Regulation of NO production by MAPK dual-specificity phosphatases (DUSP) in human neutrophils exposed to N-nitrosodimethylamine

437-444One of the enzymes responsible for nitric oxide (NO) production in neutrophils is the inducible nitric oxide synthase (iNOS). Changes in its expression may result from the activation of different signaling pathways, including MAPK, which lead to activation of various genes, including DUSP genes. DUSP induce the negative feedback loop leading to MAPK deactivation through their phosphorylation. Our study assessed the role of DUSP1, DUSP10 and DUSP16 with the participation of MAPK in the iNOS-dependent NO production by neutrophils exposed to xenobiotic, N-nitrosodimethylamine (NDMA). The obtained results suggest that N-nitrosodimethylamine enhances the expression of all tested proteins (except DUSP10) in the cytoplasmic and nuclear fractions of neutrophils. The JNK pathway inhibition resulted in an extenuation of iNOS, phospho-p38 and DUSP10 expression in the cytoplasmic fraction and DUSP1 expression in the nuclear fraction of neutrophils. Inhibition of the p38 pathway led to a lower expression of iNOS, DUSP16 and DUSP10 in the cytoplasmic fraction. No changes in the phospho-JNK and DUSP1 expressions were observed. With the results of this study we can conclude that DUSP are positive regulators of MAP kinases in NDMA-induced signaling pathway which lead to modulation of iNOS-dependent NO production in human neutrophils

Wpływ rhIL-17 na ekspresję TLR2 i białek apoptotycznych w ludzkich neutrofilach

Introduction. Neutrophils (PMNs) apoptosis occurs involving proteins of the Bcl-2 family, which control the permeability of the outer mitochondrial membrane. Proteins of this family are regulated by various factors, among others cytokine. Early observations of their own show the relationship between increased apoptosis and expression of Toll-like receptor (TLR2) in these cells stimulated by IL-17. The aim of the planned study was to investigate the effect of rhIL-17 on selected proteins of the Bcl-2 family, such as: the proapoptotic Bax protein and antiapoptotic Mcl-1 protein in relation to the expression of TLR2 receptor. Reference to the analysed results was to investigate the influence of fMLP on estimated parameters. Material and method. The study was conducted on human PMN isolated from peripheral blood. Apoptosis was assessed by cytometry, and the expression of TLR2, Bax, and Mcl-1 was estimated by Western blot. The results and conclusions. The results have shown that the enhancement of neutrophils intense apoptosis depends directly from the effect of rhIL-17 and probably indirectly on TLR2 receptor. The observed changes in the expression of Bax both under the influence of fMLP and rhIL-17 stress the important role of this protein in regulating neutrophils apoptosis.Wstęp. Apoptoza neutrofilów (PMN) zachodzi z udziałem białek rodziny Bcl-2, które kontrolują przepuszczalność zewnętrznej błony mitochondrialnej. Białka tej rodziny podlegają regulacji przez różne czynniki, m.in. cytokiny. Wcześniejsze obserwacje własne wskazują na relację między nasileniem apoptozy a ekspresją receptora TLR2 w komórkach stymulowanych IL-17. Celem zaplanowanych badań było zbadanie wpływu rhIL-17 na wybrane białka rodziny Bcl-2 szlaku wewnątrzkomórkowego, takich jak: proapoptotyczne białko Bax i antyapoptotyczne białko Mcl-1 w relacji do ekspresji receptora TLR2. Odniesieniem do analizowanych wyników było zbadanie wpływu fMLP na oceniane parametry. Materiał i metoda. Badania przeprowadzono na izolowanych z krwi obwodowej ludzkich PMN. Apoptozę oceniano metodą cytometrii przepływowej, natomiast ekspresję TLR2, Bax oraz Mcl-1 oznaczano metodą Western blot. Wyniki i wnioski. Uzyskane wyniki wykazały, że nasilona apoptoza neutrofilów zależy w sposób bezpośredni od wpływu rhIL-17 na badane białka szklaku wewnętrznego i prawdopodobnie w sposób zależny od receptora TLR2. Obserwowane zmiany ekspresji Bax zarówno pod wpływem fMLP, jak i rhIL-17 podkreślaj

Role of ERK1/2 Kinase in the expression of iNOS by NDMA in human neutrophils

73-80<span style="font-size:11.0pt;mso-bidi-font-size: 10.0pt;font-family:" times="" new="" roman";mso-fareast-font-family:"times="" roman";="" mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="" lang="EN-US">Potential role of ERK1/2 kinase in conjunction with p38 in the regulation of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, and superoxide anion generation by human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was determined. Increased synthesis of NO due to the involvement of iNOS in neutrophils exposed to NDMA was observed. In addition, intensified activation of ERK1/2 and p38 kinases was determined in these cells. Inhibition of <span style="font-size:11.0pt;mso-bidi-font-size: 10.0pt;font-family:" times="" new="" roman";mso-fareast-font-family:"times="" roman";="" mso-ansi-language:en-gb;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="" lang="EN-GB">kinase regulated by extracellular signals<span style="font-size: 11.0pt;mso-bidi-font-size:10.0pt;font-family:" times="" new="" roman";mso-fareast-font-family:="" "times="" roman";mso-ansi-language:en-us;mso-fareast-language:en-us;="" mso-bidi-language:ar-sa"="" lang="EN-US"> (ERK1/2) pathway, in contrast to p38 pathway, led to an increased production of NO and expression of iNOS in PMNs. Moreover, as a result of inhibition of ERK1/2 pathway, a decreased activation of p38 kinase was observed in neutrophils, while inhibition of p38 kinase did not affect activation of ERK1/2 pathway in these cells. An increased ability to release superoxide anion by the studied PMNs was observed, which decreased after ERK1/2 pathway inhibition. In conclusion, in human neutrophils, ERK1/2 kinase is not directly involved in the regulation of iNOS and NO production induced by NDMA; however, the kinase participates in superoxide anion production in these cells. </span

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

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):333–4

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 &lt; 0.01; 5.19, p &lt; 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

Bone Metabolism Markers and Bone Mineral Density in Patients on Long-Term Acenocoumarol Treatment: A Cross-Sectional Study

The aim of this study was to evaluate levels of osteocalcin (OC), osteoprotegerin (OPG) and total soluble receptor activator of nuclear factor-&kappa;B ligand (RANKL), and bone mineral density (BMD) in patients on long-term acenocoumarol (AC) treatment. The cross-sectional study was carried out in 42 patients treated long-term with AC and 28 control subjects. Serum concentrations of OC, OPG, and sRANKL were measured using enzyme linked immunosorbent assay (ELISA) kits, and BMD at the femoral neck and lumbar spine were assessed by dual energy X-ray absorptiometry. A significantly decreased concentration of OC was found in AC users compared to control subjects (4.94 &plusmn; 2.22 vs. 10.68 &plusmn; 4.5; p &lt; 0.001). Levels of OPG, sRANKL logarithm (log), sRANKL/OPG log ratio, and BMD were comparable between. In female AC users, positive correlations between OC and RANKL log, and between OC and RANKL/OPG log ratio (p = 0.017; p = 0.005, respectively), and a negative correlation between OC and OPG (p = 0.027) were found. Long-term AC anticoagulation significantly decreases OC concentration, but does not affect other bone metabolism markers or BMD. Our results also suggest the possibility that long-term treatment with AC may alleviate bone resorption in postmenopausal women

The Redox Balance in Erythrocytes, Plasma, and Periosteum of Patients with Titanium Fixation of the Jaw

Titanium miniplates and screws are commonly used for fixation of jaw fractured or osteotomies. Despite the opinion of their biocompatibility, in clinical practice symptoms of chronic inflammation around the fixation develop in some patients, even many years after the application of miniplates and screws. The cause of these complications is still an unanswered question. Taking into account that oxidative stress is one of the toxic action of titanium, we have evaluated the antioxidant barrier as well as oxidative stress in the erythrocytes, plasma and periosteum covering the titanium fixation of the jaw. The study group was composed of 32 patients aged 20–30 with inserted miniplates and screws. The antioxidant defense: catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase-1 (SOD1), uric acid (UA), total antioxidant capacity (TAC), as well as oxidative damage products: advanced oxidation protein products (AOPP), advanced glycation end products (AGE), dityrosine, kynurenine, N-formylkynurenine, tryptophan, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), total oxidant status (TOS), and oxidative status index (OSI) were evaluated. SOD1 activity (↓37%), and tryptophan levels (↓34%) showed a significant decrease while AOPP (↑25%), TOS (↑80%) and OSI (↑101%) were significantly elevated in maxillary periosteum of patients who underwent bimaxillary osteotomies as compared to the control group. SOD-1 (↓55%), TAC (↓58.6%), AGE (↓60%) and N-formylkynurenine (↓34%) was statistically reduced while AOPP (↑38%), MDA (↑29%), 4-HNE (↑114%), TOS (↑99%), and OSI (↑381%) were significantly higher in the mandibular periosteum covering miniplates/screw compared with the control tissues. There were no correlations between antioxidants and oxidative stress markers in the periosteum of all patients and the blood. As exposure to the Ti6Al4V titanium alloy leads to disturbances of redox balance in the periosteum surrounding titanium implants of the maxilla and the mandible so antioxidant supplementation should be recommended to the patients undergoing treatment of dentofacial deformities with the use of titanium implants. The results we obtained may also indicate a need to improve the quality of titanium jaw fixations through increase of TiO2 passivation layer thickness or to develop new, the most highly biodegradable materials for their production