| 1. |
- Elies, Rozenn, et al.
(författare)
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Immunochemical and functional characterization of an agonist-like monoclonal antibody against the M2 acetylcholine receptor.
- 1998
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Ingår i: European journal of biochemistry / FEBS. - 0014-2956. ; 251:3, s. 659-66
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Tidskriftsartikel (refereegranskat)abstract
- Monoclonal antibodies were raised against a peptide corresponding to the second extracellular loop of the M2 acetylcholine receptor. One of the monoclonal antibodies, B8E5, was selected for further characterization on the basis of its high yield, its isotype (IgG2a), its dissociation kinetics and its agonist-like activity. The epitope recognized by B8E5 corresponded to the N-terminal part of the second extracellular loop of the receptor (V-R-T-V-E-) as determined by competition immunoassays and epitope scanning. The KA of B8E5 for the target peptide was assessed by surface plasmon resonance (SPR) to be 6.5x10(7) M(-1) by equilibrium and 3.7x10(7) M(-1) by kinetic analysis. B8E5 recognized the M2 acetylcholine receptor on rat cardiac tissue. It only recognized the non-reduced receptor in immunoblots. The antibody had no effect on antagonist binding but decreased the affinity for the agonist carbachol. B8E5 decreased the beating frequency of neonatal rat cardiomyocytes. The effect was specific since it was blocked by the target peptide and the antagonist atropine. The EC50 of the antibody corresponded to the KA measured by surface plasmon resonance. The physiological effect of the antibody did not lead to desensitization. The Fab fragments had no physiological effect; subsequent addition of anti-mouse IgG however restored the physiological effect. These results confirm that the N-terminus of the second extracellular loop is a functional target for antibodies against the M2 acetylcholine receptor. They suggest that the functional epitope is only accessible in the non-reduced receptor. The antibodies act through a functional dimerization of the receptor.
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| 2. |
- Zhao, R, et al.
(författare)
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Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes.
- 1996
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Ingår i: Molecular and cellular biochemistry. - 0300-8177. ; 163-164, s. 185-93
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Tidskriftsartikel (refereegranskat)abstract
- The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K+ current and decreased the inward basal ICa significantly. The reversal potential of both carbachol- and antibody-induced extra currents were close to -80 mV, being in proximity to the calculated Ek of -90 mV. A beta-adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward ICa and outward Ik simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated ICa but not the isoprenaline-stimulated Ik. The antibody- or carbachol-induced outward K+ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated ICa were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.
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| 3. |
- Fu, Michael, 1963, et al.
(författare)
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Autoantibodies against cardiac G-protein-coupled receptors define different populations with cardiomyopathies but not with hypertension.
- 1994
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Ingår i: Clinical immunology and immunopathology. - 0090-1229. ; 72:1, s. 15-20
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Tidskriftsartikel (refereegranskat)abstract
- It was previously shown that the second extracellular loop of cardiovascular G-protein-coupled receptors is an antigenic target for pharmacologically active autoantibodies in patients with idiopathic dilated cardiomyopathy. To extend these observations to cover patients with the same disease from different geographical origins or to patients with other cardiac diseases, peptides corresponding to the sequences of the second extracellular loops of the human M2 muscarinic receptors and beta adrenoceptors were used as antigens in an enzyme immunoassay. Sera from patients from Sweden and Japan with idiopathic dilated cardiomyopathy (DCM, n = 32), hypertrophic cardiomyopathy (HCM, n = 23), malignant essential hypertension (MEH, n = 11), malignant secondary hypertension (MSH, n = 10), and sera from healthy blood donors (HBD, n = 49) were tested. Sera from patients with DCM recognized the muscarinic receptor peptide in 38% of cases and the beta 1 adrenoceptor peptide in 31% of cases. In 50% of the positive patients, autoantibodies against both peptides coexisted as shown by competition experiments using both peptides as inhibitors. In HCM patients, there was a lower frequency of autoantibodies but with a higher but not significant predominance against the M2 peptide. No autoantibodies were detected in sera from patients with MEH or MSH. Autoantibodies against the M2 muscarinic receptors, affinity-purified from positive patients, displayed pharmacological activity as demonstrated by changes in the affinity and number of radioligand binding sites. In contrast, antibodies purified from positive HBD had no effect. These results confirm that autoantibodies displaying pharmacological activity against G-protein-coupled cardiovascular receptors are mainly restricted to patients with idiopathic dilated cardiomyopathy and that different autoantibody populations are responsible for the recognition of the different receptors.
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| 4. |
- Fu, Michael, 1963, et al.
(författare)
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Autoantibodies against the angiotensin receptor (AT1) in patients with hypertension.
- 2000
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Ingår i: Journal of hypertension. - 0263-6352. ; 18:7, s. 945-53
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Tidskriftsartikel (refereegranskat)abstract
- Sera from patients with malignant essential hypertension (n = 14), malignant secondary hypertension mainly attributable to renovascular diseases (n = 12) and renovascular diseases without malignant hypertension (n = 11) and from normotensive healthy blood donors (n = 35) were studied for the presence of autoantibodies against G-protein-coupled cardiovascular receptors. Autoantibodies against the angiotensin II receptor (AT1) were detected in 14, 33, 18 and 14% of patients with malignant essential hypertension, malignant secondary hypertension, renovascular diseases and control patients, respectively. Sensitivity of the enzyme immunoassay was assessed as 5 microg/ml IgG. Patients did not show antibodies against bradykinin (B2) or angiotensin II subtype 2 (AT2) receptors. Autoantibodies affinity-purified from positive patients localized AT receptors in Chinese hamster ovary transfected cells, and displayed a positive chronotropic effect on cultured neonatal rat cardiomyocytes. These results demonstrate the existence of autoantibodies against a functional extracellular domain of human AT1 receptors in patients with malignant hypertension, and suggest that these autoantibodies might be involved in the pathogenesis of malignant hypertension.
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| 5. |
- Fu, Michael, 1963, et al.
(författare)
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Immunohistochemical localization of angiotensin II receptors (AT1) in the heart with anti-peptide antibodies showing a positive chronotropic effect.
- 1998
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Ingår i: Receptors & channels. - 1060-6823. ; 6:2, s. 99-111
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies were produced against a synthetic peptide corresponding to amino acids (165-191) of the second extracellular loop of the human angiotensin II receptor subtype 1 (AT1) in rabbits. The purified antibodies had an apparent affinity of about 1 nM and were monospecific for the AT1-receptor peptide. Chemical modification of the carboxyl groups (glu at positions 173 and 185) and the sulfhydryl group (cys at position 180) of the AT1-receptor peptide did not alter the relative affinity of the coated AT1-receptor peptide to antibodies. The antibodies specifically stained CHO cells expressing the rat AT1a receptor. Immunoblots on rat kidney revealed that the antibody recognized a protein band of 59 +/- 3 kDa in a dose-dependent manner and this band was no longer detected after preincubating the antibodies with AT1-receptor peptide. Using electron microscopic and immunofluorescence immunocytochemistry techniques, angiotensin II receptors were detected in (1) the sarcolemma, T-tubules and nuclei of rat cardiomyocytes, (2) the transluminal side of endothelial cells and (3) fibroblast cells. These localizations are specific, as the immunostaining did not appear when preimmune rabbit serum was used and was blocked after preincubating antibodies with antigenic peptide. Functionally, these antibodies did not affect the ligand binding properties of the receptors but displayed agonist-like activity as shown by dose-dependent increases in beating frequency in cultured neonatal cardiomyocytes. These results suggest that the antibodies against the second extracellular loop of human AT1 receptors were able to specifically recognize AT1 receptors. In addition, they extend the observation that the second extracellular loop of the G-protein coupled membrane receptors is a specific target for antibodies with agonist-like activity.
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| 6. |
- Matsui, S, et al.
(författare)
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Peptides derived from cardiovascular G-protein-coupled receptors induce morphological cardiomyopathic changes in immunized rabbits.
- 1997
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Ingår i: Journal of molecular and cellular cardiology. - : Elsevier BV. - 0022-2828. ; 29:2, s. 641-55
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Tidskriftsartikel (refereegranskat)abstract
- An experimental model of early-stage cardiomyopathy was created by immunizing rabbits for 1 year with synthetic peptides corresponding to the sequence of the second extracellular loop of either beta-adrenoceptors or M2-muscarinic receptors. Thirty male rabbits were used and divided into three groups: a control group (n = 10), a group immunized with the peptide corresponding to the beta-adrenoceptor (beta 1 group) (n = 10) and a group immunized with the peptide corresponding to the M2-muscarinic receptor (M2 group) (n = 10). If the sera from both groups of immunized rabbits high-titres of anti-peptide antibodies were found throughout the study period but not in the sera from control rabbits or in the preimmune sera of immunized rabbits. No significant cross-reaction with peptides other than those used for immunization was found. The myocardial receptor density of both immunized groups displayed a strong trend toward receptor up-regulation. This was significant in the beta 1 group but not in the M2 group. Both groups of immunized rabbits displayed significantly enlarged ventricles and thinner walls, as compared with the control group. However, in contrast to the beta 1 group, which showed enlarged cavities in both left and right ventricles, the M2 group was mainly affected in the right ventricles. Moreover, morphological examinations of the hearts of rabbits from both immunized groups demonstrated focal myofibrillar lysis, loss of myofilament, mitochondrial swelling and condensation, sarcoplasmic vacuolation, deposition of dense granules in the sarcoplasm and the myofibrils. One of the sex control rabbit hearts which were examined showed mild degenerative changes in the myocardium and scant mononuclear cell infiltration. However, when all the control rabbit hearts were examined by electron microscopy, no significant alterations were found. These results suggest that immunization by peptides, corresponding to the target sequences for anti-receptor autoantibodies in idiopathic dilated cardiomyopathy, induces morphological changes in the heart similar to those found in the human disease.
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| 7. |
- Mobini, Reza, 1965, et al.
(författare)
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A monoclonal antibody directed against an autoimmune epitope on the human beta1-adrenergic receptor recognized in idiopathic dilated cardiomyopathy.
- 2000
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Ingår i: Hybridoma. - : Mary Ann Liebert Inc. - 0272-457X. ; 19:2, s. 135-42
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Tidskriftsartikel (refereegranskat)abstract
- A monoclonal antibody (MAb M16) was obtained by immunizing Balb/C mice with free peptide H26R, corresponding to the second extracellular loop of the human beta1-adrenergic receptor (beta1AR), against which functional autoantibodies have been detected in patients with idiopathic dilated cardiomyopathy. The MAb was found to be of IgG2b type and directed against a conformational epitope, encompassing the sequence recognized by the human autoantibodies. BIAcore measurements yielded an equilibrium constant of 6.5 X 10(7) M1 with an association rate constant (kon) of 6.5 X 10(4) M(-1) sec(-1) and a dissociation rate constant (koff) of 1.0 X 10(-3) sec(-1). It immunoprecipitated only poorly the solubilized beta1AR of Sf9 cell membranes. Functionally, the MAb was capable of not only reducing the number of the maximal binding sites to the beta1-adrenergic receptor of transfected Sf9 cell membranes, but also of displaying a positive chronotropic effect on cultured neonatal rat cardiomyocytes. These properties, which the MAb shares with the human autoantibodies, makes it an interesting tool for passive transfer studies in mice.
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| 8. |
- Mobini, Reza, 1965, et al.
(författare)
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Hemodynamic improvement and removal of autoantibodies against beta1-adrenergic receptor by immunoadsorption therapy in dilated cardiomyopathy.
- 2003
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Ingår i: Journal of autoimmunity. - 0896-8411. ; 20:4, s. 345-50
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Tidskriftsartikel (refereegranskat)abstract
- The removal of beta(1)-adrenergic receptor (beta(1)AR) autoantibodies by immunoadsorption (IA) has been proposed as a potential mechanism for the improvement of the left ventricular function in dilated cardiomyopathy (DCM). In the present study, the possible association between removal of the autoantibodies against the human beta(1)AR with the hemodynamic improvement induced by IA was investigated.IA was performed in 22 DCM patients (n=22; NYHA III-IV, EF<30%, stable medication). The beta(1)AR autoantibodies from column eluents (CE) were detected by enzyme-linked immunosorbent assay (ELISA) and BIAcore methods. CE of 32% (7/22) of the patients was found to be antibody-positive with ELISA or BIAcore. In addition, a bioassay system was also used for the detection of this autoantibody. Seventy-three percent (16/22) of the patients were found to be antibody-positive by this method. However, independent of the beta(1)AR antibody detection method, both antibody-positive and antibody-negative groups showed similar acute and prolonged hemodynamic improvements during IA therapy. Furthermore, antibody-positive and -negative groups received a comparable improvement of left ventricular ejection fraction.These results suggest that different mechanisms are involved in the hemodynamic improvement induced by IA. The beneficial hemodynamic effects induced by IA are not directly associated with the removal of beta(1)AR autoantibodies.
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| 9. |
- Omerovic, Elmir, 1968, et al.
(författare)
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Induction of cardiomyopathy in severe combined immunodeficiency mice by transfer of lymphocytes from patients with idiopathic dilated cardiomyopathy.
- 2000
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Ingår i: Autoimmunity. - 0891-6934. ; 32:4, s. 271-80
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Tidskriftsartikel (refereegranskat)abstract
- Growing evidence suggests that autoimmune mechanisms play an important role in the pathogenesis of idiopathic dilated cardiomyopathy (DCM). The aim of the study was to evaluate the effects of transfer of lymphocytes from patients with DCM into severe combined immunodeficiency (SCID) mice on the heart structure and function. Thirty CB-17 SCID (6-8 weeks old) mice were used and divided into 3 groups (n = 10). Mice were injected intraperitoneally with up to 25 x 10(6) peripheral blood lymphocytes (PBL) from either patients with DCM which contain human autoantibodies against cardiac beta1-adrenergic receptors and M2-muscarinic receptors (DCM group) or PBL from healthy controls (control-H group). Ten mice did not receive any injections and were used as baseline controls (control-N group). Echocardiography and morphological studies were performed seventy five days after the transfer. Results showed that in DCM group, left ventricle dimensions (LVD) in diastole were increased (4.2 +/- 0.1mm) as compared to both control-H group (3.8 +/- 0.1mm) and control-N group (3.6 +/- 0.1 mm) (p < 0.01). Further, there was a trend for increased LVD in systole. Fractional shortening was not different between groups. Histological evaluation revealed accumulation of human lymphocytes in the capillaries and scarce infiltration of the lymphocytes in the hearts from DCM group. Diffuse fibrosis was significant increased in DCM mice as compared to mice receiving PBL from normal subjects (2.2 +/- 0.3% vs. 0.8 +/- 0.1%, p < 0.01). In conclusion, transfer of the PBL from the patients with DCM was able to induce early stage of heart dilatation in SCID mice. These data provide for the first time the direct evidence supporting that the autoimmune mechanism is important in the pathogenesis of human DCM.
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| 10. |
- Wang, W, et al.
(författare)
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Stimulatory activity of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptors.
- 2000
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Ingår i: Chinese medical journal. - 0366-6999. ; 113:10, s. 867-71
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To study the activity of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptors on cAMP production and inward calcium currents (Ica) in guinea pig ventricular myocytes. A comparison was also made with those of a muscarinic receptor agonist. METHODS: cAMP content was determined by radioimmunoassay and the Ica in guinea pig single ventricular cells were recorded by the whole-cell patch clamp technique. RESULTS: Both the muscarinic receptor agonist, carbachol (Carb 10 mumol/L), and anti-peptide antibodies (Abs 100 nmol/L) could decrease basal cAMP levels (by 46.9% +/- 4.2% and 60.2% +/- 4.6%, respectively) and basal Ica. Both Carb (10 mumol/L) and Abs (100 nmol/L) could also inhibit the isoprenaline-induced (Iso 0.8 mumol/L) increases in cAMP production (from 108.2 +/- 7.0 to 88.4 +/- 7.2 pmol/mg.protein/min for Carb and 88.6 +/- 5.1 pmol/mg.protein/min for Abs, respectively) and the increases in Ica. The muscarinic receptor antagonist atropine (Atr) was able to prevent these effects of Carb and Abs. CONCLUSIONS: Anti-peptide antibodies against an epitope located in the second extracellular loop of human M2 muscarinic receptors, similar to muscarinic receptor agonist, could decrease the basal Ica and beta-receptor agonist stimulated increase of Ica by decreasing the basal and beta-receptor agonist stimulated increase of cAMP production, and therefore could have an effect on their target receptor. These results further suggest that autoimmunity may participate in the pathogenesis of human cardiomyopathy and the second extracellular loop of human M2 muscarinic receptor could be the main immunodominant region.
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