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1.
J Autoimmun ; : 102469, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32362501

RESUMO

Previous studies have reported robust inflammatory cell infiltration, synthesis of IgG, B-cell clonal expansion, deposition of immune complexes and complement within cerebral cavernous malformation (CCM) lesions. B-cell depletion has also been shown to reduce the maturation of CCM in murine models. We hypothesize that antigen(s) within the lesional milieu perpetuate the pathogenetic immune responses in CCMs. This study aims to identify those putative antigen(s) using monoclonal antibodies (mAbs) derived from plasma cells found in surgically removed human CCM lesions. We produced human mAbs from laser capture micro-dissected plasma cells from four CCM patients, and also germline-reverted versions. CCM mAbs were assayed using immunofluorescence on central nervous system (CNS) tissues and immunocytochemistry on human primary cell lines. Antigen characterization was performed using a combination of confocal microscopy, immunoprecipitation and mass spectrometry. Affinity was determined by enzyme-linked immunosorbent assay, and specificity by multi-color confocal microscopy and quantitative co-localization. CCM mAbs bound CNS tissue, especially endothelial cells and astrocytes. Non-muscle myosin heavy chain IIA (NMMHCIIA), vimentin and tubulin are three cytoskeleton proteins that were commonly targeted. Selection of cytoskeleton proteins by plasma cells was supported by a high frequency of immunoglobulin variable region somatic hypermutations, high affinity and selectivity of mAbs in their affinity matured forms, and profoundly reduced affinity and selectivity in the germline reverted forms. Antibodies produced by plasma cells in CCM lesions commonly target cytoplasmic and cytoskeletal autoantigens including NMMHCIIA, vimentin and tubulin that are abundant in endothelial cells and astrocytes. Binding to, and selection on autoantigen(s) in the lesional milieu likely perpetuates the pathogenetic immune response in CCMs. Blocking this in situ autoimmune response may yield a novel treatment for CCM.

3.
Front Immunol ; 9: 1516, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30038611

RESUMO

Vimentin has been implicated in pulmonary sarcoidosis as a T-cell autoantigen, particularly in the context of HLA-DRB1*03, the Vα2.3/Vß22 T-cell receptor (TCR), and Löfgren's syndrome. As vimentin is a known antigenic target in B-cell-mediated autoimmunity, we investigated in situ humoral anti-vimentin responses in pulmonary sarcoidosis and their relationship with HLA-DRB1*03. Sarcoid and healthy control (HC) lung biopsies were analyzed by multi-color confocal microscopy for B-cells, T-cells, proliferation, and vimentin, and compared to tonsillectomy tissue. Bronchoalveolar lavage fluid (BALF) and serum from 48 sarcoidosis patients and 15 healthy volunteers were typed for HLA-DRB1*03 and titrated for antibodies to full-length vimentin, vimentin truncations, and total IgG and IgA by ELISA. Presence of extracellular vimentin in BALF was determined by mass spectrometry and T-cell populations measured by flow cytometry. Sarcoid lung samples, especially from HLA-DRB1*03+ patients, contained vimentin-rich tertiary lymphoid structures and corresponding BALF was highly enriched for both IgG and IgA anti-vimentin antibody (AVA) titers. Furthermore, sarcoidosis patient BALF AVA concentrations (expressed as arbitrary units per milligram of total immunoglobulin isotype) correlated with the percentage of CD4+ T-cells expressing the Vα2.3/Vß22 TCR. BALF antibody reactivity to the vimentin N-terminus was most prominent in HCs, whereas reactivity to the C-terminus (VimC-term) was enriched in the sarcoid lung. Specifically, HLA-DRB1*03+ patient BALF contained higher concentrations of anti-VimC-term antibodies than BALF from both HCs and HLA-DRB1*03- patients. Consistent with the lung as a site of AVA production, the concentration of AVAs in BALF was dramatically higher than in matched serum samples. Overall, there was a poor correlation between BALF and serum AVA concentrations. Together, these studies reveal the presence of linked in situ recognition of vimentin by both T- and B-cells in HLA-DRB1*03+ sarcoidosis patients, associated with a selective humoral immune response to the vimentin C-terminus.

4.
Arthritis Rheumatol ; 66(12): 3359-70, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25306868

RESUMO

OBJECTIVE: In lupus nephritis (LN), severe tubulointerstitial inflammation (TII) predicts progression to renal failure. Severe TII is associated with tertiary lymphoid neogenesis and in situ antigen-driven clonal B cell selection. The autoantigen(s) driving in situ B cell selection in TII are not known. This study was undertaken to identify the dominant driving autoantigen(s). METHODS: Single CD38+ or Ki-67+ B cells were laser captured from 7 biopsy specimens that were diagnostic for LN. Eighteen clonally expanded immunoglobulin heavy- and light-chain variable region pairs were cloned and expressed as monoclonal antibodies. Seven more antibodies were cloned from flow-sorted CD38+ cells from an eighth biopsy specimen. Antigen characterization was performed using a combination of confocal microscopy, enzyme-linked immunosorbent assay, screening protoarrays, immunoprecipitation, and mass spectrometry. Serum IgG titers to the dominant antigen in 48 LN and 35 non-nephritic lupus samples were determined using purified antigen-coated arrays. Autoantigen expression on normal and LN kidney was localized by immunohistochemistry and immunofluorescence. RESULTS: Eleven of 25 antibodies reacted with cytoplasmic structures, 4 reacted with nuclei, and none reacted with double-stranded DNA. Vimentin was the only autoantigen identified by both mass spectrometry and protoarray. Ten of the 11 anticytoplasmic TII antibodies directly bound vimentin. Vimentin was highly expressed by tubulointerstitial inflammatory cells, and the TII antibodies tested preferentially bound inflamed tubulointerstitium. Finally, high titers of serum antivimentin antibodies were associated with severe TII (P = 0.0001). CONCLUSION: Vimentin, an antigenic feature of inflammation, is a dominant autoantigen targeted in situ in LN TII. This adaptive autoimmune response likely feeds forward to worsen TII and renal damage.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Rim/imunologia , Nefrite Lúpica/imunologia , Nefrite Intersticial/imunologia , Vimentina/imunologia , Adolescente , Adulto , Biópsia , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Humanos , Imunidade Humoral , Imuno-Histoquímica , Imunoprecipitação , Inflamação , Rim/patologia , Nefrite Lúpica/patologia , Espectrometria de Massas , Microscopia Confocal , Nefrite Intersticial/patologia , Índice de Gravidade de Doença , Adulto Jovem
5.
Arthritis Rheum ; 63(12): 3818-23, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21953289

RESUMO

OBJECTIVE: To examine the hypothesis that the subset of rheumatoid arthritis (RA) characterized by antibodies to citrullinated α-enolase is mediated by Porphyromonas gingivalis enolase in the context of DR4 alleles. METHODS: Recombinant human α-enolase and P gingivalis enolase, either citrullinated or uncitrullinated, were used to immunize DR4-IE-transgenic mice and control mice (class II major histocompatibility complex-deficient [class II MHC(-/-)] and C57BL/6 wild-type mice). Arthritis was quantified by measurement of ankle swelling in the hind paws and histologic examination. Serum IgG reactivity with α-enolase and citrullinated α-enolase was assayed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Antibodies to peptide 1 of citrullinated α-enolase (CEP-1) and its arginine-bearing control peptide, REP-1, were also assessed by ELISA. RESULTS: Significant hind-ankle swelling (≥0.3 mm) occurred in DR4-IE-transgenic mice immunized with citrullinated human α-enolase (9 of 12 mice), uncitrullinated human α-enolase (9 of 12 mice), citrullinated P gingivalis enolase (6 of 6 mice), and uncitrullinated P gingivalis enolase (6 of 6 mice). Swelling peaked on day 24. None of the control groups developed arthritis. The arthritic joints showed synovial hyperplasia and erosions, but there was a paucity of leukocyte infiltration. Antibodies to human α-enolase, both citrullinated and unmodified, and to CEP-1 and REP-1 were detectable in all immunized mice except the class II MHC(-/-) control mice. CONCLUSION: This is the first animal model that links an immune response to P gingivalis enolase to an important subset of RA, defined by antibodies to citrullinated α-enolase in the context of DR4. The fact that arthritis and anti-CEP-1 antibodies were induced independent of citrullination of the immunizing antigen suggests that the unmodified form of α-enolase may be important in initiating the corresponding subset of human RA.


Assuntos
Artrite Experimental/imunologia , Autoimunidade/efeitos dos fármacos , Antígeno HLA-DR4/genética , Imunização , Fosfopiruvato Hidratase/farmacologia , Porphyromonas gingivalis/enzimologia , Animais , Artrite Experimental/sangue , Artrite Experimental/induzido quimicamente , Autoimunidade/imunologia , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosfopiruvato Hidratase/efeitos adversos , Fosfopiruvato Hidratase/imunologia , Proteínas Recombinantes/farmacologia
6.
FEBS Lett ; 585(23): 3681-8, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21704035

RESUMO

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by synovial inflammation and destruction of joints. Over 20 years ago, tumour necrosis factor alpha (TNFα) was identified as a key player in a cytokine network, whose multifunctional effects could account for both the inflammation and destruction in RA. The remarkable efficacy of TNF inhibitors in the treatment of RA has resulted in extensive research addressing the regulation of TNFα production responsible for this excessive production. The discovery of autoimmunity to citrullinated protein/peptide antigens (ACPA) has led the concept that ACPA may be the essential link between disease susceptibility factors and the production of TNFα, which ultimately accounts for the disease phenotype. In this review we will consider (1) the mechanisms of citrullination, both physiological and pathological, (2) how known genetic and environmental factors could drive this peculiar form of autoimmunity and (3) how the immune response could lead to excessive production of TNFα by the synovial cells and ultimately to the disease phenotype (Fig. 1).


Assuntos
Artrite Reumatoide/etiologia , Artrite Reumatoide/imunologia , Autoantígenos/imunologia , Citrulina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Autoanticorpos/imunologia , Humanos , Modelos Imunológicos
7.
Artigo em Inglês | MEDLINE | ID: mdl-21453269

RESUMO

Rheumatoid arthritis (RA), although widely considered to be the most commonly occurring autoimmune disease, has only truly been substantiated as a distinct autoimmune disease very recently. The lack of understanding of the specific autoimmune system/s at work in rheumatoid patients resulted in an absence of robust diagnostic tools and had meant that the rational choice for use and design of therapy was based on broad-spectrum immunosuppression. The revelation that the autoimmune response specific for patients with RA is to particular protein antigens bearing the post-translational modification 'citrulline' has therefore revolutionized diagnostics and has helped explain why patients carrying particular MHC alleles are predisposed to the disease. The last two decades have seen the characterization of citrullinated antigens targeted by both antibodies and T cells in rheumatoid patients. In more recent years, we have also witnessed the success of biological therapies in the treatment of RA that specifically target T cells and B cells. Ongoing mapping of antibody targets is increasing the percentage of patients who can be definitively diagnosed with, and prognosed to develop, RA. These advances have led to a great number of patents for citrullinated peptides that have been and may be, in the coming years, used in diagnostic test kits. More recently, characterization of T cell targets (citrullinated peptides) has resulted in the patenting of peptides that could be used in antigen specific therapy. This review focuses on the characterization of the autoimmune response to citrullinated protein targets in RA and how the community is translating this knowledge to improve diagnostics, prognostics and therapy.


Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Autoimunidade , Citrulina/imunologia , Peptídeos Cíclicos/imunologia , Processamento de Proteína Pós-Traducional , Animais , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/terapia , Biomarcadores/sangue , Humanos , Testes Imunológicos , Imunoterapia/métodos , Valor Preditivo dos Testes , Prognóstico , Kit de Reagentes para Diagnóstico , Resultado do Tratamento
8.
Expert Rev Clin Immunol ; 2(3): 365-75, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-20476908

RESUMO

In the last 10 years, the discovery that antibodies to citrullinated proteins are highly specific for rheumatoid arthritis has led to a model of pathogenesis that ties together the genetic and environmental risk factors for susceptibility and severity of disease. The authors propose that the chronic inflammation is driven by two phases of an immune response. The first phase is the priming of autoimmunity, which may occur many years before the onset of disease and is caused by environmental factors, such as smoking and infectious agents, in the context of disease susceptibility alleles. This may occur in sites outside the joint, such as the respiratory tract. The second phase is the induction of arthritis, which is associated with the generation of citrullinated proteins within the joint, which is then perpetuated as the erosive disease by a local chronic immune response. The identity of candidate synovial citrullinated antigen(s), whether fibrin, vimentin, alpha-enolase, collagen type II or others yet to be described, may be the key to the pathogenesis of the destructive disease characteristic of rheumatoid arthritis. There is emerging evidence that citrullination may already be modified by established therapy in rheumatoid arthritis, but more specific inhibitors of deimination may provide new agents for future treatments.

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