Expression of inducible nitric oxide synthase and formation of peroxynitrite in posttransplant obliterative bronchiolitis

BACKGROUND: Obliterative bronchiolitis is characterized histologically by inflammation, epithelial cell damage and loss, fibrosis, and eventual obliteration of airways. Production of high levels of the potential cytotoxin nitric oxide by inducible nitric oxide synthase has been implicated in several inflammatory diseases. The damaging effects of nitric oxide are mediated by peroxynitrite, are formed from nitric oxide and superoxide, and can be demonstrated by the detection of nitrotyrosine. Our previous finding of high inducible nitric oxide synthase expression in inflamed airway epithelium led us to hypothesize that release of nitric oxide in obliterative bronchiolitis mediates the characteristic epithelial damage. METHODS: Immunocytochemistry was carried out to seek expression of inducible nitric oxide synthase and nitrotyrosine in transplant samples from patients with obliterative bronchiolitis (n=10) and, as controls, unused donor lungs (n=5). RESULTS: Inducible nitric oxide synthase was strongly expressed in the damaged airway epithelium in obliterative bronchiolitis and in inflammatory cells, where its distribution was matched by that of nitrotyrosine. Normal controls showed little or no immunoreactivity for any of the antigens studied. CONCLUSIONS: Our findings suggest that nitric oxide may play a role in the pathogenesis of obliterative bronchiolitis and indicate that further work is essential to fully understand the processes and mechanisms involved

Increased expression of inducible nitric oxide synthase and cyclo-oxygenase-2 in the airway epithelium of asthmatic subjects and regulation by corticosteroid treatment

Background: Nitric oxide (NO) and prostanoids are mediators of vascular and bronchial tone that are postulated to be involved in asthma. Increased levels of both are found in asthmatic subjects and are synthesised by enzymes that have cytokine inducible forms: inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2), respectively. We hypothesised that the in vivo expression of iNOS and COX-2 in the airways would be increased in asthma, and that these cytokine inducible enzymes may represent targets for regulation by corticosteroid treatment.Methods: Bronchial biopsy specimens were obtained from three groups of subjects: atopic asthmatics treated with beta 2 agonists alone (n=7), atopic asthmatics additionally receiving regular treatment with corticosteroids (n=8), and non-asthmatic control subjects (n=10). Expression of iNOS and COX-2 mRNA and immunoreactive protein was studied using in situ hybridisation and quantitative immunohistochemistry.Results: Immunoreactivity and the hybridisation signal for iNOS and COX-2 were mainly localised in the airway epithelium. The proportion of epithelium immunostained was significantly greater in the non-steroid treated asthmatic subjects (iNOS 8.6 (1.8)%; COX-2 26.3 (4.6)%) than either the steroid treated asthmatics (iNOS 3.4 (1.0)%, p=0.009; COX-2 13.0 (0.6)%, p=0.0015) or the non-asthmatic controls (iNOS 4.2 (0.9)%, p=0.018; COX-2 11.6 (0.6)%, p=0.0003). Similarly, the hybridisation signal was stronger in the non-steroid treated group of asthmatic subjects than in the other two groups.Conclusions: These findings highlight the potential role of the airway epithelium both as a contributor to the inflammatory process in asthma and as a target for inhaled corticosteroid treatment in this disease

Simultaneous immunostaining method for localization of bromodeoxyuridine and calcitonin gene-related peptide

A new simultaneous double immunostaining method has been optimized to localize the DNA synthesis marker bromodeoxyuridine (BrdU) and calcitonin gene-related peptide (CGRP) in endocrine cells of Bouin's-fixed, paraffin-embedded rat lung. Nuclease pre-treatment before immunostaining is compatible with optimal tissue morphology and CGRP antigenicity preservation. Nickel-enhanced development of avidin-biotin-peroxidase staining is used to show CGRP immunoreactivity in black and alkaline phosphatase-anti-alkaline phosphatase is applied to demonstrate incorporated BrdU in red. The present methodology could be useful for studies requiring detection of incorporated BrdU in cells producing regulatory peptides or other labile antigens

Endothelin-like immunoreactivity in midgut endocrine cells of the desert locust, Locusta migratoria

Endothelin-1-like immunoreactivity has been found in endocrine cells of the midgut of the desert locust Locusta migratoria. Several antisera have been directed against the whole molecule and its C-terminal sequence. Endothelin-1-immunoreactive cells are present in the main region of the midgut (ventriculus) and in the midgut caeca but not in the ampullae through which the malpighian tubules drain. Endothelin-1-like immunoreactivity colocalizes with FMRFa immunoreactivity in the cells of the main region of the midgut but not in those in the midgut caeca. Endothelin-1-immunoreactive cells are present not only in adults but also throughout the five instars of posthatching development

Endothelin-like immunoreactivity in midgut endocrine cells of the desert locust, Locusta migratoria

Endothelin-1-like immunoreactivity has been found in endocrine cells of the midgut of the desert locust Locusta migratoria. Several antisera have been directed against the whole molecule and its C-terminal sequence. Endothelin-1-immunoreactive cells are present in the main region of the midgut (ventriculus) and in the midgut caeca but not in the ampullae through which the malpighian tubules drain. Endothelin-1-like immunoreactivity colocalizes with FMRFa immunoreactivity in the cells of the main region of the midgut but not in those in the midgut caeca. Endothelin-1-immunoreactive cells are present not only in adults but also throughout the five instars of posthatching development

Immunocytochemical localization of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in human endocrine pancreas

We studied the distribution of the enzymes that are involved in the post-translational alpha-amidation of regulatory peptides in human endocrine pancreas, using immunocytochemical methods for light and electron microscopy. Immunoreactivity for the two enzymes involved, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), was located in the periphery of the islets of Langerhans and in ductal endocrine cells. Staining of reverse-face serial sections demonstrated that these immunoreactivities co-localize with glucagon but not with pancreatic polypeptide (PP), insulin, or somatostatin. Double immunogold staining for electron microscopy confirmed the previous results and revealed a different localization for each enzyme inside the secretory granule: PHM is present in the central core of the glucagon-containing granules, whereas PAL is predominantly located near the granule membrane. The existence of an amidated peptide, GLP1, in the A-cells explains the presence of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in these cells. The absence of the enzymes in the PR-cells raises the possibility that a different form of amidating enzyme may be involved in the post-translational processing of this peptide