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1.
MAbs ; 12(1): 1685350, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31856660

RESUMO

The development of antibody therapeutics relies on animal models that accurately recapitulate disease biology. Syngeneic mouse models are increasingly used with new molecules to capture the biology of complex cancers and disease states, and to provide insight into the role of the immune system. The establishment of syngeneic mouse models requires the ability to generate surrogate mouse counterparts to antibodies designed for humans. In the field of bispecific antibodies, there remains a dearth of technologies available to generate native IgG-like mouse bispecific antibodies. Thus, we engineered a simple co-expression system for one-step purification of intact mouse IgG1 and IgG2a bispecific antibodies from any antibody pair. We demonstrated proof of concept with CD3/CD20 bispecific antibodies, which highlighted both the quality and efficacy of materials generated by this technology.

2.
Nature ; 574(7779): 565-570, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645726

RESUMO

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.


Assuntos
Antígenos B7/química , Antígenos B7/metabolismo , Glicoproteínas de Membrana/metabolismo , Linfócitos T/metabolismo , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Antígenos B7/antagonistas & inibidores , Antígenos B7/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cristalografia por Raios X , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Linfócitos T/citologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologia
3.
Bioanalysis ; 10(8): 559-576, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29701071

RESUMO

Ligand-binding assay (LBA) performance depends on quality reagents. Strategic reagent screening and characterization is critical to LBA development, optimization and validation. Application of advanced technologies expedites the reagent screening and assay development process. By evaluating surface plasmon resonance technology that offers high-throughput kinetic information, this article aims to provide perspectives on applying the surface plasmon resonance technology to strategic LBA critical reagent screening and characterization supported by a number of case studies from multiple biotherapeutic programs.


Assuntos
Bioensaio/métodos , Terapia Biológica/métodos , Ressonância de Plasmônio de Superfície/métodos , Humanos , Ligantes
4.
Anal Chim Acta ; 979: 36-44, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28599707

RESUMO

Myostatin, also known as growth differentiation factor 8 (GDF-8), is a protein acting as a negative regulator in skeletal muscle growth. Inhibition of myostatin by therapeutic agents provides opportunities for current unmet medical needs. In order to better understand drug engagement to aid the drug development, we have developed a hybrid LC-MS/MS method which can differentially measure myostatin and another protein from the same GDF family, GDF-11. Although the two proteins share high homology, the LC-MS/MS assay provided the specificity based on monitoring of unique surrogate peptide generated from enzymatic digestion. An automated sample preparation platform, Agilent AssayMap Bravo, was used for automated immunocapture. Capture antibody that is non-competing with our investigational drug and has similar binding affinity to both myostatin and GDF-11 was used. Therefore, total myostatin and GDF-11 including both free form and drug-bound form were captured and measured. The enriched sample was digested after reduction and alkylation. Two surrogate peptides (IPAMVVDR for myostatin and IPGMVVDR for GDF-11) were monitored and the lower limit of quantitation (LLOQ) was established at 1.0 ng/mL for myostatin and 0.1 ng/mL for GDF-11. The accuracy was demonstrated with recovery for IPAMVVDR between 99.2% and 103.1% and for IPGMVVDR between 90.3% and 114.5%. The developed hybrid assay exhibits sufficient sensitivity, accuracy and specificity to differentiate between the highly structurally similar myostatin and GDF-11. This analytical approach was successfully applied to a rat toxicology study, and was demonstrated to be a powerful tool for biomarker measurement in the present of a therapeutic agent.


Assuntos
Cromatografia Líquida , Fatores de Diferenciação de Crescimento/sangue , Miostatina/sangue , Espectrometria de Massas em Tandem , Animais , Ratos
5.
Bioanalysis ; 8(15): 1611-1622, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27397670

RESUMO

BACKGROUND: Isomerization of aspartic acid and deamidation of asparagine are two common amino acid modifications that are of particular concern if located within the complementarity-determining region of therapeutic antibodies. Questions arise as to the extent of modification occurring in circulation due to potential exposure of the therapeutic antibody to different pH regimes. RESULTS: To enable evaluation of site-specific isomerization and deamidation of human mAbs in vivo, immunoprecipitation (IP) has been combined with LC-MS providing selective enrichment, separation and detection of naive and modified forms of tryptic peptides comprising complementarity-determining region sequences. CONCLUSION: IP-LC-MS can be applied to simultaneously quantify in vivo drug concentrations and measure the extent of isomerization or deamidation in PK studies conducted during the drug discovery stage.


Assuntos
Anticorpos Monoclonais/química , Asparagina/análise , Ácido Aspártico/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/sangue , Cromatografia Líquida/métodos , Humanos , Imunoprecipitação/métodos , Isomerismo , Macaca fascicularis , Masculino , Espectrometria de Massas em Tandem/métodos
6.
Bioanalysis ; 8(13): 1383-401, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27277879

RESUMO

BACKGROUND: Antibody-drug conjugates (ADCs) are complex drug constructs with multiple species in the heterogeneous mixture that contribute to their efficacy and toxicity. The bioanalysis of ADCs involves multiple assays and analytical platforms. METHODS: A series of ligand binding and LC-MS/MS (LB-LC-MS/MS) hybrid assays, through different combinations of anti-idiotype (anti-Id), anti-payload, or generic capture reagents, and cathepsin-B or trypsin enzyme digestion, were developed and evaluated for the analysis of conjugated-payload as well as for species traditionally measured by ligand-binding assays, total-antibody and conjugated-antibody. RESULTS & CONCLUSION: Hybrid assays are complementary or viable alternatives to ligand-binding assay for ADC bioanalysis and PK/PD modeling. The fit-for-purpose choice of analytes, assays and platforms and an integrated strategy from Discovery to Development for ADC PK and bioanalysis are recommended.


Assuntos
Imunoconjugados/sangue , Preparações Farmacêuticas/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Cromatografia Líquida/métodos , Haplorrinos , Humanos , Imunoensaio/métodos , Imunoconjugados/análise , Limite de Detecção , Preparações Farmacêuticas/análise , Ratos
7.
Bioanalysis ; 8(8): 847-56, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27005854

RESUMO

LC-MS/MS has been investigated to quantify protein therapeutics in biological matrices. The protein therapeutics is digested by an enzyme to generate surrogate peptide(s) before LC-MS/MS analysis. One challenge is isolating protein therapeutics in the presence of large number of endogenous proteins in biological matrices. Immunocapture, in which a capture agent is used to preferentially bind the protein therapeutics over other proteins, is gaining traction. The protein therapeutics is eluted for digestion and LC-MS/MS analysis. One area of tremendous potential for immunocapture-LC-MS/MS is to obtain quantitative data where ligand-binding assay alone is not sufficient, for example, quantitation of antidrug antibody complexes. Herein, we present an overview of recent advance in enzyme digestion and immunocapture applicable to protein quantitation.


Assuntos
Cromatografia Líquida de Alta Pressão , Peptídeo Hidrolases/metabolismo , Proteínas/análise , Espectrometria de Massas em Tandem , Cromatografia de Afinidade , Humanos , Imunoglobulina G/imunologia , Proteínas/isolamento & purificação , Proteínas/metabolismo
8.
Bioanalysis ; 8(3): 193-204, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26811930

RESUMO

BACKGROUND: Therapeutic protein discovery study highlights the need for the development of quantitative bioanalytical methods for determining the levels of both the therapeutic protein and the target protein, as well. RESULTS: For the quantitation of BMS-986089, both accuracy (99-103%) and precision (2.4-12%) were obtained for the analysis of the surrogate peptide (ITYGGNSPVQEFTVPGR), in addition to the accuracy (100-108%) and precision (0.7-18%) that were obtained for the analysis of the surrogate peptide (VVSVLTVLHQDWLNGK). For Myostatin, accuracy (94-103%) and precision (2.4-14.9%) were obtained for the analysis of the surrogate peptide (IPAMVVDR). CONCLUSION: The developed method was applied to the analysis of samples following dosing of BMS-986089 to mice. This method highlights the potential of LC-MS/MS-based methods to eventually assess in vivo drug-target engagement.


Assuntos
Cromatografia Líquida/métodos , Miostatina/análise , Proteínas Recombinantes de Fusão/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Cromatografia Líquida/economia , Análise Custo-Benefício , Humanos , Imunoglobulina G/análise , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Miostatina/metabolismo , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Ratos , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Espectrometria de Massas em Tandem/economia , Tripsina/metabolismo
9.
Artigo em Inglês | MEDLINE | ID: mdl-26310897

RESUMO

Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC-MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys.


Assuntos
Cromatografia Líquida/métodos , Haplorrinos/sangue , Imunoconjugados/sangue , Espectrometria de Massas em Tandem/métodos , Animais
10.
Bioanalysis ; 7(13): 1569-82, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26226308

RESUMO

BACKGROUND: The bioanalytical strategy for antibody-drug conjugates (ADC) includes numerous measurements integrally designed to provide comprehensive characterization of PK, PD and immunogenicity. This manuscript describes the utilization of reagents specifically tailored to an ADC with a microtubule polymerization inhibitor payload and cathepsin B cleavable linker. METHODS: The PK strategy includes the evaluation of physiological levels of total antibody, active ADC, total ADC, antibody-conjugated payload and unconjugated payload. These data are evaluated in the context of target and antidrug antibody levels to elucidate bioactive ADC. RESULTS & CONCLUSION: Herein, we discuss how this strategy has been applied and present our preliminary observations. Continuously evolving to meet pipeline demands, the integrated bioanalytical data will provide critical insights into the exposure-response relationship.


Assuntos
Anticorpos Monoclonais/imunologia , Imunoconjugados/imunologia , Anticorpos Monoclonais/química , Humanos , Imunoconjugados/química
12.
Bioanalysis ; 6(22): 2985-98, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25496253

RESUMO

BACKGROUND: FGF21-AdPKE is a fusion protein and functionally inactivated in vivo by cleavage around the C-terminus. It is important to quantify the intact active protein in serum. RESULTS & DISCUSSION: Taking advantage of a uniquely acid-labile aspartyl-prolyl amide bond, we developed an acid hydrolysis procedure based on heating FGF21-AdPKE in dilute formic acid to generate a surrogate peptide encompassing the last 17 amino acids at the C-terminus. The monkey serum samples were extracted with an immunocapture procedure with an antibody specific for AdPKE. The calibration range was 200-50000 ng/ml. The assay accuracy and precision were between 92.8-99.8% and 3.9-14.5%, respectively. The method was applied to analyze incurred serum samples from a cynomolgus monkey toxicokinetic study involving administration of FGF21-AdPKE. CONCLUSION: A method of combining immunocapture and acid hydrolysis to quantify a therapeutic protein in biological fluids was developed.


Assuntos
Cromatografia Líquida/métodos , Dipeptídeos/química , Fatores de Crescimento de Fibroblastos/química , Espectrometria de Massas em Tandem/métodos , Tripsina/química , Amidas/química , Sequência de Aminoácidos , Animais , Calibragem , Fatores de Crescimento de Fibroblastos/farmacocinética , Fatores de Crescimento de Fibroblastos/toxicidade , Dados de Sequência Molecular , Mapeamento de Peptídeos
13.
J Allergy Clin Immunol ; 128(5): 1086-92.e1-3, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21762978

RESUMO

BACKGROUND: IL-5 plays a central role in the development and maintenance of eosinophilia (EO) and eosinophil activation in a wide variety of eosinophilic disorders. Although IL-5, IL-3, and GM-CSF can modulate the expression of IL-5 receptor α (IL-5Rα) on eosinophils in vitro, little is known about soluble and surface IL-5Rα levels in vivo. OBJECTIVE: To assess soluble and surface IL-5Rα levels in patients with EO and/or mastocytosis. METHODS: Surface IL-5Rα expression was assessed by flow cytometry in blood and/or bone marrow from subjects with EO (n = 39) and systemic mastocytosis (n = 8) and from normal volunteers (n = 28). Soluble IL-5Rα (sIL-5Rα) level was measured in a cohort of 177 untreated subjects and correlated with EO, eosinophil activation, and serum tryptase and cytokine levels. RESULTS: IL-5Rα expression on eosinophils inversely correlated with EO (r = -0.48; P < .0001), whereas serum levels of sIL-5Rα increased with the eosinophil count (r = 0.56; P < .0001) and serum IL-5 (r = 0.40; P < .0001) and IL-13 (r = 0.29; P = .004) levels. Of interest, sIL-5Rα level was significantly elevated in patients with systemic mastocytosis without EO. Although sIL-5Rα levels correlated with serum tryptase levels in these patients, eosinophil activation, assessed by CD69 expression on eosinophils and serum eosinophil-derived neurotoxin levels, was increased compared with that in normal subjects. CONCLUSIONS: These data are consistent with an in vivo IL-5Rα regulatory pathway in human eosinophils similar to that described in vitro and involving a balance between soluble and surface receptor levels. This may have implications with respect to the use of novel therapeutic agents targeting IL-5 and its receptor in patients with EO and/or mastocytosis.


Assuntos
Eosinofilia/metabolismo , Subunidade alfa de Receptor de Interleucina-5/biossíntese , Mastocitose Sistêmica/metabolismo , Adulto , Idoso , Separação Celular , Citocinas/análise , Citocinas/biossíntese , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Neurotoxina Derivada de Eosinófilo/análise , Neurotoxina Derivada de Eosinófilo/biossíntese , Neurotoxina Derivada de Eosinófilo/imunologia , Eosinofilia/imunologia , Eosinófilos/imunologia , Eosinófilos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Subunidade alfa de Receptor de Interleucina-5/imunologia , Masculino , Mastocitose Sistêmica/imunologia , Pessoa de Meia-Idade , Triptases/sangue , Adulto Jovem
14.
J Allergy Clin Immunol ; 125(6): 1344-1353.e2, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20513525

RESUMO

BACKGROUND: Peripheral blood eosinophilia and lung mucosal eosinophil infiltration are hallmarks of bronchial asthma. IL-5 is a critical cytokine for eosinophil maturation, survival, and mobilization. Attempts to target eosinophils for the treatment of asthma by means of IL-5 neutralization have only resulted in partial removal of airway eosinophils, and this warrants the development of more effective interventions to further explore the role of eosinophils in the clinical expression of asthma. OBJECTIVE: We sought to develop a novel humanized anti-IL-5 receptor alpha (IL-5Ralpha) mAb with enhanced effector function (MEDI-563) that potently depletes circulating and tissue-resident eosinophils and basophils for the treatment of asthma. METHODS: We used surface plasmon resonance to determine the binding affinity of MEDI-563 to FcgammaRIIIa. Primary human eosinophils and basophils were used to demonstrate antibody-dependent cell-mediated cytotoxicity. The binding epitope of MEDI-563 on IL-5Ralpha was determined by using site-directed mutagenesis. The consequences of MEDI-563 administration on peripheral blood and bone marrow eosinophil depletion was investigated in nonhuman primates. RESULTS: MEDI-563 binds to an epitope on IL-5Ralpha that is in close proximity to the IL-5 binding site, and it inhibits IL-5-mediated cell proliferation. MEDI-563 potently induces antibody-dependent cell-mediated cytotoxicity of both eosinophils (half-maximal effective concentration = 0.9 pmol/L) and basophils (half-maximal effective concentration = 0.5 pmol/L) in vitro. In nonhuman primates MEDI-563 depletes blood eosinophils and eosinophil precursors in the bone marrow. CONCLUSIONS: MEDI-563 might provide a novel approach for the treatment of asthma through active antibody-dependent cell-mediated depletion of eosinophils and basophils rather than through passive removal of IL-5.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Eosinófilos/metabolismo , Epitopos/metabolismo , Subunidade alfa de Receptor de Interleucina-5/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos Monoclonais/efeitos adversos , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Contagem de Células , Eosinófilos/efeitos dos fármacos , Eosinófilos/patologia , Mapeamento de Epitopos , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-5/genética , Subunidade alfa de Receptor de Interleucina-5/imunologia , Macaca fascicularis , Masculino , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Ressonância de Plasmônio de Superfície
15.
Am J Respir Crit Care Med ; 181(9): 917-27, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20133931

RESUMO

RATIONALE: Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation and remodeling. High-mobility group box 1 (HMGB1), a nuclear protein that is released during inflammation and repair, interacts with proinflammatory cytokines and with the receptor for advanced glycation end products (RAGE), which is highly expressed in the lung. OBJECTIVES: To determine whether HMGB1 is augmented in COPD and is associated with IL-1beta and RAGE. METHODS: HMGB1 was assessed in the bronchoalveolar lavage (BAL) of 20 never-smokers, 20 smokers, and 30 smokers with COPD and it was correlated with inflammatory and clinical parameters. In parallel, HMGB1 and RAGE immunolocalization was determined in bronchial and lung tissues. Last, binding of HMGB1 to IL-1beta in human macrophages and in BAL fluid was examined. MEASUREMENTS AND MAIN RESULTS: BAL levels of HMGB1 were higher in smokers with COPD than in smokers and never-smokers (P < 0.0001 for both comparisons), and similar differences were observed in epithelial cells and alveolar macrophages. BAL HMGB1 correlated positively with IL-1beta (r(s) = 0.438; P = 0.0006) and negatively with FEV(1) (r(s) = -0.570; P < 0.0001) and transfer factor of the lung for carbon monoxide (r(s) = -0.382; P = 0.0026). HMGB1-IL-1beta complexes were found in BAL supernatant and alveolar macrophages from smokers and patients with COPD, as well as in the human macrophage cell line, THP-1, where they enhanced the synthesis of tumor-necrosis factor-alpha. RAGE was overexpressed in the airway epithelium and smooth muscle of patients with COPD and it colocalized with HMGB1. CONCLUSIONS: Elevated HMGB1 expression in COPD airways may sustain inflammation and remodeling through its interaction with IL-1beta and RAGE.


Assuntos
Proteína HMGB1/metabolismo , Interleucina-1beta/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptores Imunológicos/metabolismo , Remodelação das Vias Aéreas/fisiologia , Brônquios/metabolismo , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular , Feminino , Imunofluorescência , Fluxo Expiratório Forçado , Humanos , Imuno-Histoquímica , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Masculino , Pessoa de Meia-Idade , Receptor para Produtos Finais de Glicação Avançada , Fumar/metabolismo
16.
Am J Pathol ; 176(2): 638-49, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20042671

RESUMO

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation and emphysematous alveolar destruction. In this study, we have investigated whether chitotriosidase (ChTRase) and acidic mammalian chitinase, two chitinases with chitinolytic activity, are selectively augmented in COPD and contribute to its pathogenesis. We found that smokers with COPD, but not asthmatics, had higher chitinolytic activity and increased levels of ChTRase in bronchoalveolar lavage, more ChTRase-positive cells in bronchial biopsies, and an elevated proportion of alveolar macrophages expressing ChTRase than smokers without COPD or never-smokers. ChTRase accounted for approximately 80% of bronchoalveolar lavage chitinolytic activity, while acidic mammalian chitinase was undetectable. Bronchoalveolar lavage chitinolytic activity and ChTRase were associated with airflow obstruction and emphysema and with the levels of interleukin (IL)-1beta, IL-8, tumor-necrosis factor (TNF)-alpha, and its type II soluble receptor. Tumor necrosis factor-alpha stimulated ChTRase release only from alveolar macrophages from smokers with COPD, and exposure of these cells to ChTRase promoted the release of IL-8, monocyte-chemoattractant protein-1, and metalloproteinase-9. Finally, ChTRase overexpression in the lung of normal mice promoted macrophage recruitment and the synthesis of the murine homologue of IL-8, keratinocyte-derived cytokine, and of monocyte-chemoattractant protein-1. We conclude that pulmonary ChTRase overexpression may represent a novel important mechanism involved in COPD onset and progression.


Assuntos
Quitinases/metabolismo , Hexosaminidases/metabolismo , Pulmão/enzimologia , Pneumonia/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Animais , Asma/metabolismo , Asma/patologia , Líquido da Lavagem Broncoalveolar/química , Células Cultivadas , Quitinases/fisiologia , Citocinas/análise , Citocinas/metabolismo , Feminino , Hexosaminidases/fisiologia , Humanos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia/metabolismo , Doença Pulmonar Obstrutiva Crônica/enzimologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Receptores de Citocinas/análise , Receptores de Citocinas/metabolismo , Fumar/metabolismo , Estudos de Validação como Assunto
17.
J Exp Med ; 206(5): 1149-66, 2009 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-19414556

RESUMO

Mouse breast regression protein 39 (BRP-39; Chi3l1) and its human homologue YKL-40 are chitinase-like proteins that lack chitinase activity. Although YKL-40 is expressed in exaggerated quantities and correlates with disease activity in asthma and many other disorders, the biological properties of BRP-39/YKL-40 have only been rudimentarily defined. We describe the generation and characterization of BRP-39(-/-) mice, YKL-40 transgenic mice, and mice that lack BRP-39 and produce YKL-40 only in their pulmonary epithelium. Studies of these mice demonstrated that BRP-39(-/-) animals have markedly diminished antigen-induced Th2 responses and that epithelial YKL-40 rescues the Th2 responses in these animals. The ability of interleukin13 to induce tissue inflammation and fibrosis was also markedly diminished in the absence of BRP-39. Mechanistic investigations demonstrated that BRP-39 and YKL-40 play an essential role in antigen sensitization and immunoglobulin E induction, stimulate dendritic cell accumulation and activation, and induce alternative macrophage activation. These proteins also inhibit inflammatory cell apoptosis/cell death while inhibiting Fas expression, activating protein kinase B/AKT, and inducing Faim 3. These studies establish novel regulatory roles for BRP-39/YKL-40 in the initiation and effector phases of Th2 inflammation and remodeling and suggest that these proteins are therapeutic targets in Th2- and macrophage-mediated disorders.


Assuntos
Apoptose/fisiologia , Glicoproteínas/genética , Inflamação/genética , Interleucina-13/genética , Adipocinas , Animais , Apoptose/genética , Asma/genética , Proteína 1 Semelhante à Quitinase-3 , Sequência Conservada , Doença das Coronárias/genética , Glicoproteínas/deficiência , Glicoproteínas/uso terapêutico , Humanos , Imunoglobulina E/imunologia , Inflamação/induzido quimicamente , Inflamação/patologia , Lectinas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina
18.
J Infect Dis ; 198(12): 1783-93, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-18980502

RESUMO

Although respiratory syncytial virus (RSV) infection is the most important cause of bronchiolitis in infants, the pathogenesis of RSV disease is poorly described. We studied histopathologic changes in a panel of lung tissue specimens obtained from infants with fatal cases of primary RSV infection. In these tissues, airway occlusion with accumulations of infected, apoptotic cellular debris and serum protein was consistently observed. Similar observations were found after RSV infection in New Zealand black (NZB) mice, which have constitutive deficiencies in macrophage function, but not in BALB/c mice. A deficiency in the number of alveolar macrophages in NZB mice appears to be central to enhanced disease, because depletion of alveolar macrophages in BALB/c mice before RSV exposure resulted in airway occlusion. In mice with insufficient numbers of macrophages, RSV infection yielded an increased viral load and enhanced expression of type I interferon-associated genes at the height of disease. Together, our data suggest that innate, rather than adaptive, immune responses are critical determinants of the severity of RSV bronchiolitis.


Assuntos
Obstrução das Vias Respiratórias/patologia , Obstrução das Vias Respiratórias/virologia , Bronquiolite/complicações , Macrófagos/fisiologia , Infecções por Vírus Respiratório Sincicial/complicações , Animais , Ácido Clodrônico/farmacologia , Humanos , Imunidade Inata , Recém-Nascido , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NZB , Vírus Sincicial Respiratório Humano
19.
J Immunol ; 181(7): 5167-73, 2008 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-18802121

RESUMO

YKL-40 is a chitin-binding protein that is elevated in patients with various inflammatory conditions associated with ongoing remodeling. We investigated whether the levels of YKL-40 were up-regulated in the circulation and the airways of patients with chronic obstructive pulmonary disease (COPD), and whether it promoted the production of inflammatory mediators from macrophages. Serum, bronchoalveolar lavage (BAL), bronchial biopsies, lung tissue specimens, and alveolar macrophages from never-smokers (n = 15), smokers without COPD (n = 20), and smokers with COPD (n = 30) were assessed for YKL-40 levels and immunolocalization. In addition, YKL-40-induced mediator release from alveolar macrophages was examined. We found that smokers with COPD had elevated levels of YKL-40 in serum (p

Assuntos
Glicoproteínas/biossíntese , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Adipocinas , Idoso , Brônquios/imunologia , Brônquios/metabolismo , Brônquios/patologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Linhagem Celular , Proteína 1 Semelhante à Quitinase-3 , Feminino , Glicoproteínas/sangue , Glicoproteínas/fisiologia , Humanos , Lectinas , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/patologia , Índice de Gravidade de Doença
20.
Mol Immunol ; 41(10): 985-1000, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15302161

RESUMO

A panel of anti-human CD2 monoclonal antibodies (mAb) and soluble human CD58 (LFA-3) were tested for binding to human peripheral blood mononuclear cells (PBMCs), recombinant human CD2 and mononuclear cells from Cynomolgus, Rhesus and African green monkey, Stump-tail, Pig-tail and Assamese macaque, Chimpanzee and Baboon. This analysis revealed that whilst some antibodies recognized all species, there were differential binding profiles with others. Three antibodies, MEDI-507, 6F10.3 and 4B2, recognized CD2 from human and Chimpanzee but not that from the other primates. We have cloned eight of the previously unknown primate CD2 molecules and report here their sequences for the first time. This analysis revealed that 12 amino acids formed a common set of residues in the extra cellular domain of human and Chimpanzee CD2. Using a "knock-in" mutagenesis approach starting with Baboon CD2, which does not bind MEDI-507, 6F10.3 and 4B2, we have identified three residues in the adhesion domain of human CD2 which are critical for its binding to these mAbs. These residues, N18, K55 and T59 define a region located outside of the previously described binding regions on CD2. Affinity measurements of the mutants revealed a variety of degrees of binding restoration for MEDI-507, 6F10.3 and 4B2, indicating that there are fine differences within a given epitope. Furthermore, the analysis of the competition of several of the anti-human CD2 antibodies with each other and CD58 demonstrated the existence of a continuum of overlapping epitopes on human CD2, which is in contrast to the commonly held belief that epitopes on human CD2 are clearly segregated.


Assuntos
Anticorpos/imunologia , Antígenos CD2/imunologia , Animais , Antígenos CD2/química , Antígenos CD2/genética , Antígenos CD58/imunologia , Mapeamento de Epitopos , Haplorrinos/genética , Haplorrinos/imunologia , Humanos , Pan troglodytes/genética , Pan troglodytes/imunologia , Análise de Sequência de Proteína , Linfócitos T/imunologia
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