Biomedical subjects

Vincent Sapin

Publications and source records attributed to Vincent Sapin.

27 records · Page 2Linked to original sources

PubMed2004

Developmentally regulated expression of KLF6 in the mouse cornea and lens.

PURPOSE: To examine the expression of transcription factor Kruppel-like factor-6 (KLF6) in the cornea and lens of mouse eyes throughout developmental stages. METHODS: C57BL/6 mice were set up for timed mating. Embryos on embryonic day (E)10.5, E12.5, E15.5, and E18.5 and eyes from mice on postnatal day (P)0, P7, P11, P15, P30, and P60 were collected for immunofluorescence staining. Transcript of KLF6 was detected by in situ hybridization with digoxigenin-labeled RNA probes. Relative quantitative reverse transcription (RT)-PCR was performed to measure the KLF6 transcript level in eyes enucleated at embryonic stages, and the corneas and lenses isolated at postnatal stages. RESULTS: Staining for the KLF6 protein was observed at E10.5 in the lens pit and at E12.5 in the ectoderm, the mesenchyme, and the lens epithelium. Nuclear staining for KLF6 protein was evident at E15.5 in the corneal epithelium and the stroma. The staining was abundant between E18.5 and P60. In the lens epithelium, nuclear staining was detected at P0, P7, and P11. The staining intensity declined subsequently. The KLF6 transcript was found in the lens pit at E10.5 and in the ectoderm and mesenchyme at E12.5. The KLF6 mRNA level in corneal layers remained relatively constant until P60. In the lens epithelium, it was high during embryonic stages but decreased with development. CONCLUSIONS: KLF6 is expressed in both the cornea and the lens in mouse eyes. Its expression in the lens is temporally regulated, suggesting that it has a central role in lens development.

Animals↗

PubMed2003

Detection of trisomy 21 by quantitative fluorescent-polymerase chain reaction in uncultured amniocytes.

Prenatal diagnosis of fetal trisomy 21 is usually performed by cytogenetic analysis. This requires lengthy laboratory procedures, high costs and is unsuitable for large-scale screening of pregnant women. Today, trisomy 21 can be rapidly diagnosed within 24 h by molecular analysis of uncultured fetal cells using the semi-quantification of fluorescent PCR products from short tandem repeat (STR) polymorphic markers. The aim of our study was to test a chromosome quantification method on the basis of the analysis of fluorescent PCR products derived from non-polymorphic target genes. Co-amplification of a portion of DSCR1 (Down syndrome Critical Region 1) and the reference gene, CFTR (cystic fibrosis transmembrane regulator) enabled molecular detection of trisomy 21. Our method was successfully tested on a total of 154 amniotic fluids in a blind prospective study. Calculation of the DSCR1/CFTR ratio allowed us to distinguish between 152 normal amniotic fluids (mean ratio 0.99) and 2 amniotic fluids presenting a trisomy 21 status (DSCR1/CFTR ratio of 1.53 and 1.61, respectively). The results obtained by conventional cytogenetic analysis and our quantitative PCR method were concordant in every case. Our gene-based fluorescent PCR approach represents an alternative molecular method for rapid and reliable detection of trisomy 21, which can be helpful in the prenatal diagnosis of women at high risk of fetal trisomy 21.

Adult↗

PubMed2003

Identification of biologic markers of the premature rupture of fetal membranes: proteomic approach.

In obstetrics, premature rupture of the membranes (PROM) is a frequent observation which is responsible for many premature deliveries. PROM is also associated with an increased risk of fetal and maternal infections. Early diagnosis is mandatory in order to decrease such complications. Despite that current biological tests allowing the diagnosis of PROM are both sensitive and specific, contamination of the samples by maternal blood can induce false positive results. Therefore, in order to identify new potential markers of PROM (present only in amniotic blood, and absent in maternal blood), proteomic studies were undertaken on samples collected from six women at terms (pairs of maternal plasma and amniotic fluid) as well as on four samples of amniotic fluid collected from other women at the 17(th) week of gestation. All samples (N = 16) were analyzed by two-dimensional (2-D) high-resolution electrophoresis, followed by sensitive silver staining. The gel images were studied using bioinformatic tools. Analyses were focused on regions corresponding to pI between 4.5 and 7 and to molecular masses between 20 and 50 kDa. In this area, 646 +/- 113 spots were detected, and 27 spots appeared to be present on the gels of amniotic fluid, but were absent on those of maternal plasma. Nine out of these 27 spots were also observed on the gels of the four samples of amniotic fluids collected at the 17(th) week of pregnancy. Five of these 9 spots were unambiguously detected on preparative 2-D gels stained by Coomassie blue, and were identified by mass spectrometry analyses. Three spots corresponded to fragments of plasma proteins, and 2 appeared to be fragments of proteins not known to be present in plasma. These 2 proteins were agrin (SWISS-PROT: O00468) and perlecan (SWISS-PROT: P98160). Our results show that proteomics is a valuable approach to identify new potential biological markers for future PROM diagnosis.

Biomarkers↗

PubMed2003

Trophoblastic remodeling in normal and preeclamptic pregnancies: implication of cytokines.

OBJECTIVE: To summarize the recent knowledge on the implications of placenta and cytokines in normal and preeclamptic pregnancies. DATA SOURCES: A literature search was conducted of applicable articles related to interactions between trophoblast and cytokines in generating preeclampsia. CONCLUSIONS: The initiating event in preeclampsia has been postulated to be the reduced uteroplacental perfusion as a result of abnormal extravillous cytotrophoblast invasion and remodeling of the uterine spiral arteries. Focal ischemia and hypoxia, deportation of hypoxemic trophoblast cells and abnormal expression of various placental biologic molecules, particularly the cytokines, are thought to lead to widespread dysfunction of the maternal vascular endothelium resulting in overproduction of endothelin and thromboxane, enhanced vascular sensitivity to angiotensin II, and reduced secretion of vasodilators such as nitric oxide and prostacyclin. These alterations, in turn, cause hypertension, proteinuria and edema, and pathologies in many organ systems (kidney, lung, liver, brain).

Cytokines↗

PubMed2003

Frequency, risk factors, and outcome of hyperlactatemia in HIV-positive persons: implications for the management of treated patients.

BACKGROUND: The nucleoside reverse transcriptase inhibitors used for the treatment of HIV-positive persons are now clearly associated with metabolic disorders. We determined the prevalence of and risk factors for hyperlactatemia in HIV-positive persons to assess the relevance of lactate venous blood concentrations during antiretroviral therapy. METHODS: We conducted a prospective cross-sectional study of venous lactate determinations with 282 consecutive HIV-positive persons who, in addition to a physical examination, had blood samples taken every 3-4 months for routine biochemical, immunologic, and viral assessment. The frequencies of hyperlactatemia and lactic acidosis were determined, and the risk factors were analyzed by a multivariate logistic regression model. The effect of modification of antiretroviral therapy in patients with moderate hyperlactatemia was also assessed. RESULTS: From 782 blood lactate determinations, we identified 65 (23%) patients with moderate hyperlactatemia and 5 (1.8%) with lactate concentrations >5 mmol/L (2 with severe lactic acidosis; 0.7%). Older age, drug regimens containing stavudine [adjusted odds ratio (OR) = 2.5] or a combination of stavudine-didanosine (adjusted OR = 3.1), and the use of buprenorphine (adjusted OR = 14.7) were independent predictors of hyperlactatemia. Among 65 patients with moderate hyperlactatemia, 39 did not have their treatments changed, and 26 had a new combination therapy that was associated with a clinical improvement and a more pronounced decrease in lactate (-1.66 vs -0.99 mmol/L; P <0.05). CONCLUSIONS: Chronic compensated and moderate hyperlactatemia was common in our population study. Measurement of lactate, under standardized conditions, may be useful in optimizing management of HIV-positive persons on antiretroviral therapy.

Acidosis, Lactic↗

PubMed2002

Treatment of the rat hepatic stellate cell line, PAV-1, by retinol and palmitic acid leads to a convenient model to study retinoids metabolism.

The main site of vitamin A storage in the liver is the hepatic stellate cells (HSC). Involvement of HSC in vitamin A metabolism has mainly been studied using primary culture, which represents the most physiological model but technically suffers several drawbacks (yield, low reproducibility, etc.). To circumvent these problems, we have previously established and characterised an immortalised rat HSC line named PAV-1. This study aimed to investigate in PAV-1 and in primary HSC (i) the incorporation of retinol and its esterification, (ii) the cellular retinol-binding protein (CRBP) content, (iii) the acid retinyl ester hydrolase activity (aREH), (iv) the thermal susceptibility and (v) the lipid composition of the membranes, which may play a crucial role in retinol transport across cellular membrane. In routine conditions of culture, the rate of retinol esterification in PAV-1 was low (5.2%) compared to that obtained with primary HSC (69.9%). Retinol pre-treatment doubled this esterification rate (10.7%) and the CRBP content in PAV-1. The co-incubation with retinol and palmitic acid enabled PAV-1 to esterify retinol with a rate close to that of primary HSC (66.2% vs. 69.9%) and with similar retinyl ester profiles. aREH activity was higher in primary HSC than in PAV-1. Thermal susceptibility and phospholipid composition of membranes in PAV-1 treated cells were similar to those of primary HSC. In conclusion, our study shows that PAV-1 cells treated with retinol and palmitic acid is a sound and convenient model for studying vitamin A mobilisation, a fundamental physiological event occurring in HSC.

Animals↗

PubMed2002

PAV-1, a new rat hepatic stellate cell line converts retinol into retinoic acid, a process altered by ethanol.

During liver fibrogenesis or long term culture, hepatic stellate cells (HSCs) evolved from "quiescent" to activated phenotype called "myofibroblast-like", a transition prevented by retinoic acid (RA). Little is known about RA generation by HSCs. Our study aimed to check the ability of these cells to produce RA from retinol (Rol) and the alterations of this metabolic step by ethanol. To study this metabolic pathway, primary cultures of HSCs represent the most physiological model but technically suffer several drawbacks. To circumvent these problems, an immortalized rat HSC line (named PAV-1) has been established. We validated PAV-1 cell line as a convenient model to study retinoids metabolism by HSCs. Then, we showed that PAV-1 cells express Rol-binding proteins (RBPs), enzymes and nuclear receptors involved in RA signaling pathway. We also demonstrated in situ generation of functional all-trans-RA (ATRA), using transient transfections with a RA-sensitive reporter gene, in situ modulation of tissue transglutaminase (tTG) activity and HPLC experiments. This production was Rol dose-dependent; 4-methylpyrazole, citral, and ethanol-inhibited which argues in favor of an enzymatic process.In conclusion, we first demonstrate in situ RA generation from Rol in a newly immortalized rat HSC line, named PAV-1. Inhibition of RA production by ethanol in PAV-1 and recent data, suggesting fundamental role of RA to prevent fibrosis development in the liver, allow us to hypothesize that Rol metabolism could be a primary target for ethanol during development of hepatic fibrosis.

Acyclic Monoterpenes↗

PubMed2002

Human choriocarcinoma cell line JEG-3 produces and secretes active retinoids from retinol.

Vitamin A (retinol) and its active derivatives (the retinoids) are essential for growth and development of the mammalian fetus. Maternally-derived retinol has to pass through the placenta to reach the developing fetus. Despite its apparent importance, little is known about placental metabolism of retinol, and particularly placental production and/or secretion of active retinoids. It has been previously considered that retinoids are recruited from the uterine environment to influence placental development and function during gestation. We have studied retinoid metabolism in the human choriocarcinoma cell line JEG-3 and demonstrate, for the first time, that active retinoids are produced endogenously by the JEG-3 cell line from retinol. These retinoids induce gene expression from a retinoic acid-responsive enhancer element reporter plasmid and modulate placental transglutaminase activity. Furthermore, retinoids are secreted from JEG-3, as shown by the activation of retinoic acid-responsive beta lacZ reporter cells grown in conditioned media. These results suggest that there could be an active role for trophoblast-derived retinoids during human development.

Alcohol Dehydrogenase↗

PubMed2002

Regulation of corneal keratin-12 gene expression by the human Krüppel-like transcription factor 6.

PURPOSE: The keratin-12 (K12) protein is essential for the integrity of the corneal epithelium. This study was conducted to investigate the possible involvement of Krüppel-like factor 6 (KLF6) in the corneal regulation of K12 gene expression, in view of the presence of one KLF6 potential binding site in the human K12 promoter and the known role of KLF6 in regulating keratin gene expression. METHODS: RT-PCR, Western blot analysis, and immunolocalization experiments were used to investigate the expression of KLF6 mRNA and protein in five human total corneas. The same experimental design was used to explore human corneal epithelial (HCE) cells in 20 patients and a HCE cell line. The ability of the KLF6 protein to modulate K12 promoter activity was studied in the HCE cell line, by transient transfections with a KLF6 expression plasmid and promoter-reporter gene assays. Gel-shift assays were performed to confirm the interactions between the KLF6 protein and specific sequences of the K12 promoter. RESULTS: The presence of KLF6 transcripts and proteins in human total corneal extracts was demonstrated. Immunohistofluorescence experiments showed positive staining specifically present in the corneal epithelial layer. KLF6 transcripts and proteins were also present in corneal epithelial cells in 20 patients and the HCE cell line. Transient transfections of KLF6 showed statistical transactivation of the K12 promoter in HCE cells. The gel-shift assay showed a physical interaction between KLF6 and the K12 promoter. CONCLUSIONS: The expression of KLF6 in HCE cells and its role in the regulation of K12 gene expression were demonstrated.

Base Sequence↗