Jelena Ostojić

Chicken heterophil extracellular traps (HETs): Novel defense mechanism of chicken heterophils

Recent findings in mammals and fish have revealed that neutrophil nuclear material associated with cytoplasmic granular content is released in the form of neutrophil extracellular traps (NETs) that can trap and kill invading microorganisms in vitro and in vivo. To determine if a similar mechanism is present in chicken heterophils, hydrogen peroxide (H(2)O(2)) and phorbol myristate acetate (PMA) were used for stimulation of blood-derived heterophils. Stimulated heterophils produced structures that were characterized using immunocytochemistry and confocal microscopy as heterophil extracellular traps (HETs). Released HETs contained DNA, histone-DNA complex and elastase from heterophil cytoplasmic granules. HETs released from chicken heterophils are structurally similar to NETs found in mammalian and fish neutrophils. Extracellular DNA released from heterophils was quantified by Picogreen assay. Stimulation with PMA or H(2)O(2) significantly increased the HET-DNA release index in vitro compared to non-stimulated heterophils (1.11+/-0.04 and 1.55+/-0.10, respectively), and H(2)O(2) stimulation induced significantly higher HET-DNA release than PMA (P<0.001). Thus, HETs are now characterized as an important heterophil-mediated defense mechanism in chickens.

Patterns of Distribution of Oxygen-Binding Globins, Neuroglobin and Cytoglobin in Human Retina

OBJECTIVE To determine the distribution of 2 intracellular oxygen-carrying molecules, neuroglobin (NGB) and cytoglobin (CYGB), in specific retinal cell types of human retinas. METHODS Specific antibodies against NGB and CYGB were used in immunohistochemical studies to examine their distribution patterns in human retinal sections. Double-labeling studies were performed with the anti-NGB and anti-CYGB antibodies along with antibodies against neuronal (microtubule-associated protein 2, class III beta-tubulin [TUJ1], protein kinase C alpha, calretinin) and glial (vimentin, glial fibrillary acid protein) markers. Confocal microscopy was used to examine the retinal sections. RESULTS Immunohistochemical analysis of human retinal tissue showed NGB and CYGB immunoreactivity in the ganglion cell layer, inner nuclear layer, inner and outer plexiform layers, and retinal pigment epithelium. Neuroglobin immunoreactivity was also present in the outer nuclear layer and photoreceptor inner segments. Neuroglobin and CYGB were coexpressed in the neurons in the ganglion cell layer and inner nuclear layer but not within glial cells. CONCLUSION Neuroglobin and CYGB are colocalized within human retinal neurons and retinal pigment epithelium but not within glial cells. Clinical Relevance Our results suggest that NGB and CYGB may serve a neuroprotective role as scavengers of reactive oxygen species and therefore should be considered when developing therapeutic strategies for treatment of hypoxia-related ocular diseases.

Neuroglobin and Cytoglobin Distribution in the Anterior Eye Segment: A Comparative Immunohistochemical Study

This study provides a detailed description of immunolocalization of two oxygen-binding proteins, neuroglobin (Ngb) and cytoglobin (Cygb), in the anterior segment of healthy human and canine eyes. Specific antibodies against Ngb and Cygb were used to examine their distribution patterns in anterior segment structures including the cornea, iris, trabecular meshwork, canal of Schlemm, ciliary body, and lens. Patterns of immunoreactivity (IR) were imaged with confocal scanning laser and conventional microscopy. Analysis of sectioned human and canine eyes showed Ngb and Cygb IR in the corneal epithelium and endothelium. In the iris, Ngb and Cygb IR was localized to the anterior border and the stroma, iridal sphincter, and dilator muscle. In the iridocorneal angle, Ngb and Cygb were detected in endothelial cells of the trabecular meshwork and canal of Schlemm in human. In the ciliary body, Ngb and Cygb IR was localized to the non-pigmented ciliary epithelium of the pars plana and pars plicata and in ciliary body musculature. Ngb and Cygb distribution was similar and colocalized within the same structures of healthy human and canine anterior eye segments. Based on their immunolocalization and previously reported biochemical features, we hypothesize that Ngb and Cygb may function as scavengers of reactive oxygen species. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.