Mechanism of atopic cataract caused by eosinophil granule major basic protein.

Atopic cataracts develop under the ages of 40 years, after which visual acuity rapidly declines. However, the mechanism underlying the development of atopic cataracts is not yet clear. We focused on the eosinophil granule major basic protein (MBP), which was detected in the aqueous humor of atopic cataracts previously, and which was cytotoxic. Specifically, we investigated its origin in this fluid and its effects on lens epithelial cells (LECs). MBP immunostaining was positive in atopic cataract-derived LECs, but negative in age-related cataract-derived LECs. MBP mRNA was not detected in either type of cataract, but protein was detected in the aqueous humor. Furthermore, the flare values associated with atopic cataracts were higher than those with age-related cataracts. When MBP was purified from eosinophils or recombinant MBP was added to LEC culture medium, cell viability decreased in a concentration-dependent manner, but an MBP antibody neutralized the cytotoxic effect of this protein towards these cells. These results were consistent with the flow of MBP into the aqueous humor from the blood due to a compromised blood-aqueous barrier. Thus, MBP could further penetrate the lens capsule and adhere to LECs, resulting in decreased cell viability and the development of atopic cataracts.

Extracellular Eosinophil Granule Protein Deposition in Ringed Esophagus with Sparse Eosinophils.

BACKGROUND: Eosinophilic esophagitis (EoE) remains difficult to classify because of varying presentations. Not uncommonly, patients present with symptoms of esophageal dysfunction and have esophageal changes on endoscopy resembling EoE but without >15 eosinophils/HPF. Patients with low numbers of eosinophils in esophageal biopsy specimens may have esophageal changes and symptomatic disease brought about by eosinophil granule protein deposition without recognizable intact cells. AIM: To determine whether extracellular eosinophil granule protein deposition is present in the esophagi of patients with low eosinophil numbers who have clinical symptoms and characteristic endoscopic esophageal changes of EoE including ringed esophagus (RE). METHODS: Esophageal biopsy specimens were studied from eight EoE patients with >15 eosinophils per high power field (HPF) and nine patients with RE (<15 eosinophils/HPF). The specimens were analyzed for eosinophil granule proteins, major basic protein 1 (eMBP1) and eosinophil-derived neurotoxin (EDN), by indirect immunofluorescence. RESULTS: Both EoE and RE showed positive EDN and eMBP1 extracellular deposition; control esophagus showed minimal or none. Comparing EoE and RE, extracellular EDN and eMBP1 were similar except that EDN in EoE was greater in the distal esophagus. CONCLUSIONS: This study highlights the importance of assessing eosinophil granule protein deposition in esophageal disease with potential eosinophil involvement. Persistent/progressive esophageal changes may be brought about by eosinophil granule proteins despite low numbers of intact cells. The meaning of "resolution" in EoE may need to be redefined based on numbers of esophageal eosinophils, extracellular eosinophil granule protein deposition, and subsequent clinical course of patients.

Longitudinal changes in C-reactive protein, proform of eosinophil major basic protein, and pregnancy-associated plasma protein-A during weight changes in obese children.

BACKGROUND: Childhood obesity is associated with several complications, including cardiovascular comorbidity. Several biomarkers, such as high-sensitive C-reactive protein (hs-CRP), proform of eosinophil major basic protein (Pro-MBP) and pregnancy associated plasma protein-A (PAPP-A), have equally been linked to increased cardiovascular susceptibility. This study investigates these biomarkers during weight loss and regain in obese children. MATERIALS AND METHODS: A longitudinal study during a 12-week weight loss program with a 28 months follow-up was conducted. Anthropometrics and plasma concentrations of hs-CRP, Pro-MBP, and PAPP-A were measured at baseline; at days 14, 33 and 82 during weight loss; and at months 10, 16, and 28 during follow-up. RESULTS: Fifty-three boys and 62 girls aged 8-15 years with a median body mass index (BMI) standard deviation score (SDS) at baseline of 2.78 (boys), and 2.70 (girls) were included. Ninety children completed the weight loss program and 68 children entered the follow-up program. Pro-MBP and PAPP-A, but not hs-CRP, exhibited individual-specific levels (tracking) during weight loss and regain. The PAPP-A/Pro-MBP correlation was strong, whereas the hs-CRP/PAPP-A correlation was weak during weight fluctuations. CONCLUSION: Hs-CRP changes reflect weight changes. PAPP-A and Pro-MBP exhibited tracking during weight perturbations and may contribute as early risk markers of cardiovascular susceptibility.

Prostaglandin D2 receptor D-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.

Surgical management and immunohistochemical study of corneal plaques in vernal keratoconjunctivitis.

Two children with shield ulcer in vernal keratoconjunctivitis unresponsive to steroid therapy received plaque removal by superficial keratectomy, followed by amniotic membrane transplantation (AMT). Hematoxylin and eosin staining of the excised corneal specimen revealed a thick layer of eosinophilic material attached to the Bowman's layer. These deposits were positive for eosinophil granule major basic protein, as confirmed by an immunohistochemical study. The shield ulcer healed after the amniotic membrane was removed. No recurrent corneal plaque developed, although corneal opacity complicated in both cases. Lamellar keratectomy with AMT offers an effective management by removing the cytotoxic plaques and protecting the denuded stroma from deposition of inflammatory debris.

Increased numbers of eosinophils, rather than only etiology, predict histologic changes in patients with esophageal eosinophilia.

BACKGROUND & AIMS: It can be a challenge to differentiate individuals with eosinophilic esophagitis (EoE) from those with gastroesophageal reflux disease (GERD). We investigated differences in histologic and eosinophil patterns and numbers of mast cells between patients with these disorders. METHODS: We performed histologic analyses and immunohistochemical assays for eosinophil-derived neurotoxin (EDN), major basic protein (MBP), and tryptase, using biopsy samples from 10 patients with GERD (positive results from a pH study and response to proton pump inhibitors), Barrett's esophagus, or EoE (negative results from a pH study and positive response to budesonide). Patients were matched for degree of eosinophilia. RESULTS: Samples from patients with EoE, GERD, or Barrett's esophagus had similar increases in concentrations of eosinophils. Patients with GERD or EoE did not differ in amount of basal zone hyperplasia, microabscesses, spongiosis, eosinophil distribution, maximum eosinophils/high-power field (HPF), or composite histologic scores. Samples from all 3 groups had high levels of EDN and MBP; the levels of eosinophil products were correlated (ρ = 0.93). Extracellular staining for EDN was greater than intracellular staining (2.67 of 3 vs 1.86 of 3); levels tended to be greater in samples from patients with EoE than GERD (P = .05) or Barrett's esophagus (P = .06). Detection of EDN correlated with peak numbers of eosinophils/HPF (ρ = 0.6 for intracellular and extracellular staining). Peak numbers of tryptase-positive mast cells/HPF were significantly greater in samples from patients with EoE than GERD or Barrett's esophagus (P = .01 and .005, respectively). The Spearman correlation between eosinophil and mast cell density was a ρ value of 0.2. CONCLUSIONS: Biopsy samples from patients with GERD and EoE, matched for esophageal eosinophilia, have similar changes in histology and levels of EDN and MBP, whereas mast cells from patients with EoE have higher levels of these products. The presence of esophageal eosinophils, rather than etiology, could be the most important determinant of epithelial response.

Eosinophil degranulation is more important than eosinophilia in identifying asthma in chronic cough.

OBJECTIVE: We investigated whether eosinophil degranulation is a distinctive feature of asthma and can distinguish between chronic cough patients with asthma and those without. METHODS: Thirty-seven patients, with a chronic cough for more than 1 month, and nine normal individuals (controls) were enrolled. Subjects were divided into two groups: one group with asthma and positive bronchial hyperresponsiveness (BHR) (Asthma group, n = 18) and the other group without asthma and negative BHR (Non-Asthma group, n = 19). From induced sputum, total cell counts and differentials were determined. Myeloperoxidase levels were measured by Enzyme-Linked Immunosorbent Assay (ELISA), and eosinophil-derived neurotoxin (EDN) and major basic protein (MBP) levels were measured by radioimmunoassay. RESULTS: The percentage of sputum eosinophils was increased in the Asthma (p < .001) and Non-Asthma (p < .05) groups compared with the Control group and when comparing the Asthma and Non-Asthma (p < .001) groups. Sputum EDN and MBP levels were increased in the Asthma group compared with the Non-Asthma (p < .05 and p < .05, respectively) and Control groups (p < .05 and p = .055, respectively). However, EDN and MBP levels were not increased in the Non-Asthma group compared with the Control group. The percentage of sputum eosinophils in the Asthma group correlated positively with sputum EDN (Rs = 0.921, p < .001) and MBP (Rs = 0.882, p < .0001) levels and negatively with maxΔFEV(1) (Rs = -0.501, p < .05) (FEV(1), forced expiratory volume in 1 second). Unexpectedly, the percentage of eosinophils in the Non-Asthma group did not correlate significantly with any of these markers. Increased EDN and MBP levels and significant correlations between the percentage of eosinophils and EDN and MBP were only observed in asthma patients. CONCLUSIONS: These findings suggest that eosinophil degranulation is more important than eosinophilia in identifying asthma.

Pregnancy-associated plasma protein A and proform eosinophilic major basic protein in the detection of different types of coronary artery disease.

Kryptor system was proven to be a rapid, standard method for pregnancy-associated plasma protein A and proform eosinophilic major basic protein (PAPP-A/proMBP) complex detection in coronary artery disease (CAD). No age and/or gender differences in 51 controls and 110 stable coronary artery disease (SCAD) patients were found. SCAD patients did not differ from controls and no difference in PAPP-A/proMBP levels with regards to the number of affected vessels was found. In 21 unstable angina pectoris (UAP), in 35 without and 66 with ST elevation acute myocardial infarctions (NSTEMI, STEMI respectively) patients PAPP-A/proMBP levels were increased (P=0.004 and P<0.0005, respectively). PAPP-A/proMBP levels did not correlate with cardiac troponin I (cTnI) in STEMI and NSTEMI patients. PAPP-A/ proMBP increase was more frequent than cTnI (P=0.036) within the early phase of STEMI. In NSTEMI patients PAPP-A/proMBP positivity was present in 50% of cTnI negative cases. Receiver operating characteristic (ROC) analysis revealed the highest diagnostic accuracy of PAPP-A/proMBP (0.919) in STEMI cTnI positive cases. The highest specificity/sensitivity PAPP-A/proMBP levels for particular acute coronary syndrome (ACS) types were 10.65-14.75 mIU/l. Combination of PAPP-A/proMBP with cTnI increases their diagnostic efficacy within the early phase of ACS. Our results suggest that PAPP-A/proMBP complex is involved in processes preceding vulnerable plaque development in ACS.

Expression of prostaglandin D2 synthase in activated eosinophils in nasal polyps.

OBJECTIVE: To clarify the relationship between prostaglandin D2 production and eosinophil accumulation. DESIGN: Screening and diagnostic tests. SUBJECTS: Nineteen patients with chronic rhinosinusitis. INTERVENTIONS: Nasal polyps were obtained from 19 patients at endoscopic sinus surgery. Eosinophils in nasal polyps were counted after hematoxylin-eosin staining and immunostaining with antibodies against 2 eosinophil markers-major basic protein and EG2. Hematopoietic prostaglandin D2 synthase (HPGDS) expression was examined by semiquantitative Western blot analysis and by immunohistochemical staining with anti-HPGDS antibody. RESULTS: Nasal polyps were divided into 3 groups by the degree of eosinophilic infiltration. Western blot analysis revealed that HPGDS was more intensely and frequently expressed in the group with high infiltration than in the groups with low or medium infiltration. Hematopoietic prostaglandin D2 synthase was immunohistochemically found in a subpopulation of EG2-positive eosinophils that had accumulated in the nasal polyps but not in the EG2-negative resting eosinophils. The ratio of HPGDS-positive eosinophils to EG2-positive eosinophils in the group with high eosinophil infiltration (mean+/-SD, 64.8%+/-19.2%) was twice that in the group with low eosinophil infiltration (30.5%+/-13.8%). CONCLUSION: Prostaglandin D2 was actively produced by an EG2 and HPGDS double-positive subpopulation of activated eosinophils that had infiltrated into nasal polyps.

Neonatal eosinophilic pustulosis.

A pre-term, 7-week-old male infant presented with a recurrent pustular eruption involving his face and scalp with associated peripheral blood eosinophilia. Skin biopsy revealed spongiosis with numerous dermal and epidermal eosinophils without predominant follicular involvement. Immunohistology showed deposition of eosinophil granule major basic protein and eosinophil derived neurotoxin in the dermis and epidermis. He responded to conservative management. We discuss the differential diagnosis of neonatal eosinophilic pustular eruptions and suggest the term 'neonatal eosinophilic pustulosis' to best describe our case.

[Activated eosinophils: techniques to characterize them]. / Etude de l'activation du polynucléaire éosinophile. Apport des méthodologies classiques et récentes à la caractérisation d'une cellule complexe.

The deleterious role thought to be played by eosinophils in many situations is linked to their ability to secrete various inflammatory substances, mainly toxic proteins and lipid mediators, in body tissue. This ability is a particular feature of activated eosinophils, which have undergone numerous metabolic, functional, and phenotypic changes from their resting state. Characterizing the properties of these activated cells is an essential step in improving our understanding of their contributions to local inflammatory response, as both regulatory and effector cells. Improvements in existing methods as well as the development of new technical approaches have facilitated the ex vivo and in vitro study of activated eosinophils and their contribution to various disease states.

Airway remodeling in allergen-challenged Brown Norway rats: distribution of proteoglycans.

Proteoglycans (PG) have important effects on the mechanical properties of tissues and the phenotype of various structural cells. Little is known about changes in PG deposition in the airways in animal models of asthma. We studied changes in PG in the airway wall of Brown Norway rats sensitized to ovalbumin (OA) and exposed to repeated OA challenge. Control (Sal) animals were sensitized and challenged with saline. After the 3rd challenge, animals were killed and lungs fixed in formalin. Tissue sections were incubated with antibodies to the small, leucine-rich PG, decorin, and biglycan and collagen type I. Airways were classified according to basement membrane perimeter length (< or =0.99, 1-2.99, and > or =3 mm). Decorin, biglycan, and collagen type I were increased in the airways of OA vs. Sal rats. Remodeling was most prominent in central airways. The distribution of PG differed with respect to the subepithelial vs. airway smooth muscle (ASM) vs. adventitial layer. Whereas biglycan was readily detected within the ASM, decorin and collagen were detected outside the ASM and especially in the adventitial layer. Differences in the distribution of these molecules within the layers of the airway wall may reflect their specific functional roles.

Striking deposition of toxic eosinophil major basic protein in mucus: implications for chronic rhinosinusitis.

BACKGROUND: The mechanisms by which eosinophilic inflammation damages the epithelium and contributes to recurrent acute exacerbations in chronic rhinosinusitis (CRS) have not been fully elucidated. OBJECTIVE: We tested the hypotheses that eosinophils deposit toxic major basic protein (MBP) in the mucus and that MBP reaches concentrations able to damage the sinonasal epithelium. METHODS: Tissue specimens with mucus attached to the tissue were carefully collected from 22 patients with CRS and examined by using immunofluorescence staining for MBP. This immunofluorescence was digitally analyzed to determine the area covered by MBP and the intensity of the staining (estimating MBP concentration). Levels of MBP in extracts from nasal mucus were quantitated by means of RIA. RESULTS: Heterogeneous eosinophilia was evident within tissue and mucus specimens. All tissue specimens showed intact eosinophils, but diffuse extracellular MBP deposition, as a marker of eosinophil degranulation, was rare. In contrast, all mucus specimens showed diffuse MBP throughout and abundant diffuse extracellular MBP deposition within clusters of eosinophils. Digitized analyses of MBP immunofluorescence revealed increased area coverage (P < .0001) in mucus compared with that seen in tissue. Estimated concentrations of MBP within the clusters suggested toxic levels. MBP concentrations in mucus extract reached 11.7 microg/mL; MBP was not detectable in healthy control subjects. CONCLUSION: In patients with CRS, eosinophils form clusters in the mucus where they release MBP, which is diffusely deposited on the epithelium, a process not observed in the tissue. Estimated MBP levels far exceed those needed to damage epithelium from the luminal side and could predispose patients with CRS to secondary bacterial infections.

In chronic idiopathic urticaria autoantibodies against Fc epsilonRII/CD23 induce histamine release via eosinophil activation.

BACKGROUND: Chronic idiopathic urticaria is a common skin disorder characterized by recurrent, transitory, itchy weals for more than 6 weeks. An autoimmune origin has been suggested based on the findings of auto-antibodies (Abs) directed against either the alpha subunit of the high-affinity IgE receptor or the IgE molecule in nearly half of the patients. OBJECTIVE: To identify other autoantigen targets in patients with chronic idiopathic urticaria. METHODS: We used pooled IgG derived from 133 patients with chronic idiopathic urticaria to screen a random peptide library to identify disease-relevant autoantigen peptides. Among the identified peptides, one was recognized by the vast majority of patients' sera. Abs against this peptide were affinity purified from the patients' sera and assayed for their ability to induce histamine release from basophils. RESULTS: We identified a peptide that showed similarity with the low-affinity IgE receptor (Fc epsilonRII/CD23) expressed on lymphomonocytes and eosinophils. Anti-peptide IgG Abs purified from the patients' sera bound cell surface CD23 and were able to induce histamine release from basophils. This effect appeared to be mediated by the release of major basic protein from eosinophils upon engagement of CD23. The same effects were obtained with the sera from mice immunized with the CD23 peptide. CONCLUSION: Our results indicate that patients with chronic idiopathic urticaria have Abs against CD23 and that eosinophils, which infiltrate the skin of these patients, play a crucial role in maintaining the disease through the release of major basic protein upon engagement of the low-affinity IgE receptor by such auto-Abs.

Chronic rhinosinusitis: an enhanced immune response to ubiquitous airborne fungi.

BACKGROUND: Chronic rhinosinusitis (CRS) is one of the most common long-term illnesses in the United States. The etiology of CRS is unknown, and no effective treatment has been established. OBJECTIVE: We investigated the hypothesis that abnormal immunologic responses to ubiquitous airborne fungi contribute to the pathogenesis of this disease. METHODS: The proliferative and cytokine responses of PBMCs to extracts from 4 common airborne fungi-including Alternaria , Aspergillus , Cladosporium , and Penicillium -were examined by in vitro culture. Serum specimens were tested for specific IgE and IgG to these fungi. RESULTS: PBMCs from approximately 90% of the patients with CRS, but not those from normal individuals, produced both IL-5 and IL-13 when exposed to Alternaria. Furthermore, PBMCs from patients with CRS produced significantly more IFN-gamma than PBMCs from normal individuals in response to Alternaria (median, 553 pg/mL vs 98 pg/mL; P < .01). Levels of serum IgG antibodies to Alternaria and Cladosporium were clearly increased in patients with CRS compared with normal individuals ( P < .01). Less than 30% of the patients with CRS had specific IgE antibodies to Alternaria or Cladosporium. The increased humoral (serum IgG) response strongly correlated with the increased cellular (IL-5 production) response to Alternaria ( r = 0.619; P < .01). CONCLUSION: Patients with CRS show exaggerated humoral and cellular responses, both T(H)1 and T(H)2 types, to common airborne fungi, particularly Alternaria. The anomalous immune and inflammatory responses to ubiquitous fungi may explain the chronicity of airway inflammation in CRS.