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C Bohuon

Publications and source records attributed to C Bohuon.

At least 73 records · Page 4Linked to original sources
PubMed1990

Changes in brain neuropeptide Y induced by cholecystokinin peptides.

Cholecystokinin (CCK) and neuropeptide Y (NPY) are two peptides with opposite effects on the regulation of feeding behaviour. The possible interaction between these two systems has always been controversial. In this study, rat brain NPY levels were assayed after treatment with CCK 8 S and with a potent CCK agonist (Boc-(Nle 28-Nle 31)-CCK 26-33). CCK 8 S and its agonist analogue (50 micrograms/kg i.p.) both decreased hypothalamic and hippocampal NPY levels. This result suggests a negative relationship between NPY and CCK-peptides which is not surprising given their opposite role in the control of feeding. The hypothalamus and secondarily the hippocampus appear to be the site of this interaction; no change in NPY levels was observed in other brain areas (striatum and cortex). The same pattern of variation was found in the plasma, suggesting a direct release from the brain via a mechanism which remains to be investigated. The effect appeared later with the CCK analogue than with CCK 8 S itself; this is not surprising with regard to other behavioural and biochemical effects of the analogue and provides further characterization of its action.

Animals↗
PubMed1990

Structural probing of human lutropin using antibodies raised against synthetic peptides constructed by classical and multiple antigen peptide system approaches.

Antibodies were elicited against a synthetic peptide which encompassed two different regions of the human lutropin beta-subunit (hLH-beta). These antibodies were raised against either the peptide which was assembled using a conventional approach and conjugated to the tetanus toxoid, or with the peptide assembled using the multiple antigen peptide system approach. Automated simultaneous synthesis of the two forms of the immunizing peptide was successfully achieved. Animal injected with the peptide conjugated to tetanus toxoid produced high titers of antibodies to the synthetic peptide, but did not bind to the native hLH-beta subunit. In contrast, antisera induced by the peptide in its MAP form displayed reactivity with both the peptide and the native hLH-beta subunit; these latter antisera appeared to preferentially recognize the beta 47-55 portion of the molecule and were able to bind to the beta-subunit of human choriogonadotropin. Present results demonstrate that the beta 47-55 region is accessible to antibody binding and appears to be located at the surface of both hLH-beta and hLH. Moreover, this study confirms that the MAP approach provides a chemically unambiguous method for obtaining antibodies of predetermined specificity, capable of recognizing cognate sequences of various native proteins.

Amino Acid Sequence↗
PubMed1990

Occurrence of fragmentation of free and combined forms of the beta-subunit of human chorionic gonadotropin.

In an attempt to further study various fragments of free and combined forms of hCG beta present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCG beta-(111-116) portion (monoclonal antibody FB12) antiserum to the hCG beta(8-16) portion or antiserum which was specific for fragments ending at residue 47. Results observed in a crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCG beta nicked subunit (beta NS) [hCG beta-(48-145)], three other fragments of mol wt 18,000 (F1), 16,500 (F2), and 12,000 (F3) were detectable after cleavage of disulfide bonds. Both the immunoreactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCG beta-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the beta NS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free beta-subunit contained beta NS. Furthermore, free hCG beta present in those urine samples appeared to be extensively, if not totally, nicked. Results observed in urine were confirmed using separation of hCG from its beta-subunit by a two-step chromatography procedure, identification of hCG and hCG beta immunoreactive peaks by specific monoclonal immunoradiometric assay, and analysis of resulting preparations by one-dimensional electrophoresis under reducing conditions, followed by Western immunoblotting with FB12. This latter protocol was also used to investigate the presence of beta NS in sera of four patients with choriocarcinoma tumors. In those sera, hCG appeared to be nicked. This study demonstrates that the beta-subunit of hCG is modified by multiple fragmentations.

Amino Acid Sequence↗
PubMed1990

[Use of monoclonal antibodies in the treatment of cancer of the pancreas: towards new progress?].

Pancreatic cancers rank amongst the most deadly malignant diseases with a 5 year-survival percentage less than 2% and few therapeutic approaches are hitherto available. This study presents the recent attempts to construct antibodies for therapy. The characterization of pancreatic tumor-associated antigens which might serve as target antigens for antibody therapy is the limiting factor before considering the treatment of pancreatic cancer with antibodies. Antigens such as CA 19-9, BW 494 and DU-PAN-2 have been reported to be associated with pancreatic cancers. However, monoclonal antibodies directed to these antigens was not proven to be specific enough to warrant therapeutic utilization and new tumor-associated antigens must be identified. Remarkable progress has been made recently in the construction of antibodies for therapy. Amongst these antibodies are "chimeric" antibodies, antibody heteroconjugates or hybrid antibodies. The in vivo utilization of those antibodies may well result in effective tumor-cell destruction.

Antibodies, Monoclonal↗
PubMed1989

Identification and measurement of calcitonin precursors in serum of patients with malignant diseases.

Previous studies have suggested that molecular species larger than the mature calcitonin (CT) are produced by tumors of different origin. In order to study these species, we developed a monoclonal immunoradiometric assay for calcitonin precursors (CT-pr). This assay was based on both monoclonal antibody KC01 directed to the 1-11 region of katacalcin and monoclonal antibody CT08 directed to the 11-17 portion of CT. The sensitivity of this monoclonal immunoradiometric assay for CT-pr was less than 100 pg/ml. Only one of 131 healthy subjects had CT-pr serum levels greater than 100 pg/ml; this value was therefore selected as the standard serum value in healthy individuals. CT-pr was present in the serum of seven of ten patients with advanced renal failure and in that of 21 of 52 patients (40%) with benign liver disease but was undetectable in sera of patients with other benign diseases. The serum CT-pr level was correlated with that of mature CT in patients with medullary carcinoma of the thyroid. In contrast, the serum CT-pr level was frequently elevated in the absence of a detectable CT level in patients with various malignant tumors and, particularly, in those with either tumors of the neuroendocrine system (60%) or hepatocellular carcinomas (62%). CT-pr was detected in tumor extract from a patient with a hepatocellular carcinoma. Moreover, hybridization experiments with total RNA extracted from this tumor demonstrated the presence of RNAs hybridizing with complementary DNA encoding for common region, calcitonin, and katacalcin sequences. These results show that CT precursors are excreted by numerous cancers and might well be useful biological markers for the follow-up of productive tumors.

Antibodies, Monoclonal↗
PubMed1989

In vivo toxicity of cisplatin and carboplatin on the Leydig cell function and effect of the human choriogonadotropin.

In order to characterize the respective toxicity of cisplatin (CDDP) and carboplatin (CBDCA) on the male reproductive system, we have investigated their in vivo effects on the steroidogenesis function of rat leydig cells. Animals were treated at the respective LD50 of platin compounds, and we analyzed plasmatic testosterone level, microsomal cytochrome P-450 and platinum concentrations in the testis. CDDP induces a dramatic change in both the testosterone level and the microsomal cytochrome P-450 concentration. In contrast, CBDCA was found to be less toxic than CDDP, probably due to its different accumulation at the testis level. We also investigated the potential action of human chorionic gonadotropin which allows a full restoration of the steroidogenesis function.

Animals↗
PubMed1989

Monoclonal antibodies directed to human and equine chorionic gonadotropins as probes for the topographic analysis of epitopes on the human alpha-subunit.

To improve our knowledge of the structural features of the alpha-subunit of hCG we have studied the antigenic site recognized by monoclonal antibody (MAb) ECG01 raised against equine CG (eCG) which binds to hormones and alpha-subunits from human and equine species. We have also delineated regions of hCG alpha comprising the epitope recognized by HT13 which was raised against hCG and binds to hCG and hCG alpha. To define the residues involved in the antigenic sites recognized by ECG01 and HT13, we have studied the reactivities of these two MAbs with native or chemically modified LH and CG with subunits from equine, human, or ovine (o) species or with synthetic peptides analogous to various portions of hCG alpha. We have also compared these reactivities with those displayed by MAbs AHT20 and FA 36, whose epitopes have been previously described; anti-hCG alpha MAb AHT20 is specific for the free alpha-subunits of various species and recognizes residues localized to the 36-41 region of hCG alpha, whereas antipeptide MAb FA36 binds to the 87-92 carboxyl-terminal part of hCG alpha. Our results show that the epitopes of HT13 and ECG01 are 1) probably discontinuous, as these MAbs did not bind to the reduced and S-carboxymethylated hCG alpha; and 2) constituted by residues borne on the 1-35 and 52-86 sequences, as they do recognize the hCG alpha core missing the 36-51 portion, yet do not recognize hCG alpha-(87-92) region recognized by FA36. The comparative studies performed with specific two-site immunoradiometric assays to determine the interspecies cross-reactivities of the MAbs allow us hypothetical assignment of residues on the primary structure of hCG alpha. The antigenic site recognized by ECG01 might include two to six amino acids, four of these residues being located at inverted places compared to those of oLH alpha (Asp6/Gly22 and Arg67/Lys75). These residues present important charged functional groups highly conserved among evolutionarily related variants, and it is likely that they are located on the surface of both the intact hormone and its alpha-subunit. Three peptidic portions of hCG alpha, 16-17, 64-66, and 73-76, respectively, might be involved in the epitope recognized by HT13, although we could not rule out the possibility that other residues were also involved in the antigenic site. These observations allow us to identify several residues as potentially constituting the epitopes recognized by two MAbs on both hCG and hCG alpha.

Amino Acid Sequence↗
PubMed1989

Plasma neuropeptide Y concentrations in patients with neuroendocrine tumors.

In order to develop an immunoradiometric assay for human neuropeptide (hNPY), a recently discovered and potent vasoconstrictor 36-amino-acid peptide, we used hNPY and some of its subpeptides to prepare monoclonal anti-NPY antibodies. Two monoclonal antibodies with high affinity for hNPY, that is affinity constants in the range of 10(10) mol/L-1, which respectively, reacted with the 9-18 portion and the 32-36 portion of hNPY were used in the immunoradiometric system. The assay was highly specific, NPY-related peptides such as pancreatic polypeptide and peptide YY not being detected. The lower limit of sensitivity was 0.5 pmol/L. In 303 normal subjects, plasma NPY concentrations were less than 0.5 pmol/L in 67%, 0.5 to 5.0 pmol/L in 25% and 5.1 to 30 pmol/L in the remaining 8%. A value of 7.5 pmol/L (95th percentile value in the normal group) was considered as the upper limit of normal. Among 111 patients with various neuroendocrine tumors, elevated plasma NPY concentrations were found in patients with pheochromocytomas and neuroblastomas, the highest plasma levels being found in patients with malignant pheochromocytomas. We conclude that patients with neuroendocrine tumors, especially secreting and or malignant tumors of the sympathochromaffin system, often have elevated plasma NPY concentrations.

Adolescent↗
PubMed1989

The development of a two-site enzyme immunometric assay (EIA) for calcitonin and its application in the measurement of the hormone in normal subjects, MTC-patients and post-menopausal women.

We have developed a sensitive, two-site enzyme immunometric assay (EIA) using high affinity monoclonal antibodies for the measurement of immunoreactive human calcitonin (i-hCT) and compared the performance of the assay with that of the conventional radioimmunoassay (RIA). The sensitivity of an overnight EIA (2 pmol/l) was comparable with the long incubation (7 days) RIA. Both, the EIA and the RIA have been applied for the measurement of i-hCT in three groups: normal volunteers (n = 53), post-menopausal women (n = 25) and patients with MTC (n = 20). The results showed that in all three groups, the level of i-hCT was somewhat lower than that measured by RIA due to its greater specificity. The EIA can provide an attractive alternative to the conventional RIA for both routine diagnostic work and physiological studies.

Antibodies, Monoclonal↗
PubMed1989

[The value of tumor markers in digestive oncology].

Over the last few years, many tumor markers have been proposed to clinicians but only a limited number of them meet the necessary criteria to be useful for either screening, diagnosis, prognosis or follow-up of gastrointestinal (GI) tumors. Both CEA and Ca 19-9 have proven to be clinically useful for the detection of recurrent tumors. AFP remains the most useful marker for the follow-up of hepatocellular carcinoma (HCC). Its interest for the early detection of primary tumor is debated. Recent data suggest that assays based on monoclonal antibodies to AFP could be used for detection HCC in high risk populations. Decarboxy-prothrombin assay may be a complement to the AFP test in this localization. In addition to GI hormones, serotonin and urinary 5HIAA, Neuron Specific Enolase appears to be a valuable marker for the follow-up of neuroendocrine tumors of the GI tract. Only a few of the new tumor-associated antigens detected by monoclonal antibodies, appear to be promising clinical ly e.g. Ca50 TAG-72, PAO. Monoclonal antibodies to tumor-associated markers have also been used with other techniques: Immunohistochemistry: this technique is useful to the pathologist for the diagnosis of undifferentiated tumors by demonstrating the presence of specific antigens on tissue samples. Immunoscintigraphy: it can be useful for the detection of either metastases of recurrences of colorectal cancer by using anti-ACE antibodies labeled with Iodine 131 iodine 123 or indium 111. However immunoscintigraphy is less sensitive than both ultrasonography and CT scan for localizing hepatic metastases. At the present time the best indication of this method remains the diagnosis of pelvic recurrences.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1988

Monoclonal antipeptide antibodies as tools to dissect closely related gene products. A model using peptides encoded by the calcitonin gene.

We have explored the possibility of using mAb as tools for distinguishing between closely related gene products. We utilized calcitonin (CT) gene products as a model, because this 32-amino-acid-amidated hormone is biosynthesized by post-translational processing of a larger precursor. By using CT as a hapten, we had previously identified a mAb (CT07) with restricted specificity to mature CT, and had shown that another mAb (CT08) directed to a different epitope bound to both CT and the CT precursor. In this study, we used synthetic peptides analogous to various regions of biosynthetic intermediates of CT as haptens, and generated a library of mAb which define distinct epitopes. First, we identified two separate epitopes located in either the 1-11 or the 12-21 region of the C-terminal flanking peptide of CT (katacalcin, KC), and which were recognized by mAb KC01 and KC04, respectively. Second, we identified a conformational epitope in the C-terminal region of the putative glycine-extended form of CT (CT-Gly). This epitope was recognized by mAb CT19 and was shared by mature CT but not by CT precursors. Third, we identified an epitope restricted to CT-Gly and recognized by mAb CT20. For dissecting between related products of the CT gene, we designed different monoclonal immunoradiometric assays (m-IRMA) based on CT08 as the radiolabeled indicator antibody. A first m-IRMA based on CT07 as the capture antibody specifically recognized mature CT and did not cross-react with CT precursors. Conversely, another m-IRMA with KC01 as the capture antibody was specific for CT precursors and did not cross-react with either mature CT or CT-Gly. A third assay based on CT20 as the capture mAb was specific for CT-Gly and was not affected by the presence of either CT precursors or mature CT. We also used these antibodies to demonstrate that neoplastic C cells incompletely released processed CT precursors in serum, in addition to mature CT. This study demonstrates that mAb can be used as tools to selectively recognize closely related gene products. These findings might be applied to the study of other molecules biosynthesized by enzymatic modifications of a larger precursor.

Amino Acid Sequence↗
PubMed1988

Characterization of a cleavage product in the human choriogonadotropin beta-subunit.

The various molecular forms of human chorionic gonadotropin present in a crude preparation of urine from pregnant women were analyzed by two-dimensional gel electrophoresis and immunoblotting with monoclonal antibodies directed to synthetic peptides corresponding to the carboxyl-terminal part of either the alpha or beta-subunit. Under reducing conditions, immunoblotting with antibodies directed to the beta-subunit revealed the presence of a low-molecular-weight material of 22 kDa. This molecular form had large heterogeneity, as analyzed by isoelectrofocusing; it was immunoreactive with antibodies directed to the 111-145 region. Using microsequencing techniques, we found that the fragment had a NH2 terminal portion corresponding to the sequence of the beta-subunit appearing from residue 48. Thus, the 22-kDa fragment comprises the 48-145 portion of the beta-subunit and is probably a cleavage product of the native protein with intrachain nicking.

Amino Acid Sequence↗
PubMed1988

Construction and clinical validation of a sensitive and specific assay for serum mature calcitonin using monoclonal anti-peptide antibodies.

Using calcitonin (CT) as an hapten, we have generated a library of monoclonal antibodies. Six monoclonal antibodies were developed and analyzed with respect to affinity and specificity for epitopes on CT by enzyme linked immunosorbent assay and radioimmunoassay. These antibodies were used in the construction and the optimization of a two-site monoclonal immunoradiometric assay (m-IRMA) for CT. We used a combination of two monoclonal antibodies to develop an assay which is rapid (overnight incubation), sensitive (10 pg/ml) and strictly specific for the mature form of calcitonin, ie the 32 amino acid-long polypeptide bearing a prolineamide as the C-terminal residue. This assay was utilized to demonstrate that mature calcitonin circulates as heterogeneous molecular species resulting from polymerization of calcitonin by disulphide linkage and/or by aggregation on irrelevant proteins. The clinical relevance of this assay was studied on a series of patients with medullary carcinoma of the thyroid (MCT) and the characteristics of the assay was compared with those of a conventional polyclonal radioimmunoassay. The m-IRMA for CT proved to be useful for both the diagnosis and the follow-up of MCT.

Adult↗
PubMed1988

Identification of a CD2- CD3+ T cell receptor-gamma+ peripheral blood lymphocyte subpopulation.

We have identified, in a healthy individual, a sub-population of human peripheral lymphocytes which surface express a CD3-TCR-gamma complex recognized by anti-Ti gamma A mAb, while being unreactive with a phycoerythrin-conjugated anti-CD2 antibody with T11/1 specificity. Further immunofluorescence analyses performed on uncultured cells indicated that such a putative CD2-CD3+ phenotype was restricted to a fraction of those T lymphocytes which carry a surface receptor of the "second family" (gamma/delta). The actual lack of CD2 expression was confirmed by a subsequent series of cloning experiments which showed that none of the three well characterized CD2 epitopic clusters, namely T11/1, T11/2, and T11/3, were detectable on the surface of the relevant cells. The cultured CD2-, CD3+/TCR gamma + lymphocytes were found to display, as well as their CD2+ counterparts, both non-MHC-restricted cytotoxic function and proliferative responses induced via the gamma receptor complex. In contrast, the proliferative capacity of the CD2-, CD3+/TCR-gamma + cells observed in a culture system designed for in vitro expansion of lymphocytes with undefined specificity was extremely limited. This may relate to an impaired interaction of the CD2- cloned lymphocytes with lymphocyte function-associated (LFA)3+ irradiated cells present in the feeder layer. Further characterization of such minor CD2- T lymphocytes subsets may help to better understand the biologic relevance of the CD2/LFA3 pathway of cell-cell interaction.

Adult↗
PubMed1988

Conjugates of elliptinium acetate with mouse monoclonal anti-alpha-fetoprotein antibodies or Fab fragments: in vitro cytotoxic effects upon human hepatoma cell lines.

Elliptinium acetate (EA) is a new anti-cancer compound displaying cytostatic activity against various malignancies including hepatoma. Using 3 hepatoma cell lines, we compared the in vitro activity of doxorubicin (reference drug), of EA and of conjugates made up with this latter drug and monoclonal antibodies (MAbs). The linkage was performed by a direct oxidation method. Specific immunoconjugates were prepared with an anti-alphafetoprotein (AFP) MAb (AF01) or its Fab fragment (Fab AF01). Non-specific conjugates were obtained with an anti-thyroglobulin MAb (TG01) or its Fab (Fab TG01). Direct membrane injury (51Cr-release), DNA and protein synthesis as well as AFP release were investigated for all compounds. Free EA displayed only weak activity on DNA and protein synthesis, at 10-fold higher molar concentration than doxorubicin. Conjugation of EA with whole AF01 allowed significant potentiation of protein synthesis inhibition without affecting the 3 other tests. In contrast, Fab AF01 x EA conjugates displayed a marked effect in the 4 tests; in particular, this conjugate was at least 100 times more efficient than any other compound when tested in the 51Cr-release test. Neither Fab AF01 nor free EA alone or in combination exhibited such an effect. Fab TG01 x EA conjugate was not directly cytotoxic but potentiated inhibition of DNA and protein synthesis between 2- and 10-fold. The mechanism of the direct cytotoxic effect of anti-AFP Fab x EA conjugate, which has never been described in any other immunodrug model, was investigated.

Alkaloids↗
PubMed1988

A sensitive and specific two-site enzyme-immunoassay for human calcitonin using monoclonal antibodies.

A highly sensitive, specific and rapid two-site enzyme-immunometric assay (EIA) for the measurement of immunoreactive (ir) human calcitonin (hCT) in human plasma was developed using high-affinity monoclonal antibodies. The assay was validated in terms of sensitivity, specificity and reproducibility and its performance compared with that of a radioimmunoassay (RIA) employing a polyclonal antiserum. The sensitivity of the overnight EIA (2 pmol/l) was comparable with the long-incubation (7 days) RIA. The overnight RIA had a sensitivity of 10 pmol/l. The inter- and intra-assay variations of the EIA were less than 12%. Some related and non-related peptides were compared with synthetic hCT for cross-reactivity in the assay and were found to be negative. The mean recovery of added synthetic hCT from plasma of normal volunteers was 96%. Both RIA and EIA have been applied to the measurement of ir-hCT in normal volunteers and in patients with medullary carcinoma of the thyroid. In both groups, the level of ir-hCT measured by EIA was found to be lower than that measured by RIA, presumably due to the ability of the more specific EIA to detect only the 'mature' form of the hormone. EIA offers an attractive alternative to the more cumbersome and lengthy RIA in current usage, with the added advantage of employing a non-isotopic label.

Alkaline Phosphatase↗