Improved serological detection of rheumatoid arthritis: a highly antigenic mimotope of carbonic anhydrase III selected in a murine model by phage display

© 2015 Araujo et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects around 1% of the human population worldwide. RA diagnosis can be difficult as there is no definitive test for its detection. Therefore, the aim of this study was to identify biomarkers that could be used for RA diagnosis. Methods: Sera from a collagen-induced arthritis mouse model were used to select potential biomarkers for RA diagnosis by phage display technology. In silico and in vitro analyses were performed to characterize and validate the selected peptides. Samples were classified into three groups: RA; two other immune-mediated rheumatic diseases (systemic lupus erythematosus (SLE) and ankylosing spondylitis (AS)); and healthy controls (HC). Enzyme-linked immunosorbent assay (ELISA) was carried out to determine antibody levels, and diagnostic parameters were determined by constructing receiver operating characteristic curves. Mass spectrometry and Western blot were performed to identify the putative autoantigen that was mimicked by a highly reactive mimotope. Results: After three rounds of selection, 14 clones were obtained and tested for immunoreactivity analysis against sera from RA and HC groups. The phage-fused peptide with the highest immunoreactivity (M12) was synthesized, and was able to efficiently discriminate RA patients from SLE, AS and HCs (p < 0.0001) by ELISA. The specificity and sensitivity of anti-M12 antibodies for RA diagnosis were 91 % and 84.3 %, respectively. The M12 peptide was identified as one that mimics a predicted antigenic site of the carbonic anhydrase III (CAIII) protein, a ubiquitous biomarker that has been identified in patients with other diseases. Conclusion: M12 is the first peptide associated with the CAIII protein that may be used as an antigen for antibody detection to aid in RA diagnosis with high sensitivity and specificity.This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Foundation, Ministry of Education of Brazil (CSF SDW-2027/13-5) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).info:eu-repo/semantics/publishedVersio

Isolamento e identificação de mimetopos de auto-antígenos para utilização em imunodiagnóstico da artrite idiopática juvenil

CHAPTER 2: Juvenile Idiopathic Arthritis (JIA) is a set of chronic diseases characterized by persistent inflammation of joints. "Idiopathic" means that no one knows the cause of illness and "Juvenile" in this case means that the onset of symptoms occurs before 16 years of age. The diagnosis of JIA is clinical and is based on the finding of arthritis in one or more joints, lasting less than six weeks. It is essential that several diseases such as infections are investigated and removed, since arthritis is a common manifestation of various rheumatic and non-rheumatic diseases. Therefore, studies that are aimed at investigating new biomarkers become of great importance for the diagnosis of JIA. The aim of this study was to select and identify peptides recognized by antibodies purified from the serum of patients with JIA by Phage Display technology. For selection of peptides was performed a bioppaning using a peptide library Ph.D.-C7C expressed on the surface of filamentous phage M13. The DNA from selected clones was sequenced, translated, and several clones were subjected to ELISA assays and bioinformatics. Among the 192 clones sequenced, 100 had valid sequences, which were identified 40 different sequences. The bioinformatics analysis showed that there are similarities between most of the selected peptides and proteins highly expressed in patients with JIA. ELISA allowed the pre-validation of a clone with a potential biomarker for the serological immunodiagnosis of JIA.Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorMestre em Genética e BioquímicaCAPITULO 1: A Artrite Idiopática Juvenil (AIJ) é uma doença auto-imune caracterizada por artrite persistente, de causa desconhecida, que se inicia antes dos 16 anos de idade e está presente há pelo menos 6 semanas após exclusão de outras doenças. Longos períodos de AIJ ativa podem prejudicar o desenvolvimento do músculo, resultando em um retardo do crescimento generalizado, uso limitato de alguns membros, erosões nas articulações e menor capicidade aeróbica. É uma das mais prevalentes entre as doenças reumáticas crônicas da infância, podendo acarretar incapacidade motora permanente, afetando a qualidade de vida das crianças afetadas. O exame mais importante para o diagnóstico da doença hoje é o exame físico que o pediatra ou especialista faz na hora da consulta. O diagnóstico para AIJ é clínico, não depende de exames laboratoriais porque ainda não existe nenhum teste que confirme a presença da doença, não há exames laboratoriais considerados específicos para a sua definição diagnóstica. Testes laboratoriais bioquímicos e marcadores sorológicos são úteis para auxiliar no diagnóstico diferencial, classificar o subgrupo da doença, avaliar a extensão da inflamação, determinar o prognóstico e resposta à terapia. Nesse sentido, a busca por novos biomarcadores podem levar a novos métodos para o diagnóstico e tratamento da AIJ. Essa revisão visa descrever alguns aspectos da Artrite Idiopática Juvenil e como a tecnologia de Phage Display tem sido utilizada no mapeamento de epitopos de diversos antígenos, constituintes de vários agentes causadores de doenças. CAPITULO 2: As Artrites Idiopáticas Juvenis (AIJ) são um conjunto de doenças crônicas caracterizadas por inflamação persistente das articulações, sendo os sinais típicos desta inflamação a dor, inchaço e limitação dos movimentos. Idiopática significa que não se sabe a causa da doença e juvenil , neste caso, significa que o início dos sintomas acontece antes dos 16 anos de idade. O diagnóstico da AIJ é clínico e baseia-se no achado de artrite em uma ou mais articulações, com duração igual ou superior a 6 semanas. É fundamental que várias doenças, como as infecções, sejam pesquisadas e afastadas, uma vez que a artrite é a manifestação comum de várias doenças reumáticas e não-reumáticas. Por isso, trabalhos que são direcionados a investigar novos biomarcadores tornam-se de grande importância para o diagnóstico da AIJ. O objetivo deste estudo foi selecionar e identificar através da metodologia de Phage Display, peptídeos reconhecidos por anticorpos purificados a partir do soro de pacientes com AIJ. Para seleção dos peptídeos foi realizado um bioppaning utilizando uma biblioteca de peptídeos Ph.D.-C7C expressa na superfície do fago filamentoso M13. O DNA dos clones selecionados foi sequenciado, traduzido, e os diversos clones foram submetidos aos ensaios de ELISA e bioinformática. Dentre os 192 clones sequênciados, 100 apresentaram sequências válidas, onde foram identificadas 40 sequências diferentes. As análises de bioinformática demonstraram que há similaridades entre a maioria dos peptídeos selecionados e proteínas altamente expressas em pacientes portadores de AIJ. O ensaio ELISA permitiu a prévalidação de um clone com potencial biomarcador para o imunodiagnóstico sorológico da Artrite Idiopática Juvenil

Epítopos miméticos e autoantígenos aplicados ao imunodiagnóstico da artrite reumatoide e artrite idiopática Juvenil oligoarticular e poliarticular

Introduction: Rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA) are autoimmune rheumatic diseases of unknown cause characterized by inflammation, which can cause joint damage, and in some cases, damage to other parts of the body. To date, there is no single, definitive test for the diagnosis of RA and JIA, which is based on patient history, and various imaging and laboratory tests. In this context, the search for novel biomarkers with high sensitivity and specificity for RA and JIA is of great interest. Objectives: The aim of this study was to select, characterize and validate targets recognized by circulating antibodies that could be potentially described as autoantigens of RA or JIA. Methods: Phage Display technology was used to select peptides binding to circulating antibodies from mice presenting signs of arthritis after collagen type II induction (CIA model), and circulating antibodies from patients with JIA. In silico analysis, mass spectrometry and western blot were used to assist in the characterization of selected peptides. ELISA assays, ELISA avidity and differential pulse voltammetry was used as platform for detection of antibodies directed against the targets proposed in this study. Receiver Operating Characteristics (ROC) curve was determined for diagnostic accuracy for JIA and RA. Results: The M12 peptide selected against circulating IgGs from mice with signs of arthritis was able to discriminate RA patients from patients with other autoimmune diseases and healthy individuals (p < 0.0001) with high accuracy, presenting 91% specificity and 84.3% sensitivity. The M12 peptide was identified in an antigenic region of the protein carbonic anhydrase III, which has been previously identified as a RA autoantigen. The levels of antibodies to type II collagen were significantly higher in patients with JIA compared to levels obtained in patients with ankylosing spondylitis (p = 0.006) and healthy individuals (p < 0.0001). Moreover, the antibodies detection to type II collagen was more frequent in patients with ≤ 6 months duration (p = 0.0007). Antibodies displaying high avidity to collagen type II were associated with disease activity (p = 0.004). The PRF+1 peptide, selected from circulating IgGs from patients with JIA, was able to discriminate patients with JIA and RA from patients with other autoimmune diseases and healthy individuals (p < 0.0001) with high accuracy, presenting 91% specificity and 61% sensitivity for JIA, and 93% specificity and 94% sensitivity for RA. The electrochemical biosensor designed to detect antibodies against the peptide PFR+1 proved to be a fast, cheap and effective way of discriminating serum samples from patients with RA or JIA from healthy individuals. Conclusion: In a general analysis of the studies presented here, we conclude that the antigens used for the detection of circulating antibodies in patients with RA or JIA can be used with high accuracy to assist in diagnosis.Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorDoutor em Genética e BioquímicaIntrodução: Artrite reumatoide (AR) e artrite idiopática juvenil (AIJ) são doenças reumáticas, de origem autoimune, sem causa conhecida, caracterizadas por inflamação que pode provocar danos articulares e, em alguns casos, danos em outras partes do corpos. Não há, até o momento, um teste único para o diagnóstico da AR e AIJ, que é baseado na história clínica do paciente, e em vários exames laboratoriais e de imagem. Neste contexto, a busca por novos biomarcadores com alta sensibilidade e especificidade para a AR e AIJ é de grande interesse. Objetivos: O foco do presente estudo foi selecionar, caracterizar e validar alvos reconhecidos por anticorpos circulantes e que poderiam ser potencialmente descritos como autoantígenos da AR ou AIJ. Metodologia: A tecnologia de Phage Display foi empregada para selecionar peptídeos ligantes a anticorpos circulantes de camundongos apresentando sinais de artrite após indução com colágeno tipo II (modelo CIA), e anticorpos circulantes de pacientes com AIJ. Análises in silico, espectrometria de massas e Western blot foram utilizadas para auxiliar na caracterização de peptídeos selecionados. Ensaios de ELISA, ELISA avidez e voltametria de pulso diferencial foram utilizados como plataforma para detecção de anticorpos direcionados contra os alvos propostos neste estudo. A acurácia no diagnóstico da AR e AIJ foi determinada pela curva ROC (Receiver Operating Characteristics). Resultados: O peptídeo M12, selecionado contra IgGs circulantes de camundongos com sinais de artrite, foi capaz de discriminar pacientes com AR de pacientes com outras doenças autoimunes e indivíduos saudáveis (p < 0.0001) com alta acurácia, apresentando 91% de especificidade e 84.3% de sensibilidade. O peptídeo M12 foi identificado como mimético de uma região antigênica da proteína anidrase carbônica III, que já foi previamente identificada como um autoantígeno da AR. Os níveis de anticorpos contra o colágeno tipo II foram significativamente maiores em pacientes com AIJ quando comparados aos níveis obtidos por pacientes com espondilite anquilosante (p = 0.006) e indivíduos saudáveis (p < 0.0001). Além do mais, a detecção de anticorpos contra o colágeno tipo II foi mais frequente em pacientes com ≤ 6 meses de duração (p =0.0007). Anticorpos apresentando alta avidez para o colágeno tipo II foram associados com a atividade da doença (p = 0.004). O peptídeo PRF+1, selecionado contra IgGs circulantes de pacientes com AIJ, foi capaz de discriminar pacientes com AIJ e AR de pacientes com outras doenças autoimunes e indivíduos saudáveis (p < 0.0001) com alta acurácia, apresentando 91% de especificidade e 61% de sensibilidade para AIJ, e 93% de especificidade e 94% de sensibilidade para AR. O biosensor eletroquímico desenvolvido para detecção de anticorpos contra o peptídeo PFR+1 provou ser uma forma rápida, barata e eficaz de discriminar amostras de soro de pacientes com JIA e RA de indivíduos saudáveis. Conclusão: Em uma análise geral do conjunto de estudos aqui apresentados, é possível concluir que os antígenos utilizados para a detecção de anticorpos circulantes em pacientes com AIJ ou AR podem ser utilizados com alta acurácia para auxiliar no diagnóstico

Pediatric Allergy and Immunology / How relevant is panallergen sensitization in the development of allergies?

Panallergens comprise various protein families of plant as well as animal origin and are responsible for wide IgE crossreactivity between related and unrelated allergenic sources. Such crossreactivities include reactions between various pollen sources, pollen and plantderived foods as well as invertebratederived inhalants and foodstuff. Here, we provide an overview on the most clinically relevant panallergens from plants (profilins, polcalcins, nonspecific lipid transfer proteins, pathogenesisrelated protein family 10 members) and on the prominent animalderived panallergen family, tropomyosins. In addition, we explore the role of panallergens in the sensitization process and progress of the allergic disease. Emphasis is given on epidemiological aspects of panallergen sensitization and clinical manifestations. Finally, the issues related to diagnosis and therapy of patients sensitized to panallergens are outlined, and the use of panallergens as predictors for crossreactive allergy and as biomarkers for disease severity is discussed.(VLID)268683

Context matters: TH2 polarization resulting from pollen composition and not from protein-intrinsic allergenicity

(VLID)360017

Leishmania infantum -Tubulin Identified by Reverse Engineering Technology through Phage Display Applied as Theranostic Marker for Human Visceral Leishmaniasis

Two Leishmania infantum mimotopes (B10 and C01) identified by phage display showed to be antigenic and immunogenic for visceral (VL) and tegumentary (TL) leishmaniasis; however, their biological targets in the parasites have not been identified. The aim of the present study was to investigate the native antigens expressing both mimotopes, and to use them in distinct immunological assays. For this, a subtractive phage display technology was used, where a combinatorial library of single-chain variable fragments (scFv) was employed and the most reactive monoclonal antibodies for each target were captured, being the target antigens identified by mass spectrometry. Results in immunoblotting and immunoprecipitation assays showed that both monoclonal scFvs antibodies identified the -tubulin protein as the target antigen in L. infantum. To validate these findings, the recombinant protein was cloned, purified and tested for the serodiagnosis of human leishmaniasis, and its immunogenicity was evaluated in PBMC derived from healthy subjects and treated or untreated VL patients. Results showed high diagnostic efficacy, as well as the development of a specific Th1 immune response in the cell cultures, since higher IFN- and lower IL-10 production was found.(VLID)361478

A Short Peptide That Mimics the Binding Domain of TGF-β1 Presents Potent Anti-Inflammatory Activity.

The transforming growth factor beta 1 (TGF-β1) is a pleiotropic cytokine with multiple roles in development, wound healing, and immune regulation. TGF-β1-mediated immune dysfunction may lead to pathological conditions, such as inflammation. Chronic inflammatory process is characterized by a continuous release of pro-inflammatory cytokines, and the inhibition or the blockage of these cytokines signaling pathways are considered a target treatment. In this context, despite the high numbers of TGF-β-targeted pathways, the inducible regulatory T cells (iTreg) to control inflammation seems to be a promising approach. Our aim was to develop novel peptides through phage display (PhD) technology that could mimic TGF-β1 function with higher potency. Specific mimetic peptides were obtained through a PhD subtraction strategy from whole cell binding using TGF-β1 recombinant as a competitor during elution step. We have selected a peptide that seems to play an important role on cellular differentiation and modulation of TNF-α and IL-10 cytokines. The synthetic pm26TGF-β1 peptide tested in PBMC significantly down-modulated TNF-α and up-regulated IL-10 responses, leading to regulatory T cells (Treg) phenotype differentiation. Furthermore, the synthetic peptide was able to decrease leukocytes rolling in BALB/C mice and neutrophils migration during inflammatory process in C57BL/6 mice. These data suggest that this peptide may be useful for the treatment of inflammatory diseases, especially because it displays potent anti-inflammatory properties and do not exhibit neutrophils' chemoattraction

Analysis of cytokines production by PBMC after stimulation for 48 hours.

<p>Analysis of TNF-α (A) and IL-10 (B) cytokines released in the absence of inflammatory stimulus. Analysis of TNF-α (C) and IL-10 (D) cytokines released after PBMC stimulation with LPS. TNF-α production was decreased after PBMC pretreated with the pm26TGF-β1 peptide (1 μM and 10 μM; P < 0.05) and 100 μM (P < 0.0001). PBMC pretreated with the peptide followed by stimulation with LPS showed no difference compared to LPS/TGF-β1.</p

Three-dimensional analysis showing the region of interaction between the pm26TGF-β1 peptide and TβRII.

<p>The interaction between the Glu and Lys residues present in the pm26TGF-β1 peptide (purple) and the Ser⁴⁹ Glu¹¹⁹ residues of the TβRII (red). The binding site shared by TGF-β1 and pm26TGF-β1 peptide is represented in yellow. The Arg residue present in the pm26TGF-β1 peptide is represented in cyan.</p