Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 599
Filtrar
1.
JCI Insight ; 6(22)2021 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-34806648

RESUMO

Human islet antigen reactive CD4+ memory T cells (IAR T cells) play a key role in the pathogenesis of autoimmune type 1 diabetes (T1D). Using single-cell RNA sequencing (scRNA-Seq) to identify T cell receptors (TCRs) in IAR T cells, we have identified a class of TCRs that share TCRα chains between individuals ("public" chains). We isolated IAR T cells from blood of healthy, new-onset T1D and established T1D donors using multiplexed CD154 enrichment and identified paired TCRαß sequences from 2767 individual cells. More than a quarter of cells shared TCR junctions between 2 or more cells ("expanded"), and 29/47 (~62%) of expanded TCRs tested showed specificity for islet antigen epitopes. Public TCRs sharing TCRα junctions were most prominent in new-onset T1D. Public TCR sequences were more germline like than expanded unique, or "private," TCRs, and had shorter junction sequences, suggestive of fewer random nucleotide insertions. Public TCRα junctions were often paired with mismatched TCRß junctions in TCRs; remarkably, a subset of these TCRs exhibited cross-reactivity toward distinct islet antigen peptides. Our findings demonstrate a prevalent population of IAR T cells with diverse specificities determined by TCRs with restricted TCRα junctions and germline-constrained antigen recognition properties. Since these "innate-like" TCRs differ from previously described immunodominant TCRß chains in autoimmunity, they have implications for fundamental studies of disease mechanisms. Self-reactive restricted TCRα chains and their associated epitopes should be considered in fundamental and translational investigations of TCRs in T1D.


Assuntos
Diabetes Mellitus Tipo 1/genética , Células Germinativas/metabolismo , Cadeias alfa de Imunoglobulina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Adolescente , Adulto , Feminino , Humanos , Masculino , Adulto Jovem
5.
PLoS One ; 13(8): e0201567, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30089177

RESUMO

In mammals, the most striking IgA system belongs to Lagomorpha. Indeed, 14 IgA subclasses have been identified in European rabbits, 11 of which are expressed. In contrast, most other mammals have only one IgA, or in the case of hominoids, two IgA subclasses. Characteristic features of the mammalian IgA subclasses are the length and amino acid sequence of their hinge regions, which are often rich in Pro, Ser and Thr residues and may also carry Cys residues. Here, we describe a new IgA that was expressed in New Zealand White domestic rabbits of IGHVa1 allotype. This IgA has an extended hinge region containing an intriguing stretch of nine consecutive Ser residues and no Pro or Thr residues, a motif exclusive to this new rabbit IgA. Considering the amino acid properties, this hinge motif may present some advantage over the common IgA hinge by affording novel functional capabilities. We also sequenced for the first time the IgA14 CH2 and CH3 domains and showed that IgA14 and IgA3 are expressed.


Assuntos
Cadeias alfa de Imunoglobulina/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Serina/química , Motivos de Aminoácidos , Animais , Evolução Molecular , Alótipos de Imunoglobulina/química , Alótipos de Imunoglobulina/genética , Cadeias alfa de Imunoglobulina/química , Nova Zelândia , Filogenia , Domínios Proteicos , Coelhos
6.
Folia Histochem Cytobiol ; 55(3): 95-106, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28994094

RESUMO

Cytogenetic lesions do not completely explain clinical heterogeneity of chronic lymphocytic leukemia (CLL). The 2016 revision of the World Health Organization classification 2008 indicated that molecular lesions of TP53, NOTCH1, SF3B1 and BIRC3 have potential clinical relevance and could be integrated into an updated risk profile. The negative clinical implications of TP53 disruptions are well constituted and patients with these mutations should be considered for novel, small molecule signal transduction inhibitors therapies. Mutations of NOTCH1, SF3B1 and BIRC3 are associated with poor prognosis. Patients with mutated SF3B1 or NOTCH1 genes present shorter time to first treatment compared to unmutated group. NOTCH1 mutations are related to a high risk of Richter's syndrome transformation, especially in case of TP53 disruptions' coexistence. Large studies on MYD88 mutations in CLL have not explained clearly their clinical importance.The aim of this paper is to provide a comprehensive review on novel molecular aberrations identified in CLL.


Assuntos
Leucemia Linfocítica Crônica de Células B/diagnóstico , ADP-Ribosil Ciclase 1/genética , Computadores Moleculares , Humanos , Cadeias alfa de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/fisiopatologia , Mutação , Prognóstico , Proteína-Tirosina Quinase ZAP-70/genética
7.
Environ Toxicol ; 32(6): 1775-1783, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28181387

RESUMO

Synthetic nanomaterials have many unique chemical and physical properties, mainly due to their high specific surface area and quantum confinement effect. Specifically, titanium dioxide (TiO2 ) nanomaterial has high stability, anticorrosive, and photocatalytic properties. However, there are concerns over adverse biological effects resulting from bioeffects. This study was to investigate adverse effects associated with acute ingestion of TiO2 nanofiber (TDNF). TDNF was fabricated via electrospinning method, followed by dissolution in water. Six- to seven-week-old male Sprague Dawley rats were exposed to a total of 0, 40, and 60 ppm of TDNF for 2 weeks via oral gavage. Serum total protein and weight gain during the course of this study displayed marginal concentration-dependent alterations. These findings were followed by a global gene expression analysis to identify which transcripts might be responsive to TNDF toxicity. Differentially expressed mRNA levels were dose-dependently higher in animals exposed to TNDF. The majority of the affected genes were biochemically involved in immune response and inflammation. We believe this is due to the fact that TNDF is unable to penetrate the cell and forms phagocytosis sites that trigger inflammatory and immune response. All results taken together, short-term ingestion of TNDF produced marginal effects indicative of inflammation. Finally, the broad gene expression data were validated through quantification of immunoglobulin heavy chain alpha (Igha). Igha gene was upregulated in treated groups, showing similar expression patterns to the global gene expression data.


Assuntos
Expressão Gênica/efeitos dos fármacos , Cadeias alfa de Imunoglobulina/genética , Nanofibras/toxicidade , Pneumonia/virologia , Titânio/toxicidade , Administração Oral , Animais , Relação Dose-Resposta a Droga , Estudo de Associação Genômica Ampla , Masculino , Pneumonia/imunologia , Ratos , Ratos Sprague-Dawley
8.
Kidney Int ; 91(3): 720-728, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28069266

RESUMO

Monoclonal gammopathy of renal significance (MGRS) regroups renal disorders caused by a monoclonal immunoglobulin without overt hematological malignancy. MGRS includes tubular disorders, glomerular disorders with organized deposits, and glomerular disorders with non-organized deposits, such as proliferative glomerulonephritis with monoclonal IgG deposits. Since glomerular involvement related to monotypic IgA deposits is poorly described we performed retrospective analysis and defined clinico-biological characteristics, renal pathology, and outcome in 19 referred patients. This analysis allowed distinction between 2 types of glomerulopathies, α-heavy chain deposition disease (5 patients) and glomerulonephritis with monotypic IgA deposits (14 patients) suggestive of IgA-proliferative glomerulonephritis with monoclonal immunoglobulin deposits in 12 cases. Clinicopathologic characteristics of α-heavy chain deposition disease resemble those of the γ-heavy chain disease, except for a higher frequency of extra-capillary proliferation and extra-renal involvement. IgA-proliferative glomerulonephritis with monoclonal immunoglobulin deposits should be differentiated from diseases with polytypic IgA deposits, given distinct clinical, histological, and pathophysiological features. Similarly to IgG-proliferative glomerulonephritis with monoclonal immunoglobulin deposits, overt hematological malignancy was infrequent, but sensitive serum and bone marrow studies revealed a subtle plasma cell proliferation in most patients with IgA-proliferative glomerulonephritis with monoclonal immunoglobulin deposits. Anti-myeloma agents appeared to favorably influence renal prognosis. Thus, potential progression towards symptomatic IgA multiple myeloma suggests that careful hematological follow-up is mandatory. This series expands the spectrum of renal disease in MGRS.


Assuntos
Glomerulonefrite por IGA/imunologia , Glomerulonefrite/imunologia , Doença das Cadeias Pesadas/imunologia , Imunoglobulina A/análise , Rim/imunologia , Mieloma Múltiplo/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Biópsia , Proliferação de Células , Diagnóstico Diferencial , Progressão da Doença , Feminino , Imunofluorescência , França , Glomerulonefrite/tratamento farmacológico , Glomerulonefrite/patologia , Glomerulonefrite por IGA/tratamento farmacológico , Glomerulonefrite por IGA/patologia , Doença das Cadeias Pesadas/tratamento farmacológico , Doença das Cadeias Pesadas/patologia , Humanos , Cadeias alfa de Imunoglobulina/análise , Cadeias gama de Imunoglobulina/análise , Rim/efeitos dos fármacos , Rim/ultraestrutura , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Fatores de Tempo
9.
Kidney Int ; 91(2): 423-434, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27773425

RESUMO

Randall-type heavy chain deposition disease (HCDD) is a rare disorder characterized by tissue deposition of a truncated monoclonal immunoglobulin heavy chain lacking the first constant domain. Pathophysiological mechanisms are unclear and management remains to be defined. Here we retrospectively studied 15 patients with biopsy-proven HCDD of whom 14 presented with stage 3 or higher chronic kidney disease, with nephrotic syndrome in 9. Renal lesions were characterized by nodular glomerulosclerosis, with linear peritubular and glomerular deposits of γ-heavy chain in 12 patients or α-heavy chain in 3 patients, without concurrent light chain staining. Only 2 patients had symptomatic myeloma. By serum protein electrophoresis/immunofixation, 13 patients had detectable monoclonal gammopathy. However, none of these techniques allowed detection of the nephrotoxic truncated heavy chain, which was achieved by immunoblot and/or bone marrow heavy chain sequencing in 14 of 15 patients. Serum-free kappa to lambda light chain ratio was abnormal in 11 of 11 patients so examined. Immunofluorescence studies of bone marrow plasma cells showed coexpression of the pathogenic heavy chain with light chain matching the abnormal serum-free light chain in all 3 tested patients. Heavy chain sequencing showed first constant domain deletion in 11 of 11 patients, with high isoelectric point values of the variable domain in 10 of 11 patients. All patients received chemotherapy, including bortezomib in 10 cases. Renal parameters improved in 11 patients who achieved a hematological response, as assessed by normalization of the free light chain ratio in 8 cases. Tissue deposition in HCDD relates to physicochemical peculiarities of both variable and constant heavy chain domains. Early diagnosis and treatment with bortezomib-based combinations appear important to preserve renal prognosis. Thus, monitoring of serum-free light chain is an indirect but useful method to evaluate the hematological response.


Assuntos
Doença das Cadeias Pesadas/imunologia , Doença das Cadeias Pesadas/patologia , Cadeias gama de Imunoglobulina/análise , Nefropatias/imunologia , Rim/imunologia , Rim/patologia , Idoso , Idoso de 80 Anos ou mais , Biópsia , Bortezomib/uso terapêutico , Quimioterapia Combinada , Feminino , Imunofluorescência , França , Glomerulonefrite/tratamento farmacológico , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Doença das Cadeias Pesadas/tratamento farmacológico , Doença das Cadeias Pesadas/genética , Humanos , Cadeias alfa de Imunoglobulina/análise , Cadeias gama de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/análise , Cadeias lambda de Imunoglobulina/análise , Rim/efeitos dos fármacos , Nefropatias/tratamento farmacológico , Nefropatias/patologia , Masculino , Pessoa de Meia-Idade , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/imunologia , Síndrome Nefrótica/patologia , Paraproteinemias/tratamento farmacológico , Paraproteinemias/imunologia , Reação em Cadeia da Polimerase , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/imunologia , Insuficiência Renal Crônica/patologia , Estudos Retrospectivos , Resultado do Tratamento
10.
Plant Biotechnol J ; 14(8): 1695-704, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26834022

RESUMO

Transforming growth factor beta (TGF-ß) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF-ß isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF-ß3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF-ß1 in Nicotiana benthamiana plants. We successfully expressed mature TGF-ß1 in the absence of the latency-associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP-TGF-ß1, we were able to show that processing of the latent complex by a furin-like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP-TGF-ß1, and co-expression of human furin enabled the proteolytic processing of latent TGF-ß1. Engineering the plant post-translational machinery by co-expressing human furin also enhanced the accumulation of biologically active TGF-ß1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.


Assuntos
Furina/metabolismo , Nicotiana/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Animais , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Furina/genética , Humanos , Cadeias alfa de Imunoglobulina/genética , Cadeias alfa de Imunoglobulina/metabolismo , Vison , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Redobramento de Proteína , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Nicotiana/metabolismo , Fator de Crescimento Transformador beta1/genética
11.
Clin Chem Lab Med ; 54(6): 1053-7, 2016 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-26485749

RESUMO

Accurate measurement of IgA monoclonal proteins presents a significant challenge to laboratory staff. IgA heavy/light chain (Hevylite, HLC) analysis is an alternative methodology for monoclonal protein assessment, giving an independent measure of IgAκ and IgAλ concentrations. Clonality is assessed by calculating the ratio of involved immunoglobulin to background uninvolved immunoglobulin concentrations (e.g. IgAκ/IgAλ in an IgAκ patient). Here we discuss the challenges faced by the laboratory in IgA monoclonal protein assessment, and compare the performance of Hevylite assays with electrophoresis and total IgA results. We present data which validates the use of Hevylite for response assessment: in most cases, Hevylite provides comparable response assignment to that provided by serum protein electrophoresis (SPE) and total IgA; in other cases Hevylite provides additional information, such as detection of residual disease or relapse.


Assuntos
Imunoensaio/métodos , Imunoglobulina A/sangue , Humanos , Cadeias alfa de Imunoglobulina/sangue , Cadeias kappa de Imunoglobulina/sangue , Cadeias lambda de Imunoglobulina/sangue , Paraproteinemias/diagnóstico , Paraproteinemias/imunologia , Paraproteínas/análise , Recidiva
12.
Int J Sports Med ; 37(1): 63-70, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26528943

RESUMO

Moderate exercise enhances resistance to pathogen-associated infections. However, its influence on intestinal IgA levels and resistance to Salmonella typhimurium in mice has not been reported. The aim of this study was to assess the impact of moderate exercise on bacterial resistance and the intestinal-IgA response in a murine typhoid model. Sedentary and exercised (under a protocol of moderate swimming) BALB/c mice were orally infected with Salmonella typhimurium and sacrificed on days 7 or 14 post-infection (n=5 per group). Compared with infected sedentary mice, infected exercised animals had i) lower intestinal and systemic bacterial loads; ii) higher total and specific intestinal-IgA levels, iii) a higher percentage of IgA plasma cells in lamina propria; iv) a higher level on day 7 and lower level on day 14 of intestinal α- and J-chain mRNA and plasma corticosterone, v) unchanged mRNA expression of intestinal pIgR, and vi) a higher mRNA expression of liver pIgR, α-chain and J-chain on day 7. Hence, it is likely that an increase in corticosterone levels (stress response) induced by moderate exercise increased intestinal IgA levels by enabling greater liver expression of pIgR mRNA, leading to a rise in IgA transcytosis from the liver to intestine. The overall effect of these changes is an enhanced resistance to infection.


Assuntos
Resistência à Doença/fisiologia , Imunoglobulina A/metabolismo , Mucosa Intestinal/metabolismo , Condicionamento Físico Animal/fisiologia , Infecções por Salmonella/prevenção & controle , Salmonella typhimurium , Animais , Carga Bacteriana , Corticosterona/sangue , Modelos Animais de Doenças , Cadeias J de Imunoglobulina/metabolismo , Cadeias alfa de Imunoglobulina/metabolismo , Intestinos/microbiologia , Fígado/metabolismo , Masculino , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Receptores de Imunoglobulina Polimérica/metabolismo , Natação/fisiologia
13.
BMC Immunol ; 15: 45, 2014 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-25391515

RESUMO

BACKGROUND: In the immune system, the serum levels of immunoglobulin (Ig) increase gradually during ageing. Through B cell development, the Ig heavy chain expression is modulated by a regulatory region at the 3' of the constant alpha gene (3'RR), in single copy in rodents and, due to a large duplication, in two copies in apes. The human 3'RR1 and 3'RR2 are both characterized by three enhancers, the central of which, namely hs1.2, is highly polymorphic. Human hs1.2 has four different variants with unique binding sites for transcription factors (e.g. NF-kB and SP1) and shows variable allelic frequencies in populations with immune disorders. In previous works, we have reported that in several autoimmune diseases the *2 allele of hs1.2 is genetically associated to high level of IgM in peripheral blood. In subjects with altered levels of circulating Ig, an increased level was associated to *2 allele of hs1.2 and low levels corresponded to high frequency of *1 allele. RESULTS: We have correlated the allelic frequencies of hs1.2 with IgM, IgG and IgA serum concentrations in two cohorts of healthy people of different age and after three years follow-up in children homozygous for the allele. Here we show that when the expression levels of Ig in children are low and medium, the frequencies of *1 and *2 alleles are the same. Instead, when the Ig expression levels are high, there is a significantly higher frequency of the allele *2. The follow-up of children homozygous for *1 and *2 alleles showed that the increase or decrease of circulating Ig was not dependent on the number of circulating mature B cells. CONCLUSIONS: These data support the idea that under physiologic condition there is a switch of regulative pathways involved in the maturation of Ig during ageing. This mechanism is evidenced by hs1.2 variants that in children but not in adults participate to Ig production, coordinating the three class levels.


Assuntos
Elementos Facilitadores Genéticos/genética , Cadeias alfa de Imunoglobulina/genética , Polimorfismo Genético , Adulto , Criança , Pré-Escolar , Feminino , Seguimentos , Frequência do Gene , Humanos , Cadeias alfa de Imunoglobulina/sangue , Masculino
14.
PLoS One ; 9(6): e100902, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24979072

RESUMO

Risk stratification of patients with pulmonary embolism (PE) may identify patients at high risk of early death who may benefit from more intensive surveillance or aggressive therapy. Nontargeted proteomics may identify biomarkers useful for the risk stratification of patients with acute symptomatic pulmonary embolism (PE). We studied 6 patients presenting with low-risk PE and 6 patients presenting with intermediate (n = 3) or high-risk (n = 3) PE. Two-dimensional difference gel electrophoresis was used to compare their plasma protein abundances. Candidate protein markers were identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry. A panel of four biomarkers (haptoglobin, hemopexin, α2-macroglobulin, and Ig α1-chain C region) showed differences in plasma abundance among patients with acute symptomatic PE of different severity. Haptoglobin and hemopexin were decreased, whereas α2-macroglobulin and Ig α1-chain C region were increased, in patients with high or intermediate-risk PE compared with low-risk PE patient. In a separate clinical population consisting of 104 adults with acute PE, serum haptoglobin concentrations had an 85% chance of correctly identifying patients with high-risk PE according to receiving operating characteristics curve analysis. Moreover, serum haptoglobin concentrations ≤1 g/l showed an 80% sensitivity and a 96% specificity for the diagnosis of high-risk PE. Nontargeted proteomics identified protein biomarkers for the severity of PE that are involved in iron metabolism pathways and acute-phase response. Among them, reduced serum haptoglobin concentrations show a high accuracy for the biochemical detection of high-risk PE.


Assuntos
Haptoglobinas/metabolismo , Embolia Pulmonar/sangue , Embolia Pulmonar/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Eletroforese em Gel Bidimensional , Feminino , Hemopexina/metabolismo , Humanos , Cadeias alfa de Imunoglobulina/sangue , Masculino , Prognóstico , Proteoma/metabolismo , Embolia Pulmonar/patologia , Risco , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , alfa-Macroglobulinas/metabolismo
15.
J Clin Periodontol ; 41(8): 733-47, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24738839

RESUMO

AIM: Application of quantitative stable isotope-labelling chemistries and mass spectrometry (MS) to determine alterations in gingival crevicular fluid (GCF) proteome in periodontal disease. MATERIAL AND METHODS: Quantitative proteome of GCF from 40 healthy individuals versus 40 patients with periodontal disease was established using 320 GCF samples and stable isotope-labelling reagents, ICAT and mTRAQ, with MS technology and validated by enzyme-linked immunosorbent methods. RESULTS: We have identified 238 distinct proteins of which 180 were quantified in GCF of both healthy and periodontal patients with additional 26 and 32 distinct proteins that were found only in GCF of healthy or periodontal patients. In addition, 42 pathogenic bacterial proteins and 11 yeast proteins were quantified. The data highlighted a series of proteins not quantified previously by large-scale MS approaches in GCF with relevance to periodontal disease, such as host-derived Ig alpha-2 chain C, Kallikrein-4, S100-A9, transmembrane proteinase 13, peptidase S1 domain, several collagen types and pathogenic bacterial proteins, e.g. formamidase, leucine aminopeptidase and virulence factor OMP85. CONCLUSIONS: The innovative analytical approaches provided detailed novel changes in both host and microbial derived GCF proteomes of periodontal patients. The study defined 50 host and 16 pathogenic bacterial proteins significantly elevated in periodontal disease most of which were novel with significant potential for application in the clinical arena of periodontal disease.


Assuntos
Líquido do Sulco Gengival/química , Doenças Periodontais/metabolismo , Proteoma/análise , Adulto , Albuminas/análise , Amidoidrolases/análise , Proteínas da Membrana Bacteriana Externa/análise , Proteínas de Bactérias/análise , Calgranulina B/análise , Cromatografia Líquida , Colágeno/análise , Eletroforese em Gel de Poliacrilamida , Feminino , Proteínas Fúngicas/análise , Humanos , Cadeias alfa de Imunoglobulina/análise , Isótopos , Calicreínas/análise , Leucil Aminopeptidase/análise , Masculino , Espectrometria de Massas , Proteínas de Membrana/análise , Pessoa de Meia-Idade , Doenças Periodontais/microbiologia , Serina Endopeptidases/análise , Albumina Sérica/análise , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Adulto Jovem
16.
Cell Mol Immunol ; 11(2): 197-205, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24185710

RESUMO

Immunoglobulins (Igs) are known to be synthesized and secreted only by B lymphocytes. Class switch recombination (CSR) is a key event that enables B cells to express Igs, and one of the crucial steps for CSR initiation is the germline transcription of Ig genes. Surprisingly, recent studies have demonstrated that the Ig genes are also expressed in some epithelial cancer cells; however, the mechanisms underlying how cancer cells initiate CSR and express Igs are still unknown. In this study, we confirmed that the Ig Iα1 promoter in cancer cell lines was activated by the Ets-1 transcription factor, and the activity of the Ig Iα1 promoter and Ig Iα1-Cα1 germline transcription were attenuated after knockdown of Ets-1 by specific small interfering RNAs (siRNA). Furthermore, the expression of Ets-1 and Igα heavy chain in cancer cells was dose dependently upregulated by TGF-ß1. These results indicate that activation of the Ig Iα1 promoter by the transcription factor Ets-1 is a critical pathway and provides a novel mechanism for Ig expression in non-B cell cancers.


Assuntos
Carcinoma/imunologia , Imunoglobulina A/metabolismo , Regiões Constantes de Imunoglobulina/metabolismo , Cadeias alfa de Imunoglobulina/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Células Epiteliais/imunologia , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos , Imunoglobulina A/genética , Regiões Constantes de Imunoglobulina/genética , Cadeias alfa de Imunoglobulina/genética , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Proteína Proto-Oncogênica c-ets-1/genética , RNA Interferente Pequeno/genética , Ativação Transcricional/genética , Fator de Crescimento Transformador beta1/imunologia , Regulação para Cima
17.
Nat Immunol ; 14(9): 966-75, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23913047

RESUMO

The adaptor Nck links receptor signaling to cytoskeleton regulation. Here we found that Nck also controlled the phosphatidylinositol-3-OH kinase (PI(3)K)-kinase Akt pathway by recruiting the adaptor BCAP after activation of B cells. Nck bound directly to the B cell antigen receptor (BCR) via the non-immunoreceptor tyrosine-based activation motif (ITAM) phosphorylated tyrosine residue at position 204 in the tail of the immunoglobulin-α component. Genetic ablation of Nck resulted in defective BCR signaling, which led to hampered survival and proliferation of B cells in vivo. Indeed, antibody responses in Nck-deficient mice were also considerably impaired. Thus, we demonstrate a previously unknown adaptor function for Nck in recruiting BCAP to sites of BCR signaling and thereby modulating the PI(3)K-Akt pathway in B cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/metabolismo , Proteínas Oncogênicas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/imunologia , Feminino , Cadeias alfa de Imunoglobulina/química , Cadeias alfa de Imunoglobulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Fosforilação , Ligação Proteica , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tirosina/metabolismo
18.
J Immunol Methods ; 378(1-2): 95-101, 2012 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-22570865

RESUMO

Immunoglobulins of isotype A (IgA) mediate a key role in mucosal immunity and are promising new immunotherapeutic candidates, but difficulties in obtaining enough material often hamper their in vivo exploration. We established recombinant Chinese hamster ovary (CHO) cells which stably expressed two IgA1 antibodies under serum-free conditions. The two cell lines achieved significantly different specific productivities of 16 pg per cell and day and 100 times less, a common phenomenon in recombinant antibody expression which challenges the production and purification process. Polymeric IgA in crude culture supernatants was assembled with J chain and showed expected specificity. We employed an immobilized camelid anti-human alpha-chain VHH ligand and isolated both recombinant IgAs at high purity and yield in a single chromatographic step. The described method was irrespective of the light chain and specificity and may be used as a generic capture step for the isolation of any IgA. Results were compared with a multistep purification process consisting of an affinity step based on immobilized jacalin followed by anion exchange and hydrophobic interaction chromatography.


Assuntos
Cromatografia de Afinidade/métodos , Imunoglobulina A/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Células CHO , Linhagem Celular , Cromatografia por Troca Iônica/métodos , Cricetinae , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Imunoglobulina A/isolamento & purificação , Cadeias Leves de Imunoglobulina/imunologia , Cadeias Leves de Imunoglobulina/isolamento & purificação , Cadeias alfa de Imunoglobulina/imunologia , Cadeias alfa de Imunoglobulina/isolamento & purificação , Ligantes , Lectinas de Plantas/imunologia , Plasmídeos/genética , Plasmídeos/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
19.
Immunology ; 136(1): 54-63, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22250990

RESUMO

Class switching and plasma cell differentiation occur at a high level within all mucosa-associated lymphoid tissues. The different classes of membrane immunoglobulin heavy chains are associated with the Igα/Igß heterodimer within the B-cell receptor (BCR). Whether BCR isotypes convey specific signals adapted to the corresponding differentiation stages remains debated but IgG and IgA membranes have been suggested to promote plasma cell differentiation. We investigated the impact of blocking expression of the IgA-class BCR through a 'αΔtail' targeted mutation, deleting the Cα immunoglobulin gene membrane exon. This allowed us to evaluate to what extent class switching and plasma cell differentiation can be concurrent processes, allowing some αΔtail(+/+) B cells with an IgM BCR to directly differentiate into IgA plasma cells and yield serum secreted IgA in spite of the absence of membrane IgA(+) B lymphocytes. By contrast, in secretions the secretory IgA was very low, indicating that J-chain-positive plasma cells producing secretory IgA overwhelmingly differentiate from previously class-switched membrane IgA(+) memory B cells. In addition, although mucosa-associated lymphoid tissues are a major site for plasma cell accumulation, αΔtail(+/+) mice showed that the gut B-cell lineage homeostasis is not polarized toward plasma cell differentiation through a specific influence of the membrane IgA BCR.


Assuntos
Membrana Celular/imunologia , Cadeias alfa de Imunoglobulina/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diferenciação Celular , Linhagem da Célula , Polaridade Celular , Cadeias alfa de Imunoglobulina/genética , Lipopolissacarídeos/imunologia , Camundongos , Receptores de Antígenos de Linfócitos B/imunologia , Fator de Crescimento Transformador beta/imunologia
20.
Biochim Biophys Acta ; 1823(2): 206-14, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22182704

RESUMO

The SH2-containing inositol 5'-phosphatase, SHIP1, negatively regulates signal transduction from the B cell antigen receptor (BCR). The mode of coupling between SHIP1 and the BCR has not been elucidated so far. In comparison to wild-type cells, B cells expressing a mutant IgD- or IgM-BCR containing a C-terminally truncated Ig-α respond to pervanadate stimulation with markedly reduced tyrosine phosphorylation of SHIP1 and augmented activation of protein kinase B. This indicates that SHIP1 is capable of interacting with the C-terminus of Ig-α. Employing a system of fluorescence resonance energy transfer in S2 cells, we can clearly demonstrate interaction between the SH2-domain of SHIP1 and Ig-α. Furthermore, a fluorescently labeled SH2-domain of SHIP1 translocates to the plasma membrane in an Ig-α-dependent manner. Interestingly, whereas the SHIP1 SH2-domain can be pulled-down with phospho-peptides corresponding to the immunoreceptor tyrosine-based activation motif (ITAM) of Ig-α from detergent lysates, no interaction between full-length SHIP1 and the phosphorylated Ig-α ITAM can be observed. Further studies show that the SH2-domain of SHIP1 can bind to the C-terminus of the SHIP1 molecule, most probably by inter- as well as intra-molecular means, and that this interaction regulates the association between different forms of SHIP1 and Ig-α.


Assuntos
Cadeias alfa de Imunoglobulina/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Domínios de Homologia de src , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Membrana Celular/metabolismo , Dimerização , Inositol Polifosfato 5-Fosfatases , Camundongos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais Cultivadas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA