R. Wes Leid

Aggregation of equine platelets by PAF (platelet-activating factor)

Platelet-activating factor (PAF), a lipid released as a result of immediate allergic reactions from basophils and mast cells as well as by a variety of other cell types and stimuli, is one of the most potent platelet agonists and hypotensive agents known. Equine platelets stimulated over a wide range of PAF concentrations aggregated in a time- and dose-dependent manner. Maximum aggregation was observed at concentrations of PAF as low as 3.58×10−14 M with platelet-rich plasma (PRP) and 3.58×10−16 M with washed platelets. Furthermore, the aggregation observed did not appear to be breed-dependent. Finally, the platelet arachidonate pathway appeared to play no role in PAF-induced aggregation as exogenous arachidonate did not enhance the reaction, nor were equine platelets pretreated with 38 μM aspirin inhibited in their response to PAF. This level of aspirin totally inhibited the equine platelet aggregation response to arachidonate.

A superoxide dismutase of metacestodes of Taenia taeniaeformis

Superoxide dismutase was purified from Taenia taeniaeformis metacestodes by sequential ion exchange chromatography on quaternary-amino-ethyl-cellulose, gel filtration chromatography on ACA 44 and ion exchange chromatography on DEAE-cellulose, followed by chromatofocusing on polybuffer exchanger 94. This isolation procedure resulted in the detection of a single protein-staining band on alkaline gels, coincident with enzyme activity. We have, however, detected what appear to be two peaks of enzyme activity within this single protein-staining band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis using gradient slab gels and analysis under reducing conditions, resulted in the detection of only one protein at an apparent Mr of 16,600, while analysis under non-reducing conditions, gave a single protein of an apparent Mr of 64,000. The isoelectric point of the purified protein is 6.6. Boiling for 3 min completely destroyed the enzyme, whereas incubation for 2 h at 37 degrees C resulted in the loss of 56% of the enzymic activity. Incubation with 10 mM KCN resulted in 83% inhibition of the enzyme. We have detected up to 168 U ml-1 of enzyme activity in the cyst fluid surrounding the parasite in situ. This is the first instance in which any parasite superoxide dismutase has been purified to apparent homogeneity. Parasite-mediated enzymic destruction of superoxide anion can not only protect against oxygen toxicity as a result of normal parasite respiratory processes but also may serve as yet another mechanism used by tissue-dwelling parasites to evade host immunologic attack.

Parasite defense mechanisms for evasion of host attack; A review

This review covers some of the basic mechanisms whereby parasites evade host responses. These mechanisms include; antigenic variation, repeated antigenic determinants, induction of suppressor cells, acquisition of host proteins or molecular mimicry, proteinase destruction of host effector molecules, proteinase inhibitor-mediated inhibition of humoral and cellular immune effector arms and immunosuppressive products of parasite arachidonic acid metabolism. Vet. Parasitol.

The susceptibility of the goat to Fasciola hepatica infections

Mixed breed goats were infected with metacercariae of Fasciola hepatica and the resulting worm burdens were quantitated after primary and secondary exposure of the goats to the parasite. Mean length and width of the parasite recovered after all primary exposures were 1.91 +/- 0.2 cm and 0.91 +/- 0.2 cm, respectively. A mean of 71.8 +/- 5.9% of the flukes were recovered from all of the primary infections. In the secondary infections, the mean length and width of the flukes from the physically smaller population was 0.88 +/- 0.27 cm and 0.53 +/- 0.19 cm, respectively. A mean of 67 +/- 6.7% of the flukes were recovered from this secondary infection. It appears that the goat is susceptible to challenge infections with F. hepatica and that its response to this infection is much like that of sheep.

Isolation and partial characterization of a Taenia taeniaeformis metacestode proteinase inhibitor

Abstract Suquet C. , Green-Edwards C. and Wes Leid R. 1984. Isolation and partial characterization of a Taenia taeniaeformis metacestode proteinase inhibitor. International Journal for Parasitology 14 : 165–172. A proteinase inhibitor from the metacestode of Taenia taeniaeformis was purified 136-fold to apparent homogeneity as evidenced by one Coomassie Blue protein staining band on 10% SDS slab gels under both reducing and non-reducing conditions. The apparent molecular weight under dissociating conditions was 19,500. This parasite protein inhibited esterolysis of TAME and BTEE by bovine pancreatic trypsin and chymotrypsin respectively in a time and dose-dependent manner. Proteolysis of casein by both enzymes was also inhibited in a time and dose-dependent manner. The parasite inhibitor was stable from pH 2.2 to 10.5 and was fully active after heating at 56 °C for 3 h. The proteases pronase and thermolysin, at concentrations of 1 mg ml −1 , completely inactivated the metacestode inhibitor. Two sulfhydryl proteases, papain and chymopapain, used at concentrations of 1 mg ml −1 were without effect. The irreversible proteinase inhibitors TLCK, TPCK and PMSF at concentrations up to 10 mM had no effect on the parasite inhibitor.

Stimulation of arachidonic acid metabolism in silica-exposed alveolar macrophages

The molecular events involved in both the initiation and development of silicosis are at present poorly defined, although mediators released from macrophages exposed to silica particles are believed to play a role. We have investigated the in vitro production of arachidonic acid (AA) metabolites in adherent bovine alveolar macrophages (BAM) incubated with crystalline silica. BAM were prelabeled with 3H-AA and incubated with 0.5-5.0 mg silica. Lipid metabolites released into the culture medium were analyzed by high-performance liquid chromatography. Simultaneously, lactate dehydrogenase (LDH) was assayed to provide an indication of cell injury. No 5-lipoxygenase metabolites were detected at the lowest silica dose tested (0.5 mg/well), but 5-hydroxyeicosatetraenoic acid (5-HETE) was the major AA metabolite detected between 1.5 and 5.0 mg of silica. A fivefold increase in the production of leukotriene B4 (LTB4) and its two nonenzymatic diastereomers (Isomers I and II) was observed as the silica concentration was increased from 1.0 to 5.0 mg. In contrast, the release of cyclooxygenase products declined with increasing concentrations of silica. LDH release increased in a linear, dose-dependent fashion in the range of silica doses used. The kinetics of eicosanoid release was investigated over a 3-h interval and LDH release was assayed for each time point. Within 15 min following silica addition, a shift to the production of 5-lipoxygenase metabolites was observed, accompanied by a reduction in cyclooxygenase products. This rapid alteration in AA metabolism preceded cell injury as measured by LDH release. These results demonstrate that silica is a powerful stimulator of arachidonic acid metabolism in BAM. Moreover, silica selectively stimulates the 5-lipoxygenase pathway as the dose of silica increases. Our results suggest that dysfunction in arachidonate metabolism could contribute to the pathogenesis of silicosis.

The Effects of Different Silicas on Arachidonic Acid Metabolism in Alveolar Macrophages

Bovine alveolar macrophages (BAM) prelabeled with 3H-arachidonic acid (AA) were exposed in vitro to different doses of DQ-12, Minusil-5, and Sigma silicas, or carbonyl iron beads. Arachidonic acid metabolites released into the culture medium by BAM were identified and quantitated using high performance liquid chromatography (HPLC). Cytotoxicity was assessed by the release of lactate dehydrogenase (LDH). At doses of 0.1 or 0.25 mg of DQ-12 silica and of 0.25 or 0.5 mg of Minusil-5 and Sigma silica, the release of cyclooxygenase metabolites (TXB2, PGE2, PGF2, and HHT) comprised greater than 95% of the total released AA metabolites. Silica doses above 0.5 mg led to 5-lipoxygenase metabolite release (LTB4, its two nonenzymatic isomers, and 5-HETE). This shift to 5-lipoxygenase metabolite release paralleled increased cellular cytotoxicity and was observed for each of the silicas. In contrast to silica stimulation, carbonyl iron beads elicited only small quantities of cyclooxygenase metabolites, no 5-lipoxygenase metabolites, and showed little cytotoxicity toward BAM. The relative potency of each particulate for stimulating the release of AA metabolites and LDH was calculated with DQ-12 greater than Minusil-5 greater than Sigma much greater than carbonyl iron beads. Our results indicate that the cytotoxic and presumed fibrogenic potential of a silica may be correlated with the potency to stimulate the release of 5-lipoxygenase metabolites from AM.

Thromboxane A2 generation by the larval cestode Taenia taeniaeformis

The larval stage of the tapeworm, Taenia taeniaeformis, was incubated in different concentrations of arachidonic acid. At various times after incubation, aliquots were removed for bioassay on either 38 microM aspirin or non-aspirin-treated equine platelets and by radioimmunoassay for the thromboxane A2 breakdown product, thromboxane B2 (TXB2). Platelet agonist activity was detected as early as 30 sec after addition of the arachidonic acid. In aspirin-treated platelets, this agonist activity peaked at 1 to 4 min and thereafter decayed rapidly with a time course that was both worm and arachidonic acid dependent. When assayed on non-aspirin-treated platelets the agonist activity was again detectable as early as 30 sec after addition of arachidonic acid, peaked at 1 to 3 min and then decayed very slowly over a 30-min period of incubation. It was found that levels of TXB2 were detected which increased over time concomitant with the decay of the platelet agonist activity. The most consistent detection of TXB2 generation was at 30 min with a mean of 49.6 pg and a range of 22.1 to 84.8 pg for four experiments. This report presents the first evidence for arachidonic acid utilization by a cestode or trematode and could in part provide an explanation for the marked cellular inflammation noted around dead or dying parasites.

A review of the antigens of Fasciola hepatica

The interaction between the antigens of Fasciola hepatica and the host immune response are reviewed. This paper evaluates not only more recent work, but the older literature as well. Antigens from each stage in the life cycle are considered with the idea of identifying those antigens with a potential for use in an effective vaccine. Antigens which cross-react with other parasite species are detailed as well as those that cross-react between different stages in the life cycle of F. hepatica. The objective of the review is to demonstrate for other investigators that vaccination against F. hepatica is a distinct possibility. We hope to encourage more investigators to initiate work on this aspect of an economically-important cosmopolitan parasite.

Arachidonic acid metabolism in bovine alveolar macrophages: effect of calcium ionophore on lipoxygenase products

The production of lipoxygenase metabolites of arachidonic acid was studied in bovine alveolar macrophages (BAM). Unstimulated macrophages produced small amounts of LTB4 (0.2±0.2 ng/106 BAM) but monohydroxyeicosatetraenoic acids (5t-, 12-, and 15-HETE) usually were not detectable. Both exogenous arachidonic acid and the calcium ionophore A23187 induced production of LTB4, 5-, 12-, and 15-HETE, of which 60–80% was 5-HETE. Combined challenge of BAM with both exogenous arachidonic acid and A23187 was more effective in the production of these metabolites than with either stimulus alone. The generation of the peptidoleukotrienes LTC4, LTD4, and LTE4 by BAM could not be detected under these in vitro conditions. Our results demonstrate that bovine alveolar macrophages produce similar lipoxygenase metabolites of arachidonic acid in response to A23187, as do human alveolar macrophages stimulated with the same agonist.