Investigations on apocynin, a potent NADPH oxidase inhibitor

Polymorphonuclear neutrophils (PMNs) play a major role in inflammatory diseases. They act as a first line of defense against invading infectious microorganisms. For this purpose, PMNs contain granules filled with proteolytic and other cytotoxic enzymes. Besides releasing enzymes, PMNs are also able to phagocytose and to convert oxygen into highly reactive oxygen species (ROS). Following phagocytosis, ingested microorganisms may be killed inside the phagosome by a combined action of enzyme activity and ROS production. Although the formation of ROS by stimulated PMNs is a physiological response which is advantageous to the host, it can also be detrimental in many inflammatory states in which these radicals give rise to excessive tissue damage. Therefore, there is an ongoing search for anti-inflammatory compounds which are able to prevent this damaging ROS production without affecting the other killing capacities of the PMN. In 1971, the isolation of apocynin from the roots of Picrorhiza kurroa Royle ex Benth. was reported. Picrorhiza kurroa is a small, perennial plant growing at high altitudes in the western Himalayas and which has been used extensively for ages and is still in use in the Ayurvedic system of medicine in India and Sri Lanka. Following experiments showed that apocynin was a potent anti-inflammatory agent, based on the selective inhibition of the production of ROS by activated human PMNs. Although proven to be an active anti-inflammatory compound in several experimental animal models, the exact mechanism of action of apocynin was still not fully understood. In this thesis, experiments are described that have led to a better understanding of the mode of action by which apocynin inhibits the ROS production by activated human PMNs. One of the conclusions is that apocynin itself is not active, but that it is converted into an active dimer inside the phagosomes of activated PMNs

Expression of haem oxygenase-1 and inducible nitric oxide synthase in horses with lower airway inflammation

The heat shock protein haem oxygenase-1 (HO-1) and the enzyme inducible nitric oxide synthase (iNOS) are both induced during conditions of oxidative stress and by pro-inflammatory mediators. Both HO-1 and iNOS may therefore be induced by a range of disease states that result in tissue inflammation. Both have been demonstrated to have potentially beneficial anti-inflammatory effects. Both HO-1 and iNOS and their respective gaseous products carbon monoxide and nitric oxide have being the subject of investigation as potential therapeutic targets for human respiratory disease, most notably asthma. The pathogenesis of inflammatory diseases of the equine lower respiratory tract has many similarities with human asthma and both HO-1 and iNOS have been demonstrated in fixed equine pulmonary tissue. The aim of the study was to investigate the expression of HO-1 and iNOS in the equine respiratory tract. The primary objective was to investigate whether HO-1 and iNOS were expressed within leukocytes from equine BALF and to semi-quantify expression in different cell types. If it proved possible to characterise and quantify expression, additional aims were to investigate associations between HO-1 expression, iNOS expression, clinical signs of respiratory disease and markers of oxidative stress in exhaled breath condensate. We were successful in demonstrating the expression of both HO-1 and iNOS in leukocytes from equine BALF. Expression was most intense in macrophages and neutrophils which was consistent with previous studies performed in other species. We did not identify an association between the degree of HO-1 or iNOS staining in leukocytes from BALF and the severity of clinical respiratory disease. We also failed to identify an association between expression of HO-1 and iNOS in leukocytes from BALF and markers of oxidative stress in exhaled breath condensate. Further investigations ought to be performed into the expression and activity of both HO-1 and iNOS in the equine respiratory tract