Attenuated in vitro effects of IFN-α, IL-2 and IL-12 on functional and receptor characteristics of peripheral blood lymphocytes in metastatic melanoma patients.

Considering tumor-induced suppression of lymphocytes the aim of this study was to investigate in vitro effects of IFN-α, IL-2, IL-12 and IL-18 as immunomodulating agents on the functional and receptor characteristics of peripheral blood lymphocytes (PBL) in metastatic melanoma (MM) patients compared to healthy controls (HC). In HC IFN-α, IL-2 and IL-12 enhanced mRNA level of perforin by inducing pSTAT-1 and pSTAT-5 signaling molecules. Additionally, the expression of NKG2D activating receptor and its DAP10 signaling molecule was upregulated by IL-2. Contrary to this, in MM patients only IL-2 by upregulating pSTAT-5 increased perforin-mediated cytotoxicity of lymphocytes. Furthermore, there was significantly negative correlation between the percentage of CD4+CD25bright+CD27+ regulatory T (Treg) cells and NK cell cytotoxicity, as well as the expression of NKG2D receptor on PBL in HC and MM patients. Therefore, the absence of IL-2 effect on the increase of NKG2D/DAP10 level in MM patients could be the consequence of the increased percentage of immunosuppressive CD4+CD25bright+CD27+ cells after this cytokine treatment in patients. However, in MM IL-12 significantly decreases the percentage of these inhibitory cells. Although IL-2 as a single agent has numerous side effects, it remains the important cytokine for PBL activation in melanoma immunotherapy. Additionally, the removal of Treg cells from patient PBL by IL-12 before in vitro stimulation with IL-2, may lead to the generation of more potent cytotoxic lymphocytes against tumor cells. Therefore, lymphocyte based therapy for MM patients should integrate not only the choice of appropriate immunostimulatory cytokine, but also the removal of inhibitory cells from tumor microenvironment.

Is there a link between Hashimoto's thyroiditis and primary hyperparathyroidism? A study of serum parathormone and anti-TPO antibodies in 2267 patients.

According to various authors, thyroid disorders like Hashimoto's thyroiditis (HT), diffuse goiter or multinodular goiter, Graves' disease, medullary or papillary carcinoma could be found in a number of patients with primary hyperparathyroidism (PHPT). This association is more common in elderly women. Neck irradiation, lithium treatment and elevated TSH levels have been suggested as some of the possible causes of this co-existance. The aim of this study was to investigate and determine the prevalence of patients having both HT and PHPT, and the possible relation between these two diseases. We conducted a prospective study during three and a half years. This study included 45,231 patients, which were referred by their general practitioner or endocrinologist, under suspicion of having thyroid and/or parathyroid disease. In these patients we measured serum levels of the following parameters: anti-thyroid peroxidase antibodies (antiTPO-Ab), anti-thyroglobulin antibodies (Tg-Ab), anti-TSH-receptor antibodies (TSHR-Ab), thyroid-stimulating hormone (TSH), parathyroid hormone (PTH) and calcium (Ca). In 2,267 of these 45,231 patients (5.01%) we noticed elevated antiTPO-Ab (3542±3407IU/mL), with statistical significant difference from normal values (normal range 0-70IU/mL), P<0.05, and normal levels of other antithyroid antibodies (Tg-Ab, TSHR-Ab). All patients with elevated antiTPO-Ab were assumed to have HT. Within this group, 43 patients (1.89%) also had elevated serum levels of PTH (112.4±33.2pg/mL, normal range 8-76pg/mL) as well as elevated serum levels of calcium (2.92±0.06mmol/L, normal range 2.2-2.65mmol/L). These laboratory findings, accompanied with clinical symptoms, satisfied the criteria for PHPT. The mean age in this subgroup was 60.5±12.2 years. All 2,267 patients had normal or slightly elevated TSH levels. In conclusion, although the reported rate of prevalence of PHPT in the general population is about 0.3%, our results indicated a 1.89% occurrence of PHPT in 2267 patients with HT in central Serbia. This may be due to the autoimmune inflammatory process in HT supporting PHPT to PTH or calcium supporting HT or to common genetical predisposition of both entities.

Beneficial in-vitro effects of interleukin-2, interleukin-12, and their combination on functional and receptor characteristics of natural killer cells in metastatic melanoma patients with normal serum lactate dehydrogenase levels.

Considering tumor-mediated suppression of natural killer (NK) cells, the aim of this study was to investigate the in-vitro effects of interleukin (IL)-2 and IL-12, as immunostimulatory cytokines, on the functional and receptor characteristics of NK cells and their subsets in healthy control (HC) and metastatic melanoma (MM) patients. Peripheral blood mononuclear cells of 27 HC and 35 MM patients were stimulated in vitro with IL-2, IL-12, and their combination for functional and phenotypic analysis. IL-2, IL-12, and primarily their combination, significantly induced NK cell activity, CD107a degranulation marker, and perforin expression in NK cells and their subsets in HC and MM patients. Furthermore, the combination of IL-2 and IL-12 was significantly more efficient than IL-12 alone in the augmentation of NK cell cytotoxicity and CD107a expression. Also, IL-2 and IL-12 reciprocally upregulated each other's receptors, IL-2Rα and IL-12Rß1/ß2, on NK cells and their subsets in MM and HCs. In addition, the priming of NK cells with IL-2 before IL-12 treatment led to an increase in the expression of both IL-12 receptors. In contrast to IL-12, IL-2 increased activating NKG2D and DNAM-1, as well as inhibitory CD158a and CD158b KIRs. In addition, the cytokines investigated exerted a more potent effect on the increase in NK cell activity and the expression of various NK cell receptors in MM patients with normal lactate dehydrogenase (LDH) serum levels. Therefore, serum LDH could represent a predictor of response to cytokine immunotherapy in MM patients. The optimization of combined IL-2/IL-12 therapy is needed to enhance NK cell functions in MM patients stratified by their LDH levels.

Decreased expression of NKG2D, NKp46, DNAM-1 receptors, and intracellular perforin and STAT-1 effector molecules in NK cells and their dim and bright subsets in metastatic melanoma patients.

Although natural killer (NK) cells play an important antitumor role, melanoma cells may affect their effector functions. In this study, we analyzed the expression of various receptors and effector molecules in NK cells and their subsets in metastatic melanoma (MM) patients compared with healthy controls (HCs). In HC and MM patients, we analyzed NK cell activity using a chromium release assay and the expression of CD107a degranulation marker, activating NKG2D, NKp46, DNAM-1, and inhibitory CD158a and CD158b receptors, IL-12R beta 1, IL-12R beta 2, intracellular interferon (IFN)-γ, perforin, and STAT-1 in CD3-CD56+ NK cells, and cytotoxic CD3-CD56 and immunoregulatory CD3-CD56 subsets by flow cytometry. MM patients compared with HC not only had significantly decreased NK cell activity, lower expression of CD107a, and impaired IFN-γ production but also had decreased expression of activating NKG2D, NKp46, and DNAM-1 receptors, which was followed by lower expression of perforin, STAT-1, and both IL-12R subunits in NK cells. In MM patients only, there was a positive correlation between NKG2D expression and degranulation capacity, as well as IFN-γ production in NK cells. Analysis of the expression of various parameters of NK cell effector functions between MM patients with different localization of distant metastases showed that patients in the unfavorable M1c subclass had decreased expression of NKG2D and NKp46 on NK cells compared with patients in the M1a+b group. Downregulated NKG2D, NKp46, and DNAM-1 receptors associated with impaired NK cell effector function are important biomarkers of advanced disease with a poor prognosis in melanoma patients.