Cytokine Network & NETs.

The process of forming and releasing neutrophil extracellular traps (NETs) can be regulated by exogenous and endogenous factors, including cytokines. The study aimed to assess the impact of proinflammatory cytokines, IL-15, IL-17, IL-18, and anti-inflammatory IL-10 on the formation of NETs, all in comparison to IL-8 and pathogenic factors: LPS, fMLP. Also, the expression of myeloperoxidase (MPO), one of the main elements of neutrophil traps, was evaluated. After isolating the neutrophils with Polymorphprep™, the cells were sorted using CD16 MACS® microbeads and incubated with selected factors. The formation of NETs was registered using a BD Pathway 855 microscope system and the expression of MPO was evaluated using flow cytometry. The amounts of circulating DNA in cell supernatants was fluorescently quantified. Microscopic photographs indicated that rhIL-15, rhIL-17, rhIL-18 and fMLP induce formation and release of NETs at a similar timespan, while in the presence of rhIL-10, the formation of the traps was delayed. The presence of the studied cytokines indicated two populations of neutrophils displaying differing MPO expression (MPOlow and MPOhigh). Moreover, stimulation of neutrophils with LPS and fMLP revealed two populations of these cells that differed not only in the expression of MPO, but also in size.

New Aspects of the Biology of Neutrophil Extracellular Traps.

The formation and release of neutrophil extracellular traps (NETs) discovered in 2004 by Volker Brinkmann and Arturo Zychlinsky cast a new light on the role of neutrophils in the non-specific immune response of the body. This discovery has resulted in the rapid development of neutrophil studies in different bacterial and autoimmune diseases as well as neoplasms. Research is also being performed on the role of different signalling pathways engaged in the induction of NETosis, a unique form of a programmed cell death leading to the creation of NETs. The literature provides information on the structure and composition of neutrophil extracellular traps. This review presents the latest data on NET formation and the role of their key components, as well as describing the intracellular signalling pathways leading to the generation of NETs that have been discovered.

DNA damage and oxidative stress response to selenium yeast in the non-smoking individuals: a short-term supplementation trial with respect to GPX1 and SEPP1 polymorphism.

PURPOSE: Selenium, both essential and toxic element, is considered to protect against cancer, though human supplementation trials have generated many inconsistent data. Genetic background may partially explain a great variability of the studies related to selenium and human health. The aim of this study was to assess whether functional polymorphisms within two selenoprotein-encoding genes modify the response to selenium at the level of oxidative stress, DNA damage, and mRNA expression, especially in the individuals with a relatively low selenium status. METHODS: The trial involved 95 non-smoking individuals, stratified according to GPX1 rs1050450 and SEPP1 rs3877899 genotypes, and supplemented with selenium yeast (200 µg) for 6 weeks. Blood was collected at four time points, including 4 weeks of washout. RESULTS: After genotype stratification, the effect of GPX1 rs1050450 on lower GPx1 activity responsiveness was confirmed; however, in terms of DNA damage, we failed to indicate that individuals homozygous for variant allele may especially benefit from the increased selenium intake. Surprisingly, considering gene and time interaction, GPX1 polymorphism was observed to modify the level of DNA strand breaks during washout, showing a significant increase in GPX1 wild-type homozygotes. Regardless of the genotype, selenium supplementation was associated with a selectively suppressed selenoprotein mRNA expression and inconsistent changes in oxidative stress response, indicating for overlapped, antioxidant, and prooxidant effects. Intriguingly, DNA damage was not influenced by supplementation, but it was significantly increased during washout. CONCLUSIONS: These results point to an unclear relationship between selenium, genotype, and DNA damage.

The impact of TNF superfamily molecules on overall survival in acute myeloid leukaemia: correlation with biological and clinical features.

B cell-activating factor (BAFF), a proliferation-inducing ligand (APRIL) and apoptosis-inducing ligand (TRAIL) were demonstrated in several haematological diseases including acute myeloid leukemia (AML). Those cytokines are capable of activating a broad spectrum of intracellular signalling cascades that can either induce apoptosis or protect from programmed cell death. We have analysed BAFF, APRIL and TRAIL serum concentrations in 76 patients with newly diagnosed AML and 40 healthy volunteers. The values were significantly higher for APRIL and BAFF but lower for TRAIL compared to healthy volunteers. Induction therapy significantly reduced the values for BAFF and increased them for TRAIL. Moreover, the concentration of BAFF and APRIL was significantly lower and the concentration of TRAIL higher in a group of patients with complete remission compared to non-respondent AML patients. In addition, higher concentrations of BAFF and lower of TRAIL predicted a shorter overall survival, suggesting thereby an important prognostic marker and possible therapeutic target in AML.

BAFF and APRIL as TNF superfamily molecules and angiogenesis parallel progression of human multiple myeloma.

Tumour necrosis factor alpha (TNF-α) is an inflammatory cytokine with a wide spectrum of biological activity, including angiogenesis. B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are members of the TNF-α family. Vascular endothelial growth factor (VEGF), on the other hand, is one of the most characteristic pro-angiogenic cytokines produced by multiple cell types in multiple myeloma (MM). We have analysed BAFF and APRIL concentrations in parallel with pro-angiogenic cytokines in serum and trephine biopsy, and the bone marrow microvascular density (MVD) in 50 patients with newly diagnosed IgG MM and 24 healthy volunteers. The study showed statistically higher concentrations of BAFF, APRIL and TNF-α, as well as VEGF and its receptor, in MM patients compared to healthy volunteers and patients in advanced stages of the disease. A statistically positive correlation between the concentration of TNF-α and the expression of VEGF was demonstrated, and so was a positive link between BAFF, APRIL, MVD and lactate dehydrogenase (LDH). Furthermore, we observed a significant decrease in all studied cytokines after anti-angiogenic therapy, with meaningful differences between responders (at least partial remission) and patients with stable disease. It was also established that APRIL, but not BAFF, correlated with pro-angiogenic cytokines such as VEGF with its receptor, MVD and syndecan-1. Finally, our results showed that serum BAFF and APRIL levels could be useful biomarkers of MM disease activity and its progression which suggests that APRIL could be a possible novel therapeutic target in MM.

Glucocorticoids upregulate decreased IL-7 receptor expression in asthmatic patients and simian immunodeficiency virus-infected non-human primates.

Signaling through interleukin-7 receptor (IL-7R) is essential for regulation of T-cell homeostasis and survival. Previously, we and others have found diminished IL-7R levels in simian immunodeficiency virus (SIV) - infected non-human primates and human immunodeficiency virus (HIV) - infected patients. To date, it remains unknown whether changes in IL-7R expression could also be linked to non-infectious inflammatory diseases such as asthma or anti-inflammatory drug use. Here, we investigated through flow cytometry the levels of IL-7R expression on CD4+ and CD4- T-cells in asthmatic patients in relation to disease severity, immune status and glucocorticoid (GC) use. In addition, we sought to evaluate the effects of in vivo and in vitro GC treatment on IL-7R expression in both asthmatic patients and SIV-infected non-human primates. We demonstrated that expression of IL-7R on peripheral blood CD4+ T-cells was significantly decreased in clinically stable GC-naive mild and moderate asthmatic patients. Accordingly, the development of asthmatic reaction following bronchial allergen challenge performed in sensitized subjects was associated with a significant drop in levels of IL-7R on circulating CD4+ T-cells. In contrast, CD4+ T-cells from both, mild and moderate, but not severe asthmatics, treated with inhaled GC displayed levels of IL-7R similar to that seen in healthy controls. We did not find significant differences with serum or sputum interleukin-7 levels among healthy controls and GC-naïve and GC-treated asthmatic patients. Furthermore, both in vitro GC treatment and short-term oral GC administration to asthmatic patients resulted in a significant enhancement of IL-7R. Similarly, we demonstrated that GC-stimulated T-cells from SIV-infected non-human primates up-regulated IL-7R expression. Accordingly, experimental short-term systemic in vivo administration of GC to SIV-infected macaques led to enhancement of IL-7R expression on circulating T-cells. Our data indicate that GC bear potential to recover diminished IL-7R expression, as well in asthma as in lentiviral infection.

Expression of NRF2 and NRF2-modulated genes in peripheral blood leukocytes of bladder cancer males.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is an oxidant-responsive transcription factor involved in induction of antioxidant genes. We assessed NRF2 and selected NRF2-modulated gene expression: glutathione S-transferase A1 and P1 (GSTA1 and GSTP1), mitochondrial superoxide dismutase (SOD2) in blood leukocytes of 51 bladder cancer patients and 90 control males. A significant up-regulation of SOD2 expression (P=0.002) was observed in leukocytes of patients. NRF2 expression was positively correlated with GSTP1 and with SOD2 mRNA level, both in patients and controls. These data suggest disturbances in SOD2 transcription in circulating blood leukocytes of males with bladder cancer. Moreover, concomitant constitutive expression of NRF2 and its target genes may suggest important role of NRF2 transcription factor in positive regulation of antioxidant genes, resulted in enhanced cytoprotection in human peripheral blood leukocytes.

Evaluation of TNF superfamily molecules release by neutrophils and B leukemic cells of patients with chronic B - cell lymphocytic leukemia.

It was demonstrated that TNF superfamily proteins may affect significantly the time of leukemic cells' survival in the course of B-cell chronic lymphocytic leukemia (B-CLL). The aim of our study was to evaluate the expression and release of BAFF (B-cell activating factor), APRIL (a proliferation-inducing ligand) and TRAIL (TNF-related apoptosis inducing ligand) molecules belonging to the cytokines of the superfamily of the tumor necrosis factor (TNF) by neutrophils (PMNs) and, for comparison, B cells isolated from the blood of patients with B-CLL vs. their concentration in the blood serum. 40 patients suffering from B-CLL and a control group of 15 healthy subjects were included in the study. Cytoplasmic fractions of PMNs and B cells were analyzed with the use of western blotting for the presence of TRAIL, BAFF and APRIL. Soluble TRAIL, BAFF and APRIL in the culture supernatants and the serum were assessed using ELISA kits. PMNs and B cells of patients with B-CLL before treatment demonstrated the statistically significantly higher expression of APRIL and BAFF proteins when compared with the control group of healthy subjects. In contrast, the expression of TRAIL protein in both types of cells of patients was statistically significantly lower than its expression in the control cells. In the supernatants of PMN and B lymphocytes of patients the decreased concentrations of sBAFF, unchanged of APRIL and increased of sTRAIL molecules were demonstrated. The results of studies carried out in patients with B-CLL before treatment indicate that the relations demonstrated between APRIL, BAFF and TRAIL molecules, released by neutrophils and B cells and relations between their concentrations in the serum can significantly influence the development of B-CLL.

Overexpression of B cell-activating factor (BAFF) in neutrophils of oral cavity cancer patients - preliminary study.

In the present study the expression of tumor-promoting B cell-activating factor (BAFF), a member of the tumor necrosis factor superfamily (TNF) in neutrophils from oral cavity cancer patients, was examined by real-time PCR. For the purpose of comparison, the expression of BAFF protein was assessed in autologous peripheral blood mononuclear cells (PBMCs). An important question of this study has also been to explain the role of NF-κB in the induction of BAFF molecule. The increased expression of BAFF at the mRNA and protein levels in neutrophils and mononuclear cells of patients before and after treatment were accompanied by the increased expression of phospho-IκB protein level. Demonstrated excessive expression and secretion of BAFF by examined leukocytes suggest a tumor-promoting activity of those cells in oral cavity cancer patients. The overexpression of BAFF, observed at mRNA and protein levels in PMNs and PBMCs, as well as the secretion of soluble form of sBAFF by these cells, accompany the increased concentrations of sBAFF in the serum of patients. Observations above suggest that the modulation of BAFF molecules in examined leukocytes and the levels thereof in the serum may have future implications for immunotherapy of oral cavity cancer patients.

Association between GPx1 Pro198Leu polymorphism, GPx1 activity and plasma selenium concentration in humans.

BACKGROUND: Glutathione peroxidase 1 (GPx1) is an antioxidant selenoenzyme that protects the cells against reactive oxygen species. Its activity depends on the concentration of selenium (Se) which is present in the active centre of the enzyme. The genetic polymorphism of GPx1 encoding gene (GPx1) associated with the proline (Pro) to leucine (Leu) change at codon 198 is supposed to be functional. An in vitro study performed on human breast carcinoma cell line showed that GPx1Leu allele was associated with a lower responsiveness of the enzyme to Se added to the culture medium. Some authors observed a decrease in GPx1 activity associated with GPx1 Leu allele in humans; however, there were no findings on how GPx1 activity changes with Se concentration in individuals with different GPx1 genotypes. AIM OF THE STUDY: To assess whether GPx1 activity that depends on the Se status may be influenced by GPx1 polymorphism through studying this relationship in the blood of healthy individuals. METHODS: The association between the Se status, GPx1 activity and GPx1 genotype was assessed in 405 individuals of Polish origin. GPx1 activity in red blood cells was measured by the spectrophotometric method by Paglia and Valentine, using t-butylhydroperoxide as the substrate. Plasma Se concentration was measured using graphite furnace atomic absorption spectrometry. GPx1 Pro198Leu polymorphism was determined with the Molecular Beacon Real-Time PCR assay. RESULTS: In the subjects examined, the mean plasma Se concentration was 54.4 +/- 14.2 mcg/L. The mean GPx1 activity was 15.1 +/- 4.7 U/g Hb. No difference regarding both the parameters was found between individuals with different GPx1 genotype. However, the association between GPx1 activity and Se concentration, analyzed separately for each genotype group, was not the same. The correlation coefficients amounted to r = 0.44 (p < 0.001) for Pro/Pro, r = 0.35 (p < 0.001) for Pro/Leu and r = 0.25 (p = 0.45) for Leu/Leu group, which indicates that the correlation strength was as follows: Pro/Pro > Pro/Leu > Leu/Leu. Notably, statistically significant difference in this relationship (analyzed as difference between correlation coefficients for linear trends) was found between genotypes Pro/Pro and Leu/Leu (p = 0.034). CONCLUSIONS: The findings of the present study provide evidence for the hypothesis based on in vitro studies which assumes that GPx1 Pro198Leu polymorphism has a functional significance for the human organism and that this functionality is associated with a different response of GPx1 activity to Se. They also point to the importance of the genetic background in the assessment of the Se status with the use of selenoprotein biomarkers such as GPx1 activity.

IL-17 family cytokines in neutrophils of patients with oral epithelial squamous cell carcinoma.

UNLABELLED: Epithelial squamous cell carcinoma is the predominant histological type among cancers of the head and neck. It is characterised by high growth dynamics and a low survival rate of patients. The usefulness of various markers and prognostic factors is assessed to improve treatment results in patients with epithelial squamous cell carcinoma.

The involvement of neutrophils (PMNs) in the neoplastic process and scarce evidence for the role of interleukin 17 (IL-17) family cytokines in these reactions encouraged us to undertake a study in this field. We assessed the expression and capability of neutrophils in patients with oral epithelial squamous cell carcinoma to release IL-17E and IL-17A in relation to their serum levels and expression of the specific receptors, i.e. IL-17R and IL-17BR. For the sake of comparison, the expressions of the proteins examined were assessed in autologous peripheral blood mononuclear cells (PBMCs). The levels were determined in the patients prior to treatment, taking into consideration the stage of the disease according to TNM classification.

Western blot analysis revealed no differences in the expressions of IL-17E and IL-17A either in PMNs or PBMCs of the patients as compared to the healthy subjects. However, the expressions of IL-17BR and IL-17R were found to be higher in both groups of cells in cancer patients as compared to the control. The use of ELISA method revealed that the levels of IL-17E and IL-17A were higher in cell supernatants and blood serum of the patients than of the healthy subjects. No differences were noted in the protein expression in the cells or concentration in supernatants of the patients with different stages of the disease.

Our findings as well as observations reported by other authors seem to indicate some new aspects of the biological role of IL-17 family cytokines, not only as markers of the inflammatory process but also as indicators of leukocyte activity in IL-17A and IL-17E-dependent reactions in patients with oral epithelial squamous cell carcinoma.

N-nitrosodimethylamine (NDMA) induced apoptosis dependent on Fas/FasL complex in human leukocytes.

OBJECTIVE: To investigate the mechanism of apoptosis dependent on the Fas/FasL (Fas ligand) complex in the presence of N-nitrosodimethylamine (NDMA) in human leukocytes. METHODS: Polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated form whole blood by density centrifugation. The concentration of NDMA was assessed by cellular toxicity assay. Apoptotic cells were assessed with flow cytometry and the expression of pro- and antiapoptotic proteins was investigated by Western blotting in PMNs and PBMCs treated with NDMA and/or FasL. RESULTS: PMNs showed a higher ratio of apoptotic cells than PBMCs after exposure to NDMA and/or FasL. Enhanced apoptosis was related to the increased expression of proapoptotic proteins in neutrophils following exposure to either NDMA or FasL. In PBMCs, the relation was observed after exposure to FasL only. PMNs and PBMCs incubated with NDMA and FasL simultaneously demonstrated the highest increase in protein expression. CONCLUSIONS: NDMA shows a stronger proapoptotic effect with PMNs than with PBMCs. The Fas/FasL complex, along with other proapoptotic proteins of the receptor (Fas, FADD) and mitochondrial pathway (Noxa, Puma, Bim), plays a key role in the induction of neutrophil apoptosis. Synergic effects of NDMA and FasL which lead to higher induction of apoptosis in PMNs than in PBMCs indicates a multistage and varied regulation of apoptosis in different populations of leukocytes.

Sex-specific differences in the regulation of inducible nitric oxide synthase by bisphenol A in neutrophils.

The aim of the study was to evaluate the effect of bisphenol A (BPA) on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression by neutrophils with regard to sex and nuclear factor-κB (NF-κB) pathway participation in this process. This study demonstrated that BPA intensifies the production of NO and the expression of iNOS in the cytoplasmic fraction of neutrophils of women as well as men. In addition, an enhanced expression of NF-κB in the cytoplasmic and nuclear fraction of neutrophils exposed to BPA was observed in the cells of both sexes. The lipopolysaccharide (LPS) stimulation of neutrophils of both sexes led to an intensification of NO production and expression of all tested proteins. However, simultaneous stimulation of neutrophils of both men and women with LPS and BPA decreased the production of NO and expression of iNOS and NF-κB in both fractions compared to the cells exposed only to xenoestrogen. Moreover, expression of iNOS and NF-κB was higher in female neutrophils than in male cells. This study demonstrated that BPA affects the production of NO with the participation of iNOS by human polymorphonuclear neutrophils. This process is associated with the activation of the NF-κB pathway. In addition, different activity of NF-κB in neutrophils, observed with respect to sex, indicates a different role of this pathway in female and male cells.

TRAIL receptors in the serum of patients with B-cell chronic lymphocytic leukemia.

Great importance in the course of chronic B-cell lymphocytic leukemia (B-CLL) has been ascribed to cytokines belonging to the superfamily of the tumor necrosis factor (TNF), including TRAIL (TNF-related apoptosis inducing ligand) and its specific receptors: TRAIL receptor 1 (TRAIL-R1), TRAIL receptor 2 (TRAIL-R2), TRAIL receptor 3 (TRAIL-R3), TRAIL receptor 4 (TRAIL-R4) and osteoprotegerin (OPG). Both the molecule and the receptors may occur in membrane and soluble forms, except for OPG which has only a soluble form. The aim of the study was to assess the levels of sTRAIL molecule and soluble TRAIL receptors - sTRAIL-R2 and OPG in the serum of patients with B-CLL. The findings revealed reduced concentrations of sTRAIL both before and after treatment and elevated levels of sTRAIL-R2 and OPG in patients before treatment. After treatment with CC (2CdA/Cladrybin and Cyklofosfamid) and FC (Fludarabin and Cyklofosfamid) we observed an increase in sTRAIL and a decrease in sTRAIL-R2. OPG levels were found to increase after treatment with CHOP (Vincristini, Cyklofosfamid, Adriamycin and Prednisol) and they decreased after administration of Leukeran (Chlorambucyl) and CMC (2CdA/Cladrybin, Mitoxanton and Cyklofosfamid). The relationships between TRAIL and its natural regulators in the serum of BCLL patients prior to treatment may impair apoptosis of leukemic B cells. Changes in these relationships after treatment with CC and FC seem to promote enhancement of apoptosis in these cells.

Cadmium, arsenic, selenium and iron- Implications for tumor progression in breast cancer.

The aim of this study was to determine Cd (cadmium) and As (arsenic) contents in human breast cancer tissues, investigate their interactions with Se (selenium) and Fe (iron), and assess their further implications for tumor progression. Metal contents were determined in 42 tissue sets (tumor and adjacent tissue) collected from 42 women diagnosed with primary breast cancer. Analytical methods included AAS and ICP-MS techniques. Significantly higher contents of Cd (p=0.0003), Se (p<0.0001) and Fe (p=0.0441) whereas significantly lower content of As (p<0.0001) were observed in tumors as compared to adjacent tissues. There was a significant positive correlation between Cd and As contents in tumor tissue. However, only Cd was significantly associated with histological type of tumor, its size, grading and progesterone receptor status. This study support the role of Cd in breast cancer risk and progression. The possible link between As exposure and breast cancer is still not clear.

Pentobarbital in tobacco.

The spectrometric analysis of extracts from tobacco and tobacco smoke revealed the presence of pentobarbital in the analyzed substances. Tobacco samples and tobacco smoke were extracted with chloroform, determinations were performed with the Perkin-Elmer Autosystem XL system, on a Turbo Mass spectrometer. Subject to analysis were 4 cigarette brands manufactured in Poland and raw, unprocessed tobacco. The presence of pentobarbital in the analyzed samples was confirmed by the analysis of the mass spectrum of the substance, as well as by comparison of retention time with standard of pentobarbital. The determined pentobarbital concentrations in tobacco amounted to 3-6 microg/cigarette, and in tobacco smoke they were approximately 45% lower. In case of tobacco extracts it can with high probability be excluded that pentobarbital is synthesized during chromatographical analysis. The presence of pentobarbital in tobacco is thus beyond question.

Effect of IL-18 on leukocyte expression of iNOS and phospho-IkB in patients with squamous cell carcinoma of the oral cavity.

The inducible synthase of nitric oxide (iNOS) is responsible for the synthesis of nitric oxide (NO) in neutrophils (PMN) and in peripheral blood mononuclear cells (PBMC). This enzyme is controlled by a number of cytokines which accomplish their biological effect via e.g. activation of NF-kB pathway. The aim of the present study was to assess the expression of iNOS and production of NO by PMN and PBMC in patients with oral cavity squamous cell carcinoma (SCC), and to examine the role of the NF-kB pathway in the IL-18-stimulated activation of this enzyme. The production of NO and iNOS expression in PMN were reduced, while iNOS expression in PBMC was increased but NO production by these cells remain unchanged. Patients after treatment showed lower intensity of iNOS expression compared to that observed before treatment. Moreover, both before and after treatment iNOS expression was inversely correlated with phospho-IkB expression in PMN and in PBMC. Significantly higher levels of total NO were observed in the serum of Stage IV patients before and after treatment as compared to the control group. Altered expression of iNOS and NO generation by PMN and PBMC may have an unfavorable effect on the course of antineoplastic response in patients with squamous cell carcinoma of the oral cavity. Intensification of iNOS expression and NO production in Stage IV patients, induced by rhIL-18, suggests its beneficial effect on the activity of leukocytes in patients with squamous cell carcinoma of the oral cavity.

Structure, mechanical characteristics and in vitro degradation, cytotoxicity, genotoxicity and mutagenicity of novel biodegradable Zn-Mg alloys.

Zn-(0-1.6)Mg (in wt.%) alloys were prepared by hot extrusion at 300 °C. The structure, mechanical properties and in vitro biocompatibility of the alloys were investigated. The hot-extruded magnesium-based WE43 alloy was used as a control. Mechanical properties were evaluated by hardness, compressive and tensile testing. The cytotoxicity, genotoxicity (comet assay) and mutagenicity (Ames test) of the alloy extracts and ZnCl2 solutions were evaluated with the use of murine fibroblasts L929 and human osteosarcoma cell line U-2 OS. The microstructure of the Zn alloys consisted of recrystallized Zn grains of 12 µm in size and fine Mg2Zn11 particles arranged parallel to the hot extrusion direction. Mechanical tests revealed that the hardness and strength increased with increasing Mg concentration. The Zn-0.8 Mg alloys showed the best combination of tensile mechanical properties (tensile yield strength of 203 MPa, ultimate tensile strength of 301 MPa and elongation of 15%). At higher Mg concentrations the plasticity of Zn-Mg alloys was deteriorated. Cytotoxicity tests with alloy extracts and ZnCl2 solutions proved the maximum safe Zn(2+) concentrations of 120 µM and 80 µM for the U-2 OS and L929 cell lines, respectively. Ames test with extracts of alloys indicated that the extracts were not mutagenic. The comet assay demonstrated that 1-day extracts of alloys were not genotoxic for U-2 OS and L929 cell lines after 1-day incubation.

Treatment of Persistent Hypercalcemia and Hyperparathyroidism With Cinacalcet After Successful Kidney Transplantation.

INTRODUCTION: Hypercalcemia caused by persistent hyperparathyroidism after successful kidney transplantation (KT) is a common problem and may negatively affect graft function, bone metabolism and the cardiovascular system. Cinacalcet is a novel, available tool to control hypercalcemia after KT. The aim of the study was to examine the efficacy of cinacalcet in lowering calcium in KT recipients with persistent hypercalcemia owing to hyperparathyroidism. METHODS: In this retrospective observational study, we analyzed 30 patients with persistent hypercalcemia >10.8 mg/dL. All patients in the study were started on cinacalcet at different points after KT, with the mean time of 43 ± 37 months. The initial dose of 30 mg/d was adapted progressively based on serum calcium levels. RESULTS: During the observation period, graft function in all patients was stable (estimated glomerular filtration rate [Chronic Kidney Disease Epidemiology Collaboration formula] 64 ± 25 mL/min/1.73 m(2)). The mean baseline calcemia was 11.9 ± 0.7 mg/dL, the intact parathyroid hormone value was 490 ± 228 pg/mL and phosphorus concentration was 2.2 ± 0.5 mg/dL. Treatment with cinacalcet resulted in a significant decrease in serum calcium level (mean, 9.9 ± 0.7 mg/dL; P < .001), a reduction in intact parathyroid hormone level (308 ± 199 pg/dL; P < .001), and an increase in phosphorus concentration (mean, 2.8 ± 0.6 mg/dL; P < .001). In 5 females, gastrointestinal side effects were observed, requiring withdrawal of cinacalcet in 1 case. CONCLUSIONS: Cinacalcet administered after KT seems to be an effective option for the management of persistent hypercalcemia owing to hyperparathyroidism with satisfactory tolerability and may be considered as a therapeutic alternative to surgical parathyroidectomy or as a bridging therapy to parathyroidectomy.

FOXO1 is a TXN- and p300-dependent sensor and effector of oxidative stress in diffuse large B-cell lymphomas characterized by increased oxidative metabolism.

Molecular profiling has led to identification of subtypes of diffuse large B-cell lymphomas (DLBCLs) differing in terms of oncogenic signaling and metabolic programs. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation. As increased oxidative metabolism leads to overproduction of potentially toxic reactive oxygen species (ROS), we sought to identify mechanisms responsible for adaptation of OxPhos cells to these conditions. Herein, we describe a mechanism involving the FOXO1-TXN-p300 redox-dependent circuit protecting OxPhos-DLBCL cells from ROS toxicity. We identify a BCL6-dependent transcriptional mechanism leading to relative TXN overexpression in OxPhos cells. We found that OxPhos cells lacking TXN were uniformly more sensitive to ROS and doxorubicin than control cells. Consistent with this, the overall survival of patients with high TXN mRNA expression, treated with doxorubicin-containing regimens, is significantly shorter than of those with low TXN mRNA expression. TXN overexpression curtails p300-mediated FOXO1 acetylation and its nuclear translocation in response to oxidative stress, thus attenuating FOXO1 transcriptional activity toward genes involved in apoptosis and cell cycle inhibition. We also demonstrate that FOXO1 knockdown in cells with silenced TXN expression markedly reduces ROS-induced apoptosis, indicating that FOXO1 is the major sensor and effector of oxidative stress in OxPhos-DLBCLs. These data highlight dynamic, context-dependent modulation of FOXO1 tumor-suppressor functions via acetylation and reveal potentially targetable vulnerabilities in these DLBCLs.