Clinical biomarkers of cardiac injury: cardiac troponins and natriuretic peptides

Laboratory and clinical medicine groups are actively collaborating and optimizing their individual expertise to potentially prove standardized biomarker assays that will optimize patient care. It is critical as new biomarkers are discovered, that quality specifications be developed prior to approval by regulatory agencies that give supported endorsement for the worldwide marketplace. The laboratory medicine and clinical communities of scientists continues to challenge the biomarker field, working towards developing standard reference materials, and providing quality analytical specifications that, hopefully, will be endorsed by our clinical, in vitro diagnostic and pharmaceutical industry colleagues. This paper will specifically address cardiac troponins and natriuretic peptides.     

Lethal immunoglobulins: Autoantibodies and sudden cardiac death

Sudden cardiac death (SCD) is an unexpected death due to cardiac causes that occurs in a short time period (generally within 1?h of symptom onset) in a person with known or unknown cardiac disease. Patients with cardiomyopathies, myocarditis, ischemic heart disease and cardiac channelopathies are at risk of SCD. However, a certain percentage of autopsy-negative cases of SCD in the young (<35?years) remain unexplained even after a post-mortem genetic testing. Autoantibodies against cardiac proteins may be potentially involved in the pathogenesis of different heart diseases and in the occurrence of unexplained SCD. In this review we analyze clinical and animal studies that elucidate the prevalence of these autoantibodies in patients with different cardiac diseases and their pathophysiological relevance. We propose a classification of the autoantibodies associated with heart diseases and focus on their molecular and cellular effects. Anti-beta adrenergic receptor antibodies and anti-muscarinic acetylcholine receptor antibodies affect myocardial electrophysiological properties and were reported to be the independent predictors of SCD in patients with different heart diseases. Autoimmune mechanism is proposed for cardiac-related adverse reactions following human papillomavirus (HPV) vaccination. The pentapeptid sharing between HPV's antigens, adrenergic receptors and muscarinic acetylcholine receptors supports this assumption. The dysregulating effects of the autoantibodies against calcium and potassium ion channels can be the basis for autoimmune phenocopies of genetic cardiac channelopathies, which are also associated with SCD.     

Autoantibodies to cardiac myosin in mouse cytomegalovirus myocarditis

Myocarditis accompanies sublethal mouse cytomegalovirus (MCMV) infection in susceptible BALB/c mice and persists beyond the acute phase of infection, in the absence of demonstrable virus antigen but in the continuing presence of autoantibodies to cardiac muscle. Heart tissue autoantibodies of the IgG class were first detected by ELISA in sera at Days 3-5 post-infection (PI) and persisted to Day 100, in two strains of MCMV-infected mice which are susceptible (BALB/c) and resistant (C57BL/10) to MCMV-induced myocarditis. Analysis by immunoblot showed that autoantibodies in early immune sera (Day 10) from both mouse strains reacted with the contractile proteins troponin, tropomyosin and myosin, as well as with other unidentified polypeptides within normal mouse organ homogenates. However, the dominant reactivity of late immune sera (Day 100) was to a 200,000 molecular weight (MW) polypeptide in muscle homogenates identified as the heavy chain of myosin. Autoantibodies reacting with the cardiac or striated muscle isoforms of myosin were assessed by ELISA in BALB/c and C57BL/10 mice. At Days 28, 56 and 100 PI only the susceptible BALB/c strain had high titres of autoantibodies reacting with the cardiac isoform of myosin. Increasing the virus dose given to C57BL/10 mice resulted in slight increases in titres of anti-myosin antibody; however, the peak antibody titres did not approach those of BALB/c mice and persisting myocarditis did not develop. Absorption experiments showed that cardiac myosin-specific antibodies were present in immune sera from susceptible BALB/c mice at Day 100 but not in resistant C57BL/10 mice by ELISA and immunoblot. These results demonstrate that autoimmunity to myosin is a prominent feature of the humoral autoimmune response following MCMV infection, and that there are differences both in fine isoform specificity and titre of anti-myosin antibodies between strains of mice that develop persisting myocarditis and strains that do not. Cardiac myosin-specific autoantibodies may play an immunopathogenic role in CMV-induced myocarditis.     

Autoantibodies against G-protein-coupled receptors modulate heart mast cells

Mast cells are believed to be involved in myocardial tissue remodelling under pathophysiological conditions. We examined the effects of autoantibodies against G-protein-coupled receptors in sera of patients with heart diseases on myocardial mast cells in the cultured neonatal Sprague-Dawley rat heart cells. Cells collected at day 3 and 10 of the culture were preincubated with autoantibodies against α-adrenoceptor and angiotensin Ⅱ ATl-receptor, agonist phenylephrine and angiotensin Ⅱ, and control IgG. The pretreated cultured cells were stained for selected mast cell markers tryptase, chymase and TNF-α The cultured cells were also processed for observation with electron microscopy. The autoantibodies-treatment of the 3-day cultured cells caused both increased intensity of immunofluorescence （p 〈 0.05） and their enlarged diameters of the mast cells when compared to age-matched ones. In contrast, the fluorescence of preincubated 10-day-old mast cells was decreased compared with controls （p 〈 0.01）. In control samples, the fluorescence of 10-day-old mast cells was significantly higher than that of 3-day-old ones （p 〈 0.001）. Results of electron microscopy examination demonstrated there was an increased granulation of treated 3-day-old mast cells, while a degranulation of mast cells at day 10 of application. The results suggest the modulation effect of the autoantibodies against G-protein-coupled receptors on mast cells, indicating a potential functional link between the autoantibodies against G-protein-coupled receptors and the mast cells in progression of heart disease.     

Autoantibodies against mannose-binding lectin in systemic lupus erythematosus

In systemic lupus erythematosus (SLE), autoantibodies directed against complement components of the classical pathway, especially against C1q, are associated with severe disease and are of prognostic value for flares of lupus nephritis. Mannose-binding lectin (MBL), the recognition unit of the MBL pathway of complement activation, has structural similarities to C1q. Deficiencies of MBL have been shown to predispose to the development of SLE and to influence the course of the disease. We hypothesized that the presence of autoantibodies to MBL, analogous to autoantibodies to C1q in patients with SLE, may contribute to disease development. The occurrence of anti-MBL autoantibodies was assessed by enzyme-linked immunosorbent assay (ELISA) of 68 serum samples from 20 patients with SLE and in serum from 70 healthy controls. Levels of antibodies directed against MBL were significantly higher in patients with SLE compared to healthy subjects. No significant difference was found between patients with active disease compared to those with inactive disease. While the occurrence of anti-C1q autoantibodies was associated with renal involvement, no such relationship was found for anti-MBL autoantibodies. A significant correlation was found between anti-MBL and anti-C1q antibody levels. The level of anti-MBL antibodies was negatively correlated with MBL-complex activity of circulating MBL. Anti-MBL autoantibodies were of the immunoglobulin G (IgG) isotype and the binding site of IgG anti-MBL was located in the F(ab')2 portion. We conclude that anti-MBL are present in sera from SLE patients and influence the functional activity of MBL.     

Autoantibodies to vimentin cause accelerated rejection of cardiac allografts

Autoimmune responses to vimentin occur after solid organ transplantation, but their pathogenic effects are unclear. The aim of these studies was to investigate the effects of vimentin preimmunization on allogeneic and isografted hearts in a murine transplant model. Immunization of C57BL/6 mice with murine vimentin in complete Freund's adjuvant resulted in anti-vimentin antibodies and vimentin-reactive Th-1 cells. Transplantation of 129/sv hearts into vimentin-immunized C57BL/6 recipients resulted in accelerated rejection (8.4 +/- 1.5 days; n = 18), compared with hen egg lysozyme-immunized C57BL/6 (13.3 +/- 2.2 days; n = 10; P < 0.0001, log-rank test). In contrast, isografts continued to beat beyond 90 days. Immunohistochemical analysis of allografts from vimentin/complete Freund's adjuvant mice demonstrated increased numbers of T cells and enhanced microvascular deposition of C3d, CD41, and P-selectin compared with controls. Antibodies were necessary for accelerated rejection, shown by the fact that vimentin-immunized B-cell-deficient IgH6 mice did not show accelerated rejection of 129/sv allografts, but rejection was restored by adoptive transfer of serum containing anti-vimentin antibodies. Eluates from donor hearts placed in vimentin/complete Freund's adjuvant recipients contained anti-vimentin antibodies, shown by Western blotting. Confocal imaging of rejected hearts de-monstrated presence of vimentin and C3d on apoptosed leukocytes, endothelial cells, and platelet/leukocyte conjugates. These results demonstrate that autoantibodies to vimentin, in conjunction with the alloimmune response, have a pathogenic role in allograft rejection.     

Autoantibodies directed against the UDP-glucuronosyltransferases in human autoimmune hepatitis

Liver-Kidney Microsomes Type 3 (LKM3) autoantibodies (aAbs) have been described in chronic hepatitis D virus infection in 1983. The detection of such aAbs in autoimmune hepatitis (AIH) Type 2 was thereafter reported. The molecular targets of LKM3 aAbs have been identified as enzymes belonging to the UDP-glucuronosyltransferase family 1. Since 20-30% of suspected AIH are negative for the classical autoimmune serological markers, such as aAbs directed against antinuclear autoantibodies, smooth muscle autoantibodies and Liver-Kidney Microsomes Type 1 aAbs, LKM3 aAbs could be of great interest in the diagnosis of such negative AIH. In this review, we discuss the sensitivity and specificity of these aAbs in AIH in order to stress out their potential clinical use as a marker.     

Autoantibodies against muscarinic cholinergic receptor in chronic fatigue syndrome

The disturbance of the central nervous system and immunological abnormalities have been suggested in patients with chronic fatigue syndrome (CFS). We focused on immunological abnormalities against neurotransmitter receptors in CFS. Using a sensitive radioligand assay, we examined serum autoantibodies to recombinant human muscarinic cholinergic receptor 1 (CHRM1), mu-opioid receptor (OPRM1), 5-hydroxytryptamine receptor 1A (HTR1A), and dopamine receptor D2 (DRD2) in patients with CFS (n=60) and results were compared with those in patients with autoimmune disease (n=33) and in healthy controls (n=30). The mean anti-CHRM1 antibody index was significantly higher in patients with CFS (p<0.0001) and autoimmune disease (p<0.05) than that in healthy controls, and positive reaction was found in 53.3% of patients with CFS. Anti-OPRM1 antibodies, anti-HTR1A antibodies, and anti-DRD2 antibodies were found in 15.2, 1.7, and 5.0% of patients with CFS, respectively. Anti-nuclear antibodies were found in 56.7% (34/60) of patients with CFS, but anti-nuclear antibody titers did not correlate with the activities of the above four autoantibodies. The patients with positive autoantibodies to CHRM1 had a significantly higher mean score (1.81) of 'feeling of muscle weakness' than negative patients (1.18) among CFS patients (p<0.01). Higher scores on 'painful node', 'forgetfulness', and 'difficulty thinking' were also found in CFS patients with anti-CHRM1 antibodies but did not reach statistical significance. In conclusion, autoantibodies to CHRM1 were detected in a large number of CFS patients and were related to CFS symptoms. Our findings suggested that subgroups of CFS are associated with autoimmune abnormalities of CHRM1.     

Autoantibodies directed against the protease inhibitor calpastatin in psoriasis

Psoriasis is believed to be a T cell-mediated autoimmune disease, but also exhibits autoantibody production. Calpastatin is an endogenous inhibitor of calpain, a ubiquitous protease that regulates inflammatory processes. Anti-calpastatin autoantibody was first identified as an autoantibody specific to rheumatoid arthritis, but has been also detected in other autoimmune diseases. In this study, we examined the presence and levels of anti-calpastatin antibody in 77 psoriasis patients by enzyme-linked immunosorbent assay. Compared with normal controls, psoriasis patients exhibited significantly elevated IgG anti-calpastatin antibody levels that were similar to those found in rheumatoid arthritis patients. Remarkably, IgG anti-calpastatin autoantibody in sera from psoriasis patients inhibited calpastatin activity. Calpain II expression was up-regulated in psoriasis skin lesions compared with normal skin while calpastatin expression was normal. The results of this study reveal the presence of anti-calpastatin autoantibody in psoriasis.     

Autoantibodies against CD28 are associated with atopic diseases

The B7-1/B7-2-CD28/CTLA-4 pathway is crucial in regulating T cell activation and tolerance. Autoantibodies to surface molecules on lymphocytes have already been described in various immune conditions, such as autoimmune diseases, infections and blood transfusions. The objective of this study was to test sera from healthy individuals and from patients for association of CD28 autoantibodies with inflammatory and non-inflammatory diseases. First, CD28 was obtained by digestion of CD28-Ig fusion protein with trypsin. The cleavage products were separated by sodium dodecyl sulphate-page gel electrophoresis. Additionally, a CD28/GST fusion protein was expressed in Escherichia coli and was used to establish an enzyme-linked immunosorbent assay for detection of autoantibodies against CD28. Sera from healthy individuals (n = 72) and patients with different inflammatory and non-inflammatory skin diseases (n = 196) were tested for the presence of autoantibodies against CD28. Using mixed lymphocyte reaction (MLR), purified autoantibodies against CD28 were tested for their effects on CTLA-4-Ig-induced T cell anergy. In this study, for the first time, we describe the existence of autoantibodies against CD28 in humans which are associated with atopic diseases, e.g. allergic rhinitis and asthma. These antibodies stimulate T cells and overcome the CTLA-4-Ig-induced anergy of T cells in an MLR. The existence of autoantibodies against CD28, which may have a T cell-stimulating function, has been shown. The data indicate that autoantibodies against CD28 could be a new immunological mechanism in allergic inflammation. Additionally, autoantibodies against CD28 could be an important new marker to discriminate between atopic diseases and other inflammatory skin diseases.     

The Occurrence of Serum Autoantibodies against Enolase in Cancer-Associated Retinopathy

Cancer-associated retinopathy (CAR) is an uncommon paraneoplastic disease in which degeneration of the retina occurs as a remote effect of cancer in a distant part of the body. Immunoreactivity of sera from CAR patients and controls have been analyzed. Immunostaining of human retinal proteins showed that a soluble protein ofM_r∼46 kDa (p46) is labeled by antibodies from several CAR patients with various types of cancer (lung, breast, bladder, prostate, salivary gland, and gastrointestinal tract cancer and chronic lymphocytic leukemia). These sera didnotshow reactivity with the 23-kDa protein previously associated with CAR. To identify and further characterize p46, the retinal protein was purified to homogeneity by anion-exchange chromatography and preparative gel electrophoresis. Protein sequence analysis of the peptides from p46 revealed a high homology with human enolase, an important glycolytic enzyme. Although enolase has been previously identified as a product of several types of tumors, and enolase activity has been detected in the sera of some cancer patients, the existence of autoantibodies directed to enolase has not been described. This is the first report of the presence of serum antibodies to retinal enolase in the patients with cancer and the CAR syndrome. When antibodies of specific isotypes (IgG, IgM, and IgA) were measured, IgG1 isotype was dominant. The significance of these antibodies for the disease process is under investigation.     

Autoantibodies against small nucleolar ribonucleoprotein complexes and their clinical associations

Sera from patients suffering from systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) have been shown to contain reactivities to nuclear components. Autoantibodies specifically targeting nucleolar antigens are found most frequently in patients suffering from SSc or SSc overlap syndromes. We determined the prevalence and clinical significance of autoantibodies directed to nucleolar RNA-protein complexes, the so-called small nucleolar ribonucleoprotein complexes (snoRNPs). A total of 172 patient sera with antinucleolar antibodies were analysed by immunoprecipitation. From 100 of these patients clinical information was obtained by chart review. Autoantibodies directed to snoRNPs were detected not only in patients suffering from SSc and primary Raynaud's phenomenon (RP), but also in patients suffering from SLE, rheumatoid arthritis (RA) and myositis (PM/DM). Antibodies against box C/D small snoRNPs can be subdivided in antifibrillarin positive and antifibrillarin negative reactivity. Antifibrillarin-positive patient sera were associated with a poor prognosis in comparison with antifibrillarin negative (reactivity with U3 or U8 snoRNP only) patient sera. Anti-Th/To autoantibodies were associated with SSc, primary RP and SLE and were found predominantly in patients suffering from decreased co-diffusion and oesophagus motility and xerophthalmia. For the first time autoantibodies that recognize box H/ACA snoRNPs are described, identifying this class of snoRNPs as a novel autoantigenic activity. Taken together, our data show that antinucleolar patient sera directed to small nucleolar ribonucleoprotein complexes are found frequently in other diseases than SSc and that categorization of diagnoses and clinical manifestations based on autoantibody profiles seems particularly informative in patient sera recognizing box C/D snoRNPs.     

Autoantibodies against complement C1q in acute post-streptococcal glomerulonephritis

Autoantibodies against complement C1q (anti-C1q) strongly correlate with the occurrence of severe lupus nephritis. Recent data suggest that anti-C1q might also correlate with more severe forms of acute post-streptococcal glomerulonephritis (APSGN). Therefore, we prospectively investigated the role of anti-C1q in 50 children with newly diagnosed APSGN. Associations between anti-C1q and disease manifestations as well as serum complement concentrations were analyzed. Nineteen of the 50 children (38%) with APSGN were positive for anti-C1q compared to 0/40 healthy controls. Levels of anti-C1q correlated negatively with serum C1q and C3 concentrations. Anti-C1q positive patients had significantly higher proteinuria and serum creatinine as well as more often oliguria, hypertension and delayed resolution of the disease than patients without anti-C1q. The data point to a potential pathogenic role of anti-C1q in APSGN. Determination of anti-C1q might help to identify patients at risk for prolonged courses of the disease.     

Prescription, assay and interpretation of cardiac troponins tests: guidelines from SFBC-CNBC troponin working group

Troponin (I or T) has become the gold-standard marker in acute coronary syndromes during the last few years, as confirmed by a national survey realized within french clinical chemists, cardiologists and emergency practitioners. The importance of this marker and the heterogeneousness of circulating forms of troponin after myocardial necrosis fully justify international studies about standardization of this assay, which is a central bulk to reach a global market coherence. Checking analytical problems, although necessary, must be absolutely associated with an informed clinical interpretation. The knowledge of the crucial thresholds of each assay, the kinetic curves and the specificity limits of troponin assays allow the best use of their potential in diagnosis and prognosis together with an optimal patient care in very different clinical settings, in addition to others clinical and technical arguments. The quality improvement through successive generations of assay kits must nowadays persuade the physicians never to ignore a significant and valid troponin increase, which mainly reveals a cardiac injury, whatever its origin.     

Autoantibodies against Forssman glycolipids in Graves'' disease and Hashimoto''s thyroiditis.

Sera from patients with Graves' disease and Hashimoto's thyroiditis have been shown to react with the Forssman glycolipid antigen (Gb5) using the techniques of high performance thin-layer chromatography (HPTLC) immunostaining and ELISA. Human monoclonal antibodies (MoAbs) have been prepared by fusion of human myeloma with peripheral lymphocytes from patients with Graves' disease. A MoAb, TRMo-4, reacted strongly and specifically with Gb5. These results suggest that anti-Forssman antibody may be involved in the pathogenesis of these autoimmune diseases. The detection of anti-Forssman glycolipid antibody may provide a useful means for clinical diagnosis and therapy.