RESUMO
BACKGROUND: Control of Lyme disease is attributed predominantly to innate and adaptive T-helper 1 cell (TH1) immune responses, whereas the role of T-helper 17 cell (TH17) responses is less clear. Here we characterized these inflammatory responses in patients with erythema migrans (EM) or Lyme arthritis (LA) to elucidate their role early and late in the infection. METHODS: Levels of 21 cytokines and chemokines, representative of innate, TH1, and TH17 immune responses, were assessed by Luminex in acute and convalescent sera from 91 EM patients, in serum and synovial fluid from 141 LA patients, and in serum from 57 healthy subjects. Antibodies to Borrelia burgdorferi or autoantigens were measured by enzyme-linked immunosorbent assay. RESULTS: Compared with healthy subjects, EM patients had significantly higher levels of innate, TH1, and TH17-associated mediators (P ≤ .05) in serum. In these patients, the levels of inflammatory mediators, particularly TH17-associated cytokines, correlated directly with B. burgdorferi immunoglobulin G antibodies (P ≤ .02), suggesting a beneficial role for these responses in control of early infection. Late in the disease, in patients with LA, innate and TH1-associated mediators were often >10-fold higher in synovial fluid than serum. In contrast, the levels of TH17-associated mediators were more variable, but correlated strongly with autoantibodies to endothelial cell growth factor, matrix metalloproteinase 10, and apolipoprotein B-100 in joints of patients with antibiotic-refractory LA, implying a shift in TH17 responses toward an autoimmune phenotype. CONCLUSIONS: Patients with Lyme disease often develop pronounced TH17 immune responses that may help control early infection. However, late in the disease, excessive TH17 responses may be disadvantageous by contributing to autoimmune responses associated with antibiotic-refractory LA.
Assuntos
Anticorpos Antibacterianos/imunologia , Autoanticorpos/imunologia , Borrelia burgdorferi/imunologia , Citocinas/metabolismo , Doença de Lyme/imunologia , Doença de Lyme/metabolismo , Células Th17/metabolismo , Imunidade Adaptativa , Anticorpos Antibacterianos/sangue , Artrite/etiologia , Artrite/patologia , Autoantígenos/imunologia , Autoimunidade , Biomarcadores , Citocinas/sangue , Feminino , Glossite Migratória Benigna/etiologia , Glossite Migratória Benigna/metabolismo , Humanos , Imunidade Inata , Mediadores da Inflamação/sangue , Mediadores da Inflamação/metabolismo , Doença de Lyme/complicações , Doença de Lyme/microbiologia , Masculino , Células Th17/imunologiaRESUMO
Niemann-Pick Types A and B (NPA/B) diseases are autosomal recessive lysosomal storage disorders caused by the deficient activity of acid sphingomyelinase (ASM) because of the mutations in the SMPD1 gene. Here, we provide a comprehensive updated review of already reported and newly identified SMPD1 variants. Among them, 185 have been found in NPA/B patients. Disease-causing variants are equally distributed along the SMPD1 gene; most of them are missense (65.4%) or frameshift (19%) mutations. The most frequently reported mutation worldwide is the p.R610del, clearly associated with an attenuated NP disease type B phenotype. The available information about the impact of 52 SMPD1 variants on ASM mRNA and/or enzymatic activity has been collected and whenever possible, phenotype/genotype correlations were established. In addition, we created a locus-specific database easily accessible at http://www.inpdr.org/genes that catalogs the 417 SMPD1 variants reported to date and provides data on their in silico predicted effects on ASM protein function or mRNA splicing. The information reviewed in this article, providing new insights into the genotype/phenotype correlation, is extremely valuable to facilitate diagnosis and genetic counseling of families affected by NPA/B.
Assuntos
Bases de Dados Genéticas , Mutação , Doença de Niemann-Pick Tipo A/genética , Doença de Niemann-Pick Tipo B/genética , RNA Mensageiro/genética , Esfingomielina Fosfodiesterase/genética , Éxons , Expressão Gênica , Genes Recessivos , Estudos de Associação Genética , Genótipo , Humanos , Íntrons , Doença de Niemann-Pick Tipo A/diagnóstico , Doença de Niemann-Pick Tipo A/patologia , Doença de Niemann-Pick Tipo B/diagnóstico , Doença de Niemann-Pick Tipo B/patologia , Fases de Leitura Aberta , Fenótipo , Splicing de RNARESUMO
Infection-induced autoimmunity is thought to be a contributing factor in antibiotic-refractory Lyme arthritis, but studies of autoimmunity have been hindered by difficulty in identifying relevant autoantigens. We developed a novel approach that begins with the identification of T cell epitopes in synovial tissue using tandem mass spectrometry. Herein, we identified an immunogenic HLA-DR-presented peptide (T cell epitope) derived from the source protein matrix metalloproteinase-10 (MMP-10) from the synovium of a patient with antibiotic-refractory arthritis. This finding provided a bridge for the identification of autoantibody responses to MMP-10, the "first autoimmune hit" in a subgroup of patients with erythema migrans, the initial skin lesion of the infection. Months later, after priming of the immune response to MMP-10 in early infection, a subset of patients with antibiotic-responsive or antibiotic-refractory arthritis had MMP-10 autoantibodies, but only patients with antibiotic-refractory arthritis had both T and B cell responses to the protein, providing evidence for a "second autoimmune hit". Further support for a biologically relevant autoimmune event was observed by the positive correlation of anti-MMP-10 autoantibodies with distinct synovial pathology. This experience demonstrates the power of new, discovery-based methods to identify relevant autoimmune responses in chronic inflammatory forms of arthritis.
Assuntos
Linfócitos B/imunologia , Doença de Lyme/etiologia , Doença de Lyme/patologia , Metaloproteinase 10 da Matriz/imunologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Linfócitos T/imunologia , Antibacterianos/uso terapêutico , Apresentação de Antígeno , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoimunidade , Linfócitos B/metabolismo , Borrelia burgdorferi/imunologia , Resistência a Medicamentos , Epitopos de Linfócito T/imunologia , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Imuno-Histoquímica , Doença de Lyme/tratamento farmacológico , Doença de Lyme/metabolismo , Metaloproteinase 10 da Matriz/química , Metaloproteinase 10 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/imunologia , Peptídeos/imunologia , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Linfócitos T/metabolismoRESUMO
Glycogen storage disease type II is a lysosomal storage disorder due to mutations of the GAA gene, which causes lysosomal alpha-glucosidase deficiency. Clinically, glycogen storage disease type II has been classified in infantile and late-onset forms. Most late-onset patients share the leaky splicing mutation c.-32-13T>G. To date, the mechanism by which the c.-32-13T>G mutation affects the GAA mRNA splicing is not fully known. In this study, we demonstrate that the c.-32-13T>G mutation abrogates the binding of the splicing factor U2AF65 to the polypyrimidine tract of exon 2 and that several splicing factors affect exon 2 inclusion, although the only factor capable of acting in the c.-32-13 T>G context is the SR protein family member, SRSF4 (SRp75). Most importantly, a preliminary screening using small molecules described to be able to affect splicing profiles, showed that resveratrol treatment resulted in a significant increase of normal spliced GAA mRNA, GAA protein content and activity in cells transfected with a mutant minigene and in fibroblasts from patients carrying the c-32-13T>G mutation. In conclusion, this work provides an in-depth functional characterization of the c.-32-13T>G mutation and, most importantly, an in vitro proof of principle for the use of small molecules to rescue normal splicing of c.-32-13T>G mutant alleles.
Assuntos
Doença de Depósito de Glicogênio Tipo II/genética , Mutação , Splicing de RNA , alfa-Glucosidases/genética , Células Cultivadas , Éxons , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Processamento de Serina-Arginina , Fator de Processamento U2AF , alfa-Glucosidases/metabolismoRESUMO
To discover novel autoantigens associated with Lyme arthritis (LA), we identified T-cell epitopes presented in vivo by human leukocyte antigen (HLA)-DR molecules in patients' inflamed synovial tissue or joint fluid and tested each epitope for autoreactivity. Using this approach, we identified the highly expressed human protein, apolipoprotein B-100 (apoB-100), as a target of T- and B-cell responses in a subgroup of LA patients. Additionally, the joint fluid of these patients had markedly elevated levels of apoB-100 protein, which may contribute to its autoantigenicity. In patients with antibiotic-refractory LA, the magnitude of apoB-100 antibody responses correlated with increased numbers of plasma cells in synovial tissue, greater numbers and activation of endothelial cells, and more synovial fibroblast proliferation. Thus, a subset of LA patients have high levels of apoB-100 in their joints and autoreactive T- and B-cell responses to the protein, which likely contributes to pathogenic autoimmunity in patients with antibiotic-refractory LA.
Assuntos
Apolipoproteína B-100/imunologia , Autoantígenos/imunologia , Doença de Lyme/imunologia , Linfócitos B/imunologia , Borrelia burgdorferi , Estudos de Coortes , Feminino , Humanos , Doença de Lyme/epidemiologia , Pessoa de Meia-Idade , Linfócitos T/imunologiaRESUMO
In this study, autoantibody responses to annexin A2 were found in 11-15% of 278 patients with Lyme disease, including in those with erythema migrans (EM), an early sign of the illness, and in those with antibiotic-responsive or antibiotic-refractory Lyme arthritis (LA), a late disease manifestation. In contrast, robust T cell reactivity to annexin A2 peptides was found only in patients with responsive or refractory LA. In LA patients, annexin A2 protein levels, which were higher in the refractory group, correlated with annexin A2 antibody levels in sera and synovial fluid. In addition, in patients with antibiotic-refractory LA who had anti-annexin A2 antibodies, synovial tissue had intense staining for annexin A2 protein, greater synovial fibroblast proliferation and more tissue fibrosis. Thus, a subset of LA patients had T and B cell responses to annexin A2, and in the refractory group, annexin A2 autoantibodies were associated with specific pathologic findings.
Assuntos
Anexina A2/imunologia , Autoanticorpos/imunologia , Linfócitos B/imunologia , Proliferação de Células , Fibroblastos/imunologia , Doença de Lyme/imunologia , Membrana Sinovial/patologia , Linfócitos T/imunologia , Antibacterianos/uso terapêutico , Autoimunidade , Borrelia burgdorferi , Estudos de Casos e Controles , Farmacorresistência Bacteriana , Ensaio de Imunoadsorção Enzimática , Eritema Migrans Crônico/tratamento farmacológico , Eritema Migrans Crônico/imunologia , Humanos , Imunoglobulina G/imunologia , Imuno-Histoquímica , Doença de Lyme/tratamento farmacológico , Doença de Lyme/patologia , Membrana Sinovial/citologiaAssuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Alelos , Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Deformidades Congênitas dos Membros/diagnóstico , Deformidades Congênitas dos Membros/genética , Sulfotransferases/genética , Sinostose/diagnóstico , Sinostose/genética , Criança , Mapeamento Cromossômico , Hibridização Genômica Comparativa , Genótipo , Humanos , Masculino , FenótipoRESUMO
OBJECTIVE: Obliterative microvascular lesions are found in the synovial tissue of ~50% of patients with post-antibiotic Lyme arthritis (LA) and correlate with autoantibodies to certain vascular antigens. In this study, we identified lymphocytes with cytotoxic potential that may also mediate this feature of synovial pathology. METHODS: The cytotoxic potential of lymphocytes and their T cell receptor (TCR) Vß gene usage were determined using samples of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with antibiotic-responsive or post-antibiotic LA. Cell phenotypes were analyzed using flow cytometry and intracellular cytokine staining. Immunohistochemistry was performed on post-antibiotic synovial tissue samples. RESULTS: In SFMC and PBMC samples, the percentages of CD8+ T cells and double-negative T cells (primarily γδ T cells) were greater among 22 patients with post-antibiotic LA than in 14 patients with antibiotic-responsive LA. Moreover, CD8+ T cells and γδ T cells often expressed cytotoxic mediators, granzyme A/granzyme B, and perforin. The same 3 TCR Vß segments were over-represented in both CD4+ T cells and CD8+ T cells in SFMC samples from post-antibiotic LA patients. In synovial tissue samples from 3 patients with post-antibiotic LA, CD8+ T cells intermixed with CD4+ T cells were seen around blood vessels, and 2 patients with microvascular damage had autoantibodies to vascular-associated antigens. One of these 2 patients, the one in whom cytotoxicity appeared to be active, had complement (C5b-9) deposition on obliterated vessels. Very few natural killer cells or γδ T cells were seen. CONCLUSION: We propose that CD8+ T cell-mediated cytotoxicity, CD4+ T cell help, autoantibodies to vascular antigens, and complement deposition may each have a role in microvasculature damage in post-antibiotic LA.
Assuntos
Leucócitos Mononucleares , Doença de Lyme , Humanos , Líquido Sinovial , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Doença de Lyme/tratamento farmacológico , Receptores de Antígenos de Linfócitos T alfa-beta , Antibacterianos/uso terapêutico , AutoanticorposRESUMO
BACKGROUNDAutoimmune diseases often have strong genetic associations with specific HLA-DR alleles. The synovial lesion in chronic inflammatory forms of arthritis shows marked upregulation of HLA-DR molecules, including in postinfectious Lyme arthritis (LA). However, the identity of HLA-DR-presented peptides, and therefore the reasons for these associations, has frequently remained elusive.METHODSUsing immunopeptidomics to detect HLA-DR-presented peptides from synovial tissue, we identified T cell epitopes from 3 extracellular matrix (ECM) proteins in patients with postinfectious LA, identified potential Borreliella burgdorferi-mimic (Bb-mimic) epitopes, and characterized T and B cell responses to these peptides or proteins.RESULTSOf 24 postinfectious LA patients, 58% had CD4+ T cell responses to at least 1 epitope of 3 ECM proteins, fibronectin-1, laminin B2, and/or collagen Vα1, and 17% of 52 such patients had antibody responses to at least 1 of these proteins. Patients with autoreactive T cell responses had significantly increased frequencies of HLA-DRB1*04 or -DRB1*1501 alleles and more prolonged arthritis. When tetramer reagents were loaded with ECM or corresponding Bb-mimic peptides, binding was only with the autoreactive T cells. A high percentage of ECM-autoreactive CD4+ T cells in synovial fluid were T-bet-expressing Th1 cells, a small percentage were RoRγt-expressing Th17 cells, and a minimal percentage were FoxP3-expressing Tregs.CONCLUSIONAutoreactive, proinflammatory CD4+ T cells and autoantibodies develop to ECM proteins in a subgroup of postinfectious LA patients who have specific HLA-DR alleles. Rather than the traditional molecular mimicry model, we propose that epitope spreading provides the best explanation for this example of infection-induced autoimmunity.FUNDINGSupported by National Institute of Allergy and Infectious Diseases R01-AI101175, R01-AI144365, and F32-AI125764; National Institute of Arthritis and Musculoskeletal and Skin Diseases K01-AR062098 and T32-AR007258; NIH grants P41-GM104603, R24-GM134210, S10-RR020946, S10-OD010724, S10-OD021651, and S10-OD021728; and the G. Harold and Leila Y. Mathers Foundation, the Eshe Fund, and the Lyme Disease and Arthritis Research Fund at Massachusetts General Hospital.
Assuntos
Artrite , Borrelia burgdorferi , Doença de Lyme , Humanos , Autoimunidade , Proteínas da Matriz Extracelular , Cadeias HLA-DRB1 , Peptídeos , Epitopos de Linfócito TRESUMO
OBJECTIVE: We previously identified HLA-DR-presented epitopes from a 27-kd protein of Prevotella copri (Pc) obtained from peripheral blood mononuclear cells (PBMCs) from 1 rheumatoid arthritis (RA) patient. Herein, we sought to identify other HLA-DR-presented Pc peptides and source proteins in PBMCs from additional patients to better understand Pc immune responses and RA disease pathogenesis. METHODS: Using tandem mass spectrometry, we searched for HLA-DR-presented Pc peptides in PBMCs from RA and Lyme arthritis (LA) patients. The identified peptides and source proteins were tested for reactivity in RA patients, those with other arthritides, and the general population. These results were assessed for correlation with clinical findings. RESULTS: Including Pc-p27, we identified 5 HLA-DR-presented Pc peptides, each derived from a different Pc protein, in 3 of 4 RA patients, but none in 2 LA patients. When tested in our RA cohort, 14 of 19 patients (74%) had T cell responses, and 47 of 89 patients (53%) had IgG or IgA responses to ≥1 of the 5 Pc peptides or proteins, most commonly IgA reactivity with Pc-p27. Additionally, 74% of RA patients with IgA antibodies to ≥1 Pc protein had anti-citrullinated protein antibodies (ACPAs) compared with 49% of patients who lacked IgA Pc antibody responses (P = 0.05), and IgA Pc antibody levels correlated with ACPA values. CONCLUSION: The majority of the RA patients had Pc immune responses. The correlation of IgA Pc antibody responses, particularly to Pc-p27, with ACPA supports the hypothesis that specific microbial antigens in the mucosa have a role in shaping or amplifying immune responses in RA joints.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Epitopos/imunologia , Antígenos HLA-DR/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Prevotella , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
Nucleophosmin (NPM) is a protein that contributes to several cell functions. Depending on the context, it can act as an oncogene or tumor suppressor. No data are available on NPM expression in thyroid cells. In this work, we analyzed both NPM mRNA and protein levels in a series of human thyroid tumor tissues and cell lines. By using immunohistochemistry, NPM overexpression was detected in papillary, follicular, undifferentiated thyroid cancer, and also in follicular benign adenomas, indicating it as an early event during thyroid tumorigenesis. In contrast, various levels of NPM mRNA levels as detected by quantitative RT-PCR were observed in tumor tissues, suggesting a dissociation between protein and transcript expression. The same behavior was observed in the normal thyroid FRTL5 cell lines. In these cells, a positive correlation between NPM protein levels, but not mRNA, and proliferation state was detected. By using thyroid tumor cell lines, we demonstrated that such a post-mRNA regulation may depend on NPM binding to p-Akt, whose levels were found to be increased in the tumor cells, in parallel with reduction of PTEN. In conclusion, our present data demonstrate for the first time that nucleophosmin is overexpressed in thyroid tumors, as an early event of thyroid tumorigenesis. It seems as a result of a dysregulation occurring at protein and not transcriptional level related to an increase of p-Akt levels of transformed thyrocytes.
Assuntos
Biomarcadores Tumorais/biossíntese , Transformação Celular Neoplásica/metabolismo , Proteínas Nucleares/biossíntese , Neoplasias da Glândula Tireoide/metabolismo , Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Humanos , Proteínas Nucleares/genética , Nucleofosmina , Proteínas Proto-Oncogênicas c-akt/metabolismo , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Nucleophosmin 1 (NPM1) is an abundant phosphoprotein mainly located in the nucleolus but also shuttling between the nucleus and cytoplasm. NPM1 has been proposed to be involved in synthesis and processing of ribosomal RNA, regulation of chromatin structure and transport of rRNA and ribosomal proteins. NPM1 gene is considered to be implicated in human cancer as it is a frequent target of genetic alterations, primarily in haematopoietic neoplasms. We describe a case of a therapy-responder acute myeloid leukaemia (AML) patient bearing two novel NPM1 mutations. Cells' transfection studies indicate that the presence of one of these mutations is associated to an abnormal nucleolar structure, suggesting that NPM1 may contribute to the control of nucleolar integrity.
Assuntos
Leucemia Mieloide Aguda/genética , Mutação , Proteínas Nucleares/genética , Nucléolo Celular/genética , Éxons , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Pessoa de Meia-Idade , Nucleofosmina , TransfecçãoRESUMO
PURPOSE: PAX6 mutations cause aniridia as well as other various congenital eye abnormalities. Aniridia can be due to both point mutations and chromosomal deletions/rearrangements. Therefore, a complete search for PAX6 gene alterations in aniridia subjects requires a technically complex approach involving the comprehension of fluorescence in situ hybridization (FISH) analysis. In the present study, an Italian casistic of aniridia patients has been investigated and a quantitative polymerase chain reaction (PCR) assay to detect PAX6 gene deletions was set up. METHODS: Twenty-one aniridia patients were screened for point mutations (missense, nonsense, splicing-affecting, and short insertion/deletion) by using single-stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (dHPLC). To reveal deletions not detectable by SSCP or dHPLC, a quantitative PCR approach was set up for the PAX6 structural gene and for regions 5' and 3' to it at the level of WT1 and ELP4, respectively. RESULTS: Point mutations were found in 7 out of 21 patients. Three out of twenty-one patients showed deletions at the level of the PAX6 structural gene. In addition, two familial cases showed an undamaged PAX6 gene but a deletion in the region 3' to it at level of the ELP4 gene. In one of the families, the presence of the deletion has been confirmed by linkage analysis of polymorphic markers. CONCLUSIONS: In our casistic, a significant fraction of familial aniridia patients appears to be caused by a 3' deletion to PAX6, suggesting that evaluation of this alteration should be included in routine procedures of aniridia patients analysis. The quantitative PCR assay described here represents a simple approach to accomplish this task.
Assuntos
Aniridia/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Deleção de Sequência , Bases de Dados de Ácidos Nucleicos , Feminino , Ligação Genética , Genoma Humano/genética , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Fator de Transcrição PAX6 , Linhagem , Mutação Puntual/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: Prevotella copri, an intestinal microbe, may overexpand in stool samples from patients with new-onset rheumatoid arthritis (RA), but it is not yet clear whether the organism has immune relevance in RA pathogenesis. METHODS: HLA-DR-presented peptides (T cell epitopes) from P copri were sought directly in the patients' synovial tissue or peripheral blood mononuclear cell (PBMC) samples using tandem mass spectrometry. The antigenicity of peptides or their source proteins was examined in samples from the RA patients or comparison groups. T cell reactivity was determined by enzyme-linked immunospot assay; antibody responses were measured by enzyme-linked immunosorbent assay, and cytokine/chemokine determinations were made by bead-based assays. Serum and synovial fluid samples were examined for 16S ribosomal DNA for P copri using nested polymerase chain reaction analysis. RESULTS: In PBMCs, we identified an HLA-DR-presented peptide from a 27-kd protein of P copri (Pc-p27), which stimulated Th1 responses in 42% of patients with new-onset RA. In both new-onset RA patients and chronic RA patients, 1 subgroup had IgA antibody responses to either Pc-p27 or the whole organism, which correlated with Th17 cytokine responses and frequent anti-citrullinated protein antibodies (ACPAs). The other subgroup had IgG P copri antibodies, which were associated with Prevotella DNA in synovial fluid, P copri-specific Th1 responses, and less frequent ACPAs. In contrast, P copri antibody responses were rarely found in patients with other rheumatic diseases or in healthy controls. CONCLUSION: Subgroups of RA patients have differential IgG or IgA immune reactivity with P copri, which appears to be specific for this disease. These observations provide evidence that P copri is immune-relevant in RA pathogenesis.
Assuntos
Artrite Reumatoide/imunologia , Proteínas de Bactérias/imunologia , Epitopos de Linfócito T/imunologia , Microbioma Gastrointestinal/imunologia , Leucócitos Mononucleares/imunologia , Prevotella/imunologia , Membrana Sinovial/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/microbiologia , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Ensaio de Imunoadsorção Enzimática , ELISPOT , Feminino , Microbioma Gastrointestinal/genética , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Compostos Organotiofosforados/imunologia , Peptídeos/imunologia , Reação em Cadeia da Polimerase , Prevotella/genética , Líquido Sinovial/metabolismo , Espectrometria de Massas em Tandem , Células Th1/imunologia , Adulto JovemRESUMO
In rheumatoid arthritis (RA), immunological triggers at mucosal sites, such as the gut microbiota, may promote autoimmunity that affects joints. Here, we used discovery-based proteomics to detect HLA-DR-presented peptides in synovia or peripheral blood mononuclear cells and identified 2 autoantigens, N-acetylglucosamine-6-sulfatase (GNS) and filamin A (FLNA), as targets of T and B cell responses in 52% and 56% of RA patients, respectively. Both GNS and FLNA were highly expressed in synovia. GNS appeared to be citrullinated, and GNS antibody values correlated with anti-citrullinated protein antibody (ACPA) levels. FLNA did not show the same results. The HLA-DR-presented GNS peptide has marked sequence homology with epitopes from sulfatase proteins of the Prevotella sp. and Parabacteroides sp., whereas the HLA-DR-presented FLNA peptide has homology with epitopes from proteins of the Prevotella sp. and Butyricimonas sp., another gut commensal. Patients with T cell reactivity with each self-peptide also had responses to the corresponding microbial peptides, and the levels were directly correlated. Furthermore, HLA-DR molecules encoded by shared-epitope (SE) alleles were predicted to bind these self- and microbial peptides strongly, and these responses were more common in RA patients with SE alleles. Thus, sequence homology between T cell epitopes of 2 self-proteins and a related order of gut microbes may provide a link between mucosal and joint immunity in patients with RA.
Assuntos
Artrite Reumatoide/imunologia , Autoantígenos/imunologia , Autoimunidade/imunologia , Microbioma Gastrointestinal , Articulações/imunologia , Leucócitos Mononucleares/imunologia , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Filaminas/imunologia , Antígenos HLA-DR/imunologia , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Articulações/patologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Sulfatases/imunologia , Membrana Sinovial/metabolismoRESUMO
Pax6 controls eye, pancreas and brain morphogenesis. In humans, heterozygous PAX6 mutations cause aniridia and various other congenital eye abnormalities. Most frequent PAX6 missense mutations are located in the paired domain (PD), while very few missense mutations have been identified in the homeodomain (HD). In the present report, we describe a molecular analysis of the human PAX6 R242T missense mutation, which is located in the second helix of the HD. It was identified in a male child with partial aniridia in the left eye, presenting as a pseudo-coloboma. Gel-retardation assays revealed that the mutant HD binds DNA as well as the wild-type HD. In addition, the mutation does not modify the DNA-binding properties of the PD. Cell transfection assays indicated that the steady-state levels of the full length mutant protein are higher than those of the wild-type one. In cotransfection assays a PAX6 responsive promoter is activated to a higher extent by the mutant protein than by the wild-type protein. In vitro limited proteolysis assays indicated that the presence of the mutation reduces the sensitivity to trypsin digestion. Thus, we suggest that the R242T human phenotype could be due to abnormal increase of PAX6 protein, in keeping with the reported sensitivity of the eye phenotype to increased PAX6 dosage.
Assuntos
Aniridia/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Mutação de Sentido Incorreto , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Animais , Aniridia/metabolismo , Linhagem Celular , DNA/metabolismo , Proteínas do Olho/metabolismo , Dosagem de Genes/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Organogênese/genética , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Proteínas Repressoras/metabolismoRESUMO
Inhibitors of histone deacetylases (HDACs) activate the sodium iodide symporter (NIS) expression in thyroid tumor cells. In this study, mechanisms accounting for these effects were investigated. Various human thyroid tumor cell lines (ARO, BCPAP, FRO, TPC-1) were treated with the HDAC inhibitors Na butyrate (NaB) and tricostatin A (TSA), and the effects on the expression of NIS and several thyroid-specific transcription factors together with the activity of NIS promoter were evaluated. TSA and NaB increased NIS mRNA levels in all cell lines. Among thyroid-specific transcription factors, only expression of PAX8 in ARO cells was increased. Down-regulation of thyroid-specific transcription factor-1 expression was observed in BCPAP and TPC-1 cell lines. Thyroid-specific transcription factor-2 mRNA was reduced in FRO, BCPAP, and TPC-1 cells. Histone acetylation had no significant effects on HEX expression. Altogether, these data indicate that the increase of NIS expression is not mediated by modification of expression of thyroid-specific transcription factors. Accordingly, in transfection experiments performed in the HeLa cell line (which does not express thyroid-specific transcription factors), treatment with TSA and NaB increased NIS promoter activity. Stimulation of NIS promoter activity was also obtained by overexpressing histone acetylating proteins pCAF and p300 in HeLa cells. Conversely, overexpression of the HDAC 1 enzyme inhibited basal activity of the NIS promoter. Effects of TSA and NaB on NIS expression were also evaluated in nonthyroid cell lines MCF-7, Hep-G2, and SAOS-2. In all cell lines TSA and NaB greatly increased NIS mRNA levels. We concluded that control of NIS expression by inhibition of HDAC appears not to be mediated by cell-specific mechanisms, suggesting it as a potential strategy to induce radioiodine sensitivity in different human tumors.
Assuntos
Histonas/metabolismo , Simportadores/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Acetilação , Neoplasias da Mama , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Expressão Gênica/fisiologia , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Humanos , Neoplasias Hepáticas , Osteossarcoma , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: ABCG2 protein overexpression and FLT3 internal tandem duplication (ITD) correlate with higher relapse rate and shorter disease-free survival (DFS) in acute myeloid leukemia (AML), but no data are available on the possible effect of concomitant presence of these 2 factors. METHODS: The authors analyzed the outcome of 166 cases of adult AML patients who were homogeneously treated with a fludarabine-based induction therapy. RESULTS: ABCG2 overexpression and FLT3-ITD were detected in 83 (50%) and 47 (28%) patients, respectively. A significant correlation was found between ABCG2 positivity and FLT3 mutation, with 33 (40%) ITD in 83 ABCG2-positive patients compared with 14 (17%) ITD in 83 ABCG2-negative patients (P = .002). Complete remission (CR) after induction therapy was achieved in 95 (57%) patients. Neither ABCG2 overexpression nor FLT3-ITD had any impact on achievement of CR. Relapse occurred in 42 of 95 (44%) patients at a median time of 28 months. Time to relapse was shortened in patients overexpressing ABCG2 (P = .0004). DFS was not affected by FLT3-ITD alone, but FLT3 mutation significantly worsened long-term outcome of ABCG2-positive patients. DFS at 1 and 3 years in patients with overexpression of both ABCG2 and FLT3-ITD was only 36% and 28%, respectively; in ABCG2-positive/FLT3-negative patients, DFS at 1 and 3 years was 65% and 48%, respectively; and in ABCG2-negative cases (regardless of FLT3 status), DFS at 1 and 3 years was greater than 85% and 75%. CONCLUSIONS: Concomitant overexpression of ABCG2 and FLT3-ITD is relatively frequent and identifies a subgroup of AML patients with a significantly worse prognosis. The possible interactions between these 2 prognostic factors need to be defined.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Sequências de Repetição em Tandem , Tirosina Quinase 3 Semelhante a fms/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Intervalo Livre de Doença , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/metabolismo , Prognóstico , Recidiva , Indução de Remissão , Vidarabina/análogos & derivados , Vidarabina/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Nucleophosmin (NPM) is a multifunctional nucleolar protein that, depending on the context, can act as oncogene or tumour suppressor. Mutations of the NPM1 gene induce delocalization of NPM in acute myeloid leukaemia. Differently, in solid tumours, only NPM overexpression, but not delocalization, has been so far reported. Here, NPM localization in thyroid tumours was investigated. By using immunohistochemistry, we show increase of NPM cytoplasmic localization in follicular adenomas and papillary carcinomas compared to normal thyroid tissue (p = 0.0125 and <0.0001, respectively). NPM1 mutations commonly found in human leukaemia are not present in thyroid tumours. Immunofluorescence in cultured cell lines was utilized to discriminate between nucleolar and nuclear localization. We show that in thyroid cancer cell lines NPM localizes both in the nucleolus and in nucleus, while in non-tumorigenic thyroid cell lines localizes only in nucleolus. Either presence of the histone deacetylase inhibitor trichostatin A or absence of thyroid-stimulating hormone induces NPM nuclear localization in non-tumorigenic thyroid cell lines.