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1.
Cells ; 10(3)2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799494

RESUMO

In this retrospective, monocentric cohort study, we tested if an intrathecal free light chain kappa (FLC-k) synthesis reflects not only an IgG but also IgA and IgM synthesis. We also analysed if FLC-k can help to distinguish between an inflammatory process and a blood contamination of cerebrospinal fluid (CSF). A total of 296 patient samples were identified and acquired from patients of the department of Neurology, University Medicine Greifswald (Germany). FLC-k were analysed in paired CSF and serum samples using the Siemens FLC-k kit. To determine an intrathecal FLC-k and immunoglobulin (Ig) A/-M-synthesis we analysed CSF/serum quotients in quotient diagrams, according to Reiber et al. Patient samples were grouped into three cohorts: cohort I (n = 41), intrathecal IgA and/or IgM synthesis; cohort II (n = 16), artificial blood contamination; and the control group (n = 239), no intrathecal immunoglobulin synthesis. None of the samples had intrathecal IgG synthesis, as evaluated with quotient diagrams or oligoclonal band analysis. In cohort I, 98% of patient samples presented an intrathecal synthesis of FLC-k. In cohort II, all patients lacked intrathecal FLC-k synthesis. In the control group, 6.5% presented an intrathecal synthesis of FLC-k. The data support the concept that an intrathecal FLC-k synthesis is independent of the antibody class produced. In patients with an artificial intrathecal Ig synthesis due to blood contamination, FLC-k synthesis is lacking. Thus, additional determination of FLC-k in quotient diagrams helps to discriminate an inflammatory process from a blood contamination of CSF.


Assuntos
Imunoglobulina A , Imunoglobulina M , Cadeias kappa de Imunoglobulina , Inflamação/diagnóstico , Adulto , Idoso , Artefatos , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Feminino , Humanos , Imunoglobulina A/biossíntese , Imunoglobulina A/sangue , Imunoglobulina A/líquido cefalorraquidiano , Imunoglobulina M/biossíntese , Imunoglobulina M/sangue , Imunoglobulina M/líquido cefalorraquidiano , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/sangue , Cadeias kappa de Imunoglobulina/líquido cefalorraquidiano , Inflamação/sangue , Inflamação/líquido cefalorraquidiano , Focalização Isoelétrica , Masculino , Pessoa de Meia-Idade , Nefelometria e Turbidimetria , Bandas Oligoclonais/sangue , Bandas Oligoclonais/líquido cefalorraquidiano , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Estudos Retrospectivos
2.
J Pharm Biomed Anal ; 162: 91-100, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30227357

RESUMO

Metal ions can be enzyme cofactors and can directly influence the kinetics of biochemical reactions that also influence the biological production and quality attributes of therapeutic proteins, such as glycan formation and distribution. However, the concentrations of metals in commercially available chemically defined media can range from 1 to 25,000 ppb. Because such concentration changes can impact cell growth, manufacturing yield and product quality the alteration/fluctuation in media composition should be well controlled to maintain product quality. Here, we describe a platform of analytical methods to determine the composition of several metals in different sample matrices using an advanced automated Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). These methods, validated to ICH Q2R1 regulatory validation parameters, were successfully applied to- (a) screen cell culture media; (b) determine changes in the metal concentration during cell growth in spinner flasks, and, (c) determine effect on the glycosylation pattern and homogeneity of an IgG3:κ produced from a murine-hybridoma cell line in bench-top parallel bioreactors due to a spike in copper and iron concentration. Our results show that maintenance of metal content in the cell culture media is critical for product consistency of the IgG3:κ produced.


Assuntos
Anticorpos Monoclonais/biossíntese , Cobre/metabolismo , Meios de Cultura/metabolismo , Glucuronidase/biossíntese , Imunoglobulina G/biossíntese , Cadeias kappa de Imunoglobulina/biossíntese , Ferro/metabolismo , Espectrometria de Massas/métodos , Animais , Anticorpos Monoclonais/genética , Reatores Biológicos , Células CHO , Proliferação de Células , Cricetulus , Glucuronidase/genética , Glicosilação , Hibridomas , Imunoglobulina G/genética , Cadeias kappa de Imunoglobulina/genética , Espectrometria de Massas/normas , Camundongos , Controle de Qualidade , Reprodutibilidade dos Testes , Fatores de Tempo , Transfecção
3.
Clin Chim Acta ; 489: 109-116, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30529605

RESUMO

BACKGROUND: Detection of cerebrospinal fluid (CSF) specific oligoclonal bands (OCB) supports the diagnosis of multiple sclerosis (MS), but the method is technically demanding and gives only qualitative information. Kappa free light chains (KFLC) quantification could represent a convenient alternative. We evaluated the diagnostic accuracy of OCB and KFLC in our cohort to further estimate the gain in diagnostic performance when combining both of them. METHODS: KFLC were measured in paired serum and CSF samples of 80 patients with MS and 50 patients with non-inflammatory neurological disorders. OCB were detected using an in-house alkaline phosphatase assay. Likelihood ratio (LR) was used to explore the benefit of the combined KFLC and OCB test. RESULTS: Sensitivity of KFLC index (≥5.3) and intrathecal KFLC fraction (≥10%) was 96% and 95% respectively, compared to 91% sensitivity of OCB assay. Specificity was 96% for intrathecal KFLC synthesis and 98% for OCB. Probability of MS in the absence of OCB was further reduced with concurrently normal KFLC index. CONCLUSIONS: Normal KFLC parameters allow confident exclusion of intrathecal inflammation, but probability of MS is greater with positive OCB. Use of KFLC as an adjunct test might be beneficial in specialized MS centers with larger pretest probability.


Assuntos
Análise Química do Sangue , Cadeias kappa de Imunoglobulina/biossíntese , Adulto , Estudos de Coortes , Análise Custo-Benefício , Feminino , Humanos , Cadeias kappa de Imunoglobulina/sangue , Cadeias kappa de Imunoglobulina/líquido cefalorraquidiano , Masculino , Esclerose Múltipla/sangue , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/diagnóstico , Sensibilidade e Especificidade , Fatores de Tempo
4.
Immunology ; 155(4): 491-498, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30098214

RESUMO

The rearrangement and expression of immunoglobulin genes are regulated by enhancers and their binding transcriptional factors that activate or suppress the activities of the enhancers. The immunoglobulin κ (Igκ) gene locus has three important enhancers: the intrinsic enhancer (Ei), 3' enhancer (E3'), and distal enhancer (Ed). Ei and E3' are both required for Igκ gene rearrangement during early stages of B-cell development, whereas optimal expression of the rearranged Igκ gene relies on both E3' and Ed. The transcription factor YY1 affects the expression of many genes involved in B-cell development, probably by mediating interactions between their enhancers and promoters. Herein, we found that YY1 binds to the E3' enhancer and suppresses Igκ expression in B lymphoma cells by epigenetically modifying the enhancer. Knocking down YY1 enhanced Igκ expression, which was associated with increased levels of E2A (encoded by the TCF3 gene) and its binding to the E3' enhancer. Moreover, in germinal centre B cells and plasma cells, YY1 expression was reversely associated with Igκ levels, implying that YY1 might facilitate antibody affinity maturation in germinal centre B cells through the transient attenuation of Igκ expression.


Assuntos
Linfócitos B/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Elementos Facilitadores Genéticos/genética , Cadeias kappa de Imunoglobulina/biossíntese , Linfoma de Células B/imunologia , Fator de Transcrição YY1/metabolismo , Linfócitos B/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Centro Germinativo/imunologia , Células HEK293 , Humanos , Cadeias kappa de Imunoglobulina/genética , Linfoma de Células B/patologia , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transcrição Gênica/genética , Fator de Transcrição YY1/genética
6.
Exp Hematol ; 57: 42-49.e1, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29030084

RESUMO

The genetic abnormalities underlying multiple myeloma (MM) are notoriously complex and intraclonal heterogeneity is a common disease feature. In the current study, we describe the establishment of a monoclonal immunoglobulin A (IgA) kappa (κ) MM cell line designated MC-B11/14. Cytogenetic and fluorescence in situ hybridization analyses of the original and relapse patient samples revealed that the MM clone was nonhyperdiploid and possessed an 11;14 chromosomal translocation. The MC-B11/14 cell line, established from the relapse sample, is tetraploid and houses the t(11;14) abnormality. Given our long-standing interest in Ig function and secretion, we next used CRISPR technology to knock out IgA heavy-chain expression in the MC-B11/14 cells to assess the biological consequences of converting this cell line to one only expressing κ light chains. As expected, secretion of intact IgA was undetectable from MC-B11/14IgA- cells. Sensitivity to pomalidomide treatment was similar between the MC-B11/14WT and MC-B11/14IgA- cells; however, MC-B11/14IgA- cells were found to be significantly more resistant to bortezomib treatment. This study describes the establishment of a new human MM cell line tool with which to study disease biology and the use of CRISPR technology to create a potentially useful model with which to study MM light-chain escape.


Assuntos
Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Genes de Imunoglobulinas , Imunoglobulina A/genética , Cadeias Pesadas de Imunoglobulinas/genética , Mieloma Múltiplo/patologia , Sequência de Aminoácidos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Sequência de Bases , Transplante de Medula Óssea , Bortezomib/administração & dosagem , Bortezomib/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 11/ultraestrutura , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 14/ultraestrutura , Terapia Combinada , Evolução Fatal , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/terapia , Proteínas do Mieloma/biossíntese , Proteínas do Mieloma/genética , Alinhamento de Sequência , Tetraploidia , Talidomida/análogos & derivados , Talidomida/farmacologia , Translocação Genética
7.
Sci Rep ; 7(1): 12713, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28983085

RESUMO

In response to immunization, B-cells generate a repertoire of antigen-specific antibodies. Antibody-based immunotherapies hold great promise for treating a variety of diseases in humans. Application of antibody-based immunotherapy in cats is limited by the lack of species-specific complete sequences for mRNAs encoding rearranged heavy and light chain immunoglobulins in B cells. To address this barrier, we isolated mRNAs from feline peripheral blood mononuclear cells (PBMCs), and used available immunoglobulin sequences and 5' and 3' RACE to clone and sequence heavy and light chain immunoglobulin mRNAs. We recovered mRNA from PBMCs from two cats, cloned and sequenced the variable and constant domains of the feline heavy chains of IgG1a (IGHG1a), IgG2 (IGHG2), and IgA (IGHA), and the light chains (lambda and kappa). Using these sequences, we prepared two bicistronic vectors for mammalian expression of a representative feline heavy (IGHG1a) together with a light (lambda or kappa) chain. Here we report novel feline Ig sequences, a technique to express antigen-specific felinized monoclonal antibodies, and the initial characterization of a functional felinized monoclonal antibody against feline panleukopenia virus.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Vírus da Panleucopenia Felina/imunologia , Panleucopenia Felina/terapia , Imunoglobulina A/genética , Imunoglobulina G/genética , RNA Mensageiro/genética , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Linfócitos B/imunologia , Gatos , Imunoglobulina A/biossíntese , Imunoglobulina G/biossíntese , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/genética , Análise de Sequência de RNA
8.
Protein Expr Purif ; 132: 27-33, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28089882

RESUMO

Signal peptides (SPs) are key elements in the production of recombinant proteins; however, little information is available concerning different SP in mammalian cells other than CHO. In order to study the efficiency of different SPs to direct the traffic along the secretory pathway of the green fluorescence protein (GFP) and a scFv-Fc fusion protein; CHO-K1, HEK293 and NS0 cell lines were transfected in a transient and stable way. SP of human azurocidin (AZ), modified human albumin (mSA), modified Cricetulus griseus Ig kappa chain V III region MOPC 63 like (mIgκ C) and modified human Ig kappa chain V III region VG (mIgκ H) were evaluated. The efficiency of SPs to translocate a propeptide across the ER membrane was evaluated by fluorescence microscopy and flow cytometry for the GFP inside the secretory pathway, and by antigen-specific indirect ELISA for the scFv-Fc outside the cell. The mSA SP was successful in directing the secretion of the active proteins in these different types of mammalian cells, regardless of the transgene copy number. The goal of this work was to demonstrate that a modified version of SA SP might be used in different mammalian cells employing the same expression vector.


Assuntos
Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Sinais Direcionadores de Proteínas , Animais , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/genética , Albumina Sérica
9.
Biotechnol Appl Biochem ; 64(3): 327-336, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-26790760

RESUMO

A truncated precursor form of prostate-specific antigen (PSA), [-2]proPSA, is a well-known biomarker for prostate cancer. To develop a biomarker assay, highly purified [-2]proPSA is required as a standard reference and for generation of a specific antibody. In this study, we generated an efficient mammalian expression system for producing a recombinant [-2]proPSA-human kappa constant domain (Cκ ) fusion protein. N-terminal amino acid sequencing using Edman degradation demonstrated that over 95% of the recombinant protein produced is [-2]proPSA, thereby showing for the first time that recombinant [-2]proPSA can be produced as a major fraction. We also generated a recombinant chicken antibody specific to [-2]proPSA but not cross-reactive to recombinant [-7]proPSA-Cκ , [-5]proPSA-Cκ , and PSA purified from human seminal fluid in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Also, the recombinant chicken antibody reacted to recombinant [-2]proPSA protein bound to an anti-PSA antibody coated on the micrometer plate in a sandwich ELISA. All of these results suggest that the N-terminus of the [-2]proPSA-Cκ fusion protein resides on the exterior of the protein, thus allowing exposure to the antibody.


Assuntos
Anticorpos Monoclonais/química , Proteínas Aviárias/química , Cadeias kappa de Imunoglobulina/biossíntese , Calicreínas/biossíntese , Antígeno Prostático Específico/biossíntese , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Animais , Anticorpos Monoclonais/imunologia , Proteínas Aviárias/imunologia , Galinhas , Células HEK293 , Humanos , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/imunologia , Calicreínas/análise , Calicreínas/genética , Calicreínas/imunologia , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia
10.
MAbs ; 9(2): 231-239, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28001485

RESUMO

When production of bispecific antibodies requires the co-expression and assembly of three or four polypeptide chains, low expression of one chain can significantly limit assembly and yield. κλ bodies, fully human bispecific antibodies with native IgG structure, are composed of a common heavy chain and two different light chains, one kappa and one lambda. No engineering is applied to force pairing of the chains, thus both monospecific and bispecific antibodies are secreted in the supernatant. In this context, stoichiometric expression of the two light chains allows for maximal assembly of the bispecific antibody. In this study, we selected a κλ body with suboptimal characteristics due to low kappa chain expression. Codon optimization to increase expression of the kappa chain did not improve bispecific yield. Surprisingly, progressive introduction of non-optimal codons into the sequence of the lambda chain resulted in lowering its expression for an optimal tuning of the relative distribution of monospecific and bispecific antibodies. This codon de-optimization led to doubling of the κλ body yield. These results indicate that assembly of different proteins into a recombinant complex is an interconnected process and that reducing the expression of one polypeptide can actually increase the overall yield.


Assuntos
Anticorpos Biespecíficos/biossíntese , Engenharia de Proteínas/métodos , Animais , Códon , Humanos , Imunoglobulina G/biossíntese , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/genética
11.
PLoS One ; 11(11): e0166556, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27846293

RESUMO

OBJECTIVES: We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. METHODS: We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. RESULTS: Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. CONCLUSIONS: Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.


Assuntos
Doenças Desmielinizantes/diagnóstico , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/biossíntese , Esclerose Múltipla/diagnóstico , Estudos de Casos e Controles , Doenças Desmielinizantes/sangue , Doenças Desmielinizantes/líquido cefalorraquidiano , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Immunoblotting/instrumentação , Immunoblotting/métodos , Cadeias kappa de Imunoglobulina/sangue , Cadeias kappa de Imunoglobulina/líquido cefalorraquidiano , Cadeias lambda de Imunoglobulina/sangue , Cadeias lambda de Imunoglobulina/líquido cefalorraquidiano , Focalização Isoelétrica/instrumentação , Focalização Isoelétrica/métodos , Esclerose Múltipla/sangue , Esclerose Múltipla/líquido cefalorraquidiano , Nefelometria e Turbidimetria/instrumentação , Nefelometria e Turbidimetria/métodos , Variações Dependentes do Observador , Curva ROC , Reprodutibilidade dos Testes
12.
Ann Clin Biochem ; 53(Pt 1): 174-6, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25753032

RESUMO

BACKGROUND: The results of free light chains quantitation in the cerebrospinal fluid were recently compared with the presence of cerebrospinal fluid-restricted oligoclonal IgG, but not oligoclonal free kappa light chains and oligoclonal free lambda light chains. We therefore aimed to compare the performance of the quantitative tests with the qualitative one for the same molecule. METHODS: Seventy-five paired cerebrospinal fluid and serum samples were analysed for oligoclonal IgG, oligoclonal free kappa light chains and oligoclonal free lambda light chains. Cerebrospinal fluid and serum free kappa and lambda light chains were quantified using Freelite™ kits on SPA Plus analyzer. ROC curves were analysed for the prediction of intrathecal synthesis and compared for cerebrospinal fluid concentration, cerebrospinal fluid/serum quotient (QfLC) and index (QfLC/QAlbumin). The presence of cerebrospinal fluid-restricted oligoclonal free kappa light chains and oligoclonal free lambda light chains bands was used as reference. RESULTS: No statistically significant differences were observed among cerebrospinal fluid concentration, QfLC and index for the prediction of free light chain intrathecal synthesis. Each parameter was able to predict the occurrence of cerebrospinal fluid-restricted oligoclonal free light chain bands (AUCs 0.932-0.999). However, we noted elevated cerebrospinal fluid free light chain concentrations in the absence of cerebrospinal fluid-restricted oligoclonal free light chain bands in two patients with very high serum free light chain values. CONCLUSIONS: Quantitation of cerebrospinal fluid free light chains reliably predicts their intrathecal synthesis. Yet, cerebrospinal fluid/serum quotient may still be preferred to correct for high serum free light chain concentrations. An appropriate formula should be sought to correct for blood-cerebrospinal fluid barrier status.


Assuntos
Testes de Química Clínica/métodos , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/líquido cefalorraquidiano , Cadeias lambda de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/líquido cefalorraquidiano , Humanos , Cadeias kappa de Imunoglobulina/sangue , Cadeias lambda de Imunoglobulina/sangue , Canal Medular/metabolismo
13.
Sci Rep ; 4: 5885, 2014 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-25073855

RESUMO

Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.


Assuntos
Clonagem Molecular/métodos , Vetores Genéticos/metabolismo , Imunoglobulina E/biossíntese , Kit de Reagentes para Diagnóstico , Proteínas Recombinantes de Fusão/biossíntese , Animais , Antígenos de Plantas/biossíntese , Antígenos de Plantas/genética , Sequência de Bases , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Primers do DNA/síntese química , Epitopos/química , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Humanos , Imunoglobulina E/genética , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/genética , Dados de Sequência Molecular , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética
15.
J Neuroimmunol ; 263(1-2): 116-20, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23916392

RESUMO

Intrathecal immunoglobulin synthesis is observed in several disorders of the central nervous system, but its detection by current laboratory tests is relatively insensitive and operator depending. We assessed the diagnostic accuracy of a nephelometric assay for k free light chain determination in cerebrospinal fluid and serum. The patients were grouped according to clinical and laboratory criteria. ROC curves for all methods were performed to find the best cut-off value. kFLC Index seems to be more accurate than other parameters. Our data indicate that nephelometric assay for kFLCs in CSF reliably detect intrathecal immunoglobulin synthesis and discriminate multiple sclerosis patients.


Assuntos
Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/líquido cefalorraquidiano , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/imunologia , Bandas Oligoclonais , Adulto , Idoso , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Feminino , Humanos , Cadeias kappa de Imunoglobulina/sangue , Injeções Espinhais , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/diagnóstico , Bandas Oligoclonais/biossíntese , Bandas Oligoclonais/sangue , Bandas Oligoclonais/líquido cefalorraquidiano , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Adulto Jovem
16.
Mol Immunol ; 55(3-4): 329-36, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23618164

RESUMO

Kappa transcripts from fetal piglets were compared to the recently reported kappa genome. Although five IGKV gene families are present in the genome, only IGKV1 and IGKV2 family genes are transcribed; the latter comprises >95% of the repertoire, in which two genes account for ~80%. We provisionally identified a new sequence allele of IGKV2-10 and two new IGKV genes that were not present in the genome of a single Duroc sow. One of these (IGKV2-1) accounted for 39% of the total pre-immune repertoire. The discovery of new IGKV genes and alleles in only 90 transcripts from mixed breeds, suggests considerable polymorphism and polygeny in the kappa locus of swine. Similar to lambda rearrangements, CDR3 length and diversity is restricted. The somatic mutation frequency is low and accumulates in especially CDR1. This transcriptional analysis of the pre-immune kappa repertoire completes a comparative study of all three Ig loci which has allowed the potential and actual combinatorial repertoire to be determined. Calculations show that combinatorial diversity in all three loci contribute comparatively little to the swine pre-immune antibody repertoire. Compared to humans that can potentially generate a million binding site variants, only 16-48 variant comprise 70% of the swine repertoire and 224 account for 95-100%. The frequency of somatic mutation does not differ among rearrangements from all three loci and the CDR3 diversity index shows that swine overwhelmingly generate their pre-immune repertoire by junctional diversity in heavy chain rearrangements.


Assuntos
Diversidade de Anticorpos/genética , Genes de Cadeia Pesada de Imunoglobulina , Genes de Cadeia Leve de Imunoglobulina , Cadeias kappa de Imunoglobulina/genética , Sus scrofa , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Feminino , Feto/imunologia , Rearranjo Gênico de Cadeia Leve de Linfócito B , Humanos , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/biossíntese , Dados de Sequência Molecular , Gravidez
18.
J Immunol ; 188(5): 2305-15, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22287713

RESUMO

Previous estimates of the diversity of the mouse Ab repertoire have been based on fragmentary data as a result of many technical limitations, in particular, the many samples necessary to provide adequate coverage. In this study, we used 5'-coding end amplification of Igκ mRNAs from bone marrow, splenic, and lymph node B cells of C57BL/6 mice combined with amplicon pyrosequencing to assess the functional and nonfunctional Vκ repertoire. To evaluate the potential effects of receptor editing, we also compared V/J associations and usage in bone marrows of mouse mutants under constitutive negative selection or an altered ability to undergo secondary recombination. To focus on preimmune B cells, our cell sorting strategy excluded memory B cells and plasma cells. Analysis of ~90 Mbp, representing >250,000 individual transcripts from 59 mice, revealed that 101 distinct functional Vκ genes are used but at frequencies ranging from ~0.001 to ~10%. Usage of seven Vκ genes made up >40% of the repertoire. A small class of transcripts from apparently nonfunctional Vκ genes was found, as were occasional transcripts from several apparently functional genes that carry aberrant recombination signals. Of 404 potential V-J combinations (101 Vκs × 4 Jκs), 398 (98.5%) were found at least once in our sample. For most Vκ transcripts, all Jκs were used, but V-J association biases were common. Usage patterns were remarkably stable in different selective conditions. Overall, the primary κ repertoire is highly skewed by preferred rearrangements, limiting Ab diversity, but potentially facilitating receptor editing.


Assuntos
Rearranjo Gênico de Cadeia Leve de Linfócito B , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Edição de RNA/genética , Edição de RNA/imunologia , Recombinação Genética/imunologia , Animais , Diversidade de Anticorpos/genética , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Feminino , Região de Junção de Imunoglobulinas/biossíntese , Região de Junção de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/metabolismo , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência de DNA
19.
J Immunol ; 188(1): 47-56, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22131331

RESUMO

Secondary Ab V region gene segment rearrangement, termed receptor editing, is a major mechanism contributing to B lymphocyte self-tolerance. However, the parameters that determine whether a B cell undergoes editing are a current subject of debate. We tested the role that the level of BCR expression plays in the regulation of receptor editing in a polyclonal population of B cells differentiating in vivo. Expression of a short hairpin RNA for κ L chain RNA in B cells resulted in reduction in levels of this RNA and surface BCRs. Strikingly, fully mature and functional B cells that developed in vivo and efficiently expressed the short hairpin RNA predominantly expressed BCRs containing λ light chains. This shift in L chain repertoire was accompanied by inhibition of development, increased Rag gene expression, and increased λ V gene segment-cleavage events at the immature B cell stage. These data demonstrated that reducing the translation of BCRs that are members of the natural repertoire at the immature B cell stage is sufficient to promote editing.


Assuntos
Regulação da Expressão Gênica/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Cadeias lambda de Imunoglobulina/imunologia , Edição de RNA/imunologia , RNA Mensageiro/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Linhagem Celular , Regulação da Expressão Gênica/genética , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/genética , Camundongos , Edição de RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos B/biossíntese , Receptores de Antígenos de Linfócitos B/genética
20.
Cell Mol Immunol ; 8(6): 479-85, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21860405

RESUMO

Accumulating evidence has shown that immunoglobulin (Ig) is 'unexpectedly' expressed by epithelial cancer cells and that it can promote tumor growth. The main purpose of this study was to explore the components of the cancerous Ig and its possible function. The presence of cancerous Ig in the Golgi apparatus was confirmed by immunofluorescence, indirectly suggesting that the cancerous Ig was processed and packaged in cancer cells. Western blot analysis and ELISA results indicated that cancer cells produced membrane Ig and secreted Ig into the supernatant fraction. The cancerous Ig consists of an α heavy chain and a κ light chain. Finally, by analyzing the Ig components pulled down by protein A beads, the cancerous Ig was found to be structurally distinct from normal Ig. The cancerous Ig was truncated or aberrant. Although the underlying mechanism that causes the abnormalities has not been determined, our current discoveries strengthen our previous findings and promise fruitful future explorations.


Assuntos
Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Expressão Gênica/imunologia , Complexo de Golgi/metabolismo , Cadeias kappa de Imunoglobulina/biossíntese , Western Blotting , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Carcinoma , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/imunologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/citologia , Células Epiteliais/imunologia , Feminino , Heterogeneidade Genética , Complexo de Golgi/genética , Complexo de Golgi/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/química , Cadeias kappa de Imunoglobulina/química , Imunoprecipitação , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/imunologia , Neoplasias Nasofaríngeas/patologia , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/patologia , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/patologia
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