Citations

Abstract Paradigms of eosinophil effector function in the lungs of asthma patients invariably depend on activities mediated by cationic proteins released from secondary granules during a process collectively referred to as degranulation. In this study, we generated knockout mice deficient for eosinophil peroxidase (EPO) to assess the role(s) of this abundant secondary granule protein in an OVA-challenge model. The loss of EPO had no effect on the development of OVA-induced pathologies in the mouse. The absence of phenotypic consequences in these knockout animals extended beyond pulmonary histopathologies and airway changes, as EPO-deficient animals also displayed OVA-induced airway hyperresponsiveness after provocation with methacholine. In addition, EPO-mediated oxidative damage of proteins (e.g., bromination of tyrosine residues) recovered in bronchoalveolar lavage from OVA-treated wild-type mice was <10% of the levels observed in bronchoalveolar lavage recovered from asthma patients. These data demonstrate that EPO activities are inconsequential to the development of allergic pulmonary pathologies in the mouse and suggest that degranulation of eosinophils recruited to the lung in this model does not occur at levels comparable to those observed in humans with asthma.

Abstract Ecalectin, produced by Ag-stimulated T lymphocytes, is a potent eosinophil-specific chemoattractant in vitro as well as in vivo and thus is implicated in allergic responses. Ecalectin differs structurally from other known eosinophil chemoattractants (ECAs); ecalectin belongs to the galectin family defined by their affinity for β-galactosides and by their conserved carbohydrate recognition domains. These characteristic features suggest that ecalectin has unique activities associated with allergic inflammation besides ECA activity. Conversely, ecalectin may mediate ECA activity by binding to a receptor of a known ECA via affinity for the β-galactosides present on this receptor. In this study, we have tested whether ecalectin mediates ECA activity by binding to a receptor of a known ECA, and we have assessed its effects on eosinophils. Ecalectin did not mediate ECA activity by binding to the IL-5R or to CCR3. Also, the ECA activity of ecalectin was mainly chemokinetic. In addition, ecalectin induced concentration-dependent eosinophil aggregation, a marker for eosinophil activation. Ecalectin induced concentration-dependent superoxide production from eosinophils but did not induce degranulation; usually these two events are coupled in eosinophil activation. Moreover, ecalectin directly prolonged eosinophil survival in vitro and did not trigger eosinophils to secrete cytokines that prolong eosinophil survival. These results demonstrate that ecalectin has several unique effects on eosinophils. Therefore, we conclude that ecalectin is a novel eosinophil-activating factor. Presumably, these effects allow ecalectin to play a distinctive role in allergic inflammation.

Abstract We recently described a novel H2E class II-transgenic model (A−E+) of experimental autoimmune thyroiditis (EAT) that permits disease induction with heterologous thyroglobulin (Tg), but unlike conventional susceptible strains, precludes self-reactivity to autologous mouse Tg. In transgenic E+B10 (A+E+) mice, the presence of endogenous H2A genes is protective against H2E-mediated thyroiditis, inhibiting EAT development. The suppressive effect of H2A genes on H2E-mediated thyroiditis mirrors previous reports of H2E suppression on H2A-mediated autoimmune diseases, including EAT. The mechanism of the reciprocal-suppressive effect between class II genes is unclear, although the involvement of regulatory T cells has been proposed. We have recently reported that CD4+CD25+ regulatory T cells mediate peripheral tolerance induced with mouse Tg in CBA mice. To determine whether these cells play a role in our E+-transgenic model, we first confirmed the existence of CD4+CD25+ T cells regulating thyroiditis in E+B10.Ab0 (A−E+) and B10 (A+E−) mice by i.v. administration of CD25 mAb before EAT induction. The depletion of CD4+CD25+ T cells enhanced thyroiditis induction in the context of either H2E or H2A. Moreover, reconstitution of CD4+CD25+ T cells from naive B10 mice restored resistance to EAT. E+B10 (A+E+) mice were also depleted of CD4+CD25+ T cells before the challenge to determine their role in thyroiditis in the presence of both H2A and H2E genes. Depletion of CD4+CD25+ regulatory T cells offset the suppression of H2E-mediated thyroiditis by H2A. Thus, these regulatory T cells may be involved in the reciprocal-suppressive effect between class II genes.

Abstract Human eosinophil granule major basic protein (MBP1) is an exceedingly basic (isoelectric point >11) 14-kDa protein, comprising the core of the secondary eosinophil granule. Recently, a less cationic homolog of MBP, termed MBPH or simply, MBP2, has been discovered. We prepared a panel of mAbs to MBP2 and used these Abs to localize and quantitate this molecule in leukocytes and biological fluids. Specific mAbs for MBP2 were selected using slot-blot analyses and used in a two-site immunoassay, Western blotting, and immunofluorescence microscopy. The sensitivity of the immunoassay was markedly improved by reduction and alkylation of MBP2. MBP1 is more abundant than MBP2 in lysates of eosinophils and their granules, as judged by immunoassay and Western blotting. By immunofluorescence, MBP1 is present in eosinophils, basophils, and a human mast cell line (HMC1), whereas MBP2 is only detected in eosinophils. Neither MBP1 nor MBP2 could be detected in any other peripheral blood leukocyte. MBP2 levels measured in plasma and serum were essentially identical. In contrast to past measurements for MBP1, MBP2 was not detected above normal levels in sera from pregnant donors. However, measurement of serum MBP2 discriminated patients with elevated eosinophils from normal subjects, and MBP2 was also detectable in other biological specimens, such as bronchoalveolar lavage, sputum, and stool. These results indicate that MBP2 is present only in eosinophils and that it may be a useful biomarker for eosinophil-associated diseases.

AbstractBackgroundAsthma guidelines suggest reducing controller medications when asthma is stable.MethodsThe purpose of the study is to estimate the risk of asthma exacerbation in stable asthmatics who reduce inhaled corticosteroids (ICS) compared to those who maintain a stable ICS dose. We identified articles from a systematic review of English and non‐English articles using MEDLINE, EMBASE, Web of Science, and CENTRAL (inception to May 25, 2013). We included randomized controlled trials (RCTs) with a stable asthma run‐in period of 4 weeks or more, an intervention to reduce ICS, and a follow‐up period of at least 3 months.ResultsThe search strategy identified 2253 potential articles, of which 206 were reviewed at the full‐text level and 6 met criteria for inclusion. The relative risk of an asthma exacerbation in individuals who reduced ICS compared to those who maintained the same ICS dose was 1.25 (95% CI 0.96, 1.62; P = 0.10; I2 = 0%) in studies with a mean follow‐up of 22 weeks. Individuals who reduced ICS had a decreased% predicted FEV1 of 0.87% (95% CI −1.58%,3.33%; P = 0.49, I2 = 58%) and a decreased mean morning peak expiratory flow of 9.57 l/min (95% CI 1.25, 17.90; P = 0.02; I2 = 74%) compared to those individuals who maintained a stable ICS dose.ConclusionsAsthma exacerbations were statistically no more likely among individuals who reduced ICS compared to those who maintained their ICS dose, supporting current guidelines which recommend decreasing ICS by 50% after a period of asthma stability.

AbstractBackgroundRecent data suggest that acute coronary syndromes (ACS) and acute myocardial infarction (AMI) are characterized by an inflammatory subset of thrombosis. We have previously described the accumulation of neutrophils at the coronary culprit lesion site. In this work, we assessed the prognostic value of culprit site (CS) neutrophil accumulation on long‐term mortality in patients with AMI.Materials and methodsIn this prospective study, 417 AMI patients were enrolled after thrombectomy during primary percutaneous coronary intervention. The optimal cut‐off for CS neutrophil accumulation for predicting 4‐year all‐cause mortality was calculated using time‐dependent receiver operator characteristic curve analyses.ResultsThe median follow‐up time was 39 months interquartile range (IQR 21·4–54·6 months) corresponding to 1217 patient years of follow‐up. The cut‐off for CS neutrophil accumulation (difference between culprit neutrophil counts and systemic neutrophil counts) was 0·25 Giga/l. CS neutrophil accumulation occurred in 195 patients (47%) and was independently associated with mortality (hazard ratio 1·88 (95%CI 1·02–3·41, P = 0·043)). In patients with CS neutrophil accumulation, 1‐year mortality (10·8% vs. 7·2%) and 4‐year mortality (19·8% vs. 10·4%) were markedly increased compared with patients without local neutrophil accumulation. Concordance index for CS neutrophil accumulation and mortality was 0·64 (95% CI 0·51–0·77; P = 0·035). Patients with CS neutrophil accumulation had significantly more often nonobstructive lesions compared with patients without neutrophil accumulation (32·6% vs. 22·4%; P = 0·024).Conclusions Neutrophil accumulation at the coronary culprit lesion site is a strong and independent predictor of mortality in patients with ACS/AMI.

Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents γ-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm microtubule-nucleating capability on reconstituted centrosomes. γ-Tubulin is biparentally inherited in humans (maternal >> than paternal): Western blots detect the presence of paternal γ-tubulin. Recruitment of maternal γ-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm “priming” induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosome phosphorylation is detected after exposure of primed sperm to egg extracts as well as during the early stages of sperm incorporation after fertilization. Finally, centrosome reconstitution in cell-free extracts permits sperm aster microtubule assembly in vitro. Collectively, these results support a model of a blended zygotic centrosome composed of maternal constituents attracted to an introduced paternal template after insemination.