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Discover how PEPperPRINT Peptide Microarray products have been used in different fields of research.

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Autoimmune Atrial Fibrillation

Maguy, Ange; Mahendran, Yuvaraj; Tardif, Jean-Claude; Busseuil, David; Li, Jin
Circulation.
Aug 2023
10.1161/CIRCULATIONAHA.122.062776
BACKGROUND: Atrial fibrillation (AF) is by far the most common cardiac arrhythmia. In about 3% of individuals, AF develops as a primary disorder without any identifiable trigger (idiopathic or historically termed lone AF). In line with the emerging field of autoantibody-related cardiac arrhythmias, the objective of this study was to explore whether autoantibodies targeting cardiac ion channels can underlie unexplained AF. METHODS: Peptide microarray was used to screen patient samples for autoantibodies. We compared patients with unexplained AF (n=37 pre-existent AF; n=14 incident AF on follow-up) to age- and sex-matched controls (n=37). Electrophysiological properties of the identified autoantibody were then tested in vitro with the patch clamp technique and in vivo with an experimental mouse model of immunization. RESULTS: A common autoantibody response against K ir 3.4 protein was detected in patients with AF and even before the development of clinically apparent AF. K ir 3.4 protein forms a heterotetramer that underlies the cardiac acetylcholine-activated inwardly rectifying K + current, I KACh . Functional studies on human induced pluripotent stem cell–derived atrial cardiomyocytes showed that anti-K ir 3.4 IgG purified from patients with AF shortened action potentials and enhanced the constitutive form of I KACh , both key mediators of AF. To establish a causal relationship, we developed a mouse model of K ir 3.4 autoimmunity. Electrophysiological study in K ir 3.4-immunized mice showed that K ir 3.4 autoantibodies significantly reduced atrial effective refractory period and predisposed animals to a 2.8-fold increased susceptibility to AF. CONCLUSIONS: To our knowledge, this is the first report of an autoimmune pathogenesis of AF with direct evidence of K ir 3.4 autoantibody-mediated AF.

Antigen discovery by bioinformatics analysis and peptide microarray for the diagnosis of cystic echinococcosis

Batisti Biffignandi, Gherard; Vola, Ambra; Sassera, Davide; Najafi-Fard, Saeid; Gomez Morales, Maria Angeles; Brunetti, Enrico; Teggi, Antonella; Goletti, Delia; Petrone, Linda; Tamarozzi, Francesca
PLoS Negl Trop Dis.
Apr 2023
10.1371/journal.pntd.0011210
Background Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato, is a neglected zoonosis. Its diagnosis relies on imaging, supported by serology, while only imaging is useful for staging and follow-up. Since diagnostic tools and expertise are not widely available, new accurate and easily implementable assays for the diagnosis and follow-up of CE are highly needed. Methodology/Principal Findings We aimed to identify new E . granulosus antigens through a bioinformatics selection applied to the parasite genome, followed by peptide microarray screening and validation in ELISA, using independent panels of sera from patients with hepatic CE and clinically relevant controls. From 950 proteins selected in silico , 2,379 peptides were evaluated by microarray for IgG reactivity and eight candidates selected for validation. Reactivity to one peptide was significantly higher in the CE group (p = 0.044), but had suboptimal diagnostic accuracy. Conclusions/Significance Here we performed bioinformatics analysis and peptide microarray for antigen discovery, useful for the diagnosis of CE. Eight candidates were selected and validated. Reactivity to one peptide associated to CE but had suboptimal diagnostic accuracy. Importantly, the database developed in this study may be used to identify other antigenic candidates for CE diagnosis and follow-up.

IgE and IgG4 Epitopes of Dermatophagoides and Blomia Allergens before and after Sublingual Immunotherapy

Figo, Daniele Danella; Cordeiro Macedo, Priscilla Rios; Gadermaier, Gabriele; Remuzgo, Cesar; Castro, Fábio Fernandes Morato; Kalil, Jorge; Galvão, Clovis Eduardo Santos; Santos, Keity Souza
IJMS.
Feb 2023
10.3390/ijms24044173
Sublingual immunotherapy (SLIT) is used worldwide to treat house dust mites (HDM) allergy. Epitope specific immunotherapy with peptide vaccines is used far less, but it is of great interest in the treatment of allergic reactions, as it precludes the drawbacks of allergen extracts. The ideal peptide candidates would bind to IgG, blocking IgE-binding. To better elucidate IgE and IgG4 epitope profiles during SLIT, sequences of main allergens, Der p 1, 2, 5, 7, 10, 23 and Blo t 5, 6, 12, 13, were included in a 15-mer peptide microarray and tested against pooled sera from 10 patients pre- and post-1-year SLIT. All allergens were recognized to some extent by at least one antibody isotype and peptide diversity was higher post-1-year SLIT for both antibodies. IgE recognition diversity varied among allergens and timepoints without a clear tendency. Der p 10, a minor allergen in temperate regions, was the molecule with more IgE-peptides and might be a major allergen in populations highly exposed to helminths and cockroaches, such as Brazil. SLIT-induced IgG4 epitopes were directed against several, but not all, IgE-binding regions. We selected a set of peptides that recognized only IgG4 or were able to induce increased ratios of IgG4:IgE after one year of treatment and might be potential targets for vaccines.

Analysis of Plasmablasts from Children with Kawasaki Disease Reveals Evidence of a Convergent Antibody Response to a Specific Protein Epitope

Rowley, Anne H; Arrollo, David; Shulman, Stanford T; Torres, Abigail; O’Brien, Amornrat; Wylie, Kristine; Kim, Kwang-Youn A; Baker, Susan C
Feb 2023
10.1093/infdis/jiad048
Abstract Background Kawasaki disease (KD) is a febrile illness of young childhood that can result in coronary artery aneurysms and death. COVID mitigation strategies resulted in a marked decrease in KD cases worldwide, supporting a transmissible respiratory agent as the cause. We previously reported a peptide epitope recognized by monoclonal antibodies (MAbs) derived from clonally expanded peripheral blood plasmablasts from 3 of 11 KD children, suggesting a common disease trigger in a subset of patients with KD. Methods We performed amino acid substitution scans to develop modified peptides with improved recognition by KD MAbs. We prepared additional MAbs from KD peripheral blood plasmablasts and assessed MAb characteristics that were associated with binding to the modified peptides. Results We report a modified peptide epitope that is recognized by 20 MAbs from 11 of 12 KD patients. These MAbs predominantly use heavy chain VH3-74; two-thirds of VH3-74 plasmablasts from these patients recognize the epitope. The MAbs were nonidentical between patients but share a common CDR3 motif. Conclusions These results demonstrate a convergent VH3-74 plasmablast response to a specific protein antigen in children with KD, supporting one predominant causative agent in the etiopathogenesis of the illness.

Human antibody profiling technologies for autoimmune disease

Carlton, Lauren H.; McGregor, Reuben; Moreland, Nicole J.
Immunol Res.
Jan 2023
10.1007/s12026-023-09362-8
Abstract Autoimmune diseases are caused by the break-down in self-tolerance mechanisms and can result in the generation of autoantibodies specific to human antigens. Human autoantigen profiling technologies such as solid surface arrays and display technologies are powerful high-throughput technologies utilised to discover and map novel autoantigens associated with disease. This review compares human autoantigen profiling technologies including the application of these approaches in chronic and post-infectious autoimmune disease. Each technology has advantages and limitations that should be considered when designing new projects to profile autoantibodies. Recent studies that have utilised these technologies across a range of diseases have highlighted marked heterogeneity in autoantibody specificity between individuals as a frequent feature. This individual heterogeneity suggests that epitope spreading maybe an important mechanism in the pathogenesis of autoimmune disease in general and likely contributes to inflammatory tissue damage and symptoms. Studies focused on identifying autoantibody biomarkers for diagnosis should use targeted data analysis to identify the rarer public epitopes and antigens, common between individuals. Thus, utilisation of human autoantigen profiling technology, combined with different analysis approaches, can illuminate both pathogenesis and biomarker discovery.

Antibody Properties Associate with Clinical Phenotype in LGI1 Encephalitis

Ludewig, Susann; Salzburger, Leonie; Goihl, Alexander; Rohne, Jana; Leypoldt, Frank; Bittner, Daniel; Düzel, Emrah; Schraven, Burkhart; Reinhold, Dirk; Korte, Martin; Körtvélyessy, Péter
Cells.
Jan 2023
10.3390/cells12020282
Autoimmune encephalitis (AE) associated with autoantibodies against leucine-rich glioma-inactivated protein-1 (LGI1) can present with faciobrachial dystonic seizures (FBDS) and/or limbic encephalitis (LE). The reasons for this heterogeneity in phenotypes are unclear. We performed autoantibody (abs) characterization per patient, two patients suffering from LE and two from FBDS, using isolated antibodies specified with single amino acid epitope mapping. Electrophysiological slice recordings were conducted alongside spine density measurements, postsynaptic Alpha-amino-3-hydoxy-5-methyl-4-isoaxole-proprionate-receptors (AMPA-R) and N-methyl-D-aspartate-receptors receptor (NMDA-R) cluster counting. These results were correlated with the symptoms of each patient. While LGI1 abs from LE patients mainly interacted with the Leucine-rich repeat section of LGI1, abs from both FBDS patients also recognized the Epitempin section as well. Six-hour incubation of mouse hippocampal slices with LE patients autoantibodies but not from the FBDS patients resulted in a significant decline in long-term potentiation (p = 0.0015) or short-term plasticity at CA3-CA1 neurons and in decreased hippocampal synaptic density. Cluster differentiation showed no decrease in postsynaptic AMPA-R and NMDA-R. LGI1 autoantibodies selected by phenotype show an almost distinct epitope pattern, elicit disparate functional effects on hippocampal neurons, and cause divergent effects on spine density. This data illuminates potential pathomechanisms for disease heterogeneity in LGI1 AE.

Deciphering the Autoantibody Response to the OJ Antigenic Complex

Fritzler, Marvin J.; Bentow, Chelsea; Satoh, Minoru; McHugh, Neil; Ghirardello, Anna; Mahler, Michael
Diagnostics.
Jan 2023
10.3390/diagnostics13010156
(1) Background: Myositis specific antibodies (MSA) are important diagnostic biomarkers. Among the rarest and most challenging MSA are anti-OJ antibodies which are associated with anti-synthetase syndrome (ASS). In contrast to the other tRNA synthetases that are targets of ASS autoantibodies (e.g Jo-1, PL-7, PL-12, EJ, KS, Zo), OJ represents a macromolecular complex with several ribonucleoprotein subunits. Therefore, the choice of the antigen in autoantibody assays can be challenging. (2) Methods: We collected two independent cohorts with anti-OJ antibodies, one based on a commercial line immunoassay (LIA) (n = 39), the second based on protein immunoprecipitation (IP) (n = 15). Samples were tested using a particle-based multi-analyte technology (PMAT) system that allows for the simultaneous detection of antibodies to various autoantigens. For the detection of anti-OJ antibodies, two different antigens were deployed (KARS, IARS) on PMAT. The reactivity to the two antigens KARS and IARS was analyzed individually and combined in a score (sum of the median fluorescence intensities). (3) Results: In the cohort selection based on LIA, 3/39 (7.7%) samples were positive for anti-KARS and 7/39 (17.9%) for anti-IARS and 14/39 (35.9%) when the two antigens were combined. In contrast, in samples selected by IP the sensitivity of anti-KARS was higher: 6/15 (40.0%) samples were positive for anti-KARS, 4/15 (26.7%) for anti-IARS and 12/15 (80.0%) for the combination of the two antigens. 18/39 (46.2%) of the LIA samples generated a cytoplasmic IIF pattern (compatible with anti-synthetase antibodies), but there was no association with the antibody levels, neither with LIA nor with PMAT. (4) Conclusions: The combination of IARS and KARS might represent a promising approach for the detection of anti-OJ antibodies on a fully automated platform.

Molecular mimicry, genetic homology, and gene sharing proteomic “molecular fingerprints” using an EBV (Epstein-Barr virus)-derived microarray as a potential diagnostic method in autoimmune disease

Dreyfus, David H.; Farina, Antonella; Farina, Giuseppina Alessandra
Immunol Res.
Dec 2018
10.1007/s12026-018-9045-0
EBV (Epstein-Barr Virus) and other human DNA viruses are associated with autoimmune syndromes in epidemiologic studies. In this work, immunoglobulin G response to EBV-encoded proteins which share regions with human immune response proteins from the human host including ZEBRA (BZLF-1 encoded protein), BALF-2 recombinase expressed primarily during the viral lytic replication cycle, and EBNA-1 (Epstein-Barr Virus Nuclear Antigen) expressed during the viral latency cycle respectively were characterized using a laser-printed micro-array (PEPperprint.com). IgG response to conserved “A/T hooks” in EBV-encoded proteins such as EBNA-1 and the BALF-2 recombinase related to host DNA-binding proteins including RAG-1 recombinase and histones, and EBV-encoded virokines such as the IL-10 homologue BCRF-1 suggest further directions for clinical research. The author suggests that proteomic “molecular fingerprints” of the immune response to viral proteins shared with human immune response genes are potentially useful in early diagnosis and monitoring of autoantibody production and response to therapy in EBV-related autoimmune syndromes.

Regulatory T-cell deficiency leads to pathogenic bullous pemphigoid antigen 230 autoantibody and autoimmune bullous disease

Haeberle, Stefanie; Wei, Xiaoying; Bieber, Katja; Goletz, Stephanie; Ludwig, Ralf J.; Schmidt, Enno; Enk, Alexander H.; Hadaschik, Eva N.
Journal of Allergy and Clinical Immunology.
Dec 2018
10.1016/j.jaci.2018.04.006
Background Autoimmune bullous diseases/dermatoses (AIBDs) are severe autoantibody-mediated skin diseases. The pathogenic relevance of autoreactive CD4+ T cells for the induction of autoantibody production remains to be fully evaluated. Scurfy mice lack functional regulatory T (Treg) cells, experience spontaneous activation of autoreactive CD4+ T cells, and display severe erosive skin lesions suggestive of AIBDs. Objective We sought to determine whether AIBDs develop in Treg cell–deficient scurfy mice. Methods Histology, indirect immunofluorescence (IF) microscopy, direct IF, and ELISA were used to prove the presence of AIBDs in scurfy mice. Monoclonal autoantibodies from sera of scurfy mice were screened by using indirect IF on murine skin, and immunoprecipitation and mass spectrometry were used for target antigen identification, followed by confirmation in modified human embryonic kidney cells and murine keratinocytes. Pathogenicity was determined by injecting the autoantibody into neonatal mice and transferring scurfy CD4+ T cells into nu/nu mice. Results Autoantibodies against different known autoantigens of AIBDs spontaneously develop in scurfy mice. Histology reveals subepidermal blisters, and direct IF of skin of scurfy mice shows a predominant linear staining pattern. The mAb 20B12 shows a linear staining pattern in indirect IF, recognizes the murine hemidesmosomal protein bullous pemphigoid antigen 230 (BP230) as the target antigen, and cross-reacts with human BP230. Purified mAb 20B12 induces subepidermal blisters in neonatal mice. Transfer of scurfy CD4+ T cells is sufficient to induce antibodies with reactivity to AIBD autoantigens and subepidermal blisters in the skin of recipient T cell–deficient nu/nu mice. Conclusion We show that the absence of Treg cells leads to AIBDs by pathogenic autoantibodies targeting BP230.

Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

Lee, Yeon-Ah; Hahm, Dae-Hyun; Kim, Jung Yeon; Sur, Bonjun; Lee, Hyun Min; Ryu, Chun Jeih; Yang, Hyung-In; Kim, Kyoung Soo
Arthritis Res Ther.
Oct 2018
10.1186/s13075-018-1736-3
Background Different adiponectin isoforms appear to be differentially involved in the pathogenesis of various diseases. The purpose of this study was to generate monoclonal antibodies (mAbs) specific to different adiponectin isoforms and investigate whether these mAbs have potential as therapeutic agents for such diseases. Methods Hybridoma cells producing monoclonal antibodies were generated and screened using enzyme-linked immunosorbent assay and Western blotting for the production of mAbs recognizing human adiponectin isoforms. Results The mAb from hybridoma clone KH7–41 recognized both the middle molecular weight (MMW) (hexamer) and low molecular weight (LMW) (trimer) isoforms of adiponectin in human serum, whereas the KH7–33 mAb detected only MMW (hexamer) adiponectin. The KH4–8 clone recognized both the high molecular weight (HMW) (multimer) and MMW adiponectin isoforms. However, in mouse and rat sera, the abovementioned antibodies recognized only the MMW isomer. These mAbs also recognized adiponectin in various human tissues, such as lung, kidney, and adipose tissues, although the three mAbs had different staining intensities. The mAb from clone KH4–8 effectively inhibited increases in interleukin-6 (IL-6) and IL-8 expression in recombinant adiponectin-stimulated human osteoblasts and human umbilical vein endothelial cells. Also, the mAbs KH7–33 and KH4–8 significantly ameliorated rheumatic symptoms in a collagen-induced arthritis mouse model. This result suggests that these mAb treatments may ameliorate adiponectin-mediated inflammatory response. Conclusions mAbs against human adiponectin isomers can potentially be developed as therapeutic antibodies to target specific detrimental isoforms of adiponectin while maintaining the functions of beneficial isoforms.

The immunome of soy bean allergy: Comprehensive identification and characterization of epitopes

Kern, Karolin; Havenith, Heide; Delaroque, Nicolas; Rautenberger, Paul; Lehmann, Jörg; Fischer, Markus; Spiegel, Holger; Schillberg, Stefan; Ehrentreich-Foerster, Eva; Aurich, Stefanie; Treudler, Regina; Szardenings, Michael
Clin Exp Allergy.
Sep 2018
10.1111/cea.13285
Background The precise mapping of multiple antibody epitopes recognized by patients’ sera allows a more detailed and differentiated understanding of immunological diseases. It may lead to the development of novel therapies and diagnostic tools. Objective Mapping soy bean specific epitopes relevant for soy bean allergy patients and persons sensitized to soy bean, and analysis of their IgE/IgG binding spectrum. Methods Identification of epitopes using sera, applying an optimized peptide phage display library followed by next-generation sequencing, specially designed in silico data analysis and subsequent peptide microarray analysis. Results We were able to identify more than 400 potential epitope motifs in soy bean proteins. More than 60% of them have not yet been described as potential epitopes. Eighty-three peptides, representing the 42 most frequently found epitope candidates, were validated by microarray analysis using 50 sera from people who have been tested positive in skin prick test (SPT). Of these peptides, 56 were bound by antibodies, 55 by serum IgE, 43 by serum IgG and 30 by both. Person-specific epitope patterns were found for each individual and protein. Conclusions For individuals with clinical symptoms, epitope resolved analyses reveal a high prevalence of IgE binding to a few soy bean specific epitopes. Evaluation of individual immune profiles of patients with soy bean sensitization allows the identification of peptides that do facilitate studying individual IgE/IgG epitope binding patterns. This enables discrimination of sensitization from disease, such assay test has the potential to replace SPT assays

Combinatorial Synthesis of Macromolecular Arrays by Microchannel Cantilever Spotting (µCS)

Atwater, Jordyn; Mattes, Daniela S.; Streit, Bettina; von Bojničić-Kninski, Clemens; Loeffler, Felix F.; Breitling, Frank; Fuchs, Harald; Hirtz, Michael
Adv. Mater..
Aug 2018
10.1002/adma.201801632
Surface-bound microarrays of multiple oligo- and macromolecules (e.g., peptides, DNA) offer versatile options in biomedical applications like drug screening, DNA analysis, or medical diagnostics. Combinatorial syntheses of these molecules in situ can save significant resources in regard to processing time and material use. Furthermore, high feature densities are needed to enable high-throughput and low sample volumes as generally regarded in combinatorial chemistry. Here, a scanning-probe-lithography-based approach for the combinatorial in situ synthesis of macromolecules is presented in microarray format. Feature sizes below 40 µm allow for the creation of high-density arrays with feature densities of 62 500 features per cm2. To demonstrate feasibility of this approach for biomedical applications, a multiplexed array of functional protein tags (HA- and FLAG-tag) is synthesized, and selective binding of respective epitope recognizing antibodies is shown. This approach uses only small amounts of base chemicals for synthesis and can be further parallelized, therefore, opening up a route to flexible, highly dense, and cost-effective microarrays.

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