Walking a thin line between fixation and epitope binding - characterization of antigen retrieval methods suitable for eosinophil and HSV-2 staining in formalin-fixed female reproductive tissue.

Antibody-based fluorescence analysis of female reproductive tissues in research of sexually transmitted diseases allows for an in-depth understanding of protein localization, interactions, and pathogenesis. However, in many cases, cryosectioning is not compatible with biosafety regulations; at all times, exposure of lab personnel and the public to potentially harmful pathogens from biological infectious material must be avoided; thus, formaldehyde fixation is essential. Due to formaldehyde's cross-linking properties, protein detection with antibodies can be impeded. To allow effective epitope binding during immunofluorescence of formalin-fixed paraffin-embedded vaginal tissue, we investigated two antigen retrieval methods. We tested these methods regarding their suitability for automated image analysis, facilitating reproducible quantitative microscopic data acquisition in sexually transmitted disease research. Heat-based retrieval at 80°C in citrate buffer proved to increase antibody binding to eosinophil protein and HSV-2 visibly and tissue morphology best, and was the most efficient for sample processing and quantitative analysis.

Exposure to organophosphate flame retardants and plasticizers is positively associated with wheeze and FeNO and eosinophil levels among school-aged children: The Hokkaido study.

Exposure to organophosphate flame retardants and plasticizers (PFRs) increases the risk of asthma and allergies. However, little is known about its association with type 2 inflammation (T2) biomarkers used in the management of allergies. The study investigated associations among urinary PFR metabolite concentrations, allergic symptoms, and T2 biomarkers. The data and samples were collected between 2017 and 2020, including school children (n = 427) aged 9-12 years living in Sapporo City, Japan, among the participants of "The Hokkaido Study on Environment and Children's Health." Thirteen urinary PFR metabolites were measured by LC-MS/MS. Allergic symptoms were assessed using the International Study of Asthma and Allergies in Childhood questionnaire. For T2 biomarkers, the peripheral blood eosinophil counts, fraction of exhaled nitric oxide level (FeNO), and serum total immunoglobulin E level were measured. Multiple logistic regression analysis, quantile-based g-computation (qg-computation), and Bayesian kernel machine regression (BKMR) were used to examine the associations between the health outcomes of the individual PFRs and the PFR mixtures. The highest concentration of PFR was Σtris(1-chloro-isopropyl) phosphates (ΣTCIPP) (Median:1.20 nmol/L). Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) was significantly associated with a high odds ratio (OR, 95%CI:1.36, 1.07-1.72) for wheeze. TDCIPP (OR, 95%CI:1.19, 1.02-1.38), Σtriphenyl phosphate (ΣTPHP) (OR, 95%CI:1.81, 1.40-2.37), and Σtris(2-butoxyethyl) phosphate (ΣTBOEP) (OR, 95%:1.40, 1.13-1.74) were significantly associated with increased odds of FeNO (≥35 ppb). ΣTPHP (OR, 95%CI:1.44, 1.15-1.83) was significantly associated with high eosinophil counts (≥300/µL). For the PFR mixtures, a one-quartile increase in all PFRs (OR, 95%CI:1.48, 1.18-1.86) was significantly associated with high FeNO (≥35 ppb) in the qg-computation model. The PFR mixture was positively associated with high FeNO (≥35 ppb) and eosinophil counts (≥300/µL) in the BKMR models. These results may suggest that exposure to PFRs increases the probability of asthma, allergies, and T2 inflammation.

Comparison of CRP, Procalcitonin, Neutrophil Counts, Eosinophil Counts, sTREM-1, and OPN between Pneumonic and Nonpneumonic Exacerbations in COPD Patients.

Introduction: The patients with community-acquired pneumonia (CAP) and acute exacerbations of COPD (AECOPD) could have a higher risk of acute and severe respiratory illness than those without CAP in AECOPD. Consequently, early identification of pneumonia in AECOPD is quite important. Methods. 52 subjects with AECOPD + CAP and 93 subjects with AECOPD from two clinical centers were enrolled in this prospective observational study. The values of osteopontin (OPN), soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), C-reactive protein (CRP), procalcitonin (PCT), eosinophil (EOS) counts, and neutrophil (Neu) counts in blood on the first day of admission and clinical symptoms were compared in AECOPD and AECOPD + CAP. In addition, subgroup analyses of biomarker difference were conducted based on the current use of inhaled glucocorticoids (ICS) or systemic corticosteroids (SCS). Results: Patients with AECOPD + CAP had increased sputum volume, sputum purulence, diabetes mellitus, and longer hospital stays than AECOPD patients (p < 0.05). A clinical logistic regression model showed among the common clinical symptoms, purulent sputum can independently predict pneumonia in AECOPD patients after adjusting for a history of diabetes. At day 1, AECOPD + CAP patients had higher values of Neu, CRP, PCT, and OPN, while serum sTREM-1 levels and EOS counts were similar in the two groups. CRP fared best at predicting AECOPD with CAP (p < 0.05 for the test of difference), while OPN had similar accuracy with Neu, PCT, and purulent sputum (p > 0.05 for the test of difference). Multivariate analysis, including clinical symptoms and biomarkers, suggested that CRP ≥15.8 mg/dL at day 1 was a only promising predictor of pneumonia in AECOPD. CRP and OPN were not affected by ICS or SCS. Conclusions: CRP ≥15.8 mg/dL is an ideal promising predictor of pneumonia in AECOPD, and its plasma level is not affected by ICS or SCS. The diagnostic performance of CRP is not significantly improved when combined with clinical symptoms or other markers (OPN, PCT, and Neu).

Role of Eosinophils in Intestinal Inflammation and Fibrosis in Inflammatory Bowel Disease: An Overlooked Villain?

Eosinophils are leukocytes which reside in the gastrointestinal tract under homeostatic conditions, except for the esophagus which is normally devoid of eosinophils. Research on eosinophils has primarily focused on anti-helminth responses and type 2 immune disorders. In contrast, the search for a role of eosinophils in chronic intestinal inflammation and fibrosis has been limited. With a shift in research focus from adaptive to innate immunity and the fact that the eosinophilic granules are filled with inflammatory mediators, eosinophils are becoming a point of interest in inflammatory bowel diseases. In the current review we summarize eosinophil characteristics and recruitment as well as the current knowledge on presence, inflammatory and pro-fibrotic functions of eosinophils in inflammatory bowel disease and other chronic inflammatory conditions, and we identify research gaps which should be covered in the future.

Mixed Sputum Granulocyte Longitudinal Impact on Lung Function in the Severe Asthma Research Program.

Rationale: Some reports indicate longitudinal variability in sputum differential cell counts, whereas others describe stability. Highly variable sputum eosinophil percentages are associated with greater lung function loss than persistently elevated eosinophil percentages, but elevated neutrophils are linked to more severe asthma.Objectives: To examine sputum granulocyte stability or variability longitudinally and associations with important clinical characteristics.Methods: The SARP III (Severe Asthma Research Program III) cohort underwent comprehensive phenotype characterization at baseline and annually over 3 years. Adult subjects with acceptable sputum levels were assigned to one of three longitudinal sputum groups: eosinophils predominantly <2%, eosinophils predominantly ≥2%, or highly variable eosinophil percentages (>2 SDs determined from independent, repeated baseline eosinophil percentages). Subjects were similarly assigned to one of three longitudinal neutrophil groups with a 50% cut point.Measurements and Main Results: The group with predominantly <2% sputum eosinophils had the highest lung function (prebronchodilator FEV1% predicted, P < 0.01; FEV1/FVC ratio, P < 0.001) at baseline and throughout 3 years compared with other eosinophil groups. Healthcare use did not differ, although the highly variable eosinophil group reported more asthma exacerbations at Year 3. Longitudinal neutrophil groups showed few differences. However, a combination of predominantly ≥2% eosinophil and ≥50% neutrophil groups resulted in the lowest prebronchodilator FEV1% predicted (P = 0.049) compared with the combination with predominantly <2% eosinophils and<50% neutrophils.Conclusions: Subjects with predominantly ≥2% sputum eosinophils in combination with predominantly ≥50% neutrophils showed greater loss of lung function, whereas those with highly variable sputum eosinophils had greater healthcare use.

A FACS-based approach to obtain viable eosinophils from human adipose tissue.

Eosinophils have been widely investigated in asthma and allergic diseases. More recently, new insights into the biology of these cells has illustrated eosinophils contribute to homeostatic functions in health such as regulation of adipose tissue glucose metabolism. Human translational studies are limited by the difficulty of obtaining cells taken directly from their tissue environment, relying instead on eosinophils isolated from peripheral blood. Isolation techniques for tissue-derived eosinophils can result in unwanted cell or ribonuclease activation, leading to poor cell viability or RNA quality, which may impair analysis of effector activities of these cells. Here we demonstrate a technique to obtain eosinophils from human adipose tissue samples for the purpose of downstream molecular analysis. From as little as 2 g of intact human adipose tissue, greater than 104 eosinophils were purified by fluorescence-activated cell sorting (FACS) protocol resulting in ≥ 99% purity and ≥ 95% viable eosinophils. We demonstrated that the isolated eosinophils could undergo epigenetic analysis to determine differences in DNA methylation in various settings. Here we focused on comparing eosinophils isolated from human peripheral blood vs human adipose tissue. Our results open the door to future mechanistic investigations to better understand the role of tissue resident eosinophils in different context.

The Effects of Indoor Pollutants Exposure on Allergy and Lung Inflammation: An Activation State of Neutrophils and Eosinophils in Sputum.

BACKGROUND: To explore the inflammation phenotypes following indoor pollutants exposure based on marker expression on eosinophils and neutrophils with the application of chemometric analysis approaches. METHODS: A cross-sectional study was undertaken among secondary school students in eight suburban and urban schools in the district of Hulu Langat, Selangor, Malaysia. The survey was completed by 96 students at the age of 14 by using the International Study of Asthma and Allergies in Children (ISAAC) and European Community Respiratory Health Survey (ECRHS) questionnaires. The fractional exhaled nitric oxide (FeNO) was measured, and an allergic skin prick test and sputum induction were performed for all students. Induced sputum samples were analysed for the expression of CD11b, CD35, CD63, and CD66b on eosinophils and neutrophils by flow cytometry. The particulate matter (PM2.5 and PM10), NO2, CO2, and formaldehyde were measured inside the classrooms. RESULTS: Chemometric and regression results have clustered the expression of CD63 with PM2.5, CD11b with NO2, CD66b with FeNO levels, and CO2 with eosinophils, with the prediction accuracy of the models being 71.88%, 76.04%, and 76.04%, respectively. Meanwhile, for neutrophils, the CD63 and CD66b clustering with PM2.5 and CD11b with FeNO levels showed a model prediction accuracy of 72.92% and 71.88%, respectively. CONCLUSION: The findings indicated that the exposure to PM2.5 and NO2 was likely associated with the degranulation of eosinophils and neutrophils, following the activation mechanisms that led to the inflammatory reactions.

Eosinophils synthesize trihydroxyoctadecenoic acids (TriHOMEs) via a 15-lipoxygenase dependent process.

Trihydroxyoctadecenoic acids (TriHOMEs) are linoleic acid-derived lipid mediators reported to be dysregulated in obstructive lung disease. In contrast to many other oxylipins, TriHOME biosynthesis in humans is still poorly understood. The association of TriHOMEs with inflammation prompted the current investigation into the ability of human granulocytes to synthesize the 16 different 9,10,13-TriHOME and 9,12,13-TriHOME isomers and of the TriHOME biosynthetic pathway. Following incubation with linoleic acid, eosinophils and (to a lesser extent) the mast cell line LAD2, but not neutrophils, formed TriHOMEs. Stereochemical analysis revealed that TriHOMEs produced by eosinophils predominantly evidenced the 13(S) configuration, suggesting 15-lipoxygenase (15-LOX)-mediated synthesis. TriHOME formation was blocked following incubation with the 15-LOX inhibitor BLX-3887 and was shown to be largely independent of soluble epoxide hydrolase and cytochrome P450 activities. TriHOME synthesis was abolished when linoleic acid was replaced with 13-HODE, but increased in incubations with 13-HpODE, indicating the intermediary role of epoxy alcohols in TriHOME formation. In contrast to eosinophils, LAD2 cells formed TriHOMEs having predominantly the 13(R) configuration, demonstrating that there are multiple synthetic routes for TriHOME formation. These findings provide for the first-time insight into the synthetic route of TriHOMEs in humans and expand our understanding of their formation in inflammatory diseases.

Eosinophil peroxidase oxidizes isoniazid to form the active metabolite against M. tuberculosis, isoniazid-NAD.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD+) by the mycobacterial catalase-peroxidase enzyme, KatG, was known to be the major component of the mode of action of isoniazid (INH), an anti-tuberculosis drug. However, there are other enzymes that may catalyze this reaction. We have previously reported that neutrophil myeloperoxidase (MPO) is capable of metabolizing INH through the formation of INH-NAD+ adduct, which could be attributed to being a possible mode of action of INH. However, eosinophilic infiltration of the lungs is more pronounced and characteristic of granulomas in Mycobacterium tuberculosis-infected patients. Thus, the aim of the present study is to investigate the role of eosinophil peroxidase (EPO), a key eosinophil enzyme, during INH metabolism and the formation of its active metabolite, INH-NAD+ using purified EPO and eosinophils isolated from asthmatic donors. UV-Vis spectroscopy revealed INH oxidation by EPO led to a new product (λmax = 326 nm) in the presence of NAD+. This adduct was confirmed to be INH-NAD+ using LC-MS analysis where the intact adduct was detected (m/z = 769). Furthermore, EPO catalyzed the oxidation of INH and formed several free radical intermediates as assessed by electron paramagnetic resonance (EPR) spin-trapping; a carbon-centred radical, which is considered to be the reactive metabolite that binds with NAD+, was found when superoxide dismutase was included in the reaction. Our findings suggest that eosinophilic EPO may also play a role in the pharmacological activity of INH through the formation of INH-NAD+ adduct, and supports further evidence that human cells and enzymes are capable of producing the active metabolite involved in tuberculosis treatment.

Analytical validation of fecal 3-bromotyrosine concentrations in healthy dogs and dogs with chronic enteropathy.

Studies that have used serum 3-bromotyrosine (3-BrY) to investigate eosinophil activation in dogs have found elevated 3-BrY levels in clinical patients with chronic enteropathy (CE). To our knowledge, a method to measure 3-BrY concentrations in feces has not been reported. We developed and analytically validated an electron ionization gas chromatography-mass spectrometry method to measure fecal 3-BrY concentrations in dogs. The mean and maximum fecal 3-BrY concentrations in healthy dogs ( n = 40) and dogs with CE ( n = 40) over 3 consecutive days were compared. Analytical validation had a limit of blank and a limit of detection of 2.5 and 3.7 mmol/g of feces, respectively. The mean coefficients of variation for precision and reproducibility for 3-BrY were 11.2% (range: 7.5-14.2%) and 10.1% (4.8-15.2%), respectively. The ranges of observed-to-expected ratios for linearity and accuracy were 81.3-125% and 85.4-120%, respectively. The reference intervals for mean and maximum fecal 3-BrY concentrations in 40 healthy dogs were 3.7-23.0 and 3.7-37.8 mmol/g of feces. Mean and maximum fecal 3-BrY concentrations in dogs with CE were significantly higher than those of healthy dogs ( p < 0.001). Further research is warranted to determine the clinical usefulness of fecal 3-BrY concentrations in dogs with CE.

Proteomic and Phosphoproteomic Changes Induced by Prolonged Activation of Human Eosinophils with IL-3.

Purified human eosinophils treated for 18-24 h with IL-3 adopt a unique activated phenotype marked by increased reactivity to aggregated immunoglobulin-G (IgG). To characterize this phenotype, we quantified protein abundance and phosphorylation by multiplexed isobaric labeling combined with high-resolution mass spectrometry. Purified blood eosinophils of five individuals were treated with IL-3 or no cytokine for 20 h, and comparative data were obtained on abundance of 5385 proteins and phosphorylation at 7330 sites. The 1150 proteins that were significantly up-regulated ( q < 0.05, pairwise t test with Benjamini-Hochberg correction) by IL-3 included the IL3RA and CSF2RB subunits of the IL-3 receptor, the low-affinity receptor for IgG (FCGR2B), 96 proteins involved in protein translation, and 55 proteins involved in cytoskeleton organization. Among the 703 proteins that decreased were 78 mitochondrial proteins. Dynamic regulation of protein phosphorylation was detected at 4218 sites. These included multiple serines in CSF2RB; Y694 of STAT5, a key site of activating phosphorylation downstream of IL3RA/CSF2RB; and multiple sites in RPS6KA1, RPS6, and EIF4B, which are responsible for translational initiation. We conclude that IL-3 up-regulates overall protein synthesis and targets specific proteins for up-regulation, including its own receptor.

Morphologic and Cytochemical Characteristics of the Blood Cells of the Yellow-Bellied Slider (Trachemys scripta scripta).

The increasing prevalence of yellow-bellied sliders (Trachemys scripta scripta) as pets in the European Union and also its utilization as animal models for experimental purposes makes crucial an accurate classification of their blood cells. The aim of this work was to provide a morphologic classification based on the cytochemical characteristics of the blood cells of 15 yellow-bellied sliders. Cytochemical stains included benzidine peroxidase, chloroacetate esterase, alpha-naphthyl butyrate esterase (with and without sodium fluoride), acid phosphatase (with and without tartaric acid), Sudan black B, periodic acid-Schiff and toluidine blue. Nuclear and cellular dimensions were also measured based on quick Romanowsky-type stained smears. Besides erythrocytes and thrombocytes, five types of white blood cells were identified: heterophils, eosinophils, basophils, lymphocytes and monocytes. The cytochemical patterns of heterophils, eosinophils and basophils were unique compared to those described for other chelonians. This paper provides a useful guideline for clinical settings and further haematological studies of this species.

[Increased expressions of substance P and neurokinin/tachykinin receptor 1 in eosinophils of patients with psoriasis].

Objective To investigate the expressions of substance P (SP) and its receptor neurokinin/tachykinin receptor 1 (NK1R) in peripheral blood eosinophils of patients with psoriasis. Methods The levels of SP and NK1R in the peripheral blood of both patients with psoriasis and healthy people were detected by flow cytometry. This method was again used to detect the levels of SP and NK1R in the peripheral blood eosinophils of patients with psoriasis after stimulated with the crude extracts of Artemisia pollen, dust mite and Platanus pollen (all at concentrations of 0.1 and 1.0 µg/mL). Results Compared with the healthy controls, the percentages of SP+ and NK1R+ eosinophils in psoriasis patients increased up to 2.7 and 0.5 folds, respectively. Moreover, the mean fluorescence intensity (MFI) of SP+ and NK1R+ eosinophils of psoriasis patients were elevated by 1.5 and 0.2 folds, respectively. The percentage of SP+ eosinophils in psoriasis were down-regulated by 60% after the stimulation with Platanus pollen extract (1 µg/mL), while 0.1 µg/mL Platanus pollen extract induced a 0.6-fold increase in the percentage of NK1R+ eosinophis. Conclusion The expressions of SP and NK1R are up-regulated in peripheral blood eosinophils of patients with psoriasis.

Functional and phenotypic evaluation of eosinophils from patients with the acute form of paracoccidioidomycosis.

BACKGROUND: Eosinophilia is a typical finding of the acute/juvenile form of paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America. This clinical form is characterized by depressed cellular immune response and production of Th2 cytokines. Moreover, it has been shown that the increased number of eosinophils in peripheral blood of patients returns to normal values after antifungal treatment. However, the role of eosinophils in PCM has never been evaluated. This study aimed to assess the phenotypic and functional characteristics of eosinophils in PCM. METHODS/PRINCIPAL FINDINGS: In 15 patients with the acute form of the disease, we detected expression of MBP, CCL5 (RANTES) and CCL11 (eotaxin) in biopsies of lymph nodes and liver. In addition, there were higher levels of chemokines and granule proteins in the peripheral blood of patients compared to controls. Isolation of eosinophils from blood revealed a higher frequency of CD69+ and TLR2+ eosinophils in patients compared to controls, and a lower population of CD80+ cells. We also evaluated the fungicidal capacity of eosinophils in vitro. Our results revealed that eosinophils from PCM patients and controls exhibit similar ability to kill P. brasiliensis yeast cells, although eosinophils of patients were less responsive to IL-5 stimulation than controls. CONCLUSION/PRINCIPAL FINDINGS: In conclusion, we suggest that eosinophils might play a role in the host response to fungi and in the pathophysiology of PCM by inducing an intense and systemic inflammatory response in the initial phase of the infection.

Differences in eosinophil molecular profiles between children and adults with eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) afflicts both children and adults. It has been debated whether pediatric EoE and adult EoE represent different disease entities. The objectives of this study were to determine whether the blood eosinophil molecular pattern of children with EoE is (i) distinct from that of healthy children; and (ii) different from that of adults with EoE. METHODS: Blood eosinophils from children and adults with EoE, and healthy controls, were analyzed with flow cytometry regarding levels of CD23, CD44, CD54, CRTH2, FOXP3, and galectin-10. Eosinophil FOXP3 and galectin-10 mRNA levels were determined by qPCR. The data were analyzed using a multivariate method of pattern recognition. RESULTS: An eosinophil molecular pattern capable of distinguishing children with EoE from control children was identified. A smaller fraction of eosinophils from children with EoE expressed CD44 and a larger fraction expressed CRTH2 than the controls. Eosinophils from children with EoE also had higher levels of galectin-10 mRNA and lower levels of FOXP3 mRNA. The eosinophils from children with EoE had lower levels of surface CD54 and of FOXP3 mRNA compared with the eosinophils from the adult patients. A key finding was the detection in healthy individuals of age-related differences in the levels of several eosinophil markers. CONCLUSIONS: Children with EoE can be distinguished from healthy children based on the molecular patterns of their blood eosinophils. Age-related physiologic differences in eosinophil molecular patterns may partly explain the different blood eosinophil phenotypes in children vs adults with EoE.

Heparin reduces nonspecific eosinophil staining artifacts in mass cytometry experiments.

The analysis of heterogeneous cell samples by mass cytometry (CyTOF) relies on the assumption that metal labeled antibodies accurately bind to their target antigens. We report a previously unappreciated experimental artifact of non-specific antibody binding by eosinophils during intracellular CyTOF analysis of human whole blood samples. We hypothesized that this non-specific binding results from a charge-based interaction between the metal-labeled antibodies and highly cationic proteins found in eosinophillic granules and found that this non-specific staining artifact could be reduced to background levels with a simple blocking protocol using heparin as a competing anionic protein. This protocol eliminates a potential source of erroneous data interpretation in all experiments involving intracellular staining of human whole blood samples, and allows accurate assessment of dynamic changes in intracellular proteins in eosinophils by CyTOF. © 2016 International Society for Advancement of Cytometry.

The Peripheral Blood Eosinophil Proteome.

A system-wide understanding of biological processes requires a comprehensive knowledge of the proteins in the biological system. The eosinophil is a type of granulocytic leukocyte specified early in hematopoietic differentiation that participates in barrier defense, innate immunity, and allergic disease. The proteome of the eosinophil is largely unannotated with under 500 proteins identified. We now report a map of the nonstimulated peripheral blood eosinophil proteome assembled using two-dimensional liquid chromatography coupled with high-resolution mass spectrometry. Our analysis yielded 100,892 unique peptides mapping to 7,086 protein groups representing 6,813 genes as well as 4,802 site-specific phosphorylation events. We account for the contribution of platelets that routinely contaminate purified eosinophils and report the variability in the eosinophil proteome among five individuals and proteomic changes accompanying acute activation of eosinophils by interleukin-5. Our deep coverage and quantitative analyses fill an important gap in the existing maps of the human proteome and will enable the strategic use of proteomics to study eosinophils in human diseases.