Olivier Thellin

Housekeeping genes as internal standards: use and limits

Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their comparison to the expression of the housekeeping gene transcripts like glyceraldehyde-3-phosphate dehydrogenase (G3PDH), albumin, actins, tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), L32. 28S, and 18S rRNAs are also used as internal standards. In this paper, it is recalled that the commonly used internal standards can quantitatively vary in response to various factors. Possible variations are illustrated using three experimental examples. Preferred types of internal standards are then proposed for each of these samples and thereafter the general procedure concerning the choice of an internal standard and the way to manage its uses are discussed.

Pregnancy and the immune system: between tolerance and rejection

Interactions between the conceptus and the mother are bi-directional: the feto-placental tissues need nutrition and a suitable environment in homeostatic condition whereas the mother influenced by the placental factors adapts her metabolism and immune system. Many different mechanisms acting locally or at distance ensure tolerance of the semi-allogeneic graft by the maternal natural and adaptive immune defences. In front of this tolerance, mechanisms exist ensuring rejection of the conceptus by the mother (spontaneous abortion) through rupture of one or more tolerance mechanisms, notably in stress situations endangering the mother. Thus outcome of a pregnancy is dependent on efficiently working tolerance mechanisms, and rupture of such mechanisms can lead to rejection. The balance of influence leading either to tolerance or rejection is under control of internal (maternal and fetal) and external (environmental) factors. Rejection, if triggered, mainly occurs through immune-induced inflammation, tissue degradation and coagulation.

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.

Demonstration of the expression of CD95 ligand transcript and protein in human placenta

Tolerance of the fetal allograft enables the human conceptus to implant itself into the maternal uterus and survive and grow there. This tolerance phenomenon remains largely obscure, notably because it appears to be controlled by multiple mechanisms. CD95 ligand (CD95-L), which can trigger death of CD95-positive cells by apoptosis, may participate in inducing anti-fetus-sensitized CD95-positive T lymphocytes to enter apoptosis. Using immunohistochemistry (first trimester and term placentae), FACS assays (term placenta) and RT-PCR assays (term placenta), the presence of CD95-L protein and mRNA has been shown in crude placental tissue preparations and isolated placental cells. Among the latter, CD95-L expression was detected in trophoblastic cells, fetal blood cells (mRNA only) and also the Hofbauer macrophages. No CD95-L was detected in fibroblasts or fetal endothelial cells. Thus trophoblastic cells, Hofbauer macrophages, and perhaps also fetal blood cells could form a sequential barrier blocking maternal activated defence cells bearing CD95 molecules.

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.

Oral scrapie infection modifies the homeostasis of Peyer’s patches’ dendritic cells

In transmitted prion diseases the immune system supports the replication and the propagation of the pathogenic agent (PrPSc). DCs, which are mobile cells present in large numbers within lymph organs, are suspected to carry prions through the lymphoid system and to transfer them towards the peripheral nervous system. In this study, C57Bl/6 mice were orally inoculated with PrPSc (scrapie strain 139A) and sacrificed at the preclinical stages of the disease. Immunolabelled cryosections of Peyer’s patches were analysed by confocal microscopy. Membrane prion protein expression was studied by flow cytometry. In Peyer’s patches (PP), dissected at day one and day 105 after oral exposure to scrapie, we observed an increased population of DCs localised in the follicular-associated epithelium. On day 105, PrPSc was found in the follicles inside the PP of prion-infected mice. A subset of Peyer’s patches DCs, which did not express cellular prion protein on their surface in non-infected mice conditions, was prion-positive in scrapie conditions. Within Peyer’s patches oral scrapie exposure thus induced modifications of the homeostasis of DCs at the preclinical stages of the disease. These results give new arguments in favour of the implication of DCs in prion diseases.

Influence of the Ixodes ricinus tick blood-feeding on the antigen-specific antibody response in vivo

The blood meal of hard ticks such as Ixodes ricinus lasts several days. This is made possible by tick salivary factors that inhibit inflammation, haemostasis and the host immune response. We assessed the latter on a model of immune response in vivo. A significant reduction of specific IgM and IgG levels was observed in BALB/c mice infested 5 days before injection with bovine serum albumin (BSA) and QuilA but not in mice infested 5 days after the immunization. This effect was not observed in mock-infested mice and could not be attributed to the use of anesthetics. The antibody response was not merely delayed and the Th(1)/Th(2) balance appeared not altered. T-dependent zones and germinal centers in lymph nodes draining the tick bite site showed no apparent morphological alterations or shift in T cell subpopulations. However, the spleens of tick-infested mice had also an enlarged red pulp, indicating an increased extramedullary haematopoietic activity.

Expression of Growth Hormone Receptors by Lymphocyte Subpopulations in the Human Tonsil

The ability of human tonsillar lymphoid cells to express growth hormone receptor (hGH-N-R) was analyzed by flow cytometry. FITC-coupled recombinant human growth hormone (hGH-N) was used to reveal the receptors, in combination with phenotype markers. Unlike T cells, tonsillar B cells constitutively express the hGH-N receptor. Quiescent cells separated from activated cells by Percoll-gradient centrifugation bear fewer receptors than activated ones. Activated T cells express hGH-N-R, but the typical germinal centre CD4+CD57+ T cells do not. These latter thus appear not to be fully activated. Inside the lymph follicles, the germinal centre CD38+ B-cell population and the mantle-zone CD39+ B-cell population display similar levels of hGH-N-R expression, but receptor density is lower on dividing dark-zone CD38+CD10+ B cells. Different lymphoid-cell populations thus differ markedly in their ability to express the growth hormone receptor, in relation notably to their activation status. This highlights the link between the neuroendocrine system and the active immune defense.

In vitro approach to study the synergistic effects of tobramycin and clarithromycin against Pseudomonas aeruginosa biofilms using prokaryotic or eukaryotic culture media

Recurrent Pseudomonas aeruginosa infections involving biofilm formation are frequent in cystic fibrosis, aggravating the respiratory distress. Co-administration of clarithromycin and classical tobramycin could improve the health status of patients. Antibiotic toxicity was assessed on epithelial (CFBE41o(-)) and macrophagic (THP-1) cell lines. Non-toxic concentrations of antibiotics alone or in combination were applied twice daily for 12 days on mature (12-day-old) biofilms of three P. aeruginosa strains, developed either in prokaryotic culture broth [tryptic soy broth (TSB)] or in a eukaryotic cell culture medium (RPMI-FCS) more similar to an in vivo environment. The antibiofilm and bactericidal effects of antibiotics were assessed. No toxicity of tobramycin was observed on eukaryotic cell lines at concentrations up to 500μg/mL, whilst 100μg/mL was selected as the clarithromycin upper safe limit. The amount of biofilm was strongly reduced by 100μg/mL and 500μg/mL tobramycin for each strain in both media, whilst clarithromycin was only effective in RPMI-FBS, with synergistic (PAO1 strain) and additive (PYO2 strain) effects detected when combining tobramycin 4μg/mL and clarithromycin 100μg/mL. Finally, tobramycin at ≥100μg/mL exerted strong bactericidal effects on each strain in both media. Clarithromycin also exerted bactericidal effects on each strain in both media; its effect was weaker than tobramycin in TSB but was similar in RPMI-FBS. Synergistic effects were observed on PAO1 and MUCO biofilms, e.g. when combining tobramycin 4μg/mL and clarithromycin 100μg/mL. These in vitro data show that co-administration of clarithromycin and tobramycin acts synergistically against in vitro P. aeruginosa biofilms.

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.