Major Basic Protein Research Articles

Association of eosinophil-mediated inflammatory biomarkers with the presence of the Schatzki ring

PurposeThe study aimed to assess the level of inflammatory biomarkers related to eosinophilia: interleukins 5 (IL-5) and 13 (IL-13), eotaxin 3, major basic protein (MBP) and transforming growth factor β1 (TGF-β1) in patients with dysphagia and Schatzki ring (SR), as well as the characteristics of this group of patients in terms of the features differentiating gastroesophageal reflux disease (GERD) and eosinophilic esophagitis (EoE). Patients and methodsWe analyzed 42 patients with dysphagia, each of whom underwent panendoscopy with an assessment of the occurrence of SR, retrospectively assessed EoE Endoscopic Reference Score (EREFS) total, inflammatory and fibrostenotic and serum concentrations of IL-5 and 13, TGF-β1, eotaxin 3 and MBP. All of them completed a symptom and comorbid questionnaire. Patients diagnosed with SR constituted the SR group (n ​= ​8), the rest – the non-SR group. The quantification of the biomarkers was performed by enzyme immunoassay (ELISA). In the data analysis, p ​≤ ​0.05 was considered statistically significant. ResultsWe demonstrated a significant increase in terms of exceeding the reference values of TGF-β1 (37.5% vs 8.8%) and MBP (75% vs 35.3%) in patients with SR compared to the non-SR group (qualitative analysis). There was also a statistically significant increase in the concentration of each of the determined biomarkers (quantitative analysis) in the SR group. ConclusionsThe increase in TGF-β1 and MBP concentrations indicates the inflammatory and probably fibrostenotic pathogenesis of SR. Obtained results do not allow for an unequivocal classification of SR as a complication typical only for GERD or EoE.

Involvement of inflammatory cells in chronic rhinosinusitis with nasal polyps.

Inflammation plays an important role in the pathogenesis of nasal polyps. Understanding the biomolecular action mechanisms of inflammatory elements can contribute to improving the prognosis of these lesions. The study analyzed the distribution and immunohistochemically quantified eosinophils [eosinophil major basic protein (BMK-13)], lymphocytes [cluster of differentiation (CD) 4, CD8, CD20] and plasmocytes (CD138) in both the epithelial and stromal compartment in relation to composite scores, which included specific histopathological parameters for 50 sinonasal polyps. Inflammatory elements predominated at stromal level, the high histological composite scores being frequently associated with increased expression of inflammatory elements. Also, the numerical distribution of inflammatory elements indicated positive linear relations within the groups BMK-13/CD8 and CD4/CD20/CD138, and a negative linear relation between the two groups. This aspect can support the existence of alternative or sequential pathogenic mechanisms involved in the pathogenesis of sinonasal polyps, and the results obtained can be used for a better stratification of patients in order to optimize the therapy.

Eosinophils in the pathogenesis of pancreatic disorders.

Eosinophils comprise approximately 1-4% of total blood leukocytes that reside in the intestine, bone marrow, mammary gland, and adipose tissues to maintain innate immunity in healthy individuals. Eosinophils have four toxic granules known as major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), and upon degranulation, these granules promote pathogenesis of inflammatory diseases like allergy, asthma, dermatitis, and gastrointestinal disorders. Additionally, the role of eosinophils is underscored in exocrine disorders including pancreatitis. Chronic pancreatitis (CP) is an inflammatory disorder that occurs due to the alcohol consumption, blockage of the pancreatic duct, and trypsinogen mutation. Eosinophil levels are detected in higher numbers in both CP and pancreatic cancer patients compared with healthy individuals. The mechanistic understanding of chronic inflammation-induced pancreatic malignancy has not yet been reached and requires further exploration. This review provides a comprehensive summary of the epidemiology, pathophysiology, evaluation, and management of eosinophil-associated pancreatic disorders and further summarizes current evidence regarding risk factors, pathophysiology, clinical features, diagnostic evaluation, treatment, and prognosis of eosinophilic pancreatitis (EP) and pancreatic cancer.

Development and application of novel immunoassays for eosinophil granule major basic proteins to evaluate eosinophilia and myeloproliferative disorders

BackgroundDuring eosinophil differentiation, the granule eosinophil major basic protein 1 (eMBP1) is synthesized as a 32-kDa precursor form, referred to as proMBP1, which is processed into the 14-kDa mature form of eMBP1. The prevalence of these two forms of MBP1 in most pathological conditions remains unknown. ObjectiveTo develop the immunoassays that differentiate mature eMBP1 and proMBP1 and apply them to analyze their levels in biological fluids from patients with eosinophilia and hematologic disorders. MethodsWe produced a series of monoclonal antibodies and selected pairs capable of discriminating between the two molecular forms of eMBP1. Radioimmunoassay (RIA) was performed to simultaneously quantitate the levels of mature eMBP1 and proMBP1 in secretions from patients with chronic rhinosinusitis (CRS) and sera from patients with hypereosinophilic syndrome (HES) and other myeloproliferative disorders. ResultsThe novel immunoassays possessed less than 1% crossreactivity between mature eMBP1 and proMBP1. Mature eMBP1, but not proMBP1, was found in nasal secretions of CRS patients. In contrast, elevated serum levels of mature eMBP1 and proMBP1 were observed in approximately 60% and 90% of HES patients, respectively, with proMBP1 present in greater quantities than mature eMBP1. Patients with several myeloproliferative disorders also showed high serum levels of proMBP1 while mature eMBP1 remained at basal levels. ConclusionThe novel immunoassays successfully differentiated mature eMBP1 and proMBP1 in human biological fluids. Further studies addressing the clinical correlates of these assays will help to develop biomarkers to diagnose and monitor patients with eosinophilia and myeloproliferative disorders.

Eosinophil: a central player in modulating pathological complexity in asthma.

Eosinophils are the major inflammatory cells which play a crucial role in the development of allergic and non-allergic asthma phenotypes. Eosinophilic asthma is the most heterogeneous phenotype where activated eosinophils are reported to be significantly associated with asthma severity. Activated eosinophils display an array of cell adhesion molecules that not only act as an activation marker, suitable for assessing severity, but also secrete several tissue factors, cytokines and chemokines which modulate the clinical severity. Eosinophil activations are also strictly associated with activation of other hetero cellular populations like neutrophils, macrophages, mast cells, and platelets which culminate in the onset and progression of abnormal phenotypes such as bronchoconstriction, allergic response, fibrosis instigated by tissue inflammation, epithelial injury, and oxidative stress. During the activated state, eosinophils release several potent toxic signaling molecules such as major basic proteins, eosinophil peroxidase, eosinophil cationic protein (ECP), and lipid mediators, rendering tissue damage and subsequently leading to allergic manifestation. The tissue mediators render a more complex manifestation of a severe phenotype by activating prominent signaling cross-talk. Here, in the current review with the help of search engines of PubMed, Medline, etc, we have tried to shed light and explore some of the potent determinants regulating eosinophil activation leading to asthma phenotype.

Morphologic, cytochemical, and ultrastructural features of gray eosinophils in nine cats.

Gray eosinophils, resembling those in sighthound dog breeds, have not previously been reported in cats. We aimed to provide a morphologic, cytochemical, and ultrastructural description of gray eosinophils in cats. Blood films examined as part of routine hematology profiles in cats from May 2015 to July 2018 were evaluated for the presence of gray eosinophils. When identified with modified Wright stain, cells were morphologically assessed and additionally stained with Diff-Quik, ALP, Luna, and Luxol fast blue stains and compared with feline controls. Two cases were prepared for transmission electron microscopy (TEM) and compared with a feline control. Gray eosinophils were identified in 9 of 2641 cats during the study period. Compared with typical feline eosinophils, these cells contained abundant round granules instead of the characteristic rod-shaped specific granules. These granules lacked the characteristic intense pink/red staining with Romanowsky stains and did not stain with ALP, Luna, or Luxol fast blue stains. On TEM, the classical electron-dense core of these granules was replaced by a core with fragmented or amorphous internal material. Typical eosinophils were not identified in any cat in which gray eosinophils were identified. The distinct morphologic, cytochemical, and ultrastructural changes in gray feline eosinophils might be associated with a reduction or lack of major basic protein (MBP) in specific granule cores. Similar to canine gray eosinophils, accurate recognition of these cells is essential to prevent their misclassification as toxic neutrophils.

Experimental Modeling of Eosinophil-Associated Diseases.

Eosinophils are an important subtype of leukocytes derived from bone marrow multipotent hematopoietic stem cells and represent about 1% of leukocytes in circulating blood. In homeostatic conditions, eosinophils reside in the intestine to maintain the balance of immune responses by communicating with gut microbes without causing inflammation. However, under the stressed or diseased condition, eosinophils degranulate, releasing their granule-derived cytotoxic proteins that are involved in inflammatory responses. Various eosinophil-associated inflammatory diseases are eosinophilic esophagitis (EoE), eosinophilic gastroenteritis (EG), and eosinophilic colitis (EC), together called EGID, asthma, hypereosinophilic syndrome, and eosinophilic pneumonia (EP). Eosinophildegranulation results in thereleaseof their four toxic proteins [major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN)]which promote disease pathogenesis. Pancreatitis is the inflammatory disease of the pancreas that arises due to blockage of the pancreatic duct, trypsinogen mutation, alcohol consumption, and repeated occurrence of pancreatitis leading to chronic pancreatitis (CP); subsequently some CP patients may also develop pancreatic cancer. The presence of eosinophils is now shown in various case reports with acute, recurrent acute, and chronic pancreatitis and pancreatic cancer indicating the role of eosinophils in the pathogenesis of various pancreatic inflammatory disorders. However, the details of eosinophil accumulation during pancreatic diseases are not well explored and need further attention. Overall, the chapter provides the current understanding of reported eosinophils associated with inflammatory diseases like EGID diseases, asthma, and pancreatic disorders, i.e., acute, chronicpancreatitis, and pancreatic cancer. This knowledge will be helpful for future studies to develop novel treatment options for the eosinophils associated diseases. Therefore, more efforts are needed to perform preclinical and clinical studies in this field for the successful development of eosinophil-targeting treatments for a variety of eosinophil-associated diseases.

Eosinophilic esophagitis and asymptomatic esophageal eosinophilia display similar immunohistological profiles.

Patients with asymptomatic esophageal eosinophilia (aEE) do not exhibit clinical symptoms because of esophageal dysfunction, although they have endoscopic and histological findings similar to those of eosinophilic esophagitis (EoE). The cause of the symptoms and the differences between aEE and EoE are unclear. The aim of this study is to determine whether aEE and EoE are same disease entities by comparing immune-related tissue biomarkers using immunohistological staining. Esophageal biopsy specimens from 61 patients, including 18 with aEE and 43 with EoE, were analyzed. Immunofluorescence staining was performed to quantify the immune-related tissue biomarkers such as major basic protein, eosinophil-derived neurotoxin, eotaxin-3, and immunoglobulin G4. Data are presented as median (interquartile range). There were no significant differences in clinical, endoscopic, or histological features, between patients with aEE and EoE, with the exception of body mass index. There were no significant differences in all immune-related tissue biomarkers between both groups. In conclusions, EoE and aEE displayed similar immunohistological profiles. Hence, they may be similar disease entities with some common pathogenic mechanisms. Our findings suggest that patients with aEE also have histopathological esophageal inflammation.

Intrauterine inflammation induced white matter injury protection by fibrinogen-like protein 2 deficiency in perinatal mice

BackgroundWhite matter injury (WMI) induced by intrauterine inflammation can cause adverse neurological outcomes. Fibrinogen-like protein 2 (FGL2)/fibroleukin is an important trigger of inflammatory responses and is involved in some cerebral diseases. However, the role of FGL2 in intrauterine inflammation-induced WMI remains unclear.MethodsLipopolysaccharide (LPS) was intraperitoneally injected into wild-type and FGL2 knockout mice to induce intrauterine inflammation. Body weight and brain weight of offspring were monitored. Major basic protein (MBP) expression was evaluated to demonstrate the myelination of offspring. To investigate the regulatory mechanism of FGL2, cytokine expression, microglial polarization, and the activation of mitogen-activated protein kinase (MAPK) signaling pathway in the offspring were analyzed.ResultsUpon LPS exposure, FGL2 knockout offspring showed a significant increase in body weight loss. MBP reduction induced by LPS was prevented in FGL2 knockout offspring. Expression levels of proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α, and M1 marker CD86 were suppressed, while the expression levels of anti-inflammatory cytokines IL-10 and M2 marker CD206 were increased. FGL2 deficiency significantly inhibited the phosphorylation of p38MAPK and c-Jun N-terminal kinase (JNK) protein.ConclusionsFGL2 deficiency can ameliorate WMI induced by intrauterine inflammation, reducing inflammatory cascade and improving hypomyelination, through the regulation of microglial polarization and MAPK signaling pathways.ImpactIntrauterine inflammation induces WMI leading to severe neurological sequelae. FGL2 plays an important role in the progression of WMI induced by intrauterine inflammation.FGL2 deficiency can protect against WMI by inhibiting p38 MAPK and JNK phosphorylation, regulating microglia polarization, and reducing inflammation response.FGL2 could be a novel molecular target for protecting against WMI induced by intrauterine inflammation.

Proton-pump Inhibitor Response Prediction Using Esophageal microRNAs in Children With Eosinophilic Esophagitis.

ABSTRACTObjectives:Eosinophilic esophagitis (EoE) is a chronic esophageal disease characterized by eosinophilic inflammation. Proton-pump inhibitors (PPI) induce disease remission but no predictive factors of PPI-responsiveness have been identified yet. So, a biomarker must be found to differentiate between responders (PPI-R) and nonresponder patients (PPI-NR) to PPI. Aims were to identify any molecular biomarker that could predict PPI responsiveness and to study molecular remission after PPI therapy.Methods:This prospective study enrolled 39 controls and 43 pediatric children with EoE from 2 hospitals, and they were treated with esomeprazole for 8 to 12 weeks. After therapy, patients were classified as either PPI-R or PPI-NR. Biopsies were collected and RNA, microRNAs, and proteins were isolated from them, measuring levels by qPCR and Western blot (WB). Also, miRNAs were evaluated in serum.Results:We found several esophageal miRNAs with different expression values between PPI-R and PPI-NR children, which can be used to discriminate them (area under curve = 0.90). No useful serum miRNAs were, however, identified. Also, these miRNAs were dysregulated in responder patients before and after PPI therapy. Moreover, we corroborated in this child population, that PPI-R displayed a significant decrease in eotaxin-3, IL-5, IL-13, periostin, and major basic protein (P < 0.05) and a significant increase in filaggrin levels after PPI treatment (P < 0.01).Conclusions:Esophageal miRNA levels found are able to discriminate between both PPI-R and PPI-NR at baseline, and before and after treatment in PPI-R, so they could be used as biomarkers. Furthermore, we observed clinical and esophageal molecular restoration in PPI-R patients after PPI therapy.

MicroRNA miR-155-5p knockdown attenuates Angiostrongylus cantonensis-induced eosinophilic meningitis by downregulating MMP9 and TSLP proteins

Angiostrongylus cantonensis infection is a major cause of eosinophilic meningitis (EM). Severe cases or cases that involve infants and children present poor prognoses. MicroRNAs (miRNAs), which are important regulators of gene expression in many biological processes, were recently found to be regulators of the host response to infection by parasites; however, their roles in brain inflammation caused by A. cantonensis are still unclear. The current study confirmed that miR-155-5p peaked at 21 days after A. cantonensis infection, and its expression was positively correlated with the concentration of excretory and secretory products (ESPs). We found that miR-155-5p knockdown lentivirus successfully ameliorated brain injury and downregulated the expression of major basic protein (MBP) in vivo, and the number of eosinophils in CSF (and the percentage of eosinophils in peripheral blood were also decreased in the miR-155-5p knockdown group. Moreover, the expression of several eosinophilic inflammation cytokines such as CCL6/C10, ICAM-1, and MMP9, declined after the miR-155-5p knockdown. SOCS1 protein, which is an important negative regulator of inflammation activation, was identified as a direct miR-155-5p target. We further detected the effect of miR-155-5p knockdown on phosphorylated-STAT3 and phosphorylated-p65 proteins, which were found to be negatively regulated by SOCS1 and play an important role in regulating the inflammatory response. We found that miR-155-5p knockdown decreased the activity of p-STAT3 and p-p65, thereby leading to lower expression of MMP9 and TSLP proteins, which were closely related to the chemotaxis and infiltration of eosinophils. Interestingly, the inhibition of p-STAT3 or p-p65 was found to induce the downregulation of miR-155-5p in an opposite manner. These observations suggest that a positive feedback loop was formed between miR-155-5p, STAT3, and NF-κB in A. cantonensis infection and that miR-155-5p inhibition might provide a novel strategy to attenuate eosinophilic meningitis.

Utility of major basic protein, eotaxin-3, and mast cell tryptase staining for prediction of response to topical steroid treatment in eosinophilic esophagitis: analysis of a randomized, double-blind, double dummy clinical trial.

Inflammatory factors in eosinophilic esophagitis (EoE), including major basic protein (MBP), eotaxin-3 (EOT3) and mast cell tryptase (TRP), may predict treatment response to topical corticosteroids (tCS). We aimed to determine whether baseline levels of these markers predict response to tCS for EoE. To do this, we analyzed data from a randomized trial comparing two topical steroids for treatment of newly diagnosed EoE (NCT02019758). A pretreatment esophageal biopsy was stained for MBP, EOT3, and TRP to quantify tissue biomarker levels (cells/mm2). Levels were compared between histologic responders (<15 eos/hpf) and nonresponders (the primary outcome), and endoscopic responders (EREFS<2) and nonresponders. Complete histologic response (<1 eos/hpf) was also assessed, and area under the receiver operator characteristic curve (AUC) was calculated. We also evaluated whether baseline staining predicted symptom relapse in the trial's off-treatment observation phase. Baseline samples were evaluable in 110/111 subjects who completed the randomized trial. MBP levels were higher in nonresponders (n = 36) than responders (704 vs. 373 cells/mm2; P = 0.007), but EOT3 and TRP levels were not statistically different. The combination of all three stains had an AUC of 0.66 to predict response. For complete histologic response, baseline TRP levels were higher in nonresponders (n = 69) than responders (370 vs. 268 mast cells/mm2; P = 0.01), with an AUC of 0.65. The AUC for endoscopic response was 0.68. Baseline staining did not predict symptom recurrence after remission. Pretreatment MBP, EOT3, and TRP levels were not strongly or consistently associated with histologic or endoscopic response to topical steroids. While elevated TRP levels may be associated with nonresponse compared with complete response, the magnitude and predictive utilities were modest. Novel methods for predicting steroid response are still required.

Strongyloides stercoralis Infection in Solid Organ Transplant Patients Is Associated With Eosinophil Activation and Intestinal Inflammation: A Cross-sectional Study.

Strongyloidiasis can cause devastating morbidity and death in immunosuppressed patients. Identification of reliable biomarkers for strongyloidiasis in immunosuppressed patients is critical for the prevention of severe disease. In this cross-sectional study of solid organ transplant (SOT) candidates and recipients, we quantified Strongyloides-specific IgG to the recombinant NIE-Strongyloides antigen and/or to a soluble extract of S. stercoralis somatic antigens ("crude antigen") using enzyme-linked immunosorbent assays (ELISAs). We also measured peripheral eosinophilia, 4 different eosinophil granule proteins, and intestinal fatty acid-binding protein (IFABP). We evaluated serum biomarkers in 149 individuals; 77 (52%) pre-SOT and 72 (48%) post-SOT. Four percent (6/149) tested positive by NIE ELISA and 9.6% (11/114) by crude antigen ELISA (overall seropositivity of 9.4% [14/149]). Seropositive patients had higher absolute eosinophil counts (AECs) than seronegative patients (P = .004). AEC was positively correlated to the levels of eosinophil granule proteins eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) (P < .05), while IFABP was positively related to the 2 other eosinophil granule proteins (major basic protein [MBP] and eosinophil-derived neurotoxin [EDN]; Spearman's r = 0.3090 and 0.3778, respectively; P < .05; multivariate analyses slopes = 0.70 and 2.83, respectively). This study suggests that, in SOT patients, strongyloidiasis triggers both eosinophilia and eosinophil activation, the latter being associated with intestinal inflammation. These data provide insight into the pathogenesis of S. stercoralis infection in the immunocompromised population at high risk of severe strongyloidiasis syndromes.

16 CCL11 EXACERBATES COLITIS VIA MODULATION OF EPITHELIAL WOUND REPAIR

Abstract Background We have shown that CCL11 (eotaxin-1), an eosinophil chemoattractant, is significantly increased in the serum of ulcerative colitis and Crohn’s disease patients vs controls and is also increased in dextran sulfate sodium (DSS)-induced murine colitis. In response to azoxymethane (AOM)-DSS, Ccl11–/– mice have significantly decreased colonic tumor number and burden, histologic injury, and colonic eosinophil infiltration vs wild-type (WT) mice. Ccl11 is expressed by isolated colonic epithelial and lamina propria immune cells. Studies in bone marrow chimera mice revealed that both hematopoietic- and epithelial-cell derived CCL11 were important for protection in the AOM-DSS model. Our aim was to assess the role of CCL11 in an injury and recovery colitis model and in an epithelial wound restitution model. Methods C57BL/6 WT and Ccl11–/– mice were exposed to 4% DSS in the drinking water for 5 days followed by 5 days of water for an injury and recovery model. Body weights were assessed daily. Histologic injury was assessed by a validated scoring system including depth and percent involvement of inflammation and crypt damage. Colonic eosinophil and neutrophil infiltration was determined by immunohistochemistry for major basic protein (MBP) and myeloperoxidase (MPO), respectively. The number of MBP- and MPO-positive cells per high-powered field (HPF) were quantified in a blinded manner. Young Adult Mouse Colon (YAMC) epithelial cells were stained with antibodies to the CCL11 receptors CCR2, CCR3, and CCR5 for assessment by flow cytometry. YAMC cell monolayers were wounded and followed for 24 h in normal media or with the addition of 100ng/mL recombinant CCL11 (rCCL11). The wound area was measured at 0 and 24 h to determine percent restitution. RNA was isolated and assessed for Ccl11 mRNA expression. Results In the injury and recovery model, Ccl11–/– mice exhibited decreased body weight loss (p&amp;lt;0.05), histologic injury (20.6 ± 1.5 vs 12.4 ± 1.6, p&amp;lt;0.001), and fewer MBP-positive cells/HPF (23.5 ± 1.4 vs 2.4 ± 0.2, p&amp;lt;0.001) vs WT mice. While colonic MPO-positive cells were increased with DSS exposure, there was no difference in Ccl11–/– mice. Naïve/unstimulated YAMC cells expressed both CCR3 and CCR5. Wounding the YAMC monolayer alone did not lead to increased Ccl11 mRNA expression. However, in the presence of rCCL11, there was a decrease in wound restitution at 24 h (48.0% ± 1.7 vs 31.3% ± 2.1, p&amp;lt;0.001). Conclusions Loss of CCL11 leads to clinical and histologic improvement in an injury and recovery colitis model. Colonic epithelial cells express both CCR3 and CCR5, and epithelial wound restitution is decreased in the presence of rCCL11. These data suggest that CCL11 has a deleterious effect on epithelial restitution leading to an exacerbation of colitis. Therefore, treatment with anti-CCL11 antibodies may be a therapeutic strategy in IBD patients.

Fibrinogen Is a Specific Trigger for Cytolytic Eosinophil Degranulation.

In inflamed human tissues, we often find intact eosinophilic granules, but not eosinophils themselves. Eosinophils, tissue-dwelling granulocytes with several homeostatic roles, have a surprising association with fibrinogen and tissue remodeling. Fibrinogen is a complex glycoprotein with regulatory roles in hemostasis, tumor development, wound healing, and atherogenesis. Despite its significance, the functional link between eosinophils and fibrinogen is not understood. We tested IL-5-primed mouse bone marrow-derived and human blood-sorted eosinophil activity against FITC-linked fibrinogen substrates. The interactions between these scaffolds and adhering eosinophils were quantified using three-dimensional laser spectral, confocal, and transmission electron microscopy. Eosinophils were labeled with major basic protein (MBP) Ab to visualize granules and assessed by flow cytometry. Both mouse and human eosinophils showed firm adhesion and degraded up to 27 ± 3.1% of the substrate area. This co-occurred with active MBP-positive granule release and the expression of integrin CD11b. Mass spectrometry analysis of fibrinogen proteolytic reactions detected the presence of eosinophil peroxidase, MBP, and fibrin α-, β-, and γ-chains. Eosinophil activity was adhesion dependent, as a blocking Ab against CD11b significantly reduced adhesion, degranulation, and fibrinogenolysis. Although adhered, eosinophils exhibited no proteolytic activity on collagen matrices. Cytolytic degranulation was defined by loss of membrane integrity, cell death, and presence of cell-free granules. From transmission electron microscopy images, we observed only fibrinogen-exposed eosinophils undergoing this process. To our knowledge, this is the first report to show that fibrinogen is a specific trigger for cytolytic eosinophil degranulation with implications in human disease.

Increased CD69 expression on activated eosinophils in eosinophilic chronic rhinosinusitis correlates with clinical findings

BackgroundEosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis associated with asthma. CD69 is an important marker of activation for eosinophils. But, whether a correlation exist between the CD69 expression on eosinophils and clinical findings is unclear. MethodsWe performed quantitative PCR and/or flow cytometry using tissue and purified eosinophils from the blood and nasal polyps of 12 patients with ECRS and from 8 patients without ECRS (controls). We assessed clinical findings including nasal polyp (NP) scores, sinus CT findings, and pulmonary function test results, and examined their possible association with the CD69 expression. We also performed CD69 cross-linking experiments in mouse eosinophils to investigate the functional role of CD69. ResultsLevels of cytokine mRNAs (IL-4, -5, -10, and -13) were significantly higher in purified NP eosinophils and tissues from patients with ECRS than the levels of those in controls. The expressions of major basic protein (MBP), eosinophilic cationic protein (ECP), eosinophilic-derived neurotoxin (EDN), eosinophil peroxidase (EPX) in cytotoxic granules, and CD69 mRNA were significantly higher in purified eosinophils from NPs than in those from blood. We also found a correlation between expression of CD69 and clinical findings. Moreover, we found EPX release from mouse eosinophils following CD69 cross-linking. ConclusionsThese data suggest that increased CD69 expression by eosinophils is not only a biomarker for nasal obstruction and pulmonary dysfunction, but also a potential therapeutic target for patients with ECRS and asthma.

Lockdown measures and relative changes in the age-specific incidence of SARS-CoV-2 in Spain.

During the first months of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) epidemic in 2020, Spain implemented an initial lockdown period on 15 March followed by a strengthened lockdown period on 30 March when only essential workers continued to commute to work. However, little is known about the epidemic dynamics in different age groups during these periods.We used the daily number of coronavirus 2019 cases (by date of symptom onset) reported to the National Epidemiological Surveillance Network among individuals aged 15-19 years through 65-69 years. For each age group g, we computed the proportion PrE(g) of individuals in age group g among all reported cases aged 15-69 years during the pre-lockdown period (1-10 March 2020) and the corresponding proportion PrL(g) during two lockdown periods (initial: 25 March-3 April; strengthened: 8-17 April 2020). For each lockdown period, we computed the proportion ratios PR(g) = PrL(g)/PrE(g). For each pair of age groups g1, g2, PR(g1)>PR(g2) implies a relative increase in the incidence of detected SARS-CoV-2 infection in the age group g1 compared with g2 for the lockdown period vs. the pre-lockdown period.For the initial lockdown period, the highest PR values were in age groups 50-54 years (PR = 1.21; 95% CI: 1.12,1.30) and 55-59 years (PR = 1.19; 1.11,1.27). For the second lockdown period, the highest PR values were in age groups 15-19 years (PR = 1.26; 0.95,1.68) and 50-54 years (PR = 1.20; 1.09,1.31).Our results suggest that different outbreak control measures led to different changes in the relative incidence by age group. During the initial lockdown period, when non-essential work was allowed, individuals aged 40-64 years, particularly those aged 50-59 years, had a higher relative incidence compared with the pre-lockdown period. Younger adults/older adolescents had an increased relative incidence during the later, strengthened lockdown. The role of different age groups during the epidemic should be considered when implementing future mitigation efforts.

Peptides of major basic protein and eosinophil cationic protein activate human mast cells

The eosinophil granule proteins, major basic protein (MBP) and eosinophil cationic protein (ECP), activate mast cells during inflammation; however the mechanism responsible for this activity is poorly understood. We found that some theoretical tryptase-digested fragments of MBP and ECP induced degranulation of human cord blood-derived mast cells (HCMCs). The spectrum of activities of these peptides in HCMCs coincided with intracellular Ca2+ mobilization activities in Mas-related G-protein coupled receptor family member X2 (MRGPRX2)-expressing HEK293 cells. Two peptides corresponding to MBP residues 99–110 (MBP (99–110)) and ECP residues 29–45 (ECP (29–45)), respectively, induced degranulation of HCMCs and intracellular Ca2+ mobilization in MRGPRX2-expressing HEK293 cells in a concentration-dependent manner. Stimulation with MBP (99–110) or ECP (29–45) induced the production of prostaglandin D2 by HCMCs. The activities of MBP (99–110) and ECP (29–45) in both HCMCs and MRGPRX2-expressing HEK293 cells were inhibited by MRGPRX2-specific antagonists. In conclusion, these results indicated that MBP and ECP fragments activate HCMCs, and it may occur via MRGPRX2. Our findings suggest that tryptase-digested fragments of eosinophil cationic proteins acting via the MRGPRX2 pathway may further our understanding of mast cell/eosinophil communication.

Bioinformatic analysis of eosinophil activity and its implications for model and target species.

Eosinophils are important immune cells that have been implicated in resistance to gastrointestinal nematode (GIN) infections in both naturally and experimentally infected sheep. Proteins of particular importance appear to be IgA-Fc alpha receptor (FcαRI), C-C chemokine receptor type 3 (CCR3), proteoglycan 3 (PRG3, major basic protein 2) and EPX (eosinophil peroxidase). We used known human nucleotide sequences to search the ruminant genomes, followed by translation to protein and sequence alignments to visualize differences between sequences and species. Where a sequence was retrieved for cow, but not for sheep and goat, this was used additionally as a reference sequence. In this review, we show that eosinophil function varies among host species. Consequently, investigations into the mechanisms of ruminant immune responses to GIN should be conducted using the natural host. Specifically, we address differences in protein sequence and structure for eosinophil proteins.

Poor Correlation of Oral Swabs with Esophageal Eosinophil Counts.

Eosinophilic esophagitis (EoE) is a chronic condition that requires repeated endoscopies/biopsies to track the disease and treatment response. This invasive procedure involves risk to the patient and has significant costs. We studied whether the detection of specific proteins (cytokines and eosinophil degranulation products) from oral swabs could serve as a minimally invasive test for EoE. Swabs of the oral cavity (buccal and oropharyngeal) were obtained prior to endoscopy/biopsies in patients with EoE, possible EoE, and non-EoE patients in addition to obtaining additional esophageal biopsy tissue. ELISAs measuring the levels of cytokines IL-5, IL-8, IL-13, and eosinophil degranulation products including major basic protein (MBP), eosinophil derived neurotoxin (EDN), and eosinophil peroxidase (EPO) were performed on the samples. Comparisons were made to peak esophageal eosinophil counts. Tolerability of the swabs was evaluated. 43 patients, 4-17years old, participated in the study. Swabs were well tolerated and all showed measurable protein. 26 patients had EoE [14 active (> 15 eosinophils/high power field), 12 non-active], 17 patients did not have EoE. Results obtained from oral swabs showed poor correlation with those from esophageal tissue. Only measurement of eosinophil degranulation products EDN and EPO from esophageal tissues showed strong correlations with eosinophil counts. In this study, the levels of cytokines and eosinophil degranulation products detected from oral swabs did not correlate with esophageal eosinophilia, and their detection would be insufficient to displace endoscopy/biopsies.