Activation and modulation of antiviral and apoptotic genes in pigs infected with classical swine fever viruses of high, moderate or low virulence

The immune response to CSFV and the strategies of this virus to evade and suppress the pigs’ immune system are still poorly understood. Therefore, we investigated the transcriptional response in the tonsils, median retropharyngeal lymph node (MRLN), and spleen of pigs infected with CSFV strains of similar origin with high, moderate, and low virulence. Using a porcine spleen/intestinal cDNA microarray, expression levels in RNA pools prepared from infected tissue at 3 dpi (three pigs per virus strain) were compared to levels in pools prepared from uninfected homologue tissues (nine pigs). A total of 44 genes were found to be differentially expressed. The genes were functionally clustered in six groups: innate and adaptive immune response, interferon-regulated genes, apoptosis, ubiquitin-mediated proteolysis, oxidative phosphorylation and cytoskeleton. Significant up-regulation of three IFN-γ-induced genes in the MRLNs of pigs infected with the low virulence strain was the only clear qualitative difference in gene expression observed between the strains with high, moderate and low virulence. Real-time PCR analysis of four response genes in all individual samples largely confirmed the microarray data at 3 dpi. Additional PCR analysis of infected tonsil, MRLN, and spleen samples collected at 7 and 10 dpi indicated that the strong induction of expression of the antiviral response genes chemokine CXCL10 and 2′–5′ oligoadenylate synthetase 2, and of the TNF-related apoptosis-inducing ligand (TRAIL) gene at 3 dpi, decreased to lower levels at 7 and 10 dpi. For the highly and moderately virulent strains, this decrease in antiviral and apoptotic gene expression coincided with higher levels of virus in these immune tissues

Microcalcification after excitotoxicity is enhanced in transgenic mice expressing parvalbumin in all neurones, may commence in neuronal mitochondria and undergoes structural modifications over time

AIMS: Parenchymal microcalcification in the brain coincides with neurodegenerative diseases, but is also frequently found in neurologically normal individuals. The origin and role of this process are still under debate. Parvalbumin (PV) is a protein acting as a Ca(2+) buffer and Ca(2+) shuttle towards intracellular Ca(2+) sinks, like mitochondria and the endoplasmic reticulum. Constitutively, it is present in a subset of inhibitory neurones. In transgenic mice expressing pan-neuronal PV, the mitochondrial volume is reduced. We tested whether elevated levels of intraneuronal [Ca(2+)] and reduced mitochondrial volume in the neurone interfere with the generation of parenchymal microcalcification. METHODS: The striatum of wild type and transgenic mice was injected with the glutamate receptor agonist ibotenic acid (IBO), which is known to induce not only excitotoxic neurodegeneration, but also parenchymal calcification. Sections were studied by light and electron microscopy at various time points after IBO application. RESULTS: Morphometric analysis 2, 4 and 20 weeks after IBO application revealed microcalcification in transgenic and wild type mice; the calcification process, however, was enhanced and accelerated in the transgenic animals. Ultrastructural analyses suggest neuronal mitochondria as the nucleators of the deposits which consist of hydroxyapatite. The time-dependent changes in size and surface structure of the deposits indicate the presence of biological mechanisms in the brain promoting regression of bioapatites. CONCLUSIONS: The overload of intraneuronal [Ca(2+)] in combination with impaired mitochondrial function activates neuronal microcalcification. It is hypothesized that this process is an alternative/adaptive mechanism of the neurone to reduce further brain damage

Preservation of transendothelial glucose transporter 1 and P-glycoprotein transporters in a cortical slice culture model of the blood-brain barrier

A variety of neurological diseases are characterized by disturbances of the blood-brain barrier (BBB) and its transporters. We recently introduced fibroblast growth factor treated cortical organotypic slice cultures of mice as a model for in vitro studies of the blood-brain barrier and have now further characterized the maintenance and function of transport-proteins typically expressed in the endothelium of cerebral blood vessels. The glucose transporter GLUT-1 is present in blood vessels of slice cultures derived from postnatal day 4 to 21 mice after 3 days in vitro. The endothelial multidrug resistance P-glycoprotein (P-gp) which is involved in the control of pharmacological substance transport across the blood-brain barrier is also maintained in blood vessels, most prominently in slice cultures derived from postnatal day 14 and 21 mice. To assess P-gp function, we tested rhodamine 123 transport in presence or absence of the P-gp inhibitor verapamil. Rhodamine 123-fluorescence accumulated rapidly in the vascular lumen both in acute slices and in slices cultured for 3 days in vitro. Our results provide evidence that endothelial transporters and their functional properties can be maintained in organotypic cortical slices cultures, thus making it an attractive model system for the study of the blood-brain barrier