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Anti-fibrillarin autoantibodies are useful for the diagnosis and prognosis of systemic sclerosis (SSc). Anti-fibrillarin produces a clumpy nucleolar pattern in indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA). Here we develop and validate a reliable cell-based anti-fibrillarin assay (Fibrillarin/CBA) for use in clinical diagnostic laboratories. A TransMembrane Signal was fused to the human fibrillarin gene (TMS-fibrillarin). HEp-2 cells overexpressing transgenic TMS-fibrillarin at the cytoplasmic membrane were used as IFA substrate in the Fibrillarin/CBA. Sixty-two serum samples with nucleolar pattern in the HEp-2 IFA (41 clumpy; 21 homogeneous/punctate) were tested for anti-fibrillarin using Fibrillarin/CBA, immunoprecipitation (IP), line-blot and ELISA. In addition, samples from 106 SSc-patients were evaluated with Fibrillarin/CBA and the results were correlated with disease phenotypes. Thirty-eight of 41 samples with the clumpy nucleolar pattern (92.7%) were positive in the Fibrillarin/CBA, while all 21 samples with other nucleolar patterns were negative. Fibrillarin/CBA results agreed 100% with IP results. Among the 38 Fibrillarin/CBA-positive samples, only 15 (39.5%) and 11 (29%) were positive for anti-fibrillarin in line-blot and ELISA, respectively. Higher frequency of diffuse cutaneous SSc (dcSSc) phenotype (72.7% vs 36.8%; p=0.022), cardiac involvement (36.4% vs 6.5%; p=0.001) and scleroderma renal crisis (18.2% vs 3.3% p = 0.028) was observed in SSc patients with positive compared to negative Fibrillarin/CBA result. Performance of Fibrillarin/CBA in the detection of anti-fibrillarin autoantibodies was comparable to the gold standard IP. Positive Fibrillarin/CBA results correlated with disease phenotypes known to be associated with anti-fibrillarin autoantibodies, underscoring the clinical validation of this novel assay.

Agregação de enzimas em corpos grandes e sem membrana é uma característica comum entre as células eucarióticas. Nos últimos cinco anos, várias publicações têm descrito a capacidade de Citidina Trifosphato Sintase (CTPS) e Inosina Monofosfato Desidorgenase 2 (IMPDH2), enzimas que são críticas nas vias de biossíntese dos nucleotídeos citidina e guanina, respectivamente, de formar agregados chamados de anéis e bastões (RR) ou cytoophidia ("cobras celulares"). Curiosamente, uma fração considerável de pacientes com hepatite C (HCV) tratados com IFN-? e ribavirina, um inibidor da IMPDH2, desenvolvem autoanticorpos contra as estruturas de RR. Neste trabalho, estudamos aspectos da formação de estruturas RR in vitro e in vivo, bem como a evolução temporal da produção dos autoanticorpos anti-RR. Ao estudar o processo de agregação da IMPDH2 e CTPS em RR, na presença dos respectivos inibidores, demonstramos a formação de estruturas RR independentes compostas por IMPDH2 ou CTPS em duas linhagens celulares de duas espécies de mamíferos. Também relatamos que ambas as enzimas podem interagir na formação de estruturas RR mistas, como um mosaico de IMPDH2 e CTPS. Ao estudar o comportamento temporal das estruturas RR, ricas em IMPDH2, em células vivas in vitro, observamos que estas se desmontam na presença de um anticorpo contra IMPDH2, um componente molecular do RR. Também demonstramos que as drogas ribavirina e MPA, ambas inibidoras da IMPDH2, produzem estruturas RR ricas em IMPDH2 in vivo em PBMC de pacientes HCV e lúpicos, respectivamente, mas apenas os pacientes HCV apresentam os autoanticorpos anti-RR. Os autoanticorpos que geram o padrão de imunofluorescência indireta RR reconhecem predominantemente a enzima IMPDH2 como alvo. Ao analisar o comportamento temporal da resposta autoimune anti-RR/IMPDH2, observamos que tais anticorpos foram induzidos após 3 a 6 meses de tratamento com IFN-?+ribavirina, com um pico em torno do décimo segundo mês, e diminuição ou desaparecimento após a conclusão do tratamento. O cenário acima descrito caracteriza um modelo humano de quebra de tolerância imunológica e representa uma oportunidade única para estudar vários aspectos do desenvolvimento de uma resposta autoimune. Enzyme aggregation into non-membrane bound large bodies is a common feature among eukaryotic cells. In the last five years, many reports have described the ability of Cytidine Triphosphate Synthase (CTPS) and Inosine Monophosphate Dehydrogenase (IMPDH2), enzymes that are critical in the cytidine and guanine nucleotide biosynthesis pathways, respectively, to form aggregates named rods and rings (RR) or cytoophidia (?cellular snakes?). Interestingly, a considerable fraction of hepatitis C (HCV) patients treated with IFN-? plus ribavirin, an inhibitor of IMPDH2, develops autoantibodies against the RR structures. We study aspects of the formation of RR structures in vitro and in vivo, as well as explore the temporal evolution of anti-RR autoantibodies production. By studying the process of aggregation of IMPDH2 and CTPS into RR structures in the presence of their respective inhibitors, we demonstrated the independent in vitro formation of IMPDH2-based and CTPS-based RR structures in cell lines of two mammalian species, and reported that both enzymes can interact in the formation of mixed RR structures as a mosaic of IMPDH2 and CTPS aggregates. By studying the temporal behavior of RR IMPDH2-based structures in living cells in vitro, we show the disassembly of RR structures in the presence of an antibody targeting IMPDH2, a molecular constituent of RR. We also demonstrate that the IMPDH2-inhibitory drugs ribavirin and MPA generate IMPDH2-rich RR structures in vivo in PBMC from HCV and SLE patients, respectively, but only ribavirin-treated HCV patients present anti-RR autoantibodies. The autoantibodies that elicit the anti-RR indirect immunofluorescence pattern recognized predominantly the IMPDH2 enzyme. By analyzing the temporal behavior of the anti-RR/IMPDH2 autoimmune response, we report that such autoantibodies were induced at 3 to 6 months of IFN-?+ribavirin treatment, reached a plateau around the twelfth month, and decreased/disappeared after treatment conclusion. The above described scenario characterizes a human model of immune tolerance breakdown and represents a unique opportunity to study various aspects of the autoimmune response development in humans. Dados abertos - Sucupira - Teses e dissertações (2013 a 2016)