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Background and Objective: Post-translational modifications of antibodies, with a specific focus on galactosylation, have garnered increasing attention in the context of understanding the pathogenesis and therapeutic implications of autoimmune diseases. However, the comprehensive scope and the clinical significance of antibody galactosylation in the context of Neuromyelitis Optica Spectrum Disorder (NMOSD) remain enigmatic.The primary aim of this research was to discern disparities in serum IgG galactosylation levels between individuals in the acute stage of NMOSD relapse and their age- and sex-matched healthy counterparts. Methods: A total of fourteen untreated NMOSD patients experiencing an acute relapse phase, along with thirteen patients under medication, were enrolled, and an additional twelve healthy controls of the same age and gender were recruited for this investigation. Western blot and lectin enzyme techniques were used to determine the level of IgG galactosylation in the serum samples from these subjects. The expression of CD45+, CD3+, CD3+CD4+, CD3+CD8+, CD19+, and CD16+CD56+ in peripheral blood leukocytes was measured by flow cytometry. The enzyme-linked immunosorbent assay (ELISA) was also used to quantify the amounts of IgG. Magnetic particle luminescence assays are used to detect cytokines. Robust statistical analysis was executed to ascertain the potential associations between IgG galactosylation and the aforementioned immune indices. Results: In the context of NMOSD relapses, serum IgG galactosylation exhibited a notable decrease in untreated patients (0.2482 ± 0.0261), while it remained comparatively stable in medicated patients when contrasted with healthy controls (0.3625 ± 0.0259) (p=0.0159). Furthermore, a noteworthy inverse correlation between serum IgG galactosylation levels and the Expanded Disability Status Scale (EDSS) score during NMOSD relapse was observed (r=-0.4142; p=0.0317). Notably, IgG galactosylation displayed an inverse correlation with NMOSD relapse among peripheral blood CD45+, CD3+, CD3+CD8+, CD19+ cells, as well as with IL-6 and IL-8. Nevertheless, it was not determined whether IgG galactosylation and CD3+CD4+ T cells or other cytokines are statistically significantly correlated. Conclusion: Our research identified reduced IgG galactosylation in the serum of NMOSD patients during relapses, significantly correlated with disease severity, thereby providing a novel target for the diagnosis and treatment of NMOSD in the realm of medical research.
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Neuromielitis Óptica , Humanos , Inflamación , Citocinas , Inmunoglobulina G , RecurrenciaRESUMEN
OBJECTIVE: Anti-ß-2 glycoprotein I (anti-ß2GPI) antibodies, defined as primary pathogenic antibody in antiphospholipid syndrome (APS). It has been reported that IgG Fc N-glycosylation affects IgG effector, we aim to investigate the association of Fc glycosylation profiles of purified anti-ß2GP1 IgG with clinical features of APS. METHODS: We purify anti-ß2GPI IgG and total IgG from 82 APS patients including nine catastrophic antiphospholipid syndrome (CAPS) patients, as well as total IgG from 103 healthy controls to quantitatively analyse all detectable Fc N-glycanforms of all IgG subclasses with Multiple Reaction Monitoring (MRM) method based on UPLC-ESI-QqQ mass spectrometry. RESULTS: Both purified anti-ß2GPI IgG and APS total IgG showed altered N-glycan profiles when compared with healthy control (HC) IgG. Anti-ß2GPI IgG presented with lower galactosylation, increased bisection and core fucosylation compared with APS total IgG and HC IgG. We found higher galactosylation of aß2GPI IgG2 in thrombotic APS compared with the obstetric APS, and lower galactosylation of aß2GPI IgG2 associated with late pregnancy morbidity. Moreover, low galactosylation of all anti-ß2GPI IgG subclasses, increased bisection and core fucosylation of anti-ß2GPI IgG1/2 were strongly associated with CAPS and triple positivity of antiphospholipid antibodies (aPLs). CONCLUSION: We comprehensively characterize the N-Glycans landscape of both anti-ß2GP1 and total IgG in APS. Altered N-glycan profiles of anti-ß2GPI IgG enables enabled the antibodies with proinflammatory properties. Furthermore, we associated levels of IgG Fc-glycosylation with clinical features antiphospholipid syndrome. These findings could increase our understanding of anti-ß2GPI antibody mediated mechanisms in APS and be used to develop diagnostics and new target treatments.
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Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/inmunología , Inmunoglobulina G/inmunología , Complicaciones del Embarazo/inmunología , Trombosis/inmunología , beta 2 Glicoproteína I/inmunología , Femenino , Humanos , EmbarazoRESUMEN
OBJECTIVES: MRI is an important tool for evaluating inflammation levels and assessing treatment response in patients with ankylosing spondylitis (AS). However, it is expensive and requires experienced physicians. The goal of this study was to identify a biomarker correlated with the MRI score. METHODS: A total of 558 spondyloarthritis (SpA) patients including 527 AS patients, 10 psoriasis (PsA) patients, and 21 non-radiographic SpA (nr-SpA) patients and 725 controls were enrolled for the studies. Plasma IgG galactosylation (IgG-Gal) level was measured by mass spectrometry. Clinical indexes such as Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and C-reactive protein (CRP) were measured in all AS patients. MRIs and X-rays were obtained from 65 AS patients who were followed up for 6 months. RESULTS: The IgG-Gal ratio was twice as high in the AS patients compared with the controls. It correlated with inflammation indices which is evaluated by MRI according to SPARCC. (Pearson coefficient/p value was 0.6/7E10-6). In addition, AS patients with a higher IgG-Gal ratio at baseline tended to show greater improvement in inflammation scores by MRI both in 3-month follow-up and 6-month follow-up. CONCLUSION: The IgG-Gal ratio was significantly increased in AS patients. In clinical care, it may be used as a potential biomarker for diagnosis in the future. Key Points ⢠IgG galactosylation level was abnormal in SpA patients. ⢠IgG galactosylation level was associated with MRI indices.
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Galactosa/sangre , Inmunoglobulina G/sangre , Imagen por Resonancia Magnética , Espondiloartritis/diagnóstico , Adulto , Biomarcadores/sangre , Proteína C-Reactiva/análisis , Estudios de Casos y Controles , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Espondiloartritis/sangre , Espondiloartritis/diagnóstico por imagen , Adulto JovenRESUMEN
Over the past 30 years, it has been firmly established that a wide spectrum of (autoimmune) diseases such as rheumatoid arthritis, Crohn's and lupus, but also other pathologies like alcoholic and non-alcoholic steatohepatitis (ASH and NASH) are driven by chronic inflammation and are hallmarked by a reduced level of serum IgG galactosylation. IgG (under)galactosylation is a promising biomarker to assess disease severity, and monitor and adjust therapy. However, this biomarker has not been implemented in routine clinical chemistry because of a complex analytical procedure that necessitates IgG purification, which is difficult to perform and validate at high throughput. We addressed this issue by using endo-ß-N-acetylglucosaminidase from Streptococcus pyogenes (endoS) to specifically release Fc N-glycans in whole serum. The entire assay can be completed in a few hours and only entails adding endoS and labeling the glycans with APTS. Glycans are then readily analyzed through capillary electrophoresis. We demonstrate in two independent patient cohorts that IgG undergalactosylation levels obtained with this assay correlate very well with scores calculated from PNGaseF-released glycans of purified antibodies. Our new assay allows to directly and specifically measure the degree of IgG galactosylation in serum through a fast and completely liquid phase protocol, without the requirement for antibody purification. This should help advancing this biomarker toward clinical implementation.
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Enfermedades Autoinmunes/sangre , Proteínas Bacterianas/metabolismo , Glicósido Hidrolasas/metabolismo , Inmunoglobulina G/sangre , Inmunoglobulina G/metabolismo , Adulto , Anciano , Análisis Químico de la Sangre/métodos , Enfermedad Crónica , Estudios de Cohortes , Electroforesis Capilar , Glicosilación , Semivida , Humanos , Inflamación/inmunología , Persona de Mediana Edad , Polisacáridos/metabolismo , Receptores de IgG/metabolismo , Adulto JovenRESUMEN
A facile online method coupling polymer monolithic microextraction (PMME) with mass spectrometry (MS) was developed for the detection of Immunoglobulin G (IgG) galactosylation glycopeptides. A peanut agglutinin-ß-cyclodextrin (PNA-ß-CD) functionalized poly(hydroxyethyl methylacrylate-ethyleneglycol dimethacrylate) monolith was designed via a click reaction. Thanking to the specificity of PNA-ß-CD for the targets, the material exhibited enhanced enrichment selectivity and extraction efficiency for IgG galactosylation glycopeptides. Under optimal conditions, the developed method gave a linear range of 0.005-5 pmol for IgG glycopeptides with the regression coefficient greater than 0.9990, and the detection limit of IgG galactosylation glycopeptides as low as 0.5 fmol was achieved. The PMME-MS method was applied to IgG galactosylation glycopeptides in real samples including human serum and acute myelogenous leukemia (AML) cell lysate. A series of unique IgG galactosylation glycopeptides were captured by the monolith in the complex samples, indicating satisfactory enrichment ability for IgG galactosylation glycopeptides. The quick and integrated online PMME-MS method exhibited high selectivity for IgG galactosylation, demonstrating its perspectives on the development and broad applications of MS in studying galactosylation proteins regulated biological processes.