Modulation of cholesterol-related sterols during Eimeria bovis macromeront formation and impact of selected oxysterols on parasite development.

Obligate intracellular apicomplexan parasites are considered as deficient in cholesterol biosynthesis and scavenge cholesterol from their host cell in a parasite-specific manner. Compared to fast proliferating apicomplexan species producing low numbers of merozoites per host cell, (e. g. Toxoplasma gondii), the macromeront-forming protozoa Eimeria bovis is in extraordinary need for cholesterol for offspring production (≥ 170,000 merozoites I/macromeront). Interestingly, optimized in vitro E. bovis merozoite I production occurs under low foetal calf serum (FCS, 1.2%) supplementation. To analyze the impact of extensive E. bovis proliferation on host cellular sterol metabolism we here compared the sterol profiles of E. bovis-infected primary endothelial host cells grown under optimized (1.2% FCS) and non-optimized (10% FCS) cell culture conditions. Therefore, several sterols indicating endogenous de novo cholesterol synthesis, cholesterol conversion and sterol uptake (phytosterols) were analyzed via GC-MS-based approaches. Overall, significantly enhanced levels of phytosterols were detected in both FCS conditions indicating infection-triggered sterol uptake from extracellular sources as a major pathway of sterol acquisition. Interestingly, a simultaneous induction of endogenous cholesterol synthesis based on increased levels of distinct cholesterol precursors was only observed in case of optimized parasite proliferation indicating a parasite proliferation-dependent effect. Considering side-chain oxysterols, 25 hydroxycholesterol levels were selectively found increased in E. bovis-infected host cells, while 24 hydroxycholesterol and 27 hydroxycholesterol contents were not significantly altered by infection. Exogenous treatments with 25 hydroxycholesterol, 27 hydroxycholesterol, and 7 ketocholesterol revealed significant adverse effects on E. bovis intracellular development. Thus, the number and size of developing macromeronts and merozoite I production was significantly reduced indicating that these oxysterols bear direct or indirect antiparasitic properties. Overall, the current data indicate parasite-driven changes in the host cellular sterol profile reflecting the huge demand of E. bovis for cholesterol during macromeront formation and its versatility in the acquisition of cholesterol sources.

2-Aminoethoxydiphenyl borate (2-APB) reduces alkaline phosphatase release, CD63 expression, F-actin polymerization and chemotaxis without affecting the phagocytosis activity in bovine neutrophils.

2-Aminoethoxydiphenyl borate (2-APB) interferes with the Ca(2+) influx and reduces the ROS production, gelatinase secretion and CD11b expression in bovine neutrophils. Moreover, it has been suggested that inhibition of the Ca(2+) channel involved in the store operated Ca(2+) entry (SOCE) is a potential target for the development of new anti-inflammatory drugs in cattle, however it is unknown whether 2-APB affects neutrophil functions associated with the innate immune response. This study describes the effect of 2-APB, a putative SOCE inhibitor, on alkaline phosphatase activity a marker of secretory vesicles, CD63 a marker for azurophil granules, F-actin polymerization and in vitro chemotaxis in bovine neutrophils stimulated with platelet-activating factor (PAF). Also, we evaluated the effect of 2-APB in the phagocytic activity against Escherichia coli and Staphylococcus aureus bioparticles. We observed that doses of 2-APB ≥10 µM significantly reduced alkaline phosphatase activity and in vitro chemotaxis, whereas concentrations of 2-APB ≥50 µM reduced CD63 expression and F-actin polymerization. Finally, we observed that 2-APB did not affect the phagocytic activity in neutrophils incubated with E. coli and S. aureus bioparticles. We concluded that inhibition of Ca(2+) influx could be a useful strategy to reduce inflammatory process in cattle.

Ultrastructural localization of fructose-1,6-bisphosphatase in mouse brain.

Fructose-1,6-bisphosphatase has been studied in adult mouse brain of different ages using an antibody directed against the liver isoform. The presence of liver fructose-1,6-bisphosphatase in cerebellum, cerebral cortex, and hippocampus was assayed using Western blot and different immunocytochemical techniques. Immunocytochemistry with peroxidase reaction product was used to locate fructose-1,6-bisphosphatase in both neurons and astrocytes in the same areas, as well as in the rest of the brain, at light and electron microscope levels. Double immunofluorescence with neuronal or astrocytic markers confirmed the neuronal and astrocytic location of fructose-1,6-bisphosphatase in confocal microscope images. At the subcellular level, fructose-1,6-bisphosphatase was located in the nuclear and cytoplasmic compartments of both neurons and astrocytes, at all ages studied. Ultrastructurally, immunostaining appeared as small patches in the nucleus and the cytosol. In addition, immunostaining was present over the outer mitochondrial membrane, the plasma membrane, and the membranes of the rough endoplasmic reticulum and nuclear envelope, but not over Golgi membranes. In the neuropil fructose-1,6-bisphosphatase was located in dendritic spines, as well as in abundant astrocytic processes that, in some cases, surrounded immunopositive synapses. The possible role of fructose-1,6-bisphosphatase in neurons and astrocytes is discussed.