The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63-Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63-Lys68 of NT-proBNP exhibits O-glycosylation-independent binding.
Amino Acid Sequence ; Animals ; Antibodies/*immunology ; Antigen-Antibody Reactions ; Epitope Mapping ; Epitopes/chemistry/genetics/*immunology ; Glycosylation ; HEK293 Cells ; Heart Failure/immunology ; Humans ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Natriuretic Peptide, Brain/chemistry/genetics/*immunology ; Peptide Fragments/chemistry/genetics/*immunology ; Rabbits ; Recombinant Fusion Proteins/chemistry/genetics/immunology

Heart transplantation is the last treatment option in refractory end stage heart failure, which can prolong survival. The number of heart transplantations has increased and the survival rate has improved during the last few decades which was contributed by advanced understanding of immunologic mechanism of rejection, pharmaceutical development and clinical management of donors and recipients. However, only a fraction of patients can be offered to transplantation due to shortage of donor heart and many patients suffer high mortality while waiting. Meanwhile, technical advancement of mechanical assist device in recent years enabled long term implantable left ventricular assist devices (LVAD) to bridge the patients with high mortality in the waiting list to transplantation and to assist as a long term destination therapy for patients who are not eligible for transplantation. Development of solid phase assay increased the sensitivity and the specificity of detection of anti-human leukocyte antigen (HLA) antibodies in the recipient. It enabled identifying unacceptable HLA antigens, acquire calculated Panel Reactive Antibodies and perform virtual cross match that can enhance the efficacy of donor allocation system to decrease the waiting time, obviate prospective cross match to decrease ischemic time and to assess the risk of rejection in presensitized patients. Antibody mediated rejection is a challenging entity in diagnosis and management. However, standardized classification of histology and immunology of endomyocardial biopsies was made recently and immunotherapy is moving toward targeted therapies directed at antibody production and function. This review focuses on those major changes in the heart transplantation field in the last decade.
Allergy and Immunology ; Antibodies ; Antibody Formation ; Biopsy ; Classification ; Diagnosis ; Graft Rejection ; Heart ; Heart Failure ; Heart Transplantation* ; Heart-Assist Devices* ; HLA Antigens ; Humans ; Immunotherapy ; Leukocytes ; Mortality ; Sensitivity and Specificity ; Survival Rate ; Tissue Donors ; Waiting Lists

OBJECTIVEAutoimmunity participates in chronic heart failure (CCI), it is CD4+ T lymphocytes that mainly induces myocardial infiltration and the progression of the disease. The purpose of this research is to assess changes of CD4+, CD8+ T lymphocyte subset, and phenotype of primary T cell (CD4+ CD45RA+) and memory T cells (CD4+ CD45RO+) in peripheral blood in aged male patients with CCI. And to investigate the immunomodulatory effects on subsets of CD4+, CD8+ and phenotype of CD4+ CD45RA+ and CD4+ CD45RO+ and the possible therapeutic mechanism.

METHODSThe participant were 155 aged men among whom 94 cases were diagnosed as CCI and heart function of the rest 41 cases were normal. All patients underwent echocardiography examination and were collected peripheral blood before and after treatment. Serum N terminal pro-brain natriuretic peptide (NT-proBNP) levels were detected by heterogeneous immunoassay. Serum C reactive protein (CRP) were measured by immunoturbidimetry assay. T lymphocytes in peripheral blood were separated and determined distribution of CD4+, CD8+, CD4+ CD45RA+, CD4+ CD45RO+ using flow cytometry. Participants were divided into 3 groups: the CCI intervention group, who received regular therapy and thymopentin (20 mg intramuscular injection, once every other day for 3 month; n = 60) , the CCI control group, who received regular therapy (n = 54) and 41 healthy individual older than 57 years of age, who served as normal controls.

RESULTSCompared with the control group, left ventricular ejection fraction (LVEF) and CD4+ CD45RO+ levels decreased, left ventricular end diastolic diameter (LVEDD), NT-proBNP, CRP, CD4+, CD4+ CD45RA+, CD4+/CD8+, CD4+ CD45RA+/CD4+ CD45RO+ levels were obviously higher in CCI group. Distribution of CD8+ was not significantly changed. The level of NT-proBNP, CRP, CD4+/CD8+, CD4+ CD45RA+/CD4+ CD45 RO+ was negatively correlated with LVEF. LVEF could be much improved via decreasing distribution of CD4+/CD8+, CD4+ CD45RA+/CD4+ CD45RO in CCI intervention group than in CCI control group.

CONCLUSIONThe changes of CD4+/CD8+ and CD4+ CD45RA+/CD4+ CD45RO+ suggest that CD4+ T lymphocyte subset and its phenotype play an important role in the process of CCI. The regulation of CD4+ T lymphocyte and its phenotype may be one of the strategy in the treatment of CCI.

Aged ; Aged, 80 and over ; CD4-CD8 Ratio ; CD4-Positive T-Lymphocytes ; CD8-Positive T-Lymphocytes ; Heart Failure ; blood ; immunology ; Humans ; Immunomodulation ; Leukocyte Common Antigens ; immunology ; Male ; Phenotype ; T-Lymphocyte Subsets ; immunology

Angiotensin II ; immunology ; Chronic Disease ; Heart Failure ; drug therapy ; Humans ; Renin-Angiotensin System ; immunology ; Vaccines ; therapeutic use

At present ventricular remodeling (VR) is regarded as the main pathological basis of chronic heart failure after acute myocardial infarction (AMI), and preventing VR after AMI is of great importance for the prevention of heart failure. Previously, it has not been paid enough attention to the role of inflammation and autoimmune injury during the process of VR after AMI. This topic was discussed in the paper and the treating strategies with Chinese and Western medicine were also explored.
Animals ; Anti-Inflammatory Agents ; therapeutic use ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Heart Failure ; prevention & control ; Humans ; Inflammation ; drug therapy ; etiology ; immunology ; Myocardial Infarction ; complications ; physiopathology ; Prednisone ; therapeutic use ; Ventricular Remodeling ; drug effects

OBJECTIVETo detect the serum autoantibodies against the cardiac beta(1)-adrenergic receptor and observe the clinical characteristics and response to carvedilol use in patients with chronic heart failure (CHF).

METHODSCardiac function was examined by echocardiography and levels of autoantibodies against cardiac beta(1)-adrenergic receptor were detected in 65 patients with CHF by means of enzyme linked immune assay. Carvedilol was added on ACEI, diuretics and digitalis regimen for a target dose of 50 mg/d. All patients were followed up for 6 months.

RESULTSAutoantibodies against cardiac beta(1)-adrenergic receptor were detected in 30 patients (group 1) and not detected in the remaining 35 patients (group 2). The achieved target dose of carvedilol was significantly higher in group 1 than that in group 2 [(36.25 +/- 14.31) mg/d vs. (25.97 +/- 8.83) mg/d, P < 0.01]. Heart rate was significantly higher in group 1 compared to group 2 [(94.19 +/- 14.46) beats/min vs. (86.56 +/- 15.88) beats/min, P < 0.05] before treatment and heart rate and blood pressure of both groups decreased significantly (P < 0.01) and there was no significant difference between two group (P > 0.05) after 6 months treatment. LVEDD and LVESD were significantly larger while LVEF significantly lower in group 1 patients than those in group 2 patients (all P < 0.05) before treatment and LVEDD and LVESD decreased and LVEF increased significantly in both groups (all P < 0.01 vs. before treatment) and there was on significant difference in LVEDD, LVESD and LVEF between two groups (all P > 0.05) post 6 months treatment. Moreover, average titer of autoantibodies against the cardiac beta(1)-adrenergic receptors significantly decreased after 6 months treatment (1:119.35 vs. 1:72.21, P < 0.01).

CONCLUSIONThe detection of autoantibodies against the cardiac beta(1)-adrenergic receptors is related to severer cardiac dysfunction and autoantibodies title decrease was found with improved cardiac function after standard therapy (ACEI, digitalis, betablocker) in patients with CHF.

Adrenergic beta-Antagonists ; therapeutic use ; Adult ; Aged ; Autoantibodies ; blood ; Carbazoles ; therapeutic use ; Female ; Follow-Up Studies ; Heart Failure ; blood ; immunology ; physiopathology ; Humans ; Male ; Middle Aged ; Propanolamines ; therapeutic use ; Receptors, Adrenergic, beta-1 ; immunology

OBJECTIVETo investigate the effect of serum autoantibodies against the human M(2) muscarinic acetylcholine receptors (M(2)-receptors, Abs) from patients with congestive heart failure on L-Type Ca(2+) channel activity in guinea pig cardiac myocytes.

METHODUsing whole cell patch-clamp technique, we quantitatively measured the ionic intensity and density of L-Type Ca(2+) channel (I(Ca-L)).

RESULTSThe M(2)-receptors agonist (carbachol) could decrease the I(Ca-L) peak intensity and density stimulated by isoprenaline from (2111.65 +/- 203.13) pA and (18.83 +/- 1.14) pA/pF to (1230.87 +/- 208.14) pA (P < 0.01) and (10.72 +/- 1.06) pA/pF (P < 0.01). The serum Abs could also decrease I(Ca-L) peak intensity and density [from (1995.21 +/- 195.13) pA and (18.13 +/- 1.03) pA/pF to (636.42 +/- 110.07) pA (P < 0.01) and (5.54 +/- 0.81) pA/pF, P < 0.01]. The M(2)-receptors antagonist, atropine was able to block these effects of carbachol and Abs.

CONCLUSIONSThe circulating serum autoantibodies against the M(2)-receptors has similar effect as M(2)-receptors agonist on decreasing the isoprenaline stimulated I(Ca-L) in guinea pig cardiac myocytes and possess negative inotropic effect. These results further suggest that serum autoantibodies against the human M(2) muscarinic acetylcholine receptors may participate in the pathophysiological processes in patients with heart failure.

Adult ; Aged ; Animals ; Autoantibodies ; pharmacology ; Calcium Channels, L-Type ; drug effects ; Female ; Guinea Pigs ; Heart Failure ; immunology ; Humans ; Male ; Middle Aged ; Myocytes, Cardiac ; drug effects ; metabolism ; Patch-Clamp Techniques ; Receptor, Muscarinic M2 ; immunology ; Serum ; immunology

OBJECTIVETo investigate the effect of the positive sera of autoantibodies against the human beta1-adrenoceptor from patients with congestive heart failure on activity of L-Type Ca2+ channel in guinea pig cardiac myocytes.

METHODUsing whole cell patch-clamp technique, we quantitatively researched the ionic intensity and density of L-type Ca2+ channel (ICa-L).

RESULTSThe beta-adrenocepter agonist isoprenaline increased the ICa-L peak intensity and density from (997.09 +/- 227.5) pA and (8.20 +/- 0.86) pA/pF to (2241.01 +/- 348.5) pA and (18.98 +/- 1.18) pA/pF, respectively (P < 0.01). The positive sera of autoantibodies against the beta1-adrenoceptor could also increase ICa-L peak intensity and density from (963.57 +/- 207.56) pA and (8.14 +/- 0.72) pA/pF to (1382.41 +/- 241.36) pA and (11.70 +/- 1.03) pA/pF (P < 0.01). Esmolol, a beta1-adrenoceptor antagonist blocked these effects of isoprenaline and autoantibodies.

CONCLUSIONSHuman cardiac positive sera of autoantibodies against the beta1-adrenoceptor has an isoproterenol-like effect on cardiac myocytes receptor. It may participate in the pathophysiologic process of cardiac myocytes.

Animals ; Autoantibodies ; blood ; Calcium Channels, L-Type ; metabolism ; Female ; Guinea Pigs ; Heart Failure ; immunology ; Male ; Myocytes, Cardiac ; metabolism ; Patch-Clamp Techniques ; Receptors, Adrenergic, beta ; immunology

OBJECTIVETo investigate the biological effects of anti-beta(3) adrenoceptor (beta(3)-AR) autoantibody in the serum of patients with heart failure, which may contribute to a new therapeutic clue for heart failure.

METHODSThe synthetic peptide of the second extracellular loop of the beta(3)-AR was used as the antigen to screen sera of patients with heart failure and of healthy controls by using enzyme-linked immunosorbent assay. IgG in the patients group of positive autoantibody sera was prepared by using a MabTrap Kit (Amersham) following the manufacturer's instructions. The effects of IgG per each group both on contractile response of adult isolated cardiomyocytes and on beating frequency of cultured neonatal rat cardiomyocytes were observed.

RESULTSThe positive rate of anti-beta(3)-AR autoantibody was 26.7% (mean antibody titer: 1:43.27 +/- 2.71) or 11.0% (mean antibody titer: 1:14.59 +/- 1.61) in patients or healthy subjects, respectively P < 0.05. Compared with the control group, the autoantibody against beta(3)-AR from the patients group decreased cell shortening amplitude/cell shortening 3.84% +/- 0.33%, the velocity of shortening -0.47 microm/s +/- 0.07 microm/s and relengthening 0.17 microm/s +/- 0.02 microm/s in adult isolated cardiac myocytes, respectively. The autoantibody in the patients group decreased the beating rate in cultured neonatal rat cardiac myocytes by 47.1 beats/min +/- 8.11 beats/min, which could have a 6-hour continuance. This decreasing was not modified by Nadolol (beta(1)-AR and beta(2)-AR antagonist) in pretreating myocytes, but was nearly prevented by Bupranolol (nonselective beta-AR antagonist) or beta(3)-AR specific antigen.

CONCLUSIONIt seems reasonable to state that a high titer of the autoantibody against beta(3)-AR in the serum in patients with heart failure, which could have a negative inotropic and chronotropic effect, may be a part of pathophysiological mechanisms of heart failure.

Adult ; Animals ; Autoantibodies ; immunology ; metabolism ; Case-Control Studies ; Cells, Cultured ; Female ; Heart Failure ; immunology ; metabolism ; Humans ; Immunoglobulin G ; immunology ; Male ; Middle Aged ; Myocytes, Cardiac ; immunology ; Rats ; Rats, Wistar ; Receptors, Adrenergic, beta-3 ; immunology

OBJECTIVETo study the significance of Th1/Th2 function imbalance in patients with post-infarction cardiac insufficiency.

METHODSForty-three MI (myocardial infarction) patients were divided into 2 groups one month after the onset according to the New York Heart Association (NYHA) classification system: group MI 1 (I, II) 25 patients and group MI 2 (III, IV) 18 patients. At the same time, the heart function was evaluated by two-dimensional echocardiography. Peripheral blood mononuclear cells (PBMCs) were collected from these patients. Cytokine-producing CD4 + T cells were quantified by 3-color flow cytometry after being stimulated with phorbol myristate acetate (PMA) and ionomycin. After being stimulated with PHA, the levels of IFN-gamma and IL-4 in culture supernatants were measured by ELISA.

RESULTSThe frequencies of IFN-gamma-producing T cells were found to be significantly higher in group MI 2 (16.8%) than that in group MI 1 (13.1%). There was no significant difference on the frequencies of IL-4-producing peripheral T cells between the two groups. The IFN-gamma level and the ratios of IFN-gamma/IL-4 in group MI 2 were significantly higher than those in group MI 1, while there was no significant difference in IL-4 levels between the two groups.

CONCLUSIONSThe Th-cell function was associated with heart function in post MI patients. The up-regulation of Th1 cell function was consistent with poor heart function, suggesting that Th1/Th2 cell function imbalance may participate in ventricular remodelling after MI.

Aged ; CD4-Positive T-Lymphocytes ; metabolism ; Female ; Heart Failure ; etiology ; immunology ; physiopathology ; Humans ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Male ; Middle Aged ; Myocardial Infarction ; complications ; immunology ; Th1 Cells ; Th2 Cells