Biomedical subjects

Sophie Iochmann

Publications and source records attributed to Sophie Iochmann.

6 recordsLinked to original sources

PubMed2003

Human choriocarcinoma cell resistance to natural killer lysis due to defective triggering of natural killer cells.

The trophoblast, the outermost layer of the human placenta, lacks expression of the classical human leukocyte antigen (HLA) class I molecules. This prevents allorecognition by T cells but raises the question of what protects the trophoblast from natural killer (NK) cells. In a previous study, we have shown that choriocarcinoma cell (CC) resistance to NK lysis was mainly independent of HLA class I molecules. In the present study, we postulated that CC may prevent activation of NK cells by failing to stimulate their triggering receptors (TR). To test this hypothesis, we evaluated the lysis of JAR and JEG-3 CC after effective cross-linking and activation of NK cells by means of lectins or antibodies. Our results show that NK-resistant CC were sensitive to lysis by unstimulated peripheral blood lymphocytes in the presence of phytohemagglutin (PHA), to antibody-dependent cell cytotoxicity in presence of anti-Tja antibodies, and to monoclonal antibody redirected killing using anti-TR antibodies anti-CD16 and anti-CD244/2B4. Finally, CC fail to express CD48, the ligand for CD244/2B4. These results indicate that the resistance of CC to lysis results primarily from defective NK cell activation, at least partially due to the lack of expression of ligands, such as CD48, involved in the triggering of NK cells.

Antibodies, Monoclonal↗

PubMed2003

Transcriptional silencing of the TFPI-2 gene by promoter hypermethylation in choriocarcinoma cells.

Tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type serine proteinase inhibitor associated with the extracellular matrix, has been shown to reduce tumor invasion. In the present study we identified the presence of a complete CpG island region spanning exon 1 and the three transcription initiation sites. We demonstrate that DNA demethylation by 5'-aza-2'-deoxycytidine restores TFPI-2 transcription in JAR choriocarcinoma cells. The effect of in vitro DNA methylation on TFPI-2 promoter function was also confirmed with TFPI-2/luciferase promoter constructs. Finally, we determined the precise methylation status of CpG sites of the TFPI-2 promoter in normal and tumor trophoblast cells using the bisulfite genomic sequencing method. We conclude that hypermethylation of the TFPI-2 gene is correlated with transcriptional silencing and that the TFPI-2 gene may be a candidate tumor suppressor gene.

Cell Line, Tumor↗

PubMed2002

Demonstration of a tissue factor pathway inhibitor 2 messenger RNA synthesis by pure villous cytotrophoblast cells isolated from term human placentas.

Tissue factor pathway inhibitor 2 (TFPI-2), a Kunitz-type proteinase inhibitor, might play an important role during placenta growth by regulating trophoblast invasion and differentiation. Many TFPI-2 transcripts have been detected in syncytiotrophoblast cells, but conflicting results have been reported concerning TFPI-2 synthesis by the cytotrophoblast. To address this issue, we developed a method to isolate pure preparations of human villous cytotrophoblast cells from normal term placentas, and the synthesis of tissue factor, TFPI-1, and TFPI-2 mRNAs was then evaluated. Cells were isolated by trypsin-DNase-EDTA digestion, followed by Percoll gradient separation and immunodepletion of human leukocyte antigen-positive cells. The quality of villous cytotrophoblast cells was verified by electron microscopy. Purity of cell preparations was assessed by labeling cells with GB25, a monoclonal antibody specific to villous trophoblast cells, and by checking the absence of contaminating cells using anti-CD9 antibody. The lack of hCG, CD32 mRNA, and tissue factor mRNA also indicated the absence of contaminating cells. Using competitive reverse transcription polymerase chain reaction, we showed that freshly isolated villous cytotrophoblast cells synthesized significant levels of TFPI-1 mRNA and larger amounts of TFPI-2 mRNA. TFPI-1 and TFPI-2 mRNA synthesis remained unchanged when cytotrophoblast cells were cultured in complete medium and evolved as a multinucleated syncytiotrophoblast. These results indicate that the villous cytotrophoblast and syncytiotrophoblast are both important sites of TFPI-2 synthesis in the human placenta. This study also indicates that tissue factor detection should be used systematically to check the purity of cytotrophoblast cell preparations because it allows detection of contamination by monocytes/macrophages and by syncytial fragments.

Adult↗

PubMed2002

Demonstration of inducible TFPI-2 mRNA synthesis in BeWo and JEG-3 trophoblast cells using a competitive RT-PCR.

Tissue factor pathway inhibitor-2 (TFPI-2) displays structural similarities with TFPI-1, the major inhibitor of tissue factor (TF)/, factor VIIa. It is synthesized mostly by syncytiotrophoblast in the placenta, but its physiological functions are not fully understood. We studied the synthesis of TFPI-2 mRNA and that of TFPI-1 and TF in three human trophoblast cell lines, JAR, BeWo, and JEG-3. We first developed specific competitive reverse transcription-polymerase chain reaction (RT-PCR) assays for each gene studied using human umbilical vein endothelial cells (HUVEC). The three trophoblast cell lines strongly synthesized TF mRNA whereas the synthesis of TFPI-1 mRNA was very low. TFPI-2 mRNA was not detected in unstimulated or stimulated JAR cells. In contrast, JEG-3 and, to a lesser extent, BeWo produced significant amounts of TFPI-2 mRNA, which were significantly increased after stimulation with phorbol 12-myristate 13-acetate (PMA). However, tumor necrosis factor-alpha (TNF-alpha) had no effect on this synthesis. JEG-3 and BeWo are thus two cell lines that could be used to study TFPI-2 gene regulation and to investigate the role of TF, TFPI-1, and TFPI-2 during trophoblast differentiation.

Cell Line↗

PubMed2002

FasL-transfected endothelial cells decrease the proliferative response of allogeneic PBL.

Graft endothelium has a key role in organ transplantation because it regulates graft infiltration by allogeneic activated T cells. Overexpression of death molecules that could induce apoptosis of alloreactive T cells might be an alternative to the immunosuppressive treatment currently used in graft transplantation. Several studies have shown that immune-privileged sites express Fas ligand (FasL) and induce apoptosis of activated T-cells. We propose that endothelial cells engineered to express FasL could inhibit alloreactive T cell-proliferation by inducing apoptosis. An expression vector was constructed with human FasL cDNA and used to transfect an endothelial cell line (ECV304 cells). We demonstrated that FasL-transfected ECV304 cells were effective in inducing apoptosis of Jurkat T cell lymphoma as an agonist anti-Fas antibody. Using a mixed lymphocyte-endothelial cell culture model we observed that FasL-transfected ECV304 cells which conserved their two principal costimulatory pathways inhibited alloreactive T cell-proliferation by inducing activated T-cell apoptosis. These results suggest that endothelial cells could be interesting candidates to convey a death signal and induce hyporesponsiveness of alloreactive T cells during organ transplantation.

Apoptosis↗

PubMed2002

Phosphatidylinositol 3-kinases are involved in the all-trans retinoic acid-induced upregulation of CD38 antigen on human haematopoietic cells.

All-trans retinoic acid (ATRA) is a specific inducer of CD38 antigen on marrow CD34+ cells as well as on blast cells in acute promyelocytic and myeloblastic leukaemia. The CD38 antigen contributes to the control of blast cell proliferation, and the upregulation of CD38 might constitute an element in the pathogenesis of retinoic acid syndrome. The aim of this study was to determine whether phosphoinositide 3-kinase (PI3-K) is involved in the modification of CD38 antigen expression on myeloid cells, as PI3-K plays a major role in the ATRA-induced granulocytic differentiation of HL-60 cells. We evaluated the effects of PI3-K inhibitors (wortmannin and LY294002) on the levels of CD38 antigen and mRNA in HL-60 and normal marrow CD34+ cells exposed to ATRA (1 micromol/l). The inhibitors prevented increase in CD38 mRNA expression and the overexpression of membrane CD38 antigen, without modification of the cytoplasmic level of this antigen. Interestingly, PI3-K activity was also necessary for CD38 expression on normal marrow CD34+ cells and for the ATRA-induced upregulation of CD157, a CD38-related antigen. In conclusion, PI3-K activity plays an essential role in the regulation of CD38 expression on human haematopoietic cells, and might constitute an interesting therapeutic target in haematological disorders involving CD38 overexpression.

ADP-ribosyl Cyclase↗