Immunoaging - the effect of age on serum levels of NET biomarkers in men: a pilot study.

OBJECTIVES: The study aimed to evaluate the impact of aging on the formation of neutrophil extracellular traps (NETs). The impaired formation of NETs is the cause of an abnormal innate immune response. MATERIAL AND METHODS: The study included a total of 45 healthy male subjects of different age groups. Whole blood was collected from the subjects, and the concentration of myeloperoxidase (MPO), the main biocidal protein in NETs, was determined in serum using ELISA. The serum levels of circulating free DNA (cfDNA), which are the structural basis of NETs, were also measured by fluorescence. In addition, the white blood cell count was determined, whole blood smear was evaluated, and the neutrophillymphocyte ratio was calculated. The variations in the levels of NET biomarkers were analyzed in different age groups. RESULTS: The low levels of MPO (243.70 ng/ml) and cfDNA (6.24 ng/100 µl) in boys indicated neutrophil insufficiency for NETosis in children. A progressive increase in the levels of MPO and cfDNA with age was observed among adolescents (420.91, p = 0.04; 13.55, p = 0.03, respectively), with the highest level noted in the healthy adult group (466.58, p = 0.01; 14.07, p = 0.01, respectively). The levels of the studied parameters were comparable in adolescents and young adults, which proved that the NETosis process was appropriate and suggested the attainment of neutrophil maturity for the release of NETs in adolescence. The levels of MPO and cfDNA were low in older men (225.46, p < 0.01; 5.19, p < 0.01, respectively) indicating impaired NET formation. CONCLUSIONS: Data on the generation of NETs in different age groups obtained in this study can allow a better understanding of the ontogenesis of the immune system in terms of the course of NETosis, and also indicate the need to support nonspecific responses in children and adults. Further research should be performed to determine the possibility of regulating the NETosis process. Int J Occup Med Environ Health. 2023;36(3):333-48.

The diagnostic potential of novel biomarkers of hypertension in men.

Introduction: The present study aimed to evaluate the diagnostic usefulness of selected novel parameters as biomarkers of hypertension: miR-145-5p, miR-1-3p, miR-423-5p, PCSK9, MyBPC3, NOX1, and CYBb, and NCF2, DNase 1, anti-MPO and anti-PR3 antibodies. Methods: We present the data of men with normal blood pressure, diagnosed hypertension, confirmed hypertension, and hypertension and coexisting coronary artery disease. Results: Elevated levels of miR-145-5p, miR-1-3p, and miR-423-5p and high levels of PCSK9, MyBPC3, and DNase 1 were observed in all groups of hypertensive men. We showed decreased levels of NOX1 and CYBb, and an elevated level of NCF2. Conclusions: PCSK9 shows the greatest potential as an early biomarker of screening-detected hypertension.

Bisphenol A: Potential Factor of Miscarriage in Women in the Context of the Phenomenon of Neutrophil Extracellular Traps.

Humans are exposed to a number of environmental pollutants every day. Among them, endocrine disruptors are particularly harmful to human health. Bisphenol A (BPA) is a xenoestrogen that has been shown to disrupt the endocrine system and cause reproductive toxicity. In this study, we aimed to verify the potential relationship between BPA and miscarriage involving the formation of neutrophil extracellular traps (NETs). Blood samples were collected from healthy women and women who had miscarriage in the first trimester of pregnancy. The serum levels of cytoplasmic anti-PR3 antibody and perinuclear anti-MPO antibody were determined using an immunoenzymatic method. The concentrations of key proinflammatory proteins TNF-α and MCP-1, as well as NADPH oxidase subunits NOX1 and NCF2, were also measured in the serum samples. The serum concentration of BPA was determined using gas chromatography. The results showed that the concentrations of BPA were significantly elevated in the serum of women who had miscarriage compared to the control group, with the highest concentration found in the "NETs-positive" group. The levels of MCP-1 and TNF-α were significantly higher in the "NETs-positive" group compared to the "NETs-negative" and control group. The levels of NOX1 and NCF2 were also higher in the "NETs-positive" group compared to the "NETs-negative" group. The study showed that BPA could play a role in the course of miscarriage through the formation of NETs. The results indicate the need to limit the exposure of women planning pregnancy to xenoestrogens, including BPA.

The promoting effect of neutrophil-derived BAFF molecule on the proliferation and life span of CAL-27 oral squamous carcinoma cells.

Tumor-associated neutrophils (TANs) are the major cellular component of the tumor microenvironment and have been shown to release of different bioactive molecules such as B-cell activating factor (BAFF). The data on the interactions between OSCC cells and neutrophils are limited and do not explain the actual role of the BAFF in the development of the OSCC. In the present study we examined the direct effect of neutrophils-derived BAFF on the OSCC cell line CAL-27 proliferation and apoptosis. PMNs of OSCC patients and healthy control were isolated from whole blood and separated by magnetic selection with monoclonal anti-human CD16 antibodies. CD-16 - positive neutrophils were incubated in the presence of TGF-ß and/or LPS as well as flavonoids (luteolin and quercetin). CAL-27 cells were co-incubated with supernatants of neutrophils. BAFF expression in neutrophils, BAFF-R expression on CAL-27 cells and apoptosis of CAL-27 cells were assessed by flow cytometry. To determine the CAL-27 cells proliferation, the MTT test was used. Expression of select mitochondrial proteins in CAL-27 cells were measured by Western blot. Neutrophils from OSCC patients showed significantly higher expression of BAFF than those from the healthy controls. The results obtained revealed upregulation of the proliferation and downregulation of the apoptosis of the CAL-27 cells in the presence of the supernatants of TGF-ß-treated neutrophils. Flavonoids reduced BAFF expression in neutrophils of patients with OSCC and control group. Lower intensity of apoptosis in CAL-27 cells was associated with the increased expression of anti-apoptotic Bcl-2, Mcl-1 and activated form of PI3K kinase (pPI3K) and simultaneously reduced expression of pro-apoptotic Bax protein in the presence of rhBAFF, as well as of supernatants of neutrophils derived from OSCC patients. In conclusion, the data presented confirm the previously suggested role of neutrophil-derived BAFF in OSCC development. The favorable effects of examined flavonoids on tumor-promoting BAFF expression in neutrophils suggest that they might be promising candidates as chemo-preventive agents in the therapy of patients with OSCC.

Investigation of estrogen-like effects of parabens on human neutrophils.

This study investigated the estrogen-like effects and mechanism of action most commonly used parabens: methyl- (MeP), ethyl- (EtP), propyl- (PrP) and butylparaben (BuP) in human neutrophils. Neutrophils were isolated from 50 blood donors, pre-incubated with antagonists of estrogen receptor α (ERα), ERß and G-protein coupled estrogen receptor 1 (GPER), then incubated with MeP, EtP, PrP, BuP and 17ß-estradiol (E2; 10 nM). Cytotoxic effect was evaluated by MTT test. Neutrophils apoptosis, necrosis and NETs formation were assessed in flow cytometry and confocal microscopy. The ability of the neutrophils for chemotaxis, phagocytosis, NADPH oxidase activity and generation of superoxide anion was assessed in Boyden's chamber, Park's method with latex, the NBT test, and reduction of cytochrome C, respectively. The total nitric oxide concentration was measured in neutrophils supernatants by the Griess reaction. The expression of cathepsin G, neutrophil elastase, proteinase 3, ERα, ERß and GPER was assessed in Western blot method. In our research, parabens did not cause a cytotoxic effect on human neutrophils nor affect their lifespan. Parabens exposure did not change neutrophils functions (chemotaxis, phagocytosis, NETs formation and oxygen-dependent killing mechanism) and expression of estrogen receptors. Our results suggest that parabens do not cause estrogen receptor-mediated neutrophils-related effects at concentrations measured in the plasma of individuals using products preserved with parabens.

NETs biomarkers in saliva and serum OSCC patients: One hypothesis, two conclusions.

PURPOSE: The actual role of neutrophils and neutrophil extracellular traps (NETs) in the course of cancer has not been clearly defined. The aim of this study was to determine the clinical usefulness of NETs biomarkers in saliva in confrontation with the blood serum and tumor tissue as a potential prognostic and therapeutic target in patients with oral squamous cell carcinoma (OSCC). MATERIAL AND METHODS: Expression of myeloperoxidase (MPO), and histones H2A, H2B, H3 in the tumor tissue, was investigated using immunohistochemistry. The expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits: p47-phox, p67-phox (neutrophil cytosolic factor 2, NCF2) and panRac, as well as citrullinated histone H3 (CitH3) in peripheral blood neutrophil lysates, was assessed via Western blot. ELISA tests were employed to measure the concentrations of circulating free DNA (cfDNA) and MPO in saliva only, and NOX1, NCF2, DNASE1 in saliva and serum. RESULTS: Extracellular expression of MPO and histones was localized within tumor tissue. Significantly lower expression of p67-phox, panRac, and CitH3 was determined in OSCC patients. Considerably lower concentrations of NOX1, NCF2, and DNASE1 in the saliva samples of cancer patients were observed. However, the levels of NOX1, NCF2, and DNASE1 in the serum of patients with cancer were substantially higher. CONCLUSIONS: The results obtained from the saliva of cancer patients suggest an impairment of the immunological homeostasis within the oral cavity related to NET formation, the causes of which should be sought in deficient activation of NADPH oxidase.

Novel Dual PI3K/mTOR Inhibitor, Apitolisib (GDC-0980), Inhibits Growth and Induces Apoptosis in Human Glioblastoma Cells.

Deregulated PI3K/AKT/mTOR signalling commonly exists in glioblastoma, making this axis an attractive target for therapeutic manipulation. Given that activation of PI3K/AKT/mTOR promotes tumour growth, metastasis, and resistance to anticancer therapies, mTOR inhibitors show promise in the treatment of cancer. The aim of this study was to investigate the underlying mechanism of novel dual PI3K/mTOR inhibitor, Apitolisib (GDC-0980), in A-172 and U-118-MG GBM tumour cell line suppression. It has been demonstrated that GDC-0980 induces time- and dose-dependent cytotoxicity and apoptosis in investigated glioma cell lines. In our study, the strongest induction of apoptosis was exhibited in the A-172 line after 48 h of incubation with 20 µM GDC-0980, where we observed 46.47% of apoptotic cells. In conclusion, we first discovered that dual PI3K/mTOR blockade by GDC-0980 markedly suppressed survival of human GBM cells and induced apoptosis, independent of the ER stress-mediated DR5 activation. We suggest that GDC-0980, by exerting an inhibitory effect on PERK expression, may thus block its inhibitory effect on protein synthesis, leading to intensification of translation, and this may result in an increase in apoptosis. On the other hand, CHOP stimulates protein synthesis and increases apoptosis. These findings suggest that GDC-0980 may be a candidate for further evaluation as a chemotherapeutic agent for anti-GBM therapy.

Methylparaben-induced regulation of estrogenic signaling in human neutrophils.

Parabens, including the most common methylparaben (MeP), are popular preservatives, which possess estrogenic activity. The aims of this study were to assess the impact of MeP on estrogen receptors (ERs) and/or NF-κB-dependent generation of IL-8 and production of nitric oxide (NO), and also to verify the hypothesis about the crosstalk of ERs with NF-κB in xenoestrogen-exposed neutrophils. Human neutrophils were incubated for 20-h with MeP (0.06 µM) and/or ER antagonist (1 µM) and/or NF-κB inhibitor (100 µM). After the isolation of cell lysates and cytoplasmic and nuclear fraction, the expression of ERα, ERß, p-IKKα/ß, p65 NF-κB, and inducible nitric oxide synthase (iNOS) was measured by Western blot analysis, The concentration of NO was evaluated by Griess reaction, and that of IL-8 was measured by ELISA. The results showed that MeP modulated the expression of ERα, but not ERß. Exposure to paraben activated iKKα/ß-dependent NF-κB pathway, but translocation of p65 NF-κB into the cell nucleus was inhibited by ERs. MeP also decreased the iNOS-dependent production of NO, but did not influence the secretion of IL-8 by neutrophils. The study indicates that MeP may affect the functioning of human neutrophils by modulating intracellular signal transduction pathways, including ERs and NF-κB pathway.

NF-κB-An Important Player in Xenoestrogen Signaling in Immune Cells.

The proper functioning of the immune system is critical for an effective defense against pathogenic factors such as bacteria and viruses. All the cellular processes taking place in an organism are strictly regulated by an intracellular network of signaling pathways. In the case of immune cells, the NF-κB pathway is considered the key signaling pathway as it regulates the expression of more than 200 genes. The transcription factor NF-κB is sensitive to exogenous factors, such as xenoestrogens (XEs), which are compounds mimicking the action of endogenous estrogens and are widely distributed in the environment. Moreover, XE-induced modulation of signaling pathways may be crucial for the proper development of the immune system. In this review, we summarize the effects of XEs on the NF-κB signaling pathway. Based on our analysis, we constructed a model of XE-induced signaling in immune cells and found that in most cases XEs activate NF-κB. Our analysis indicated that the indirect impact of XEs on NF-κB in immune cells is related to the modulation of estrogen signaling and other pathways such as MAPK and JAK/STAT. We also summarize the role of these aspects of signaling in the development and further functioning of the immune system in this paper.

Levels of Biological Markers of Nitric Oxide in Serum of Patients with Mandible Fractures.

BACKGROUND: Nitric oxide is a small gaseous molecule with significant bioactivity. It has been observed that NO may have a dual role dependent on its production and concentrations in the bone microenvironment. The objective of the study was to assess the concentration of total nitric oxide malonyldialdehyde, nitrotyrosine, and asymmetric dimethylarginine in the serum of patients with mandibular fractures and to understand the relationship between these compounds, in order to expand the knowledge base of the role of nitric oxide and its activity indicators in the process of bone fracture healing. MATERIAL AND METHODS: The study included 20 patients with mandibular fractures who were undergoing inpatient and outpatient treatments and a control group of 15 healthy people. Results were analyzed with respect to the measurement time. Total nitric oxide concentration in the blood serum was determined according to the Griess reaction, while the concentration of malonyldialdehyde, nitrotyrosine, and asymmetric dimethylarginine was estimated using the immunoenzymatic method (i.e., enzyme-linked immunosorbent assay). RESULTS: Before the procedure, as well as on the first day and 2 and 6 weeks after the procedure, higher concentrations of total nitric oxide and lower concentrations of malonyldialdehyde were observed in the blood serum of patients with mandibular fractures compared to the control group. No statistically significant differences were found in nitrotyrosine concentrations in the blood serum of patients throughout the measurement period. However, a significantly higher asymmetric dimethylarginine concentration was observed in the patient serum before the procedure and on the first day of operation as compared with the control group. Analysis of the results observed in patient serum with respect to the number of fractures within the mandible demonstrated the same trend of concentrations for the tested compounds for the entire study group. CONCLUSIONS: In summary, our results revealed that the intensity of local processes resulting from mandibular fractures is associated with the concentration of nitric oxide, confirming its significant role, as well as that of its indicators, in the process of bone fracture healing in this patient population.

Profile of new vascular damage biomarkers in middle-aged men with arterial hypertension.

PURPOSE: Normal endothelial function is important for the homeostasis of the cardiovascular (CV) system. The aim of the present study was to determine the profile of key parameters of endothelial dysfunction in middle-aged men that play a significant role in the functioning of endothelial vessels, which seems to be crucial for the early diagnosis of cardiovascular disorders. MATERIALS AND METHODS: The study included 53 men, 20 with hypertension (HTN), 18 with HTN and related diseases, 15 healthy controls Apart from general testing (BMI, biochemical analysis, SBP, DBP), we used the Griess reaction to assess the total amount of nitric oxide (NO), and used ELISA to verify the concentrations of malondialdehyde (MDA), nitrotyrosine (NT), asymmetric dimethylarginine (ADMA), tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein 1 (MCP-1), and myeloperoxidase (MPO). Furthermore, we assessed the concentration of circulating free DNA (cfDNA) using the fluorescence method. RESULTS: The values of MDA, ADMA, cfDNA, and MPO observed in samples from men with HTN were determined to be higher compared to those from men without HTN. In the group of men with HTN and other concomitant cardiovascular disorders, we observed low concentrations of NO, MDA, and ADMA with high concentrations of cfDNA. CONCLUSIONS: The results obtained for parameters selected for the study, should be considered by cardiologists as a prompt to include in the diagnostic profile the assessment of NO and cfDNA concentrations for risk evaluation and/or diagnosis of endothelial dysfunction in patients suffering from HTN or related complications.

Sex-dependent dysregulation of human neutrophil responses by bisphenol A.

BACKGROUND: In the present study, we aimed to investigate selected functions of human neutrophils exposed to bisphenol A (BPA) under in vitro conditions. As BPA is classified among xenoestrogens, we compared its action and effects with those of 17ß-estradiol (E2). METHODS: Chemotaxis of neutrophils was examined using the Boyden chamber. Their phagocytosis and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase activity were assessed via Park's method with latex beads and Park's test with nitroblue tetrazolium. To assess the total concentration of nitric oxide (NO), the Griess reaction was utilized. Flow cytometry was used to assess the expression of cluster of differentiation (CD) antigens. The formation of neutrophil extracellular traps (NETs) was analyzed using a microscope (IN Cell Analyzer 2200 system). Expression of the investigated proteins was determined using Western blot. RESULTS: The analysis of results obtained for both sexes demonstrated that after exposure to BPA, the chemotactic capacity of neutrophils was reduced. In the presence of BPA, the phagocytic activity was found to be elevated in the cells obtained from women and reduced in the cells from men. Following exposure to BPA, the percentage of neutrophils with CD14 and CD284 (TLR4) expression, as well as the percentage of cells forming NETs, was increased in the cells from both sexes. The stimulatory role of BPA and E2 in the activation of NADPH oxidase was observed only in female cells. On the other hand, no influence of E2 on the expression of CD14 and CD284, chemotaxis, phagocytosis, and the amount of NET-positive neutrophils was found for both sexes. The study further showed that BPA intensified NO production and iNOS expression in the cells of both sexes. In addition, intensified expression of all tested PI3K-Akt pathway proteins was observed in male neutrophils. CONCLUSIONS: The study demonstrated the influence of BPA on neutrophil functions associated with locomotion and pathogen elimination, which in turn may disturb the immune response of these cells in both women and men. Analysis of the obtained data showed that the effect of this xenoestrogen on the human neutrophils was more pronounced than E2.

Controversy around parabens: Alternative strategies for preservative use in cosmetics and personal care products.

Parabens usage as preservatives in cosmetics and personal care products have been debated among scientists and consumers. Parabens are easy to production, effective and cheap, but its safety status remains controversial. Other popular cosmetics preservatives are formaldehyde, triclosan, methylisothiazolinone, methylchloroisothiazolinone, phenoxyethanol, benzyl alcohol and sodium benzoate. Although their high antimicrobial effectiveness, they also exhibit some adverse health effects. Lately, scientists have shown that natural substances such as essential oils and plant extracts present antimicrobial potential. However, their use in cosmetic is a challenge. The present review article is a comprehensive summary of the available methods to prevent microbial contamination of cosmetics and personal care products, which can allow reducing the use of parabens in these products.

LDGs versus NDGs in patients with oral squamous cell carcinoma (OSCC).

BACKGROUND: The significant role played by neutrophils in cancer biology is indisputable; yet, their subpopulations may exhibit a contrasting role. The phenomenon of polarization of neutrophils and signaling modulators in the course of a neoplastic process has gained increased attention in recent times. The present study's objective was to quantitatively assess low-density neutrophils (LDNs) and normal-density neutrophils (NDNs) populations including IL-17 expression in confrontation with Th17 lymphocytes in patients with oral squamous cell carcinoma (OSCC). The neutrophil to lymphocyte ratio (NLR) biomarker value was determined. Besides, the influence of rhIL-17 on the proliferation level of the squamous cell carcinoma (SCC) malignant line cells was tested. METHODS: Leukocytes were isolated in the density gradient and the CD16+ population was magnetically sorted. The percentages of neutrophil subpopulations, lymphocyte Th17, and IL-17 expression in the studied cells were determined on a flow cytometer. Squamous cell carcinoma proliferation was assessed with the MTT test. RESULT: The existence of two populations of human neutrophils was determined: LDNs and NDNs. A higher percentage of LDNs and Th17 was observed with the concomitant lower percentage of NDNs in patients with OSCC as compared with the control group. NLR was elevated in patients with cancer. The highest IL-17 expression was obtained in the LDNs population in these patients. However, no influence of IL-17 on SCC proliferation could be determined. CONCLUSION: The present study demonstrated a strong relationship between IL-17 concentration and the count of LDNs or Th17 in the course of OSCC, which may serve as a reference point for new therapies. Moreover, the obtained LDNs/NDNs and NLR values in patients with cancer prove their usefulness in diagnostic and prognostic in patients with OSCC.

Cancers Cells in Traps? The Pathways of NETs Formation in Response to OSCC in Humans-A Pilot Study.

The aim of the experiment was to evaluate the process of neutrophil extracellular traps (NETs) formation in patients with oral squamous cell carcinoma (OSCC) in response to direct or indirect contact with SCC cells in comparison to results obtained in the cells of healthy subjects. To fulfill study objectives CAL 27 cell line and blood were obtained from cancer patients and control subjects. Parameters related to NETs formation were analyzed utilizing flow cytometry, fluorescence microscopy, and ELISA-type tests. The expression of selected phosphorylated proteins of the PI3K/Akt/PBK pathway in neutrophils was evaluated using the Western blot method. An increase in NETs formation was observed in a coculture of neutrophils with SCC cells, with the largest amount of NETs formed after stimulation with a supernatant obtained from the SCC culture. The enhanced process of NETs formation was accompanied by changes in the expression of proteins from the PI3K/Akt/PBK pathway. The obtained results prove the existence of interactions between neutrophils and cancer cells resulting in NETosis with the participation of the PI3K/Akt/PBK pathway in patients with OSCC.

A Proliferation-Inducing Ligand Regulation in Polymorphonuclear Neutrophils by Panax ginseng.

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor superfamily that was first identified as a factor favoring tumorigenesis. APRIL is important fitness and survival factors for B cells and plasma cells in the periphery. Considering this, as well as the quantitative predominance of neutrophils among the peripheral blood leukocytes, we carried out the first study assessing the influence of the transforming growth factor (TGF)-ß signaling pathway on APRIL expression in these cells. Furthermore, as the Rb1 ginsenoside is known to exhibit multiple pharmacological activities, we verified if the saponin is capable of modulating the process. The present study shows that TGF-ß increased the expression of APRIL and the level of phospho-p38, phospho-Akt(T308), and phospho-Akt(S473) in the cytoplasmic fraction, as well as the expression of Fra1, c-Fos, and c-Jun in the nuclear fraction, of neutrophils. However, exposure of these cells to Rb1 reduced the expression and level of the investigated proteins. No changes were found in the expression of APRIL and the level of p-p38 in the cytoplasmic fraction of neutrophils following the application of Rb1 alone, as well as in the neutrophils incubated first with Rb1 and then with TGF-ß, whereas a higher level of phosphorylation was observed for Akt and PI3 kinases in the cells. Moreover, a higher expression of all the studied transcription factors was observed in the nuclear fraction of neutrophils. Based on the observed changes, it may be assumed that the expression of APRIL molecule in TGF-ß-induced neutrophils and its regulation by Rb1 are associated with PI3K/AKT signaling pathways and transcription factors Fra-1, Fra-2, c-Jun, and c-Fos. Rb1 appears to be a favorable factor that may be potentially used in the modulation of tumor-promoting APRIL expression.

Biomarkers of neutrophil extracellular traps (NETs) and nitric oxide-(NO)-dependent oxidative stress in women who miscarried.

Pregnancy loss is a multidisciplinary problem which concerns researchers from the fields of medicine, epidemiology, psychology, and public health. The primary objective of the present study was to explain the potential role of neutrophil extracellular traps (NETs) in the process of spontaneous miscarriage. Enzyme-linked immunosorbent assay to assess the levels of biomarkers of NETs in the serum of examined women was conducted. Furthermore, levels of nitric oxide (NO) and late markers of its action were measured in serum samples. Analyses results demonstrated the existence of NETs in the placental tissue of women who miscarried as well as a simultaneous increase in the levels of myeloperoxidase and pentraxin 3. This clearly confirms the participation of NETs in the course of pregnancy loss. Women who have had a miscarriage but did not show the presence of NETs in their placenta exhibited the highest contents of NO, nitrotyrosine, and malondialdehyde suggesting a different pathway leading to pregnancy loss associated with disturbed oxidative-antioxidative processes. Although study results demonstrate new aspects associated with the formation of NETs they are not, however, sufficient to unambiguously determine the role of NETs in the course of miscarriage.

Effect of bisphenol A on human neutrophils immunophenotype.

Neutrophils (PMN) play a key role in eliciting congenital immune response. These cells are equipped with specific receptors that are located on the surface of their cell membrane. These receptors produce various signals which in turn help in the effective functioning of PMN. The activity of these cells may be modified by factors of endo- and exogenous origin, including xenoestrogens such as bisphenol A (BPA). The aim of this study was to evaluate the effect of BPA on the expression of CD11c, CD14, CD15, CD16, CD62L and CD284 compounds on the surface of neutrophils in women and men. The study material included PMN isolated from the whole blood. The cells were incubated in the presence of BPA and/or LPS. Flow cytometry technique was used to evaluate the expression of CD antigens. Studies of these receptors indicate that BPA, at a concentration corresponding to the serum level of this compound in healthy subjects as well as at higher doses, induces changes in the immunophenotype of PMN, which may lead to immunity disorders associated with the dysfunction of these cells. Moreover, the observed effects of xenoestrogen on the expression of CD11c, CD14, CD15, CD16, CD62L and CD284 differentiation markers on these cells are sex-independent.

Neutrophil extracellular traps (NETs) formation induced by TGF-ß in oral lichen planus - Possible implications for the development of oral cancer.

Oral Potentially Malignant Disorders (OPMDs) including Oral Lichen Planus (OLP) are associated with risk of transformation to oral squamous cell carcinoma (OSCC). Available data show that innate immune cells involving polymorphonuclear neutrophils (PMNs) with their ability to neutrophil extracellular traps (NETs) formation are likely to be directly involved in development of cancer. Examination of NETs generation by TGF-ß - induced neutrophils of OLP patients showed increased amounts of traps with MPO, H3Cit and cfDNA, known to be released with NETs. The presence of excessive amounts of NETs components may lead to numerous adverse consequences associated with potential transformation to OSCC. Bacterial-related infection may enhance the NETs formation and lead to consequences resulting from the excessive number of individual elements of these networks. It is likely that regulating NETs release by the flavonoids presented herein may be beneficial not only for inhibiting OLP development, but also in reducing risk of transformation to OSCC.

Identification of a novel target for the action of endocrine disrupting chemicals: inhibitory effect of methylparaben on human neutrophil functions.

This study was conducted to verify a hypothesis that immune cells are a target for the action of endocrine disrupting chemicals (EDCs) by investigating whether methylparaben (MeP) modulates human neutrophil functions. Neutrophils isolated from 15 donor samples were studied. Cells were incubated in the presence of increasing MeP concentrations (0.06, 0.8, 10, and 20 µM). The cytotoxic effect of MeP on neutrophils was evaluated by the MTT test. The ability of the neutrophils for chemotaxis, phagocytosis, NADPH oxidase activity, and superoxide anion generation was assessed in Boyden's chamber, Park's method with latex, the NBT test, and the cytochrome C reduction test, respectively. The total nitric oxide (NO) concentration was measured by the Griess reaction. There was no observable cytotoxic effect of MeP on human neutrophils. MeP (10 and 20 µM) exposure decreased neutrophilic ability for the tested functions, except for NO production. In neutrophils incubated with MeP (0.8 µM as well as 0.06 and 0.8 µM, respectively), we observed a decreased activity of NADPH oxidase as well as decreased generation of superoxide anion. Our results suggest a suppressive effect of MeP on the tested functions of human neutrophils, which confirms that immune cells are vulnerable to EDC action. Therefore, the disturbance of neutrophils' oxygen-dependent phagocytic function as a result of exposure to environmental doses of MeP action could lead to impairment of innate immune responses in humans exposed to xenoestrogens.