Biomedical subjects

J Hoebeke

Publications and source records attributed to J Hoebeke.

At least 19 recordsLinked to original sources

PubMed1995

Molecular mimicry between the immunodominant ribosomal protein P0 of Trypanosoma cruzi and a functional epitope on the human beta 1-adrenergic receptor.

Sera from chagasic patients possess antibodies recognizing the carboxy-terminal part of the ribosomal P0 protein of Trypanosoma cruzi and the second extracellular loop of the human beta 1-adrenergic receptor. Comparison of both peptides showed that they contain a pentapeptide with very high homology (AESEE in P0 and AESDE in the human beta 1-adrenergic receptor). Using a competitive immunoenzyme assay, recognition of the peptide corresponding to the second extracellular loop (H26R) was inhibited by both P0-14i (AAAESEEEDDDDDF) and P0-beta (AESEE). Concomitantly, recognition of P0-beta was inhibited with the H26R peptide. Recognition of P0 in Western blots was inhibited by P0-14i, P0-beta, and H26R, but not by a peptide corresponding to the second extracellular loop of the human beta 2-adrenergic receptor or by an unrelated peptide. Autoantibodies affinity purified with the immobilized H26R peptide were shown to exert a positive chronotropic effect in vitro on cardiomyocytes from neonatal rats. This effect was blocked by both the specific beta 1 blocker bisoprolol and the peptide P0-beta. These results unambiguously prove that T. cruzi is able to induce a functional autoimmune response against the cardiovascular human beta 1-adrenergic receptor through a molecular mimicry mechanism.

Amino Acid Sequence↗

PubMed1995

Analysis of human CD4-antibody interaction using the BIAcore system.

Interaction between CD4 cell surface protein and HIV-bearing gp120 has been described as the initial step for HIV entry into host cells. Some anti-CD4 antibodies were shown to inhibit this interaction. Biosensor studies using the BIAcore were performed to determine kinetic and thermodynamic parameters of the interaction of one of these antibodies (i.e. IOT4a, clone 13B8-2) with immobilized recombinant soluble CD4 (rsCD4). A non-linear regression method was used to analyze the sensorgrams, showing the existence of a double exponential time curve. A KA of 5.2 x 10(7) M-1 was calculated at 25 degrees C. The complex formation was exothermic (-4.5 kcal.mol-1( and entropically positive (+20 cal.mol-1.K-1). The reaction rate (0.234 x 10(5) M-1.s-1 at 25 degrees C) as well as the enthalpy change of the activated complex (+9.7 kcal.mol-1) are not compatible with a diffusion controlled reaction. The thermodynamic values calculated from equilibrium data corresponded to those calculated from kinetic data confirming the validity of the theoretical approach. As for most antigen-antibody interactions, complex formation was enthalpy driven. The overall positive entropy contribution to the stabilization of the complex is in contrast to that observed for the lysozyme-anti-lysozyme model and is probably due to electrostatic interaction between the epitope and the antibody combining site.

Antibodies, Monoclonal↗

PubMed1995

High prevalence of antibodies against beta 1- and beta 2-adrenoceptors in patients with primary electrical cardiac abnormalities.

OBJECTIVES: This study sought to determine the prevalence of autoantibodies directed against the beta-adrenoceptors in patients with primary electrical cardiac abnormalities, including atrial arrhythmias, ventricular arrhythmias and conduction disturbances, in the absence of any other cardiac abnormality. BACKGROUND: Using synthetic peptides corresponding to the predicted sequences for the second extracellular loop of the human beta 1- and beta 2-adrenoceptors as antigenic targets, autoantibodies directed against the beta-adrenoceptors were recently shown to occur in patients with idiopathic dilated cardiomyopathy and Chagas' heart disease. METHODS: Eighty-six patients (57 with primary electrical abnormalities, 29 with idiopathic dilated cardiomyopathy) and 101 healthy and cardiopathic control subjects were studied. Antibodies against the beta 1- and beta 2-peptides were detected with an enzyme immunoassay performed in blinded manner. In nine selected (seropositive) cases, the immunoglobulin G (IgG) fraction was tested for functional effects on the rate of beating of cultured neonatal rat cardiomyocytes. RESULTS: Antibodies recognizing the beta 1- and beta 2-peptides were found in 11 (52.3%) of 21 patients with ventricular arrhythmias (p < 0.01), 5 (35.7%) of 14 patients with conduction disturbances (p < 0.05), 3 (13.6%) of 22 patients with atrial arrhythmias (p > 0.05) and 11 (37.9%) of 29 patients with dilated cardiomyopathy (p < 0.05) compared with 15 (14.8%) of 101 control subjects. A rapid increase in the rate of beating of the cultured cardiomyocytes was induced by IgG from a selected group of patients, suggesting an agonist-like interaction with a functional epitope. This response was mediated by stimulation of both the beta 1- and beta 2-adrenoceptors in the patients with primary ventricular arrhythmias but only the beta 1-adrenoceptors in the patients with idiopathic dilated cardiomyopathy. CONCLUSIONS: Primary ventricular arrhythmias and conduction disturbances, like idiopathic cardiomyopathy, show a high prevalence of antibodies interacting with functional epitopes of the beta-adrenoceptors, suggesting a common or similar abnormal immunoregulatory process.

Adult↗

PubMed1995

Functional epitope analysis of the second extracellular loop of the human heart muscarinic acetylcholine receptor.

Two synthetic peptides corresponding to amino acids 172-181 and 169-193 of the second extracellular loop of the human M2 muscarinic receptor respectively were used to raise antibodies in rabbits. Affinity-purified antibodies were able not only to recognize a major band with a molecular weight of about 80 kDa on the electrotransferred membrane proteins of rat ventricular membranes but also to localize the muscarinic receptors on the sarcolemma and t-tubules of rat cardiomyocytes. Antibodies were also able to mimic muscarinic agonist stimulation as demonstrated by a negative chronotropic effect on cultured neonatal cardiomyocytes. In contrast with the antibodies raised against the peptide 169-193, the antibodies against the peptide 172-181 were unable to inhibit muscarinic ligand binding. These results suggest that the decapeptide 172-181 contains the B-cell epitope responsible for the functional effect of antibodies directed against the second extracellular loop of the receptor. Coupling this peptide by cystein 177 blocks the induction of antibodies with pharmacological effects but induces antibodies which are able to recognize the denatured receptor protein and to exert a negative chronotropic effect.

Amino Acid Sequence↗

PubMed1995

Characterization of pharmacologically active anti-peptide antibodies directed against the first and second extracellular loops of the serotonin 5-HT1A receptor.

The immunological properties and the functional role of the first (loop I) and second (loop II) extracellular loops of the human serotonin 5-HT1A receptor were studied with three populations of anti-peptide antibodies: Ab-1 (loop I; sequence Y-Q-V-L-N-K-W-T-L-G-Q-V-T-C-D-L; residues 96-111), Ab-2 (loop II; sequence G-W-R-T-P-E-D-R-S-D-P-D-A-C-T-I-S-K-D-H-G; residues 173-193), and Ab-12 (produced against loop I but cross-reacting with loop II). Chemical modification of peptide amino acid residues revealed the importance of the polyanionic stretch near the N-terminal domain of loop II for Ab-2 antibody binding and the role of the cysteine residues in both loops for the binding of Ab-1 and Ab-12 antibodies. Antibodies Ab-2 and Ab-12 recognized only the nonglycosylated form of the receptor (42 kDa) on immunoblots with transfected HeLa cells expressing the human 5-HT1A receptor but recognized the glycosylated forms (55 and 65 kDa) of rat 5-HT1A receptor from hippocampus membranes. The Ab-1 antibodies recognized no protein band from any cell type studied. Preincubation of transfected HeLa cell membranes with Ab-2 antibodies revealed two affinity binding sites of the 5-HT1A receptor (KDH = 0.54 +/- 0.09 nM and KDL = 13.74 +/- 4.9 nM) for the agonist 8-hydroxy-2-(di-n-[3H]propylamino) tetralin ([3H]8-OH-DPAT) binding, but Ab-1 and Ab-12 revealed only one site (KD of approximately 2.5 nM). In contrast to the Ab-2 antibodies, Ab-1 and Ab-12 antibodies decreased the Bmax of the [3H]8-OH-DPAT binding to 42 and 31%, respectively. These findings suggest that there are at least two epitopes on the extracellular loops: one inducing a high-affinity state for agonist binding and the other interfering with the accessibility of the ligand binding pocket.

Amino Acid Sequence↗

PubMed1995

[A new alpha chain of jacalin from two wild species of jack-fruit seeds].

Jacalins, from jack-fruit seeds of 2 wild species (Artocarpus asperulus, Artocarpus masticata) were purified by mucine-sepharose 4B affinity chromatography. The alpha and beta chains were separated by reverse phase high pressure liquid chromatography (HPLC). Analysis by HPLC with a C8 column and the determination of the N-terminal sequence of the alpha-chain of these jacalins allowed the identification of a new alpha-chain. Immunological cross-reactivity and carbohydrate specificity indicate that jacalins possessing the new alpha-chain conserve structural and functional properties of the other members of Artocarpus genus.

Amino Acid Sequence↗

PubMed1994

Functional autoimmune epitope on alpha 1-adrenergic receptors in patients with malignant hypertension.

Because of the growing evidence that hypertensive disease is accompanied by immunological dysfunction, we have investigated autoimmunity in patients with malignant hypertension. Peptides corresponding to the sequence of the second extracellular loops of the human alpha 1-adrenergic receptor and the M2-muscarinic receptor were used as antigens in an ELISA. Serum from 4 (12%) of 33 healthy controls, 3 (20%) of 15 patients with malignant essential hypertension, and 7 (64%) of 11 with secondary hypertension showed positive responses in the ELISA for the alpha 1-adrenergic receptor peptide. Positive responses were significantly more common among the patients with secondary hypertension than in the other two groups (p < 0.01). By contrast, no autoantibodies against the M2-muscarinic receptor peptide were detected in either hypertensive group. Autoantibodies against the alpha 1-adrenergic receptor, affinity-purified from patients with positive responses, specifically recognised bands with molecular masses of 68, 40, and 37 kDa on immunoblotted membrane proteins of rat ventricles. The patients' autoantibodies caused a decrease in tritiated prazosin binding sites and an increase in heart beating frequency of neonatal cultured rat cardiomyocytes; antibodies purified from the controls had no effect. Circulating autoantibodies against the alpha 1-adrenergic receptor are present in a subgroup of patients with malignant hypertension. These autoantibodies have pharmacological activity in vitro, which suggests that they may be involved in the pathogenesis of malignant hypertension.

Adult↗

PubMed1994

Epitopic analysis of the Toxoplasma gondii major surface antigen SAG1.

T and B cell epitopes of the major Toxoplasma gondii surface antigen SAG1 were studied following CNBr fragmentation. Three fragments, F1, F2 and F3, were obtained, of 19, 16.5 and 14 kDa, respectively. The positions of F1 F2 and F3 within the SAG1 protein were identified by N-terminal sequence determination. The F1 fragment located on residues 125-269 contains the C-terminus, and the fragment F2 (residues 1-124) is located at the N-terminal region. F3 is a C-terminal peptide about 40 amino acids shorter than the F1 fragment (residues 165-269). Polyclonal antibodies obtained from infected animals or humans and a monoclonal anti-SAG1 antibody did not recognize either the reduced protein or the reduced fragments on immunoblotting. The monoclonal antibody 1E5 did not recognize fragment F1. Mouse IgA and IgG antibodies from infected mouse sera and intestinal secretions, as well as human IgG antibodies, only recognized the whole protein and the F1 fragment. These results suggest that the fragment F1 encompasses all B cell epitopes recognized on the SAG1 protein after infection with the parasite and that the sequence 125-165 is essential for the structural integrity of these B cell epitopes. Murine anti-SAG1 T cell proliferation was observed in SAG1 immunized CBA/J mice (H-2k) and BALB/c mice (H-2d), but not in C57BL/6 mice (H-2b). The three fragments F1, F2 and F3 were able to induce specific proliferation of anti-SAG1 T cells from CBA/J mice, while only the F1 and F2 fragments induced specific blastogenesis of anti-SAG1 T cells from BALB/c mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Epitope analysis of T- and B-cell response against the human beta 1-adrenoceptor.

Several reports have recently raised the possible significance of the presence of autoantibodies against the beta 1-adrenoceptor in patients with idiopathic dilated cardiomyopathy. An investigation was thus initiated to study the immune response against this receptor at the T-cell and the B-cell level. Using membranes of E coli transfected with the human beta 1-adrenoceptor gene as immunogen, T-helper cells of the immunized mice were stimulated with synthetic peptides derived from the receptor and predicted to be immunogenic to assess the T-cell immunodominant regions of the receptor. Three peptides derived from the second transmembrane region, from the second extracellular loop and from the C-terminal domain were shown to be stimulatory. Synthetic peptides, derived from two domains of the receptor which could be potential targets for autoantibodies, yielded an antibody response after immunization with the free peptides. The peptide derived from the N-terminal region yielded antibodies which recognized the receptor in immunoblot and by immunoprecipitation but they had no functional effect on the receptor. The peptide derived from the second extracellular loop yielded antibodies which recognized the receptor in immunoblot and by immunoprecipitation of the free receptor and which had a pharmacological effect on the receptor. The second extracellular loop thus contains T- and B-cell epitopes which could be involved in the autoimmune process.

Animals↗

PubMed1994

Production of anti-peptide antibodies directed against the first and the second extracellular loop of the human serotonin 5-HT1A receptor.

The second extracellular loop of the beta-adrenergic and muscarinic acetylcholine receptors was shown to be an autoimmune target for antibodies in several autoimmune diseases. These autoantibodies and the antibodies induced against synthetic peptides corresponding to this loop have pharmacological and physiological properties upon receptor recognition which could explain their pathophysiological role. We here describe the immune properties of the first and second extracellular loops of another G protein-coupled receptor, the serotonin 5-HT1A receptor. The injection in rabbits of the free peptides Y16L and G21G corresponding to the first and second extracellular loops respectively induced anti-peptide antibodies with high titer, demonstrating the presence of a T-cell epitope on each peptide. Interestingly, in contrast to the G21G peptide that induced only anti-G21G antibodies (Ab-2 antibodies), the Y16L peptide induced two populations of antibodies. One recognized only the Y16L peptide (Ab-1 antibodies), the other recognized both peptides (Ab-12 antibodies). This reflects the presence on the two peptides of at least two B-cell epitopes. The fact that the G21G peptide induces only one antibody population might indicate that it possesses one immunodominant epitope involved in the Ab-2 antibody production and one cryptic epitope involved in the cross-reaction with the anti-Y16L antibodies. But only Ab-2 antibodies were able to recognize specifically the human protein receptor expressed in E coli in immunoblot.

Amino Acid Sequence↗

PubMed1994

Characterization of anti-peptide antibodies directed against an extracellular immunogenic epitope on the human alpha 1-adrenergic receptor.

A synthetic peptide corresponding to amino acids 192-218 of the second extracellular loop of the human alpha 1A-adrenergic receptor was used to raise antibodies in rabbits. Affinity-purified antibodies specifically recognized main bands with a molecular weight of about 68, 40 and 37 kD on the electrotransferred membrane proteins of rat ventricle membranes. The incubation of these antibodies with rat myocardial membranes resulted in a decrease in the number of binding sites for the specific radiolabelled alpha 1-antagonist prazosin. These antibodies were also able to mimic the effects of agonist stimulation as demonstrated by a positive chronotropic effect on cultured cardiomyocytes. These results constitute the first immunochemical evidence of the presence of both the A and B subtypes of the alpha 1-adrenergic receptor in the heart. They also confirm that the second extracellular loop of the alpha 1-adrenergic receptors is an immunologically and functionally important domain.

Amino Acid Sequence↗

PubMed1994

Gi-mediated muscarinic adenylyl cyclase inhibition in timolol-treated stunned porcine myocardium.

The Gi-mediated muscarinic receptor-adenylyl cyclase system was examined in stunned myocardium induced by either three or five brief ischaemic periods after beta-adrenoceptor blockade by timolol (0.1 mg kg-1). The mid-left anterior descending coronary artery was occluded for 2, 10 and 2 min in four pigs, and for 2, 2, 5, 10 and 2 min in four other pigs. All the ischaemic periods were separated by 30 min of reperfusion and the biopsies were obtained 60 min after the last ischaemic period. Segment length function was measured in the ischaemic region and in the control region supplied by the left circumflex artery. In the two groups, the percentage systolic shortening was reduced equally, to 59 +/- 9 and 58 +/- 10% of control in the region subjected to ischaemia and only minimally in the control region. The biopsies from the stunned region from both groups showed: (1) no change in either the affinity for carbachol or the number of binding sites of the muscarinic receptors; (2) no alterations in messenger RNA encoding for the alpha subunit-2 of the inhibitory guanine nucleotide binding protein, as demonstrated by northern blot and solution hybridization; (3) no change in membrane-bound inhibitory guanine nucleotide binding protein, as shown by enzyme immunoassay utilizing a specific anti-peptide antibody, and (4) unchanged inhibition of stimulated adenylyl cyclase activity. These results suggest that there is an intact inhibitory guanine nucleotide binding protein-mediated muscarinic receptor adenylyl cyclase system in the stunned porcine myocardium.

Adenylyl Cyclase Inhibitors↗

PubMed1994

Distribution of bradykinin B2 receptors in target cells of kinin action. Visualization of the receptor protein in A431 cells, neutrophils and kidney sections.

Peptides corresponding to sequences derived from predicted extra- and intracellular loops of the rat bradykinin receptor were analyzed for interspecies homology as well as for matches within the present dataset of protein sequences to provide a theoretical basis for the specific recognition of the native cognate protein by antibodies raised against these antigens. Application of polyclonal antibodies raised against the selected peptides allowed the immunocytochemical localization of the native receptor protein in cells of rat and human origin. The detection of the molecule was achieved by different immunohisto- and immunocytochemical methods in combination with light, fluorescence, confocal optical laser and electron microscopy. These results were compared to localization studies by autoradiography. Distribution and subcellular localization were determined in human neutrophils, human epithelial carcinoma cells (A431) and in rat kidney tissue.

Amino Acid Sequence↗

PubMed1993

Decreased density of mesenteric arteries but not of myocardial endothelin receptors and function in rats with chronic ischemic heart failure.

Mesenteric artery and cardiac ventricular endothelin receptors and endothelin-1-induced pressor responses were studied in normal rats and rats with chronic congestive heart failure induced by myocardial ischemia (4 weeks after coronary artery ligation). In mesenteric arteries of rats with chronic ischemic heart failure, endothelin receptor density was significantly decreased by 59%, whereas the dissociation constant was increased 2.8-fold, as compared with controls. There were, however, no changes in endothelin-receptor density or the dissociation constant in cardiac ventricular membrane preparations from rats with congestive heart failure as compared with controls. In pithed rats with congestive heart failure there was a reduced pressor response to a bolus injection of endothelin-1 (800 pmole/kg body weight), while the vasodilatory response was unaltered as compared with sham-operated controls. These results demonstrate that there is a decreased vascular endothelin-receptor function due to a down-regulated endothelin receptor. The in vivo data indicate that this is due to impaired endothelin A but not endothelin B receptor function. Thus, there is an impaired arterial but not cardiac ventricular endothelin receptor-mediated signalling system in the rat with chronic ischemic heart failure.

5'-Nucleotidase↗

PubMed1993

Localization of a functional autoimmune epitope on the muscarinic acetylcholine receptor-2 in patients with idiopathic dilated cardiomyopathy.

A peptide corresponding to the sequence 169-193 of the second extracellular loop of the human muscarinic acetylcholine receptor-2 was used as an antigen to screen sera from patients with idiopathic dilated cardiomyopathy (DCM, n = 36) and healthy blood donors (HBD, n = 40). The sera from 14 patients with DCM (38.8%) and 3 HBD (7.5%) recognized the muscarinic receptor peptide at dilutions varying from 1:20 to 1:160 in ELISA. A highly significant correlation (P = 0.006) was found between the presence of antimuscarinic receptor-2 autoantibodies and anti-beta-adrenoceptor-1 autoantibodies in the patients' sera. Affinity-purified autoantibodies from positive sera of patients with DCM recognized on the electrotransferred protein of rat ventricular membrane a major band of about 80 kD. Incubation of autoantibodies with membrane resulted not only in a decrease in the maximal binding sites (Bmax) but also in an increase in Kd of radioligand binding in a concentration-dependent manner. This suggests a mixed-type of inhibition. Moreover, preincubation with atropine abolished the inhibitory effect of autoantibodies on the receptor binding whereas carbachol appeared to have no effect on the activity of the autoantibodies. These data define a subgroup of patients with idiopathic DCM who have in their sera functionally active autoantibodies against muscarinic receptor-2.

Adult↗

PubMed1992

An immunochemical approach to investigating the mechanism of inhibition of cysteine proteinases by members of the cystatin superfamily.

Antibodies were raised against a synthetic dodecameric peptide KGAGQVVAGPWK (K12K), encompassing sequences thought to be important for the function of the cysteine proteinase inhibitors of the cystatin superfamily. These antibodies specifically recognized molecules of family 3, i.e., kininogens, in the serum of seven mammalian species tested in this study. The only notable exception was that of rat thiostatin (T kininogen) which is structurally related to the kininogen family. The antibodies also discriminated between family 2 (cystatins) and family 3 (kininogens) of the cystatin superfamily, since neither chicken cystatin nor human and rat cystatins C and S, which all belong to family 2 were recognized. The cystatin-like inhibitory domains resulting from fragmentation of human low molecular weight kininogen by bovine trypsin, were still recognized by antibodies, indicating that discrimination does not require two neighbouring inhibitory sites on the kininogen heavy chain. The antibodies blocked the capacity of kininogens to inhibit papain, suggesting that they recognize a conformational epitope at or near the kininogen inhibitory sites. The inhibitory properties of family 2 cystatins remained unchanged, confirming that members of this family do not interact with anti K12K antibodies. These antibodies are thus a new tool able to discriminate functionally and structurally between the members of the cystatin superfamily.

Amino Acid Sequence↗

PubMed1992

Anti-idiotypic antibodies against the kinin receptor.

Three sets of monoclonal antibodies against bradykinin (MBK1, MBK2, MBK3) were generated by somatic cell fusion, characterized by their peptide specificity and compared to the known ligand specificity of the kinin receptor subtypes. By these criteria the paratope of MBK3 resembled the B2 receptor binding site whereas MBK1 shared principal binding characteristics with the B1 recrptor. Anti-idiotypic antibodies against MBK1, MBK2 and MBK3 were raised in rabbit and sheep. Specificity of the network components was verified by inhibition experiments on the level of peptide, idiotype and anti-idiotype. Anti-idiotypic antibodies against MBK3 recognized a conformation-dependent epitope which was binding site-related. Binding studies on human foreskin fibroblasts and guinea pig ileum showed mutual displacement of the anti-idiotypic antibody and bradykinin at the binding site pointing to a specific interaction of the antibody with the receptor from various species. An agonist activity of the antibodies, demonstrated in human (inositolphosphate pathway) and mouse (prostaglandin pathway) fibroblasts indicated that the anti-idiotypes bear an internal image of the ligand epitope. This molecular mimicry which was further substantiated by the detection of bradykinin specific anti-idiotypic antibodies, provides the structural basis for the observed cross-reactivity over species borders.

Animals↗

PubMed1992

Beta 2-adrenergic receptor antibodies in myasthenia gravis.

Although autoantibodies against the nicotinic acetylcholine receptor are the characteristic feature of the autoimmune disease myasthenia gravis (MG), no strong correlation is found between the autoantibody titer and the degree of clinical severity. Numerous studies have attempted to detect the presence of other autoantibody populations that might have a role in the pathology of the disease. We report, for the first time, that 18% of the MG patients we screened have antibodies in their serum to a peptide corresponding to the second extracellular loop of the human beta 2-adrenergic receptor (residues 172-197). Affinity purified antibodies to the beta 2-adrenergic receptor peptide 172-197 reacted with the human beta 2-adrenergic receptor protein obtained from transfected E. coli cell membrane extracts, but did not cross-react with the human AChR. Sufficient material was obtained from nine MG patients and it was found that the gamma globulin fraction from these patients immunoprecipitated the receptor, and that affinity purified IgG to peptide 172-197 competed for receptor binding with the beta-antagonist iodo-cyanopindolol. Using truncated peptides or amino acid modification procedures, no immunodominant B-cell epitope could be detected within region 172-197. Thus, a subpopulation of MG patients possesses anti-beta 2-adrenergic receptor antibodies which are a distinct set of autoantibodies with possible pharmacological activity.

Amino Acid Sequence↗