RESUMO
BACKGROUND: The interaction of allergens and allergen-specific IgE initiates the allergic cascade after crosslinking of receptors on effector cells. Antibodies of other isotypes may modulate such a reaction. Receptor crosslinking requires binding of antibodies to multiple epitopes on the allergen. Limited information is available on the complexity of the epitope structure of most allergens. OBJECTIVES: We sought to allow description of the complexity of IgE, IgG4, and IgG epitope recognition at a global, allergome-wide level during allergen-specific immunotherapy (AIT). METHODS: We generated an allergome-wide microarray comprising 731 allergens in the form of more than 172,000 overlapping 16-mer peptides. Allergen recognition by IgE, IgG4, and IgG was examined in serum samples collected from subjects undergoing AIT against pollen allergy. RESULTS: Extensive induction of linear peptide-specific Phl p 1- and Bet v 1-specific humoral immunity was demonstrated in subjects undergoing a 3-year-long AIT against grass and birch pollen allergy, respectively. Epitope profiles differed between subjects but were largely established already after 1 year of AIT, suggesting that dominant allergen-specific antibody clones remained as important contributors to humoral immunity following their initial establishment during the early phase of AIT. Complex, subject-specific patterns of allergen isoform and group cross-reactivities in the repertoires were observed, patterns that may indicate different levels of protection against different allergen sources. CONCLUSIONS: The study highlights the complexity and subject-specific nature of allergen epitopes recognized following AIT. We envisage that epitope deconvolution will be an important aspect of future efforts to describe and analyze the outcomes of AIT in a personalized manner.
Assuntos
Alérgenos/metabolismo , Antígenos de Plantas/metabolismo , Dessensibilização Imunológica/métodos , Epitopos de Linfócito B/metabolismo , Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Adulto , Alérgenos/imunologia , Antígenos de Plantas/imunologia , Betula , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Imunoglobulina E/metabolismo , Isotipos de Imunoglobulinas/metabolismo , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Peptídeos/imunologia , Proteínas de Plantas/imunologia , Poaceae , Rinite Alérgica Sazonal/terapiaRESUMO
Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system and also is regarded as an autoimmune condition. However, the antigenic targets of the autoimmune response in MS have not yet been deciphered. In an effort to mine the autoantibody repertoire within MS, we profiled 2,169 plasma samples from MS cases and population-based controls using bead arrays built with 384 human protein fragments selected from an initial screening with 11,520 antigens. Our data revealed prominently increased autoantibody reactivity against the chloride-channel protein anoctamin 2 (ANO2) in MS cases compared with controls. This finding was corroborated in independent assays with alternative protein constructs and by epitope mapping with peptides covering the identified region of ANO2. Additionally, we found a strong interaction between the presence of ANO2 autoantibodies and the HLA complex MS-associated DRB1*15 allele, reinforcing a potential role for ANO2 autoreactivity in MS etiopathogenesis. Furthermore, immunofluorescence analysis in human MS brain tissue showed ANO2 expression as small cellular aggregates near and inside MS lesions. Thus this study represents one of the largest efforts to characterize the autoantibody repertoire within MS. The findings presented here demonstrate that an ANO2 autoimmune subphenotype may exist in MS and lay the groundwork for further studies focusing on the pathogenic role of ANO2 autoantibodies in MS.
Assuntos
Autoanticorpos/sangue , Canais de Cloreto/imunologia , Proteínas de Membrana/imunologia , Esclerose Múltipla/imunologia , Adolescente , Adulto , Idoso , Anoctaminas , Autoantígenos/sangue , Autoantígenos/imunologia , Autoantígenos/metabolismo , Encéfalo/imunologia , Encéfalo/metabolismo , Estudos de Casos e Controles , Canais de Cloreto/sangue , Canais de Cloreto/metabolismo , Mapeamento de Epitopos , Feminino , Cadeias HLA-DRB1/genética , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Membrana/sangue , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Esclerose Múltipla/sangue , Esclerose Múltipla/genética , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/imunologia , Adulto JovemRESUMO
The underlying molecular mechanisms of autoimmune diseases are poorly understood. To unravel the autoimmune processes across diseases, comprehensive and unbiased analyses of proteins targets recognized by the adaptive immune system are needed. Here we present an approach starting from high-density peptide arrays to characterize autoantibody repertoires and to identify new autoantigens. A set of ten plasma and serum samples from subjects with multiple sclerosis, narcolepsy, and without any disease diagnosis were profiled on a peptide array representing the whole proteome, hosting 2.2 million 12-mer peptides with a six amino acid lateral shift. On the basis of the IgG reactivities found on these whole-proteome peptide microarrays, a set of 23 samples was then studied on a targeted array with 174â¯000 12-mer peptides of single amino acid lateral shift. Finally, verification of IgG reactivities was conducted with a larger sample set (n = 448) using the bead-based peptide microarrays. The presented workflow employed three different peptide microarray formats to discover and resolve the epitopes of human autoantibodies and revealed two potentially new autoantigens: MAP3K7 in multiple sclerosis and NRXN1 in narcolepsy. The presented strategy provides insights into antibody repertoire reactivity at a peptide level and may accelerate the discovery and validation of autoantigens in human diseases.
Assuntos
Autoanticorpos/sangue , Esclerose Múltipla/sangue , Narcolepsia/sangue , Análise Serial de Proteínas/métodos , Proteoma/análise , Adolescente , Adulto , Idoso , Autoantígenos/sangue , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular Neuronais/sangue , Criança , Feminino , Humanos , MAP Quinase Quinase Quinases/sangue , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/sangue , Moléculas de Adesão de Célula Nervosa , Adulto JovemRESUMO
BACKGROUND: Previous studies and own clinical observations of patients with systemic lupus erythematosus (SLE) suggest that SLE harbors distinct immunophenotypes. This heterogeneity might result in differences in response to treatment in different subgroups and obstruct clinical trials. Our aim was to understand how SLE subgroups may differ regarding underlying pathophysiology and characteristic biomarkers. METHODS: In a cross-sectional study, including 378 well-characterized SLE patients and 316 individually matched population controls, we defined subgroups based on the patients' autoantibody profile at inclusion. We selected a core of an antiphospholipid syndrome-like SLE (aPL+ group; positive in the lupus anticoagulant (LA) test and negative for all three of SSA (Ro52 and Ro60) and SSB antibodies) and a Sjögren's syndrome-like SLE (SSA/SSB+ group; positive for all three of SSA (Ro52 and Ro60) and SSB antibodies but negative in the LA test). We applied affinity-based proteomics, targeting 281 proteins, together with well-established clinical biomarkers and complementary immunoassays to explore the difference between the two predefined SLE subgroups. RESULTS: The aPL+ group comprised 66 and the SSA/SSB+ group 63 patients. The protein with the highest prediction power (receiver operating characteristic (ROC) area under the curve = 0.89) for separating the aPL+ and SSA/SSB+ SLE subgroups was integrin beta-1 (ITGB1), with higher levels present in the SSA/SSB+ subgroup. Proteins with the lowest p values comparing the two SLE subgroups were ITGB1, SLC13A3, and CERS5. These three proteins, rheumatoid factor, and immunoglobulin G (IgG) were all increased in the SSA/SSB+ subgroup. This subgroup was also characterized by a possible activation of the interferon system as measured by high KRT7, TYK2, and ETV7 in plasma. In the aPL+ subgroup, complement activation was more pronounced together with several biomarkers associated with systemic inflammation (fibrinogen, α-1 antitrypsin, neutrophils, and triglycerides). CONCLUSIONS: Our observations indicate underlying pathogenic differences between the SSA/SSB+ and the aPL+ SLE subgroups, suggesting that the SSA/SSB+ subgroup may benefit from IFN-blocking therapies while the aPL+ subgroup is more likely to have an effect from drugs targeting the complement system. Stratifying SLE patients based on an autoantibody profile could be a way forward to understand underlying pathophysiology and to improve selection of patients for clinical trials of targeted treatments.
Assuntos
Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/imunologia , Autoanticorpos/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Síndrome de Sjogren/imunologia , Adulto , Síndrome Antifosfolipídica/terapia , Autoanticorpos/sangue , Biomarcadores/sangue , Estudos Transversais , Feminino , Humanos , Imunoensaio , Imunoglobulina G/sangue , Lúpus Eritematoso Sistêmico/classificação , Lúpus Eritematoso Sistêmico/terapia , Masculino , Pessoa de Meia-Idade , Proteômica/métodos , Síndrome de Sjogren/terapiaRESUMO
Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann-Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (padjusted = 3 × 10-9, 3 × 10-6, and 5 × 10-6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials.
Assuntos
Biomarcadores/sangue , Fatores Reguladores de Interferon/sangue , Lúpus Eritematoso Sistêmico/imunologia , Transportador 2 de Cátion Orgânico/sangue , Proteína S100A12/sangue , Adulto , Análise por Conglomerados , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Proteômica , Proteínas Proto-Oncogênicas c-ets/metabolismoRESUMO
Plasmodium falciparum invasion into red blood cells (RBCs) is a complex process engaging proteins on the merozoite surface and those contained and sequentially released from the apical organelles (micronemes and rhoptries). Fundamental to invasion is the formation of a moving junction (MJ), a region of close apposition of the merozoite and the RBC plasma membranes, through which the merozoite draws itself before settling into a newly formed parasitophorous vacuole (PV). SURFIN4.2 was identified at the surface of the parasitized RBCs (pRBCs) but was also found apically associated with the merozoite. Using antibodies against the N-terminus of the protein we show the presence of SURFIN4.2 in the neck of the rhoptries, its secretion into the PV and shedding into the culture supernatant upon schizont rupture. Using immunoprecipitation followed by mass spectrometry we describe here a novel protein complex we have named SURGE where SURFIN4.2 forms interacts with the rhoptry neck protein 4 (RON4) and the Glutamate Rich Protein (GLURP). The N-terminal cysteine-rich-domain (CRD) of SURFIN4.2 mediates binding to the RBC membrane and its interaction with RON4 suggests its involvement in the contact between the merozoite apex and the RBC at the MJ. Supporting this suggestion, we also found that polyclonal antibodies to the extracellular domain (including the CRD) of SURFIN4.2 partially inhibit merozoite invasion. We propose that the formation of the SURGE complex participates in the establishment of parasite infection within the PV and the RBCs.
Assuntos
Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Proteínas de Membrana/metabolismo , Merozoítos/patogenicidade , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/metabolismo , Animais , Eritrócitos/metabolismo , Humanos , Malária Falciparum/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/genética , CoelhosRESUMO
Naturally acquired antibodies to proteins expressed on the Plasmodium falciparum parasitized red blood cell (pRBC) surface steer the course of a malaria infection by reducing sequestration and stimulating phagocytosis of pRBC. Here we have studied a selection of proteins representing three different parasite gene families employing a well-characterized parasite with a severe malaria phenotype (FCR3S1.2). The presence of naturally acquired antibodies, impact on rosetting rate, surface reactivity and opsonization for phagocytosis in relation to different blood groups of the ABO system were assessed in a set of sera from children with mild or complicated malaria from an endemic area. We show that the naturally acquired immune responses, developed during malaria natural infection, have limited access to the pRBCs inside a blood group A rosette. The data also indicate that SURFIN4.2 may have a function at the pRBC surface, particularly during rosette formation, this role however needs to be further validated. Our results also indicate epitopes differentially recognized by rosette-disrupting antibodies on a peptide array. Antibodies towards parasite-derived proteins such as PfEMP1, RIFIN and SURFIN in combination with host factors, essentially the ABO blood group of a malaria patient, are suggested to determine the outcome of a malaria infection.
Assuntos
Anticorpos Antiprotozoários/sangue , Malária Falciparum/imunologia , Proteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Sistema ABO de Grupos Sanguíneos/análise , Criança , Pré-Escolar , Eritrócitos/parasitologia , Humanos , Lactente , Malária Falciparum/parasitologia , Proteínas Opsonizantes/sangue , Fagocitose , Formação de RosetaRESUMO
Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing.
Assuntos
Anticorpos Antiprotozoários/imunologia , Epitopos/imunologia , Proteínas de Membrana/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Mapeamento de Epitopos , Perfilação da Expressão Gênica , Cabras , Plasmodium falciparum/genética , Coelhos , Análise de Sequência de RNARESUMO
Narcolepsy is a chronic sleep disorder with a yet unknown cause, but the specific loss of hypocretin-producing neurons together with a strong human leukocyte antigen (HLA) association has led to the hypothesis that autoimmune mechanisms might be involved. Here, we describe an extensive effort to profile autoimmunity repertoires in serum with the aim to find disease-related autoantigens. Initially, 57 serum samples from vaccine-associated and sporadic narcolepsy patients and controls were screened for IgG reactivity towards 10 846 fragments of human proteins using planar microarrays. The discovered differential reactivities were verified on suspension bead arrays in the same sample collection followed by further investigation of 14 antigens in 176 independent samples, including 57 narcolepsy patients. Among these 14 antigens, methyltransferase-like 22 (METTL22) and 5'-nucleotidase cytosolic IA (NT5C1A) were recognized at a higher frequency in narcolepsy patients of both sample sets. Upon sequence analysis of the 14 proteins, polymerase family, member 3 (PARP3), acyl-CoA-binding domain containing 7 (ARID4B), glutaminase 2 (GLS2) and cyclin-dependent kinase-like 1 (CDKL1) were found to contain amino acid sequences with homology to proteins found in the H1N1 vaccine. These findings could become useful elements of further clinical assays that aim towards a better phenotypic understanding of narcolepsy and its triggers.