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Profiling humoral autoimmune repertoire of dilated cardiomyopathy (DCM) patients and development of a disease-associated protein chip
Abstract
Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37,200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.
... Selection of included autoantigen proteins was based on our previous autoantibody screens in prostate cancer [31] and autoimmune diseases [33][34][35], and on published autoantigens found associated with cancer [36][37][38][39][40][41][42]. In addition, protein production efficacy, protein quality, and assay performance criteria were considered for the final selection of autoantigen proteins. ...
... The bead protein array was established as a broadly applicable autoantibody analysis tool and consisted of 3065 individual protein beads grouped into 8 subarrays. As for the planar protein array, selection of autoantigen proteins was based on published data [31,[33][34][35][36][37][38][39][40][41][42] considering also autoantigen protein production and performance. The final autoantigen panel comprised putative biomarkers for autoimmune diseases and tumor-related processes (S1 Table). ...
... We hypothesized that patients with chronic inflammation might have a specific immune response to inflammation-related and prostate-associated proteins. We therefore screened prostate cancer patients grouped into low or high numbers of prostate infiltrating immune cell cases for the presence of circulating autoantibodies directed against a previously identified prostate cancer, other cancer type and autoimmune disease-associated autoantigen panel [31,[33][34][35][36][37][38][39][40][41][42]. Focusing on a cohort of cancer patients who underwent radical prostatectomy allowed to inspect the whole gland for infiltrating immune cells and guaranteed an accurate discrimination of high and low prostate inflammation patients. ...
Background:
Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens.
Methods:
Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology.
Results:
Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076).
Conclusions:
We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation.
... An early approach was to generate cDNA expression libraries from human tissue such as human fetal brain to create a redundant set of cDNA clones (37,200), which were then expressed in E. coli, and spotted onto large filters without purification. These arrays were then probed either with pooled sera from patients with alopecia areata or with plasma from individual patients with dilated cardiomyopathy (DCM) [54][55][56][57]. The authors identified 23 antigens which elicited an immune response in two or more pools of patient sera with alopecia areata and 48 antigens in two or more patients with DCM [55]. ...
... These arrays were then probed either with pooled sera from patients with alopecia areata or with plasma from individual patients with dilated cardiomyopathy (DCM) [54][55][56][57]. The authors identified 23 antigens which elicited an immune response in two or more pools of patient sera with alopecia areata and 48 antigens in two or more patients with DCM [55]. To confirm the responses, the antigens were purified and printed on a focused array to test individual sera from patients and controls. ...
The search for new biomarkers for diagnosis, prognosis, and therapeutic monitoring of diseases continues in earnest despite dwindling success at finding novel reliable markers. Some of the current markers in clinical use do not provide optimal sensitivity and specificity, with the prostate cancer antigen (PSA) being one of many such examples. The emergence of proteomic techniques and systems approaches to study disease pathophysiology has rekindled the quest for new biomarkers. In particular the use of protein microarrays has surged as a powerful tool for large-scale testing of biological samples. Approximately half the reports on protein microarrays have been published in the last two years especially in the area of biomarker discovery. In this review, we will discuss the application of protein microarray technologies that offer unique opportunities to find novel biomarkers.
... A high-density multiprotein array (MPA) (hEXselect, Engine, Berlin, Germany, Order No. 1003) derived from a cDNA bank of two first trimester human fetal brain samples, containing 23,806 E. coli expression clones representing a total of 10,000 human proteins (Büssow et al. 1998;Horn et al. 2006), was incubated with either α-HPy or α-CJe according to the manufacturer's protocol. In brief, prior to the first use of these arrays, protein spots were fixed on the PVDF membrane for 10 min with 70% ethanol and rinsed twice with distilled water. ...
Due to molecular mimicry, maternal antibacterial antibodies are suspected to promote neurodevelopmental changes in the offspring that finally can cause disorders like autism and schizophrenia. Using a human first trimester prenatal brain multiprotein array (MPA), we demonstrate here that antibodies to the digestive tract bacteria Helicobacter pylori (α-HPy) and Campylobacter jejuni (α-CJe) interact with different synaptic proteins, including the calcium sensor synaptotagmin 5 (Syt5). Interactions of both antisera with Syt5 were confirmed by Western blot with a HEK293-cells overexpression lysate of this protein. Immunofluorescence and Western blotting revealed SiMa cells to express Syt5, which also co-migrated with a band/spot labeled by either α-HPy or α-CJe. Functionally, a 12-h pretreatment of SiMa cells with 10 μg/ml of either α-HPy or α-CJe resulted in a significant reduction of acetylcholine(ACh)-dependent calcium signals as compared to controls. Also ACh-dependent vesicle recycling was significantly reduced in cells pretreated with either α-HPy or α-CJe. Similar effects were observed upon pretreatment of SiMa cells with Syt5-specific antibodies. In conclusion, the present study supports the view that prenatal maternal antibacterial immune responses towards HPy and by this to Syt5 are able to cause functional changes, which in the end might contribute also to neurodevelopmental disorders.
... Each array contains 16,000 Escherichia coli His-tagged expression clones from a variety of expression libraries arrayed in a 3 × 3 pattern on PVDF membranes. The expression vector used is pQE30NST (GenBank Accession No. AF074376) and it is transformed into E. coli strain SCS1 (Stratagene) [43,44]. Sequence information for individual clones recognized by antibodies was provided by Imagenes. ...
... Song et al. discovered 3 novel autoantigens, namely RPS20, Alba-like and dUTPase for autoimmune hepatitis (AIH) using a protein microarray containing 5011 nonredundant proteins [98]. Horn et al. profiled the repertoire of IgG autoantibodies in plasma samples from Dilated Cardiomyopathy (DCM) patients with a redundant protein microarray containing 37,200 total proteins and identified 26 autoreactive proteins to IgG (with 6 of them reactive specifically to the IgG3 subclass) [39]. Autoantigens were also identified for the chronic disease alopecia areata by protein microarray technology [61]. ...
Our understanding of human disease and potential therapeutics is improving rapidly. In order to take advantage of these developments it is important to be able to identify disease markers. Many new high-throughput genomics and proteomics technologies are being implemented to identify candidate disease markers. These technologies include protein microarrays, next-generation DNA sequencing and mass spectrometry platforms. Such methods are particularly important for elucidating the repertoire of molecular markers in the genome, transcriptome, proteome and metabolome of patients with diseases such as cancer, autoimmune diseases, and viral infections, resulting from the disruption of many biological pathways. These new technologies have identified many potential disease markers. These markers are expected to be valuable to achieve the promise of truly personalized medicine.
... This approach lacks the specificity and accuracy needed for the precise identification of autoantibody targeted antigens. In search of an antibody detection method avoiding these pitfalls, antigen microarrays are a promising approach (Robinson et al., 2002; Lueking et al., 2005; Horn et al., 2006). Over the past years antigen microarrays have more and more been used for antibody based biomarker detection (Quintana et al., 2008). ...
Glaucoma is a chronic neurodegenerative disease and one of the leading causes of blindness. Autoantibody based immune processes are assumed to be involved in its pathogenesis. However, it is still unclear to what extent autoantibody patterns found in the eye (aqueous humor) are congruent to systemic autoantibodies (blood). Consistency would underline the specificity of known serum antibody markers for glaucoma. In this study we used antigen microarrays to analyze autoantibody reactivities in sera and corresponding aqueous humor samples of primary open-angle glaucoma patients (N=37) and non-glaucomatous controls (N=31). Compared to control subjects several divergent immunoreactivities were identified for the glaucoma group in both body fluids. Interestingly, 20% of the tested antigens revealed increased immunoreactivities (e.g., against HSP27, MBP, and α-1-antitrypsin) and 7.5% decreased immunoreactivities (e.g., against GFAP and β-L-crystallin), thus demonstrating a significant alteration of the autoantibody profiles in glaucoma patients. Using an artificial neural network in combination with a unique serum autoantibody pattern on prospective sera we were able to detect glaucoma with a specificity and sensitivity of approximately 93%. The intraindividual comparison revealed a strong correlation of detected immunoreactivities in sera and comparative aqueous humor samples in both study groups. These results emphasize the specificity of immunoreactions found in blood samples of glaucoma patients. Furthermore they indicate the necessity of analyzing not only up-regulated but also down-regulated antibody reactivities, which might be likewise relevant for the understanding of other diseases.
... " Type II " tumours include high-grade serous and endometrioid carcinomas. Our laboratory has developed protein array based technologies and methodologies since first developing the hEx1 protein expression library [18] with which we have performed studies on the binding of autoantibodies to arrayed proteins in alopecia areata and dilated cardiomyopathy [19,20] , binding of antibodies to proteins identified from tumour neovasculature in humans [21], context independent motif identification in the human proteome [22] and of identification of novel protein– protein interaction networks [23,24] . We have employed this library screening method as part of an ovarian cancer research consortium, Discovary, performing a pilot study on autoantibody identification screening the hEx1 protein library with ovarian cancer serum samples from a well characterised patient cohort with stage I ovarian cancer of mixed histology, stage III serous papillary adenocarcinoma, primary peritoneal carcinoma and normal/healthy individuals. ...
Autoantibodies represent an attractive biomarker for diagnostic assays principally due to the stability of immunoglobulin in patient serum facilitating measurement with conventional assays. Immune responses to tumorigenesis may facilitate detection of ovarian cancer in the early stages of the disease with identification of a panel of tumour specific autoantibodies. Despite the reporting of many tumour associated autoantibodies using arrays of tumour antigens, this has not led to the advance in diagnostic capability as rapidly as was initially expected. Here we examine the potential diagnostic utility of candidate autoantibody biomarkers identified via screening of serum samples on a high content human protein array from a unique cohort of early stage and late stage ovarian cancer patients. We analyse the performance of autoantibodies to the tumour suppressor protein p53 and the novel autoantigens alpha adducin and endosulfine alpha identified in our array screen. Each antigen has different performance characteristics using conventional ELISA format and Western blot immunoassay. The high attrition rate of promising autoantigens identified by array screening can in part be explained by the presentation of the epitope of the antigen in the subsequent method of validation and this study provides directions on maximising the potential of candidate biomarkers. This article is part of a Special Issue entitled: Translational Proteomics.
... Protein chip technology is becoming an increasingly established technique, not only for characterizing specific proteins or even proteomes, but also for clinical applications. Although routine clinical use of microarray technology still is in its early phase, antibody microarrays have already been developed for a number of clinical diagnostic applications [1][2][3][4][5][6]. ...
The antibody microarrays have become widespread, but their use for quantitative analyses in clinical samples has not yet been established. We investigated an immunoassay based on nanoporous silicon antibody microarrays for quantification of total prostate-specific-antigen (PSA) in 80 clinical plasma samples, and provide quantitative data from a duplex microarray assay that simultaneously quantifies free and total PSA in plasma. To further develop the assay the porous silicon chips was placed into a standard 96-well microtiter plate for higher throughput analysis. The samples analyzed by this quantitative microarray were 80 plasma samples obtained from men undergoing clinical PSA testing (dynamic range: 0.14-44ng/ml, LOD: 0.14ng/ml). The second dataset, measuring free PSA (dynamic range: 0.40-74.9ng/ml, LOD: 0.47ng/ml) and total PSA (dynamic range: 0.87-295ng/ml, LOD: 0.76ng/ml), was also obtained from the clinical routine. The reference for the quantification was a commercially available assay, the ProStatus PSA Free/Total DELFIA. In an analysis of 80 plasma samples the microarray platform performs well across the range of total PSA levels. This assay might have the potential to substitute for the large-scale microtiter plate format in diagnostic applications. The duplex assay paves the way for a future quantitative multiplex assay, which analyzes several prostate cancer biomarkers simultaneously.
... With the development of etiological detection technology, microarrays have rapidly expanded into the area of biological research, including gene expression, signal transection, genome mismatch scanning, and protein trafficking. Among the many types of microarrays, the protein microarray offers an opportunity to study the full spectrum of protein attributes in a parallel, miniaturized, and automated fashion representing a significant shift from the traditional ''one protein at a time'' methods [58,59]. Furthermore, the method not only aids in improved diagnosis, but also identifies autoantibody signatures that may represent disease subgroups, early diagnostics [60,61] and facilitates the analysis of the outcome of vaccine trials [62]. ...
Food-borne helminthiases (FBHs) have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches.
In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI): 95.3-98.7%) to 100.0% (95% CI: 100.0%) in the protein microarray and from 97.7% (95% CI: 96.2-99.2%) to 100.0% (95% CI: 100.0%) in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1-96.3%) to 92.1% (95% CI: 83.5-100.0%) in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4-92.6%) to 92.1% (95% CI: 83.5-100.0%). Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used.
The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening.
... Then the arrays are treated with a secondary fluorescence labeled antibody (Cy3, Cy5) for visualizing the antibodyeantigen interactions. The subsequent data analysis and the statistical evaluation of this highly sensitive antigen microarray approach are used for the quantification of specific antibody levels Horn et al., 2006;Robinson et al., 2002). The advantage of this highly sensitive and specific technique is a simultaneous screening of numerous protein targets for the rapid confirmation of potential biomarkers making the approach highly interesting for high throughput clinical studies. ...
The main focus in clinical proteomics is the discovery of new protein or peptide biomarkers which are correlated with a certain disease. Tear proteins have been investigated extensively in the past and distinct relations between the levels of certain tear proteins to different disorders have been demonstrated. In this review we attempt to summarize proteomic technologies for biomarker identification in tears and some disease related biomarkers in tear fluids that were discovered through different proteomic techniques in different conditions like dry eye, Sjogren's syndrome, contact lens wearers, glaucoma, diabetic retinopathy or cancer. Proteomic analysis of tear fluid has proven to be e promising to gain more information about the pathogenesis of diseases and lead to new diagnostic possibilities. Furthermore, biomarkers represent promising targets for drug development and can be used to monitor the disease state or treatment responses, and accordingly improve the standards of patient care.
... Several formats of protein arrays are available, but those featuring antigens are promising tools to prole antibody biosignatures in monitoring the outcome of immunotherapeutic strategies, such as vaccination, and in a wide variety of diseases, including infections, 64 cancer 65 and autoimmunity disorders. 66 In this particular class of diseases, antigen arrays play an important role because they can act as a discovery tool to identify an autoantigen from a large set and can later be used as capture probes in a diagnostic chip. Recently, an approach to identify novel autoantigens and to describe the prole of the corresponding autoantibody reactivity was developed for celiac disease (CD). ...
The nature of protein microarray platforms is favorable for multiplexing, leading to the development of tools for personalised medicine and highly precise diagnostics. However, to date, only a limited number of protein microarrays are available in the in vitro diagnostics (IVD) market. This review article will focus on the following operational challenges that are crucial for the use of microarrays in clinical settings: (1) probe printing and quality control; (2) procurement of bio-reagents and antibody cross-reactivity; (3) mass transport limitations and assay automation; (4) calibration and quantification. A selection of microarray assays applicable to IVD and a summary of the diagnostic products currently available on the market are provided.
... To characterize how the variable strengths and durations of cyclic vortex shear and squeeze yield different myocardial "phenomes," we need specialized research methodologies. High-density DNA microarrays (e.g., Affymetrix GeneChip), Northern blotting and serial analysis of gene expression (SAGE) for mRNA profiling, Western blotting for detection of specific proteins in tissue homogenates or cell extracts, and other gene expression and protein identification methods, afford powerful tools to study the expression/regulation of myocardial genes [116,[200][201][202][203][204][205][206][207][208][209][210][211][212][213][214]. Moreover, since 2005, Next Generation Sequencing (NGS) technologies for genome, epigenome and RNA sequencing have become available commercially [215]. ...
Epigenetic mechanisms are fundamental in cardiac adaptations, remodeling, reverse remodeling, and disease. This two-article series proposes that variable forces associated with diastolic RV/LV rotatory intraventricular flows can exert physiologically and clinically important, albeit still unappreciated, epigenetic actions influencing functional and morphological cardiac adaptations and/or maladaptations. Taken in toto, the two-part survey formulates a new paradigm in which intraventricular diastolic filling vortex-associated forces play a fundamental epigenetic role, and examines how heart cells react to these forces. The objectives are to provide a perspective on vortical epigenetic effects, to introduce emerging ideas, and to suggest directions of multidisciplinary translational research. The main goal is to make pertinent biophysics and cytomechanical dynamic systems concepts accessible to interested translational and clinical cardiologists. I recognize that the diversity of the epigenetic problems can give rise to a diversity of approaches and multifaceted specialized research undertakings. Specificity may dominate the picture. However, I take a contrasting approach. Are there concepts that are central enough that they should be developed in some detail? Broadness competes with specificity. Would, however, this viewpoint allow for a more encompassing view that may otherwise be lost by generation of fragmented results? Part 1 serves as a general introduction, focusing on background concepts, on intracardiac vortex imaging methods, and on diastolic filling vortex-associated forces acting epigenetically on RV/LV endocardium and myocardium. Part 2 will describe pertinent available pluridisciplinary knowledge/research relating to mechanotransduction mechanisms for intraventricular diastolic vortex forces and myocardial deformations and to their epigenetic actions on myocardial and ventricular function and adaptations.
... B. die Autoimmunkrankheit DCM (engl. dilated cardiomyopathy) zu diagnostizieren [28]. In der DCM-Arbeit wurden mehr als 3.7×10 4 Proteine aus einer cDNA-Bibliothek von humanen, embryonalen Gehirnzellen hergestellt und auf Glasträger aufgetragen. ...
Ein charakteristisches Merkmal des humoralen Immunsystems ist die Produktion vieler verschiedener Antikörper. Geläufige diagnostische Tests für den Nachweis von Krankheit-spezifischen Serumantikörpern nutzen Antigene zum Nachweis der Krankheit via Antikörperbindung. Derartige diagnostische Tests setzen jedoch die Kenntnis von Krankheit-spezifischen Antigenen voraus. Die vorliegende Arbeit berücksichtigt die unterschiedlichen Bindungsreaktivitäten ganzer Antikörperrepertoires verschiedener Gruppen von Individuen. Dazu werden die Serumantikörperbindungen gegenüber Zufallsbibliotheken gemessen. Die Moleküle dieser beliebigen Bibliotheken müssen keine Verwandtschaft zu den Antigenen der Krankheit haben. Mit modernen Herstellungsverfahren von Peptidarrays auf Glasträgern können mit einmal synthetisierten Peptiden hunderte von Träger-Replikas produziert werden. Die Suche nach hochaffinen Bindern zur Diagnose von Krankheiten mit unbekannten Antigenen oder mit Kreuzreaktivitäten zwischen gesunden und kranken Individuen könnte überflüssig werden. Gestützt auf die beschriebenen Voraussetzungen zeigt die vorliegende Arbeit, dass die Messung von Serumantikörper-Bindungen gegenüber Peptidbibliotheken mit zufälligen Sequenzen die Unterscheidung zwischen Gruppen von gesunden und kranken Individuen für unterschiedliche Krankheiten ermöglicht. Eine unerwartet kleine Anzahl von Peptiden ist ausreichend für eine zuverlässige Vorhersage der untersuchten Gruppen. Der unvoreingenommene Ansatz ermöglicht eine ebenso gute Unterscheidung von gesund und krank, wie sie auch mit voreingenommenen Bibliotheken gezeigt worden sind. Wir vermuten, dass der vorliegende Ansatz ein wichtiger Schritt in Richtung zuverlässiger Diagnose darstellt, insbesondere für Krankheiten mit noch unbekannten Antigenen. Ausserdem bietet die hohe Spezifizität der Detektone und deren kleinen Mitgliederzahl eine Grundlage für die gleichzeitige Diagnose von verschiedenen Krankheiten auf einem einzigen Peptid-Mikroarray.
... In particular, ImmunoCAP® ISAC by Phadia (now Thermo Scientific) is already available in the market for clinical testing of more than 100 allergens. Other antigen arrays have been developed to detect autoantibodies in several different diseases such as celiac disease, [88] multiple sclerosis [89] and cardiomyopathies. [90] Proximal fluids on the other hand are less ideal for large-scale clinical screening or monitoring procedures due to more cumbersome collection procedures. ...
Aiming at clinical studies of human diseases, antibody-assisted assays have been applied to biomarker discovery and toward a streamlined translation from patient profiling to assays supporting personalized treatments. In recent years, integrated strategies to couple and combine antibodies with mass spectrometry-based proteomic efforts have emerged, allowing for novel possibilities in basic and clinical research. Described in this review are some of the field's current and emerging immunocapture approaches from an affinity proteomics perspective. Discussed are some of their advantages, pitfalls and opportunities for the next phase in clinical and translational proteomics.
... Detection of a positive binding event was performed using mouse-anti-human IgG as secondary antibody and the Cy3-labeled rabbit-anti-mouse IgG as tertiary antibody. Using a similar design, the same group subsequently developed a disease-associated protein chip for the profiling of autoantibodies in plasma of dilated cardiomyopathy consisting of 37,200 redundant, recombinant human proteins [57]. Combination of a protein expression library with protein microarray technology led to 26 proteins recognized by IgG antibodies in the patient's samples. ...
The analysis of autoantibodies in human serum plays a crucial role in the diagnosis and follow-up of autoimmune diseases. The analytical tools available for the determination of these analytes, however, are still far from mature, lack standardization, and give low negative predictive values. Approaches using biosensor technology for analysis are an attractive alternative to classical techniques. In special applications, biosensors already have been proven to be effective for clinical diagnostics. This is due to the fact that real-time monitoring of the antigen/antibody interaction gives valuable information on autoantibody affinity to the respective antigenic structure. The potentials of biosensors for the serological analysis of autoantibodies are evident from the increasing number of publications on the subject. Thus, this review focuses on underlying biosensor techniques and published clinical trials. The advantages of multiplexed analyses of autoantibodies by use of microarrays are also emphasized. This promising bioanalytical technique is also particularly important for the structural identification of novel antigens.
... Similarly, Hudson et al. utilized arrays with 5005 full-length human proteins to identify tumor-associated antigens within ovarian cancer [115]. The strategy of utilizing human fetal brain cDNA expression libraries to generate antigen arrays, originally described by Lueking et al. [115], was also applied in the context of dilated cardiomyopathy [116] and the autoimmune condition alopecia areata [117], where arrays with 37,200 redundant, recombinant human proteins were utilized. This setup, later commercialized by Protagen Diagnostics, and containing 3101 proteins or protein fragments was utilized within multiple sclerosis for autoantibody profiling in cerebrospinal fluid [118]. ...
Autoantibodies are a key component for the diagnosis, prognosis and monitoring of various diseases. In order to discover novel autoantibody targets, highly multiplexed assays based on antigen arrays hold a great potential and provide possibilities to analyze hundreds of body fluid samples for their reactivity pattern against thousands of antigens in parallel. Here, we provide an overview of the available technologies for producing antigen arrays, highlight some of the technical and methodological considerations and discuss their applications as discovery tools. Together with recent studies utilizing antigen arrays, we give an overview on how the different types of antigen arrays have and will continue to deliver novel insights into autoimmune diseases among several others.
... Both up-and downregulation of proteins are reported in diseased hearts analyzed by 2DE or gel-free separation of partially purified subfractions from F and NF human heart homogenates [15][16][17][18]. High throughput, gel-free approaches utilizing LC-MS and/or protein microarray are available for studying human tissue [19,20], but few studies have utilized this MS-based approach to analyze ischemic human heart failure. The present study utilizes multidimensional protein identification technology (MudPIT) to compare protein expression in ischemic F versus NF human heart tissue. ...
Purpose:
A goal of this study was to identify and investigate previously unrecognized components of the remodeling process in the progression to heart failure by comparing protein expression in ischemic failing (F) and nonfailing (NF) human hearts.
Experimental design:
Protein expression differences were investigated using multidimensional protein identification and validated by Western analysis. This approach detected basal lamina (BL) remodeling, and further studies analyzed samples for evidence of structural BL remodeling. A rat model of pressure overload (PO) was studied to determine whether nonischemic stressors also produce BL remodeling and impact cellular adhesion.
Results:
Differential protein expression of collagen IV, laminin α2, and nidogen-1 indicated BL remodeling develops in F versus NF hearts Periodic disruption of cardiac myocyte BL accompanied this process in F, but not NF heart. The rat PO myocardium also developed BL remodeling and compromised myocyte adhesion compared to sham controls.
Conclusions and clinical relevance:
Differential protein expression and evidence of structural and functional BL alterations develop during heart failure. The compromised adhesion associated with this remodeling indicates a high potential for dysfunctional cellular integrity and tethering in failing myocytes. Therapeutically targeting BL remodeling could slow or prevent the progression of heart disease. This article is protected by copyright. All rights reserved.
... These arrays were screened with sera from patients with various human diseases including cancer and inflammatory diseases, as well as blood sera from healthy controls. The screening was performed with minor variations as described in [107]. ...
Cancer is the consequence of genetic alterations that influence the behavior of affected cells. While the phenotypic effects of cancer like infinite proliferation are common hallmarks of this complex class of diseases, the connections between the genetic alterations and these effects are not always evident. The growth of information generated by experimental high-throughput techniques makes it possible to combine heterogeneous data from different sources to gain new insights into these complex molecular processes. The demand on computational biology to develop tools and methods to facilitate the evaluation of such data has increased accordingly. To this end, we developed new approaches and bioinformatics tools for the analysis of high-throughput data. Additionally, we integrated these new approaches into our comprehensive C++ framework GeneTrail. GeneTrail presents a powerful package that combines information retrieval, statistical evaluation of gene sets, result presentation, and data exchange. To make GeneTrail';s capabilities available to the research community, we implemented a graphical user interface in PHP and set up a webserver that is world-wide accessible. In this thesis, we discuss newly integrated algorithms and extensions of GeneTrail, as well as some comprehensive studies that have been performed with GeneTrail in the context of cancer research. We applied GeneTrail to analyze properties of tumor-associated antigens to elucidate the mechanisms of antigen candidate selection. Furthermore, we performed an extensive analysis of miRNAs and their putative target pathways and networks in cancer. In the field of differential network analysis, we employed a combination of expression values and topological data to identify patterns of deregulated subnetworks and putative key players for the deregulation. Signatures of deregulated subnetworks may help to predict the sensitivity of tumor subtypes to therapeutic agents and, hence, may be used in the future to guide the selection of optimal agents. Furthermore, the identified putative key players may represent oncogenes, tumor suppressor genes, or other genes that contribute to crucial changes of regulatory and signaling processes in cancer cells and may serve as potential targets for an individualized tumor therapy. With these applications, we demonstrate the usefulness of our GeneTrail package and hope that our work will contribute to a better understanding of cancer.
... A high-density multiprotein array (MPA) (hEXselect, Engine, Berlin, Germany, Order No. 1003) derived from a cDNA bank of two first trimester human fetal brain samples, containing 23,806 Escherichia coli expression clones representing a total of 10,000 human proteins [11,93], was incubated with α-CT or α-CS according to the manufacturer's protocol. In brief, prior to the first use of the array, protein spots were fixed on the PVDF-membrane for 10 min with 70% ethanol and rinsed twice with distilled water. ...
Chlamydia trachomatis (CT) and the Chlamydophila species (CS) Chlamydophila pneumoniae (CPn), and Chlamydophila psittaci (CPs) are suggested to induce autoantibodies causative of several human autoimmune disorders like rheumatoid arthritis and systemic lupus erythematosus (SLE). The aim of the present study was therefore to identify cellular protein interaction partners with antisera to CT (α-CT) or CS (α-CS) and to identify functional consequences of such interaction in vitro. As detected with a commercial first trimester human prenatal brain multiprotein array (hEXselect, Engine, Germany), the most frequent interaction partner with both α-CT and α-CS was the ribosomal small subunit protein RPS27a. This could be confirmed by Western blot analysis with a recombinant RPS27a sample. In addition, immunocytochemistry with both antisera in the human choroid plexus papilloma cell line HIBCPP revealed a granular cytoplasmic staining, and Western blot analysis with whole-cell protein samples of HIBCPP cells revealed both antisera to label protein bands of different molecular weights and intensity. By 2D Western blot analysis and mass spectrometry, one of the protein spots interacting with α-CT could be identified as the RPS27a. Finally, two different methods for the detection of protein synthesis activity, the SUnSET technique and an HPG fluorescence assay revealed both antisera to cause reduced translational activity in HIBCPP cells. Together with previous findings of RPS27a as an autoimmune target in a mouse model of systemic lupus erythematosus (SLE), these results suggest that infections with CT and/or CS could induce SLE-associated immune modifications. However, direct evidence for a pathogenic role of these interactions for SLE demands further investigations.
... no. 1003) derived from a cDNA bank of two first trimester human fetal brain samples, containing 22,000 Escherichia coli expression clones representing a total of 10,000 human proteins (Büssow et al. 1998, Horn et al. 2006, was incubated with α-NG according to the manufacturer's protocol. Prior to the first use of the array, protein spots were fixed on the polyvinyl difluoride (PVDF) membrane for 10 min with 70% ethanol and rinsed twice with distilled water. ...
Early maternal infections with Neisseria gonorrhoeae (NG) correlate to an increased lifetime schizophrenia risk for the offspring, which might be due to an immune-mediated mechanism. Here, we investigated the interactions of polyclonal antisera to NG (α-NG) with a first trimester prenatal brain multiprotein array, revealing among others the SNARE-complex protein Snap23 as a target antigen for α-NG. This interaction was confirmed by Western blot analysis with a recombinant Snap23 protein, whereas the closely related Snap25 failed to interact with α-NG. Furthermore, a polyclonal antiserum to the closely related bacterium Neisseria meningitidis (α-NM) failed to interact with both proteins. Functionally, in SH-SY5Y cells, α-NG pretreatment interfered with both insulin-induced vesicle recycling, as revealed by uptake of the fluorescent endocytosis marker FM1-43, and insulin-dependent membrane translocation of the glucose transporter GluT4. Similar effects could be observed for an antiserum raised directly to Snap23, whereas a serum to Snap25 failed to do so. In conclusion, Snap23 seems to be a possible immune target for anti-gonococcal antibodies, the interactions of which seem at least in vitro to interfere with vesicle-associated exocytosis. Whether these changes contribute to the correlation between maternal gonococcal infections and psychosis in vivo remains still to be clarified.
Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires necessitates the use of extensively validated secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. Despite the identified non-specific binding, the tested antibodies are well suited for use in protein array experiments as the cross-reactive binding partners can be readily excluded from further analysis. The evident cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Furthermore, secondary antibody characterisation using protein arrays enables the generation of reference lists of cross-reactive proteins, which can be then marked as potential false positives in follow-up experiments. Providing such cross-reactivity reference lists accessible to the wider research community may help to interpret data generated with the same antibodies in applications not only related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.
The humoral immune response to tumor-associated antigens in cancer patients can be used as the basis for diagnosing and monitoring
the disease. Moreover, identification of the protein targets of such a response can serve as the basis for developing antigen-specific
anticancer vaccines. This review discusses the main methods for identifying and studying tumor-associated antigens, recognized
by serum antibodies, and also notes the problems facing researchers, which must be solved to introduce serologic antigen kits
into the clinical diagnosis of cancer.
Serum antibodies against amyloid-β peptide (Aβ) in humans with or without diagnosis of Alzheimer's disease (AD) indicate the possibility of immune responses against brain antigens. In an unbiased screening for antibodies directed against brain proteins, we found in AD patients high serum levels of antibodies against the neuronal cytoskeletal protein ankyrin G (ankG); these correlated with slower rates of cognitive decline. Neuronal expression of ankG was higher in AD brains than in nondemented age-matched healthy control subjects. AnkG was present in exosomal vesicles, and it accumulated in β-amyloid plaques. Active immunization with ankG of arcAβ transgenic mice reduced brain β-amyloid pathology and increased brain levels of soluble Aβ(42). AnkG immunization induced a reduction in β-amyloid pathology, also in Swedish transgenic mice(.) Anti-ankG monoclonal antibodies reduced Aβ-induced loss of dendritic spines in hippocampal ArcAβ organotypic cultures. Together, these data established a role for ankG in the human adaptive immune response against resident brain proteins, and they show that ankG immunization reduces brain β-amyloid and its related neuropathology.Molecular Psychiatry advance online publication, 12 June 2012; doi:10.1038/mp.2012.70.
Recent developments in proteomic technologies have enabled the high-throughput, multiplex measurement of large panels of antibodies in biological fluids of patients with immune-driven diseases. Antigen microarrays are increasingly being used to delineate the natural history of autoantibody formation and epitope spread, and thus gain insight into the pathogenesis of autoimmune diseases, as well as into host immunity and its shortcomings. Characterization of autoimmunity that precedes the onset of clinically apparent disease has the potential to guide disease prevention using either conventional immunosupression or novel, antigen-specific tolerizing therapies. In addition, autoantibody profiling has the potential to identify molecular subtypes of a disease, which could allow for prediction of disease outcomes such as severity, tissue damage, and response to therapy.
The currently used prostate cancer serum marker has a low cancer specificity and improved diagnostics are needed. Here we evaluated whether autoantibodies are present in sera of prostate cancer patients and whether they are useful diagnostic markers for prostate cancer.
Sera from 20 prostate cancer patients and 20 healthy controls were incubated on expression clone arrays containing more than 37,000 recombinant human proteins. Functional annotation clustering of the identified autoantigens was performed using the DAVID database. Autoantigens identified in the prostate cancer group were validated on microarrays using sera of 40 prostate cancer patients, 40 patients with elevated PSA levels but prostate cancer negative biopsies (benign disease), and 40 healthy controls.
We detected autoantibodies against 408 different antigens in sera of prostate cancer patients. One hundred seventy-four of these were exclusively detected in the cancer group compared to the healthy control group. Functional annotation clustering revealed an enrichment of RNA-associated, cytoskeleton, and nuclear proteins. The autoantibody panel was validated in serum samples of independent prostate cancer patients. Autoantibody profiles discriminated between prostate cancer patients and benign disease patients with an ROC curve AUC of 0.71. TTLL12, a protein recently described to be over-expressed in prostate cancer, was the highest ranked discrimination autoantigen.
A variety of autoantibodies were identified in sera of prostate cancer patients and provide a first step towards autoantibody diagnostics. Serum autoantibodies reflect the disease and represent valuable tools not only for prostate cancer, but also for other diseases affecting the immune response.
Antigen arrays are becoming widely used tools for the characterization of the complexity of humoral immune responses. Current antibody profiling techniques provide modest and indirect information about the effector functions of the antibodies that bind to particular antigens. Here we introduce an antigen array-based approach for obtaining immune profiles reflecting antibody functionality. This technology relies on the parallel measurement of antibody binding and complement activation by features of the array. By comparing sera from animals immunized against the same antigen under different conditions, we show that identifying the position of an antigen in a 2-D space, derived from antibody binding and complement deposition, permits distinction between immune profiles characterized by diverse antibody isotype distributions. Additionally, the technology provides a biologically interpretable graphical representation of the relationship between antigen and host. Our data suggest that 2-D immune profiling could enrich the data obtained from proteomic scale serum profiling studies.
Protein-based microarrays is a novel, rapidly evolving proteomic technology with great potential for analysis of complex biological
samples. The technology will provide miniaturized set-ups enabling us to perform multiplexed profiling of minute amounts of
biological samples in a highly specific, selective, and sensitive manner. In this review, we describe the potential and specific
use of protein microarray technology, including both functional protein microarrays and affinity protein microarrays, for
the detection and identification of bacteria, bacterial proteins as well as bacterial diseases. To date, the first generations
of a variety of set-ups, ranging from small-scale focused biosensors to large-scale semi-dense array layouts for multiplex
profiling have been designed. This work has clearly outlined the potential of the technology for a broad range of applications,
such as serotyping of bacteria, detection of bacteria and/or toxins, and detection of tentative diagnostic biomarkers. The
use of the protein microarray technology for detection and identification of bacterial and protein analytes is likely to increase
significantly in the coming years.
Current approaches within affinity-based proteomics are driven both by the accessibility and availability of antigens and capture reagents, and by suitable multiplexed technologies onto which these are implemented. By combining planar microarrays and other multiparallel systems with sets of reagents, possibilities to discover new and unpredicted protein-disease associations, either via directed hypothesis-driven or via undirected hypothesis-generating target selection, can be created. In the following stages, the discoveries made during these screening phases have to be verified for potential clinical relevance based on both technical and biological aspects. The use of affinity tools throughout discovery and verification has the potential to streamline the introduction of new markers, as transition into clinically required assay formats appears straightforward. In this article, we summarize some of the current building blocks within array- and affinity-based proteomic profiling with a focus on body fluids, by giving a perspective on how current and upcoming developments in this bioscience could enable a path of pursuit for biomarker discovery.
Current efforts to identify antibodies that are biomarkers of disease rely on knowing the antigens they target. In many diseases, however, the relevant antigens are unknown. Reddy et al. (2010) now present an approach for discovering antibody biomarkers that avoids the need for antigen identification.
Transplant glomerulopathy (TG), a form of chronic renal transplant rejection, carries a poor prognosis. It must be differentiated from the entity defined by the Banff '05 classification, interstitial fibrosis/tubular atrophy (IF/TA). Sequential transplant biopsies have shown that these lesions are subclinical long before clinical manifestations. The availability of biomarkers may provide an earlier diagnosis and subsequent treatment. The aim of our study was to identify serum biomarkers in kidney recipients showing TG compared with IF/TA or stable patients, using protein microarray technology. This technology detects auto- or alloantibodies in patient sera. With a high degree of statistical significance, we identified 18 antibody reactivities specific for TG; 11 for IF/TA; and 10 among stable patients. Target proteins were involved in signal transduction, transcription regulation, DNA replication and repair, cell cycle, endocytosis, cell redox, as well as glycolysis. Some markers, such as podocan and collagen XXIII among TG and tubular cell ion channels among IF/TA, possibly provide insights into the pathogenesis of the lesions.
The aim of this study was to identify dysregulated pathways for the diagnosis and treatment of dilated cardiomyopathy (DCM) using pathway interaction network analysis. Methods: Transcriptome data of DCM, protein-protein interaction (PPI) data, and pathway data were recruited and preprocessed. Then, the pathway interaction network was constructed based on the gene expression analysis and gene co-expression analysis. Meanwhile, pathway activity analysis was performed, and the pathway with the greatest activity change was defined as the seed pathway. Staring from the seed pathway, the dysregulated pathways that could serve as diagnostic biomarker was extracted from the pathway interaction network using support vector machines. Results: Combining gene expression and co-expression data, we constructed the pathway interaction network, covering 4175 pathway interactions. Via pathway activity analysis, cap-dependent translation initiation with the greatest activity change was defined as the seed pathway. Staring from cap-dependent translation initiation, a total of 21 dysregulated pathways were obtained, which could discriminate DCM samples from controls with the area under the curve value of 0.95. Conclusion: A pathway interaction network was implemented to identify dysrgulated pathways that can best discriminate DCM samples from controls. We identified a total of 21 dysregulated pathways in DCM, which can serve as diagnostic biomarkers for DCM.
Within genomics, spotted microarrays have become an increasingly important research tool for the biotechnology
industry, molecular diagnostics, drug screening, nucleic acid sequencing, and mutation analysis. Working
with proteins on arrays is especially difficult, because the native state of proteins is extremely sensitive
to pH, ionic strength, temperature, and protein concentration. Since optimized conditions differ from protein
to protein, the development of in vitro protein-based assays always presents abig challenge. In this
chapter, the design and applications of peptide and protein arrays will be addressed. Different labeling
chemistries for the analytes and signal detection procedures, with special emphasis dedicated to quality
assurance related issues, are discussed. Possible applications of protein arrays, such as epitope mapping
of autoantibodies, binding assays, analysis of protein expression, and protein modification, as well as
monitoring of enzymatic activity, shed light on the high versatility of protein arrays. However, much more
research and development will be necessary, until they meet the requirements of laboratories involved in
high-throughput screenings and diagnostics.
The authors have developed a porous silicon (P-Si) based duplex antibody microarray platform for simultaneous quantitation of the biomarkers prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) in serum. Pore size-controlled P-Si surfaces have an extremely enlarged surface area that enables high-density immobilization of fluorescently labeled antibodies by physical adsorption. Automated microarraying of the antibodies provides a fast and reproducible duplex format of antibody arrays on the P-Si chips placed in the wells of a microtiter plate. The assay platform showed a 100 fg·mL−1 limit of detection for both PSA and hK2, and a dynamic range that extends over five orders of magnitude. After optimization of the density of both capture antibodies, neither the PSA nor the hK2 array showed cross-sensitivity to non-target proteins or other plasma proteins. The microarray was evaluated by titration of PSA and hK2, respectively, in the same serum samples. In our perception, this highly sensitive and selective platform holds promise for improved detection of tumor markers in an early diagnostic stage, but also to monitor the recurrence of prostate cancer. Graphical AbstractA porous silicon microarray platform is presented for simultaneous detection of biomarkers prostate specific antigen and human glandular kallikrein 2 in human blood sample. The platform shows highly sensitive and selective biomarker detection features.
Abnormalities of the cellular and humoral immune system have been described in patients with dilated cardiomyopathy (DCM).
Various circulating cardiac autoantibodies have been detected in DCM patients. Circulating antibodies are extractable by immunoadsorption
(IA). Recent open controlled pilot studies have shown that removal of circulating antibodies by IA induces improvement of
cardiac function in DCM. IA, furthermore, decreases myocardial inflammation. In vitro data indicate that removal of negative inotropic antibodies may represent the essential mechanism of IA in DCM. These antibodies
belong to immunoglobulin G subclass 3 and may play an important role in cardiac dysfunction of DCM patients. Recent data indicate
that the Fc fragments of the immunoglobulins that bind to newly detected sarcolemma-specific Fcg receptors IIa are involved
in the functional effects of cardiac autoantibodies. Removal of various cardiac antibodies through unspecific IA could therefore
offer a hopeful treatment approach in DCM for intervention into this autoimmune process.
Characterising tumour-associated antigens (TAAs) not only represents an important approach to the identification of new diagnostic/prognostic markers, but can also provide information on disease processes and additional potential therapeutic targets. Preliminary screening of a protein macroarray, containing more than 12,000 different proteins, with sera from anaplastic lymphoma kinase (ALK)-negative and ALK-positive anaplastic large cell lymphoma (ALCL) patients identified ribonuclease and tumour suppressor protein Ribonuclease T2 (RNASET2), phosphatase lipid phosphate phosphatase-related protein type 3 (LPPR3) and apoptotic adaptor molecule Fas-associating protein (FADD) as ALK-negative ALCL-associated TAAs. Further validation of these observations was confirmed using the ALCL sera in reverse ELISAs. The circulating anti-RNASET2 autoantibodies present in ALCL patients' sera also recognised eukaryotically expressed RNASET2 protein. RNASET2 expression was then investigated in normal tissues and in lymphomas to explore its clinical potential. RNASET2 protein and mRNA levels showed highest expression in the spleen, leucocytes and pancreas. RNASET2 protein expression was not restricted to ALK-negative ALCL (81%), being expressed in ALK-positive ALCL (65%) as well as in a number of other lymphomas. The immunological recognition of RNASET2, its expression in ALCL and other lymphomas together with its known tumourigenic properties suggest that further studies on this autoantigen are warranted.
Clinical Proteomics: Sample Preparation and StandardizationReferencesStem Cell ProteomicsReferences
In this chapter we provide an overview of bottom-up proteomic approaches. These allow the identification and characterization of proteins and their amino acid sequences, including post-translational modifications, by proteolytic digestion prior to mass spectrometry (MS) analysis. Intact proteins can be separated by gel electrophoresis followed by in-gel protein digestion to generate peptides which are then analyzed by MS. Alternatively, complex protein mixtures can be digested directly (an approach referred to as “shotgun”) and the resulting peptides can be separated by liquid chromatography prior to MS. Following MS analysis, the comparison of the peptides’ spectra with those predicted from genomics/proteomics sequence databases, or annotated peptide spectral libraries, allows the identification of peptides which are finally assigned to corresponding proteins. After a description of the separation methods and MS acquisition modes, a relevant part of the chapter will be dedicated to data processing pointing to algorithms, computational tools and strategies useful for researchers in the discovery process. In particular, liquid-chromatography (LC) based approaches, including Multidimensinal Protein Identification Technology (MudPIT), will be taken as reference and different aspects, ranging from database search engines to protein-protein interaction (PPI) network analysis, will be addressed. Potential issues will be discussed in the context of cardiovascular research, and specifically the last section will focus on the translational applications (clinical proteomics) of cardiovascular proteomics.
Integrated biochips exploit a multi-disciplinary approach to produce portable point-of-care medical diagnostic systems that uncouple diagnosis from centralized laboratories. These portable devices are cost effective and have several advantages including broader accessibility to health care worldwide. Fluorescence detection of a disease-specific probe excited by an optical source is one of the most diffused methods for quantitative analysis on biochips. Here we designed and characterized a miniaturized biochip based on a novel deep-blue organic light-emitting diode. The molecular design of the diode was optimized to excite a fluorophore-conjugated antibody and tested on a protein microarray configuration with good sensitivity and specificity. These findings will be instrumental for the development of next generation point-of-care biochips.
Although the number of patients receiving successful transplantation as a means of treatment for end-stage organ failure continues to increase, rejection, in particular chronic rejection still poses a major risk factor. Crucially, only 50% of allografted hearts and kidneys survive for 10 years. Currently, biopsy is the most common and, in some instances, the only means of diagnosing rejection: it is both invasive and costly. A blood or urine biomarker would overcome these problems and conceivably enable diagnosis of rejection far earlier than a biopsy because it could be diagnosed before clinical symptoms. Furthermore, development of a biomarker could lead to new insights into the disease process. In recent years, proteomics has provided a novel means of examining potential biomarkers. We discuss here the various proteomic platforms available, including 2-dimensional gel electrophoresis, mass spectrometry, and protein arrays, and the various studies exploring protein profiling in heart, lung, kidney, and liver transplantation.
Protein microarrays make it possible to detect molecular interactions with various partners (proteins, peptides, nucleic acids, sugars, etc.). Their advantages are crucial for high-throughput analysis of proteomes of different organisms. Moreover, the recent data reveal the performance of microarrays over current immunological methods. Therefore, the antigen and antibody microarrays become indispensable for medical applications, in particular, for diagnosis and prognosis of microbial infections, autoimmune and allergic diseases. The further technological progress might provide the extension of the miniaturized assays for multiparametric monitoring of human pathologies in practical medicine.
Protein microarrays is a technology with great promise for high-throughput proteomics. Designing high-performance protein microarrays for global proteome analysis has, however, turned out to be challenging. To this end, major efforts are under way to design novel array formats capable of harboring the tremendous range of probes required to target complex proteomes composed of more than 10000 analytes. By adopting nanotechnology, the first generation of miniaturized nanoarrays has recently emerged, which opens up new avenues for global proteome analysis and disease proteomics. This review describes the progress and key issues in designing miniaturized protein arrays.
Here, we developed Sol-gel based antibody microarray platform for quantitative detection of five different cancer biomarkers in clinical sample simultaneously. This kit called Ci-5, Multiplex Tumor Marker Quantitative Kit, is developed based on proprietary SG Cap™ technology. The Ci-5 has 3×5 format of microarray that contains five different antibodies for capturing multi-targets cancer biomarkers, AFP (Liver cancer), CEA (Colon cancer), PSA (Prostate cancer), CA125 (Ovarian cancer) and CA19-9 (Pancreatic cancer), which are approved by CE (Registration No. HL 60135402 0001). Using the Ci-5 kit, we tested 440 clinical specimens for multiplex detection of five different cancer biomarkers simultaneously. The results show high clinical sensitivity (86.7% for CA19-9, 94.7% for AFP, 97% for CEA, 89.8% for PSA and 69.6% for CA125) and specificity (96.2% for CA19-9, 81.1% for AFP, 92.3% for CEA, 98.4% for PSA and 98.7% for CA125). Additionally, Ci-5 shows strong correlation with state-of-art CLIA methods (R²=0.9282, 0.9718, 0.9847, 0.9707 and 0.9219 for CA19-9, AFP, CEA, PSA and CA125). The highly sensitive and selective multiplex sol-gel microarray platform holds promise for improved accuracy of early cancer stage diagnosis as well as recurrence monitoring of cancer patients. Moreover, Ci-5 is the first approved biochip for tumor marker quantitative detection kit which can be used for both manual and automatic liquid handling system.
Auto-antibodies against cardiac proteins have been described in patients with dilated cardiomyopathy. Antibodies against the C-terminal part of KChIP2 (anti-KChIP2 [C-12]) enhance cell death of rat cardiomyocytes. The underlying mechanisms are not fully understood. Therefore, we wanted to explore the mechanisms responsible for anti-KChIP2-mediated cell death. Rat cardiomyocytes were treated with anti-KChIP2 (C-12). KChIP2 RNA and protein expressions, nuclear NF-κB, mitochondrial membrane potential Δψm, caspase-3 and -9 activities, necrotic and apoptotic cells, total Ca(2+) and K(+) concentrations, and the effects on L-type Ca(2+) channels were quantified. Anti-KChIP2 (C-12) induced nuclear translocation of NF-κB. Anti-KChIP2 (C-12)-treatment for 2 h significantly reduced KChIP2 mRNA and protein expression. Anti-KChIP2 (C-12) induced nuclear translocation of NF-κB after 1 h. After 6 h, Δψm and caspase-3 and -9 activities were not significantly changed. After 24 h, anti-KChIP2 (C-12)-treated cells were 75 ± 3% necrotic, 2 ± 1% apoptotic, and 13 ± 2% viable. Eighty-six ± 1% of experimental buffer-treated cells were viable. Anti-KChIP2 (C-12) induced significant increases in total Ca(2+) (plus 11 ± 2%) and K(+) (plus 18 ± 2%) concentrations after 5 min. Anti-KChIP2 (C-12) resulted in an increased Ca(2+) influx through L-type Ca(2+) channels. In conclusion, our results suggest that anti-KChIP2 (C-12) enhances cell death of rat cardiomyocytes probably due to necrosis. J. Cell. Biochem. 115: 678-689, 2014. © 2013 Wiley Periodicals, Inc.
FAS-associated protein with death domain (FADD) is a major adaptor protein involved in extrinsic apoptosis, embryogenesis, and lymphocyte homeostasis. Although abnormalities of the FADD/death receptor apoptotic pathways have been established in tumorigenesis, fewer studies have analyzed the expression and role of phosphorylated FADD (pFADD). Our identification of FADD as a lymphoma-associated autoantigen in T-cell lymphoma patients raises the possibility that pFADD, with its correlation with cell cycle, may possess role(s) in human T-cell lymphoma development. This immunohistochemical study investigated pFADD protein expression in a range of normal tissues and lymphomas, particularly T-cell lymphomas that require improved therapies. Whereas pFADD was expressed only in scattered normal T cells, it was detected at high levels in T-cell lymphomas (eg, 84% anaplastic large cell lymphoma and 65% peripheral T cell lymphomas, not otherwise specified). The increased expression of pFADD supports further study of its clinical relevance and role in lymphomagenesis, highlighting phosphorylation of FADD as a potential therapeutic target.
Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. The cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Secondary antibody characterisation using protein arrays enables generation of reference lists of cross-reactive proteins, which can be then excluded from analysis in follow-up experiments. Furthermore, making such cross-reactivity lists accessible to the wider research community may help to interpret data generated by the same antibodies in applications not related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.
This chapter starts with general considerations on wound healing; the main cells and their structures and functions; and methods of in vitro and in vivo evaluation. Implants with thin calcium phosphate (CaP) coatings can enhance or inhibit wound healing mechanisms on various levels of gene expression and protein production. Therefore, some techniques to elucidate the material and tissue interactions are mentioned. Bone development, the various types of bone, and bone fracture healing are presented. Thin CaP coatings of implant material can be investigated in in vitro test systems using primary cells or cell lines in culture. More reliable results can be expected from in vivo investigations with animal models. Important results of such investigations are presented. The prediction of the clinical performance of thin CaP coatings on the basis of in vitro and in vivo models is limited. Therefore, retrieval studies of surgically explanted or postmortem uncovered implants should be obligatory.
Complex repertoires of IgG autoantibodies have been detected against ocular antigens in patients with glaucoma. The goal was to identify and characterize the IgG autoantibody repertoires in sera of patients with pseudoexfoliation glaucoma (PXFG) with protein macroarrays.
Serum samples of 21 patients with PXFG and 19 age- and sex-matched control subjects were profiled on high-density colony protein macroarrays expressing His-tagged recombinant human proteins derived from a human fetal brain cDNA library. Statistically prevalent expression clones in the PXFG group were sequenced. mRNA expression of identified antigens was examined in the rat ganglion cell line RGC-5 and in human brain and optic nerve cDNA. The IgG immunoreactivity of the sera of 20 control and 26 PXFG patients to purified C6orf129 was analyzed in a reverse enzyme-linked immunosorbent assay.
An increased prevalence was detected among the PXFG patients of serum antibodies to seven proteins: C6orf129; stathmin-like 4; transmembrane protein 9 domain family, member B; fibroblast growth factor receptor 3; cleft lip and palate transmembrane protein 1; EH-domain-containing protein 1; and eukaryotic translation elongation factor 2. All antigens were expressed in the RGC-5 cells and in cDNA from human brain and optic nerve, with the exception of stathmin-like 4, which was not expressed in the RGC-5 cells. The patients with PXFG had increased anti-C6orf129 IgG immunoreactivity compared with that in the control subjects (P < 0.05).
Screening high-density protein arrays identifies unique antibody profiles that may discriminate between patients with and without PXFG. Characterization of the autoantibody repertoire may provide new insights into the pathophysiology of PXFG.
The present study demonstrates the presence of natural autoantibodies of the IgG isotype directed against heat shock protein 90 (HSP90). The binding properties of af®nity-puri®ed anti-HSP antibodies were compared with those of natural antibodies speci®c for other self antigens, including anti-thyroglobulin and anti-myoglobin autoantibodies, by using semiquantitative immunoblotting, with solubilized proteins from normal liver tissue as antigens, and cross-blot analysis using puri®ed self proteins. Af®nity-puri®ed anti-HSP90 antibodies were polyreactive and the non-HSP90-speci®c fraction of normal IgG was depleted in its natural autoantibody content. We further observed that self antigens including HSP, myosin, tubulin and aldolase with highly conserved structures show similar patterns of binding with natural antibodies, and form a well-de®ned cluster as demonstrated by cluster analysis of immunoreactivity data, whereas the less-conserved self and non-self antigens remained unclustered. The results favor the hypothesis that HSP90 belongs to a subset of highly conserved and immunodominant self antigens that are the primary target for natural autoantibodies in normal human IgG.
Today, dilated cardiomyopathy (DCM) represents the main cause of severe heart failure and disability in younger adults and thus is a challenge for public health. About 30% of DCM cases are genetic in origin; however, the large majority of cases are sporadic, and a viral or immune pathogenesis is suspected. Following the established postulates for pathogenesis of autoimmune diseases, here we provide direct evidence that an autoimmune attack directed against the cardiac beta(1)-adrenergic receptor may play a causal role in DCM. First, we immunized inbred rats against the second extracellular beta(1)-receptor loop (beta(1)-EC(II); 100% sequence identity between human and rat) every month. All these rats developed first, receptor-stimulating anti-beta(1)-EC(II) Ab's and then, after 9 months, progressive severe left ventricular dilatation and dysfunction. Second, we transferred sera from anti-beta(1)-EC(II)-positive and Ab-negative animals every month to healthy rats of the same strain. Strikingly, all anti-beta(1)-EC(II)-transferred rats also developed a similar cardiomyopathic phenotype within a similar time frame, underlining the pathogenic potential of these receptor Ab's. As a consequence, beta(1)-adrenergic receptor-targeted autoimmune DCM should now be categorized with other known receptor Ab-mediated autoimmune diseases, such as Graves disease or myasthenia gravis. Although carried out in an experimental animal model, our findings should further encourage the development of therapeutic strategies that combat harmful anti-beta(1)-EC(II) in receptor Ab-positive DCM patients.
Tubulin and FtsZ share a common fold of two domains connected by a central helix. Structure-based sequence alignment shows that common residues localize in the nucleotide-binding site and a region that interacts with the nucleotide of the next tubulin subunit in the protofilament, suggesting that tubulin and FtsZ use similar contacts to form filaments. Surfaces that would make lateral interactions between protofilaments or interact with motor proteins are, however, different. The highly conserved nucleotide-binding sites of tubulin and FtsZ clearly differ from those of EF-Tu and other GTPases, while resembling the nucleotide site of glyceraldehyde-3-phosphate dehydrogenase. Thus, tubulin and FtsZ form a distinct family of GTP-hydrolyzing proteins.
Distinct profiles of autoantibodies directed to intracellular antigens can be detected in the systemic connective tissue diseases. They aid in establishing the correct diagnosis and are included in many sets of diagnostic criteria, such as the ones developed for systemic lupus erythematosus (anti-Smith antigen and anti-double-strand DNA antibodies), mixed connective tissue disease (anti-U1-nuclear ribonucleoprotein antibodies), and Sjögren's syndrome (SS) (anti-SS-A/Ro and anti-SS-B/La antibodies). They are useful prognostic markers in some situations and facilitate clinical and treatment follow-up. Autoantibodies have also been used as probes to gain insights into cell biology, helping to isolate and purify intracellular proteins involved in key cellular functions. We give detailed information on two of the most useful techniques for the detection of autoantibodies in the clinical and research laboratory settings, indirect immunofluorescence and immunoblotting. We also discuss several of the antigen-autoantibody systems found in systemic lupus erythematosus (Smith antigen, U1-nuclear ribonucleoprotein, SS-A/Ro, SS-B/La, proliferating cell nuclear antigen ribosomal ribonucleoprotein, double-strand DNA, histones, antiphospholipids, Ku, Ki/SL), systemic sclerosis (centromere, topo I, RNA polymerases, fibrillarin, polymyositis-Scl, Th/To), polymyositis/dermatomyositis (transferRNA synthetases, signal recognition particle, and others), and SS (SS-A/Ro, SS-B/La, nucleolar organizing region-90, p80-coilin), addressing their clinical significance, common detection methods, immunogenetic associations, and the molecular and cellular biology of the cognate antigens.
Myocardial protection and changes in gene expression follow whole body heat stress. Circumstantial evidence suggests that an inducible 70-kD heat shock protein (hsp70i), increased markedly by whole body heat stress, contributes to the protection. Transgenic mouse lines were constructed with a cytomegalovirus enhancer and beta-actin promoter driving rat hsp70i expression in heterozygote animals. Unstressed, transgene positive mice expressed higher levels of myocardial hsp70i than transgene negative mice after whole body heat stress. This high level of expression occurred without apparent detrimental effect. The hearts harvested from transgene positive mice and transgene negative littermates were Langendorff perfused and subjected to 20 min of warm (37 degrees C) zero-flow ischemia and up to 120 min of reflow while contractile recovery and creatine kinase efflux were measured. Myocardial infarction was demarcated by triphenyltetrazolium. In transgene positive compared with transgene negative hearts, the zone of infarction was reduced by 40%, contractile function at 30 min of reflow was doubled, and efflux of creatine kinase was reduced by approximately 50%. Our findings suggest for the first time that increased myocardial hsp70i expression results in protection of the heart against ischemic injury and that the antiischemic properties of hsp70i have possible therapeutic relevance.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
Brief ischemic periods lead to myocardial dysfunction without myocardial infarction. It has been shown that expression of inducible HSP70 in hearts of transgenic mice leads to decreased infarct size, but it remains unclear if HSP70 can also protect against myocardial dysfunction after brief ischemia. To investigate this question, we developed a mouse model in which regional myocardial function can be measured before and after a temporary ischemic event in vivo. In addition, myocardial function was determined after brief episodes of global ischemia in an isolated Langendorff heart. HSP70-positive mice and transgene negative littermates underwent 8 min of regional myocardial ischemia created by occlusion of the left descending coronary artery, followed by 60 min of reperfusion. This procedure did not result in a myocardial infarction. Regional epicardial strain was used as a sensitive indicator for changes in myocardial function after cardiac ischemia. Maximum principal strain was significantly greater in HSP70-positive mice with 88+/-6% of preischemic values vs. 58+/-6% in transgene-negative mice (P < 0.05). Similarly, in isolated Langendorff perfused hearts of HSP70-positive and transgene-negative littermates exposed to 10 min of global ischemia and 90 min of reperfusion, HSP70 transgenic hearts showed a better-preserved ventricular peak systolic pressure. Thus, we conclude that expression of HSP70 protects against postischemic myocardial dysfunction as shown by better preserved myocardial function.
Tubulin and FtsZ share a common fold of two domains connected by a central helix. Structure-based sequence alignment shows that common residues localize in the nucleotide-binding site and a region that interacts with the nucleotide of the next tubulin subunit in the protofilament, suggesting that tubulin and FtsZ use similar contacts to form filaments. Surfaces that would make lateral interactions between protofilaments or interact with motor proteins are, however, different. The highly conserved nucleotide-binding sites of tubulin and FtsZ clearly differ from those of EF-Tu and other GTPases, while resembling the nucleotide site of glyceraldehyde-3-phosphate dehydrogenase. Thus, tubulin and FtsZ form a distinct family of GTP-hydrolyzing proteins.
Antibodies that are present in the serum of healthy individuals in the absence of deliberate immunization with any antigen, are refered to as natural antibodies. A vast majority of natural antibodies react with one or more self antigens and are termed as natural autoantibodies. The importance of natural autoantibodies in immune regulation has long been neglected, since tolerance to self was thought to be primarily dependent on the deletion of autoreactive clones, rather than on peripheral suppressive mechanisms. Clonal deletion and energy cannot account, however, for the prevalence of natural autoreactivity among healthy individuals. It is now well established that autoreactive antibodies and B cells, and autoreactive T cells, are present in healthy individuals, and in virtually all vertebrate species. Autoreactive repertoires are predominantly selected early in ontogeny. Questions pertaining to the role of natural antibodies in the regulation of the immune response and maintenance of immune homeostasis and to the distinction between natural autoreactivity and pathological autoimmunity have not been adequately addressed. Here, we focus on the current knowledge on the physicochemical and functional properties of NAA in man, and the use of NAA for therapeutic intervention. reserved.
We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation
or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible
expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded
onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6 sequence of expressed proteins (RGS·His antibody, Qiagen) detected 20% of the library as putative expression clones. Two
example genes, GAPDH and HSP90α, were identified on high-density filters using DNA probes and antibodies against their proteins.
We have previously identified NPDC-1, a neural factor involved in the control of proliferation and differentiation, and we have shown that the stable introduction of NPDC-1 into transformed cells down-regulates cell proliferation both by increasing the generation time and by suppressing transformed properties. The data presented here indicate that, in vitro, NPDC-1 is able to interact with the transcription factor E2F-1 and some cell cycle proteins, such as D-cyclins and cdk2. In addition, two-hybrid experiments in mammalian cells show that the interaction between NPDC-1 and E2F-1 can also occur in vivo. This interaction reduces the binding of E2F-1 to DNA and its transcriptional activity. Taken together, the data suggest that NPDC-1 could influence cell cycle progression and neural differentiation through its association with E2F-1.
Numerous clinical and epidemiological studies link enteroviruses such as the Coxsackie virus group with the autoimmune disease type 1 diabetes mellitus (DM). In addition, there are reports that patients with type 1 DM are characterized by skewing of TCR Vbeta chain selection among peripheral blood and intraislet T lymphocytes. To examine these issues, we analyzed TCR Vbeta chain-specific up-regulation of the early T cell activation marker, CD69, on CD4 T cells after incubation with Coxsackievirus B4 (CVB4) Ags. CD4 T cells bearing the Vbeta chains 2, 7, and 8 were the most frequently activated by CVB4. Up-regulation of CD69 by different TCR families was significantly more frequent in new onset type 1 DM patients (p = 0.04), 100% of whom (n = 8) showed activation of CD4 T cells bearing Vbeta8, compared with 50% of control subjects (n = 8; p = 0.04). T cell proliferation after incubation with CVB4 Ags required live, nonfixed APCs, suggesting that the selective expansion of CD4 T cells with particular Vbeta chains resulted from conventional antigen processing and presentation rather than superantigen activity. Heteroduplex analysis of TCR Vbeta chain usage after CVB4 stimulation indicated a relatively polyclonal, rather than oligo- or monoclonal response to viral Ags. These results provide evidence that new-onset patients with type 1 DM and healthy controls are primed against CVB4, and that CD4 T cell responses to the virus have a selective TCR Vbeta chain usage which is driven by viral Ags rather than a superantigen.
Our study shows that antibodies, specific to the ADP/ATP carrier of the inner mitochondrial membrane, crossreact with the cell surface of cardiac myocytes, where the calcium channel seems to be the antigenic determinant. The antibodies enhanced the calcium current and suppressed its inactivation. Affinity-purified antibodies (IgG) exhibit an acute cytotoxic effect, which required extracellular calcium and was prevented by calcium channel blockers. Our findings suggest that antibody-mediated cytotoxicity results secondary to calcium overload caused by enhanced cellular calcium permeability, requiring no complement-dependent process.
Thanks to the results of the multiple completed and ongoing genome sequencing projects and to the newly available recombination-based cloning techniques, it is now possible to build gene repositories with no precedent in their composition, formatting, and potential. This new type of gene repository is necessary to address the challenges imposed by the post-genomic era, i.e., experimentation on a genome-wide scale. We are building the FLEXGene (Full Length EXpression-ready) repository. This unique resource will contain clones representing the complete ORFeome of different organisms, including Homo sapiens as well as several pathogens and model organisms. It will consist of a comprehensive, characterized (sequence-verified), and arrayed gene repository. This resource will allow full exploitation of the genomic information by enabling genome-wide scale experimentation at the level of functional/phenotypic assays as well as at the level of protein expression, purification, and analysis. Here we describe the rationale and construction of this resource and focus on the data obtained from the Saccharomyces cerevisiae project.
A staggering amount of genetic information is contained within the DNA of complex organisms such as humans. Of these vast tracts of DNA code, only very small regions actually constitute the 'genes'; with the information ultimately translated into proteins, the basic machinery of cells. However, a complex organism has a cell or tissue type and developmental specific pattern of gene expression which interact in complex networks within and between these cells or tissues. Such a combination of vast tracts of genomic DNA, tissue and developmental specific transcription of small subsets of this information, plus complex interactions between gene products provides a daunting scientific and logistical challenge if a global picture of gene function within complex organisms is to be obtained. This objective can be achieved only through an interdisciplinary combination of biology, computer image analysis, and robotic engineering. The development of a global approach to the understanding of complex genomes is not just an academic challenge. For example, the ability to understand the complex network of gene interactions, or to be able rapidly to screen many genetic samples in parallel, would have enormous consequences for the pharmaceutical and diagnostic industries. The systems described are used in several genetic screening or drug discovery centres that require rapid, reliable, high density screening of small quantities of biological or biologically active material.
The prognosis for patients suffering from advanced stages of dilated cardiomyopathy (DCM) is poor. Recent studies have shown that immunoadsorption (IA) may represent an effective alternative therapeutic approach for other kinds of autoimmune diseases with circulating autoantibodies. The objective of this pilot study was to ascertain the short-term hemodynamic effects of IA in patients with idiopathic DCM and circulating autoantibodies. Our study included 9 patients with circulating β1-adrenoreceptor antibodies who suffered from idiopathic DCM as well as severe heart failure (left ventricular ejection fraction <30%). Immunoadsorption was performed on 5 consecutive days using an adsorber against immunoglobulins (Ig Therasorb, Baxter, Unterschleissheim, Germany). Substitution of 0.5 g/kg of polyclonal immunoglobulin took place after the final IA session. During IA, the cardiac index and stroke volume index increased from 2.0 ± 0.42 to 2.9 ± 0.79 L/min−1/ m−2, p < 0.01, and from 24.0 ± 7.4 to 35.9 ± 10.3 ml/m2, p < 0.05, respectively. In addition to drug therapy, IA may represent a promising alternative therapeutic possibility for hemodynamic stabilization of patients with severe idiopathic DCM.
We report the molecular characterization of a novel human homologue of mouse npdc1 (neural proliferation, differentiation and control, 1) gene, designated human npdc1 (hnpdc1). hnpdc1 was identified by large-scale sequencing of fetal liver cDNA libraries and the full-length cDNA was obtained by PCR amplification. The hnpdc1 gene, which contains nine exons, was mapped to human chromosome 15. It encodes a polypeptide of 325 amino acids, which shows high homology (77% identity) to the mouse NPDC1. Sequence analysis has shown that hNPDC1 protein contains a putative signal peptide of 34 amino acids, a transmembrane segment, and a typical bipartite nuclear localization signal. Northern blot and dot blot hybridization indicates that, just like mnpdc1, hnpdc1 mRNA is strongly expressed in adult brain (especially in hippocampus, frontal lobe and temporal lobe) and about 1.82-fold higher in adult brain than that in fetal brain. Unlike mnpdc1, however, hnpdc1 contains two transcripts instead of only one (1.5 kb), and has high expression levels in prostate, pituitary gland, and mammary glands. These results support that hNPDC1 plays a role in the control of neural cell proliferation and differentiation, and suggest that it may be involved in the development of several secretion glands.
Immunization with cardiac myosin induces experimental autoimmune heart disease in genetically predisposed mice. These mice produce heart-specific autoantibodies, some of which are directed against the cardiac myosin isoform.
We have reported the presence of circulating heart-specific autoantibodies in 26% of patients with idiopathic dilated cardiomyopathy (DCM) using indirect immunofluorescence. To identify the autoantigen(s) recognized by heart-specific autoantibodies in human disease, we tested, by Western blotting, sera from 26 DCM patients, 14 of whom were cardiac antibody-positive and 12 antibody-negative, as well as sera from 12 patients with cardiac failure from ischemic or valvular heart disease and from 13 normal subjects who were cardiac antibody-negative. Crude myofibrillar proteins and myosin preparations extracted from human atrial or ventricular specimens were used as antigens. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed. The proteins were electrophoretically transferred to nitrocellulose sheets. The paper strips were incubated in sera from patients or controls at 1:100 dilution; the reaction was revealed with a peroxidase-labeled second antibody against human immunoglobulin. Twelve of the 14 DCM sera (86%) containing heart-specific antibodies reacted with both the alpha- (atrial specific) and beta- (ventricular and slow skeletal) myosin heavy chain isoforms; none of the 13 normal sera (p = 0.0001) and one of the 24 heart failure-negative control sera (4%, p = 0.0001) contained antibodies against myosin heavy chain.
These findings indicate that alpha- and beta-cardiac myosin heavy chain isoforms as in the murine model of autoimmune heart disease are major autoantigens in patients with idiopathic DCM.
The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3, was identified by the ability of its gene product to interact with pRB. RBP3 bound to pRB both in vitro and in vivo, and this binding was competed by viral proteins known to disrupt pRB-E2F association. RBP3 bound to E2F recognition sequences in a sequence-specific manner. Furthermore, transient expression of RBP3 caused a 10-fold transactivation of the adenovirus E2 promoter, and this transactivation was dependent on the E2F recognition sequences. These properties suggest that RBP3 encodes E2F, or an E2F-like protein.
Although the immediate receptors (immunophilins) of the immunosuppressants cyclosporin A (CsA) and FK506 are distinct, their similar mechanisms of inhibition of cell signaling suggest that their associated immunophilin complexes interact with a common target. We report here that the complexes cyclophilin-CsA and FKBP-FK506 (but not cyclophilin, FKBP, FKBP-rapamycin, or FKBP-506BD) competitively bind to and inhibit the Ca(2+)- and calmodulin-dependent phosphatase calcineurin, although the binding and inhibition of calcineurin do not require calmodulin. These results suggest that calcineurin is involved in a common step associated with T cell receptor and IgE receptor signaling pathways and that cyclophilin and FKBP mediate the actions of CsA and FK506, respectively, by forming drug-dependent complexes with and altering the activity of calcineurin-calmodulin.
The adenosine diphosphate (ADP)-adenosine triphosphate (ATP) carrier of the inner mitochondrial membrane is identified as an autoantigen in myocarditis and dilated cardiomyopathy. Sera of patients with these diseases contain autoantibodies to the ADP-ATP carrier capable of inhibiting nucleotide transport in vitro. Recently, an antibody-related infringement of energy metabolism was shown in intact perfused hearts isolated from guinea pigs immunized with the ADP-ATP carrier. A decreased cytosolic-mitochondrial difference of the phosphorylation potential of ATP was measured that originated from a reduction in mitochondrial-cytosolic nucleotide transport. Nonimmunized animals did not show these changes in energy metabolism, despite being in a comparable metabolic state and performing equal external heart work. To establish whether antibodies to the ADP-ATP carrier can also alter cardiac function, hemodynamic parameters of isolated hearts of guinea pigs that were preimmunized with the carrier protein were measured. Cardiac metabolism was stimulated by exposing the hearts to a high calcium concentration in conjunction with a maximum elevation of the afterload. Mean aortic pressure, stroke volume, stroke work, and external heart work were found to be lowered significantly (p less than 0.005). The external heart work of the immunized hearts reached only about 20% of the level performed by control hearts. Myocardial oxygen consumption was lowered 2.5-fold, whereas the extent of lactate production was found to be more than doubled. These results show a diminished cardiac performance of hearts from animals immunized with the ADP-ATP carrier. Our findings demonstrate that autoimmunity to the ADP-ATP carrier may contribute to the pathophysiology of dilated cardiomyopathy as a subsequent stage of myocarditis by causing an autoantibody-mediated reduction in cardiac function on the basis of an imbalance between energy delivery and demand.
Although it is recognized that the number of cardiac beta-adrenoceptors is reduced in human dilated cardiomyopathy, the mechanisms involved have not been defined. We examined the possible role of altered humoral immunity by comparing the effect of sera from patients with idiopathic dilated cardiomyopathy (n = 20), ischemic or valvular heart disease (n = 28), or controls with no known cardiac disease (n = 18) on the binding of radioligands to cardiac beta-receptors. The ability of sera from cardiomyopathic patients to inhibit the binding of [3H]dihydroalprenolol to rat cardiac membranes was significantly higher than that of the other two patient groups (40 +/- 5% at 50-fold serum dilution compared to 14 +/- 3% for the ischemic/valvular heart disease group, and 14 +/- 4% for the normal control group, p less than 0.001). A similar inhibition was exerted by IgG from cardiomyopathic patients. Only the number, not the affinity, of the beta-receptors was decreased by cardiomyopathic sera. This decrease could be prevented by preincubating the sera with anti-human IgG, indicating the presence of autoantibodies. Furthermore, the sera were ineffective against cardiac alpha 1-adrenoceptors and considerably less effective against lung beta 2-receptors. In addition to ligand binding inhibition, sera from cardiomyopathic patients could immunoprecipitate beta-adrenoceptors quantitatively from solubilized cardiac membranes. Positive sera inhibited significantly isoproterenol-stimulated adenylate cyclase with no effect on basal or NaF-stimulated activities. These results document the presence in sera from patients with idiopathic dilated cardiomyopathy of autoantibodies directed against the cardiac beta 1-adrenoceptor which may play an important role in the regulation of inotropic responsiveness to beta-agonists.
Our study shows that antibodies, specific to the ADP/ATP carrier of the inner mitochondrial membrane, crossreact with the cell surface of cardiac myocytes, where the calcium channel seems to be the antigenic determinant. The antibodies enhanced the calcium current and suppressed its inactivation. Affinity-purified antibodies (IgG) exhibit an acute cytotoxic effect, which required extracellular calcium and was prevented by calcium channel blockers. Our findings suggest that antibody-mediated cytotoxicity results secondary to calcium overload caused by enhanced cellular calcium permeability, requiring no complement-dependent process.
Anti-tubulin antibodies were studied in normal human serum either maintained at neutral pH to measure the free antibody activity (FAA) or treated at pH 2.8 to measure the total antibody activity (TAA): FAA + the antibody activity in the form of immune complexes (ICAA). Anti-tubulin antibody activities were assessed by measurements of the capacity of serum immunoglobulins to bind pure 125I-labelled tubulin in a liquid phase radioimmune assay or to immunoprecipitate unlabelled tubulin revealed by Western blot using anti-alpha- or anti-beta-tubulin monoclonal antibodies. Acid buffer-treated serum and untreated serum at a 1:200 dilution immunoprecipitated about 35% and 4% of labelled tubulin, respectively. TAA was therefore 8- to 10-fold higher than FFA. Anti-tubulin antibody titres corresponding to TAA and FAA were about 1:20,000 and 1:500, respectively. The Western blot analysis confirmed that the acid buffer-treatment of the serum dramatically increased the capacity of serum immunoglobulins to immunoprecipitate tubulin. TAA was studied in patients with Graves' disease with elevated FAA. TAA of the sera of control subjects and patients with Graves' disease were not significantly different, so an increase of FAA was related to a decrease of ICAA. These results indicate that (a) normal human serum contains high levels of anti-tubulin antibodies in the form of immune complexes which are dissociated by an acid buffer treatment, (b) these immune complexes exist in the presence of a small excess of free anti-tubulin antibodies, (c) the equilibrium between free and immune complex-bound anti-tubulin antibodies could be altered in patients with autoimmune diseases.
Peroxisomes are found in almost all eukaryotic cells. Two major functions of the organelle are in lipid metabolism: peroxisomes catalyze the initial steps in the biosynthesis of plasmalogens, which are phospholipids that are present in large amounts in myelin. Peroxisomes also catalyze the beta-oxidation of fatty acids; this pathway is essential for the catabolism of a variety of substrates that are not oxidized by mitochondria. A third important function is in cellular respiration, involving the metabolism of H2O2, for which the peroxisome is named. Peroxisomes increase in size by the post-translational import of newly synthesized proteins from the cytosol; these pre-existing peroxisomes divide to form new peroxisomes. Proteins are targeted to peroxisomes by three different types of topogenic sequences, and it is hypothesized that a receptor exists for each type. The newly made proteins are translocated through the peroxisomal membrane into the interior by a machinery that is energized by ATP hydrolysis. Human patients and yeast mutants have remarkably similar defects in peroxisome biogenesis. Some such mutants are defective in the import of a subset of peroxisomal proteins that share a topogenic sequence type; other mutants fail to import all newly made proteins into peroxisomes, regardless of the type of targeting sequence they possess. These mutants might be defective in receptors and in translocation machinery components, respectively. Cloned genes that are essential for peroxisome biogenesis encode diverse proteins: some likely receptors, some transmembrane proteins possibly involved in translocation, and others hydrophilic proteins that may play other roles in peroxisome assembly.
The role of increased dispersion of repolarization in the genesis of torsade de pointes and ventricular fibrillation has been well recognized generally, but not in the genesis of monomorphic ventricular tachycardia (VT). Monophasic action potentials (MAP) were therefore recorded simultaneously from the right ventricular (RV) apex (RVA) and outflow tract (RVOT) during sinus rhythm, RV pacing and programmed extra stimulation (PES) in 24 patients with VT. The activation time (AT), MAP duration at 90% repolarization (MAPd), and repolarization time (RT) were measured and their dispersions, defined as the differences in these parameters between RVA and RVOT, were calculated. During sinus rhythm and RV pacing, the dispersions of AT, MAPd and RT (dispersions) were significantly larger in the 17 patients with a VT induced than in those without. During PES, the dispersions were further augmented in the S2 beats in the seven patients with a sustained VT induced, the maximal dispersion of RT being 85 +/- 22 ms. Both the dispersion of AT and that of MAPd contributed to the dispersion of RT. In both of our two patients with a sustained VT induced during MAP recording, a marked increase in dispersions of RT (140 and 190 ms, respectively) was observed immediately before the initiation of the VT. A link between the dispersions and the inducibility of a monomorphic VT was found in our patients, which suggests that the increased dispersions play an important role in the genesis of a monomorphic VT.
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.
Following induction of experimental encephalomyelitis with a T-cell clone, L10C1, that is specific for the myelin basic protein epitope p87-99, the inflammatory infiltrate in the central nervous system contains a diverse collection of T cells with heterogeneous receptors. We show here that when clone L10C1 is tolerized in vivo with an analogue of p87-99, established paralysis is reversed, inflammatory infiltrates regress, and the heterogeneous T-cell infiltrate disappears from the brain, with only the T-cell clones that incited disease remaining in the original lesions. We found that antibody raised against interleukin-4 reversed the tolerance induced by the altered peptide ligand. Treatment with this altered peptide ligand selectively silences pathogenic T cells and actively signals for the efflux of other T cells recruited to the site of disease as a result of the production of interleukin-4 and the reduction of tumour-necrosis factor-alpha in the lesion.
Autoimmune disease is characterized by the presence of organ- and disease-specific autoantibodies in patients and first degree relatives; antibody detection may precede disease onset by several years. We investigated potential involvement of organ-specific autoimmunity in dilated cardiomyopathy (DCM). Using indirect immunofluorescence and absorption studies, organ- and disease-specific IgG cardiac antibodies were found in one-third of DCM patients. Antibody status at diagnosis was associated with better exercise capacity; at 1-year follow-up two-thirds of antibody-positive patients became negative. These findings suggest that antibodies are early markers; their absence in the majority of patients at diagnosis may relate to long-standing pre-clinical DCM. Antibody screening was performed in asymptomatic DCM relatives, 177 from 33 families with > 1 affected individual (familial DCM) and 165 from 31 pedigrees with non-familial DCM. Antibodies were detected in 37 (58%) pedigrees and were more common among relatives than in normals (20% vs 3.5%, P = 0.0001). Antibody-positive relatives were younger, had larger left ventricular end-systolic dimension and reduced % fractional shortening compared to antibody-negative relatives. These findings provide evidence for autoimmunity in a subset (58%) including both familial and non-familial DCM; cardiac-specific antibodies may identify relatives at risk of developing DCM.
Understanding transmembrane signalling process is one of the major challenge of the decade. In most tissues, since Fisher and Krebs's discovery in the 1950's, protein phosphorylation has been widely recognized as a key event of this cellular function. Indeed, binding of hormones or neurotransmitters to specific membrane receptors leads to the generation of cytosoluble second messengers which in turn activate a specific protein kinase. Numerous protein kinases have been so far identified and roughly classified into two groups, namely serine/threonine and tyrosine kinases on the basis of the target amino acid although some more recently discovered kinases like MEK (or MAP kinase kinase) phosphorylate both serine and tyrosine residues.
Protein kinase C is a serine/threonine kinase that was first described by Takai et al. [1] as a Ca- and phospholipid-dependent protein kinase. Later on, Kuo et al. [2] found that PKC was expressed in most tissues including the heart. The field of investigation became more complicated when it was found that the kinase is not a single molecular entity and that several isoforms exist. At present, 12 PKC isoforms and other PKC-related kinases [3] were identified in mammalian tissues. These are classified into three groups. (1) the Ca-activated α-, β-,and γ-PKCs which display a Ca-binding site (C2); (2) the Ca-insensitive δ-, ε-, θ-, η-, and μ-PKCs. The kinases that belong to both of these groups display two cystein-rich domains (C1) which bind phorbol esters (for recent review on PKC structure, see [4]). (3) The third group was named atypical PKCs and include ζ, λ, and τ-PKCs that lack both the C2 and one cystein-rich domain. Consequently, these isoforms are Ca-insensitive and cannot be activated by phorbol esters [5]. In the heart. evidence that multiple PKC isoforms exist was first provided by Kosaka et al. [6] who identified by chromatography at least two PKC-related isoenzymes. Numerous studies were thus devoted to the biochemical characterization of these isoenzymes (see [7] for review on cardiac PKCs) as well as to the identification of their substrates.
This overview aims at updating the present knowledge on the expression, activation and functions of PKC isoforms in cardiac cells. (Mol Cell Biochem 157: 65–72, 1996)
The aim of this article is to briefly review the immunological features of the autoantibodies against ADP/ATP carrier from dilated cardiomyopathy and the potential role of the autoantibodies in cardiac tissue injury. The autoantibodies against ADP/ATP carrier were found with very high frequency in patients with dilated cardiomyopathy, and had disease- and organ-specificity. The autoantibodies cross-reacted with subunits of the calcium channel on the cardiac cell membrane. There is a close correlation between the autoantibody-titer and the hemodynamic function in patients with dilated cardiomyopathy. It can be postulated that after an autoimmune response is initiated, the circulating autoantibodies against ADP/ATP carrier disturb myocardial energy metabolism, and enhance calcium influx and calcium overload in cardiac myocytes, resulting in progressive myocyte damage. These findings indicate a new immunopathological mechanism in dilated cardiomyopathy.
Previous studies have demonstrated that induction of heat shock protein (HSP) 72 by whole-body hyperthermia reduces infarct size in an in vivo model of ischemia and reperfusion. Furthermore, hearts obtained from transgenic mice that overexpress HSP72 demonstrate improved functional recovery and decreased infarct size in vitro after global ischemia and reperfusion.
To test the hypothesis that overexpression of HSP72 in transgenic mice reduces infarct size in vivo, transgenic mice that were heterozygous for a rat HSP70i gene ([+]HSP72) and transgene-negative littermate controls ([-]HSP72) were subjected to 30 minutes of left coronary artery occlusion followed by 120 minutes of reperfusion. Core body temperature was monitored with a rectal thermometer and maintained between 36.5 degrees C and 37.0 degrees C with a heating pad. Infarct size, determined by dual staining with triphenyltetrazolium chloride and phthalocyanine blue dye, was smaller in [+]HSP72 mice compared with [-]HSP72 mice (12.7 +/- 2.8% [n = 7] versus 33.4 +/- 4.5% [n = 6], infarct size/risk area, respectively; P < .05; mean +/- SEM).
Overexpression of HSP72 reduces infarct size in this in vivo transgenic mouse model of myocardial ischemia and reperfusion.
To examine autoantibodies present in patients with active systemic lupus erythematosus (SLE), sera, circulating immune complexes (CIC), and antibodies purified on DNA-immunoadsorbent were tested by enzyme immunoassay. A panel of self-antigens, including DNA, histones (HIS), glomerular basal membrane (GBM), thymus cell extract (TCE), actin (ACT), myosin (MS), and tubulin (TUB), was used to define their specificities. IgM antibodies against all antigens of the panel were detected in sera, CIC, and in antibodies eluted from the DNA-immunoadsorbent and demonstrated a large polyreactivity. IgG antibodies showed restricted activities against DNA, HIS, GBM, and TCE in sera and a large polyreactivity in CIC. Inhibition experiments were performed to assess their mono- or polyreactivities. Among the IgG autoantibody population recognizing DNA, two populations of IgG antibodies were detected in the sera and in the affinity purified anti-DNA: one recognizes DNA, HIS, and GBM, and the other binds to DNA and to cytoskeletal proteins. These autoantibody populations were found in CIC, which also often contained high amounts of IgG antibodies recognizing ACT and MS. A third population of IgG antibody that recognizes only TCE and could not be inhibited by DNA or other antigens was found in serum and CIC. Our data demonstrate the existence of several populations of autoantibody in serum and CIC of SLE patients: (1) IgM polyreactive autoantibodies, (2) IgG polyreactive autoantibodies recognizing DNA and cytoskeletal proteins, (3) IgG specific to DNA, which cross react with HIS and GBM, and (4) IgG specific to TCE antigens.
The effects of autoantibodies against the ADP/ATP carrier, from sera of patients with dilated cardiomyopathy, on calcium channel current (ICa) were studied in enzymatically-isolated guinea pig ventricular myocytes by using a whole cell patch-clamp method. The results showed that the autoantibodies enhanced ICa in a concentration-dependent manner. Verapamil inhibited enhancement of ICa induced by the autoantibodies. Control sera (without the autoantibodies) did not affect ICa. This study suggests that anti-ADP/ATP carrier autoantibodies from sera of patients with dilated cardiomyopathy may enhance ICa and cause calcium overload. Disturbing Ca-channel gating by autoantibodies may contribute to the pathogenesis of dilated cardiomyopathy.
1. The properties and subcellular distribution of phosphatidate phosphohydrolase (PAP) were studied in rat heart. A Mg2(+)-activated activity (PAP1) which was inhibited by N-ethylmaleimide was found mainly in a 105,000 x g soluble fraction. Isolation of the membranes in a medium containing KCl increased the proportion of PAP1 that was associated. Translocation of PAP1 from these membranes occurred on subsequent incubation in a low-ionic strength medium from which KCI was omitted. Incubation of cardiac myocytes with palmitate promoted translocation of PAP activity to cellular membranes. A second activity which was insensitive to N-ethylmaleimide (PAP2) was found in the 105,000 x g membrane fraction. PAP2 was inhibited by concentrations of Mg2+ known to occur in ischaemia. Specific activities of PAP1 and PAP2 in ventricle muscle homogenates were similar. The specific activity of PAP2 in homogenates of cardiac myocytes was only 42% of that in homogenates of ventricle muscle. 2. A glycerolphosphate acyltransferase (GPAT) activity with properties similar to the GPAT found in microsomes from liver or adipose tissue was enriched in the sarcoplasmic reticulum fraction from ventricle muscle. This GPAT had a significantly higher K(m) for glycerol 3-phosphate than the GPAT found in adipose tissue microsomes. The possible physiological significance of this 'high K(m)' GPAT in heart, particularly in ischaemia, is discussed. 3. Comparisons were made of the specific activities of fatty acyl-CoA synthetase, monoacylglycerolphosphate acyltransferase, diacylglycerol acyltransferase and the mitochondrial and microsomal forms of GPAT in homogenates from cardiac myocytes and ventricle muscle.
The purpose of the present study was to investigate the expression and functional relevance of sarcolemmal L-type Ca2+-channels in failing and non-failing human myocardium. The protein expression of sarcolemmal L-type Ca2+-channels was determined with 3H-(+)-PN 200-110-binding experiments and Western blot analysis using a specific antibody against the alpha1-subunit in membrane preparations of ventricular and atrial myocardium from both failing (n = 15) and non-failing hearts (n = 8). The gene expression of the ion conducting pore of the L-type Ca2+-channel was examined with Northern blot technique in human failing and non-failing RNA. For normalization the RNA expression of calsequestrin was used. In electrically driven ventricular papillary muscle strips and auricular trabeculae, the responses to nifedipine and Ca2+ as parameters of myocardial function were studied. The protein expression as measured by 3H-(+)-PN 200-110-binding (Bmax) and Western Blot analysis with calsequestrin as reference was similar in left ventricular failing and non-failing myocardium. However, both were reduced in atrial compared to ventricular tissue in failing and non-failing hearts. The KD remained unchanged. Calsequestrin levels were unaltered in failing and non-failing hearts. The gene expression of the alpha1-subunit was similar in human failing and non-failing hearts. The L-type Ca2+-channel antagonist nifedipine reduced force of contraction with the same potency and efficiency in ventricular failing and non-failing myocardium. In contrast, the potency of nifedipine was higher in atrial than in ventricular tissue. Consistently, atrial myocardium from patients with dilated cardiomyopathy was more sensitive towards Ca2+ than those of the control group. In conclusion, the altered Ca2+-homeostasis in failing human myocardium may be less due to changes in sarcolemmal L-type Ca2+-channel expression or function than due to an altered intracellular Ca2+-handling.
Heat shock proteins (hsps) have been shown to be important antigens in a number of autoimmune diseases. We have previously shown the presence of autoantibodies against hsp60 in a high proportion of patients with dilated cardiomyopathy (DCM). This study set out to investigate the expression of hsp60 in the myocardium of 30 patients with DCM and a control group of 30 normal donors.
The expression of hsp60 was quantitated at the protein and mRNA level by Western blotting and RT-PCR, respectively, and its distribution was investigated by immunocytochemistry.
Quantitation of hsp60 showed a 5-fold increase in the DCM hearts over that in the donor hearts. By immunocytochemistry 13 patients with DCM showed increased positive staining localised to the connective tissue. Semi-quantitative RT-PCR analysis of hsp60 showed a significantly increased amount of hsp60 at the mRNA level in the DCM hearts than in the controls.
These results unequivocally demonstrate a raised level of expression of endogenous hsp60 in the myocardium of patients with DCM. The increased expression of hsp60 in the myocardium of patients with DCM may render such cells susceptible to react with circulating autoantibodies to hsp60 and hsp65 found in a high proportion of patients with this disease.
Proteins translate genomic sequence information into function, enabling biological processes. As a complementary approach to gene expression profiling on cDNA microarrays, we have developed a technique for high-throughput gene expression and antibody screening on chip-size protein microarrays. Using a picking/spotting robot equipped with a new transfer stamp, protein solutions were gridded onto polyvinylidene difluoride filters at high density. Specific purified protein was detected on the filters with high sensitivity (250 amol or 10 pg of a test protein). On a microarray made from bacterial lysates of 92 human cDNA clones expressed in a microtiter plate, putative protein expressors could be reliably identified. The rate of false-positive clones, expressing proteins in incorrect reading frames, was low. Product specificity of selected clones was confirmed on identical microarrays using monoclonal antibodies. Cross-reactivities of some antibodies with unrelated proteins imply the use of protein microarrays for antibody specificity screening against whole libraries of proteins. Because this application would not be restricted to antigen-antibody systems, protein microarrays should provide a general resource for high-throughput screens of gene expression and receptor-ligand interactions.
Detection of antimyosin antibodies in non-inflammatory cardiac disease undermines their disease specificity as a sensitive marker of damage in dilated cardiomyopathy (DCM) patients. Antibody subclass specificity could provide a more sensitive marker of disease and possibly discriminate the humoral autoimmune responses in different cardiac diseases. Frequency and reactivity of autoantibodies against alpha- and beta-isoforms of myosin heavy chain (mhc) were evaluated by ELISA for IgG, IgM, and subclasses IgG1, IgG2, and IgG3 in patients with DCM (NYHA III/IV, n = 82), end stage ischemic heart disease (E-IHD: NYHA III/IV, n = 62), mild ischemic heart disease (NYHA I/II, n = 27), and controls (n = 54). Autoantibodies against atrial and ventricular myosin were raised in heart failure patients compared to mild-IHD and controls but with different antigen affinities. Reactivity in E-IHD was significantly raised against (ventricular) beta-mhc compared with only mild-IHD patients, suggesting a relative increase in ventricular specific antibodies in IHD patients with a higher NYHA class. IgG subclass analysis for IgG1, IgG2, and IgG3 against alpha- and beta-mhc showed statistically raised levels of IgG3 only in DCM patients and a significantly higher reactivity of IgG2 in heart failure patients versus controls. The results demonstrate immunological heterogeneity of antimyosin antibodies developed in different clinical entities. Pro-inflammatory characteristics of IgG3 antibodies in a select group of patients with DCM may contribute to autoimmune mechanisms of injury in these patients.
Idiopathic dilated cardiomyopathy (IDC) frequently is a progressive disease without causative therapy options. Following the hypothesis that in certain patients autoantibodies against cardiac structures may induce, maintain, or promote the progression of the disease, we investigated whether the elimination of these autoantibodies through immunoadsorption would improve cardiac function.
This prospective case-control study included 34 patients with IDC. Each patient presented with moderate to severe heart failure and evidence of autoantibodies directed against beta(1)-adrenoceptors (beta(1)-AABs). Seventeen patients received standard medical therapy (control group), whereas 17 were also treated with immunoadsorption (treatment group) to eliminate beta(1)-AABs. A 1-year follow-up included echocardiographic assessment of left ventricular ejection fraction and internal diameters, beta(1)-AAB levels, and clinical status every 3 months. Within 1 year, the mean+/-SD left ventricular ejection fraction rose from 22.3+/-3.3% to 37.9+/-7.9% (P=0.0001) in the treatment group, with a relative increase of 69.9%. However, in the control group, no overall increase was seen (from 23.8+/-3.0% to 25.2+/-5.9%, P=0. 3154). Left ventricular diameter in diastole decreased by 14.5% from 74.5+/-7.1 to 63.7+/-6.0 mm in the treatment group (P=0.0001) and by 3.8% (P=0.2342) in the control group. In the treatment group, the NYHA functional rating improved after immunoadsorption (P=0.0001). beta(1)-AABs did not increase anew.
In IDC, the use of immunoadsorption is superior to the use of standard medical therapy. It significantly improves cardiac performance and clinical status.
We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses.
Anti-factor VIII antibodies represent a unique model to study the relationship between natural autoreactivity (natural antibodies to factor VIII of healthy individuals), disease-associated autoimmunity ("spontaneous" factor VIII inhibitors of patients with anti-factor VIII autoimmune disease) and antigen-driven immune responses (immune inhibitors in multitransfused patients with hemophilia A) to a single human protein antigen. Although natural and disease-associated anti-factor VIII antibodies are not readily distinguished based on the comparison of their isotypic distribution and epitope mapping, available studies of cross-reacting idiotypes suggest that factor VIII inhibitors in patient's plasma encompass two populations of anti-factor VIII antibodies. Some antibodies result from the clonal expansion of B lymphocytes that exist before treatment with factor VIII and secrete anti-factor VIII antibodies with properties similar to those of natural anti-factor VIII antibodies present in healthy individuals; other inhibitors are produced by B cell clones that have undergone affinity maturation and hypermutation of the V regions of the antibodies they produce. The implications for the treatment of patients with anti-factor VIII inhibitors are discussed.
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.
PD-1 deficiency causes a variety of autoimmune diseases. Inhibitory signaling through the PD-1 receptor might therefore be involved in the regulation of peripheral tolerance. The constitutive expression of PD-1 ligands (PD-L1 and PD-L2) on parenchymal cells of heart, lung and kidney suggests that the PD-1-PD-L system could provide unique negative signaling to help prevent autoimmune diseases.
RTP/Drg1/Cap43/rit42/TDD5/Ndr1/NDRG1 (referred to as NDRG1 hereafter) is a cytoplasmic protein involved in stress responses, hormone responses, cell growth, and differentiation. Recently, the mutation of this gene was reported to be causative for hereditary motor and sensory neuropathy-Lom. Here, we cloned two human cDNAs encoding NDRG3 and NDRG4, which are homologous to NDRG1. These two genes, together with NDRG1 and a previously deposited cDNA (designated NDRG2), constitute the NDRG gene family. The four members share 57-65% amino acid identity. NDRG4 was further characterized because its mRNA expression was quite specific in brain and heart, in contrast to the relatively ubiquitous expression of the other three members. NDRG4 mRNA consists of three isoforms, NDRG4-B, NDRG4-B(var), and NDRG4-H. Northern and Western blot analyses showed that NDRG4-B was expressed only in the brain, whereas NDRG4-H was expressed in both brain and heart. NDRG4-B(var) was a minor product. NDRG4 expression was more abundant in adult than fetal brain and heart and was markedly decreased in the Alzheimer's diseased brain. In situ hybridization showed that NDRG4 was localized in neurons of the brain and spinal cord. The NDRG4 gene contains 17 exons. mRNA expression of the three NDRG4 isoforms is regulated by alternative splicing and possibly by alternative promoter usage. The finely tuned expression of the NDRG gene family members suggests that they have different specific functions.
Immunoadsorption (IA) and subsequent immunoglobulin (Ig) G substitution represent an additional therapeutic approach in dilated cardiomyopathy (DCM). It remains to be elucidated whether this treatment modulates myocardial inflammation, which is possibly a causal factor of ventricular dysfunction.
From 25 DCM patients (EF <30%), 12 patients were randomized for IA therapy and subsequent IgG substitution at 1-month intervals until month 3. Before (<7 days) and after IA therapy, right ventricular biopsies were obtained from all patients. Biopsies were also obtained at intervals of 3 months from 13 patients without IA/IgG treatment (controls). IA/IgG treatment induced improvement in left ventricular ejection fraction from 21.3+/-1.7% (+/-SEM) to 27.0+/-1.3% (P<0.01 versus baseline/controls) and reduction of the beta-receptor autoantibody serum levels (P<0.01 versus baseline/controls). The number of CD3 cells decreased from 5.7+/-0.8 to 2.9+/-0.5 cells/mm(2) (P<0.01 versus baseline/controls). This decline was paralleled by a decrease in CD4 (P<0.01 versus baseline/controls) and CD8 (P<0.05 versus baseline/controls) lymphocytes. The number of leukocyte common antigen-positive cells (leukocytes) was reduced from 20.0+/-3.2 to 9.9+/-2.8 cells/mm(2) (P<0.01 versus baseline/P<0.05 versus controls). HLA class II expression decreased from 2.1+/-0.7% to 1.1+/-0.4% (P<0.05 versus controls/baseline). The number of immunopositive cells and the expression of HLA class II in controls remained stable. In both groups, the degree of fibrosis remained unchanged.
IA and subsequent IgG substitution mitigate myocardial inflammation in DCM.
Myocarditis is a poorly understood disease because it progresses through stages with distinctly different mechanisms and manifestations. The objective of this article is to better define myocarditis for both clinicians and clinical scientists by setting it in the framework of 3 phases of disease. In phase 1, the viral stage, we review recent discoveries about the way viruses gain access to target tissue and how they trigger immune responses. In the second, autoimmune phase of disease, we examine the roles of autoreactive T cells, cytokines, and cross-reacting antibodies and reconsider the relevance of recent therapy trials. In the third phase of the disease, dilated cardiomyopathy, we consider the remodeling processes. We then offer current recommendations for diagnosis and therapy and conclude with a look to the future.
Myocarditis is a continuum of 3 distinct disease processes, one evolving into the other with transitional periods of indistinctness. For each of the 3 processes, pathogenesis, diagnosis, and treatment differ considerably. Without precise knowledge of the point to which an individual patient’s myocarditis has evolved in this continuum, the clinician can only use diagnostic tools and therapeutic interventions haphazardly.
It is likely that the majority of cases of myocarditis, except in countries in which Chagas’ disease or diphtheria is common, result from viral infection, which may progress to an autoimmune phase after resolution or reduction of the initial infection, and then finally to progressive dilatation after resolution or reduction of the autoimmune injury (Figure 1). A viral cause can only be proved by direct molecular or indirect methods. Appropriate treatment at the viral stage is eradication of virus and amelioration of viral injury. The autoimmune phase can be diagnosed by endomyocardial biopsy, supplemented by serological markers of immune activation. Immune suppression is probably the most appropriate therapy in this stage, unless significant viral …
KChIP2, a gene encoding three auxiliary subunits of Kv4.2 and Kv4.3, is preferentially expressed in the adult heart, and its expression is downregulated in cardiac hypertrophy. Mice deficient for KChIP2 exhibit normal cardiac structure and function but display a prolonged elevation in the ST segment on the electrocardiogram. The KChIP2(-/-) mice are highly susceptible to the induction of cardiac arrhythmias. Single-cell analysis revealed a substrate for arrhythmogenesis, including a complete absence of transient outward potassium current, I(to), and a marked increase in action potential duration. These studies demonstrate that a defect in KChIP2 is sufficient to confer a marked genetic susceptibility to arrhythmias, establishing a novel genetic pathway for ventricular tachycardia via a loss of the transmural gradient of I(to).