[Molecular mimicry between human thyroid peroxidase, thyroglobulin, cosinophil peroxidase, IL-24 and microorganisms antigens]. / Mimetismo molecular entre peroxidasa tiroidea humana, tiroglobulina, peroxidasa de eosinófilos, IL-24 y antígenos de microorganismos.

OBJECTIVE: Identify molecular mimicry between TPO, eosinophil peroxidase (EPX), thyroglobulin and IL24 and microorganism antigens. METHODS: Through in silico analysis, we performed local alignments between human and microorganism antigens with PSI-BLAST. Proteins that did not present a 3D structure were modeled by homology through the Swiss Modeller server and epitope prediction was performed through Ellipro. Epitopes were located in the 3D models using PYMOL software. RESULTS: A total of 38 microorganism antigens (parasites, bacteria) had identities between 30% and 45%, being the highest with Anisakis simplex. The alignment between 2 candidate proteins from A. simplex and EPX presented significant values, with identities of 43 and 44%. In bacteria, Campylobacter jejuni presented the highest identity with thyroglobulin (35%). 220 linear and conformational epitopes of microorganism antigens were predicted. Peroxidasin-like proteins from Toxocara canis and Trichinella pseudospiralis presented 10 epitopes similar to TPO and EPX, as possible molecules triggering cross-reactivity. No virus presented identity with the human proteins studied. CONCLUSION: TPO and EPX antigens shared potential cross-reactive epitopes with bacterial and nematode proteins, suggesting that molecular mimicry could be a mechanism that explains the relationship between infections and urticaria/hypothyroidism. In vitro work is needed to demonstrate the results obtained in the in silico analysis.

OBJETIVO: Identificar mimetismo molecular entre TPO, eosinofil peroxidasa (EPX), tiroglobulina e IL24 y antígenos de microorganismos. MÉTODOS: A través de análisis in silico, realizamos los alineamientos locales entre los antígenos humanos y de microorganismos con PSI-BLAST. Las proteínas que no presentaban estructura 3D, fueron modeladas por homología a través del servidor Swiss Modeller y se realizó una predicción de epítopes a través de Ellipro. Los epítopes se localizaron en los modelos 3D utilizando el software PYMOL. RESULTADOS: Un total de 38 antígenos de microorganismos (parásitos y bacterias), tuvieron identidades entre 30 y 45%, siendo los más altos con Anisakis simplex. El alineamiento entre dos proteínas candidatas de A. simplex y EPX presentaron valores importantes, con identidades de 43 y 44%. En las bacterias, Campylobacter jejuni presentó la mayor identidad con tiroglobulina (35%). Se predijeron 220 epítopes lineales y conformacionales de antígenos de microorganismos. Las proteínas similares a la peroxidasina de Toxocara canis y Trichinella pseudospiralis presentaron diez epítopes similares a TPO y EPX, como posibles moléculas desencadenantes de una reactividad cruzada. Ningún virus presentó identidad con las proteínas humanas estudiadas. CONCLUSIÓN: Los antígenos TPO y EPX compartieron potenciales epítopes de reacción cruzada con proteínas bacterianas y nematodos, lo que sugiere que el mimetismo molecular podría ser un mecanismo que explique la relación entre infecciones y la urticaria/hipotiroidismo. Se necesitan trabajos in vitro que demuestren los resultados obtenidos en el análisis in silico.

The Neutralization of the Eosinophil Peroxidase Antibody Accelerates Eosinophilic Mucin Decomposition.

Eosinophilic airway inflammation, complicated by bronchial asthma and eosinophilic chronic rhinosinusitis (ECRS), is difficult to treat. The disease may become refractory when eosinophilic mucin associated with eosinophil peroxidase (EPX) and autoantibodies fills in the paranasal sinus and small airway. This study investigated the functional role of an anti-EPX antibody in eosinophilic mucin of ECRS in eosinophilic airway inflammation. Eosinophilic mucin was obtained from patients with ECRS. The effects of the anti-EPX antibody on dsDNA release from eosinophils and eosinophilic mucin decomposition were evaluated. Immunofluorescence or enzyme-linked immunosorbent assays were performed to detect the anti-EPX antibody and its supernatant and serum levels in eosinophilic mucin, respectively. The serum levels of the anti-EPX antibody were positively correlated with sinus computed tomography score and fractionated exhaled nitrogen oxide. Patients with refractory ECRS had higher serum levels of the anti-EPX antibody than those without. However, dupilumab treatment decreased the serum levels of the anti-EPX antibody. Immunoglobulins (Igs) in the immunoprecipitate of mucin supernatants enhanced dsDNA release from eosinophils, whereas the neutralization of Igs against EPX stopped dsDNA release. Furthermore, EPX antibody neutralization accelerated mucin decomposition and restored corticosteroid sensitivity. Taken together, the anti-EPX antibody may be involved in the formulation of eosinophilic mucin and be used as a clinical marker and therapeutic target for intractable eosinophilic airway inflammation.

Eosinophil Shape Change and Secretion.

The analysis of eosinophil shape change and mediator secretion is a useful tool in understanding how eosinophils respond to immunological stimuli and chemotactic factors. Eosinophils undergo dramatic shape changes, along with secretion of the granule-derived enzyme eosinophil peroxidase (EPX) in response to chemotactic stimuli including platelet-activating factor (PAF) and CCL11 (eotaxin-1). Here, we describe the analysis of eosinophil shape change by confocal microscopy analysis and provide an experimental approach for comparing unstimulated cells with those that have been stimulated to undergo chemotaxis. In addition, we illustrate two different degranulation assays for EPX using OPD and an ELISA technique and show how eosinophil degranulation may be assessed from in vitro as well as ex vivo stimulation.

Assessment of Lung Eosinophils In Situ Using Immunohistological Staining.

Eosinophils are rare white blood cells that are recruited from circulation to accumulate in the lung in mouse models of allergic respiratory inflammation. In hematoxylin-eosin (HE) stained lungs, eosinophils may be difficult to detect despite their bright eosin staining in the secondary granules. For this reason, antibody-mediated detection of eosinophils is preferable for specific and clearer identification of these cells. Moreover, eosinophils may degranulate, releasing their granule proteins into surrounding tissue, and remnants of cytolysed cells cannot be detected by HE staining. The methods here demonstrate the use of eosinophil-specific anti-mouse antibodies to detect eosinophil granule proteins in formalin-fixed cells both in situ in paraffin-embedded lungs, as well as in cytospin preparations from the lung. These antibody staining techniques enable either colorimetric or fluorescence imaging of eosinophils or their granule proteins with the potential for additional antibodies to be added for detection of multiple molecules.

Sputum autoantibodies in patients with severe eosinophilic asthma.

BACKGROUND: The persistence of eosinophils in sputum despite high doses of corticosteroids indicates disease severity in asthmatic patients. Chronic inflamed airways can lose tolerance over time to immunogenic entities released on frequent eosinophil degranulation, which further contributes to disease severity and necessitates an increase in maintenance corticosteroids. OBJECTIVES: We sought to investigate the possibility of a polyclonal autoimmune event in the airways of asthmatic patients and to identify associated clinical and molecular characteristics. METHODS: The presence of autoantibodies against eosinophil peroxidase (EPX) and anti-nuclear antibodies was investigated in patients with eosinophilic asthma maintained on high-dose corticosteroids, prednisone, or both. The ability of sputum immunoglobulins to induce eosinophil degranulation in vitro was assessed. In addition, the associated inflammatory microenvironment in patients with detectable autoantibodies was examined. RESULTS: We report a "polyclonal" autoimmune event occurring in the airways of prednisone-dependent asthmatic patients with increased eosinophil activity, recurrent pulmonary infections, or both, as evident by the concomitant presence of sputum anti-EPX and anti-nuclear antibodies of the IgG subtype. Extensive cytokine profiling of sputum revealed a TH2-dominated microenvironment (eotaxin-2, IL-5, IL-18, and IL-13) and increased signalling molecules that support the formation of ectopic lymphoid structures (B-cell activating factor and B cell-attracting chemokine 1). Immunoprecipitated sputum immunoglobulins from patients with increased autoantibody levels triggered eosinophil degranulation in vitro, with release of extensive histone-rich extracellular traps, an event unsuppressed by dexamethasone and possibly contributing to the steroid-unresponsive nature of these eosinophilic patients. CONCLUSION: This study identifies an autoimmune endotype of severe asthma that can be identified by the presence of sputum autoantibodies against EPX and autologous cellular components.

Pharmacological modulation of reactive oxygen species (ROS) improves the airway hyperresponsiveness by shifting the Th1 response in allergic inflammation induced by ovalbumin.

Asthma is an allergic inflammation driven by the Th2 immune response with release of cytokines such as IL-4 and IL-13, which contribute to the airflow limitations and airway hyperresponsiveness (AHR). The involvement of oxidative stress in this process is well-established, but the specific role of the superoxide anion and nitric oxide in asthma are poorly understood. Thus, the aim of this study was to investigate the mechanisms underlying the superoxide anion/nitric oxide production and detoxification in a murine asthma model. BALB/c male mice were sensitised and challenged with ovalbumin (OVA). Pretreatments with either apocynin (14 mg/kg) or allopurinol (25 mg/kg) (superoxide anion synthesis inhibitors), aminoguanidine (50 mg/kg) (nitric oxide synthesis inhibitor) or diethyldithiocarbamate (100 mg/kg) (superoxide dismutase inhibitor) were performed 1 h before the challenge. Our data showed that apocynin and allopurinol ameliorated AHR and reduced eosinophil peroxidase, as well as IL-4 and IL-13 levels. Apocynin also abrogated leukocyte peribronchiolar infiltrate and increased IL-1ß secretion. Aminoguanidine preserved lung function and shifted the Th2 to the Th1 response with a reduction of IL-4 and IL-13 and increase in IL-1ß production. Diethyldithiocarbamate prevented neither allergen-induced AHR nor eosinophil peroxidase (EPO) generation. All treatments protected against oxidative damage observed by a reduction in TBARS levels. Taken together, these results suggest that AHR in an asthma model can be avoided by the down-regulation of superoxide anion and nitric oxide synthesis in a mechanism that is independent of a redox response. This down-regulation is also associated with a transition in the typical immunological Th2 response toward the Th1 profile.

Frontline Science: Eosinophil-deficient MBP-1 and EPX double-knockout mice link pulmonary remodeling and airway dysfunction with type 2 inflammation.

Eosinophils and the release of cationic granule proteins have long been implicated in the development of the type 2-induced pathologies linked with respiratory inflammation. Paradoxically, the ablation of the two genes encoding the most abundant of these granule proteins, major basic protein-1 (MBP-1) and eosinophil peroxidase (EPX), results in a near collapse of eosinophilopoiesis. The specificity of this lineage ablation and the magnitude of the induced eosinopenia provide a unique opportunity to clarify the importance of eosinophils in acute and chronic inflammatory settings, as well as to identify potential mechanism(s) of action linked with pulmonary eosinophils in those settings. Specifically, we examined these issues by assessing the induced immune responses and pathologies occurring in MBP-1-/-/EPX-/- mice after 1) ovalbumin sensitization/provocation in an acute allergen-challenge protocol, and 2) crossing MBP-1-/-/EPX-/- mice with a double-transgenic model of chronic type 2 inflammation (i.e., I5/hE2). Acute allergen challenge and constitutive cytokine/chemokine expression each induced the accumulation of pulmonary eosinophils in wild-type controls that was abolished in the absence of MBP-1 and EPX (i.e., MBP-1-/-/EPX-/- mice). The expression of MBP-1 and EPX was also required for induced lung expression of IL-4/IL-13 in each setting and, in turn, the induced pulmonary remodeling events and lung dysfunction. In summary, MBP-1-/-/EPX-/- mice provide yet another definitive example of the immunoregulatory role of pulmonary eosinophils. These results highlight the utility of this unique strain of eosinophil-deficient mice as part of in vivo model studies investigating the roles of eosinophils in health and disease settings.

Safety and efficiency of active immunization with detoxified antigen against scorpion venom: side effect evaluation.

OBJECTIVE: The efficiency and safety of vaccine are the most important properties, however, as any medication, it can induce side effects. This prophylactic therapy could be used to prevent the lethal and pathophysiological effects induced after scorpion envenomation. METHODS: In this study, detoxified venom associated to alum adjuvant (V*alum) is used as a vaccine against scorpion venom for immunization of mice. We evaluate the safety and the inflammatory response of this vaccine. We also investigated the protective effect of this formulation against the toxicity of native Androctonus australis hector venom. RESULTS: Results showed no adverse events occurred after immunization of animals. This active immunization of animals did not cause change in vascular permeability, no edema formation in the studied organs. Furthermore, there are no IgE production in sera, nor change in the morphology of the mast cells in skin tissues. However, low inflammatory response triggered by activating the recruitment of eosinophils associated to IL-4 and IL-5 release was observed. All immunized animals are protected from the toxic effects of native venom until 6 LD50 and to 7 LD50 after the second challenge. CONCLUSION: This safe vaccine preparation seems to induce a long-term protection without any risk of deleterious inflammatory response.

Assessment of eosinophil peroxidase as a potential diagnostic and prognostic marker in dogs with inflammatory bowel disease.

OBJECTIVE To evaluate a method for identifying intact and degranulated eosinophils in the small intestine of dogs with inflammatory bowel disease (IBD) by use of a monoclonal antibody (mAb) against eosinophil peroxidase (EPX). ANIMALS 11 untreated dogs with IBD, 5 dogs with IBD treated with prednisolone, and 8 control dogs with no clinical evidence of gastrointestinal tract disease and no immunosuppressive treatment. PROCEDURES 4-µm-thick sections of paraffin-embedded tissues from necropsy specimens were immunostained with EPX mAb. Stained intact and degranulated eosinophils in consecutive microscopic fields (400X magnification) of the upper (villus tips) and lower (between the muscularis mucosae and crypts) regions of the lamina propria of the jejunum were manually counted. RESULTS Compared with control and treated IBD dogs, untreated IBD dogs had a significantly higher number of degranulated eosinophils in the lower region of the lamina propria. However, no significant differences were detected in the number of intact eosinophils in this region among groups. In the upper region of the lamina propria, untreated IBD dogs had a significantly higher number of degranulated and intact eosinophils, compared with control and treated IBD dogs. Number of degranulated and intact eosinophils did not differ significantly between control and treated IBD dogs. CONCLUSIONS AND CLINICAL RELEVANCE Immunohistologic analysis with EPX mAb yielded prominent granule staining that allowed reliable morphological identification of degranulated and intact eosinophils, which may provide a strategy for quantitative and selective evaluation of eosinophils in gastrointestinal biopsy specimens and a potential method to diagnose IBD and evaluate treatment outcome.

Eosinophil Peroxidase Catalyzed Protein Carbamylation Participates in Asthma.

The biochemical mechanisms through which eosinophils contribute to asthma pathogenesis are unclear. Here we show eosinophil peroxidase (EPO), an abundant granule protein released by activated eosinophils, contributes to characteristic asthma-related phenotypes through oxidative posttranslational modification (PTM) of proteins in asthmatic airways through a process called carbamylation. Using a combination of studies we now show EPO uses plasma levels of the pseudohalide thiocyanate (SCN-) as substrate to catalyze protein carbamylation, as monitored by PTM of protein lysine residues into Nϵ-carbamyllysine (homocitrulline), and contributes to the pathophysiological sequelae of eosinophil activation. Studies using EPO-deficient mice confirm EPO serves as a major enzymatic source for protein carbamylation during eosinophilic inflammatory models, including aeroallergen challenge. Clinical studies similarly revealed significant enrichment in carbamylation of airway proteins recovered from atopic asthmatics versus healthy controls in response to segmental allergen challenge. Protein-bound homocitrulline is shown to be co-localized with EPO within human asthmatic airways. Moreover, pathophysiologically relevant levels of carbamylated protein either incubated with cultured human airway epithelial cells in vitro, or provided as an aerosolized exposure in non-sensitized mice, induced multiple asthma-associated phenotypes including induction of mucin, Th2 cytokines, IFNγ, TGFß, and epithelial cell apoptosis. Studies with scavenger receptor-A1 null mice reveal reduced IL-13 generation following exposure to aerosolized carbamylated protein, but no changes in other asthma-related phenotypes. In summary, EPO-mediated protein carbamylation is promoted during allergen-induced asthma exacerbation, and can both modulate immune responses and trigger a cascade of many of the inflammatory signals present in asthma.

A perspective on point-of-care tests to detect eosinophilic bronchitis.

Approximately 50% of asthma exacerbations and a third of COPD exacerbations are associated with an eosinophilic bronchitis. Quantitative cell counts reliably identify the number of eosinophils in sputum and treatment strategies that are guided by sputum eosinophil counts lead to significantly better outcomes than strategies guided by conventional assessments of symptoms and airflow. However, cell counts are not widely available and the results are not available in real time. Similarly, more sophisticated detection methods using immunoassays or genetic analysis via polymerase chain reaction are too costly and thus not amenable to rapid point-of-care diagnosis. Blood eosinophil counts and fraction of exhaled nitric oxide correlate poorly with airway eosinophilia, particularly in patients with severe airway diseases who are on corticosteroid therapy. Point of care assessments of eosinophil-specific activity may be provided by breathomics that employ metabolomics profiling of volatile compounds in breath. However, it is too early to decide if this would provide quantitative data to monitor therapy and disease activities longitudinally. Herein we provide a perspective on the potential for developing simple point-of-care tests with special emphasis on the potential for a bio-active paper diagnostic test to quantitatively assay the amount of eosinophil peroxidase in sputum samples by employing different types of detection systems.

Eosinophil granule proteins: form and function.

Experimental and clinical data strongly support a role for the eosinophil in the pathogenesis of asthma, allergic and parasitic diseases, and hypereosinophilic syndromes, in addition to more recently identified immunomodulatory roles in shaping innate host defense, adaptive immunity, tissue repair/remodeling, and maintenance of normal tissue homeostasis. A seminal finding was the dependence of allergic airway inflammation on eosinophil-induced recruitment of Th2-polarized effector T-cells to the lung, providing a missing link between these innate immune effectors (eosinophils) and adaptive T-cell responses. Eosinophils come equipped with preformed enzymatic and nonenzymatic cationic proteins, stored in and selectively secreted from their large secondary (specific) granules. These proteins contribute to the functions of the eosinophil in airway inflammation, tissue damage, and remodeling in the asthmatic diathesis. Studies using eosinophil-deficient mouse models, including eosinophil-derived granule protein double knock-out mice (major basic protein-1/eosinophil peroxidase dual gene deletion) show that eosinophils are required for all major hallmarks of asthma pathophysiology: airway epithelial damage and hyperreactivity, and airway remodeling including smooth muscle hyperplasia and subepithelial fibrosis. Here we review key molecular aspects of these eosinophil-derived granule proteins in terms of structure-function relationships to advance understanding of their roles in eosinophil cell biology, molecular biology, and immunobiology in health and disease.

Molecular analysis of non-specific protection against murine malaria induced by BCG vaccination.

Although the effectiveness of BCG vaccination in preventing adult pulmonary tuberculosis (TB) has been highly variable, epidemiologic studies have suggested that BCG provides other general health benefits to vaccinees including reducing the impact of asthma, leprosy, and possibly malaria. To further evaluate whether BCG immunization protects against malarial parasitemia and to define molecular correlates of this non-specific immunity, mice were vaccinated with BCG and then challenged 2 months later with asexual blood stage Plasmodium yoelii 17XNL (PyNL) parasites. Following challenge with PyNL, significant decreases in parasitemia were observed in BCG vaccinated mice relative to naïve controls. To identify immune molecules that may be associated with the BCG-induced protection, gene expression was evaluated by RT-PCR in i) naïve controls, ii) BCG-vaccinated mice, iii) PyNL infected mice and iv) BCG vaccinated/PyNL infected mice at 0, 1, 5, and 9 days after the P. yoelii infection. The expression results showed that i) BCG immunization induces the expression of at least 18 genes including the anti-microbial molecules lactoferrin, eosinophil peroxidase, eosinophil major basic protein and the cathelicidin-related antimicrobial peptide (CRAMP); ii) an active PyNL infection suppresses the expression of important immune response molecules; and iii) the extent of PyNL-induced suppression of specific genes is reduced in BCG-vaccinated/PyNL infected mice. To validate the gene expression data, we demonstrated that pre-treatment of malaria parasites with lactoferrin or the cathelicidin LL-37 peptide decreases the level of PyNL parasitemias in mice. Overall, our study suggests that BCG vaccination induces the expression of non-specific immune molecules including antimicrobial peptides which may provide an overall benefit to vaccinees by limiting infections of unrelated pathogens such as Plasmodium parasites.

Comparison of three staining methods to identify eosinophils in formalin-fixed canine skin.

BACKGROUND: Studies comparing the ability of staining methods to detect eosinophils in formalin-fixed canine skin are lacking. HYPOTHESIS/OBJECTIVES: The aim of this study was to compare the effectiveness of eosinophil peroxidase monoclonal antibody (EPXmAb), Luna and haematoxylin and eosin (H&E) to detect eosinophils in fixed canine skin by assessing the following parameters: (i) specificity of eosinophil staining; (ii) extracellular eosinophil granule staining; (iii) tissue background staining; (iv) contrast between eosinophil and surrounding tissue staining; and (v) differences in the number of eosinophils detected by each stain. METHODS: Three serial sections of formalin-fixed, paraffin-embedded tissues of predominantly eosinophilic skin diseases (n = 8), noneosinophilic skin diseases (n = 7) and normal canine skin (n = 1) were stained with the three stains. Each parameter was independently assessed and scored by two investigators. RESULTS: Luna and EPXmAb were specific in detecting eosinophils. The EPXmAb was significantly more effective than Luna (P < 0.001) and H&E (P < 0.001) in its ability to detect extracellular eosinophil granules (i.e. to detect intact and released eosinophil granules). The EPXmAb showed significantly less background staining compared with Luna (P = 0.0005). Moreover, significantly more stain contrast was noted with EPXmAb compared with Luna (P = 0.003) and H&E (P < 0.001). There was no significant difference between the mean eosinophil counts among the three stains. CONCLUSION AND CLINICAL IMPORTANCE: The three stains were shown to be useful to detect and quantify eosinophils in fixed canine skin. The EPXmAb-based immunohistochemical stain proved to be a novel tool to detect eosinophils in canine skin.

A sensitive high throughput ELISA for human eosinophil peroxidase: a specific assay to quantify eosinophil degranulation from patient-derived sources.

Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these ribonucleases are expressed by leukocytes other than eosinophils, as well as cells of non-hematopoietic origin, limits the usefulness of these assays. Two novel monoclonal antibodies recognizing eosinophil peroxidase (EPX) were used to develop an eosinophil-specific and sensitive sandwich ELISA. The sensitivity of this EPX-based ELISA was shown to be similar to that of the commercially available ELISA kits for ECP and EDN. More importantly, evidence is also presented confirming that among these granule protein detection options, EPX-based ELISA is the only eosinophil-specific assay. The utility of this high throughput assay to detect released EPX was shown in ex vivo degranulation studies with isolated human eosinophils. In addition, EPX-based ELISA was used to detect and quantify eosinophil degranulation in several in vivo patient settings, including bronchoalveolar lavage fluid obtained following segmental allergen challenge of subjects with allergic asthma, induced sputum derived from respiratory subjects following hypotonic saline inhalation, and nasal lavage of chronic rhinosinusitis patients. This unique EPX-based ELISA thus provides an eosinophil-specific assay that is sensitive, reproducible, and quantitative. In addition, this assay is adaptable to high throughput formats (e.g., automated assays utilizing microtiter plates) using the diverse patient fluid samples typically available in research and clinical settings.

The development of a sensitive and specific ELISA for mouse eosinophil peroxidase: assessment of eosinophil degranulation ex vivo and in models of human disease.

Mouse models of eosinophilic disorders are often part of preclinical studies investigating the underlying biological mechanisms of disease pathology. The presence of extracellular eosinophil granule proteins in affected tissues is a well established and specific marker of eosinophil activation in both patients and mouse models of human disease. Unfortunately, assessments of granule proteins in the mouse have been limited by the availability of specific antibodies and a reliance on assays of released enzymatic activities that are often neither sensitive nor eosinophil specific. The ability to detect immunologically and quantify the presence of a mouse eosinophil granule protein in biological fluids and/or tissue extracts was achieved by the generation of monoclonal antibodies specific for eosinophil peroxidase (EPX). This strategy identified unique pairs of antibodies with high avidity to the target protein and led to the development of a unique sandwich ELISA for the detection of EPX. Full factorial design was used to develop this ELISA, generating an assay that is eosinophil-specific and nearly 10 times more sensitive than traditional OPD-based detection methods of peroxidase activity. The added sensitivity afforded by this novel assay was used to detect and quantify eosinophil degranulation in several settings, including bronchoalveolar fluid from OVA sensitized/challenged mice (an animal model of asthma), serum samples derived from peripheral blood recovered from the tail vasculature, and from purified mouse eosinophils stimulated ex vivo with platelet activating factor (PAF) and PAF + ionomycin. This ability to assess mouse eosinophil degranulation represents a specific, sensitive, and reproducible assay that fulfills a critical need in studies of eosinophil-associated pathologies in mice.

Immunodetection of occult eosinophils in lung tissue biopsies may help predict survival in acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a serious respiratory disorder for which therapy is primarily supportive once infection is excluded. Surgical lung biopsy may rule out other diagnoses, but has not been generally useful for therapy decisions or prognosis in this setting. Importantly, tissue and peripheral blood eosinophilia, the hallmarks of steroid-responsive acute eosinophilic pneumonia, are not commonly linked with ALI. We hypothesized that occult eosinophilic pneumonia may explain better outcomes for some patients with ALI. METHODS: Immunohistochemistry using a novel monoclonal antibody recognizing eosinophil peroxidase (EPX-mAb) was used to assess intrapulmonary eosinophil accumulation/degranulation. Lung biopsies from ALI patients (n = 20) were identified following review of a pathology database; 45% of which (i.e., 9/20) displayed classical diffuse alveolar damage (ALI-DAD). Controls were obtained from uninvolved tissue in patients undergoing lobectomy for lung cancer (n = 10). Serial biopsy sections were stained with hematoxylin and eosin (H&E) and subjected to EPX-mAb immunohistochemistry. RESULTS: EPX-mAb immunohistochemistry provided a >40-fold increased sensitivity to detect eosinophils in the lung relative to H&E stained sections. This increased sensitivity led to the identification of higher numbers of eosinophils in ALI patients compared with controls; differences using H&E staining alone were not significant. Clinical assessments showed that lung infiltrating eosinophil numbers were higher in ALI patients that survived hospitalization compared with non-survivors. A similar conclusion was reached quantifying eosinophil degranulation in each biopsy. CONCLUSION: The enhanced sensitivity of EPX-mAb immunohistochemistry uniquely identified eosinophil accumulation/degranulation in patients with ALI relative to controls. More importantly, this method was a prognostic indicator of patient survival. These observations suggest that EPX-mAb immunohistochemistry may represent a diagnostic biomarker identifying a subset of ALI patients with improved clinical outcomes.

Petasites extract Ze 339 (PET) inhibits allergen-induced Th2 responses, airway inflammation and airway hyperreactivity in mice.

BACKGROUND: The herbal Petasites hybridus (butterbur) extract (Ze 339, PET) is known to have leukotriene inhibiting properties, and therefore might inhibit allergic diseases. METHODS: The effect of PET was investigated in ovalbumin (OVA) immunized BALB/c mice given intranasally together with antigen challenge in the murine model of allergic airway disease (asthma) with the analysis of the inflammatory and immune parameters in the lung. RESULTS: PET given with the antigen challenge inhibited the allergic response. PET inhibited airway hyperresponsiveness (AHR) and eosinophil recruitment into the bronchoalveolar lavage (BAL) fluid upon allergen challenge, but had no effect in the saline control mice. Eosinophil recruitment was further assessed in the lung by eosinophil peroxidase (EPO) activity at a concentration of 100 microg PET. Microscopic investigations revealed less inflammation, eosinophil recruitment and mucus hyperproduction in the lung with 100 microg PET. Diminution of AHR and inflammation was associated with reduced IL-4, IL-5 and RANTES production in the BAL fluid with 30 microg PET, while OVA specific IgE and eotaxin serum levels remained unchanged. CONCLUSION: PET, which has been reported to inhibit leukotriene activity, reduced allergic airway inflammation and AHR by inhibiting the production of the Th2 cytokines IL-4 and IL-5, and RANTES.