Een EHV-1-infected, mock-infected or LPS-stimulated equine monocytes and procoagulant activity

Een EHV-1-infected, mock-infected or LPS-stimulated equine monocytes and procoagulant activity was not increased in sodium azide-treated monocytes despite substantial cell death, we could not attribute the increased procoagulant activity at 24 h after EHV-1 infection to cell death. Our results also indicate 3-Fluoro-2-(trifluoromethyl)aniline that cultured equine monocytes undergo spontaneous cell death, which increases from 4 to 24 h of culture, but was not enhanced by virus infection or LPS stimulation (10 ng/mL or 10 g/mL). Such spontaneous cell death has also been observed in human monocytes cultured in vitro [53,54]. Our results with LPS-treated equine monocytes differ from that previously reported with human monocytes, in which a lower dose of LPS (1 g/mL) induced more cell death than was observed in untreated controls [45]. This could be due to species differences in the response to LPS. The procoagulant response after LPS stimulation was also highest at 24 h versus 4? h after treatment, as reported previously for horse monocytes [36]. The latter conflicting results may be explained by different experimental methods, since a two-stage amidolytic assay was used to measure procoagulant activity on the surface of living cells in this study whereas a clotting assay was used to measure procoagulant activity in whole cell lysates of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8086425 equine monocytes in the previous study [36]. The data also revealed strain-dependent differences in generation of procoagulant activity in equine monocytes, with RacL11 consistently eliciting a stronger response than Ab4 or NY03. These strain-dependent differences were not attributable to the D752 polymorphism in the DNA polymerase. This polymorphism has been strongly associated with neuropathogenicity [25,27,32,33], possibly due to a leukocyte-associated viremia of higher magnitudeand longer duration [25,27] or more efficient infection of CD172-positive mononuclear cells after invasion through the respiratory mucosa [2,29,44]. Interestingly, the D752 polymorphism in the virus DNA polymerase did affect the maximal procoagulant response induced by RacL11 but not by Ab4. The reason for this is unclear but it 4-(Benzyloxy)-4-oxobutanoic acid is unlikely to be due to virus yields or differences in replication, since previous studies have shown that strains harboring the D752 or N752 polymorphism yield similar amounts of virus [25,26]. The result does, however, support our findings with UV-inactivated or aphidicolin-treated virus showing that virus replication contributes to procoagulant activity with EHV-1. The reason PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13485127 for the higher procoagulant activity with RacL11 is unknown, but similar results have been reported for chemokine gene expression patterns, with RacL11 causing higher levels of CCL2 and 3-b]pyrazine CCL3 mRNA in equine PBMC than NY03 or Ab4 after 24 h of infection [55]. Since the observed differences in infection efficiency between RacL11 and Ab4 were small in this study, other factors are likely causative, such as variations in gene products between the virus strains used. The rapid induction of procoagulant activity and TF mRNA transcription suggests that early events in virus infection, such as attachment, entry, intracellular trafficking or transcription of immediate-early genes [56], may be mediating the observed procoagulant responses. RacL11 differs from the other two strains by partial or complete deletions in open reading frames 1 and 2, that encode early gene products, and also has a large deletion in the early gene product, ICPO, a powerful transcriptional transactiv.