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Biomedical subjects

D Bellet

Publications and source records attributed to D Bellet.

At least 19 recordsLinked to original sources
PubMed2001

Quantification of human cytomegalovirus DNA in bone marrow transplant recipients by real-time PCR.

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA in peripheral blood leukocytes (PBLs) of bone marrow transplantation patients. Unlike other teams, we quantified CMV and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene using a plasmid containing both sequences as an external standard. Tenfold serial dilutions of this plasmid yielded overlapping standard curves that allowed the quantification of CMV and GAPDH gene copies in an efficient and accurate manner. Sequential blood samples (164 specimens) were collected from 16 patients. PBLs were tested by the pp65 antigenemia assay and quantitative CMV and GAPDH gene PCRs. CMV DNA was detected by PCR in 13 patients a mean of 15 days prior to the appearance of antigenemia. The administration of anti-CMV drugs led to a rapid decrease in the numbers of viral copies and positive nuclei. Real-time PCR assay results correlated with those of the CMV pp65 antigenemia assay (P < 0.00001). The TaqMan assay may be a useful tool for rapid quantification of CMV infection and for monitoring of CMV reactivation in bone marrow transplantation recipients.

Adolescent↗
PubMed2001

Analysis of interleukin-18, interleukin-1 converting enzyme (ICE) and interleukin-18-related cytokines in Crohn's disease lesions.

A local increase of interleukin-18 (IL-18) expression has been recently demonstrated in Crohn's disease (CD), suggesting a role for mature IL-18 (cleaved by ICE protease) in the induction of proinflammatory cytokines and Th1 polarization observed in CD lesions. The aim of this study was to investigate IL-18 modulation and its potential immune consequences in CD lesions. We showed increased IL-18 production in chronic CD lesions and identified epithelial cells and macrophages as IL-18-producing cells. A twofold increase in ICE alpha, beta, and/or gamma mRNA that encodes for the complete mature peptide was required for ICE activity, and a marked increase in IL-18R-positive immune cells was observed in chronic lesions compared to uninvolved areas or normal control samples. Chronic lesions also displayed intense transcription of IL-18-induced cytokines, IFN-gamma, IL-1beta, TNF-alpha, and IL-8. By contrast, when neither IL-18 nor ICE mRNAs were enhanced (early asymptomatic CD lesions), IL-18-induced cytokines were not up-regulated. These results are in accordance with a putative role of mature IL-18 in the pathogenesis of CD.

Base Sequence↗
PubMed2000

Defect of villous cytotrophoblast differentiation into syncytiotrophoblast in Down's syndrome.

The syncytiotrophoblast (ST) is one of the major components of the human placenta, as it is involved in feto-maternal exchanges and the secretion of pregnancy-specific hormones. The aim of this study was to elucidate the formation and function of the ST in trisomy 21 (Down's syndrome). We first used the in vitro model of cytotrophoblast differentiation into ST. Cytotrophoblasts were isolated from 15 trisomy 21-affected placentas (12-35 weeks gestation) and 10 gestational age-matched control placentas. In vitro cytotrophoblasts isolated from normal placenta fused to form the ST. This was associated with an increase in transcript levels and in the secretion of hCG, human placental lactogen, placental GH, and leptin. In trisomy 21-affected placentas, we observed a defect (or a delay) in ST formation and a dramatic decrease in the synthesis and secretion of these hormones compared to those in cultured cells isolated from control age-matched placentas. These results were confirmed by a significant (P < 0.001) decrease in gene expression in total homogenates of trisomy 21-affected placentas compared to controls. These results will be of help in understanding the maternal hormonal markers of fetal trisomy 21 and the consequences of placental defects for fetal development.

Adult↗
PubMed2000

Expression of pro-EPIL peptides encoded by the insulin-like 4 (INSL4) gene in chromosomally abnormal pregnancies.

The recent development of a specific immunoassay based on monoclonal antibodies directed to chain C and chain A of early placenta insulin-like peptide (EPIL) encoded by the INSL4 gene, has made it possible to demonstrate pro-EPIL peptide expression during normal pregnancy. In the present study, we report on the expression of pro-EPIL peptides in chromosomally abnormal pregnancies, namely trisomy 21 and 18. EPIL peptide levels were measured in amniotic fluid (AF) and maternal serum (MS) from pregnancies with trisomy 21 (n=16) or 18 (n=14) and compared to levels detected in AF and MS from 33 chromosomally normal pregnancies between 12 and 32 weeks of gestation. Pro-EPIL peptide levels were significantly higher in amniotic fluids from T21 than in AF from chromosomally normal pregnancies (mean pro-EPIL levels +/- SEM, 449+/-129.2 ng/mL vs 137+/-29.6 ng/mL, P = 0.0195), whereas there was only a trend towards an increase in pro-EPIL peptide levels in maternal serum. In a limited matched gestational age range (15 to 17 weeks), it was confirmed that pro-EPIL peptide levels were significantly higher in AF from T21 pregnancies (644.0+/-155.9 ng/mL, n = 11) than in AF from normal pregnancies (177.8+/-39.0 ng/mL, n = 12; P < 0.0001). Interestingly, the expression patterns of pro-EPIL peptides, human chorionic gonadotropin (hCG) and its free subunits were parallel in T21 pregnancies as recently observed in normal pregnancies. These results are in line with previous observations suggesting that the biosynthesis of both hCG and EPIL follows common regulation pathways.

Adult↗
PubMed2000

Expression of Na+/I- symporter and Pendred syndrome genes in trophoblast cells.

Placental iodide transport is critical for the fetal thyroid function, but the molecular mechanisms of this transport are not understood. The expression of two recently identified iodide transporters, namely the sodium/iodide symporter (NIS) and pendrin, the product of the gene responsible for the Pendred syndrome (PDS), was studied using real-time kinetic quantitative PCR and immunohistochemistry 1) in placental tissues collected at different gestational ages and 2) in primary cultures of villous cytotrophoblast cells (VCT) that differentiate and fuse over 2-3 days in vitro to form villous syncytiotrophoblast (VSCT) cells. Both NIS and PDS genes are expressed in placenta, albeit at low levels compared with those in thyroid tissue. NIS gene expression in placental samples from first trimester and term pregnancies was similar. In contrast, the expression of PDS gene was higher in term than in first trimester pregnancy samples. In vitro, NIS gene was expressed at a high level in VCT obtained from first trimester pregnancy, and its expression decreased by 3- to 4-fold during the differentiation of VCT in VSCT. Expression of NIS was lower (up to 30-fold) in VCT obtained in placental samples from third trimester than from first trimester pregnancy. In contrast, the expression of PDS gene was low in VCT and increased by 5- to 10-fold during VSCT formation; this was observed in cells isolated from placental samples of both first trimester and term pregnancies. Immunohistochemical analysis showed that NIS protein was present on the entire membrane of VCT, whereas pendrin was mainly located at the brush border membrane of VSCT, facing the mother. In conclusion, 1) NIS and PDS genes are differently expressed in the placenta during gestation; and 2) whereas pendrin is expressed at the brush border membrane of syncytiotrophoblast cells, NIS protein is mainly located in the cytotrophoblast layer.

Carrier Proteins↗
PubMed1999

Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo.

Cytotoxic T lymphocytes and natural killer cells are essential effectors of anti-tumor immune responses in vivo. Dendritic cells (DC) 'prime' tumor antigen-specific cytotoxic T lymphocytes; thus, we investigated whether DC might also trigger the innate, NK cell-mediated anti-tumor immunity. In mice with MHC class I-negative tumors, adoptively transferred- or Flt3 ligand-expanded DC promoted NK cell-dependent anti-tumor effects. In vitro studies demonstrated a cell-to-cell contact between DC and resting NK cells that resulted in a substantial increase in both NK cell cytolytic activity and IFN-gamma production. Thus, DC are involved in the interaction between innate and adaptive immune responses.

Adoptive Transfer↗
PubMed1999

Pro-EPIL forms are present in amniotic fluid and maternal serum during normal pregnancy.

Recent observations based on immunohistochemistry and RT-PCR demonstrate that early placenta insulin-like peptide (EPIL), encoded by the INSL4 gene, is present in the placenta during gestation. In the present study, we report on the development of a specific immunoassay, entirely based on two monoclonal anti-peptide antibodies (mAbs), and its use for the detection of pro-EPIL forms in biological fluids during pregnancy. One mAb directed against the C-connecting peptide was used to capture pro-EPIL forms and their binding was revealed by a radiolabeled anti-A chain mAb as the indicator. A composite synthetic peptide, encompassing the C- and A-domains, was utilized as the standard. Under these experimental conditions, the assay displays a sensitivity limit of 2 ng/mL. Pro-EPIL molecular forms were detected in both amniotic fluid and maternal serum of pregnant women. At 12 to 16 weeks of pregnancy, the pro-EPIL level was higher in amniotic fluid (246 +/- 50.8 ng/mL) than in maternal serum (5 +/- 2.0 ng/mL). As gestation advanced, so the concentration of pro-EPIL forms decreased in amniotic fluid while its level increased in maternal serum. Interestingly, in amniotic fluid, the pattern of pro-EPIL concentration during pregnancy is very similar to that observed for human chorionic gonadotropin (hCG) and its free subunits. The pattern of serum pro-EPIL concentration is similar to that of the free alpha-subunit. Together with our previous immunohistochemical observations, these results indicate that pro-EPIL is preferentially secreted by cytotrophoblasts in amniotic fluid and that the biosynthesis of hCG subunits and EPIL may be regulated by common pathways. Overall, our observations strongly suggest that EPIL may play a critical physiological role during embryonic and foetal development.

Adult↗
PubMed1998

Insulin-like 4 (INSL4) gene expression in human embryonic and trophoblastic tissues.

Polypeptide growth factors play an important role in the regulation of human embryonic development. Insulin-like 4 gene (INSL4) is a member of the insulin family, which includes insulin, IGF-I, IGF-II, relaxin, and INSL3. Using RT-PCR, we previously found abundant INSL4 mRNA in the human placenta. In this study, we examined the chronology and spatial expression of this gene in sections of human placenta and conceptus by means of in situ hybridization. Expression of the IGF-II gene was studied as a positive control. INSL4 distribution was tissue- and cell-specific. Indeed, INSL4 mRNA was most abundant in syncytiotrophoblast cells. In fetal tissues, INSL4 mRNA was identified in the perichondrium of all four limbs, vertebrae, and ribs. Moreover, INSL4 mRNA was abundant in interbone ligaments. These findings indicate that the INSL4 gene may play an important role in trophoblast development and regulation of bone formation. IGF-II mRNA, in agreement with the literature, are mainly located in the mesodermal core in the villous trophoblast and in most embryonic tissues.

Gene Expression Regulation, Developmental↗
PubMed1998

Assessment of autosome and gonosome disomy in human sperm nuclei by chromosome painting.

Disomy and diploidy frequencies for autosomes 1-22 and the gonosomes were assessed in 299,442 sperm nuclei from four normal fertile men by chromosome painting. This novel approach allowed us to perform a specific and sensitive detection of each chromosome. A minimum of 5000 sperm nuclei per subject were evaluated for each chromosome by dual colour fluorescence in situ hybridization. The disomy rate proved to be similar for all the autosomes (0.24%) and the diploidy rate varied from 0.12% to 0.15%. No interchromosomal or interindividual differences in the frequency of disomic and diploid sperm nuclei were observed between the four subjects. The mean frequency of XX-, YY- and XY-bearing spermatozoa was estimated to 0.17%, 0.17% and 0.32%, respectively. This strategy constitutes a new approach for detecting aneuploidy in human sperm nuclei and suggests an equal repartition of non-disjunction among chromosomes in male gametes.

Adult↗
PubMed1998

Characterization of human lutropin carboxyl-terminus isoforms.

Human lutropin (hLH) exhibits both carbohydrate and peptidic heterogeneities that affect its biological potency and the duration of its activity in vivo. Peptidic changes within the hLH beta-subunit are characterized as intrachain proteolytic nicking and carboxyl terminus heterogeneity. To date, the carboxyl terminus of hLHbeta appears to end at either position Gln114 or Gly117, as determined by sequencing of purified subunit. Furthermore, the complementary DNA for hLHbeta predicts a protein containing an additional peptidic stretch, which would make the beta-subunit 121 residues long. This extension may be responsible for the particular intracellular behavior of hLHbeta. To investigate the carboxyl terminus polymorphism of natural hLHbeta, monoclonal antipeptide antibodies were raised against a synthetic peptide mimicking the 104-119 portion of hLHbeta. One antibody, designated LHP09, was found to specifically react with the recombinant hLHbeta ending at position hLHbeta[Leu119] but not with other recombinant forms ending at [Ser116], [Phe120] or [Leu121]. Immunochemical analysis of hLH, either pituitary or urinary in origin, indicated that only pituitary hLH contains a Leu119-ending form of hLHbeta. Finally, immunohistochemical detection was performed using LHP09 and showed specific staining of a normal adult pituitary gland. These observations support the in vivo existence of intrapituitary molecular forms of hLHbeta ending at various positions between Gln114 and Leu121.

Amino Acid Sequence↗
PubMed1998

Prognostic value of chorionic gonadotropin beta gene transcripts in human breast carcinoma.

The beta subunit of human chorionic gonadotropin is potentially encoded by six genes, which can be categorized into two types based on a sequence change at codon 117: GCC for the type I and GAC for the type II genes. We previously showed that, whereas type I genes were exclusively expressed in normal breast tissues, expression of type II genes was associated with malignant transformation (Bellet, D., et al. Cancer Res., 57: 516-523, 1997). We designed a simple and robust test (the CG117 assay) that measures the percentage of type II over both types of chorionic gonadotropin beta mRNAs. Normal breast tissues consistently had a negative CG117 index, whereas cancer breast tissues showed indexes ranging from 0 to 100%. The prognostic significance of the CG117 index was investigated in a series of 99 unilateral invasive primary breast cancer patients with known long-term outcome (median follow-up, 9 years). The CG117 index was positive in 48 (48.5%) of the 99 tumor mRNA samples. The index was not significantly associated with standard prognostic parameters, including clinical and macroscopic tumor size, histopathological grade, and lymph node status or steroid receptor status. Patients with a positive CG117 index in primary tumor mRNA had significantly shorter metastasis-free survival (P = 0.014) and overall survival (P = 0.038) after surgery, compared to patients with a negative index. The prognostic significance of the CG117 index persisted in Cox multivariate regression analysis, both for metastasis-free survival (P = 0.008) and overall survival (P = 0.016), together with lymph node status (P = 0.027 and P = 0.009, respectively). These findings indicate that the CG117 index may contribute to the identification of high-risk breast cancer patients.

Adult↗
PubMed1997

MHC-dependent cytolysis of autologous tumor cells by lymphocytes infiltrating urothelial carcinomas.

Tumor-infiltrating lymphocytes (TIL) were grown from 23 urothelial carcinomas. Phenotyping analysis showed that the TIL cultures were mainly CD3+. Although CD4+ and CD8+ T-cell sub-sets were grown in culture, CD4+ T-cell sub-sets predominated over CD8+ T cells. Immunohistochemical studies performed on 5 tumor specimens confirmed this observation, and indicated that CD4+ T cells surrounded the tumor islets, whereas CD8+ T lymphocytes were localized among the tumor cells. Five short-term carcinoma cell lines established from these urothelial tumors were used as target cells in cytolysis assays in order to investigate the functional anti-tumor activity of autologous TIL. TIL from 4/5 tumors were lytic and 3 TIL lines displayed MHC-class-I-dependent cytotoxicity directed against autologous tumor cells. CD4+ T-cell-depletion experiments performed on TIL line 07 confirmed that CD8+ MHC-class-I-dependent CTL were the predominant effectors. Finally, experiments performed on 6 allogeneic urothelial-cancer cell lines matched for HLA-class-I molecules showed that TIL07 exhibited selective lytic activity toward tumor 07. These data indicate that CD8+ MHC-class-I-dependent CTL present in urothelial carcinomas are functional and may participate in the anti-tumor immune response.

CD3 Complex↗
PubMed1997

Antigen-presenting function of murine gonadal epithelial cell lines.

Murine Leydig (TM3) and Sertoli (TM4) cell lines were studied as nonprofessional antigen-presenting cells using the antigen model of human choriogonadotropin (hCG alpha/beta) and specific T-cell hybridomas. Both cell lines were treated with IFN-gamma to induce I-A(d) and I-E(d) molecules expression. Only the TM3 cell line, which expressed MHC-class II molecules upon IFN-gamma stimulation, was able to uptake, process, and present the human choriogonadotropin beta subunit to related T-cell hybridomas. Interestingly, the TM3 cell line was incapable of presenting the human choriogonadotropin alpha subunit, the presentation of which, by classical APC, is highly efficient. Using T-cell hybridomas directed against the immunogenic regions of hCG alpha/beta previously described in BALB/c mice, we showed that the TM3 cell line generated a narrower peptide repertoire than classical APC (i.e., B cells, macrophages, and dendritic cells). This experimental system suggests that Leydig cells could initiate, in vivo, an autoimmune process directed against gonadal tissues. In particular, such a mechanism has been evoked in experimental autoimmune orchitis.

Animals↗
PubMed1997

Malignant transformation of nontrophoblastic cells is associated with the expression of chorionic gonadotropin beta genes normally transcribed in trophoblastic cells.

The beta subunit of human chorionic gonadotropin (hCGbeta) is encoded by four nonallelic CGbeta genes. An assay was developed for distinguishing type I CGbeta allelic genes beta7 and beta6, which possess a GCC codon corresponding to an alanine at position 117 of hCGbeta, from type II CGbeta genes beta8, beta5, and beta3 and its allele beta9, which possess a GAC codon corresponding to an aspartic acid at the same position. In normal trophoblast, hCGbeta is encoded by type II CGbeta genes, whereas normal nontrophoblastic tissues of differing histological origin (breast, prostate, skeletal muscle, bladder, adrenal glands, thyroid, colon, and uterus) express only type I CGbeta genes. We studied the expression of CGbeta genes in 86 tumor specimens collected from patients with breast, bladder, prostate, and thyroid cancer and found that up to 61% of these nontrophoblastic tumors expressed type II CGbeta genes. Experiments performed on tumor tissues and their normal counterparts confirmed that the malignant transformation of nontrophoblastic cells is associated with the expression of type II CGbeta genes. These findings provide the basis for a simple test (the CG117 assay) that may be useful for the diagnosis of the most frequent malignancies.

Cell Line↗