RESUMO
Antibodies from sera of a multiple sclerosis (MS) patient subpopulation preferentially recognize the hyperglucosylated adhesin protein HMW1ct(Glc) of the pathogen Haemophilus influenzae. This protein is the first example of an N-glucosylated native antigen candidate, potentially triggering pathogenic antibodies in MS. Specific antibodies in patients' sera can be isolated exploiting their biospecific interaction with antigens by affinity chromatography. Herein, the proteins HMW1ct and HMW1ct(Glc) were first immobilized on appropriately functionalized supports and further used to purify antibodies directly from MS patients sera. We describe a protocol to obtain an antibody fraction specifically recognizing the glusosylated residues on the HMW1ct(Glc) adhesin protein depleting antibodies to the unglucosylated HMW1ct sequence. Different elution solutions have been tested to recover the purified antibody fraction, strongly bound to the immobilized HMW1ct(Glc) adhesin protein.
Assuntos
Adesinas Bacterianas , Cromatografia de Afinidade , Haemophilus influenzae , Cromatografia de Afinidade/métodos , Adesinas Bacterianas/imunologia , Adesinas Bacterianas/isolamento & purificação , Humanos , Haemophilus influenzae/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/sangue , GlicosilaçãoRESUMO
Multiple sclerosis (MS) is an inflammatory and autoimmune disorder, in which an antibody-mediated demyelination mechanism plays a critical role. We prepared two glucosylated peptides derived from the human myelin proteins, that is, oligodendrocyte-myelin glycoprotein (OMGp) and reticulon-4 receptor (RTN4R), selected by a bioinformatic approach for their conformational homology with CSF114(Glc), a designed ß-turn antigenic probe derived from myelin oligodendrocyte glycoprotein (MOG), a glycoprotein present in the CNS. This synthetic antigen is specifically recognized by antibodies in sera of MS patients. We report herein the antigenic properties of these peptides, showing, on the one hand, that MS patient antibodies recognize the two glucosylated peptides and, on the other hand, that these antibodies cross-react with CSF114(Glc) and with the previously described hyperglucosylated nontypeable Haemophilus influenzae bacterial adhesin protein HMW1ct(Glc). These observations point to an immunological association between human and bacterial protein antigens, underpinning the hypothesis that molecular mimicry triggers the breakdown of self-tolerance in MS and suggesting that RTN4R and OMGp can be considered as autoantigens.
Assuntos
Esclerose Múltipla , Humanos , Autoantígenos , Adesinas Bacterianas , Bainha de Mielina/metabolismo , Haemophilus influenzae , Autoanticorpos , Proteínas da Mielina , Peptídeos , Glicoproteína Mielina-OligodendrócitoRESUMO
Enzymatic digestion is a promising alternative in the upconversion of plastic waste compared to traditional chemical recycling methods, because it warrants the use of milder conditions. However, enzymes are hardly able to penetrate the bulk of the plastic material; thus, a pretreatment is necessary to promote the reaction. In this study we investigate hydrothermal liquefaction as a thermal pretreatment of a commercial polyurethane before performing an enzymatic digestion. The feedstock is a rigid polyurethane foam. The structure and chemical composition of the feedstock were analyzed through FTIR analysis and solid-state 13C NMR. The polyurethane was then subjected to hydrothermal liquefaction using either ultrapure water or KOH as a basic catalyst. Enzymatic digestion was then performed on the organic fraction obtained from both experiments using a lipase extracted from Candida rugosa. The LC-MS analysis of the digests shows an increase in some signal intensities due to the degradation of oligomeric fragments. This new way of recycling allows the recovery of important chemicals such as quinolines and 4,4'-methylenedianiline. With this study we demonstrate that hydrothermal liquefaction coupled with enzymatic digestion is a suitable alternative for handling polyurethane waste.
RESUMO
The involvement of Myelin Basic Protein (MBP) in Multiple Sclerosis (MS) has been widely discussed in the literature. This intrinsically disordered protein has an interesting α-helix motif, which can be considered as a conformational epitope. In this work we investigate the importance of the helical structure in antibody recognition by MBP peptides of different lengths. Firstly, we synthesized the peptide MBP (81-106) (1) and observed that its elongation at both N- and C-termini, to obtain the peptide MBP (76-116) (2) improves IgM antibody recognition in SP-ELISA, but destabilizes the helical structure. Conversely, in competitive ELISA, MBP (81-106) (1) is recognized more efficiently by IgM antibodies than MBP (76-116) (2), possibly thanks to its more stable helical structure observed in CD and NMR conformational experiments. These results are discussed in terms of different performances of peptide antigens in the two ELISA formats tested.
RESUMO
Tentacle-like polymers decorated with several copies of peptide antigens can be interesting tools for increasing the ability to capture circulating antibodies in patient sera, using cooperative effects for stronger avidity. We previously showed that antibodies from multiple sclerosis (MS) patient sera preferentially recognize hyperglucosylated adhesin protein HMW1ct of non-typeable Haemophilus influenzae (NTHi). We selected the C-terminal HMW1ct(1347-1354) minimal epitope and prepared the diglucosylated analogue Ac-KAN(Glc)VTLN(Glc)TTG-K(N3 )-NH2 to graft a 40â kDa dextran scaffold modified with glycidyl-propargyl moieties to perform a copper catalyzed alkyne-azide coupling reaction (CuAAC). Quantitative NMR measurements allowed the characterization of the peptide loading (19.5 %) on the multivalent dextran conjugate. This novel polymeric structure displayed optimal capturing properties of both IgG and, more interestingly, IgM antibodies in MS sera. Specific antibodies from a representative MS serum, were successfully depleted using a Sepharose resin bearing the new glucosylated multivalent conjugate, as confirmed by ELISA. These results may offer a promising proof-of-concept for the selective purification of high affinity autoantibodies from sera of autoimmune patients, in general, and of specific high affinity antibodies against a minimally glcosylated epitope Asn(Glc) from sera of multiple sclerosis (MS) patients, in particular.
Assuntos
Adesinas Bacterianas/efeitos dos fármacos , Antibacterianos/farmacologia , Autoanticorpos/farmacologia , Dextranos/farmacologia , Haemophilus influenzae/efeitos dos fármacos , Peptídeos/farmacologia , Antibacterianos/química , Autoanticorpos/química , Dextranos/química , Glicosilação , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Peptídeos/químicaRESUMO
Immune response to biologics treatment, while widely reported, yet fails to correlate with clinical outcomes and assay to assay comparison is often not possible. Hence, we developed a new peptide based-detection assay to stratify pediatric patients with juvenile idiopathic arthritis (JIA) or chronic non-infectious uveitis (CNU) and monitor anti-drug antibodies (ADAbs) formed as part of an immune response to treatment with the fully human monoclonal therapeutic antibody Adalimumab. Adalimumab derived synthetic peptides were optimized for maximum immunogenicity and were tested by SP-ELISA on a development cohort of 18 JIA and CNU treated patients. The two best performing peptides able to differentiate patient groups were selected for evaluation with a larger scale ELISA testing on a total of 29 sera from pediatric patients with JIA or CNU. The results of this peptide-based assay were compared to an in-house developed SPR biosensor ADAbs assay and a commercially available bridging ELISA. The first peptide, termed HC3, was able to positively detect ADAbs in 7 out of the 29 sera, while the second peptide, called LC3, was able to detect ADAbs in 11 out of 29 sera in the evaluation group. Following statistical data evaluation, it has been found that the detection of ADAbs using the peptide-based ELISA assay positively correlates with disease progression and remission. Two synthetic peptides derived from Adalimumab may provide a beneficial tool to clinicians for monitoring patient response to such treatment and taking informed decisions for treatment alternatives.
Assuntos
Adalimumab/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Artrite Juvenil/tratamento farmacológico , Produtos Biológicos/uso terapêutico , Imunidade/efeitos dos fármacos , Peptídeos/uso terapêutico , Uveíte/tratamento farmacológico , Sequência de Aminoácidos , Antirreumáticos/uso terapêutico , Fatores Biológicos/uso terapêutico , Criança , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , MasculinoRESUMO
Diagnosis of Latent Autoimmune Diabetes in Adults (LADA) is based on the adult-age, anti-islet autoantibodies, and temporary insulin-independence. As in Type-1-Diabetes (T1DM), autoimmunity may trigger LADA and enteroviruses-infections can play a role. Anti-human Glutamic-Acid-Decarboxylase (hGAD) autoantibodies are accepted clinical biomarkers, but do not discriminate LADA vs. T1DM. The hypothesis is that protein antigens detecting anti-hGAD antibodies do not expose epitopes specific for different disease forms. We investigated the diagnostic value of autoantibodies in LADA vs. T1DM to peptides of hGAD65/67 isoforms, and Enterovirus-Coxsackie-B4 (CVB4), as antigens sharing the epitope PEVKXK (X: E/T) included in CD8 T-cell CVB4 epitope restricted by diabetes-associated HLA-A2.1. Statistically significant differences of IgM and/or IgG in LADA and T1DM vs. controls were identified. In LADA IgMs to GAD65/67 peptides are diagnostics, IgGs to GAD65/67 peptides correlate with anti-CVB4 peptide antibodies. IgM and/or IgG to all tested peptides can predict LADA, monitoring CVB4 infected patients, improving LADA vs. T1DM stratification.â¢A customized SP-ELISA based on synthetic peptides Ac-hGAD65(250-273)-NH2 (1), Ac-hGAD67(258-281)-NH2 (2), and Ac-CVB4P2C(28-50)-NH2 (3) is described.â¢The method was designed to detect specific IgM and/or IgG in LADA, T1DM, vs. controlsâ¢Final aim is improvement of LADA vs. T1DM patient stratification.
RESUMO
BACKGROUND: Diagnosis of latent autoimmune diabetes in adults (LADA) is usually based on the adult age, anti-pancreatic islet cell antibodies detection, and insulin independence. This study investigates the diagnostic value of antibodies against human glutamic acid decarboxylase (hGAD) peptides in LADA and type 1 diabetes mellitus (T1DM) patients, and their cross-reactivity with an Enterovirus Coxsackie B4 (CVB4) shared epitope. METHODS: Sera from 27 LADA patients, 23 T1DM patients, and 24 controls were tested in ELISA for antibodies against hGAD peptides and a selected sequence of P2C protein of CVB4 (CVB4P2C). Diagnostic power of peptides was analyzed by ROC-curve analysis and cross-reactivity among peptides evaluated. RESULTS: IgM and IgG antibodies showed significant differences between LADA and T1DM versus controls for all peptides. Antibody responses present high agreement among peptides for IgM and IgG-isotypes in T1DM, which is not reproduced in LADA. IgM antibodies showed high predicting diagnostic power particularly in LADA (sensitivity > 85%, specificity 95.8%). CONCLUSIONS: Our study highlights the usefulness of peptides as diagnostic antigens in T1DM and LADA, and extends previous findings by comparing IgM and IgG-isotype antibodies in the same population. Additionally, results highlight the role of the entourage in the shared sequon PEVKXK in GAD and CVB4P2C particularly in IgMs identification.
Assuntos
Diabetes Mellitus Tipo 1 , Enterovirus , Diabetes Autoimune Latente em Adultos , Adulto , Autoanticorpos , Diabetes Mellitus Tipo 1/diagnóstico , Epitopos , Glutamato Descarboxilase , Humanos , PeptídeosRESUMO
Peptides mimicking antigenic epitopes targeted by antibodies can be powerful tools to be used as antigen surrogates for the specific diagnosis and treatment of autoimmune diseases. Obtaining structural insights about the nature of peptide-antibody interaction in complex mixtures such as sera is a critical goal. In multiple sclerosis (MS), we previously demonstrated that the N-linked ß-d-glucopyranosyl moieties (N-Glc) containing epitopes in nontypeable Haemophilus influenzae adhesin C-terminal portion HMW1(1205-1526) were essential for high-affinity antibody binding in a subpopulation of MS patients. With the aim of developing peptide probes and assessing their binding properties to antibodies from sera of representative patients, we performed the systematic analysis of synthetic peptides based on HMW1(1347-1354) fragment bearing one or two N-Glc respectively on Asn-1349 and/or Asn-1352. The N-glucosylated nonapeptides efficiently bind to IgG antibodies, displaying IC50 in the range 10-8 -10-10 M by competitive indirect enzyme-linked immunosorbent assay (ELISA) in three representative MS patient sera. We selected the di-N-glucosylated adhesin peptide Ac-KAN (Glc)VTLN (Glc)TT-NH2 as the shortest sequence able to inhibit high-avidity interaction with N-Glc targeting IgM antibodies. Nuclear magnetic resonance (NMR)- and circular dichroism (CD)-based characterization showed that the binding properties of these antigens could not be ascribed to structural differences induced by the presence of up to two N-glucosyl moieties. Therefore, the antibody binding is not easily correlated to the position of the sugar or to a determined conformation in water.
Assuntos
Adesinas Bacterianas/imunologia , Antígenos/imunologia , Esclerose Múltipla/imunologia , Peptídeos/imunologia , Adesinas Bacterianas/química , Glicosilação , Haemophilus influenzae/química , Humanos , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/químicaRESUMO
We report herein a novel ChemMatrix® Rink resin functionalised with two phenylboronate (PhB) moieties linked on the N-α and N-ε amino functions of a lysine residue to specifically capture deoxyfructosylated peptides, compared to differently glycosylated peptides in complex mixtures. The new PhB-Lys(PhB)-ChemMatrix® Rink resin allows for exploitation of the previously demonstrated ability of cis diols to form phenylboronic esters. The optimised capturing and cleavage procedure from the novel functionalised resin showed that only the peptides containing deoxyfructosyl-lysine moieties can be efficiently and specifically detected by HR-MS and MS/MS experiments. We also investigated the high-selective affinity to deoxyfructosylated peptides in an ad hoc mixture containing unique synthetic non-modified peptides and in the hydrolysates of human and bovine serum albumin as complex peptide mixtures. We demonstrated that the deoxyfructopyranosyl moiety on lysine residues is crucial in the capturing reaction. Therefore, the novel specifically-designed PhB-Lys(PhB)-ChemMatrix® Rink resin, which has the highest affinity to deoxyfructosylated peptides, is a candidate to quantitatively separate early glycation peptides from complex mixtures to investigate their role in diabetes complications in the clinics.
Assuntos
Ácidos Borônicos/química , Cromatografia de Afinidade/métodos , Frutose/química , Peptídeos/análise , Peptídeos/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Glicosilação , Lisina/química , Peptídeos/química , Proibitinas , Soroalbumina Bovina/análise , Soroalbumina Bovina/metabolismo , Albumina Sérica Humana/análise , Albumina Sérica Humana/metabolismo , Espectrometria de Massas em TandemAssuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Artrite Psoriásica/imunologia , Autoanticorpos/imunologia , Imunoglobulina G/imunologia , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Psoríase/imunologia , CatelicidinasRESUMO
Reliable monitoring of clinical relevant anti-drug antibodies is fundamental in the follow-up of patients under adalimumab treatment. The aim of this study is to compare anti-adalimumab antibodies by using three methods based on different technologies. A cross-sectional study was performed in 50 patients with rheumatoid arthritis (RA) treated with adalimumab. Anti-adalimumab antibodies were detected in patients' sera by different techniques: bridging ELISA, reporter gene assay (RGA), and surface plasmon resonance (SPR). Results showed that all methods recognized anti-adalimumab antibodies and the percentage of positives fluctuated among the assays. Five (10%) of the 50 patients were positive in ELISA, 4 (8%) in RGA, and 6 (12%) in SPR. Among positive patients, 4 were positive in the three assays, one patient uniquely in ELISA, and two in SPR. Spearman correlation between ELISA and RGA showed good agreement (Spearman râ¯=â¯0.800). No correlation between RGA and SPR was observed (Spearman râ¯=â¯0.108). Similar results were obtained between ELISA and SPR (Spearman râ¯=â¯- 0.241). Summarizing, ELISA, RGA and SPR recognized anti-adalimumab antibodies in few RA patients, showing good agreement among the methodology employed. On the other hand, differences observed between SPR and ELISA or RGA highlight the relevance of the employed technologies in anti-drug antibody identification.
Assuntos
Adalimumab/imunologia , Anticorpos/sangue , Antirreumáticos/imunologia , Artrite Reumatoide/terapia , Adalimumab/administração & dosagem , Adulto , Antirreumáticos/administração & dosagem , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Genes Reporter/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Ressonância de Plasmônio de SuperfícieRESUMO
Deiminated proteins are the target of diagnostic anti-citrullinated peptide/protein autoantibodies (ACPA) in rheumatoid arthritis (RA). Deiminated histone H4 contained in the neutrophil extracellular traps reacts with ACPA, becoming an interesting diagnostic antigen for RA. The identification of the ACPA binding site in histone H4 was performed experimentally by mapping the complete sequence. The method describes the synthesis of an overlapping peptide library covering the entire deiminated sequence of H4 and its further evaluation in ELISA. A detailed description of an ELISA protocol to test RA patients' sera against the synthesized peptides and ACPA is provided.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/análise , Mapeamento de Epitopos/métodos , Histonas/metabolismo , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Histonas/química , Humanos , Imunoglobulina G/metabolismo , Biblioteca de Peptídeos , Peptídeos/síntese química , Técnicas de Síntese em Fase SólidaRESUMO
BACKGROUND: Mitochondria play a role in type 1 diabetes (T1D) particularly in the treatment and prevention of disorder consequences. Due to their demonstrated role in diabetes pathology, mitochondrial proteins can be an interesting starting point to study candidate antigens in T1D. We investigated the role of relevant post-translational modifications (PTM) on a synthetic mitochondrial peptide as putative antigen. METHODS: The antibody response in T1D was evaluated by solid phase-ELISA using a collection of synthetic peptides bearing different PTMs. We investigated the role of lipoylation, phosphorylation, and glycosylation. The PTMs were introduced at position 173 of the mitochondrial pyruvate dehydrogenase E2 complex peptide PDC-E2(167-184) and at position 7 of a structure-based designed ß-turn peptide as an irrelevant sequence to investigate the role of the specific PDC-E2 peptide sequence. RESULTS: IgM titres in 31 T1D patients were higher than IgGs to all the synthetic PTM peptides. Results demonstrated the crucial role of lysine lipoamide, serine O-phosphorylation, and O-glycosylation into the PDC-E2(167-184) peptide sequence for IgM antibody recognition. CONCLUSIONS: Results highlight the importance of immune dysregulation in T1D, furthermore, if confirmed in a large number of patients, they will contribute to add novel diagnostic markers for the understanding the physiopathology of the disease.
Assuntos
Anticorpos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Proteínas Mitocondriais/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Adulto , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Glicosilação , Humanos , Masculino , Fosforilação , Estereoisomerismo , Ácido Tióctico/análogos & derivados , Ácido Tióctico/química , Ácido Tióctico/metabolismoRESUMO
Three triterpene glycosides were isolated from the roots of Weigela florida "rumba" (Bunge) A. DC.: two previously undescribed 3-O-ß-d-xylopyranosyl-(1â2)-[ß-d-xylopyranosyl-(1â4)]-ß-d-xylopyranosyl-(1â4)-ß-d-xylopyranosyl-(1â3)-α-L-rhamnopyranosyl-(1â2)-α-l-arabinopyranosyloleanolic acid (1) and 3-O-ß-d-xylopyranosyl-(1â2)-[ß-d-glucopyranosyl-(1â4)]-ß-d-xylopyranosyl-(1â4)-ß-d-xylopyranosyl-(1â3)-α-l-rhamnopyranosyl-(1â2)-α-L-arabinopyranosyloleanolic acid (2), and one isolated for the first time from a natural source 3-O-ß-d-xylopyranosyl-(1â3)-α-l-rhamnopyranosyl-(1â2)-α-l-arabinopyranosyloleanolic acid (3). Their structures were elucidated mainly by 2D NMR spectroscopic analysis (COSY, TOCSY, NOESY, HSQC, HMBC) and mass spectrometry. Compounds 2 and 3 were further evaluated as antigens in enzyme-linked immunosorbent assay (ELISA) to recognize IgM antibodies in multiple sclerosis (MS) patients' sera.
Assuntos
Caprifoliaceae/química , Glicosídeos/isolamento & purificação , Ácido Oleanólico/análogos & derivados , Raízes de Plantas/química , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina M/química , Estrutura Molecular , Esclerose Múltipla/sangue , Ácido Oleanólico/isolamento & purificaçãoRESUMO
Adalimumab is a TNF-α blocker antibody similar in structure and function to natural human IgG1. Even if adalimumab is fully humanized, the development of anti-drug antibodies has been reported in several inflammatory conditions. The objective of our study was to assess the presence of anti-adalimumab antibodies (AAA) and their clinical relevance in a cohort of juvenile idiopathic arthritis (JIA) patients on adalimumab. This is a prospective observational cohort study recruiting JIA children. Experiments were performed using a validated surface plasmon resonance (SPR)-based optical assay (Biacore® T100). Disease activity was evaluated using the Juvenile Arthritis Disease Activity Score with 10 joint count (JADAS-10). The Mann-Whitney U test, Wilcoxon signed-rank test for paired samples, chi-square, and Fisher exact test were used to compare data. Pearson's and Spearman's correlation tests were used to determine correlation coefficients for entered variables: demographic, clinical, and serological data. Ten (37%) out of 27 patients included in the study had at least one AAA-positive sample. Patients developed AAA between 3 and 38 months after starting adalimumab. Seven (70%) out of 10 children with AAA positivity experienced at least a relapse compared to 4 (23.5%) out of 17 AAA-negative children (rs 0.45, p < 0.017). In conclusion, using an innovative and accurate assay method, we found a high incidence of anti-drug antibodies in a cohort of adalimumab-treated JIA patients observed over a mean period of 40 weeks; the presence of anti-adalimumab antibodies seemed to be related to the number of relapses.
Assuntos
Adalimumab/imunologia , Anticorpos Anti-Idiotípicos , Anticorpos Monoclonais Humanizados/imunologia , Artrite Juvenil/tratamento farmacológico , Adolescente , Artrite Juvenil/imunologia , Criança , Pré-Escolar , Terapia Combinada , Feminino , Humanos , Masculino , Metotrexato/uso terapêutico , Estudos Prospectivos , Resultado do TratamentoRESUMO
The design of molecules that mimic biologically relevant glycans is a significant goal for understanding important biological processes and may lead to new therapeutic and diagnostic agents. In this study we focused our attention on the trisaccharide human natural killer cell-1 (HNK-1), considered the antigenic determinant of myelin-associated glycoprotein and the target of clinically relevant auto-antibodies in autoimmune neurological disorders such as IgM monoclonal gammopathy and demyelinating polyneuropathy. We describe a structure-activity relationship study based on surface plasmon resonance binding affinities aimed at the optimization of a peptide that mimics the HNK-1 minimal epitope. We developed a cyclic heptapeptide that shows an affinity of 1.09×10-7 m for a commercial anti-HNK1 mouse monoclonal antibody. Detailed conformational analysis gave possible explanations for the good affinity displayed by this novel analogue, which was subsequently used as an immunological probe. However, preliminary screening indicates that patients' sera do not specifically recognize this peptide, showing that murine monoclonal antibodies cannot be used as a guide to select immunological probes for the detection of clinically relevant human auto-antibodies.
Assuntos
Antígenos CD57/química , Epitopos/química , Células Matadoras Naturais/química , Oligossacarídeos/química , Oligossacarídeos/imunologia , Peptídeos/química , Peptídeos/imunologia , Ressonância de Plasmônio de Superfície , Animais , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Antígenos CD57/imunologia , Epitopos/imunologia , Humanos , Células Matadoras Naturais/imunologia , Camundongos , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
In autoimmune diseases, there have been proposals that exogenous "molecular triggers", i.e., specific 'non-self antigens' accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies. The etiology of multiple sclerosis (MS) remains unclear. However, epidemiologic data suggest that exposure to infectious agents may be associated with increased MS risk and progression may be linked to exogenous, bacterially-derived, antigenic molecules, mimicking mammalian cell surface glycoconjugates triggering autoimmune responses. Previously, antibodies specific to a gluco-asparagine (N-Glc) glycopeptide, CSF114(N-Glc), were identified in sera of an MS patient subpopulation. Since the human glycoproteome repertoire lacks this uniquely modified amino acid, we turned our attention to bacteria, i.e., Haemophilus influenzae, expressing cell-surface adhesins including N-Glc, to establish a connection between H. influenzae infection and MS. We exploited the biosynthetic machinery from the opportunistic pathogen H. influenzae (and the homologous enzymes from A. pleuropneumoniae) to produce a unique set of defined glucosylated adhesin proteins. Interestingly we revealed that a hyperglucosylated protein domain, based on the cell-surface adhesin HMW1A, is preferentially recognized by antibodies from sera of an MS patient subpopulation. In conclusion the hyperglucosylated adhesin is the first example of an N-glucosylated native antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS.
Assuntos
Adesinas Bacterianas/imunologia , Anticorpos/imunologia , Haemophilus influenzae/imunologia , Esclerose Múltipla/imunologia , Adulto , Idoso , Antígenos de Bactérias/imunologia , Asparagina/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Feminino , Glicoconjugados/imunologia , Glicopeptídeos/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Surface plasmon resonance (SPR) technique is extremely interesting in immunology because it has the potential to directly visualize biomolecular interactions in real-time monitoring antibody affinity, one of the parameters affecting pathogenicity in autoimmune diseases. Herein we describe the affinity evaluation of anti-citrullinated peptide antibodies (ACPA) to a peptide-based biosensor by SPR. The method describes the purification of ACPA isolated from rheumatoid arthritis (RA) patients using affinity columns, the strategy employed for the immobilization of citrullinated peptides onto a sensor chip, and the evaluation of the specific binding of purified ACPA to immobilized peptides.