Danièle Zorzi

Lymphoid cell apoptosis induced by trophoblastic cells: a model of active foeto-placental tolerance

To test the hypothesis that CD95-L (Fas-L) present on trophoblastic cells plays a part in establishing foeto-placental tolerance by inducing apoptosis of immune defence cells, we cocultured trophoblasts with lymphoid cells and scored the frequency of cell death in these cultures. We prepared human trophoblastic cells from term placentas removed by C-section and placed them in culture for 48 h before introducing the lymphoid cells. We added Jurkat cells, a CD3 + lymphoid cell line, or purified T cells from human blood to the cultured trophoblasts and monitored apoptosis by electron microscopy and flow cytometry after TUNEL or annexin V labelling. The frequency of cell death in the CD3 + cell population was higher when the lymphoid cells were cocultured with trophoblastic cells than when they were cultured alone. This frequency increased with time but was reduced when anti-CD95-L antibodies were added to the culture medium. Cell death was less frequent in the lymphoid cell population when trophoblasts were replaced with human fibroblasts not expressing CD95-L.

Immuno‐Quantitative Polymerase Chain Reaction for Detection and Quantitation of Prion Protein

Abstract Immuno‐polymerase chain reaction (PCR) is an extremely sensitive detection method, combining the specificity of antibody detection and the sensitivity of PCR. We have developed an immuno‐quantitative PCR (iqPCR), exploiting real‐time PCR technology, in order to improve this immuno‐detection method and make it quantitative. To illustrate the advantages of iqPCR, we have compared it with a conventional enzyme linked immuno sorbent assay (ELISA) technique in experiments aimed at detecting the cellular and the resistant form of prion protein in bovine brain extract. The iqPCR technique proved to be more sensitive than ELISA, so it could be a technique of choice for the diagnosis of infected animals both at an ante mortem and post‐mortem stage.

Protective effect of prion protein via the N-terminal region in mediating a protective effect on paraquat-induced oxidative injury in neuronal cells.

Transmissible spongiform encephalopathies are a group of neurodegenerative disorders caused by a posttranslational, conformational change in the cellular isoform of the prion protein (PrPC) into an infectious, disease‐associated form (PrPSc). Increasing evidence supports a role for PrPC in the cellular response to oxidative stress. We investigated the effect of oxidative stress mediated by paraquat exposure on SH‐SY5Y neuroblastoma cells. A loss of mitochondrial membrane potential and subsequent reduction in ATP production were demonstrated in untransfected SH‐SY5Y cells, an effect that was ameliorated by the expression of PrPC. Cells expressing either PrP‐ΔOct, which lacks the octapeptide repeats, or PrP‐DA, in which the N‐terminus is tethered to the membrane, showed increased sensitivity to paraquat compared with cells expressing wild‐type PrPC as shown by reduced viability, loss of their membrane integrity, and reduced mitochondrial bioenergetic measurements. Exposure of prion‐infected mouse SMB15S cells to paraquat resulted in a reduction in viability to levels similar to those seen in the untransfected SH‐SY5Y cells. However, “curing” the cells with pentosan sulfate restored the viability to the level observed in the SH‐SY5Y cells expressing PrPC. These data would indicate that the molecular mechanism promoting cellular resistance to oxidative stress had been compromised in the infected SMB15S cells, which could be reinstated upon curing. Our study supports the hypothesis that PrPC expression protects cells against paraquat‐induced oxidative injury, demonstrates the significance of the N‐terminal region of the protein in mediating this protective effect, and also shows that the biochemical consequences of prion infection may be reversed with therapeutic intervention.

Study on the toxic mechanism of prion protein peptide 106–126 in neuronal and non neuronal cells

A synthetic peptide corresponding to the 106–126 amyloidogenic region of the cellular human prion protein (PrPc) is useful for in vitro study of prion‐induced neuronal cell death. The aim of the present work was to examine the implication of the cellular prion protein in the toxicity mechanism induced by PrP 106–126. The effect of PrP 106–126 was investigated both on human neuroblastoma SH‐SY5Y cells and on SH‐SY5Y overexpressing murine cellular prions (wtPrP). We show by metabolic assay tests and ATP assays that PrPc expression does not modulate the toxicity of the prion peptide. Moreover, we investigated the effect of this peptide on an established non neuronal model, rabbit kidney epithelial A74 cells that express a doxycycline‐inducible murine PrPc gene. We show for the first time that the prion peptide 106–126 does not exert any toxic effect on this cell line in the presence or absence of doxycycline. Our results show that the PrP 106–126‐induced cell alteration is independent of PrPc expression. Rather, it seems to act via an interaction with lipidic components of the plasma membrane as strengthened by our results showing the differential susceptibility of neuronal and non neuronal cell lines that significantly differ by their membrane fatty acid composition.

Close interactions between sympathetic neural fibres and follicular dendritic cells network are not altered in Peyer’s patches and spleen of C57BL/6 mice during the preclinical stage of 139A scrapie infection

During preclinical stage of prion diseases, secondary lymphoid organs seem to play an important role in prion amplification prior the invasion of the associated peripheral nervous system. In mice, it was shown that the relative positioning of follicular dendritic cells (FDC) and sympathetic nervous system (SNS) affects the velocity of neuroinvasion following scrapie inoculation. In this study, we checked if scrapie infection, by oral or intraperitoneal route, could influence this neuroimmune interface between FDC and tyrosine hydroxylase (TH) positive neural fibres within Peyers patches (PP) and spleen of the C57BL/6 mouse strain. We concluded that, in vivo, scrapie 139A and ME7 strains do not modify FDC-SNS neuroimmune interface. However, age seems to alter this neuroimmune interface and thus could influence the neuroinvasion in prion pathogenesis.

Detection of biomarkers of pathogenic bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In recent years, various mass-spectrometry procedures have been developed for identifying bacteria. The accuracy and speed with which data can be obtained by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry (MALDI-TOF MS) make this an advantageous technique for environmental monitoring. However, minor variations in the sample preparation can influence the mass spectra significantly. In the present study, we have introduced a procedure to prepare bacteria by microextraction and we have optimized experimental parameters for rapid identification by MALDI-TOF MS of whole bacterial cells isolated from environmental samples such as wastewater and soil.