Improved hepatic and pancreatic localisation of the equine alpha-1-proteinase inhibitor family of serpins using an antigen enhancement technique and a monoclonal antibody

Equine alpha- l-proteinase inhibitor (API) consists of three, occasionally four, serum glycoproteins. This study investigated the immunohistochemical localisation of equine API in paraformaldehyde fixed, paraffin embedded equine tissue samples of liver, lung, stomach, pancreas, jejunum and colon in five horses using affinity purified sheep polyclonal and protein A purified mouse monoclonal antibodies, whose specificities were verified by Western blotting. Exposing tissue sections to boiling citrate buffer greatly enhanced antigen recovery and improved immunostaining with both antibodies, resulting in discovery of novel tissue distribution patterns for the horse. In the horses studied, all hepatocytes showed some degree of cytoplasmic staining, many having perinuclear intense granular inclusions. This finding is contrary to findings in human studies where hepatocytes of Pi MM phenotype have proven difficult to stain for human API, despite evidence at the molecular level suggesting hepatocytes as the major source of serum API. This discrepancy may be due to the use of different tissue fixation and antigen recovery techniques. In all other tissues examined, the distribution of equine API was similar to human studies.     

Determination of carbon tetrachloride in fumigated cereal grains during storage

Alternative methods for the extraction of unchanged carbon tetrachloride residues from fumigated whole and ground wheat and maize were examined and compared. Amounts of carbon tetrachloride extracted were determined by gas-liquid chromatography using two separate columns and an electron-capture detector. A portion of any carbon tetrachloride residue was found to be converted to chloroform by a steam distillation extraction method but not when a cold solvent extraction process was used. In addition, the effectiveness of removal of carbon tetrachloride from wheat and maize during a 3.5 h steam distillation was progressively lessened, in comparison with the cold extraction process, as the length of time that residual fumigant had been associated with the grain increased. The rate of elimination of carbon tetrachloride from wheat and maize during airing at two temperatures was determined and though partially dependent on the temperature of fumigation, airing was consistently more rapid at 25° than at 10°C. Initial residues of 200 to 400 parts/million in whole grains fell to 1 to 10 parts/million when aired for six months in thin layers at 25°C and to 5 to 50 parts/million at WC. Residues in wheat disappeared more rapidly than those in maize. Grinding initially caused a sharp reduction in carbon tetrachloride content but subsequent airing rates were little faster than those of the whole grains. It is concluded that complete elimination of trace amounts of carbon tetrachloride from products of treated grain is unlikely even after milling but the toxicological significance of such residues is uncertain.     

Determination of methyl bromide residues in stored foods using automated headspace gas chromatography

An automated gas chromatographic headspace method capable of detecting methyl bromide (MB) down to 0.01 mg kg^?1 has been developed and evaluated. The method is suitable for analysing cereals, nuts, seeds and dried fruit. Commodities are extracted with solvent and MB present is derivatised to methyl iodide (MI) which is determined by automated headspace gas chromatography. Results from the method described are compared with those using an established solvent extraction method and show good quantitative agreement. The analytical conditions recommended are likely to be suitable for examination of other commodities for MB residues.     

Kinetics of equine neutrophil elastase release and superoxide anion generation following secretagogue activation: a potential mechanism for antiproteinase inactivation

Man and horses both suffer from neutrophil mediated pulmonary diseases however there are striking species differences in the underlying pathology. In particular while pulmonary emphysema is a common pathological sequel to human respiratory disease it is not a major feature of the common equine neutrophil mediated condition, chronic obstructive pulmonary disease (COPD). The proposed reason for this difference is that equine neutrophils contain less elastase than equivalent human cells and therefore there is a reduced risk of excess and/or uninhibited elastase activity, which is considered the major cause of pulmonary emphysema in man, in the horse lung. In previous studies equine neutrophil elastase (ENE) has been assayed by measuring elastinolytic activity whereas human neutrophil elastase content has been determined using immunological techniques. Neutrophils contain several intracellular protease inhibitors therefore measurement of elastase activity may underestimate the total NE content. The aim of the current study was to develop immunological techniques to allow investigation of the cellular content, distribution and release of ENE from purified equine neutrophils. Equine neutrophil elastase 2A (ENE 2A), the most abundant elastase in equine neutrophils, and equine alpha-1-proteinase inhibitor (API), the main inhibitor of elastase were found to be present at 0.813 pg +/- 0.179 and 0.021 pg +/- 0.003 (mean +/- SEM, n = 11 individual horses) per neutrophil, respectively. This represents twice as much elastase as previously found in the equine neutrophil and a comparable amount to that reported in human neutrophils. Immunolocalisation demonstrated that ENE 2A has a granular distribution within the cytosol of neutrophils, whereas API exhibits a uniform non-granular cytoplasmic appearance. In addition the kinetics of simultaneous generation and release of superoxide anions (SOA) and release of ENE 2A from equine neutrophils, stimulated in vitro by zymosan-activated serum (ZAS) in the presence and absence of the cation chelator ethylene glycol-N,N,N',N'-tetraacetic acid (EGTA), showed a close relationship between total SOA generation and total ENE 2A release during the initial 90 min post-ZAS stimulation and the dependence of both events on extracellular cations. In conclusion these studies have shown that horse and human neutrophil elastase content and mediator release functions are more closely matched than was previously thought. This suggests that the species differences in pathology resulting from neutrophil-mediated respiratory disease are determined by other factors such as differences in the abundance and function of intra- and extra-cellular protease inhibitors.     

Canine dysautonomia: two clinical cases

Two clinical cases of canine dysautonomia are described. Two young female neutered dogs were presented with clinical signs including vomiting, diarrhoea, faecal tenesmus, dysphagia and urinary retention. Decreased tear production, dry mucous membranes, bilateral Horner's syndrome, decreased anal sphincter tone and gastrointestinal hypomotility were also observed. Presumptive diagnoses of dysautonomia were made based on the clinical presentation and investigations. Postmortem histopathological examination in one of the cases demonstrated marked depletion of neuronal cell bodies in the intestinal myenteric plexuses and parasympathetic ganglia, confirming the diagnosis in this case. Criteria for aiding the antemortem diagnosis of this rare condition based on clinical observations and diagnostic testing are proposed.     

Effectiveness of cold solvent extraction systems for the determination of fumigant residues in cereal grains

Two alternative cold solvent extraction mixtures, (a) light petroleum distillate (40–60°C boiling range) + 0.1-M hydrochloric acid (2+3 by volume) and (b) acetone + water (5+1 by volume), were examined for their effectiveness in extraction of residues of bromochloromethane, 1,1,1-trichloroethane, carbon tetrachloride and 1,1,2-trichloroethylene from cereal grains and rape seed. Apparent recoveries of fumigants from spiked solutions or spiked commodity extracts were generally in the range 90-105%, acetone + water giving the higher results. For most fumigated samples extracted shortly after treatment, comparative results obtained for the two extraction mixtures were similar to those obtained for spiked extracts and this, together with rapid rates of extraction, suggested that almost complete extraction from the commodities had been obtained. However, the rate of extraction of residues from aged samples using either solvent mixture was often slow, with extraction continuing well beyond the recommended standing periods. Agreement in results obtained was significantly improved by standing for longer periods, in order that equilibrium of residual compounds between commodity and solvent could be reached. However, low results were obtained using light petroleum distillate + hydrochloric acid to extract all four fumigants from rape seed and bromochloromethane from oats. It is recommended that the effectiveness of the solvent mixture selected for extraction of this type of volatile residue from cereal grains is carefully checked, especially when determining residues in aged samples or samples of unknown history.