Dynamic Changes in the Ability to Release Neutrophil ExtraCellular Traps in the Course of Childhood Acute Leukemias.

Acute leukemias, the most common cancers in children, are characterized by excessive proliferation of malignant progenitor cells. As a consequence of impaired blood cell production, leukemia patients are susceptible to infectious complications-a major cause of non-relapse mortality. Neutrophil extracellular traps (NETs) are involved in various pathologies, from autoimmunity to cancer. Although aberrant NETs formation may be partially responsible for immune defects observed in acute leukemia, still little is known on the NET release in the course of leukemia. Here, we present the first comprehensive evaluation of NETs formation by neutrophils isolated from children with acute leukemia in different stages of the disease and treatment stimulated in vitro with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore (CI). NETs release was measured using quantitative fluorescent method and visualized microscopically. In this setting, NETs release was significantly impaired in leukemic children both at the diagnosis and during the treatment, and full restoration of neutrophil function was achieved only after successful completion of the leukemia treatment. We suggest that neutrophil function impairment may result from both disease- and treatment-related factors. In this context, deficient innate immune response observed in acute leukemia patients may be present regardless of neutrophil count and contribute to secondary immunodeficiency observed in this population.

Neutrophil extracellular traps generation and degradation in patients with granulomatosis with polyangiitis and systemic lupus erythematosus.

Neutrophils are one of the first cells to arrive at the site of infection, where they apply several strategies to kill pathogens: degranulation, respiratory burst, phagocytosis, and release of neutrophil extracellular traps (NETs). Recent discoveries try to connect NETs formation with autoimmune diseases, like systemic lupus erythematosus (SLE) or granulomatosis with polyangiitis (GPA) and place them among one of the factors responsible for disease pathogenesis. The aim of the study was to assess the NETotic capabilities of neutrophils obtained from freshly diagnosed autoimmune patients versus healthy controls. Further investigation involved assessing NETs production among treated patients. In the latter step, NETs degradation potency of collected sera from non-treated patients was checked. Lastly, the polymorphisms of the DNASE I gene among tested subjects were checked. NETs formation was measured in a neutrophil culture by fluorometry, while degradation assessment was performed with patients' sera and extracellular source of DNA. Additionally, Sanger sequencing was used to check potential SNP mutations between patients. About 121 subjects were enrolled into this study, 54 of them with a diagnosed autoimmune disorder. Neutrophils stimulated with NETosis inducers were able to release NETs in all cases. We have found that disease affected patients produce NETs more rapidly and in larger quantities than control groups, with up to 82.5% more released. Most importantly, we showed a difference between the diseases themselves. NETs release was 68.5% higher in GPA samples when compared to SLE ones while stimulated with Calcium Ionophore. Serum nucleases were less effective at degrading NETs in both autoimmune diseases, with a reduction in degradation of 20.9% observed for GPA and 18.2% for SLE when compared with the controls. Potential therapies targeting neutrophils and NETs should be specifically tailored to the type of the disease. Since there are significant differences between NETs release and disease type, a standard neutrophil targeted therapy could prevent over-generation of traps in some cases, while in others it would deplete the cells, leaving the immune system unresponsive to primary infections.

Celiac antibodies in children with type 1 diabetes - A diagnostic validation study.

INTRODUCTION: Autoimmune diseases, such as celiac disease (CD) and diabetes mellitus type 1, tend to co-occur within the same patient. The prevalence of CD in diabetic children is higher than in the general population, and is estimated to be 0.6-16.4%. The diagnosis of CD is based on histopathological examination and serological testing, however, these methods are still imperfect and new diagnostic algorithms should be considered. AIM: The aim of the study was to assess the diagnostic value of serological tests detecting antibodies against deamidated gliadin peptide, endomysium, tissue transglutaminase, neo-epitope tissue transglutaminase and to identify HLA-related genetic predisposition to CD in patients with type 1 diabetes mellitus (DM1). METHODS: Autoantibodies were measured in the sera of 392 children suffering from DM1 aged 1-19 years old (mean 11.76 ± 4.14 years old). Additionally, PCR-based assessment of HLA DQ2/DQ8 genotyping was performed. RESULTS: A positive result of at least one serological test was obtained from 81 children (20.66%). The sensitivity and specificity were 76.47% and 91.67% for anti-DGP IgA, 70.59% and 58.33% for IgG anti-DGP, respectively. A positive predictive value was 100% for the anti-TG IgA at cutoff levels of 5 and 10 times higher than upper limit of reference values. HLA DQ2 and/or DQ8 were found in 97.6% of examined children. CONCLUSIONS: Tests based on anti-TG IgA are more accurate for detecting CD in children with type 1 diabetes than anti-DGP IgA. A high percentage of diabetic children carry HLA alleles predisposing to CD, which indicates that genetic screening in this group of patients is not obligated.

Different procedures of diphenyleneiodonium chloride addition affect neutrophil extracellular trap formation.

A unique strategy, in which invading microorganisms are being caught in web-like structures composed mainly of DNA, involves a recently described phenomenon called NETosis. This process seems to be related to the production of reactive oxygen species (ROS). In our study, the influence of diphenyleneiodonium chloride (DPI), which diminishes ROS production, was assessed in the context of neutrophil extracellular trap (NET) release. According to protocol, two distinguished procedures were compared, the first one involving DPI elimination from sample before cell activation and the second one proceeding without the step of inhibitor washout. The kinetics of DNA release was monitored by fluorometric assay, and NET formation was observed by fluorescent microscopy. The addition of DPI to the sample led to a reduction of extracellular DNA release. The strongest inhibition was noticed after treatment with 10 µM DPI, which was removed from medium before stimulation with phorbol-12-myristate-13-acetate (PMA). Our findings confirmed that DPI is able to block NET creation. However, the addition of DPI together with PMA or the addition of inhibitor initially and then washing it out before stimulation resulted in different levels of NET formation. Finally, DPI that remained in the system induced specific morphological changes in the neutrophils' nuclei that was not observed in the DPI washed out from sample.

Neutrophils in asthma--a review.

Asthma is a chronic inflammatory disease, with an array of cells involved in the pathogenesis of the disease. The role of neutrophils in the development of bronchial asthma is found to be complex, as they may trigger activation of immunocompetent cells and are a potent source of free oxygen radicals and enzymes participating in airway remodeling. The review highlights the role of neutrophils in bronchial asthma.