratin positive (CK(+)) cells according to the gate(enriched BM cells) whereas 6 patients (32%) had CK(+) cells when the gate(MCF-7 cells) was taken into account. CONCLUSIONS New strategies in nonmorphological ultrasensitive techniques might be useful to categorize patients with ITCs having different tumor morphology and characteristics.

Effect of post-polymerization heat-treatments on degree of conversion, leaching residual MMA and in vitro cytotoxicity of autopolymerizing acrylic repair resin

OBJECTIVES This study evaluated the effect of post-polymerization heat-treatments on degree of conversion (DC), residual methyl methacrylate concentration (MMA(r)) and in vitro cytotoxicity of autopolymerizing acrylic repair resin. METHODS A total of 336 specimens were prepared by bench- and hydroflask-curing and subjected to post-polymerization heat-treatments: a) water immersion at 60°C for 30 min, b) microwaving at 500 W for 3 min, c) combined use of water immersion and microwaving d) no treatment (as control). Specimens were eluted in cell culture medium for 1, 2, 5 and 7 days. DC and MMA(r) in eluates were measured by FTIR spectrometry and HPLC, respectively. In vitro cytotoxicity of eluates on L-929 fibroblasts was determined by XTT assay. Data were statistically analyzed with Dunns multiple comparison and Pearson correlation tests (p≤0.05). RESULTS DC was highest (99.9%) in bench- and hydroflask-cured groups which were subjected to water immersion. At all elution periods, MMA(r) was detected in eluates of all treatment groups and were higher in bench-cured groups than hydro-flask cured groups. Cell proliferation values indicated slightly cytotoxic effect throughout 7 days; regardless of the curing method or post-polymerization treatment. The correlation between MMA(r) and cell proliferation was negative after elution of 1, 2, 5 days and was only statistically significant (p<0.05) at 5 days. At elution of 7 days, the correlation was positive with no significance. SIGNIFICANCE Post-polymerization heat-treatment of autopolymerizing acrylic repair resin by immersion in water at 60°C for 30 min is clinically recommended to improve the DC while reducing the leaching residual MMA.

Decreased therapeutic effects of noscapine combined with imatinib mesylate on human glioblastoma in vitro and the effect of midkine

BackgroundGlioblastoma (GBM) develops resistance to the advances in chemotherapy leading to poor prognosis and life quality. Consequently, new treatment modalities are needed. Our aims were to investigate the effects of combined noscapine (NOS) and imatinib mesylate (IM) on human GBM in vitro and the role of midkine (MK) in this new combination treatment.MethodsMonolayer and spheroid cultures of T98G human GBM cell line were used to evaluate the effects of IM (10 μM), Nos (10 μM) and their combination on cell proliferation and apoptotic indexes, cell cycle, the levels of antiapoptotic MK, MRP-1, p170, PFGFR-α, EGFR, bcl-2 proteins, apoptotic caspase-3 levels, morphology (SEM) and ultrastructure (TEM) for 72 hrs. Results were statistically analyzed using the Students t-test.ResultsThe combination group induced highest decrease in cell proliferation and apoptotic indexes, caspase-3 levels, MRP-1 and PDGFR-α levels. The decrease in p170 levels were lower than IM but higher that NOS. The highest increases were in EGFR, MK, bcl-2 and cAMP levels in the combination group. The G0+G1 cell cycle arrest at the end of 72nd hr was the lowest in the combination group. Apoptotic appearence was observed rarely both in the morphologic and ultrastructural evaluation of the combination group. In addition, autophagic vacuoles which were frequently observed in the IM group were observed rarely.ConclusionsThe combination of Nos with IM showed antagonist effect in T98G human GBM cells in vitro. This antagonist effect was correlated highly with MK levels. The effects of NOS on MRP-1, MK and receptor tyrosine kinase levels were firstly demonstrated in our report. In addition, we proposed that MK is one of the modulator in the switch of autophagy to cell death or survival/resistance.

Natural Killer Cells in Allergic Inflammation

Natural killer (NK) cells are large granular lymphocytes of the innate immune system that exert a potent function against infected and tumor cells. Although NK cells were originally defined by their capacity to lyse target cells and produce interferon-gamma without prior activation, recent studies showed that NK cells also display a potent regulatory function. They are activated or inhibited through the ligation of germline-encoded receptors and are involved in mediating cytotoxicity, producing cytokines and providing costimulation to cells of the adaptive immune system. NK cells play important roles in viral infections, autoimmunity, pregnancy, cancer and bone marrow transplantation, but little is known about the role of NK cells in allergy. Recent developments in the understanding of the role of human NK cells in allergy are overviewed.

Decrease in endothelial progenitor cells associated with inflammation, but not with endothelial dysfunction in chronic hemodialysis patients.

INTRODUCTION Endothelial progenitor cells (EPC), bone marrow derived cells, are considered to have a pivotal role in maintaining the integrity and repair of the endothelium. Endothelial dysfunction, atherosclerosis and inflammation are implicated for increased CV mortality in uremia. In this study, we aimed to investigate the possible association of EPC with inflammation, endothelial dysfunction and atherosclerosis in chronic hemodialysis (HD) patients. PATIENTS AND METHODS 67 HD patients (male/female: 30/37, mean age: 58 ± 15 years) and 22 healthy controls (male/female: 13/9; mean age: 48 ± 8 years) were included. EPC were cultivated in the fibronectin-covered culture dishes and counted. Also EPC markers were studied by flow cytometry using anti-CD34, anti-CD133 and anti-vascular endothelial growth factor receptor 2 (VEGFR-2) antibodies. Serum levels of IL-6, TNF-α, intercellular cell adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM) and asymmetric dimethyl-arginine (ADMA) were measured by ELISA method. Endothelial function was investigated by measuring flow-mediated dilatation (FMD) of the brachial artery. Carotid intima-media thickness (CIMT) and ratio (CIMR) were also examined. RESULTS EPC number was decreased in HD patients when compared to controls (63.7 ± 8.9 vs. 101.5 ± 19.6/ high power field, p < 0.001). Also CD34+ cell count was significantly lower in the HD group (2.26 ± 3.52 vs. 6.03 ± 4.73%, p < 0.0001). EPC number was significantly inversely correlated with serum TNF-α levels in HD patients(r: -0.453, p < 0.001) and also in the control group (r = -0.509, p = 0.044). There was an inverse association between VEGFR-2+/CD34+cell count and serum IL-6 levels (r: -0.364, p = 0.006) in HD patients. However, EPC count was not related to FMD and CIMT/CIMR. In HD patients, there was a positive correlation between serum IL-6 levels with CIMT (r = 0.358, p = 0.01) and CIMR was positively correlated with serum ICAM (r = 0.430, p = 0.002). CONCLUSION EPC number was decreased in uremia and was associated with inflammation. TNF-α might have specific inhibitory actions on EPC in both HD patients and healthy controls. No relationship was present between EPC and endothelial dysfunction/atherosclerosis.

Sorafenib and lithium chloride combination treatment shows promising synergistic effects in human glioblastoma multiforme cells in vitro but midkine is not implicated

Abstract Objectives: The objectives of this study were to test the effects of the new combination treatment modality, sorafenib (SOR) and lithium chloride (LiCl) and to assess whether midkine (MK) protein has a role in any potential effects. Methods: Monolayer and spheroid cultures of T98G human glioblastoma multiforme (GBM) cells were treated with LiCl and SOR (inhibition concentration 50 value  =  100 μM), or their combination, or were left untreated (control). Cell proliferation and apoptotic indices, the mechanism of action, and the levels of apoptotic and anti-apoptotic proteins were evaluated in monolayer cultures and ultrastructure was evaluated by transmission electron microscopy (TEM) in spheroid cultures after for 72 hours. Results: All drug applications decreased cell numbers and increased the apoptotic index. The combination shows a synergistic effect. In the combination group, the decrease in cell numbers and the increase in the apoptotic index were significantly greater than with the individual drugs (P < 0·01). The combination treatment led to the greatest decreases in MRP-1 and p170 levels; but the greatest decreases in p-STAT-3, p-ERK (P < 0·05), p-AKT, p-GSK-3-beta (P < 0·01), EGFR (P < 0·01), NF-kappa-β levels were with SOR alone, followed by the combination. The decreases in MK levels in the SOR and combination groups were similar (P  =  0·06). Severe ultrastructural damage was more frequently observed in the combination group compared with the other groups. Conclusions: These results suggest the possibility that the addition of LiCl to SOR could improve the prognosis in at least some patients who need both cancer and psychotherapy and indicate the need for further studies.