Ahmed Igout

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.

Evidence for the expression of growth hormone receptors in human placenta

Since human placenta produces a growth hormone variant, it seemed important to search for evidence of GH receptors in that organ. Evidence for the expression of the GH receptor (GHR) gene was obtained by northern blot analysis. In addition, GHR poly A+ RNA was detected in RNA from cultured trophoblastic cells, but not from placenta fibroblasts. There was a low but significant specific binding of pituitary GH-N and placental GH-V to placenta plasma membranes. Both variants apparently bound to the same receptor, which is present in the first trimester as well as in the term placenta. These results suggest that placental GH may have paracrine or autocrine functions in the placenta.

Placental growth hormone and IGF-I in a pregnant woman with Pit-1 deficiency

The respective contributions of pituitary and placental GH to circulating IGF‐I in pregnant women have not been well established. We measured the serum concentrations of placental growth hormone (PGH) and IGF‐I in a woman with pit‐1 deficiency before, during and after pregnancy, resulting in the birth of a healthy child (not pit‐1 deficient). Both PGH and IGF‐I concentrations were below the assay detection limit before and after pregnancy. During pregnancy, PGH and IGF‐I levels increased steadily; the concentrations of PGH and IGF‐I in late pregnancy were comparable with levels previously measured in normal pregnancies. PGH and IGF‐I concentrations were strongly correlated throughout pregnancy (r = 0.90; P = 0.002). PGH was undetectable in cord serum, whilst the IGF‐I concentration was within the normal range. The findings of this case study corroborate the notion that PGH is the prime regulator of maternal serum IGF‐I during pregnancy.

Effect of oestrogen status on serum levels of growth hormone-binding protein and insulin-like growth factor-I in non-pregnant and pregnant women

OBJECTIVE Since there appears to be a relationship between circulating oestrogens and growth hormone, we have investigated the effect of the oestrogen status of adult women on serum levels of GHBP and IGF‐I.

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.

Both pituitary and placental growth hormone transcripts are expressed in human peripheral blood mononuclear cells (PBMC)

The hGH‐V gene codes for a variant of human pituitary growth hormone (hGH‐N) named placental growth hormone (hPGH). hPGH shares 93% amino acid identity with hGH‐N. Until now the hGH‐V gene was considered to be exclusively expressed in human placenta, where it replaces maternal circulating hGH‐N at the end of pregnancy. In this study we investigated by reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis hGH‐N, and hGH‐V, gene expression in PBMC in men, women and pregnant women. We have demonstrated that hGH‐N and hGH‐V transcripts are simultaneously produced by PBMC in both men and women as well as pregnant women. The PBMC of a PIT‐.1‐negative woman expressed only the hGH‐V transcript, but not the hGH‐N one as expected. In conclusion, hGH‐V mRNA is expressed by cells other than the syncytiotrophoblast, is not regulated by PIT‐1, and may be involved in immune regulation, as is pituitary GH.

Cloning and Nucleotide Sequence of Placental Hgh-V Cdna

We have previously demonstrated the presence in human placenta and maternal serum of a GH variant, called human placental growth hormone (hPGH). We have also shown that the hGH-V gene is expressed at the placental level thus possibly coding for hPGH. The hGH-V cDNA has now been isolated from a lambda gt 11 human placenta cDNA library. Its sequence has been determined which firmly establishes the GH-V gene mode of splicing as well as the GH-V protein structure. Our data give final evidence of placental hGH-V gene expression and reinforce the hypothesis of identity between the hGH-V protein and hPGH.

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.

THE HUMAN PLACENTAL GROWTH HORMONE VARIANT -A Review-

Summary The hGH/CS genes are clustered in chromosome 17 in the 5′ to 3′ order GH-N, CSL, CSA, GH-V and CS-B. These genes show a high degree of sequence identity. During pregnancy we have demonstrated that the placenta secretes into the maternal circulation a placental variant of growth hormone. This is the only growth hormone secreted in pregnant women at the end of pregnancy. Since then we have purified this hormone from human placenta and have produced it by genetic engineering in E.coli . We have also studied its expression and its physiopathology.