Amali E. Samarasinghe

Eosinophils Promote Antiviral Immunity in Mice Infected with Influenza A Virus

Eosinophils are multifunctional cells of the innate immune system linked to allergic inflammation. Asthmatics were more likely to be hospitalized but less likely to suffer severe morbidity and mortality during the 2009 influenza pandemic. These epidemiologic findings were recapitulated in a mouse model of fungal asthma wherein infection during heightened allergic inflammation was protective against influenza A virus (IAV) infection and disease. Our goal was to delineate a mechanism(s) by which allergic asthma may alleviate influenza disease outcome, focused on the hypothesis that pulmonary eosinophilia linked with allergic respiratory disease is able to promote antiviral host defenses against the influenza virus. The transfer of eosinophils from the lungs of allergen-sensitized and challenged mice into influenza virus–infected mice resulted in reduced morbidity and viral burden, improved lung compliance, and increased CD8+ T cell numbers in the airways. In vitro assays with primary or bone marrow–derived eosinophils were used to determine eosinophil responses to the virus using the laboratory strain (A/PR/08/1934) or the pandemic strain (A/CA/04/2009) of IAV. Eosinophils were susceptible to IAV infection and responded by activation, piecemeal degranulation, and upregulation of Ag presentation markers. Virus- or viral peptide–exposed eosinophils induced CD8+ T cell proliferation, activation, and effector functions. Our data suggest that eosinophils promote host cellular immunity to reduce influenza virus replication in lungs, thereby providing a novel mechanism by which hosts with allergic asthma may be protected from influenza morbidity.

Beneficial Effects of Prebiotic Saccharomyces cerevisiae Mannan on Allergic Asthma Mouse Models

One of the unmet needs for asthma management is a new therapeutic agent with both anti-inflammatory and anti-smooth muscle (ASM) remodeling effects. The mannose receptor (MR) family plays an important role in allergen uptake and processing of major allergens Der p 1 and Fel d 1. We have previously reported that ASM cells express a mannose receptor (ASM-MR) and that mannan derived from Saccharomyces cerevisiae (SC-MN) inhibits mannosyl-rich lysosomal hydrolase-induced bovine ASM cell proliferation. Using a humanized transgenic mouse strain (huASM-MRC2) expressing the human MRC2 receptor in a SM tissue-specific manner, we have demonstrated that ASM hyperplasia/hypertrophy can occur as early as 15 days after allergen challenge in this mouse model and this phenomenon is preventable with SC-MN treatment. This proof-of-concept study would facilitate future development of a potential asthma therapeutic agent with dual function of anti-inflammatory and anti-smooth muscle remodeling effects.

Humoral immune responses during asthma and influenza co-morbidity in mice

Humoral immunity serve dual functions of direct pathogen neutralization and enhancement of leukocyte function. Antibody classes are determined by antigen triggers, and the resulting antibodies can contribute to disease pathogenesis and host defense. Although asthma and influenza are immunologically distinct diseases, since we have found that allergic asthma exacerbation promotes antiviral host responses to influenza A virus, we hypothesized that humoral immunity may contribute to allergic host protection during influenza. C57BL/6J mice sensitized and challenged with Aspergillus fumigatus (or not) were infected with pandemic influenza A/CA/04/2009 virus. Negative control groups included naïve mice, and mice with only asthma or influenza. Concentrations of antibodies were quantified by ELISA, and in situ localization of IgA- and IgE-positive cells in the lungs was determined by immunohistochemistry. The number and phenotype of B cells in spleens and mediastinal lymph nodes were determined by flow cytometry at predetermined timepoints after virus infection until viral clearance. Mucosal and systemic antibodies remained elevated in mice with asthma and influenza with prominent production of IgE and IgA compared to influenza-only controls. B cell expansion was prominent in the mediastinal lymph nodes of allergic mice during influenza where most cells produced IgG1 and IgA. Although allergy-skewed B cell responses dominated in mice with allergic airways inflammation during influenza virus infection, virus-specific antibodies were also induced. Future studies are required to identify the mechanisms involved with B cell activation and function in allergic hosts facing respiratory viral infections.

Chronic features of allergic asthma are enhanced in the absence of resistin-like molecule-beta

Asthma is characterized by inflammation and architectural changes in the lungs. A number of immune cells and mediators are recognized as initiators of asthma, although therapeutics based on these are not always effective. The multifaceted nature of this syndrome necessitate continued exploration of immunomodulators that may play a role in pathogenesis. We investigated the role of resistin-like molecule-beta (RELM-β), a gut antibacterial, in the development and pathogenesis of Aspergillus-induced allergic airways disease. Age and gender matched C57BL/6J and Retnlb−/− mice rendered allergic to Aspergillus fumigatus were used to measure canonical markers of allergic asthma at early and late time points. Inflammatory cells in airways were similar, although Retnlb−/− mice had reduced tissue inflammation. The absence of RELM-β elevated serum IgA and pro-inflammatory cytokines in the lungs at homeostasis. Markers of chronic disease including goblet cell numbers, Muc genes, airway wall remodelling, and hyperresponsiveness were greater in the absence RELM-β. Specific inflammatory mediators important in antimicrobial defence in allergic asthma were also increased in the absence of RELM-β. These data suggest that while characteristics of allergic asthma develop in the absence of RELM-β, that RELM-β may reduce the development of chronic markers of allergic airways disease.

Understanding fibrosis in eosinophilic esophagitis: Are we there yet?

Eosinophilic esophagitis (EoE) is an immune/antigen‐mediated, progressive fibrostenotic disease characterized by symptoms of esophageal dysfunction and abnormal eosinophilic infiltration in the esophagus. Despite current treatment modalities of dietary antigen elimination or topical corticosteroids, a subset of patients do not have clinical or histologic response. Even with resolution of superficial epithelial eosinophilia, patients may still have progressive subepithelial fibrosis, which may lead to esophageal strictures over time. Histologic identification of subepithelial fibrosis requires deep esophageal biopsies, which are not routinely obtained. Herein, we review the challenges in diagnosing and treating fibrosis in EoE. We propose the novel concept of vitamin D supplementation to treat fibrosis in EoE through downregulation of profibrotic mediator, transforming growth factor‐beta.

Saccharomyces cerevisiae-Derived Mannan Does Not Alter Immune Responses to Aspergillus Allergens

Severe asthma with fungal sensitization predominates in the population suffering from allergic asthma, to which there is no cure. While corticosteroids are the mainstay in current treatment, other means of controlling inflammation may be beneficial. Herein, we hypothesized that mannan from Saccharomyces cerevisiae would dampen the characteristics of fungal allergic asthma by altering the pulmonary immune responses. Using wild-type and transgenic mice expressing the human mannose receptor on smooth muscle cells, we explored the outcome of mannan administration during allergen exposure on the pathogenesis of fungal asthma through measurement of cardinal features of disease such as inflammation, goblet cell number, and airway hyperresponsiveness. Mannan treatment did not alter most hallmarks of allergic airways disease in wild-type mice. Transgenic mice treated with mannan during allergen exposure had an equivalent response to non-mannan-treated allergic mice except for a prominent granulocytic influx into airways and cytokine availability. Our studies suggest no role for mannan as an inflammatory regulator during fungal allergy.

Airway Epithelial Repair by a Prebiotic Mannan Derived from Saccharomyces cerevisiae

In asthmatic airways, repeated epithelial damage and repair occur. No current therapy directly targets this process. We aimed to determine the effects of mannan derived from S. cerevisiae (SC-MN) on airway epithelial wound repair, in vitro. The presence of functional mannose receptors in bronchial epithelial cells was shown by endocytosis of colloidal gold-Man BSA via clathrin-coated pits in 16HBE cells. In primary normal human bronchial epithelial cells (NHBEC), SC-MN significantly facilitated wound closure. Treatment with SC-MN stimulated cell spreading as indicated by a significant increase in the average lamellipodial width of wound edge 16HBE cells. In addition, NHBEC treated with SC-MN showed increased expression and activation of Krüppel-like factors (KLFs) 4 and 5, transcription factors important in epithelial cell survival and regulation of epithelial-mesenchymal transition. We conclude that SC-MN facilitates wound repair in human bronchial epithelium, involving mannose receptors.

Acute allergic inflammation protects against influenza A virus induced morbidity in mice

A is a debilitating disease of the airways that affects over 200 million people worldwide. The asthma burden in the population, marked by disability and death, is highest in young and senior adults. Asthma was identified as a major risk factor associated with hospitalization during the 2009 influenza pandemic. However, retrospective analyses noted that asthmatics were less likely to suffer from complications associated with or die from influenza. Reasons for these seemingly counterintuitive findings were unclear. In order to study this effect in the laboratory setting, we aimed to generate a mouse model system of asthma and influenza co-morbidity using an Aspergillus fumigatus-induced mouse model of asthma and A/CA/04/2009 pandemic H1N1 strain of influenza virus. By varying the time of viral infection, we determined that mice infected with pH1N1 during allergic airways inflammation were protected against influenza measured by body weight loss. While early viral replication kinetics was similar between the asthma and flu mice compared to flu-controls, mice with asthma were able to clear virus from the lungs sooner than those without asthma. This clearance correlated with the infiltration of virus-specific CD8+ T cells. Influenza virus is cytopathic especially to the bronchial epithelial cells. We noted that bronchial epithelia of mice in the asthma and influenza groups retained their “healthy” morphology while cells in the flu-control animals were necrotic. Using human bronchial epithelial cells from healthy and asthmatic donors, we determined that in spite of viral replication, epithelial cells from asthmatic donors were resistant to influenza virus induced damage thereby highlighting the clinical relevance of our findings. Ongoing studies are aimed at identifying mechanisms that mediate altered outcomes during influenza in acute inflammation of allergic airways.A of vaccines by novel needle-free technology such as jet injection offers an important alternative to needle and syringe and may enhance compliance. In August 2014, needle-free vaccination against influenza was approved with Afluria using PharmaJet® Stratis® jet injection technology. The objective of this post-marketing study was to assess acceptance of and satisfaction with flu vaccination using Afluria delivered in the grocery pharmacy setting via the novel needle-free system. A total of 98 grocery store customers, ages 18-64 were administered needle-free Afluria vaccination during the 20142015 flu season using the PharmaJet device and agreed to complete a short post-administration survey. The population of was 54% female and 74% of all respondents reported receiving a flu shot the prior season. Overall, 89% of subjects reported being satisfied, very satisfied or extremely satisfied with the needle-free flu shot; 83% of subjects reported that they were likely, very likely or definitely going to choose flu vaccination by jet injection next year. The high degree of satisfaction with Afluria delivered through needle-free PharmaJet Stratis suggests that needle-free vaccination with jet injection may be widely accepted, improving compliance in the general adult population which currently has very low rates of immunization against flu.Z (Relenza®) and oseltamivir (Tamiflu®) are the current mainstays of antiviral therapy for influenza.As demonstrated in the 2008/2009 influenza season in US, when the vast majority of circulating A/H1N1 virus was resistant to Tamiflu, widespread drug resistance to an influenza antiviral can emerge rapidly. A simple and rapid influenza virus drug susceptibility assay could be useful in susceptibility surveillance and in clinical settings.Since zanamivir and oseltamivir (as well as a few in the pipeline) are inhibitors of influenza viral neuraminidase, we designed a luciferin derivatized substrate specific for this enzyme. The substrate was formulated in a master mix (Reagent I) for detection of influenza virus in a sample. Presence of influenza virus (hence viral neuraminidase) in the reaction mix leads to cleavage of the substrate, freeing the luciferin moiety which is immediately consumed by luciferase to generate detectable light signal. For detection of resistance to zanamivir or oseltamivir, we formulated a second master mix (Reagent II) which was identical to Reagent I mix except that it contained zanamivir or oseltamivir carboxylate. The signal of Reagent II in relation to that of Reagent I as determined by the signal ratio was used to measure the level of susceptibility of influenza virus tozanamivir or oseltamivir.The assay is rapid (17 min) and simple with essentially one manual step (i.e., sample addition). Use of a custom-designed analyzer which can simultaneously measure the signal of both Reagents I and II compute the signal ratio and interpret the test result further simplifies the assay.