Abstract Rationale Neurogenic inflammation is recognised as an important contributor to the pathophysiology of Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). Substance P (Sub P), a neuropeptide released from sensory nerves, is a potent inducer of mast cell degranulation through the Mas-related G protein-coupled receptor member X2 (MRGPRX2), resulting in the release of pro-inflammatory mediators that perpetuate chronic bladder inflammation. Medihoney, a medical-grade Mānuka honey, possesses well-established antimicrobial and anti-inflammatory properties, and we have recently demonstrated its ability to stabilise mast cells through inhibition of histamine release. However, its effects on Sub P-induced mast cell activation and MRGPRX2-mediated neurogenic inflammation have not previously been investigated. Aim of the study We aimed to investigate the inhibitory effects of Medihoney and a sugar-free Mānuka honey extract on Substance P-induced mast cell degranulation and MRGPRX2 activation as potential therapeutic approaches for chronic neurogenic inflammation associated with IC/BPS. In addition, we examined the expression of MRGPRX2 in bladder biopsies from patients with IC/BPS. Materials and methods Human LAD2 mast cells were stimulated with Substance P (1 μM) for 40 minutes following 20-minute pre-incubation with Medihoney or a sugar-free Mānuka honey extract. Mast cell degranulation was quantified by measuring β-hexosaminidase release. MRGPRX2 activation was assessed by intracellular calcium imaging using Fluo-4 in MRGPRX2-expressing HEK-293 cells. Bladder biopsies obtained from patients with IC/BPS and healthy controls were immunostained for mast cell tryptase, chymase and MRGPRX2. Results Medihoney at 2% and 4% markedly inhibited Substance P-induced mast cell degranulation in LAD2 cells by approximately 90%, an effect that was similarly observed with the sugar-free Mānuka honey extract. Both preparations produced a dose-dependent inhibition of Substance P-induced intracellular signalling in MRGPRX2-expressing HEK-293 cells, demonstrating suppression of MRGPRX2 activation. Furthermore, immunohistochemical analysis of bladder biopsies revealed that approximately 66% of tryptase-positive mast cells expressed MRGPRX2 in patients with IC/BPS, which was significantly higher than that observed in healthy control tissues (25%). Conclusion The present study demonstrates that mast cells within IC/BPS bladder tissue express increased levels of MRGPRX2, suggesting enhanced responsiveness to Substance P and supporting a role for neurogenic inflammation in the pathophysiology of IC/BPS. Medihoney and the sugar-free Mānuka honey extract significantly inhibit Substance P-induced mast cell degranulation through modulation of MRGPRX2-mediated intracellular signalling, highlighting their potential as novel therapeutic agents for reducing neurogenic bladder inflammation associated with IC/BPS. Impact This study provides evidence that MRGPRX2-mediated neurogenic mast cell activation is enhanced in IC/BPS and demonstrates, for the first time, that Medihoney and a sugar-free Mānuka honey extract effectively inhibit Substance P-induced mast cell degranulation through modulation of MRGPRX2 signalling. These findings provide new mechanistic insight into the anti-inflammatory actions of Mānuka honey-derived preparations and identify MRGPRX2 as a potential therapeutic target in IC/BPS. The observed inhibition of neurogenic mast cell activation suggests that these naturally derived preparations may offer a novel strategy for limiting chronic bladder inflammation. Overall, this work provides a foundation for future preclinical and clinical studies evaluating the safety and therapeutic efficacy of Medihoney and Mānuka honey-derived compounds in patients with IC/BPS.

Background: Mast cells play crucial roles in the pathogenesis of allergies through the secretion of powerful mediators, prominent among which is a group of proteases that are stored and function within the intracellular granules of mast cells. Objectives: We aimed to investigate the secretion of these proteases from mast cells during degranulation and to explore the factors that can influence the secretion and gene expression of mast cell mediators. Materials and methods: Mast cell line (LAD2) were stimulated to degranulate with calcium ionophore A23187 (CaI) or substance P (SP). Gene expressions of dipeptidyl peptidase I (DPPI) and carboxypeptidase A3 (CPA3), chymase (CHY), tryptase (TRY) were determined by quantitative RT-PCR. Alterations in gene expression were investigated at time points ranging from 0.5 to 6 hours following cell stimulation. Beta-hexosaminidase activity and DPPI levels were measured using ELISA. Results: Experimental stimulation of sensitized mast cells by CaI or SP, revealed by the release of the activation marker β-hexosaminidase in a time dependent manner (net release=63% ± 2.4 at 6 h), this was associated with significant increases in gene expression for DPPI and also for CPA3, CHY and TRY. Cal sensitization produced a significant increase in DPPI, CPA3 and CHY in the first 30m of stimulation with gradual decrease in levels with time but still significantly higher than non-stimulated cells (p≤0.05 for all). The reverse for TRY, as the most significant increase in its expression was at 2H. SP produced a gradual increase in gene expression for all studied parameters (p≤0.05 for all) with the most significant increase was for TRY gene at 2h (p˂0.01 vs cal). Conclusion: DPPI can be released from mast cells on degranulation, and the subsequent increases in gene expression would be consistent with the later replenishment of secretory granules. This protease may participate in processes of protease activation and other activities outside the cell in inflammatory disease. Thus, DPPI could be represented as an innovative marker for mast cell sensitization which may accurately diagnose the allergic responses specially when measured as panel with CPA3, CHY and TRY.

Staphylococcus aureus (S. aureus) colonizes the nasal cavities of both healthy individuals and patients with chronic rhinosinusitis (CRS) with (CRSwNP) and without (CRSsNP) nasal polyps. Treatment-resistant S. aureus biofilms and intracellular persistence are common in CRS patients, requiring the expression of specific virulence factor genes to transition into these forms. We hypothesized that S. aureus isolates from non-diseased controls, CRSsNP patients, and CRSwNP patients would exhibit distinct virulence factor patterns contributing to persistence and intracellular survival in CRS patients. Nasal swabs from seventy-seven individuals yielded S. aureus cultures in eight non-diseased controls, eight CRSsNP patients, and five CRSwNP patients. Whole-genome sequencing analyzed stress, antimicrobial resistance, and virulence genes, including plasmids and prophages. Four virulence factor gene patterns emerged: a core set (hlgA, icaC, hlgB, hlgC, hld, and aur) present in all isolates, and accessory sets, including the enterotoxin gene cluster (seo, sem, seu, sei, and sen) and a partial/complete invasive virulence factor set (splE, splA, splB, lukE, and lukD) (p = 0.001). CRSwNP isolates exhibited incomplete carriage of the core set, with frequent loss of scn, icaC, and hlgA (p < 0.05). These findings suggest that S. aureus has clusters of virulence factors that may act in concert to support the survival and persistence of the bacteria, resulting in enhanced pathogenicity. This may manifest clinically with resistant disease and refractoriness to antibiotics.

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Interstitial Cystitis (PD25)1 May 2024PD25-08 MEDIHONEY AND MĀNUKA HONEY EXTRACT INHIBIT THE ACTIVATION OF THE MAST CELL RELATED-G-PROTEIN COUPLED RECEPTOR X2 (MRGPRX2): POTENTIAL INTRAVESICAL USE IN PATIENTS WITH INFLAMMATORY BLADDER PATHOLOGY Omar Abdelwahab, Brian Birch, Oliver Miller, Prapussara Sirikhansaeng, Laurie Lau, Andrew Walls, Troy Merry, Jackie Evans, and Bashir Lwaleed Omar AbdelwahabOmar Abdelwahab , Brian BirchBrian Birch , Oliver MillerOliver Miller , Prapussara SirikhansaengPrapussara Sirikhansaeng , Laurie LauLaurie Lau , Andrew WallsAndrew Walls , Troy MerryTroy Merry , Jackie EvansJackie Evans , and Bashir LwaleedBashir Lwaleed View All Author Informationhttps://doi.org/10.1097/01.JU.0001008584.88541.ff.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Mast cells numbers and activity are significantly elevated in several inflammatory conditions including interstitial cystitis (IC/BPS)This condition is associated with the release of neuroactive substances, e.g. substance P (SP), which induce mast cell degranulation and tissue inflammationMedihoney® (MH) is a medical grade Mānuka honey with strong anti-microbial properties. Recent reports demonstrate its anti-inflammatory properties via inhibition of mast cell histamine releaseIn the present study, we investigated the anti-inflammatory effect of MH and a sugar free Mānuka honey extract (EX) against neurogenic inflammation by studying their effects on SP-induced mast cell degranulation via its associated receptor MRGPRX2. METHODS: LAD2 mast cells were activated with SP with or without pre-incubation with MH or EX at 4, 2 and 1% concentrations. 1. Degranulation was assessed via a β-hexosaminidase release assay. 2. The effect of MH and EX on MRGPRX2 activation was assessed via Fluo-4 Direct™ Calcium Assay in HEK-293 cells transfected to express MRGPRX2 using the Flexstation II 384 platform. RESULTS: 1. Pre-incubation of LAD2 cells with MH or EX induced strong inhibition of SP-induced β-hexosaminidase release (Figure 1A & B)2. In addition, both MH and EX induced 77% and 62% drops in substance P-induced Calcium flux, respectively in the transfected HEK-293 cells (Figure 1 C)These effects are independent of the sugars naturally occurring in MH and both effects were maximal at 4% concentration. CONCLUSIONS: Both MH and EX exert a strong dose-dependent inhibition of 1. degranulation in LAD2 cells and2. MRGPRX2 activation in transfected HEK-293 cells. Based on our results, MH and EX (used intravesically) could potentially be useful as anti-inflammatory agents directed against the neurogenic inflammation involved in the pathophysiology of IC/BPS. Download PPT Source of Funding: PhD degree funding © 2024 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 211Issue 5SMay 2024Page: e539 Advertisement Copyright & Permissions© 2024 by American Urological Association Education and Research, Inc.Metrics Author Information Omar Abdelwahab More articles by this author Brian Birch More articles by this author Oliver Miller More articles by this author Prapussara Sirikhansaeng More articles by this author Laurie Lau More articles by this author Andrew Walls More articles by this author Troy Merry More articles by this author Jackie Evans More articles by this author Bashir Lwaleed More articles by this author Expand All Advertisement PDF downloadLoading ...

Atopic dermatitis (AD) is a heterogenous inflammatory skin disorder. Our previous study revealed that basophil infiltration in skin is observed in approximately 60% of AD cases. However, the clinical and histological characteristics of AD associated with basophil infiltration remain unclear. We examined basophil infiltration by immunohistochemical staining of 38 specimens from 34 patients who underwent skin biopsies to diagnose AD from April 2016 to September 2021 at Tokyo Medical and Dental University Hospital. The patients/specimens were divided into two groups, 17 patients/21 specimens associated with little or no basophil infiltration (basophil-low group) and 17 patients/17 specimens associated with marked basophil infiltration (basophil-high group). The clinical characteristics of the patients (age, sex, complications, blood biomarkers, skin symptoms, and treatment) and histological features of the specimens were compared between the groups. Basophil-high patients were significantly younger than basophil-low patients. Blood basophil counts were higher in basophil-high patients than in basophil-low patients. CD4(+) T-cell infiltration was more marked in basophil-high specimens than in basophil-low specimens. CD4(+) T cells infiltrated into the dermis as well as into the epidermis only in the basophil-high specimens. Thus, basophil-high AD can be characterized by skin lesions associated with abundant helper T-cell infiltration in younger patients.

Background There is growing evidence to implicate Staphylococcus aureus ( S. aureus ) in the pathogenesis of recalcitrant chronic rhinosinusitis (CRS). Our group has demonstrated the ability of S. aureus to internalise within mast cells in nasal polyps and this may mediate disease recalcitrance. We investigated carriage of virulence genes in CRS-related S. aureus strains and its influence on the bacteria’s ability to localise and survive intracellularly. Methods S. aureus strains isolated from non-CRS controls (n=5), CRSsNP (n=4) and CRSwNP patients (n=4) were sequenced using short read paired sequencing and interrogated for carriage of virulence genes. A representative control and CRSwNP isolate were tested for intracellular survival in the LAD2 mast cell line to investigate phenotypic differences. Results Fifty percent of the CRSwNP group had deletion of the hld gene which may promote small colony variant formation or reduced enterotoxin production, and seventy-five percent expressed virulence genes associated with invasive disease. The CRSwNP isolate had a superior ability to localise intracellularly at 6 and 9 hours and showed a higher burden of S. aureus colony forming units at 24 hours. Conclusions When compared to non-CRS controls, CRS-related S. aureus strains demonstrate increased carriage of virulence genes. This appears to facilitate intracellular localisation of the bacteria conferring a survival advantage and enhancing pathogenicity. The latter may be partly due to a reduction in enterotoxin production and the acquisition of serine proteases splA and B and leukocidins E/D genes. This pathogenic S. aureus phenotype may manifest clinically with disease recalcitrance and refractoriness to antibiotics.

COPYRIGHT Copyright © 2022 Pan, Walls and Pan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author (s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Editorial PUBLISHED 07 July 2022 DOI 10.3389/fimmu.2022.975553

A decrease in the number of basophils in the peripheral blood, or basopenia, has been noted, reflecting the activity of chronic spontaneous urticaria (CSU). Infiltration of basophils into the skin has also been reported, but the mechanism of basopenia in CSU has not been clarified. The phenomenon of basopenia during the active phase of urticaria was confirmed, and basophil numbers increased following symptom improvement in 15 out of 17 patients treated with omalizumab and in 13 of 15 patients treated with antihistamines. Our examination by immunostaining also revealed basophil infiltration of the CSU lesions, as in previous reports, but since most of our patients were already taking oral steroids, it was not considered appropriate to examine the relationship between basophil numbers in tissue and peripheral blood. Then, we used mouse model of contact hypersensitivity with a single application of oxazolone, which is known to stimulate basophil infiltration, and investigated basophil counts in the skin, peripheral blood, and bone marrow. In this model, a decrease in peripheral blood basophil numbers was observed one day after challenge, but not after 2 days, reflecting supplementation from the bone marrow. Indeed, when cultured basophils expressing GFP were transplanted into the peripheral blood, GFP-positive basophil numbers in the peripheral blood remained low even after 2 days of challenge. Despite differences among species and models, these results suggest that one reason for the decrease of basophils in the peripheral blood in CSU may involve migration of circulating basophils into the skin.

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Although elevated blood or sputum eosinophils are present in many patients with COPD, uncertainties remain regarding the anatomical distribution pattern of lung-infiltrating eosinophils. Basophils have remained virtually unexplored in COPD. This study mapped tissue-infiltrating eosinophils, basophils and eosinophil-promoting immune mechanisms in COPD-affected lungs. Surgical lung tissue and biopsies from major anatomical compartments were obtained from COPD patients with severity grades Global Initiative for Chronic Obstructive Lung Disease stages I-IV; never-smokers/smokers served as controls. Automated immunohistochemistry and in situ hybridisation identified immune cells, the type 2 immunity marker GATA3 and eotaxins (CCL11, CCL24). Eosinophils and basophils were present in all anatomical compartments of COPD-affected lungs and increased significantly in very severe COPD. The eosinophilia was strikingly patchy, and focal eosinophil-rich microenvironments were spatially linked with GATA3(+) cells, including type 2 helper T-cell lymphocytes and type 2 innate lymphoid cells. A similarly localised and interleukin-33/ST2-dependent eosinophilia was demonstrated in influenza-infected mice. Both mice and patients displayed spatially confined eotaxin signatures with CCL11(+) fibroblasts and CCL24(+) macrophages. In addition to identifying tissue basophilia as a novel feature of advanced COPD, the identification of spatially confined eosinophil-rich type 2 microenvironments represents a novel type of heterogeneity in the immunopathology of COPD that is likely to have implications for personalised treatment.

Background: Surfactant protein D (SP-D) contributes to innate immunity within the lung and orchestrates cellular responses to pathogens and allergens. There have been suggestions that SP-D may inhibit allergen-induced mast cell activation through binding to allergens, but its actions on mast cells, or with other stimuli for degranulation have been little investigated. Aims: To determine the ability of SP-D to modulate mast cell degranulation, examining in particular substance P and calcium ionophore as stimuli. Methods: A recombinant fragment of SP-D was incubated with cells of the LAD2 mast cell line at various concentrations, and cells were stimulated with calcium ionophore or substance P. Degranulation was assessed by colourimetric determination of beta-hexosaminidase activity in cell supernatants. Results: SP-D inhibited substance P-induced beta-hexosaminidase release (P < 0.001) from LAD-2 cells (up to 88%) in a concentration-dependent manner in the presence of physiological concentrations of calcium. There was a trend for calcium ionophore-induced mast cell degranulation to be inhibited, but this did not reach significance. Conclusions: SP-D may contribute to lung homeostasis by suppressing mast cell activation. Degradation of SP-D as observed in the airways of asthmatics could may be a factor leading to increased mast cell activation.

Staining cells or tissues with basic dyes was the mainstay of mast cell and basophil detection methods for more than a century following the first identification of these cell types using such methods. These techniques have now been largely supplanted by immunohistochemical procedures with monoclonal antibodies directed against unique constituents of these cell types. Immunohistochemistry with antibodies specific for the granule protease tryptase provides a more sensitive and discriminating means for detecting mast cells than using the classical histochemical procedures, and using antibodies specific for products of basophils (2D7 antigen and basogranulin) has allowed detection of basophils that infiltrate into tissues. The application of immunohistochemistry to detect more than one marker in the same cell has underpinned concepts of mast cell heterogeneity based on differential expression of chymase and other proteases. The double labeling procedures employed have also provided a means for investigating the expression of cytokines and a range of other products. Protocols are here set out that have been used for immunohistochemical detection of mast cells and basophils and their subpopulations in human tissues. Consideration is given to pitfalls to avoid and to a range of alternative approaches.

The novel coronavirus disease (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2, an enveloped nonsegmented positive-sense RNA β-coronavirus. The virus can trigger a cascade of biological events that can lead to critical illness. It is urgent to gain an in-depth understanding of the critical activators that foreshadow patients at highest risk for mortality.1Zhou F. Yu T. Du R. et al.Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study.Lancet. 2020; 395: 1054-1062Abstract Full Text Full Text PDF PubMed Scopus (18995) Google Scholar Patients who have received tocilizumab for COVID-19–related cytokine storm have had improvements in C-reactive protein (CRP), lymphocytosis, and chest computed tomography findings.2Mehta P. McAuley D.F. Brown M. et al.COVID-19: consider cytokine storm syndromes and immunosuppression.Lancet. 2020; 395: 1033-1034Abstract Full Text Full Text PDF PubMed Scopus (6865) Google Scholar There exists a lack of understanding on which patients would benefit from tocilizumab administration. We aimed to characterize the group of patients who received tocilizumab and failed to improve, needed mechanical ventilation, or suffered mortality. We studied clinical and laboratory variables in patients receiving tocilizumab, comparing those who did not require mechanical ventilation with those needing mechanical ventilation including those who suffered mortality. Adult patients with polymerase chain reaction–confirmed COVID-19 infection treated with tocilizumab at Spectrum Health System Hospitals from March 20, 2020, to May 18, 2020, were studied. Laboratory indices analyzed were metabolic panel, blood count, CRP, ferritin, fibrinogen, D-dimer, lactate dehydrogenase, and triglycerides. Our institutional protocol dictated that patients received tocilizumab when they required at least 4 L of oxygen or more respiratory support and had less than 14 days of symptoms. Tocilizumab dosing was as follows: 400 mg for 50 to 60 kg, 600 mg for 60 to 85 kg, and 800 mg for greater than 85 kg. Doses could be given every 8 hours and for a maximum of 3 doses. Day 0 was defined as the day tocilizumab was started. Cytokine panels were performed on day 0. Patients were followed for a maximum of 30 days until they met the end point of hospital discharge or mortality. Quantitative data were expressed as mean plus or minus SD, whereas nominal data were expressed as percentages. The groups studied (intubated vs nonintubated or survivors vs deceased) were analyzed using the unpaired 2-tailed t test, whereas difference for nominal data was determined using the χ2 test or the Fisher's exact test. In addition, mixed-effects general linear modeling was performed. The independent variables were days following tocilizumab administration (days −1, 0, 1, and 3; reference value: day 0) and either intubation status or mortality. For all analyses, significance was assessed at P < .05. All analyses were performed using Stata version 16.1 (StataCorp LLC, College Station, Texas). Baseline characteristics revealed more type 2 diabetes in intubated patients and more smokers in the deceased group (Table 1). Neutrophil-to-lymphocyte ratio (NLR) increased from day negative 1 to 0 in intubated patients (P = .057) and decreased in the nonintubated group from 6.9 to 3 compared with 6.8 to 6.7 from day 0 to 3 in the intubated group. Similarly, in the survivors, there was a marked decrease in NLR from day 0 to day 3 from 7.1 to 3.3 (51.2% decrease), whereas in the nonsurvivors, there was an increase from 5 to 6.8 (33.6% increase). In all groups, mean absolute neutrophil count was highest on day 0. In nonsurvivors, day 0 absolute lymphocyte count was 34.1% lower (P = .046). No significant interactions were found in NLR from day negative 1 to day 0 in all groups.Table 1Patient Characteristics Between GroupsCharacteristicsIntubationMortalityNo (n = 34)Yes (n = 32)P valueNo (n = 54)Yes (n = 12)P valueAge, y (mean)56.1 ± 15.257.2 ± 11.0.74555.2 ± 13.463.3 ± 10.6.053Sex.569.512 Male20 (58.8)21 (65.6)32 (59.3)9 (75.0) Female14 (41.2)11 (34.4)22 (40.7)3 (25.0)Race or ethnicity.414.463 White11 (32.4)13 (40.6)18 (33.3)6 (50.0) African American4 (11.8)3 (9.4)5 (9.3)2 (16.7) Hispanic14 (41.2)15 (46.9)25 (46.3)4 (33.3) Asian5 (14.7)1 (3.1)6 (11.1)0 (0)BMI (mean)29.7 ± 5.031.4 ± 7.2.25930.1 ± 5.432.2 ± 9.2.304GFR >6028/31 (90.3)26/31 (83.9).70745/50 (90.0)9 (75.0).177Diabetic7 (20.6)18 (56.3).00319 (35.2)6 (50.0).348Smoking history9 (26.5)15 (46.9).08513 (24.1)11 (91.7)<.001Hypertension15 (44.1)21 (65.6).07926 (48.2)10 (83.3).051Tocilizumab dose, mg688 ± 112688 ± 113.979693 ± 108667 ± 130.471Number of tocilizumab doses.147.659 11 (2.9)0 (0)1 (1.9)0 (0) 25 (14.7)1 (3.1)6 (11.1)0 (0) 328 (82.4)31 (96.9)47 (87.0)12 (100)Additional therapies Convalescent plasma11 (32.4)14 (43.8).34020 (37)5 (41.7).754 Remdesivir01 (3.1).48501 (8.3).182 Hydroxychloroquine20 (58.8)18 (56.3).83333 (61)5 (41.7).333 Corticosteroids5 (14.7)9 (28.1).18311 (20.4)3 (25).708Infections Positive sputum cultures11 (20.2)8 (66.7)<.001 Candida in sputum2 (3.7)5 (41.7)<.001Abbreviations: BMI, body mass index; GFR, glomerular filtration rate.NOTE: Data are presented as number (percentage) unless indicated otherwise. Open table in a new tab Abbreviations: BMI, body mass index; GFR, glomerular filtration rate. NOTE: Data are presented as number (percentage) unless indicated otherwise. Only interleukin (IL)-10 and IL-6 were higher in intubated patients than in nonintubated patients (20.4 pg/mL vs 9.5, P = .001; 26.5 pg/mL vs 10.5, P = .002); there was no difference in serum soluble IL-2 receptor, IL-10, and IL-6 in patients who survived compared with deceased. A moderate correlation was observed between day 0 NLR and IL-6 (ρ = 0.307; P = .021) and between day 0 NLR and CRP (r = 0.326; P < .001). No correlation was observed between day 0 NLR and serum soluble IL-2 receptor, IL-10, or ferritin. Studies have found an increased NLR to be an independent risk factor for severity in patients with COVID-19 infections. This is congruent with mounting evidence on importance of neutrophil response in severe COVID-19. Although nonspecific, existing cohorts have revealed onetime NLR greater than 3.13 and an interquartile NLR range of 5.82 to 15.1 to be predictive of critical illness and persistent elevation correlated with death.3Hwang S.Y. Shin T.G. Jo I.J. et al.Neutrophil-to-lymphocyte ratio as a prognostic marker in critically ill septic patients.Am J Emerg Med. 2017; 35: 234-239Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar,4Liu J. Liu Y. Xiang P. et al.Neutrophil-to-lymphocyte ratio predicts critical illness patients with 2019 coronavirus disease in the early stage.J Transl Med. 2020; 18: 206Crossref PubMed Scopus (527) Google Scholar The mean ratio dropped between days 0 and 3 across all groups except in the patients suffering mortality. Furthermore, NLR correlated with IL-6 levels and may represent a proxy to monitor response to IL-6 blockade. Beyond neutrophilia, neutrophil activation and degranulation are the most biologically active processes in severe COVID-19.5Morrissey S.M. Geller A.E. Hu X. et al.Emergence of low-density inflammatory neutrophils correlates with hypercoagulable state and disease severity in COVID-19 patients.https://www.medrxiv.org/content/10.1101/2020.05.22.20106724v1Date accessed: January 12, 2020Google Scholar Increased activated neutrophil extracellular traps are associated with severity of illness in patients with severe COVID-19.6Middleton E.A. He X.Y. Denorme F. et al.Neutrophil extracellular traps (NETs) contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome.Blood. 2020; 136: 1169-1179Crossref PubMed Scopus (973) Google Scholar,7Zuo Y. Yalavarthi S. Shi H. et al.Neutrophil extracellular traps in COVID-19.JCI Insight. 2020; 5e138999PubMed Google Scholar Neutrophil extracellular trap activation contributes to neutrophilia in COVID-19 simultaneously increasing IL-1β and IL-6, giving biologic plausibility to their respective blockade being therapeutic in severe COVID-19.6Middleton E.A. He X.Y. Denorme F. et al.Neutrophil extracellular traps (NETs) contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome.Blood. 2020; 136: 1169-1179Crossref PubMed Scopus (973) Google Scholar,7Zuo Y. Yalavarthi S. Shi H. et al.Neutrophil extracellular traps in COVID-19.JCI Insight. 2020; 5e138999PubMed Google Scholar The patients who seem to have a more favorable clinical course after tocilizumab are the same patients who had a drop in neutrophil levels. This is true for other biological conditions in which patients receiving tocilizumab have clinical improvement which correlated with a drop in neutrophil count during therapy.8Ghang B. Kwon O. Hong S. Lee C.K. Yoo B. Kim Y.G. Neutrophil-to-lymphocyte ratio is a reliable marker of treatment response in rheumatoid arthritis patients during tocilizumab therapy.Mod Rheumatol. 2017; 27: 405-410Crossref PubMed Scopus (24) Google Scholar,9Moots R.J. Sebba A. Rigby W. et al.Effect of tocilizumab on neutrophils in adult patients with rheumatoid arthritis: pooled analysis of data from phase 3 and 4 clinical trials.Rheumatology (Oxford). 2017; 56: 541-549PubMed Google Scholar The elevated IL-6 and IL-10 and persistently elevated NLR in the deceased group indicate that further studies may be needed to determine whether the elevated levels suggest an advanced disease state resistant to tocilizumab therapy. We posit that immunophenotyping such differences in prospective trials will be imperative to individualize treatment for these patients. Limitations of our cohort include its retrospective nature and smaller number of patients. Because all patients in the cohort received tocilizumab owing to the severe nature of their illness, we are unable to make a statement on clinical effectiveness of tocilizumab. We instead focused on biologic markers that could be indicative of disease severity and death. Our study period encompassed a time of varying hydroxychloroquine use, and dexamethasone was not widely used; however, there was no baseline difference between the groups. This report suggests that persistently elevated NLR is associated with unfavorable outcomes, but it is unclear whether COVID-19 disease, secondary infections, or other causes are driving elevated NLR in patients with more severe disease. We suggest that an elevated NLR is one potential marker. Further prospective studies are needed to reveal the performance of NLR vs in combination with other markers (CRP, complement components, cytokines, etc). NLR should be considered as a marker for COVID-19 that is resistant to anti-inflammatory therapy, with a need for infection monitoring and aggressive life support measures. We acknowledge Dr Amanda Holsworth for her careful editing, input, and feedback during the preparation of the article.

See also: World Allergy Congress (WAC) 2019 - Posters See also: World Allergy Congress (WAC) 2019 - Posters Parts of this work were orally presented at the 2018 meeting of the Collegium Internationale Allergologicum (CIA) meeting in Mallorca, Spain (Abstract 95): http://www.ciaweb.org/UserFiles/file/2018CIAFinalScientificProgram.pdf Basophil activation tests can provide valuable information on allergic sensitivity to a range of different allergens. The most widely employed methods involve flow cytometric detection of increased expression of membrane-bound CD63 or CD203c following addition of allergen to basophils in vitro. The identification of basogranulin, a unique basic protein stored in basophil granules, has opened the way for new basophil activation methods to be explored. Blood was collected from healthy subjects and patients with a history of allergy to food, drugs, house dust and grass pollen. Basophils were stimulated with specific allergen, anti-IgE antibody, or the peptide f-met-leu-phe (FMLP). Flow cytometry was performed with non-permeabilised and permeabilised cells with antibody specific for basogranulin (BB1) or CD63, and data analysed with CellQuest software. Flow cytometry with permeablised cells indicated depletion of intracellular stores of basogranulin following basophil activation. Associated with basogranulin release was the presence of increased quantities of this marker on the basophil membrane following cellular activation. Increased membrane expression of basogranulin mirrored that for CD63; and with allergens and other stimuli tested the measurement of cell surface basogranulin represented a more sensitive means for assessing basophil activation in vitro. The flow cytometric assays for basogranulin were optimised for use with samples of whole blood so as to avoid the need for basophil purification. The rapidity, simplicity and sensitivity of basogranulin-based methods for measuring basophil activation will facilitate their application to clinical samples and allow better assessment for allergic sensitivity.

Background: Chronic rhinosinusitis (CRS) with nasal polyps is a common chronic condition. The exact cause of nasal polyps remains unknown. Recently, we made the novel observation of intracellular localization of Staphylococcus aureus within mast cells in nasal polyps. Objective: This follow-up study aimed to further characterize interactions between S aureus and mast cells in this setting and elucidate potential internalization mechanisms with particular emphasis on the role of staphylococcal enterotoxin B (SEB). Methods: A prospective study was performed using an explant tissue model with ex vivo inferior turbinate mucosa obtained from patients with chronic rhinosinusitis with nasal polyps (n = 7) and patients without CRS (n = 5). Immunohistochemistry was used to characterize S aureus uptake into mast cells and investigate the effects of SEB on this process. An in vitro cell-culture model was used to investigate mast cell-S aureus interactions by using a combination of fluorescent in situ hybridization, confocal laser scanning microscopy, scanning electron microscopy, transmission electron microscopy, and proliferation assays. Results: S aureus was captured by extracellular traps and entered mast cells through phagocytosis. Proliferating intracellular S aureus led to the expansion and eventual rupture of mast cells, resulting in release of viable S aureus into the extracellular space. The presence of SEB appeared to promote internalization of S aureus into mast cells. Conclusion: This study provides new insights into the interactions between S aureus and mast cells, including the internalization process, and demonstrates a prominent role for SEB in promoting uptake of the bacteria into these cells.

BACKGROUND:Chronic rhinosinusitis (CRS) is a chronic inflammatory condition of the upper airways, often associated with the formation of nasal polyps (CRSwNP). It is well established that macroscopically normal (non-polypoidal) sinonasal mucosa in CRSwNP patients can undergo polypoidal change over time, turning into frank polyps. However, little is known about what drives this process. This study aimed to investigate potential drivers of nasal polyp formation or growth through comparison of the immunological profiles of nasal polyps with contiguous non-polypoidal sinonasal mucosa, from the same patients.METHODS:The immune profiles of three types of tissue were compared; nasal polyps and adjacent non-polypoidal sinonasal mucosa from 10 CRSwNP patients, and sinonasal mucosa from 10 control patients undergoing trans-sphenoidal pituitary surgery. Nasal polyp and control samples were also stimulated with Staphylococcus aureus enterotoxin B (SEB) using a nasal explant model, prior to cytokine analysis. Real time quantitative polymerase chain reaction (IL-5, T-bet, IL-17A, FoxP3, TLR-4, IL-8, IL-1beta and IL-6) and Luminex (IFNgamma, IL-5 and IL-17A) were used to quantify pro-inflammatory responses.RESULTS:Nasal polyps and contiguous non-polypoidal sinonasal mucosa from CRSwNP patients displayed a very similar pro-inflammatory profile. When stimulated with SEB, nasal polyps displayed a Th2/Th17 mediated response when compared to controls.CONCLUSIONS:In CRSwNP, nasal polyps and non-polypoidal sinonasal mucosa from the same patient displayed a similar pro-inflammatory profile skewed towards the Th2/Th17 pathway in nasal polyps following SEB stimulation, with evidence of disordered bacterial clearance. These factors may contribute to enhanced survival of bacteria and development of a chronic inflammatory milieu, potentially driving new polyp formation and recurrence following surgical removal.

Summary Background Tryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR 2. Objectives To investigate the contribution of PAR 2 in the pro‐inflammatory actions mediated by tryptase in a mice model. Methods We have injected recombinant human β II ‐tryptase into the peritoneum of PAR 2‐deficient and wild‐type C57 BL /6 mice. After 6, 12 and 24 hours, mice were killed, peritoneal lavage performed and inflammatory changes investigated. Results Tryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR 2‐deficient and wild‐type mice. Heat inactivation of tryptase or pre‐incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR 2 ( SLIGRL ‐ NH 2 ). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases ( MMP ) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase‐induced MMP 2 and MMP 9. At 24 hours after tryptase injection, there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat‐inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase‐induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR 2‐deficient and wild‐type mice. Conclusions Our findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Although these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR 2.