Project description:We investigated whether mouse serum autoantibody binding patterns on random-sequence peptide microarrays (immunosignaturing) can be used for diagnosing and predicting the onset of lupus and its central nervous system (CNS) manifestations. Submitter states "We have no processed data to submit. We have no gpr files to submit." To identify possible predictive and diagnostic peptides of lupus and CNS-lupus, we carried out two studies and selected peptides in common across both studies. In the first study we tested 3-6 MRL/lpr, MRL/mp and C3H/HeJ mice at four months of age. For study two we tested 9-10 MRL/lpr and MRL/mp at 1.5 and 4 months of age. In both studies the mice sera were diluted 1/500 and analyzed using microarray peptides from platform GPL14921. We ran each sample in triplicate. The MRL/lpr and MRL/mp are the autoimmune strains and the C3H/HeJ is the control strain.
Project description:In addition to determining possible diagnostic and predictive peptides of lupus and CNS-lupus, we also used our microarray technology along with the Guitope computer program to determine possible natural protein match to five monoclonal autoantibodies that were created using one of the autoimmune MRL/lpr mouse. Submitter states "We have no processed data to submit. We have no gpr files to submit." Microarray analysis was performed on five monoclonal brain-reactive autoantibodies (F9, G10, G4, D1 and D9) that were created from one autoimmune mouse with altered behavior. These samples were tested in duplicated along with a control sample on the GPL17600 platform.
Project description:Recurrence of focal segmental glomerulosclerosis (rFSGS) after kidney transplantation is a cause of early and accelerated graft loss. Immuneadsorption can alleviate renal dysfunction and suggests that circulating antibodies (Ab) are likely implicated in disease pathogenesis. To evaluate pathogenic Ab in rFSGS, we processed 141 unique serum samples from patients with and without primary rFSGS (n=64) and 34 non-FSGS control, transplanted at five (US and EU) hospitals. 9000 antigens were screened in pre-transplant sera by protein arrays and 10 Ab targeting glomerular antigens were selected for ELISA validation. A panel of 7 Ab (CD40, PTPRO, CGB-5, FAS, P2RY11, SNRPB2 and APOL2) could predict post-transplant FSGS recurrence with 92% accuracy. Pre-transplant elevation of anti-CD40 Ab levels alone had a substantial impact (78% accuracy) on the identification of rFSGS risk after transplantation. Epitope mapping of CD40 with customized peptide arrays and rFSGS sera demonstrated altered immunogenicity of the extracellular CD40 domain in rFSGS. Immunohistochemistry of CD40 demonstrated a differential expression of these antigens in FSGS compared to non-FSGS. Anti-CD40 Ab purified from rFSGS patients were uniquely pathogenic in human podocyte cultures; injection of these Ab resulted in heightened proteinuria, independently and in combination with suPAR in a rodent model, abrogated by injection of monoclonal Ab to CD40. In conclusion, a panel of 7 Ab can identify primary FSGS patients at high risk of recurrence prior to transplantation, allowing for customized therapies and improved patient selection for transplant. Intra-renal CD40 is an important axis of disease pathogenesis, and human trials of anti-CD40 therapies are warranted to evaluate their efficacy in preventing rFSGS and improving graft survival. The purpose of the study was to identify potential auto-Abs associated with rFSGS. We used a discovery set of pre-transplant sera from 20 unique patients with biopsy confirmed diagnosis of primary FSGS as their cause of ESRD, of which 10 had progressed to rFSGS within the first post-transplant year and 10 did not have recurrence of proteinuria or histological disease after transplantation (nrFSGS).
Project description:Humoral immune responses are traditionally characterized by determining the presence and quality of antibodies specific for certain antigens. Arraying of large numbers of antigens allows the parallel measurement of antibodies, generating patterns called antibody profiles. Functional characterization of these antibodies could help draw an even more informative map of an immune response. To generate functional antibody profiles we simultaneously tested not only IgM, IgG and IgA binding to but also complement activation by a panel of endogenous and exogenous antigens printed as microarrays, using normal and autoimmune human sera. We show that complement activation by a particular antigen in a given individual cannot be predicted by the measurement of antigen specific antibodies, in spite of a general correlation between the amount of antigen-bound antibody and the deposited C3 fragments. This is due to both differences in the isotypes that dominate in the recognition of an antigen and individual variations for a given isotype, resulting in altered complement activation potential. Thus, antigen specific C3 deposition can be used as an additional parameter in immune response monitoring. This is exemplified by comparing the coordinates of antigens, used for the diagnosis of systemic lupus erythematosus, of normal and autoimmune serum samples in a two-dimensional space derived from C3 deposition and antibody binding. Since cleavage fragments of C3 mediate important immunological processes we propose that measurement of their deposition on antigen microarrays, in addition to antibody profiling, can provide useful functional signature about the tested serum. Keywords: IgM immuneprofile, antigen array IgM, IgG, IgA and C3 binding in 30 human serum samples were examined using custom-made protein arrays
Project description:Background: Most asthmatic patients have high serum levels of IgE directed against common environmental allergens such as house dust mite, animal danders and moulds. However the presence of specific IgE against individual allergens alone does not account for asthma. Many individuals are atopic but not asthmatic. Precise knowledge of the serum IgE specificity repertoire in asthmatic and non-asthmatic patients would substantially help in understanding the pathogenesis of the disease and at the same time facilitate the treatment and the implementation of preventive measures. Methods: We developed a microarray immunoassay containing 103 common allergens to study the IgE reactivity profiles of 485 asthmatic and 342 non-asthmatic individuals from families whose members had a documented history of asthma and atopy. The results were analyzed using k-means clustering to investigate whether IgE reactivity profiles correlated with asthma, disease severity and age of onset as well as with other atopic conditions such as rhinitis, conjunctivitis and eczema. We trained an artificial neural network (ANN) using the serum reactivity data to identify individuals as asthmatic and non-asthmatic. Results: Individual sera showed clear differences in the number and the combinations of allergens recognized as well as in the level of specific IgE. While the presence of specific IgE against single allergens correlated poorly with the pathological conditions examined k-means clustering analysis unraveled that a particular profile was significantly associated with asthma (p <1E-8). An ANN-based algorithm, calibrated with the profile reactivity data correctly classified as asthmatic or non-asthmatic 78% of the individual examined. Conclusions: Our analysis demonstrates that asthma may be a higher-order phenomenon related to patterns of response and not attributable to single antibody reactions. This information sheds new light on the risk of developing the disease and can be readily utilized in combination with an ANN-based tool to distinguish asthmatic and non-asthmatic individuals on the basis of their serum reactivity profile. The study consisted of a total of 872 sera samples: 485 individuals were diagnosed with asthma, 342 were classified as non asthmatic, a remaining 45 were classified as unconfirmed asthma diagnosis. The serum IgE reactivity profiles were analyzed against 103 allergens representative of 11 distinct allergen classes. Quantification of bound IgE: The fluorescence signal was acquired using ProScanArray Express™ version 3.0 software. PMC reading values of individual spots were corrected against the internal negative control to identify signals above background. Duplicate measurements of individual allergens were utilized. The signal collected from the allergens was interpolated with an external calibration curve to obtain the IU/ml value (see supplementary file GSE20020_IU_ml values.txt), and translated into a Class Score by plotting the data in a standard reactivity scale (see Sample data tables). Class Score values: (CLASS 0 (less than 0.35 IU/ml); CLASS 1 (0.35-0.7 IU/ml); CLASS 2 (0.71-3.5 IU/ml); CLASS 3 (3.51-17.5 IU/ml); CLASS 4 (17.51-50 IU/ml); CLASS 5 (50.01-100 IU/ml).
Project description:Sera of experimental autoimmune encephalomyelitis (EAE) or control mice were collected at day 18 post immunization. Comprehensive analysis of cytokine levels were performed by using commercially available RayBio Mouse Cytokine Array 2 (RayBiotech, Inc.) according to manufacturer’s protocol 4 samples.There are two groups: vehicle control group and EAE group. Each group consists of 2 replications.