Gunnur Deniz

Immune Responses in Healthy and Allergic Individuals Are Characterized by a Fine Balance between Allergen-specific T Regulatory 1 and T Helper 2 Cells

The mechanisms by which immune responses to nonpathogenic environmental antigens lead to either allergy or nonharmful immunity are unknown. Single allergen-specific T cells constitute a very small fraction of the whole CD4+ T cell repertoire and can be isolated from the peripheral blood of humans according to their cytokine profile. Freshly purified interferon-γ–, interleukin (IL)-4–, and IL-10–producing allergen-specific CD4+ T cells display characteristics of T helper cell (Th)1-, Th2-, and T regulatory (Tr)1–like cells, respectively. Tr1 cells consistently represent the dominant subset specific for common environmental allergens in healthy individuals; in contrast, there is a high frequency of allergen-specific IL-4–secreting T cells in allergic individuals. Tr1 cells use multiple suppressive mechanisms, IL-10 and TGF-β as secreted cytokines, and cytotoxic T lymphocyte antigen 4 and programmed death 1 as surface molecules. Healthy and allergic individuals exhibit all three allergen-specific subsets in different proportions, indicating that a change in the dominant subset may lead to allergy development or recovery. Accordingly, blocking the suppressor activity of Tr1 cells or increasing Th2 cell frequency enhances allergen-specific Th2 cell activation ex vivo. These results indicate that the balance between allergen-specific Tr1 cells and Th2 cells may be decisive in the development of allergy.

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.

Cytokine levels in euthymic bipolar patients

BACKGROUND The pathophysiology of bipolar disorder is not thoroughly understood. Several studies have investigated the possible role of cytokines in psychiatric disorders, based on their role in neuro-immune modulation; however, findings in studies on bipolar disorder remain limited and contradictory, and most studies have focused on either manic or depressive episodes. These studies suggest that both manic and depressive episodes could be pro-inflammatory states. The present study aimed to determine whether there are enduring differences in cytokine levels-unrelated to the effects of medication-between euthymic bipolar patients and healthy controls. METHODS The study included 31 euthymic bipolar patients-16 medication-free (MF) and 15 on lithium monotherapy (LM) and 16 healthy volunteers in whom serum cytokine levels were measured. The 3 groups were homogenous in terms of age, gender, and ethnicity. IFN-γ, TNF-α, IL-2, IL-4, IL-5, and IL-10 levels were measured in all groups using flow cytometry. RESULTS There were no differences in cytokine levels between MF euthymic bipolar patients and healthy controls. TNF-α and IL-4 levels in LM euthymic bipolar patients were higher than in both the MF euthymic bipolar patients and controls. LIMITATIONS The small and strictly selected study sample could limit the generalizability of the findings. CONCLUSIONS Cytokine production in MF euthymic bipolar patients was similar to that in healthy controls. The present study shows that the pro-inflammatory state resolves in euthymia and that lithium had an influence on the cytokine profile, which could create a confounding factor while investigating disease- related immunopathology of bipolar disorder.

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.

Bacillus Calmette–Guérin‐induced interleukin‐12 did not additionally improve clinical and immunologic parameters in asthmatic children treated with sublingual immunotherapy

Objective To evaluate the effect of bacillus Calmette–Guérin (BCG) as an adjuvant to specific sublingual immunotherapy (SLIT) on the cytokine profile of peripheral blood mononuclear cells (PBMCs) and clinical outcome.

Multicentre comparison of a diagnostic assay: aquaporin-4 antibodies in neuromyelitis optica

Objective Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab) assays in neuromyelitis optica spectrum disorders (NMOSD). Methods Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4), immunohistochemistry (n=3) and ELISA (n=1). Results Results of tests on 92 controls identified 12assays as highly specific (0–1 false-positive results). 32 samples from 50 (64%) NMO sera and 34 from 51 (67%) NMOSD sera were positive on at least two of the 12 highly specific assays, leaving 35 patients with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5–100%) of all 21 assays. The specificities (85.8–100%) were based on 92 control samples and 35 seronegative NMO/SD patient samples. Conclusions The cell-based assays were most sensitive and specific overall, but immunohistochemistry or flow cytometry could be equally accurate in specialist centres. Since patients with AQP4-Ab negative NMO/SD require different management, the use of both appropriate control samples and defined seronegative NMOSD samples is essential to evaluate these assays in a clinically meaningful way. The process described here can be applied to the evaluation of other antibody assays in the newly evolving field of autoimmune neurology.

Natural killer cells in patients with allergic diseases

Natural killer (NK) cells not only exert cytotoxic activity against tumor cells or infected cells but also act to regulate the function of other immune cells through secretion of cytokines and chemokines or cell contact-dependent mechanisms. NK cells are able to polarize in vitro into 2 functional distinct subsets, NK1 or NK2 cells, which are analogous to the T-cell subsets TH1 or TH2. In addition, a regulatory NK cell subset has been described that secretes IL-10, shows antigen-specific T-cell suppression, and suppresses IgE production. Although it has been demonstrated that NK cells play important roles in autoimmunity, cancer, transplantation, and pregnancy, the role of NK cells in allergy has not been extensively discussed. This review aims to discuss our understanding of NK cells and NK cell subsets in allergic inflammation and IgE regulation.

Effect of leaching residual methyl methacrylate concentrations on in vitro cytotoxicity of heat polymerized denture base acrylic resin processed with different polymerization cycles

Objectives Residual methyl methacrylate (MMA) may leach from the acrylic resin denture bases and have adverse effects on the oral mucosa. This in vitro study evaluated and correlated the effect of the leaching residual MMA concentrations ([MMA]r) on in vitro cytotoxicity of L-929 fibroblasts. Material and Methods A total of 144 heat-polymerized acrylic resin specimens were fabricated using 4 different polymerization cycles: (1) at 74ºC for 9 h, (2) at 74ºC for 9 h and terminal boiling (at 100ºC) for 30 min, (3) at 74ºC for 9 h and terminal boiling for 3 h, (4) at 74ºC for 30 min and terminal boiling for 30 min. Specimens were eluted in a complete cell culture medium at 37ºC for 1, 2, 5 and 7 days. [MMA]r in eluates was measured using high-performance liquid chromatography. In vitro cytotoxicity of eluates on L-929 fibroblasts was evaluated by means of cell proliferation using a tetrazolium salt XTT (sodium 3´-[1-phenyl-aminocarbonyl)-3,4-tetrazolium]bis(4-methoxy-6-nitro)benzenesulphonic acid) assay. Differences in [MMA]r of eluates and cell proliferation values between polymerization cycles were statistically analyzed by Kruskal-Wallis, Friedman and Dunns multiple comparison tests. The correlation between [MMA]r of eluates and cell proliferation was analyzed by Pearsons correlation test (p<0.05). Results [MMA]r was significantly (p≤0.001) higher in eluates of specimens polymerized with cycle without terminal boiling after elution of 1 and 2 days. Cell proliferation values for all cycles were significantly (p<0.01) lower in eluates of 1 day than those of 2 days. The correlation between [MMA]r and cell proliferation values was negative after all elution periods, showing significance (p<0.05) for elution of 1 and 2 days. MMA continued to leach from acrylic resin throughout 7 days and leaching concentrations markedly reduced after elution of 1 and 2 days. Conclusion Due to reduction of leaching residual MMA concentrations, use of terminal boiling in the polymerization process for at least 30 min and water storage of the heat-polymerized denture bases for at least 1 to 2 days before denture delivery is clinically recommended for minimizing the residual MMA and possible cytotoxic effects.