Bernard Coumans

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.

Thiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals

AbstractIn most organisms, the main form of thiamine is the coenzyme thiamine diphosphate. Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain proteins. Here we show that ThTP exists not only in vertebrates but is present in bacteria, fungi, plants and invertebrates. Unexpectedly, we found that in Escherichia coli as well as in Arabidopsis thaliana, ThTP was synthesized only under particular circumstances such as hypoxia (E. coli) or withering (A. thaliana). In mammalian tissues, ThTP concentrations are regulated by a specific thiamine triphosphatase that we have recently characterized. This enzyme was found only in mammals. In other organisms, ThTP can be hydrolyzed by unspecific phosphohydrolases. The occurrence of ThTP from prokaryotes to mammals suggests that it may have a basic role in cell metabolism or cell signaling. A decreased content may contribute to the symptoms observed during thiamine deficiency.

Disrupting the melanin-concentrating hormone receptor 1 in mice leads to cognitive deficits and alterations of NMDA receptor function

In order to investigate the physiological properties of the melanin‐concentrating hormone (MCH) we have generated and used mice from which the MCH receptor 1 gene was deleted (MCHR1Neo/Neo mice). Complementary experimental approaches were used to investigate alterations in the learning and memory processes of our transgenic model. The ability of the knockout strain to carry out the inhibitory passive avoidance test was found to be considerably impaired although no significant differences were observed in anxiety levels. This impaired cognitive property prompted us to explore modifications in N‐methyl d‐aspartate (NMDA) responses in the hippocampus. Intracellular recordings of CA1 pyramidal neurons in hippocampal slices from the MCHR1Neo/Neo mice revealed significantly decreased NMDA responses. Finally, using in situ hybridization we found a 15% reduction in NMDAR1 subunit in the CA1 region. These results show for the first time a possible role for MCH in the control of the function of the NMDA receptor.

Human immune cells express ppMCH mRNA and functional MCHR1 receptor

Melanin‐concentrating hormone (MCH) is highly expressed in the brain and modulates feeding behavior. It is also expressed in some peripheral tissues where its role remains unknown. We have investigated MCH function in human and mouse immune cells. RT‐PCR analysis revealed a low expression of prepro‐MCH and MCH receptor 1 (MCHR1) but not of MCHR2 transcript in tissular and peripheral blood immune cells. FACS and in vitro assay studies demonstrated that MCHR1 receptor expression on most cell types can trigger, in the presence of MCH, cAMP synthesis and calcium mobilization in peripheral blood mononuclear cells (PBMCs). Moreover, MCH treatment decreases the CD3‐stimulated PBMC proliferation in vitro. Accordingly, our data indicate for the first time that MCH and MCHR1 may exert immunomodulatory functions.

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.

Melanin-concentrating hormone and immune function

To date, melanin-concentrating hormone (MCH) has been generally considered as peptide acting almost exclusively in the central nervous system. In the present paper, we revise the experimental evidence, demonstrating that MCH and its receptors are expressed by cells of the immune system and directly influence the response of these cells in some circumstances. This therefore supports the idea that, as with other peptides, MCH could be considered as a modulator of the immune system. Moreover, we suggest that this could have important implications in several immune-mediated disorders and affirm that there is a clear need for further investigation.

Mutations of EFHC1, linked to juvenile myoclonic epilepsy, disrupt radial and tangential migrations during brain development

Heterozygous mutations in Myoclonin1/EFHC1 cause juvenile myoclonic epilepsy (JME), the most common form of genetic generalized epilepsies, while homozygous F229L mutation is associated with primary intractable epilepsy in infancy. Heterozygous mutations in adolescent JME patients produce subtle malformations of cortical and subcortical architecture, whereas homozygous F229L mutation in infancy induces severe brain pathology and death. However, the underlying pathological mechanisms for these observations remain unknown. We had previously demonstrated that EFHC1 is a microtubule-associated protein (MAP) involved in cell division and radial migration during cerebral corticogenesis. Here, we show that JME mutations, including F229L, do not alter the ability of EFHC1 to colocalize with the centrosome and the mitotic spindle, but act in a dominant-negative manner to impair mitotic spindle organization. We also found that mutants EFHC1 expression disrupted radial and tangential migration by affecting the morphology of radial glia and migrating neurons. These results show how Myoclonin1/EFHC1 mutations disrupt brain development and potentially produce structural brain abnormalities on which epileptogenesis is established.

Effect of ppMCH derived peptides on PBMC proliferation and cytokine expression

The mRNA encoding prepro-Melanin concentrating hormone (ppMCH) is mainly expressed in the central nervous system but has also been detected at lower amount in many peripheral tissues including spleen and thymus. At the peptide level however, several forms of the precursor can be detected in these tissues and are sometimes expressed at similar levels compared to brain. In the present work, we have studied the in vitro action of a wide range of concentration (1 nM to 1 microM) of the different peptides encoded by ppMCH i.e. neuropeptide glycine-glutamic acid (NGE), neuropeptide glutamic acid-isoleucine (NEI), Melanin concentrating hormone (MCH) and the dipeptide NEI-MCH on peripheral blood mononuclear cells (PBMC) proliferation and cytokine production following anti-CD3 stimulation. Among them only MCH decreased PBMC proliferation with a maximal effect of 35% at 100 nM. Moreover as demonstrated by using ELISA, MCH significantly decreases IL-2 production by 25% but not IL-4, INF-gamma or TNF-alpha expression. Interestingly, exogenous IL-2 decreases significantly MCH-mediated inhibition, suggesting that it is an important downstream mediator of MCH action. Finally, we showed that after 7 to 9 days of incubation, MCH also inhibits proliferation of non-stimulated PBMC. Altogether, these data demonstrate that fully mature MCH modulates proliferation of anti-CD3 stimulated PBMC partially through regulation of IL-2 production.

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.