Olga Ciepiela

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.

The effect of clindamycin and amoxicillin on neutrophil extracellular trap (NET) release.

Neutrophil extracellular traps (NETs) are threads of nuclear DNA complexed with antimicrobial proteins released by neutrophils to extracellular matrix to bind, immobilise, and kill different pathogens. NET formation is triggered by different physiological and non-physiological stimulants. It is also suggested that antibiotics could be non-physiological compounds that influence NET release. The aim of the study was to investigate the effect of clindamycin and amoxicillin on NET release and the phagocyte function of neutrophils. Neutrophils isolated from healthy donors by density centrifugation method were incubated with amoxicillin or clindamycin for two hours, and then NET release was stimulated with phorbol 12-myristate 13-acetate (PMA). After three hours of incubation with PMA NETs were quantified as amount of extracellular DNA by fluorometry and visualised by immunofluorescent microscopy. The percent of phagocyting cells was measured by flow cytometry. We showed that amoxicillin induces NET formation (increase of extracellular DNA fluorescence, p = 0.03), while clindamycin had no influence on NET release (p > 0.05), as confirmed by quantitative measurement and fluorescent microscopy. Regarding phagocyte function, both antibiotics increased bacterial uptake (43.3% and 61.6% median increase for amoxicillin and clindamycin, respectively). We concluded that the ability of antibiotics to modulate NET release depends on the antibiotic used and is not associated with their ability to influence phagocytosis.

A Comparison of Mindray BC-6800, Sysmex XN-2000, and Beckman Coulter LH750 Automated Hematology Analyzers: A Pediatric Study.

Modern automated laboratory hematology analyzers allow the measurement of over 30 different hematological parameters useful in the diagnostic and clinical interpretation of patient symptoms. They use different methods to measure the same parameters. Thus, a comparison of complete blood count made by Mindray BC‐6800, Sysmex XN‐2000 and Beckman Coulter LH750 was performed.

Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis.

Abstract Background: The eosin-5′-maleimide (EMA) binding test is a flow cytometric test widely used to detect hereditary spherocytosis (HS). EMA binds to plasma membrane proteins of red blood cells (RBCs), mainly to band 3 protein. The mean fluorescence of EMA-stained RBCs in HS patients is lower when compared with control RBCs due to the decreased amount of target proteins. EMA dye in aqueous solution is sensitive to light and high temperature. Its fluorescence can decrease when exposed to light or ambient temperatures higher than 4°C. The aim of the study was to evaluate the stability of fluorescence readings of EMA-labeled RBCs over a period of 24 h. Methods: The EMA test was performed in peripheral blood from 35 patients with microcytic anemia (five with HS, and 30 without HS). Peripheral blood samples were stained immediately after blood collection and analyzed using a flow cytometer at three time points: 0, after 1 and 24 h of storage at 4°C in the darkness. The results are presented as the percentage of normal control RBCs fluorescence. Flow cytometric studies were performed with Cytomics FC500 (Beckman Coulter, USA). Results: In HS patients the mean result of the test reached 66.72%±9.26% of normal controls, and in non-HS patients the EMA result was 99.48%±5.03% of normal control cells. The results of patients with HS were 66.72%±9.26%, 66.90%±10.24% and 67.86%±11.31% at 0 h, and after 1 and 24 h of storage, respectively. The results obtained from non-HS patients at time 0, after 1 and 24 h of storage reached 99.48%±5.03%, 99.49%±5.34% and 99.78%±6.13%, respectively. There was no difference between the results from each time point in samples from patients with or without HS. Conclusions: Results of the EMA binding test do not depend on storage time of stained samples when stored at 4°C up to 24 h after staining.

Antibiotics Modulate the Ability of Neutrophils to Release Neutrophil Extracellular Traps.

Antibiotics directly inhibit the growth and kill microorganisms, and many of them have immunomodulatory properties. We investigated the influence of cefotaxime and gentamicin on the release of neutrophil extracellular traps (NETs) - recently described strategy employed by neutrophils to fight infections. We found that gentamicin inhibits NETs release by human neutrophils, while cefotaxime did not have any impact on this process. The information that antibiotics can modulate NETs release, can be useful in the therapy of infectious diseases in patients suffering from NET-related diseases.

Pro- and antiangiogenic markers in patients with pulmonary complications of systemic scleroderma.

Systemic sclerosis (SSc) is an autoimmune disorder characterized by skin and internal organs fibrosis and concomitant vascular abnormalities. Although SSc is considered mainly fibrosing disease, underlying vascular pathology plays a fundamental role in its pathogenesis. We have focused on positive and negative serum markers of angiogenesis and fibrosis (pigment epithelium-derived factor [PEDF], vascular endothelial growth factor [VEGF], and soluble VEGF receptor [sVEGFR]), in progressive SSc patients at baseline and after follow-up in relation to cardiopulmonary complications (systemic hypertension [HT], pulmonary arterial hypertension [PAH] and pulmonary fibrosis [PF]). VEGF and PEDF but not sVEGFR were reciprocally regulated in SSc progression. Moreover, VEGF/PEDF ratio significantly increased during follow up suggesting that it might be used as a biomarker of disease progression. No correlation between the studied markers and cardiopulmonary complications was observed. In conclusion, VEGF and PEDF level, and the VEGF/PEDF ratio are significantly changed in the course of SSc progression and these markers can be used to assess SSc activity.

The prevalence of IgG and IgA against adenoviruses in serum of children aged 11-26 months, hospitalised in the Clinical Paediatric Hospital in Warsaw, Poland.

Adenoviruses are widely spread viruses causing infections related mainly to children. Contact with the pathogen may result in the presence of either immunoglobulin (Ig) A or IgG antibodies in the serum. The aim of this study was to assess the prevalence of IgA and IgG antibodies against adenoviruses in serum of children aged 11-27 months, hospitalised at the Public Childrens Clinical Hospital in Warsaw. Immunoassay tests DRG Adenovirus IgA ELISA and IgG DRG Adenovirus ELISA detecting antibodies against all pathogenic serotypes of adenoviruses were performed in 128 sera samples. A low percentage of positive results for both immunoglobulins, 17 cases (13.28%) for IgA and 16 cases (12.50%) for IgG antibodies respectively were reported. We conclude, that the prevalence of antibodies against adenoviruses does not coincide with epidemiological data on the spread of infections among young children. The assays performed may be more useful in distinguishing the active form of the infection from seropositivity due to prior infection.

Influence of Sublingual Immunotherapy on the Expression of Mac-1 Integrin in Neutrophils from Asthmatic Children

Asthma can be effectively treated with sublingual immunotherapy. The influence of -sublingual immunotherapy on the function of granulocytes in asthmatic patients is largely unknown. Mac-1 integrin is a transmembrane protein containing α (CD11b) and β (CD18) chains. High expression of the complex is found on the surface of neutrophils, NK cells, and macrophages. CD11b/CD18 may bind to CD23, ICAM-1, ICAM-2, and ICAM-4. It plays a crucial role in diapedesis of neutrophils. The aim of the present study was to assess Mac-1 expression on neutrophils from asthmatic children before and after sublingual immunotherapy. Twenty five children aged of 8.1 ± 3.1 suffering from atopic asthma and allergic rhinitis, shortlisted for specific immunotherapy, served as the study group. Fifteen healthy individuals, aged 9.8 ± 3.4, served as a control group. The assessment of CD11b and CD18 expression on cells from peripheral blood was performed with a flow cytometer. The tests were performed before and after 12 months of sublingual immunotherapy. In the asthmatic children, 98.08 (90.79-99.12)% of Mac-1 positive neutrophils were detected. The group was divided into two subgroups: of more than 98% and less than 95% of neutrophils with CD11b/CD18 expression in the sample. After immunotherapy, the percentage of Mac-1 positive granulocytes increased to 99.60 (99.29-99.68)%, p = 0.01. In the control group, 90.56 (87.08-88.86)% granulocytes were Mac-1 positive, p = 0.002. We conclude that sublingual immunotherapy strongly influences the function of the immunological system, including Mac-1 expression on neutrophils.

Flow cytometric quantification of neutrophil extracellular traps: Limitations of the methodological approach.

To the Editor: Neutrophil Extracellular Traps (NETs) are structures composed of DNA, histones, and intracellular enzymes, which are released from granulocytes to immobilize and kill pathogens in blood and tissues [1]. Several approaches are used to quantify and assess the effectiveness of NETs’ release, based on fluorescent microscopy, immunoenzymatic measurement of neutrophil elastase (NE) release, and fluorometric measurement of DNA release. Unfortunately, these widely used methods have several drawbacks. Quantification of NETs using fluorescent microscopy is laborious and susceptible to observer bias. On the contrary, the release of NE and DNA is not strictly specific for NETosis as other forms of cell death, such as necrosis, could also result in the elevated levels of extracellular NE and DNA. Thus, it would be very beneficial to develop new and reliable methods to assess NETs’ formation. Recently, Gavillet et al. published an exciting study that describes the flow cytometric method of NETs’ quantification [2]. Because we have also been working on the application of flow cytometry for NETs’ studies, we love to share our observations with scientific community. Our major concern regarding the aforementioned work is that Gavillet et al. validated their assay using mice models. But, it is disputable to use murine neutrophils as a reference for human cells—mice release NETs less efficiently than humans, and the formation lasts longer. Thus, it is easier to observe murine NETs using flow cytometry technique because NETs structures are compact and do not show typical morphology of DNA threads [3]. It has been reported that only 30% of murine neutrophils release NETs after 16 h of stimulation with 100 nM phorbol-12-myristate-13-acetate (PMA), while approximately 60–80% of human eutrophils release NETs 3 h after stimulation with PMA [3,4]. Gavillet et al. analyzed human samples after 30-min incubation, and the method seemed to be appropriate for such short-time incubation periods. However, our observations suggest that this method cannot be used when long incubation periods are required. Our studies show that human neutrophils stimulated with PMA or calcium ionophore (CI) release NETs very efficiently after 4 h incubation (Fig. 1A). Due to high viscosity of DNA, neutrophils form clumps making it difficult to distinguish single cells. Classical flow cytometry allows to analyze cells in a suspension, and it constitutes the main limitation of flow cytometry-based NETs’ assay. By analyzing human neutrophil samples, we observed a significant loss of cells from suspension after the prolonged incubation. We stimulated 5 3 10 cells in suspension with PMA or CI for 1–3-h incubation and then analyzed the samples with a flow cytometer. Our samples lost neutrophils over time—samples stimulated for 1 h with PMA or CI had ca. 8000 events, while samples stimulated with these compounds for 3 h had only 2500 cells collected during the same time of acquisition (50 s) using flow cytometer (Cytomics FC500, Beckman Coulter). There are also some doubts regarding “NETs’” gating based on side scatter/forward scatter approaches. In our opinion, the number of cells, which release NETs, could not be properly evaluated. There reason behind this assumption is that many of these cells, as completely disrupted, fall outside the gate (cell remnants) after the formation of NETs (Fig. 1C). Myeloperoxidase (MPO) and citrullinated histones are located outside the cells in fully formed NETs [1,3,4]. In our opinion, those gated by Gavillet et al., cells are rather stimulated granulocytes with typical for NETosis cellular modifications, which did not release NETs. Neutrophil gating in Fig. 1C shows that cells’ debris contains almost 40% of citrullinated histones, thus we hypothesize that excluding these remnants from the analysis may cause an error in NETs’ flow quantification. We do agree with Gavillet et al. that it is desirable to assess histone citrullination by flow cytometry as a hallmark of NETosis. We have investigated this issue and found that histone citrullination (citH3) increases before tenth minute of neutrophil stimulation—

Recurrent respiratory tract infections in children - analysis of immunological examinations.

Background Paediatric respiratory tract infections are among the most common reasons for preschool and school absences and visits to physicians. The disease mainly involves the upper respiratory tract and is associated with fever, cough, sore throat, and running nose. Children with recurrent respiratory infections (RRI), which are defined as more than six serious diseases a year, are a difficult diagnostic challenge. The aim of this study was to assess immunological deviations in laboratory tests performed in children with RRI. Material and methods In the retrospective study 25 children suffering from recurrent respiratory tract infection, aged 4.1 ±2.3 years, 13 boys and 12 girls, were involved. For all children chemiluminescence of granulocytes and immunophenotyping of lymphocytes from peripheral blood were examined. An immunophenotype of peripheral blood lymphocytes involved evaluation of T cell, B cells, and NK cells, examined with flow cytometry. Results Eleven of the studied children had decreased chemiluminescent response to stimulants, normal response was found for nine children, and five children had an increased result of the test. Five of the 25 children had decreased B cells number, and five had decreased number of T cells including decrease of CD4, as well as CD8 positive cells. Children with decreased chemiluminescence had more frequent neutropaenia than children with normal or increased chemiluminescent response, p < 0.05 (exact Fisher test). Conclusions Recurrent respiratory tract infection could be associated with improper neutrophils response to pathogens, and immunological examination should be performed to find the reason for the increased number of infections in a year.