[Duodenal duplication in adults. Laparoscopic treatment]. / Duplication duodénale chez l'adulte. Traitement coelioscopique.

The authors report a case of duodenal duplication in a adult. This lesion is rare. The clinical course is dominated by a risk of cancer. These lesions should be recognized to avoid a mutilating excision procedure in favour of a conservative procedure. The originality of this case is the laparoscopic approach which confirmed the diagnosis by the elective implantation on the first part of the duodenum. This approach, by mobilisation of the duodenum, allows excision by a very short laparotomy.

Comparison of different immunoenzymatic methods for the determination of the fine specificity and affinity constants of polyclonal antibodies against pseudopeptide haptens.

We evaluated two phosphinopeptides and one phosphonopeptide, which are transition state analogs of a proteolytic reaction, for their ability to generate murine polyclonal antibodies. The specificity of these antisera was determined by indirect and competitive ELISA. Cross-reactivity analysis by these ELISA showed that the antisera recognized selectively haptens containing a phosphate group. The pseudopeptides recognized by the antisera in the indirect ELISA were not the same, however, as those recognized in the competitive ELISA. The differences between these results are probably due to the presentation forms of the hapten, i.e., passively adsorbed in the former ELISA format and soluble in the latter. The affinity of the antibodies was then determined by using two methods based on competitive ELISA, one described by Friguet et al. and the other by Seligman. The dissociation constant (Kd) values calculated by the two methods, for an antiserum and its homologous hapten, are similar. However, only the middle portion of the inhibition scale in Seligman's method gave access to reliable values. Nevertheless, the Seligman representation allowed us to underscore the large range of affinity constants of the polyclonal antibodies.

Importance of hydropathic complementarity for the binding of the neuropeptide substance P to a monoclonal antibody: equilibrium and kinetic studies.

In a previous study (Boquet et al., 1995, Molec. Immunol. 32, 303-308) we observed remarkable inversions of hydropathic profiles between complementarity determining regions (CDRs) of an anti-substance P monoclonal antibody (SP31) and the corresponding 5 amino acid C-terminal peptide epitope. Here we demonstrate the importance this hydropathic complementarity by measuring the immunoreactivity of SP-related peptides which have been modified in their C-terminal parts so that hydropathic profile has been conserved (by substituting amino acids in the epitope) or modified (by mixing the sequence of amino acids in the epitope). Experiments performed in equilibrium conditions using a competitive enzyme immunoassay showed that most of the peptides conserving the hydropathic profile of SP epitope were recognized by mAb SP31 (even if marked variations in affinity were observed), while those for which the hydropathic profile was modified exhibited very low or undetectable affinity. The kinetic parameters (ka and kd) of peptide-antibody interactions were determined using Surface Plasmon Resonance technology (BIACORE 2000). These measurements showed that all peptides recognized by mAb SP31 had similar association rate constants (close to 2 x 10[5] M[-1] s[-1]), differences in binding affinities essentially resulting from differences in dissociation rate constants (ranging from 1.61 x 10[-4] to 1.15 x 10[-1] s[-1]). From these results, it was concluded that hydropathic complementarity between the epitope and the paratope could be a necessary but not sufficient condition for establishing high-affinity binding. We hypothesize that hydropathic interactions may play a critical role during the first contacts between antibody CDRs and the peptide, possibly by favouring reorganization of water molecules at the antibody-peptide interface.

Development and standardization of an immuno-quantified solid phase assay for HIV-1 aspartyl protease activity and its application to the evaluation of inhibitors.

The catELISA technique was modified and standardized for measuring HIV-1 aspartyl protease activity and evaluating the potency of synthetic peptide inhibitors. This immuno-quantified solid phase assay combines the use of an immobilized C-terminal biotinylated peptide as substrate, a crude enzyme preparation, and a highly specific antiserum elicited against the C-terminal product of the enzyme reaction. A standard curve of this C-terminal product was constructed to determine the enzyme activity. This assay, which requires less enzyme and substrate, is more sensitive than the conventional HPLC method. The amounts of C-terminal peptide produced in solution as determined from ELISA and HPLC standard curves were comparable. Analogues of peptidomimetics designed in our laboratory were assayed for their potency to inhibit the enzyme. One of them, H4, which is a hydroxyethylamine isostere of the Phe-Pro peptide bond, was a powerful inhibitor.

Characterization of a monoclonal antibody produced in an attempt to mimic the active site of HIV aspartyl protease using haptens based on inhibitor models.

The high binding affinity and specificity of antibodies for a great variety of ligands has been widely exploited in structure-activity relationship studies of biomolecules and more recently in the development of new catalysts for several chemical reactions. It is assumed that antibodies generated against haptenic protease inhibitors would recognize both these haptens and the substrate of the model proteolytic reaction. We have produced antibodies against HIV PRp12 aspartyl protease substrate analogues, chemically modified at the scissile bond, Phe-Pro. Identical chemical modifications have been reported for related HIV protease inhibitors. We finally selected an anti-hapten monoclonal antibody that specifically recognized the substrate and those haptens with both the phenylalanyl side chain and the prolyl pyrrolidine ring. This selectivity of recognition suggests that such an antibody might mimic the catalytic site of the model protease.

New antigenic clusters on human thyroglobulin defined by an expanded panel of monoclonal antibodies.

Twenty-seven hybridomas secreting monoclonal antibodies (mAb) directed against new antigenic clusters on human thyroglobulin (hTg) were obtained by fusion of the mouse myeloma P3-X63-Ag8 653 with spleen cells from BALB/c mice immunized with a mixture of hTg and six anti-hTg mAb with the aim of masking the corresponding antigenic clusters previously reported. Fourteen mAb were selected, produced in ascitic fluid and characterized. All these mAb were of the IgG1 subclass. Five new antigenic clusters on the hTg molecule were defined by the 14 mAb, extending the initial antigenic map of hTg to 11 clusters. These mAb were used in an attempt to probe the interaction between hTg and the autoantibodies from patients with Hashimoto's thyroiditis who do not recognize antigenic cluster II, a cluster whose recognition by anti-hTg autoantibodies is significantly associated with thyroid disorders.

Localization of a vitamin-D-binding protein interaction site in the COOH-terminal sequence of actin.

The serum vitamin D binding protein is the carrier of vitamin D and its derivatives in the plasma. One of the known roles of this protein is to sequester monomeric actin in the blood, therefore implicating this protein in actin elimination. However, its binding site at the surface of actin is poorly delimited. We report here the results of a study which locates, using several actin fragments together with immunological probes, a vitamin D binding protein site near the COOH-terminal extremity. Thus, the interface is delimited by the sequence 360-372 in subdomain I of actin.

Distribution of nonmuscle actin during Xenopus laevis development.

Using antisera specific for the N-terminal peptides of nonmuscle isoforms of actin we studied these proteins in the developing egg and embryo of Xenopus laevis. The antisera did not crossreact with muscle actin and did not react with the other nonmuscle isoform. By immunofluorescence we found a large maternal actin store in the oocyte, in the Balbiani body and the nucleoli, respectively. In the blastula, nonmuscle actin was preferentially expressed in the mitotic apparatus of the individual blastomeres. A gradual decrease in the amount of these isoforms in muscle tissue could be observed during development, apparently corresponding to an increase in the concentration of muscle specific isoforms. No differences between the distribution of the two isoforms could be found. The results would seem to indicate that development of the embryo is accompanied by a decline in the concentration of cytoskeletal actin isoforms down to an almost background level, accompanied by an increase in the concentration of the muscle specific isoforms. This points to potential function of nonmuscle actins in the early embryo.

Definition of a Ca2(+)-sensitive interface in the plasma gelsolin-actin complex.

Gelsolin is a Ca2(+)-dependent protein which severs actin filaments, caps their fast-growing ends and promotes nucleation. We report here results that delimit one of the interfaces between serum gelsolin and actin monomer. An actin-derived synthetic peptide (amino acids 305-326 of actin) coupled to a hydrophilic resin was tested for its possible interaction with gelsolin. We selected this sequence because it corresponds to a region implicated in the gelsolin-actin complex in a previous work [Boyer, Feinberg, Hue, Capony, Benyamin & Roustan (1987) Biochem. J. 248, 359-364]. We showed that this actin sequence is located at the surface of the actin molecule and observed a Ca2(+)-sensitive binding of gelsolin to this actin-derived peptide. In addition, by using a chymotryptic digest of gelsolin, we reported that only the C-terminal half of gelsolin interacts with the actin-(305-326)-peptide. These results that the Ca2(+)-sensitive interface includes both amino acids 305-326 of actin and probably amino acids 660-738 of gelsolin.

Synthesis of fragment 7-20 of human gamma-interferon linked through a methionine residue to polyacrylic resin: use of the adduct as an immunogen.

A peptide corresponding to the amino acid sequence of human gamma-interferon, residues 7-20, was synthesized by a solid phase procedure using polyacrylic resin and methionine as anchor. Fluorenemethoxycarbonyl and t-butyloxycarbonyl strategies were compared. After completion of the synthesis, side chain deprotections were performed on the peptide still attached to the resin. The purity of the peptide linked to the matrix was determined by amino acid analysis and solid phase sequencing. The presence of methionine allowed the peptide to be detached from the support by CNBr cleavage. The immune responses of the free peptide, the peptide linked to bovine serum albumin and the peptide resin adduct were compared. Peptide bound to polyacrylamide gave similar results to those obtained using classical immunization procedures.

Production of oligoclonal antibodies directed to the N-terminal of smooth muscle alpha actin using peptidyl-polyacrylic resins as direct immunogens.

Polyacrylamide-based solid-phase supports were used in the production of a synthetic peptide analogous to amino acid sequence 1-9 from bovine aortic actin. Different peptidyl-resins were prepared and directly used to induce an antibody response in rabbits. Antisera that recognized the resin-bound peptide were compared. Some of them reacted with native aortic actin; this response was highly specific for the smooth muscle alpha actin isotype. We also immunized rabbits with the 1-9 peptide linked to bovine serum albumin. In this case, antisera were less reactive with native aortic actin, and cross-reacted with the two closely related smooth muscle alpha and macrophage actin isotypes. This method provides a rapid and simple procedure for generating peptide immunogens and antibodies to them, by the combination of peptidyl-resin immunizations and resin-bound peptide immunoassays.