Normalized serum eosinophil peroxidase levels are inversely correlated with esophageal eosinophilia in eosinophilic esophagitis.

Eosinophil peroxidase is an eosinophil-specific, cytoplasmic protein stored in the secondary granules of eosinophils. While eosinophil peroxidase deposition is increased in the esophagus in eosinophilic esophagitis (EOE), its potential role as a peripheral marker is unknown. This study aims to examine the relationship between serum eosinophil peroxidase and esophageal eosinophilia in eosinophilic esophagitis. Prospectively collected serum from 19 subjects with incident EoE prior to treatment and 20 non-EoE controls were tested for serum eosinophil peroxidase, eosinophilic cationic protein, and eosinophil derived neurotoxin using ELISA. Matching esophageal tissue sections were stained and assessed for eosinophil peroxidase deposition using a histopathologic scoring algorithm. Mean peripheral blood absolute eosinophil counts in eosinophilic esophagitis subjects were significantly elevated compared to controls (363 vs. 195 cells/µL, P = 0.008). Absolute median serum eosinophil peroxidase, eosinophil cationic protein, and eosinophil derived neurotoxin did not differ between groups; however, when normalized for absolute eosinophil counts, eosinophilic esophagitis subjects had significantly lower median eosinophil peroxidase levels (2.56 vs. 6.96 ng/mL per eos/µL, P = 0.002, AUC 0.79 (0.64, 0.94 95% CI)). Multivariate analysis demonstrated this relationship persisted after controlling for atopy. Esophageal biopsies from eosinophilic esophagitis subjects demonstrated marked eosinophil peroxidase deposition (median score 46 vs. 0, P < 0.0001). Normalized eosinophil peroxidase levels inversely correlated with esophageal eosinophil density (r = -0.41, P = 0.009). In contrast to marked tissue eosinophil degranulation, circulating eosinophils appear to retain their granule proteins in EoE. Investigations of normalized serum eosinophil peroxidase levels as a biomarker of EoE are ongoing.

Nasal and pharyngeal eosinophil peroxidase levels in adults with poorly controlled asthma correlate with sputum eosinophilia.

The objective of the study was to compare nasal, pharyngeal, and sputum eosinophil peroxidase (EPX) levels with induced sputum eosinophil percentage in 10 adults with poorly controlled asthma and 10 normal controls. EPX was measured using an ELISA and normalized for grams of protein for nasal and pharynx specimens and for mL-gram of protein for sputum. Sputum EPX levels were statistically different between asthma and control subjects (P = 0.024). EPX levels measured in the nasal and pharyngeal swab samples derived from the same patients were also different between asthma and control subjects, each displaying a high degree of significance (P = 0.002). Spearman's correlation coefficients for nasal EPX and pharyngeal EPX levels compared to induced sputum eosinophil percentage were 0.81 (P = 0.0007) and 0.78 (P = 0.0017), respectively. Thus, there is a strong association in a given patient between both nasal and pharyngeal EPX levels and the eosinophil percentage of induced sputum.

Eosinophil activities modulate the immune/inflammatory character of allergic respiratory responses in mice.

BACKGROUND: The importance and specific role(s) of eosinophils in modulating the immune/inflammatory phenotype of allergic pulmonary disease remain to be defined. Established animal models assessing the role(s) of eosinophils as contributors and/or causative agents of disease have relied on congenitally deficient mice where the developmental consequences of eosinophil depletion are unknown. METHODS: We developed a novel conditional eosinophil-deficient strain of mice (iPHIL) through a gene knock-in strategy inserting the human diphtheria toxin (DT) receptor (DTR) into the endogenous eosinophil peroxidase genomic locus. RESULTS: Expression of DTR rendered resistant mouse eosinophil progenitors sensitive to DT without affecting any other cell types. The presence of eosinophils was shown to be unnecessary during the sensitization phase of either ovalbumin (OVA) or house dust mite (HDM) acute asthma models. However, eosinophil ablation during airway challenge led to a predominantly neutrophilic phenotype (>15% neutrophils) accompanied by allergen-induced histopathologies and airway hyper-responsiveness in response to methacholine indistinguishable from eosinophilic wild-type mice. Moreover, the iPHIL neutrophilic airway phenotype was shown to be a steroid-resistant allergic respiratory variant that was reversible upon the restoration of peripheral eosinophils. CONCLUSIONS: Eosinophil contributions to allergic immune/inflammatory responses appear to be limited to the airway challenge and not to the sensitization phase of allergen provocation models. The reversible steroid-resistant character of the iPHIL neutrophilic airway variant suggests underappreciated mechanisms by which eosinophils shape the character of allergic respiratory responses.

Eosinophil peroxidase in sputum represents a unique biomarker of airway eosinophilia.

BACKGROUND: Sputum eosinophilia has been shown to be a predictor of response to anti-eosinophil therapies in patients with airway diseases. However, quantitative cell counts and differentials of sputum are labor intensive. The objective of this study was to validate a novel ELISA-based assay of eosinophil peroxidase (EPX) in sputum as a rapid and reliable marker of airway eosinophils. METHODS: The utility of EPX-based ELISA as an eosinophil-specific assay was achieved through comparisons with sputum eosinophil differential counts in freshly prepared and archived patient samples from a variety of clinical settings. RESULTS: EPX levels in sputum correlated with eosinophil percentage (r(s) = 0.84) in asthma patients with varying degrees of airway eosinophilia. Significantly, unlike assays of other eosinophil granule proteins (e.g., ECP and EDN), which often detect the presence of these proteins even in asthma patients with neutrophilic bronchitis, EPX-based ELISA levels are not increased in this subset of asthma patients or in COPD patients lacking evidence of an airway eosinophilia. Moreover, sputum EPX was a surrogate marker of airway eosinophilia in other patient studies (e.g., allergen inhalation and treatment trials the anti-(IL-5) therapeutic Mepolizumab™). Finally, EPX levels in cytocentrifuged prepared sputum supernatants correlated with those from rapidly prepared noncentrifuged filtrates of sputum (r(s) = 0.94). CONCLUSION AND CLINICAL IMPLICATION: EPX-based ELISA is a valid, reliable, repeatable, and specific surrogate marker of eosinophils and/or eosinophil degranulation in the sputum of respiratory patients. The novel EPX assay is a valid and reproducible eosinophil-specific assay that can potentially be developed into a point-of-care assessment of eosinophil activity in airway secretions.

Characterization of acrosin-like activity of lake sturgeon (Acipenser fulvescens) spermatozoa.

Acipenserid fish sperm possess trypsin-like activity, resembling acrosin activity of mammalian sperm, which can be measured by hydrolysis of N-alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) (Ciereszko et al., 1994: J Exp Zool 268:486-491). We found that this activity can be preserved when sperm is frozen on dry ice with 0.6 M sucrose-10% dimethylsulfoxide (DMSO) extender (sperm:extender ratio 1:3), and subsequently stored in liquid nitrogen. However, other methods of freezing (without cryoprotectant at -18 degrees C, -80 degrees C, and -196 degrees C) did not protect this activity. Acrosin-like activity decreased in the course of storage of milt on ice; 88% decline was recorded after 13 days. Acrosin-like activity increased with temperature from 10 degrees C to 30 degrees C, but was inactivated at 40 degrees C to about 40% as compared to the optimum temperature. Triton X-100 inhibited activity by 15% and 72% at 0.01% and 0.1% concentrations, respectively. Activity was not affected by Mg2+ but was inhibited by Zn2+ (30% and 75% in the presence of 0.1 mM and 1 mM, respectively). Maximum velocity of substrate hydrolysis was observed at 2 mM of BAPNA. Acrosin-like activity was effectively inhibited by 4'-acetamidophenyl 4-guanidinobenzoate (AGB), an inhibitor of mammalian acrosin. Sperm acrosin-like activity correlated negatively with antiproteinase activity of seminal plasma. We conclude that sturgeon acrosin-like activity shares many properties with mammalian acrosin. On the other hand, it has some unique properties which may represent adaptations of this enzyme to the environment of external fertilization.

The influence of cryopreservation on parameters of energetic metabolism and motility of fowl spermatozoa.

The objective of this study was to estimate the effect of cryopreservation on the main pathways of energetic metabolism and motility of fowl spermatozoa. Sperm diluted 1:5 with the cryoprotective medium containing ethylene glycol (1.4 M final concentration) was frozen at the rate of 2-3 degrees C/min to -25 degrees C with a pause on the plateau of crystallization and then at an exponentially increasing rate to -196 degrees C. The frozen sperm was thawed in two successive water baths at 0 and at 41 degrees C. After cryopreservation, the rate of radioactive glucose oxidation to 14CO2 slightly decreased, the rate of labeled glutamate oxidation remained unchanged, and the rate of labeled succinate oxidation increased two-fold. After freeze-thawing, the rates of endogenous respiration with and without 2,4-dinitrophenol decreased; the oxidation rate of exogenous succinate in the presence of 2,4-dinitrophenol, rotenone, and digitonin slightly decreased; and the rate of respiration in the presence of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, antimycin A, 2,4-dinitrophenol, and digitonin did not differ from that seen in control. Sperm respiration was highly sensitive to rotenone; antimycin A and cyanide blocked oxygen consumption completely. Succinate, added after 2,4-dinitrophenol and rotenone, stimulated respiration of thawed spermatozoa, which indicated plasma membrane damage. The addition of exogenous malate in the presence of 2,4-dinitrophenol and digitonin restored the respiration rate of thawed spermatozoa to that of unfrozen cells. The rate of respiration of thawed spermatozoa with oligomycin was higher than that of control cells.(ABSTRACT TRUNCATED AT 250 WORDS)