Major Basic Protein Research Articles

White Blood Count Components as Risk Predictors for Coronary Heart Disease: The Role of Eosinophils

To the Editor: We read with great interest the paper by Madjid and Fatemi1 about the components of the complete blood count as indicators of inflammation and risk predictors of coronary heart disease. Although all subtypes of the white blood count have been associated with an increased risk of coronary heart disease, the role of eosinophils has been underestimated. The authors1 correctly refer to genetic analysis of the inflammatory role of eosinophils, because a single nucleotide polymorphism at 12q24, in SH2B3, was found to have a significant association with myocardial infarction (MI). Eosinophils play an important role in inflammation, and their existence denotes hypersensitivity inflammation. Hypersensitivity inflammation can induce acute MI, and the Kounis hypersensitivity acute coronary syndrome is well described.2 In patients who had vasospastic angina pectoris, eosinophil counts were significantly increased and could predict the severity of the disease. After these patients underwent medical therapy and attained relief from chest symptoms, their eosinophil counts decreased significantly, to the same level as those of the control group.3 Clinical reports have shown that the absolute number of eosinophils and the eosinophil-to-leukocyte ratio are significantly elevated in patients who have coronary artery disease.4 Coronary vasospasm associated with eosinophilia responds poorly to conventional vasodilator therapy, and although the risk of recurrent coronary events is high, most patients respond to therapy that suppresses eosinophilia, such as corticosteroids.5 Eosinophils are pleiotropic multifunctional leukocytes involved in the initiation and propagation of inflammatory responses, and they express H4 histamine receptors on their surface. These receptors facilitate eosinophil chemotaxis toward mast cells, which are a source of inflammatory soluble mediators. These mediators can induce the coronary hypersensitivity associated with Kounis syndrome that manifests itself as vasospastic angina, acute MI, or both.2 Soluble mediators secreted by mast cells and eosinophils also modulate reciprocal interactions between these 2 cells in the so-called “allergic effector unit.” Major basic cationic protein released from eosinophils can activate mast cells.6 This activation elicits exocytosis and new eicosanoid and cytokine production, both of which are prominent responses after Fc∊RI-dependent activation of mast cells. Mediators similar to those found in Kounis syndrome are found in cases with nonallergic causes, which suggests that this is a more general problem. A common pathway between allergic and nonallergic coronary syndromes seems to exist.2 Eosinophil count has emerged as a novel biomarker of risk stratification in patients who have coronary artery disease. This reality is essential for elucidating the cause of inflammation.

Mast Cells, Eosinophils and Eosinophilic Esophagitis

Eosinophilic esophagitis (EoE) is an increasingly recognized disease entity in adults. Studies show a role for eosinophils and mast cells in its pathogenesis. Upper and mid-esophageal biopsy slides from adult patients (n=36) with dysphagia were stained for eosinophil counts (H&E), and immunostained for eosinophil-derived neurotoxin (EDN) and eosinophil major basic protein (MBP). Gene expression was studied by microarray analysis. All patients were consented before the procedure. All research protocols were approved by the Mayo Clinic IRB. EoE patients (n=12; median age 36; 10 male, 2 female) had higher EDN and MBP tissue protein levels as quantified by immunofluorescence. The measured levels of intracellular and extracellular EDN and MBP correlated with eosinophil counts in the EoE patients (median eosinophil count 48±36 cells/high power field (HPF)) compared to controls (n=14; median age 47.2; median eosinophil count 2.8±4 cells/HPF). Genetic microarray analysis showed eotaxin-3 expression was 8-fold higher, eotaxin-2 was 1.3-fold higher, and Charcot-Leyden crystal protein was 2.4-fold higher than controls. For mast cell markers, cathepsin-G precursor expression was 6.2-fold higher, high affinity IgE receptor alpha chain was 2.1-fold higher, carboxypeptidase A3 was 4.5-fold higher, and both cathepsin C and tryptase were 3-fold higher than controls. All fold-changes reached statistical significance (p<0.002). Of note two control patients showed the control phenotype but matched the EoE genotype. The significant increase in eotaxin-3 gene expression in EoE patients is in agreement with previously reported expression levels in children. The increase in mast cell markers supports a role for mast cell involvement in EoE.

Eosinophilic organ infiltration without eosinophilia or direct parasite infection

Eosinophilic organ infiltration without eosinophilia or direct parasite infection

Expression levels of eosinophil granule protein mRNAs in induced sputum reflect airway hyperresponsiveness and airflow limitation.

Eosinophils are regarded as the major effector cells that produce symptoms in allergic diseases. Activation of eosinophils induces extracellular release of a number of eosinophil granule proteins, including major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin. The objective of this study was to evaluate the differences and significance of the sputum eosinophil% and expression levels of eosinophilic granule protein mRNAs in allergic airway disease. Induced sputum samples were obtained from non-smokers with 25 asthma, 54 eosinophilic bronchitis, 16 allergic rhinitis, and 19 healthy control subjects. The eosinophil granule protein mRNAs were measured with real time RT-PCR. There was no correlation between the sputum eosinophil% and the mRNA level of any of eosinophil granule proteins. However, the expression levels of MBP and ECP mRNAs were higher in subjects with each of the specified allergic diseases than those in control subjects (P < 0.05). Moreover, in the subjects with allergic sensitization, the expression levels of MBP and EPO mRNAs were significantly higher in those with airway hyperresponsiveness (13 subjects) than in those without airway hyperresponsiveness (32 subjects) (P = 0.004 and 0.010, respectively). In asthma patients, the FEV1% was negatively correlated with ECP mRNA levels (r = -0.510, P = 0.022), but showed no correlation with sputum eosinophil%. In conclusion, mRNA levels of eosinophil granule proteins, rather than sputum eosinophil%, may reflect airway hyperresponsiveness and airflow limitation. In practice, consideration for the eosinophil% as well as the eosinophil granule proteins levels in induced sputum is needed.

Eosinophil Deficiency Compromises Lung Defense against Aspergillus fumigatus

Exposure to the mold Aspergillus fumigatus may result in allergic bronchopulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, or invasive aspergillosis (IA), depending on the host's immune status. Neutrophil deficiency is the predominant risk factor for the development of IA, the most life-threatening condition associated with A. fumigatus exposure. Here we demonstrate that in addition to neutrophils, eosinophils are an important contributor to the clearance of A. fumigatus from the lung. Acute A. fumigatus challenge in normal mice induced the recruitment of CD11b+ Siglec F+ Ly-6G(lo) Ly-6C(neg) CCR3+ eosinophils to the lungs, which was accompanied by an increase in lung Epx (eosinophil peroxidase) mRNA levels. Mice deficient in the transcription factor dblGATA1, which exhibit a selective deficiency in eosinophils, demonstrated impaired A. fumigatus clearance and evidence of germinating organisms in the lung. Higher burden correlated with lower mRNA expression of Epx (eosinophil peroxidase) and Prg2 (major basic protein) as well as lower interleukin 1β (IL-1β), IL-6, IL-17A, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and CXCL1 levels. However, examination of lung inflammatory cell populations failed to demonstrate defects in monocyte/macrophage, dendritic cell, or neutrophil recruitment in dblGATA1-deficient mice, suggesting that the absence of eosinophils in dlbGATA1-deficient mice was the sole cause of impaired lung clearance. We show that eosinophils generated from bone marrow have potent killing activity against A. fumigtaus in vitro, which does not require cell contact and can be recapitulated by eosinophil whole-cell lysates. Collectively, our data support a role for eosinophils in the lung response after A. fumigatus exposure.

Prostaglandin D2 receptord-type prostanoid receptor 2 mediates eosinophil trafficking into the esophagus

Eosinophilic esophagitis is characterized by eosinophil-predominant inflammation in the esophagus. How eosinophils migrate and infiltrate into the esophagus, however, is less clear. Our previous study demonstrated that mast cell activation led to eosinophil infiltration in the esophagus. Prostaglandin D2 (PGD2) is an important mediator released from activated mast cells. The present study aims to determine whether PGD2 induces eosinophil infiltration into the esophagus via a d-type prostanoid receptor 2 (DP2) receptor-dependent mechanism. Using an in vivo guinea pig model, PGD2, d-type prostanoid receptor 1 (DP1) agonist, or DP2 agonist were injected into the esophagus. Esophageal tissues were removed 2 hours after injections and proceeded to either hematoxylin-eosin (HE) staining or immunofluorescent staining of eosinophil major basic protein (MBP) to compare each treatment-induced eosinophil infiltration in the esophagus. In a separate study, ovalbumin (OVA)-sensitized guinea pigs were pretreated with either DP2 or DP1 antagonists, followed by inhalation of OVA to induce mast cell activation. Esophageal tissues were then processed for immunofluorescent staining of MBP. PGD2 injection in the esophagus led to an increase of eosinophil infiltration in esophageal epithelium at the injection site as revealed by HE staining. Increased infiltration of eosinophils was further confirmed by the increased presence of MBP-labeled immunopositive (MBP-LI) cells in esophageal epithelium. Injection with DP2 agonist 15(R)-PGD2, but not DP1 agonist BW 245C, mimicked the PGD2-induced response. In OVA-sensitized animals, antigen inhalation increased MBP-LI cells in esophageal epithelium. Pretreatment with DP2 antagonist BAY-u3405, but not DP1 antagonist BW 868C, inhibited the antigen inhalation-induced increase of MBP-LI cells in esophageal epithelium. These data support the hypothesis that PGD2 induces eosinophil trafficking into the esophageal epithelium via a DP2-mediated pathway, suggesting a role of DP2 antagonist in the prevention of eosinophilic esophagitis.

Eosinophil major basic protein activates human cord blood mast cells primed with fibroblast membranes by integrin‐β1

Mast cell (MC) - eosinophil (Eos) activating cross-talk might be critical for the severity and chronicity of allergy. Among soluble mediators, eosinophil major basic protein (MBP), a hallmark of allergy, is particularly important because it was shown to activate specific MC subtypes. We previously demonstrated that MBP activates IgE-desensitized rat MC and human lung and cord blood-derived MC (CBMC) after priming with fibroblast membranal stem cell factor. However, a distinct mechanism for this activation was missing. Therefore, we aimed to investigate it. Major basic protein-1 activation of CBMC primed with fibroblast-derived membranes (FBM) was measured by β-hexosaminidase and tryptase release. Chemical cross-linking followed by micrometric flow cytometry probed direct interactions. Antibodies neutralized integrin-β1 and recognized its active form. Pertussis toxin (Ptx) was used to decrease integrin-β1 active form expression. Hematopoietic cell kinase (Hck) was identified by immunoprecipitation (IP) and silenced by siRNA. Major basic protein-1-induced CBMC activation is mediated partly by MBP1-integrin-β1 interaction on the MC surface. FBM prime CBMC via a G protein, as confirmed by Ptx, to shift integrin-β1 to its active form. Following MBP1 binding, integrin-β1 binds Hck that further transduces the activation signal. MC priming with FBM leads to up-regulation in Hck protein level. MC integrin-β1 neutralization inhibits MBP1-induced activation and uptake. Hck silencing results with reduced MBP1-induced activation. Fibroblast-derived membranes, integrin-β1, and Hck are involved in MBP1-induced activation of CBMC and therefore represent a distinct mechanism for this activation. This finding might implicate integrin-β1 and Hck as targets for decreasing MC - Eos activating cross-talk in allergy.

Skin-specific expression of IL-33 activates group 2 innate lymphoid cells and elicits atopic dermatitis-like inflammation in mice

Transgenic mice expressing the mouse interleukin 33 (IL-33) gene driven by a keratin 14 promoter were generated. The skin-selective expression of the IL-33 gene was enhanced, and intense immunofluorescence for IL-33 was evident in the nuclei of the epidermis. Spontaneous itchy dermatitis developed in those mice at 6-8 wk of age in specific pathogen-free conditions. In the lesional skin, the epidermis was thickened and the eosinophils were infiltrated with increased expression of the eosinophil peroxidase and major basic protein genes. Mast cells were also abundant there, and blood histamine and total IgE levels were high. Those phenotypes closely resemble the features of atopic dermatitis. In peripheral blood and lesional skin, IL-5, IL-13, regulated upon activation, normally T-expressed, and presumably secreted (RANTES)/CCL5, and Eotaxin 1/CCL11 were increased, whereas TNF-α, IFN-γ, and thymic stromal lymphopoietin (TSLP) were unaltered. Furthermore, the proportion of group 2 innate lymphoid cells (ILC2s), which produce IL-5, were significantly increased in the lesional skin, peripheral blood, and regional lymph nodes. The dermatitis with eosinophil infiltration was improved by the administration of an anti-IL-5 antibody. These results suggest that the expression of IL-33 in the skin activates an immune response involving ILC2 and that this process might play a crucial role in the pathogenesis of allergic inflammation that is characteristic of atopic dermatitis.

Expression of the secondary granule proteins major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX) is required for eosinophilopoiesis in mice

AbstractEosinophil activities are often linked with allergic diseases such as asthma and the pathologies accompanying helminth infection. These activities have been hypothesized to be mediated, in part, by the release of cationic proteins stored in the secondary granules of these granulocytes. The majority of the proteins stored in these secondary granules (by mass) are major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX). Unpredictably, a knockout approach targeting the genes encoding these proteins demonstrated that, unlike in mice containing a single deficiency of only MBP-1 or EPX, the absence of both granule proteins resulted in the near complete loss of peripheral blood eosinophils with no apparent impact on any other hematopoietic lineage. Moreover, the absence of MBP-1 and EPX promoted a concomitant loss of eosinophil lineage-committed progenitors in the marrow, identifying a specific blockade in eosinophilopoiesis as the causative event. Significantly, this blockade of eosinophilopoiesis is also observed in ex vivo cultures of marrow progenitors and is not rescued in vivo by adoptive bone marrow engraftment, suggesting a cell-autonomous defect in marrow progenitors. These observations implicate a role for granule protein gene expression as a regulator of eosinophilopoiesis and provide another strain of mice congenitally deficient of eosinophils.

99mTechnetium-labeled heparin: A new approach to detection of eosinophilic esophagitis–associated inflammation

99mTechnetium-labeled heparin: A new approach to detection of eosinophilic esophagitis–associated inflammation

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.

Relationship of Eosinophils and Plasma Cells to Biofilm in Chronic Rhinosinusitis

This study investigates the relationship of eosinophils and plasma cells to biofilm in chronic rhinosinusitis (CRS). A prospective observational study was performed at the Keck Hospital, University of Southern California, Department of Otolaryngology, Los Angeles, CA. A total of 29 patients, 20 undergoing endoscopic sinus surgery for CRS and 9 control patients undergoing septoplasty for nasal obstruction without history or evidence of CRS, were included in this study. Contiguous sinonasal mucosa sample sections were examined by hematoxylin and eosin (H&E), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC) for biofilm, microbes, eosinophil major basic protein (EMBP), and cluster designation 27 (CD27). EMBP and CD27 were used as eosinophil and plasma cell markers, respectively. Biofilm was visualized in 15 of 20 patients with CRS on H&E sections, confirmed by microbial presence using FISH. Biofilm was not identified in tissue samples of the nine control patients. On IHC analysis, CD27 and EMBP expression were significantly higher in patients with CRS compared with control (p < 0.05) and had greater expression in biofilm-positive patients compared with biofilm-negative patients. Nasal polyps correlated with higher expression of CD27 and EMBP, but in CRS patients without polyps CD27 and EMBP was also significantly greater in biofilm-positive specimens compared with biofilm-negative specimens. Biofilm presence in CRS appears to correlate to host inflammatory response involving plasma cell and eosinophil recruitment.

Activated Eosinophils in Association with Enteric Nerves in Inflammatory Bowel Disease

Enteric neural dysfunction leads to increased mucous production and dysmotility in inflammatory bowel disease (IBD). Prior studies have shown that tissue eosinophilia is related to disease activity. We hypothesized that interactions between eosinophils and nerves contribute to neural dysfunction in IBD. Tissue from patients with intractable IBD, endoscopic biopsies from patients with steroid responsive IBD, both when active and quiescent, and control tissue were studied. Immunohistochemical studies showed that eosinophils localize to nerves in the mucosal layer of patients with Crohn’s disease (CD) (p<0.001) and ulcerative colitis (UC), (p<0.01). Eosinophils localized to substance P and choline acetyltransferase (ChAT) immunostained nerves. Real time PCR of laser capture micro-dissected enteric ganglia demonstrated Intercellular Adhesion Molecule 1 (ICAM-1) mRNA was increased 7-fold in UC (n = 4), (p = 0.03), and 10-fold in CD (n = 3), (p = 0.05). Compared with controls, eotaxin-3 (CCL-26) mRNA was increased 9-fold in UC (p = 0.04) and 15-fold in CD (p = 0.06). Eosinophil numbers correlated with disease activity, while deposition of major basic protein (MBP) and eosinophil Transforming Growth Factor β -1 (TGFβ-1) expression were seen in therapeutically responsive disease. These data indicate a significant localization of eosinophils to nerves in IBD, mediated through neurally expressed ICAM-1 and eotaxin-3. This cell/neural interaction may influence the function of nerves and contribute to symptoms in IBD.

Sa1186 The Pathological Evaluation of Pancreatic Fibrosis Correlates With Apparent Diffusion Coefficient Values Based on Diffusion-Weighted MRI

Background: 99mTc-Heparin provides contrast for localized inflammation in eosinophilic esophagitis (EoE), because themarkedly basic eosinophil granule proteins deposit in inflamed regions of the esophagus and eosinophil granule proteins, such as major basic protein 1 (MBP-1), bind strongly to heparin (Swaminathan GJ, et al., Biochemistry 44 (2005) 141528). In prior experiments, we tested the ability of 99mTc-heparin to bind to cationic eosinophil granule proteins, and we demonstrated that the 99mTc-heparin binds to Macaca monkey tissues coated with MBP-1. We also demonstrated that radiolabeled heparin binds in vitro to esophageal biopsies from patients with EoE. We are now poised to determine whether radiolabeled heparin localizes specifically to the esophagus of patients with eosinophilic esophagitis by having patients swallow the reagent and image its binding by single-photon emission computed tomography (SPECT). Aim: To evaluate the radiation risk from 99mTcheparin by studying the tissue distribution and calculating the radiation dose from orally administered 99mTc-heparin to mice. Method: 99mTc-Heparin (500 μCi/mouse, 15 mg) was administered to mice (C57-B16) by oral gavage. At each time point (0.75, 1.5, 3, 6, 18, and 30 hours), mice were euthanized with CO2 and positioned prone on the scanner bed. SPECT/CT images of mice were acquired by using an Inveon trimodality PET/SPECT/ CT scanner (Siemens Preclinical Solutions, Knoxville, TN). The activity of the harvested organs was measured in a well counter. Results: 99mTc-Heparin was not absorbed significantly through theGI tract into other bodily tissue. The highest radioactivities occurredmainly in stomach, small and large intestine. Discussion: These proof-of-principle experiments clearly show that 99mTc-heparin stays in GI tract and is not strongly absorbed. Furthermore, because heparin is a physiological substance that has long been used in clinical medicine, we believe that it will be well tolerated in our patients. This finding is important because it initiates a new avenue for clinical detection of EoE associated inflammation.

409 IL-13 Promotes Esophageal Eosinophilia but Not Required for the Induction of Allergen-Induced Eosinophilic Esophagitis

Background & Aims. IL-13 is induced in EoE patients and number of reports indicates IL13 has an important role in the induction of eosinophilic esophagitis (EoE). However, it is unclear whether IL-13 play a critical role in promoting allergen-induced EoE. Accordingly, we tested the role of IL-13 in allergen-induced EoE by using IL-13 and signal transducer and activator of transcription (STAT)6 gene-deficient mice. Methods. IL-13 was delivered intratranasally to wild-type mice and mice deficient in IL-13 or (STAT)6 genes were challenged with Aspergillus antigen. The lung and esophageal eosinophils were examined by immunohistochemical techniques using anti-MBP (major basic protein) antibody, esophageal mast cells and collegen accumulation by Wright-Giemsa and trichrome staining, respectively. Results. We report that intranasal delivery of IL-13 promote IL-5 dependent eosinophil accumulation in the esophagus. However, allergen-induced EoE is not protected in the IL13 gene-deficient mice. The IL-13 gene-deficient mice demonstrated a comparable level of eosinophils, mast cells and collegen accumulation in the esophagus compared to the wild type mice following allergen-induced experimental EoE. Similarly, we found that IL-4/IL13 duble gene-deficient and STAT6 gene-deficient mice showed a moderate decrease in esophageal eosinophilia compared to wild type mice in allergen-induced experimental EoE. In contrast, lung eosinophilia was significantl protected in IL-13, IL-4/IL-13 and STAT6 gene-deficient mice. Conclusion. Our data establish that IL-13 delivery promotes esophageal eosinophilia, but not required in promoting allergen-induced EoE. Funding: NIH R01 DK067255 (AM), NIH R01 AI080581 (AM), AGA Summer Student Fellowship (Akanksha)

Su1828 Remission in Proton Pump Inhibitors-Responsive Esophageal Eosinophilia Correlates With Downregulation of Eotaxin-3 and TH2 Cytokines, Similarly to Eosinophilic Esophagitis After Steroids

Background: In eosinophilic esophagitis (EoE), eosinophils invade the esophagus where they degranulate and release very cytotoxic proteins, including major basic protein (MBP-1). Currently the diagnosis of EoE necessitates endoscopy with biopsy that characterizes less than 2% of the esophagus. Furthermore, the inflammation is patchy, necessitating numerous biopsies to ensure that the involved area has been adequately sampled. Improved methods are needed to characterize the eosinophil density throughout the esophagus and to thoroughly understand this disease. Aim: To prepare the first MBP-1 specific ultrasound contrast agents to facilitate the detection of EoE associated inflammation by tethering EoE specific antibodies to their surfaces and demonstrating the binding of these agents to eosinophil proteins on ex vivo monkey esophagi. Method: The contrast agent was synthesized by preparing 2 mg/ mL of insulin in HCl (pH 1.6) at 65C for 12 hours. The resulting particles were incubated withmaleimide activated streptavidin. Purified antibodies specific to MBP-1 were biotinylated and reacted with the streptavidin functionalized fibers to form an insulin aggregate-steptavidin-biotin antibody complex. This complex comprises the MBP-1 specific ultrasound contrast agent. A Macaca monkey esophagus, incubated overnight in MBP-1, was filled with this solution. Ultrasound images were taken before and after washing at 15 MHz. Results: Insulin aggregates were observed using ultrasound. Incubating the insulin complex in monkey esophagi coated with MBP-1 yields an extra contrast layer at the inner wall of the esophagus, demonstrating the specific binding of these agents to eosinophil granule proteins. No such layer is visualized in control samples (not treated with MBP-1). Discussion: This is the first reported use of insulin particles as contrast agents for ultrasound. These findings indicate that insulin aggregates provide clear ultrasound contrast in liquid environments. When coupled with disease specific antibodies, these novel agents have the potential to be used for detection, minimizing the need for anesthesia or radiation associated with other diagnostic or imaging modalities. This finding is important because it provides a new avenue for clinical detection of EoE.

Su1830 T-Lymphocyte Isolation and Phenotyping From Esophageal Biopsies in Children With Eosinophilic Esophagitis: Pilot Study

Background: In eosinophilic esophagitis (EoE), eosinophils invade the esophagus where they degranulate and release very cytotoxic proteins, including major basic protein (MBP-1). Currently the diagnosis of EoE necessitates endoscopy with biopsy that characterizes less than 2% of the esophagus. Furthermore, the inflammation is patchy, necessitating numerous biopsies to ensure that the involved area has been adequately sampled. Improved methods are needed to characterize the eosinophil density throughout the esophagus and to thoroughly understand this disease. Aim: To prepare the first MBP-1 specific ultrasound contrast agents to facilitate the detection of EoE associated inflammation by tethering EoE specific antibodies to their surfaces and demonstrating the binding of these agents to eosinophil proteins on ex vivo monkey esophagi. Method: The contrast agent was synthesized by preparing 2 mg/ mL of insulin in HCl (pH 1.6) at 65C for 12 hours. The resulting particles were incubated withmaleimide activated streptavidin. Purified antibodies specific to MBP-1 were biotinylated and reacted with the streptavidin functionalized fibers to form an insulin aggregate-steptavidin-biotin antibody complex. This complex comprises the MBP-1 specific ultrasound contrast agent. A Macaca monkey esophagus, incubated overnight in MBP-1, was filled with this solution. Ultrasound images were taken before and after washing at 15 MHz. Results: Insulin aggregates were observed using ultrasound. Incubating the insulin complex in monkey esophagi coated with MBP-1 yields an extra contrast layer at the inner wall of the esophagus, demonstrating the specific binding of these agents to eosinophil granule proteins. No such layer is visualized in control samples (not treated with MBP-1). Discussion: This is the first reported use of insulin particles as contrast agents for ultrasound. These findings indicate that insulin aggregates provide clear ultrasound contrast in liquid environments. When coupled with disease specific antibodies, these novel agents have the potential to be used for detection, minimizing the need for anesthesia or radiation associated with other diagnostic or imaging modalities. This finding is important because it provides a new avenue for clinical detection of EoE.

Eosinophils mediate the pathogenesis of halothane-induced liver injury in mice

Drug-induced liver injury (DILI) is a major health issue, as it remains difficult to predict which new drugs will cause injury and who will be susceptible to this disease. This is due in part to the lack of animal models and knowledge of susceptibility factors that predispose individuals to DILI. In this regard, liver eosinophilia has often been associated with DILI, although its role remains unclear. We decided to investigate this problem in a murine model of halothane-induced liver injury (HILI). When female Balb/cJ mice were administered halothane, eosinophils were detected by flow cytometry in the liver within 12 hours and increased thereafter proportionally to liver damage. Chemokines, eotaxin-1 (CCL11) and eotaxin-2 (CCL24), which are known to attract eosinophils, increased in response to halothane treatment. The severity of HILI was decreased significantly when the study was repeated in wildtype mice made deficient in eosinophils with a depleting antibody and in eosinophil lineage-ablated ΔdblGata(-/-) mice. Moreover, depletion of neutrophils by pretreating animals with Gr-1 antibody prior to halothane administration failed to reduce the severity of HILI at antibody concentrations that did not affect hepatic eosinophils. Immunohistochemical staining for the granule protein, major basic protein, revealed that eosinophils accumulated exclusively around areas of hepatocellular necrosis. Our findings indicate that eosinophils have a pathologic role in HILI in mice and suggest that they may contribute similarly in many clinical cases of DILI.

A Proposed Model to Study Immunologic Changes during Chronic Rhinosinusitis Exacerbations: Data from a Pilot Study

One way to gain insight into the pathophysiology of chronic rhinosinusitis (CRS) is to study the immunologic changes that occur with exacerbation. This study describes the immunologic changes during CRS exacerbation We performed a prospective study to investigate the immunologic changes seen during exacerbation of CRS with nasal polyposis. We recruited adult subjects who met clinical criteria for CRS with sinus CT scan within the past 5 years with Lund-Mackay score of >5 and nasal polyps. Subjects underwent a baseline visit with collection of nasal secretion and nasal wash. With acute worsening of symptoms, subjects underwent 6 near-consecutive-day collections and one follow-up collection 2 weeks later. IL-6, IL-33, eosinophil major basic protein (MBP), eosinophil-derived neurotoxin (EDN), myeloperoxidase (MPO), and uric acid were measured on the nasal samples from each visit. A total of 10 subjects were recruited and 9 had acute worsening of CRS during the study period. Eight of the nine subjects were women and ages ranged from 26 to 56 years. At baseline, most inflammatory parameters were low and eight of the nine subjects were on intranasal corticosteroids. Compared with baseline measurements, IL-6, MBP, MPO, EDN, and uric acid were significantly elevated during CRS exacerbation. Levels of IL-6 and MBP (r = 0.47) levels as well as IL-6 and MPO (r = 0.75) were both significantly correlated (p < 0.01). Prospective study of CRS exacerbations is feasible and provides insights into the immunologic mechanisms of CRS.

Immune Modulatory Effects of IL-22 On Allergen-Induced Pulmonary Inflammation

RationaleAllergen-induced pulmonary inflammation in asthma is mainly TH2 dominated. A recently discovered TH17/TH22 cell-derived cytokine, IL-22, has been found to be increased in asthma. However, several studies showed controversial findings in the immune modulatory effects of IL-22 in animal models of allergic asthma. Our objective was to determine the regulatory role of IL-22 in OVA-induced allergic inflammation using a novel lung-specific IL-22 transgenic overexpression system.MethodsInducible IL-22 transgenic (CC10-rtTA- and SPC-rtTA-TRE-tight-IL-22) mice on C57BL/6 genetic background were generated. IHC of lung tissue and ELISA of bronchoalveolar lavage (BAL) fluid for IL-22 were performed to confirm the location and quantity of IL-22 expression. OVA-induced pulmonary inflammatory responses were compared between IL-22 transgenic and wild type control mice. BAL total and differential cell counts, lung histology, mucous metaplasia, IHC of major basic protein for eosinophils, and serum OVA-specific IgE were determined.ResultsIL-22 transgene was successfully activated in the large (CC10 promoter) and small (SPC promoter) airway epithelium cells by doxycycline in the drinking water. OVA sensitization and challenge did not induce endogenous IL-22 expression. Compared to wild type mice, IL-22 transgenic mice showed decreased percentage of eosinophils in the BAL and slight reduction in eosinophilic inflammation and mucus metaplasia in the airways. Interestingly, inflammatory cell infiltration in lung parenchyma appeared slightly accentuated. In addition, there was no statistic difference in serum OVA-specific IgE.ConclusionsThese findings indicate that IL-22 may have immune modulatory effects on pulmonary inflammatory responses in the development of allergen-induced asthma. RationaleAllergen-induced pulmonary inflammation in asthma is mainly TH2 dominated. A recently discovered TH17/TH22 cell-derived cytokine, IL-22, has been found to be increased in asthma. However, several studies showed controversial findings in the immune modulatory effects of IL-22 in animal models of allergic asthma. Our objective was to determine the regulatory role of IL-22 in OVA-induced allergic inflammation using a novel lung-specific IL-22 transgenic overexpression system. Allergen-induced pulmonary inflammation in asthma is mainly TH2 dominated. A recently discovered TH17/TH22 cell-derived cytokine, IL-22, has been found to be increased in asthma. However, several studies showed controversial findings in the immune modulatory effects of IL-22 in animal models of allergic asthma. Our objective was to determine the regulatory role of IL-22 in OVA-induced allergic inflammation using a novel lung-specific IL-22 transgenic overexpression system. MethodsInducible IL-22 transgenic (CC10-rtTA- and SPC-rtTA-TRE-tight-IL-22) mice on C57BL/6 genetic background were generated. IHC of lung tissue and ELISA of bronchoalveolar lavage (BAL) fluid for IL-22 were performed to confirm the location and quantity of IL-22 expression. OVA-induced pulmonary inflammatory responses were compared between IL-22 transgenic and wild type control mice. BAL total and differential cell counts, lung histology, mucous metaplasia, IHC of major basic protein for eosinophils, and serum OVA-specific IgE were determined. Inducible IL-22 transgenic (CC10-rtTA- and SPC-rtTA-TRE-tight-IL-22) mice on C57BL/6 genetic background were generated. IHC of lung tissue and ELISA of bronchoalveolar lavage (BAL) fluid for IL-22 were performed to confirm the location and quantity of IL-22 expression. OVA-induced pulmonary inflammatory responses were compared between IL-22 transgenic and wild type control mice. BAL total and differential cell counts, lung histology, mucous metaplasia, IHC of major basic protein for eosinophils, and serum OVA-specific IgE were determined. ResultsIL-22 transgene was successfully activated in the large (CC10 promoter) and small (SPC promoter) airway epithelium cells by doxycycline in the drinking water. OVA sensitization and challenge did not induce endogenous IL-22 expression. Compared to wild type mice, IL-22 transgenic mice showed decreased percentage of eosinophils in the BAL and slight reduction in eosinophilic inflammation and mucus metaplasia in the airways. Interestingly, inflammatory cell infiltration in lung parenchyma appeared slightly accentuated. In addition, there was no statistic difference in serum OVA-specific IgE. IL-22 transgene was successfully activated in the large (CC10 promoter) and small (SPC promoter) airway epithelium cells by doxycycline in the drinking water. OVA sensitization and challenge did not induce endogenous IL-22 expression. Compared to wild type mice, IL-22 transgenic mice showed decreased percentage of eosinophils in the BAL and slight reduction in eosinophilic inflammation and mucus metaplasia in the airways. Interestingly, inflammatory cell infiltration in lung parenchyma appeared slightly accentuated. In addition, there was no statistic difference in serum OVA-specific IgE. ConclusionsThese findings indicate that IL-22 may have immune modulatory effects on pulmonary inflammatory responses in the development of allergen-induced asthma. These findings indicate that IL-22 may have immune modulatory effects on pulmonary inflammatory responses in the development of allergen-induced asthma.