RESUMO
OBJECTIVE: Thyroglobulin (Tg) measurements assess recurrence in post-thyroidectomy thyroid cancer patients. Tg measurements by enzyme immunoassays (EIA) can be falsely elevated by interference from Tg autoantibodies (TgAb). Radioimmunoassay (RIA) is less susceptible to TgAb interference and has been the standard-of-care test for TgAb positive patients. Recently developed liquid chromatography tandem mass spectrometry (LC-MS/MS) methods may eliminate TgAb interference. We assessed the performance of Tg measurements by EIA, RIA and LC-MS/MS to evaluate TgAb interference differences. RESULTS: We measured TgAb and Tg in 50 plasma samples from 40 patients in whom Tg measurement was part of their routine follow-up and 10 healthy volunteers. Discrepancy between EIA and both LC-MS/MS and RIA was observed at low Tg concentrations (≤ 7.55 ng/mL) in TgAb positive specimens (LC-MS/MS = 1.9 * EIA - 0.03, r = 0.68). RIA and LC-MS/MS Tg measurements in TgAb positive specimens with low Tg concentrations had improved correlation but demonstrated bias (LC MS/MS = 0.6 * RIA - 1.4, r = 0.90). Disagreement between methods may be attributed to LC-MS/MS reported Tg concentrations as undetectable compared to RIA. It seems likely that most discrepant cases are falsely elevated in RIA due to TgAb interference, however, some cases appear below the detection limit of LC-MS/MS; implementation of LC-MS/MS by clinicians will require lower detection limits.
Assuntos
Autoanticorpos/sangue , Cromatografia Líquida/normas , Técnicas Imunoenzimáticas/normas , Radioimunoensaio/normas , Espectrometria de Massas em Tandem/normas , Tireoglobulina/sangue , Tireoglobulina/imunologia , Neoplasias da Glândula Tireoide/diagnóstico , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Glândula Tireoide/sangueRESUMO
Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Evolução Molecular , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS , Sequência de Aminoácidos , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/isolamento & purificação , Afinidade de Anticorpos , Especificidade de Anticorpos , Sequência de Bases , Sítios de Ligação , Sítios de Ligação de Anticorpos , Antígenos CD4/metabolismo , Regiões Determinantes de Complementaridade/genética , Cristalografia por Raios X , Epitopos , Genes de Cadeia Pesada de Imunoglobulina , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/isolamento & purificação , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , HIV-1/química , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias J de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Análise de Sequência de DNARESUMO
Subjects with X-linked hyper-IgM syndrome (X-HIgM) have a markedly reduced frequency of CD27(+) memory B cells, and their Ig genes have a low level of somatic hypermutation (SHM). To analyze the nature of SHM in X-HIgM, we sequenced 209 nonproductive and 926 productive Ig heavy chain genes. In nonproductive rearrangements that were not subjected to selection, as well as productive rearrangements, most of the mutations were within targeted RGYW, WRCY, WA, or TW motifs (R = purine, Y = pyrimidine, and W = A or T). However, there was significantly decreased targeting of the hypermutable G in RGYW motifs. Moreover, the ratio of transitions to transversions was markedly increased compared with normal. Microarray analysis documented that specific genes involved in SHM, including activation-induced cytidine deaminase (AICDA) and uracil-DNA glycosylase (UNG2), were up-regulated in normal germinal center (GC) B cells, but not induced by CD40 ligation. Similar results were obtained from light chain rearrangements. These results indicate that in the absence of CD40-CD154 interactions, there is a marked reduction in SHM and, specifically, mutations of AICDA-targeted G residues in RGYW motifs along with a decrease in transversions normally related to UNG2 activity.
Assuntos
Linfócitos B/enzimologia , Citidina Desaminase/biossíntese , DNA Glicosilases/biossíntese , Regulação Enzimológica da Expressão Gênica/genética , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/genética , Cadeias Pesadas de Imunoglobulinas/genética , Hipermutação Somática de Imunoglobulina/genética , Adolescente , Adulto , Linfócitos B/imunologia , Antígenos CD40/genética , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Criança , Citidina Desaminase/genética , Citidina Desaminase/imunologia , DNA Glicosilases/genética , DNA Glicosilases/imunologia , Análise Mutacional de DNA , Regulação Enzimológica da Expressão Gênica/imunologia , Centro Germinativo/enzimologia , Centro Germinativo/imunologia , Humanos , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/enzimologia , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Memória Imunológica/genética , Masculino , Mutação , Hipermutação Somática de Imunoglobulina/imunologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
OBJECTIVE: Triptolide and tripdiolide are thought to be active components of the Chinese antirheumatic herbal remedy Tripterygium wilfordii Hook F, which has been shown to be effective in treating murine lupus nephritis. This study was undertaken to examine the therapeutic effect of triptolide and tripdiolide on established lupus nephritis in (NZB x NZW)F1 mice. METHODS: (NZB x NZW)F1 mice were treated with vehicle, triptolide, or tripdiolide for 15 weeks beginning at the age of 29 weeks (after the development of lupus nephritis). Body weight, proteinuria, and anti-double-stranded DNA (anti-dsDNA) antibodies were monitored, and the kidney and spleen were assessed histologically. Culture supernatants of spleen mononuclear cells were assayed for cytokines. RESULTS: By 28 weeks, most (NZB x NZW)F1 mice had developed lupus nephritis. Vehicle-treated mice exhibited progressive proteinuria, hypoalbuminemia, elevated blood urea nitrogen (BUN) levels, and evidence of severe nephritis. In contrast, proteinuria and BUN levels were significantly reduced in mice treated with either triptolide or tripdiolide as compared with those treated with vehicle. There was no hypoalbuminemia or apparent evidence of lupus nephritis in mice treated with either of the 2 diterpenoids. At 44 weeks of age, the survival rate in mice treated with vehicle (35.7%) was markedly lower than that in mice treated with either triptolide (87.5%) or tripdiolide (88.2%). The mean level of anti-dsDNA antibody in mice treated with tripdiolide was lower than that in the vehicle-treated mice upon completion of the treatment course. Production of tumor necrosis factor, interleukin-6, and monocyte chemoattractant protein 1 by spleen cells was also decreased after diterpenoid therapy. CONCLUSION: Therapy with triptolide or tripdiolide significantly ameliorated lupus nephritis in (NZB x NZW)F1 mice, reduced cytokine and chemokine production, and prolonged survival.
Assuntos
Diterpenos/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Imunossupressores/uso terapêutico , Nefrite Lúpica/tratamento farmacológico , Fenantrenos/uso terapêutico , Tripterygium/química , Animais , Modelos Animais de Doenças , Compostos de Epóxi/uso terapêutico , Feminino , Nefrite Lúpica/sangue , Nefrite Lúpica/patologia , Camundongos , Baço/efeitos dos fármacos , Baço/patologiaRESUMO
V(D)J recombination is essential to produce an Ig repertoire with a large range of Ag specificities. Although NF-kappaB-binding sites are present in the human and mouse IgH, Igkappa, and Iglambda enhancer modules and RAG expression is controlled by NF-kappaB, it is not known whether NF-kappaB regulates V(D)J recombination mechanisms after RAG-mediated dsDNA breaks. To clarify the involvement of NF-kappaB in human V(D)J recombination, we amplified Ig gene rearrangements from individual peripheral B cells of patients with X-linked anhidrotic ectodermal dysplasia with hyper-IgM syndrome (HED-ID) who have deficient expression of the NF-kappaB essential modulator (NEMO/Ikkgamma). The amplification of nonproductive Ig gene rearrangements from HED-ID B cells reflects the influence of the Ikkgamma-mediated canonical NF-kappaB pathway on specific molecular mechanisms involved in V(D)J recombination. We found that the CDR3(H) from HED-ID B cells were abnormally long, as a result of a marked reduction in the exonuclease activity on the V, D, and J germline coding ends, whereas random N-nucleotide addition and palindromic overhangs (P nucleotides) were comparable to controls. This suggests that an intact canonical NF-kappaB pathway is essential for normal exonucleolytic activity during human V(D)J recombination, whereas terminal deoxynucleotide transferase, Artemis, and DNA-dependent protein kinase catalytic subunit activity are not affected. The generation of memory B cells and somatic hypermutation were markedly deficient confirming a role for NF-kappaB in these events of B cell maturation. However, selection of the primary B cell repertoire appeared to be intact and was partially able to correct the defects generated by abnormal V(D)J recombination.
Assuntos
Displasia Ectodérmica Anidrótica Tipo 1/genética , Rearranjo Gênico do Linfócito B , Síndrome de Imunodeficiência com Hiper-IgM/genética , NF-kappa B/metabolismo , Sequência de Aminoácidos , Linfócitos B/imunologia , Exonucleases/metabolismo , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Rearranjo Gênico de Cadeia Leve de Linfócito B , Humanos , Imunoglobulina D/análise , Masculino , Dados de Sequência Molecular , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análiseRESUMO
Monoclonal B cell lymphocytosis (MBL) was detected in four unaffected first-degree relatives (FDR) in a familial chronic lymphocytic leukaemia (CLL) kindred. The proband remains untreated and two male siblings have died. The four unaffected siblings have been followed for a five-year period. All four FDR developed a kappa(+)CD5(+) MBL detected by flow cytometry. Poymerase chain reaction (PCR) for IGHV rearrangement showed evidence of oligoclonality in three of these individuals. Single cell PCR of flow cytometric sorted kappa(+) cells combined with Ig kappa light chain gene sequencing revealed further evidence of monoclonality in two of these individuals. Three of these individuals all showed evidence of hyper-somatic mutations. The B-cell repertoire in unaffected FDR in familial CLL offers a new area to investigate the interface between the immune system and lymphoid neoplasm.
Assuntos
Linfócitos B , Leucemia Linfocítica Crônica de Células B/genética , Linfocitose/genética , Células-Tronco Neoplásicas/patologia , Antígenos CD5/sangue , Feminino , Citometria de Fluxo/métodos , Seguimentos , Rearranjo Gênico do Linfócito B , Genes de Cadeia Pesada de Imunoglobulina , Genes de Cadeia Leve de Imunoglobulina , Predisposição Genética para Doença , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Linfocitose/imunologia , Masculino , Mutação , Reação em Cadeia da Polimerase/métodosRESUMO
Protein A (SpA) of Staphylococcus aureus is endowed with the capacity to interact with the H chain variable region (V(H)) of human Abs and to target >40% of B lymphocytes. To investigate whether this property represents a virulence factor and to determine the in vivo consequences of the confrontation of SpA with B lymphocytes, we used transgenic mice expressing fully human Abs. We found that administration of soluble SpA reduces B-1a lymphocytes of the peritoneal cavity and marginal zone B lymphocytes of the spleen, resulting in a markedly deficient type 2 humoral response. Single-cell PCR analysis and sequencing of the Ab V(H) gene repertoire revealed a significant reduction of V(H)3+ marginal zone B cells. Since the two B lymphocyte subsets targeted are involved in innate immune functions, our data suggest that crippling of humoral immunity by S. aureus represents an immune evasion mechanism that may aggravate recurrent infections.
Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Região Variável de Imunoglobulina/imunologia , Proteína Estafilocócica A/imunologia , Staphylococcus aureus/patogenicidade , Animais , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Humanos , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Transgênicos , Cavidade Peritoneal/citologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/imunologia , Baço/citologia , Baço/imunologia , Staphylococcus aureus/imunologiaRESUMO
Some pathogens have evolved to produce proteins, called B-cell superantigens, that can interact with human immunoglobulin variable regions, independently of the combining site, and activate B lymphocytes that express the target immunoglobulins. However, the in vivo consequences of these interactions on human B-cell numbers and function are largely unknown. Using transgenic mice expressing fully human immunoglobulins, we studied the consequences of in vivo exposure of protein L of Peptostreptococcus magnus with human immunoglobulins. In the mature pool of B cells, protein L exposure resulted in a specific reduction of splenic marginal-zone B cells and peritoneal B-1 cells. Splenic B cells exhibited a skewed light-chain repertoire consistent with the capacity of protein L to bind specific kappa gene products. Remarkably, these two B-cell subsets are implicated in innate B-cell immunity, allowing rapid clearance of pathogens. Thus, the present study reveals a novel mechanism that may be used by some infectious agents to subvert a first line of the host's immune defense.
Assuntos
Subpopulações de Linfócitos B/imunologia , Proteínas de Bactérias/imunologia , Imunoglobulinas/metabolismo , Peptostreptococcus/patogenicidade , Superantígenos/imunologia , Animais , Subpopulações de Linfócitos B/fisiologia , Proteínas de Bactérias/metabolismo , Humanos , Imunidade Inata , Imunoglobulina M/sangue , Cadeias kappa de Imunoglobulina/metabolismo , Imunoglobulinas/genética , Camundongos , Camundongos Transgênicos , Peptostreptococcus/metabolismo , Baço/citologia , Baço/imunologiaRESUMO
Mutations within the 5'-non-coding region of the bcl-6 gene can occur in lymphomas that originate from germinal centers (GCs), as well as in normal memory and GC B cells. Mutations in the p53 gene occur in 50% of human cancers. Since both bcl-6 and p53 can be mutated in certain circumstances, we investigated the accumulation of mutations in these genes in individual tonsillar B and T cells to determine whether the mutations exhibited a pattern anticipated from the B-cell hypermutation machinery. In tonsillar GC B cells, the overall mutational frequencies in the 5'-non-coding region of the bcl-6 gene was 0.85 x 10(-3)/bp. In contrast, there were no mutations in a region 2.8 kb downstream of the promoter. RGYW (purine, guanine, pyrimidine, A/T) targeting and a significantly lower mutational frequency in nai;ve B and GC founder B cells compared with GC B cells suggested that a similar mutator mechanism was active on Ig genes and this non-Ig gene. The mutational frequency in the exon-7-region of p53 was similar in the GC, memory and nai;ve B-cell subsets (1.02 x 10(-3) to 1.25 x 10(-3)/bp). RGYW/WRCY motifs were not targeted preferentially in the p53 gene. Moreover, a comparable mutational frequency of p53 was noted in tonsillar B and T cells. Hence, mutations in p53 do not appear to be the result of the B-cell hypermutational mechanism.