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1.
J Immunol ; 208(11): 2497-2507, 2022 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-35589126

RESUMEN

Rare immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a life-threatening disease resulting from a severe autoantibody-mediated ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motifs, member 13) deficiency. Acute iTTP episodes are medical emergencies, but when treated appropriately &gt;95% of patients survive. However, at least half of survivors will eventually experience a relapse. How remission of an initial episode is achieved and factors contributing to reemergence of anti-ADAMTS13 Abs and a relapsing course are poorly understood. In acquired hemophilia and systemic lupus erythematosus, anti-idiotypic Abs counteracting and neutralizing pathogenic autoantibodies contribute to remission. We selected and amplified the splenic anti-idiotypic IgG<sub>1</sub> Fab κ/λ repertoire of two relapsing iTTP patients on previously generated monoclonal inhibitory anti-ADAMTS13 Fabs by phage display to explore whether anti-idiotypic Abs have a role in iTTP. We obtained 27 single anti-idiotypic Fab clones, half of which had unique sequences, although both patients shared four H chain V region genes (V<sub>H</sub>1-69*01, V<sub>H</sub>3-15*01, V<sub>H</sub>3-23*01, and V<sub>H</sub>3-49*03). Anti-idiotypic Fab pools of both patients fully neutralized the inhibitor capacity of the monoclonal anti-ADAMTS13 Abs used for their selection. Preincubation of plasma samples of 22 unrelated iTTP patients stratified according to functional ADAMTS13 inhibitor titers (&gt;2 Bethesda units/ml, or 1-2 Bethesda units/ml), with anti-idiotypic Fab pools neutralized functional ADAMTS13 inhibitors and restored ADAMTS13 activity in 18-45% of those cases. Taken together, we present evidence for the presence of an anti-idiotypic immune response in iTTP patients. The interindividual generalizability of this response is limited despite relatively uniform pathogenic anti-ADAMTS13 Abs recognizing a dominant epitope in the ADAMTS13 spacer domain.


Asunto(s)
Púrpura Trombocitopénica Trombótica , Humanos , Proteína ADAMTS13 , Autoanticuerpos , Epítopos , Recurrencia , Anticuerpos Antiidiotipos
2.
J Immunol ; 197(9): 3669-3679, 2016 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-27698012

RESUMEN

Premature atherosclerosis and thrombotic complications are major causes of morbidity and mortality in patients with systemic lupus erythematosus (SLE). However, the high incidence of these complications cannot be explained by traditional risk factors alone, suggesting direct effects of an activated immune system on hemostasis. The unexpected nucleotide sequence homology between SLE patient-derived autoantibodies against complement C1q (Fab anti-C1q) and von Willebrand factor (VWF) led us to investigate a potential interaction between the complement and hemostatic systems on the level of initiating molecules. VWF was found to bind to surface-bound C1q under static conditions. The binding could specifically be inhibited by Fab anti-C1q and C1q-derived peptides. Under shear stress the C1q-VWF interaction was enhanced, resembling the binding of VWF to collagen I. Additionally, we could show that C1q-VWF complexes induced platelet rolling and firm adhesion. Furthermore, we observed VWF binding to C1q-positive apoptotic microparticles and cholesterol crystals, as well as increased VWF deposition in C1q-positive glomeruli of SLE patients compared with control nephropathy. We show, to our knowledge for the first time, binding of VWF to C1q and thus a direct interaction between starter molecules of hemostasis and the classical pathway of complement. This direct interaction might contribute to the pathogenic mechanisms in complement-mediated, inflammatory diseases.


Asunto(s)
Aterosclerosis/inmunología , Plaquetas/inmunología , Complemento C1q/metabolismo , Riñón/metabolismo , Lupus Eritematoso Sistémico/complicaciones , Trombosis/inmunología , Factor de von Willebrand/metabolismo , Complejo Antígeno-Anticuerpo/metabolismo , Apoptosis , Autoanticuerpos/inmunología , Autoanticuerpos/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Hemostasis , Humanos , Riñón/patología , Lupus Eritematoso Sistémico/inmunología , Activación Plaquetaria , Unión Proteica
3.
Blood ; 124(23): 3469-79, 2014 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-25261198

RESUMEN

Acquired thrombotic thrombocytopenic purpura (TTP) is the consequence of a severe ADAMTS13 deficiency resulting from autoantibodies inhibiting ADAMTS13 or accelerating its clearance. Despite the success of plasma exchange the risk of relapse is high. From 2 patients (A and B), splenectomized for recurrent episodes of acquired TTP, the splenic B-cell response against ADAMTS13 was characterized through generation of human monoclonal anti-ADAMTS13 autoantibodies (mAbs) by cloning an immunoglobulin G (IgG)4κ- and IgG4λ-Fab library using phage display technology and by Epstein-Barr virus transformation of switched memory B cells (CD19+/CD27+/IgG+). Sequence analysis of the anti-ADAMTS13 IgGs of both patients revealed that the VH gene use was limited in our patients to VH1-3 (55%), VH1-69 (17%), VH3-30 (7%), and VH4-28 (21%) and contained 8 unique and thus far not reported heavy-chain complementarity determining region 3 motifs, of which 4 were shared by the 2 patients. The discovery of several highly similar anti-ADAMTS13 autoantibodies in 2 unrelated TTP patients suggests that the autoimmune response is antigen driven, because the probability that such similar immunoglobulin rearrangements happen by chance is very low (< 10(-9)).


Asunto(s)
Proteínas ADAM/inmunología , Autoanticuerpos/química , Autoanticuerpos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Púrpura Trombocitopénica Trombótica/inmunología , Bazo/inmunología , Proteína ADAMTS13 , Adulto , Complejo Antígeno-Anticuerpo/química , Autoinmunidad/inmunología , Niño , Femenino , Humanos , Unión Proteica , Púrpura Trombocitopénica Trombótica/metabolismo , Púrpura Trombocitopénica Trombótica/cirugía , Inducción de Remisión , Bazo/metabolismo , Esplenectomía
4.
Arthritis Rheum ; 64(11): 3706-14, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22740328

RESUMEN

OBJECTIVE: Autoantibodies against C1q strongly correlate with the occurrence of severe nephritis in patients with systemic lupus erythematosus (SLE). We undertook this study to determine whether identification of the C1q epitope(s) recognized by these autoantibodies might lead to a better diagnostic assay and help elucidate the putative role of C1q and anti-C1q in SLE. METHODS: SLE patient-derived anti-C1q Fab were used in a microarray-based peptide scan to identify the peptide sequence recognized by anti-C1q. Anti-C1q Fab binding to the target peptide was further analyzed using real-time interaction measurements (surface plasmon resonance) and peptide-based enzyme-linked immunosorbent assays (ELISAs). RESULTS: A peptide scan of the collagen-like region of C1q identified 2 regions, 1 on the A chain and 1 on the B chain, that were the targets of the anti-C1q Fab. Binding was confirmed by surface plasmon resonance and showed nanomolar affinity. The A chain-derived peptide could specifically be detected in a peptide-based ELISA by SLE patient sera. Competition experiments suggested that this peptide represented one of the major linear epitopes of C1q that is the target of anti-C1q in SLE. Serum antibodies from most SLE patients but not from healthy individuals specifically bound to this epitope. Binding to the peptide correlated with binding of the same sera to native C1q but was found to be more sensitive for the detection of lupus nephritis. CONCLUSION: We identified a major linear epitope of C1q that is the target of anti-C1q in SLE. The ELISA using this peptide was more specific and more sensitive than a conventional anti-C1q assay for the detection of active nephritis in SLE patients.


Asunto(s)
Complemento C1q/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/inmunología , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/inmunología , Autoanticuerpos/inmunología , Complemento C1q/química , Epítopos/química , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Nefritis Lúpica/diagnóstico , Nefritis Lúpica/inmunología , Masculino , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína , Resonancia por Plasmón de Superficie
5.
J Biol Chem ; 285(51): 39914-21, 2010 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-20959443

RESUMEN

At the earliest stage of activation, human polymorphonuclear neutrophils release vesicles derived directly from the cell surface. These vesicles, called ectosomes (PMN-Ect), expose phosphatidylserine in the outer membrane leaflet. They inhibit the inflammatory response of human monocyte-derived macrophages and dendritic cells to zymosan A (ZymA) and LPS and induce TGF-ß1 release, suggesting a reprogramming toward a tolerogenic phenotype. The receptors and signaling pathways involved have not yet been defined. Here, we demonstrate that PMN-Ect interfered with ZymA activation of macrophages via inhibition of NFκB p65 phosphorylation and NFκB translocation. The MerTK (Mer receptor tyrosine kinase) and PI3K/Akt pathways played a key role in this immunomodulatory effect as shown using specific MerTK-blocking antibodies and PI3K inhibitors LY294002 and wortmannin. As a result, PMN-Ect reduced the transcription of many proinflammatory genes in ZymA-activated macrophages. In sum, PMN-Ect interacted with the macrophages by activation of the MerTK pathway responsible for down-modulation of the proinflammatory signals generated by ZymA.


Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Activación de Macrófagos/fisiología , Macrófagos/metabolismo , Neutrófilos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Androstadienos/farmacología , Cromonas/farmacología , Humanos , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor de Transcripción ReIA/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Wortmanina , Zimosan/farmacología , Tirosina Quinasa c-Mer
6.
Nephrol Dial Transplant ; 26(4): 1220-8, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20841491

RESUMEN

BACKGROUND: In systemic lupus erythematosus patients, a strong association between the occurrence of antibodies against complement C1q (anti-C1q) and lupus nephritis can be observed. However, the predictive value of anti-C1q titres for a renal flare remains to be determined. Increasing titres of anti-C1q before the occurrence of clinical apparent nephritis might not only serve as a clinical parameter but also indicate a direct pathogenic mechanism of anti-C1q. METHODS: The aim of this study was to analyse the occurrence of anti-C1q before the onset of experimental lupus nephritis in MRL/MpJ +/+ mice and to correlate anti-C1q titres with the type and severity of glomerulonephritis (GN) developing at advanced age. RESULTS: As judged by a number of morphological and immunological analyses, GN in MRL/MpJ +/+ mice resembled human lupus nephritis and occurred in variable degrees of severity. We also observed an abundant and early presence of anti-C1q. However, anti-C1q neither correlated with overall survival nor with any histological marker of severity of GN. CONCLUSIONS: The absence of a correlation between the presence of anti-C1q and the occurrence of experimental lupus nephritis contradicts the hypothesis that anti-C1q are pathogenic. However, different pathogenic mechanisms of experimental lupus nephritis and human proliferative lupus nephritis cannot be excluded.


Asunto(s)
Autoanticuerpos/sangre , Biomarcadores/sangre , Complemento C1q/inmunología , Glomerulonefritis/inmunología , Nefritis Lúpica/inmunología , Animales , Anticuerpos Antinucleares/sangre , Femenino , Técnica del Anticuerpo Fluorescente , Glomerulonefritis/sangre , Glomerulonefritis/diagnóstico , Humanos , Técnicas para Inmunoenzimas , Nefritis Lúpica/sangre , Nefritis Lúpica/diagnóstico , Ratones , Ratones Endogámicos BALB C , Tasa de Supervivencia
7.
J Immunol ; 183(12): 8225-31, 2009 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-20007586

RESUMEN

Autoantibodies against complement C1q (anti-C1q Abs) were shown to strongly correlate with the occurrence of severe nephritis in patients with systemic lupus erythematosus (SLE), suggesting a potential pathogenic role by interfering with the complement cascade. To analyze the humoral immune response against C1q at the molecular level, we screened a bone marrow-derived IgGkappa/IgGlambda Fab phage display library from a SLE patient with high anti-C1q Ab titer against purified human C1q. Six Fabs that exhibited strong binding to C1q in ELISA were isolated. The anti-C1q Fabs recognized neoepitopes that were only exposed on bound C1q and not present on soluble C1q mapping to different regions of the collagen-like region of C1q. Analysis of the genes encoding the variable H and L chains of the IgG-derived anti-C1q Fab revealed that all the variable H and L chain regions were highly mutated, with nucleotide and amino acid homologies to the closest germline in the range of 71-97% (average 85 +/- 4) and 72-92% (average 88 +/- 6), respectively. In addition, the variable region of the Fabs exhibited high replacement to silent ratios. The six anti-C1q Fabs were shown to be of high affinity, with a K(d) ranging from of 8.4 x 10(-8) M to 1.4 x 10(-7) M, comparable to an antiviral immune response. Our data underlines the notion that the development of anti-C1q Abs in SLE is the consequence of an Ag-driven, affinity-matured immune response. Those anti-C1q Fabs are unique tools to address how complement C1q is implicated in the pathogenesis of SLE.


Asunto(s)
Autoanticuerpos/biosíntesis , Autoantígenos/fisiología , Complemento C1q/fisiología , Lupus Eritematoso Sistémico/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/aislamiento & purificación , Proteínas Reguladoras de la Apoptosis/metabolismo , Autoanticuerpos/aislamiento & purificación , Autoanticuerpos/metabolismo , Autoantígenos/inmunología , Autoantígenos/metabolismo , Complemento C1q/inmunología , Complemento C1q/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fab de Inmunoglobulinas/metabolismo , Datos de Secuencia Molecular
8.
J Immunol ; 183(5): 3512-21, 2009 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-19648280

RESUMEN

Autoantibodies against complement C1q (anti-C1q) are frequently found in patients with systemic lupus erythematosus (SLE). They strongly correlate with the occurrence of severe lupus nephritis, suggesting a pathogenic role in SLE. Because anti-C1q are known to recognize a neoepitope on bound C1q, but not on fluid-phase C1q, the aim of this study was to clarify the origin of anti-C1q by determining the mechanism that renders C1q antigenic. We investigated anti-C1q from serum and purified total IgG of patients with SLE and hypocomplementemic urticarial vasculitis as well as two monoclonal human anti-C1q Fab from a SLE patient generated by phage display. Binding characteristics, such as their ability to recognize C1q bound on different classes of Igs, on immune complexes, and on cells undergoing apoptosis, were analyzed. Interestingly, anti-C1q did not bind to C1q bound on Igs or immune complexes. Neither did we observe specific binding of anti-C1q to C1q bound on late apoptotic/necrotic cells when compared with binding in the absence of C1q. However, as shown by FACS analysis and confocal microscopy, anti-C1q specifically targeted C1q bound on early apoptotic cells. Anti-C1q were found to specifically target C1q bound on cells undergoing apoptosis. Our observations suggest that early apoptotic cells are a major target of the autoimmune response in SLE and provide a direct link between human SLE, apoptosis, and C1q.


Asunto(s)
Apoptosis/inmunología , Autoanticuerpos/fisiología , Sitios de Unión de Anticuerpos , Complemento C1q/inmunología , Complemento C1q/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Complejo Antígeno-Anticuerpo/metabolismo , Autoanticuerpos/biosíntesis , Autoanticuerpos/metabolismo , Línea Celular Tumoral , Epítopos/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Células Jurkat , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Endogámicos BALB C , Vasculitis/inmunología , Vasculitis/patología
9.
Am J Kidney Dis ; 51(4): 624-9, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18371538

RESUMEN

BACKGROUND: Antinucleosome autoantibodies were previously described to be a marker of active lupus nephritis. However, the true prevalence of antinucleosome antibodies at the time of active proliferative lupus nephritis has not been well established. Therefore, the aim of this study is to define the prevalence and diagnostic value of autoantibodies against nucleosomes as a marker for active proliferative lupus nephritis. STUDY DESIGN: Prospective multicenter diagnostic test study. SETTING & PARTICIPANTS: 35 adult patients with systemic lupus erythematosus (SLE) at the time of the renal biopsy showing active class III or IV lupus nephritis compared with 59 control patients with SLE. INDEX TEST: Levels of antinucleosome antibodies and anti-double-stranded DNA (anti-dsDNA) antibodies. REFERENCE TEST: Kidney biopsy findings of class III or IV lupus nephritis at the time of sampling in a study population versus clinically inactive or no nephritis in a control population. RESULTS: Increased concentrations of antinucleosome antibodies were found in 31 of 35 patients (89%) with active proliferative lupus nephritis compared with 47 of 59 control patients (80%) with SLE. No significant difference between the 2 groups with regard to number of positive patients (P = 0.2) or antibody concentrations (P = 0.2) could be found. The area under the receiver operating characteristic curve as a marker of the accuracy of the test in discriminating between proliferative lupus nephritis and inactive/no nephritis in patients with SLE was 0.581 (95% confidence interval, 0.47 to 0.70; P = 0.2). Increased concentrations of anti-dsDNA antibodies were found in 33 of 35 patients (94.3%) with active proliferative lupus nephritis compared with 49 of 58 control patients (84.5%) with SLE (P = 0.2). In patients with proliferative lupus nephritis, significantly higher titers of anti-dsDNA antibodies were detected compared with control patients with SLE (P < 0.001). The area under the receiver operating characteristic curve in discriminating between proliferative lupus nephritis and inactive/no nephritis in patients with SLE was 0.710 (95% confidence interval, 0.60 to 0.82; P < 0.001). CONCLUSIONS: Antinucleosome antibodies have a high prevalence in patients with severe lupus nephritis. However, our data suggest that determining antinucleosome antibodies is of limited help in the distinction of patients with active proliferative lupus nephritis from patients with SLE without active renal disease.


Asunto(s)
Autoanticuerpos/sangre , Nefritis Lúpica/sangre , Nucleosomas/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , ADN/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos
10.
PLoS One ; 3(10): e3314, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19390657

RESUMEN

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrP(C)) to the infectious scrapie form (PrP(Sc)). However, the mechanism that exogenous PrP(Sc) infects cells and where pathologic conversion of PrP(C) to the PrP(Sc) form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrP(C) to the pathologic PrP(Sc) form in N2a cells exposed to strain RML PrP(Sc) infected brain homogenates, suggesting that a critical determinant of PrP(C) conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrP(Sc) infects cells by lipid raft dependent, macropinocytosis.


Asunto(s)
Microdominios de Membrana/metabolismo , Pinocitosis , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Dermatán Sulfato/metabolismo , Relación Dosis-Respuesta a Droga , Genes Reporteros , Heparina/metabolismo , Integrasas/metabolismo , Microdominios de Membrana/patología , Ratones , Datos de Secuencia Molecular , Proteínas PrPC/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Recombinación Genética , Factores de Tiempo
11.
J Biol Chem ; 282(10): 7465-71, 2007 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-17218310

RESUMEN

Direct interaction between endogenous cellular prion protein (PrP(C)) and misfolded, disease-associated (PrP(Sc)) conformers is a key event in prion propagation, which precedes templated conversion of PrP(C) into nascent PrP(Sc) and prion infectivity. Although almost none of the molecular details of this pivotal process are understood, the persistence of individual prion strains suggests that assembly of the prion replicative complex is mechanistically precise. To systematically map defined regions of PrP(C) sequence that bind tightly to PrP(Sc), we have generated a comprehensive panel of over 45 motif-grafted antibodies containing overlapping peptide grafts collectively spanning PrP residues 19-231. Grafted antibody binding experiments, performed under stringent conditions, clearly identified only three distinct and independent high affinity PrP(Sc) recognition motifs. The first of these binding motifs lies at the very N-terminal region of the mature PrP molecule within PrP-(23-33); the second motif lies within PrP-(98-110); and the third is contained within PrP-(136-158). Mutational analyses of these PrP(Sc)-binding regions revealed that reactivity of the 23-33 and 98-110 segments are largely dependent upon the presence of multiple positively charged amino acid residues. These studies yield new insight into critical peptidic components composing one side of the prion replicative interface.


Asunto(s)
Proteínas PrPC/química , Proteínas PrPSc/química , Mapeo de Interacción de Proteínas/métodos , Sitios de Unión , Humanos , Mutación Puntual , Conformación Proteica
12.
Biochem Biophys Res Commun ; 349(2): 838-45, 2006 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-16949042

RESUMEN

In K/BxN mice, anti-glucose-6-phosphate isomerase (G6PI) IgG antibodies (Abs) cause joint-specific inflammation and destruction. Anti-G6PI Abs are also present in humans with inflammatory arthritis, especially among patients with rheumatoid arthritis (RA). A contributing factor to the induction of such autoantibodies may be upregulated expression of the corresponding antigen G6PI in affected tissues and/or increased levels of G6PI in the circulation. To determine G6PI levels and the presence of free G6PI and/or G6PI-containing immune complexes in sera and synovial fluids (SF) of patients with different arthritides, serum and SF obtained concomitantly from 91 clinically well-defined arthritis patients were assessed in a blinded manner for G6PI enzymatic assay and for G6PI protein concentration by ELISA. Sera and SF from patients with immune-based inflammatory arthritis contained significantly higher levels of G6PI enzymatic activity compared to sera or SF from patients with non-immune-based inflammatory arthritis or healthy controls. In addition, significantly higher levels of total G6PI protein concentration (including both enzymatically active and inactive forms) were present in sera of RA patients vs. those with other immune-based or non-immune-based inflammatory arthritis.G6PI in sera and SF were present both as G6PI-containing immune complexes and as free G6PI, with the majority of free G6PI existing as tetramers with lesser amounts of dimers and monomers. Levels of G6PI enzymatic activity in the sera of most immune-based inflammatory arthritis patients are elevated and may reflect ongoing inflammation and cell destruction. The high serum levels of enzymatically inactive forms of G6PI in RA relative to those in other arthritic diseases are partially due to G6PI-containing immune complexes, a portion of which also contains C1q. Overall, our study supports the notion that elevated G6PI levels present in patients with immune-based inflammatory arthritis may contribute to elevated levels of anti-G6PI Abs and G6PI/anti-G6PI immune complexes. This, in turn, may trigger production of proinflammatory cytokines and perpetuate the inflammatory process.


Asunto(s)
Artritis/sangre , Glucosa-6-Fosfato Isomerasa/sangre , Inflamación/metabolismo , Articulaciones/patología , Líquido Sinovial/metabolismo , Artritis/metabolismo , Estudios de Cohortes , Complemento C1q/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Glucosa-6-Fosfato Isomerasa/metabolismo , Humanos , Sistema Inmunológico , Masculino , Proteínas Recombinantes/química
13.
Xenotransplantation ; 9(4): 252-9, 2002 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12060461

RESUMEN

Galalpha1-3Gal (anti-Gal), a polyclonal so-called natural antibody (Ab), is present in large amounts in human serum not only as IgG-, but also as IgM- and IgA-isotypes. It has gained a particular interest in the context of xenotransplantation, because the endothelial pig cells express the terminal Galalpha1-3Gal determinant on several adhesion molecules. Little is known of it's function and direct examination of the structure of the Ig genes responsible for coding anti-Gal is lacking. We used the technique of isoelectric focussing (IEF)/affinity immunoblotting for direct analysis of the clonal distribution and spectrotype analysis of IgM- and IgG anti-Gal. By single cell analysis of magnetic bead and fluorescent-activated cell sorter (FACS) isolated mature anti-Gal bearing human B cells from whole blood we analyzed the VH gene families involved in anti-Gal production. Oligoclonal and individually distinct IgG banding patterns were found with isoelectric points between 4 and 9. IgM spectrotypes revealed to be more uniform with a polyclonal banding pattern of more than 12 bands at a pH between 4.7 and 7. IgG- and IgM-banding patterns over a period of 6 months remained unchanged. Single cell polymerase chain reaction (PCR), with all family specific primers, revealed the use of the VH2f gene family for the IgG2 isoptype. The differences found in the spectrotype banding patterns of IgG and IgM could be explained by the suggestion that anti-Gal IgM were produced by the use of unmutated germline genes and the possibility of the absence of somatic mutations. The greater clonal heterogeneity in the IgG population could be explained by somatic hypermutations during the switch from IgM to IgG. The use of this VH2f gene family, which is also involved in the generation of Abs against bacterial pathogens, could mean that this is a predominant region used for the generation of such natural occurring antibodies.


Asunto(s)
Anticuerpos Antiidiotipos/sangre , Disacáridos/inmunología , Secuencia de Bases , Donantes de Sangre , Anergia Clonal/inmunología , Cartilla de ADN , ADN Complementario , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Humanos , Inmunoglobulina G/sangre , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulina M/sangre , Región Variable de Inmunoglobulina/genética , Reacción en Cadena de la Polimerasa/métodos
14.
Arthritis Rheum ; 50(2): 395-9, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14872481

RESUMEN

OBJECTIVE: In the K/BxN mouse model, autoantibodies against glucose-6-phosphate isomerase (GPI) cause arthritis. The relevance of this model for human disease remains a subject of controversy. We set out to determine whether GPI autoantibodies occur in patients with rheumatoid arthritis (RA) and, if so, at what stage of the RA. METHODS: Using an enzyme-linked immunosorbent assay, serum from 131 RA patients and 28 healthy controls was tested for autoantibodies against recombinant human GPI. Patients were grouped according to disease duration and presence of rheumatoid nodules, rheumatoid vasculitis, and Felty's syndrome, which are extraarticular complications of RA. RESULTS: Elevated levels of autoantibodies against GPI were present in 5% of patients with uncomplicated RA and 4% of controls. In RA complicated by extraarticular manifestations, anti-GPI antibodies were observed in 18% of patients with rheumatoid nodules, 45% of patients with rheumatoid vasculitis, and 92% of patients with Felty's syndrome. CONCLUSION: In patients with RA, autoantibodies to GPI are associated with the occurrence of extraarticular complications.


Asunto(s)
Artritis Reumatoide/inmunología , Autoanticuerpos/sangre , Síndrome de Felty/inmunología , Glucosa-6-Fosfato Isomerasa/inmunología , Nódulo Reumatoide/inmunología , Vasculitis/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/complicaciones , Artritis Reumatoide/patología , Ensayo de Inmunoadsorción Enzimática , Síndrome de Felty/etiología , Síndrome de Felty/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nódulo Reumatoide/etiología , Nódulo Reumatoide/patología , Vasculitis/etiología , Vasculitis/patología
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