Streptococcal Endo-β--Acetylglucosaminidase Suppresses Antibody-Mediated Inflammation .

Endo-β--acetylglucosaminidase (EndoS) is a family 18 glycosyl hydrolase secreted by . Recombinant EndoS hydrolyzes the β-1,4-di--acetylchitobiose core of the N-linked complex type glycan on the asparagine 297 of the γ-chains of IgG. Here, we report that EndoS and IgG hydrolyzed by EndoS induced suppression of local immune complex (IC)-mediated arthritis.

Collagen Autoantibodies and Their Relationship to CCP Antibodies and Rheumatoid Factor in the Progression of Early Rheumatoid Arthritis.

Serum autoantibodies to cyclic citrullinated peptides (anti-CCP) and rheumatoid factor (RF) are important markers for diagnosis and prognosis of rheumatoid arthritis (RA), but their autoantigens are not cartilage-specific. Autoantibodies to joint-specific type II collagen (CII) also occur in RA, and monoclonal antibodies of similar specificity induce collagen antibody-induced arthritis in animals, but their role in RA is uncertain. We utilized an enzyme-linked immunosorbent assay (ELISA) with the CB10 peptide of CII to compare the frequency of autoantibodies with those of anti-CCP and RF in stored sera from a prospective study of 82 patients with early RA to examine the outcome, defined as remission ( = 23), persisting non-erosive arthritis ( = 27), or erosions ( = 32).

Dominant suppression of inflammation by glycan-hydrolyzed IgG.

A unique anti-inflammatory property of IgG, independent of antigen specificity, is described. IgG with modification of the heavy-chain glycan on asparagine 297 by the streptococcal enzyme endo-β-N-acetylglucosaminidase (EndoS) induced a dominant suppression of immune complex (IC)-mediated inflammation, such as arthritis, through destabilization of local ICs by fragment crystallizable-fragment crystallizable (Fc-Fc) interactions. Small amounts (250 µg) of EndoS-hydrolyzed IgG were sufficient to inhibit arthritis in mice and most effective during the formation of ICs in the target tissue.

Type II collagen-specific antibodies induce cartilage damage in mice independent of inflammation.

Objective: Murine collagen antibody-induced arthritis (CAIA), like collagen-induced arthritis, has clinical and immunopathologic features that parallel those in human rheumatoid arthritis (RA). This study was undertaken to examine the effects of autoantibodies to type II collagen (CII) on articular cartilage in the paws of mice, under conditions in which other factors that may influence joint pathology could be excluded.

Methods: Mice of 2 different strains, B10.

Chemical changes demonstrated in cartilage by synchrotron infrared microspectroscopy in an antibody-induced murine model of rheumatoid arthritis.

Collagen antibody-induced arthritis develops in mice following passive transfer of monoclonal antibodies (mAbs) to type II collagen (CII) and is attributed to effects of proinflammatory immune complexes, but transferred mAbs may react directly and damagingly with CII. To determine whether such mAbs cause cartilage damage in vivo in the absence of inflammation, mice lacking complement factor 5 that do not develop joint inflammation were injected intravenously with two arthritogenic mAbs to CII, M2139 and CIIC1. Paws were collected at day 3, decalcified, paraffin embedded, and 5-μm sections were examined using standard histology and synchrotron Fourier-transform infrared microspectroscopy (FTIRM).

An analysis of the cross-reactivity of autoantibodies to GAD65 and GAD67 in diabetes.

Background: Autoantibodies to GAD65 (anti-GAD65) are present in the sera of 70-80% of patients with type 1 diabetes (T1D), but antibodies to the structurally similar 67 kDa isoform GAD67 are rare. Antibodies to GAD67 may represent a cross-reactive population of anti-GAD65, but this has not been formally tested.

Methodology/principal Findings: In this study we examined the frequency, levels and affinity of anti-GAD67 in diabetes sera that contained anti-GAD65, and compared the specificity of GAD65 and GAD67 reactivity.

Autoantibody heritability in thyroiditis: IgG subclass contributions.

Using a simple screening technique called regression of offspring on mid-parent (ROMP) to examine the role of IgG subclasses in affected and unaffected siblings of children and adolescents with autoimmune thyroid disease and their parents, both total-restricted and subclass-restricted autoantibodies to thyroglobulin (Tg) were assayed quantitatively for each of the IgG subclasses. There was a significant correlation of anti-Tg titer of probands with parental titers in thyrotoxicosis (TT), (R(2) = 0.569, p = 0.

Specific antibody protection of the extracellular cartilage matrix against collagen antibody-induced damage.

Objective: The type II collagen (CII)-specific monoclonal antibodies (mAb) M2139 and CIIC1 induce arthritis in vivo and degrade bovine cartilage explants in vitro, whereas mAb CIIF4 is nonarthritogenic and prevents arthritis development when given in combination with M2139 and CIIC1. To determine the nature of the protective capacity of CIIF4 antibody, we examined the effects of adding CIIF4 to cartilage explants cultured in vitro with M2139 and CIIC1.

Methods: Bovine cartilage explants were cultured in the presence of M2139 and CIIC1, with or without CIIF4.

Structural determinants of GAD antigenicity.

Our aim was to ascertain structural determinants of autoantigenicity based on the model of the diabetes autoantigen glutamic acid decarboxylase 65 kDa isoform (GAD65) in comparison with that of the non-autoantigenic isoform GAD67. This difference exists despite the two isoforms having the same fold and high sequence identity. Autoantibodies to GAD65 precede the development of type 1 diabetes and are clinical markers of this and certain neural autoimmune diseases.

The role of collagen antibodies in mediating arthritis.

This review examines evidence that rheumatoid arthritis (RA) depends on autoimmunity to articular collagen, and mechanisms whereby autoantibodies to type II collagen contribute to disease development. Three major autoantigenic reactants have been identified in RA; the corresponding autoantibodies are rheumatoid factor (RF), antibodies to citrullinated peptide antigens (ACPA), citrullinated peptides (anti-CCP), and anti-type II collagen (anti-CII). Both RF and ACPA are well-validated and predictive markers of severe erosive RA, but cannot be linked to pathogenesis.

GAD65 as a prototypic autoantigen.

The repertoire of known autoantigens is limited to a very small proportion of all human proteins, and the reason why only some proteins become autoantigens is unclear, but is likely associated with structural features. The 65kDa isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in type I diabetes, and in various neurological diseases, whereas the closely related isoform, GAD67, is rarely antigenic. Conformational epitopes of GAD65 have been mapped using human monoclonal antibodies to GAD65 and GAD mutated by GAD65/67 sequence exchanges or point mutations, but these studies have been limited by a lack of structural information.

A tribute to an outstanding immunologist - Ian Reay Mackay.

The 11th Australasian Autoimmunity Workshop was held in Melbourne, Australia from July 6-8, 2007 organized by the Monash University Autoimmunity Network. The workshops, founded by the late Kevin Lafferty, are a chance for Australasians interested in research into autoimmune disease to present and discuss their work. This workshop also was a chance to acknowledge Ian Mackay, a pioneer clinician-scientist who has made major contributions to our understanding of autoimmune diseases.

The lack of anti-idiotypic antibodies, not the presence of the corresponding autoantibodies to glutamate decarboxylase, defines type 1 diabetes.

Autoantibodies to glutamate decarboxylase 65 (GAD65Ab) are commonly believed to be a major characteristic for type 1 diabetes (T1D). We investigated the presence of GAD65Ab in healthy individuals (n = 238) and first-degree relatives (FDRs) of T1D patients (n = 27) who tested negative for GAD65Ab in conventional RIAs. Sera were applied to affinity columns coated with GAD65-specific mAbs to absorb anti-idiotypic antibodies (anti-Ids).

COOH-terminal clustering of autoantibody and T-cell determinants on the structure of GAD65 provide insights into the molecular basis of autoreactivity.

Objective: To gain structural insights into the autoantigenic properties of GAD65 in type 1 diabetes, we analyzed experimental epitope mapping data in the context of the recently determined crystal structures of GAD65 and GAD67, to allow "molecular positioning" of epitope sites for B- and T-cell reactivity.

Research Design And Methods: Data were assembled from analysis of reported effects of mutagenesis of GAD65 on its reactivity with a panel of 11 human monoclonal antibodies (mAbs), supplemented by use of recombinant Fab to cross-inhibit reactivity with GAD65 by radioimmunoprecipitation of the same mAbs.

Results: The COOH-terminal region on GAD65 was the major autoantigenic site.

Arthritogenic antibodies specific for a major type II collagen triple-helical epitope bind and destabilize cartilage independent of inflammation.

Objective: To investigate the significance and pathogenic potential of a highly conserved major type II collagen triple-helical epitope-specific antibody (U1; amino acids 494-504) in vivo and in vitro in patients with early rheumatoid arthritis (RA) and in experimental animal models of collagen-induced arthritis (CIA).

Methods: U1-specific antibodies in sera from patients with early RA (with or without joint erosions) were analyzed. Disease progression in the CIA models in mice and rats with anti-U1 antibodies was compared.

Peptide mimotopes selected with HIV-1-blocking monoclonal antibodies against CCR5 represent motifs specific for HIV-1 entry.

CCR5 is a chemokine receptor that mediates entry of human immunodeficiency virus-1 (HIV-1). Two monoclonal antibodies (mAbs) that block HIV-1 entry, 3A9 and 5C7, were used to select peptide mimotopes of sequences on CCR5 from phage displayed peptide libraries. The selected mimotofpes comprised motifs at the N-terminus and on the first and third extracellular loops (ECL1 and ECL3) of CCR5.

GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop.

Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5'-phosphate-dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form.

Molecular characterization of a disease associated conformational epitope on GAD65 recognised by a human monoclonal antibody b96.11.

Autoantibodies to the 65kDa isoform of glutamate decarboxylase (GAD65) are associated with type I diabetes and recognise highly conformational epitope(s) that remain to be defined. The human recombinant Fab from mAb b96.11 inhibits binding of most GAD65 antibody positive sera from patients and its epitope has previously been localized to the middle region of GAD65.

Characterisation of an autoreactive conformational epitope on GAD65 recognised by the human monoclonal antibody b78 using a combination of phage display, in vitro mutagenesis and molecular modelling.

Autoantibodies to the diabetes autoantigen, the 65kDa isoform of glutamic acid decarboxylase (GAD65), react with conformational epitopes defined according to linear sequences but not according to structural information, or contact sites with the antibody paratope. To ascertain such information for an exemplary human monoclonal antibody (mAb) to GAD65, b78, we combined antibody screening of phage-displayed peptide libraries, alanine mutagenesis of selected motifs, homology modelling of the PLP and C-terminal regions of GAD65, and molecular dynamics to examine for structural effects of mutagenesis. By phage display, mAb b78 selected phagotopes containing acidic residues (D, E), hydrophobic residues (Y, F or W) and LRS that localised to a possible surface-exposed conformational epitope on the combined homology model.

Amino acids critical for binding of autoantibody to an immunodominant conformational epitope of the pyruvate dehydrogenase complex subunit E2: identification by phage display and site-directed mutagenesis.

The E2 subunit of the mitochondrial multienzyme pyruvate dehydrogenase complex (PDC-E2) is the major autoantigen in the liver disease, primary biliary cirrhosis (PBC). An epitope region which has been localized to amino acids 91-227 is believed to include the residue K173 to which is attached the lipoyl cofactor. We investigated structural features of this epitope region by screening random peptide phage-displayed libraries and identified prevalent phagotopes that contained likely contact amino acids in separate regions of the linear sequence, H132M133, and F178, V180.

Destructive effects of murine arthritogenic antibodies to type II collagen on cartilage explants in vitro.

Certain monoclonal antibodies (mAbs) to type II collagen (CII) induce arthritis in vivo after passive transfer and have adverse effects on chondrocyte cultures and inhibit self assembly of collagen fibrils in vitro. We have examined whether such mAbs have detrimental effects on pre-existing cartilage. Bovine cartilage explants were cultured over 21 days in the presence of two arthritogenic mAbs to CII (CIIC1 or M2139), a non-arthritogenic mAb to CII (CIIF4) or a control mAb (GAD6).

Heritability of levels of autoantibodies using the method of plotting regression of offspring on midparent (ROMP).

The genetic control of the levels of autoantibodies has rarely been examined. We examined the heritability of autoantibodies to glutamic acid decarboxylase (GAD65) in type 1 diabetes, and to thyroglobulin (Tg) in chronic lymphocytic thyroiditis and thyrotoxicosis, using regression of offspring on midparent (ROMP) methods. Levels of autoantibodies in patients and their parents were significantly correlated in thyrotoxicosis (R2 = 0.

Arthritogenic anti-type II collagen antibodies are pathogenic for cartilage-derived chondrocytes independent of inflammatory cells.

Objective: Some monoclonal antibodies (mAb) to type II collagen (CII) are arthritogenic upon passive transfer to mice. We undertook this study to investigate whether such mAb are pathogenic in the absence of mediators of inflammation.

Methods: The arthritogenic mAb CIIC1 and M2139, and the nonarthritogenic mAb CIIF4, each reactive with a distinct and well-defined conformational epitope on CII, were compared with control mAb GAD6.

Requirement of multiple phage displayed peptide libraries for optimal mapping of a conformational antibody epitope on CCR5.

In the absence of information from crystallography, conformational epitopes can often be discerned by antibody screening of phage displayed random peptide libraries. However the context in which the peptide is displayed, and the number of copies displayed in the library, can influence results and interpretations. Here, the monoclonal antibodies 3A9 specific for the transmembrane chemokine receptor CCR5, and CII-C1 specific for type II collagen, were used to screen multiple phage-displayed peptide libraries in which peptides were displayed in either the pIII or pVIII coat proteins.