Yvonne Magnusson

Mapping of a functional autoimmune epitope on the beta 1-adrenergic receptor in patients with idiopathic dilated cardiomyopathy.

The presence and properties of serum autoantibodies against beta-adrenergic receptors in patients with idiopathic dilated cardiomyopathy were studied using synthetic peptides derived from the predicted sequences of the human beta-adrenergic receptors. Peptides corresponding to the sequences of the second extracellular loop of the human beta 1- and beta 2-adrenergic receptors were used as antigens in an enzyme immunoassay to screen sera from patients with dilated cardiomyopathy (n = 42), ischemic heart disease (n = 17), or healthy blood donors (n = 34). The sera of thirteen dilated cardiomyopathy patients, none of the ischemic heart disease patients, and four of the healthy controls monospecifically recognized the beta 1-peptide. Only affinity-purified antibodies of these patients had a inhibitory effect on radioligand binding to the beta 1 receptor of C6 rat glioma cells. They recognized the receptor protein by immunoblot and bound in situ to human myocardial tissue. We conclude that a subgroup of patients with idiopathic dilated cardiomyopathy have in their sera autoantibodies specifically directed against the second extracellular loop of the beta 1-adrenergic receptor. These antibodies could serve as a marker of an autoimmune response with physiological and/or pathological implications.

Ser49Gly of β1-adrenergic receptor is associated with effective β-blocker dose in dilated cardiomyopathy

Our objective was to evaluate the influence of polymorphisms at codons 49 and 389 of the β1‐adrenergic receptor (β1‐AR) on the response to β‐blockers and outcome in patients with dilated cardiomyopathy.

A Monoclonal Antibody Directed Against an Autoimmune Epitope on the Human β1-Adrenergic Receptor Recognized in Idiopathic Dilated Cardiomyopathy

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.

β1-Adrenoceptor autoimmunity in cardiomyopathy

Abstract A growing body of studies have confirmed that autoantibodies against β 1 -adrenoceptors are present in different types of cardiomyopathy. This suggests that they play a role in the pathophysiology of the disease. This article will review the data indicating the presence of anti- β 1 -adrenoceptor autoantibodies in cardiomyopathy. It will focus upon their structural and functional properties which could explain their possible role in the induction and development of cardiomyopathic diseases.

β2-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.

Lower levels of the host protective IL-10 in DCM—a feature of autoimmune pathogenesis?

Objectives: To investigate whether cytokine patterns differ with respect to heart failure (HF) etiology, and to study how cytokine concentrations relate to hemodynamic alterations. Methods: Plasma levels of interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α) and high sensitive-CRP (hs-CRP) were analysed with enzyme-linked-immunosorbent serologic assay and turbidimetry in 45 healthy subjects and 89 patients with HF, of whom 65 were diagnosed with dilated cardiomyopathy (DCM) and 24 had ischemic heart disease (IHD). Results: IL-6, IL-10 and hs-CRP were significantly higher in patients with HF as compared to healthy controls. The IL-10 was significantly lower in patients with DCM as compared to IHD, also when adjusting for clinical variables. Diastolic filling pressure correlated with IL-6, IL-10 and hs-CRP while heart rate (HR) correlated with IL-6 and TNF-α. Conclusions: Proinflammatory cytokines are elevated in patients with HF and display a positive correlation with filling pressures and HR. Most significant, the regulatory and protective cytokine IL-10 was much lower in patients with DCM as compared to IHD, indicating a differentiation in cytokine patterns with respect to HF etiology.

T and B lymphocytes reacting with the extracellular loop of the β2-adrenergic receptor (β2AR) are present in the peripheral blood of patients with myasthenia gravis

Eighteen percent of patients with myasthenia gravis (MG) have serum antibodies against a synthetic peptide corresponding to the second extracellular loop of the human β2AR (residues 172–197). In this study we examined T and B cell responses to the peptide, using assays to detect individual cells secreting interferon‐gamma (IFN‐γ) and IL‐4 or antibodies against the peptide, and by measuring thymidine incorporation in response to the peptide. The peptide from the β2AR induced cytokine secretion from blood mononuclear cells in 67% of MG patients, compared with 14–28% of the control groups. Cells secreting antibodies binding to the peptide were present in 54% of MG patients and in 19–28% of controls. The numbers of β2AR‐reactive cells were higher in MG patients than in controls. Peptide‐induced increase in thymidine incorporation in cells was also more frequently demonstrated in patients (26%) compared with controls (about 10%). Activation of cells was dependent on monocytes and on MHC class II DR antigen. Based on the pattern of the cytokine secretion induced, β2AR‐reactive T cells comprise both T helper type‐1 and type‐2 subsets. In addition, control peptide‐reactive T and B cells were much less frequently demonstrated in the patients, and the number of such cells did not differ between the groups. Our results show that β2AR‐reactive cells are present in most patients with MG. Such autoreactive antibodies and cells might play a role in the pathogenesis of the disease by influencing the function of skeletal muscle and immune systems.

Abnormality of adenylate cyclase regulation in human platelet membranes in renal insufficiency.

Abstract. Adenylate cyclase in human platelets is under dual control of prostaglandins (PGI2 and PGE1) and catecholamines. The adenylate cyclase complex in membranes of platelets from ten patients with uraemia was investigated. The activation of the platelet cyclase by PGEi is increased in the uraemic state, Vmax 4436 ± 607 pmol cAMP mg‐1 15 min‐1. In the normal state Vmax is 2098 ± 309 pmol cAMP mg‐1 15 min‐1. The alpha2‐adrenergic receptor was assayed with 3H‐yohimbine binding. The density of receptors was equal in the uraemic (175 fmol mg‐1 membrane protein) and the normal (170 fmol mg‐1 membrane protein) states. Norepinephrine/3H‐yohimbine competition binding revealed that catecholamines were bound with normal affinity in platelets in uraemia. Yet the inhibition of adenylate cyclase through the alpha2–adrenergic receptor was diminished since Vmax values of adenylate cyclase with PGE1 and PGE2+ norepineprine did not significantly differ. In the normal state, norepinephrine significantly (P < 0·05) inhibited the PGE1 stimulated cyclase.

Impaired activation of IFN-γ+CD4+ T cells in peripheral blood of patients with dilated cardiomyopathy

Viral persistence and autoantibodies are pathogenic components in patients with idiopathic dilated cardiomyopathy (DCM). The aim was to evaluate T-cell function in DCM using different flow cytometry based detection techniques. Following stimulation, the frequency of IFN-gamma-producing CD4+ T cells was significantly lower in patients compared with controls. In contrast, the frequency of IL-4 producing CD4+ T cells was no different. In supernatants of cultured PBMC, IFN-gamma and IL-10 were significantly lower in patients. In addition, lymphocyte proliferation was significantly lower in patients compared with controls, whereas major lymphocyte subsets were not different. IFN-gamma and IL-10 are key cytokines in the ability to mount protective immune responses and to maintain self-tolerance. A reduced activation of T-helper 1 (IFN-gamma producing) cells and a decreased capacity to produce IL-10, found in the present study, could explain parts of the autoimmune features seen in patients with DCM.

A Polymorphism at Position +874 in The IFN-γ Gene is Associated with Susceptibility for Dilated Cardiomyopathy

The etiology of idiopathic dilated cardiomyopathy (DCM) is largely unknown. One hypothesis is that immunological factors are responsible for disease development. Single nucleotide polymorphisms (SNP) at position +874 of the IFN-γ gene and at position -168 of the MHC2TA gene have previously been shown to associate with infl ammatory and autoimmune disorders. We analysed their possible infl uence on susceptibility and prognosis in patients with DCM. Genotypes were determined by real-time PCR in 442 patients and 425 controls. Genotype frequencies were found signifi cantly different between patients and controls for IFN-γ (p=0.029), but not for MHC2TA (p=0.26). In a logistic regression analysis, the high producing TT genotype of IFN-γ was signfi cantly more common in patients than controls (OR 1.55 [1.12-2.15], p=0.009). None of the polymorphisms had an infl uence on long-term outcome, 10-years mortality with a HR of 0.94 (p=0.74) and 0.85 (p=0.30), IFN-γ and MHC2TA, respectively. A 50% higher risk to develop DCM for individuals with the TT genotype of the IFN- gene is a new finding and emphasize the role of IFN- in DCM pathology.