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Biomedical subjects

D Bellet

Publications and source records attributed to D Bellet.

At least 19 recordsLinked to original sources
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↗
PubMed1996

A new series of mycobacterial expression vectors for the development of live recombinant vaccines.

Recombinant BCG (bacillus Calmette-Guérin) is a promising candidate as a live vaccine delivery system. Thus far, however, only autoreplicative plasmids carrying the heterologous genes to be expressed in BCG, together with antibiotic-resistance genes, have been successfully used. This could potentially lead to the spreading of antibiotic resistance among other bacteria, and might therefore be unsafe for the environment. In this study, we present a series of three Escherichia coli-Mycobacteria shuttle vectors which enable expression and secretion of antigens without the use of antibiotic-resistance markers. All these plasmids confer mercury resistance to the host bacteria as the only selectable marker and contain a unique restriction site to allow for single-step in-frame cloning of open reading frames downstream from the Mycobacterium tuberculosis 85A antigen promoter and export signal. The system was used to express the free beta-subunit of human chorionic gonadotropin (hCG beta), a potential target of an immunotherapeutic vaccine.

Antigens↗
PubMed1996

Characterization of a monoclonal antibody reacting with the free human luteinizing hormone beta-subunit.

Measurement of serum luteinizing hormone (hLH) is important for the detection and follow-up of patients with pathological processes of the reproductive axis. Detection of the uncombined form of the beta-subunit of hLH, or free hLH beta, has proved to be of clinical interest in the recognition of gonadotroph adenomas. As no monoclonal antibody specific for the free hLH beta is at present available, we elicited monoclonal antibodies using free hLH beta as an immunogen. An antibody, named BLH01, was selected for its specific binding to the free beta-subunit, and its antibody-binding site was characterized at the molecular level, emphasizing the importance of amino acid residues located between the disulfide-bonded Cys93 and Cys100. This region has been demonstrated as being particularly critical for the specific binding of lutropic hormones to their receptors. Topographic assignment of the epitope recognized by BLH01 was then achieved by cross-matching studies based on a library of antibodies directed to hLH beta, and the location of some epitopes on the three-dimensional model of the beta-subunit is proposed. A two-site immunoassay based on BLH01 as capture antibody was then developed. This assay, using BLH01, may constitute a simple, sensitive and highly specific procedure for assessing the clinical usefulness of measuring free hLH beta, particularly for the diagnosis and follow-up of patients presenting with pituitary adenomas.

Adenoma↗
PubMed1995

Cloning of a new member of the insulin gene superfamily (INSL4) expressed in human placenta.

A new member of the insulin gene superfamily was identified by screening a subtracted cDNA library of first-trimester human placenta and, hence, was tentatively named early placenta insulin-like peptide (EPIL). In this paper, we report the cloning and sequencing of the EPIL cDNA and the EPIL gene (INSL4). Comparison of the deduced amino acid sequence of the early placenta insulin-like peptide revealed significant overall and structural homologies with members of the insulin-like hormone superfamily. Moreover, the organization of the early placenta insulin-like gene, which is composed of two exons and one intron, is similar to that of insulin and relaxin. By in situ hybridization, the INSL4 gene was assigned to band p24 of the short arm of chromosome 9. RT-PCR analysis of EPIL tissue distribution revealed that its transcripts are expressed in the placenta and uterus.

Amino Acid Sequence↗
PubMed1995

Expression of human chorionic gonadotropin beta subunit genes in superficial and invasive bladder carcinomas.

Increased serum levels of human chorionic gonadotropin beta subunit (hCG beta) were described previously in patients with bladder cancer. To obtain insight into such production of hCG beta, the expression of hCG beta 7, 8, 5, and 3 genes in bladder carcinomas and normal urothelia was investigated by reverse transcription PCR. Surprisingly, hCG beta mRNAs were detected in both normal urothelial and carcinomatous cells. However, tumor progression was characterized by different patterns of transcription of the hCG beta genes; the beta 7 gene was the only gene transcribed in normal urothelia and Ta tumors included in this study, whereas in addition to beta 7, genes beta 5, 8, and 3 were transcribed in T1 to T4 tumors. Moreover, transcription levels of the latter three genes increased with the stage of the disease. These observations showed that dramatic modifications in the expression of hCG beta genes accompany progression of bladder carcinomas.

Base Sequence↗
PubMed1995

[Cancer vaccines: scientific bases and clinical developments].

More than 100 years ago, William Coley published the first report of tumor regression induced by immune system activation. Since that time, numerous clinical trials of cancer vaccines have been carried out. However, active immunotherapy has not yet become an established modality of cancer therapy. Recently, striking progress has been accomplished in both the understanding of antitumor immune response and the characterization of tumor rejection antigens in human species. This progress serves as a basis for the development of new cancer vaccines.

Animals↗
PubMed1994

Distribution of VRA09, an ovarian tumor-associated antigen, distinguishing borderline serous tumors.

We recently reported the characterization of an antigen designated VRA09, identified by a monoclonal antibody and overexpressed on the surface of vincristine-resistant human ovarian carcinoma cells. In the present study, we analyze the distribution of this antigen in normal and tumor tissues. Its pattern of expression appears to differ from that described for other drug-resistance- and/or tumor-associated antigens. In normal tissues, the antigen has a restricted histological distribution and appears to be localized in mesoderm-derived tissues. In tumor tissues, VRA09 expression was mainly detected in serous ovarian tumors. Indeed, VRA09 is strongly expressed in papillary serous cystadenocarcinomas and their metastases, and more specifically in the basement membranes of serous tumors of borderline malignancy. In contrast, no immunostaining was observed in normal ovarian tissue or benign tumors. The detection of this antigen may help to identify serous ovarian tumors by distinguishing tumors of low malignancy from cystadenocarcinomas.

Antibodies, Monoclonal↗
PubMed1994

Cloning and sequencing of the equine testicular follitropin receptor.

To investigate the possibility that specific structural determinants within the equine follitropin receptor (eFSHR) are critical to the enhanced specificity of this receptor compared to other FSHRs, we used the RACE-PCR technique to clone the eFSHR from equine testis. Sequence analysis revealed that the eFSHR is highly homologous to other mammal FSHRs, but it presents 10 unique amino acid residue replacements in the extracellular domain. Furthermore, a potential N-glycosylation site was detected at a position not encountered in other receptors. Northern blot analysis identified three transcripts of 4.2 kb, 2.3 kb and 1.0 kb in horse testis.

Amino Acid Sequence↗
PubMed1993

Expression of the human follicle-stimulating hormone receptor in the baculovirus system.

Human follitropin receptor (hFSHR) cDNA was inserted into the genome of Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda (Sf9) insect cells expressed the hFSHR at the membrane level, 2-3 days after infection. Ligand binding analysis demonstrated saturable, high-affinity binding of 125I-labeled recombinant hFSH (Kd = 0.17 nM) as well as binding specificity for both human and equine FSH. Interestingly, a highly purified preparation of equine choriogonadotropin was found to compete with the binding of 125I-rhFSH. Finally, Western-blot analysis, using a monoclonal antipeptide antibody directed to the 178-206 region of the hFSHR, revealed two bands migrating at M(r) 77 and 130 kDa.

Animals↗
PubMed1993

[Biological markers of cancer. Critical study].

The clinical use of tumor-associated markers still raises several problems, due to the lack of specificity of most biological markers and to insufficient evaluation of their true benefit for the patients. Only two markers, calcitonin and alpha-fetoprotein, markers of medullary thyroid carcinomas and of hepatocellular carcinomas respectively, have been proved useful in screening high risk populations for tumors. The usefulness of the prostate specific antigen in screening for prostatic cancer is still debated. Human chorionic gonadotropin and its free beta subunit are useful in the early detection of testicular cancer. Other biological makers, such as CA 15-3 for breast cancers, CA 19-9 for either gastric or pancreatic cancers, anc CA 125 for ovarian tumors are useful mostly in the follow-up of these tumors. Finally, measurements of tumor markers and analysis of their results must be performed by biologists or physicians who use tumor-associated markers routinely.

Biomarkers, Tumor↗
PubMed1993

Influence of protein-quaternary structure on antigen processing.

T cell recognition of the quaternary structure of human chorionic gonadotropin (hCG), resulting from the association between its alpha (hCG-alpha) and beta (hCG-beta) subunits, was analyzed using hCG-alpha and hCG-beta T cell hybridomas produced in BALB/c mice. First, the fine specificity of these T cell hybridomas was determined, enabling us to divide hCG-alpha-specific T cell hybridomas into two groups. Group I recognized the hCG-alpha(61-81) region, and group II responded to the hCG-alpha(50-70) part of the molecule. Two groups of hCG-beta-specific T cell hybridomas, designated groups III and IV, were analyzed and found to respond to the C- and the N-terminal parts of the hCG-beta(1-22) peptide, respectively. Moreover, we observed that the nature of APC influenced Ag recognition by hCG-beta T cell hybridomas from group IV, but not by other selected T cell hybridomas. We then showed that recognition of the hCG alpha/beta dimer by alpha-specific T cell hybridomas was dramatically reduced compared to both free hCG-alpha and heat-dissociated hCG alpha/beta molecules. In contrast, hCG-beta hybridomas exhibited comparable responses to the free beta subunit and the hCG dimer. Experiments using a dimeric molecule assembled from the alpha-subunit of human follicle-stimulating hormone, which is identical to hCG-alpha, and the beta-subunit of human follicle-stimulating hormone, which is homologous to hCG-beta, confirmed that the three-dimensional structure of the complex rather than the primary structure of the beta-subunit plays a critical role in the processing pathway. Finally, kinetic experiments showed that the presentation of hCG-alpha T cell epitopes differed depending upon whether the alpha-subunit was in its free or combined form. In contrast, the kinetic expression of hCG-beta T cell epitopes appeared to be independent of the quaternary structure of hCG. Thus, conformational alterations resulting from the alpha/beta subunit association mainly influenced processing of the alpha-subunit in its complexed form, rather than processing of the combined beta-subunit. The effect of protein-quaternary structure on T cell recognition may represent a new element in our understanding of the processing and presentation of oligomeric molecules.

Animals↗
PubMed1993

Deficient antigen processing of a protein quaternary structure can be overcome by receptor-mediated uptake.

Human chorionic gonadotropin (hCG) is a dimer of non-covalently associated alpha (hCG-alpha) and beta (hCG-beta) subunits. This molecule was used to study whether receptor-mediated uptake influences the presentation of a protein quaternary structure. Unprimed splenocytes and a B cell lymphoma were capable of presenting only the free (hCG-alpha) but not the combined (hCG) alpha subunit to hCG-alpha T cell hybridomas, while hCG-alpha-primed lymph node cells (LNC) responded to both hCG-alpha and hCG. As antigen (Ag)-specific antigen-presenting cells (APC) present in the hCG-alpha-primed LNC population may be potentially effective for presenting hCG, we investigated the role of specific Ag capture, through mIg and Fc gamma R, in the processing and presentation of hCG and hCG-alpha to HAG5, a T cell hybridoma directed against the immunodominant region (amino acids 61-81) of hCG-alpha. Results showed that only B cells bearing membrane immunoglobulin capable of recognizing hCG-alpha and hCG, and present in hCG-alpha-primed mice, were extremely effective in presenting the free as well as the combined alpha subunit. The effect of FcR-mediated uptake was analyzed using a B cell line transfected with the Fc gamma RII-B2 gene to present immune complexes of either hCG-alpha or hCG. We found that hCG-alpha and hCG were presented equally well, whatever the Ag-binding site of each antibody to hCG or its alpha subunit. Using HBG 6, an hCG-beta T cell hybridoma, we performed similar experiments with the Fc gamma RII-B2 cell line and determined that the potentiation of hCG presentation to HBG 6 was similar to that observed with HAG 5. Then kinetic experiments were performed to examine the effect of Ag uptake through FcR on processing. Results demonstrated that the uptake pathway drastically influenced the expression of alpha T cell determinants in the alpha/beta dimer. In addition, treatment with cycloheximide, a protein synthesis inhibitor, only impaired the ability of APC to present specifically captured Ag. Thus, the processing pathway for specifically captured Ag might be different from the pathway used to process nonspecifically captured Ag. This observation might explain why receptor-enhanced uptake bypasses the inefficient processing of the hCG quaternary structure and enables similar efficiency in the presentation of alpha and beta T cell specificities. These findings provide new insight into the antigenicity of oligomeric molecules, which is modified whether antigen capture is specific or not.

Animals↗
PubMed1993

Immunochemical studies of equine chorionic gonadotropin (eCG), eCG alpha, and eCG beta.

The equine (e) placental glycoprotein hormone eCG plays a critical though not completely understood role during the first trimester of gestation in mares. In the present work, we have developed immunoradiometric assays (m-IRMAs) for detection of eCG, eCG alpha, and eCG beta using combinations of monoclonal antibodies (mAbs) specific for epitopes that reside on free and/or combined subunits. The free eCG alpha m-IRMA was based on AHT20 mAb, specific for the free alpha-subunit of all species, and 125I-labeled ECG01 mAb, which recognizes both free and combined alpha-subunit from equine and primate species. The free eCG beta was measured by the combination of FBT11 mAb, which binds to free beta-subunit from human and equine species, and radiolabeled 518B7 mAb, which detects CG/LH from diverse mammalian species. This assay provided a better sensitivity for eLH beta than for eCG beta. However, after treatment with neuraminidase, the latter molecule was recognized as well as eLH beta, indicating that the carbohydrate structure influenced the binding of mAbs. Detection of eCG was based on the combination of ECG01 mAb (anti-alpha) as capture antibody and 125I-labeled 518B7 mAb (anti-beta). Using such assays, we measured plasma and urinary concentrations of both eCG and free subunits in pregnant mares from mating to day 90 of gestation. eCG was constantly detectable in the serum between days 40-90, as previously reported, but small amounts of the dimeric hormone in the urine were also detectable. Further, m-IRMA specific for the free beta-subunit showed that low levels (5-100 ng/ml) of eCG beta may coexist with eCG in serum and urine during early pregnancy in mares. In contrast, free eCG alpha subunit was undetectable during the first 10 weeks of gestation. These results suggested that eCG and free subunit production in pregnant mares at the beginning of gestation is similar to that observed in pregnant women. These immunoassays, specific for either intact hormone or its free subunits, constitute useful diagnostic tools for investigating reproductive problems in mares.

Animals↗