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1.
Adv Exp Med Biol ; 1140: 377-388, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347059

RESUMO

Identifying antigen-antibody interactions have been shown as a critical step in understanding the proteins biological functions and their involvement in various pathological conditions. While many techniques have been developed to characterize antigen-antibody interactions, one strategy that has gained considerable momentum over the last decade for the identification and quantification of antigen-antibody interactions, is immune affinity-chromatography followed by mass spectrometry. Moreover, the combination of enzymatic digestion of antigens and mass spectrometric identification of specific binding peptide(s) to the corresponding anti-antigen antibody has become a versatile and clinical relevant method for mapping epitopes by mass spectrometry. In this chapter, the development and applications of novel immunoaffinity mass spectrometric methodologies for elucidating biomedical aspects will be presented. First, a simplified mass spectrometric approach that maps an epitope from a digested antigen solution without immobilizing the anti-antigen antibody on a solid support will be reported. iMALDI (from immunoaffinity and MALDI, matrix-assisted laser desorption/ionization), a technique that involves immunoaffinity capture of specific peptides and direct MALDI measurements was used for absolute quantification of serine/threonine-specific protein kinase (AKT) peptides from breast cancer and colon cancer cell lines and flash-frozen tumor lysates. The intact transition epitope mapping (ITEM) was shown as a rapid and accurate epitope mapping method by using Ion mobility mass spectrometry (IMS-MS) for analysing the antigen peptide-containing immune complex previously generated by in solution epitope extraction/excision procedures.


Assuntos
Complexo Antígeno-Anticorpo/análise , Mapeamento de Epitopos , Peptídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Antígenos de Neoplasias/análise , Epitopos , Humanos
2.
Pharm Res ; 36(9): 129, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31254106

RESUMO

PURPOSE: Immunogenicity against biotherapeutics can lead to the formation of drug/anti-drug-antibody (ADA) immune complexes (ICs) with potential impact on safety and drug pharmacokinetics (PK). This work aimed to generate defined drug/ADA ICs, characterized by quantitative (bio) analytical methods for dedicated determination of IC sizes and IC profile changes in serum to facilitate future in vivo studies. METHODS: Defined ICs were generated and extensively characterized with chromatographic, biophysical and imaging methods. Quantification of drug fully complexed with ADAs (drug in ICs) was performed with an acid dissociation ELISA. Sequential coupling of SEC and ELISA enabled the reconstruction of IC patterns and thus analysis of IC species in serum. RESULTS: Characterization of generated ICs identified cyclic dimers, tetramers, hexamers, and larger ICs of drug and ADA as main IC species. The developed acid dissociation ELISA enabled a total quantification of drug fully complexed with ADAs. Multiplexing of SEC and ELISA allowed unbiased reconstruction of IC oligomeric states in serum. CONCLUSIONS: The developed in vitro IC model system has been properly characterized by biophysical and bioanalytical methods. The specificity of the developed methods enable discrimination between different oligomeric states of ICs and can be bench marking for future in vivo studies with ICs.


Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/análise , Animais , Anticorpos Monoclonais/sangue , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/química , Cromatografia Líquida , Dimerização , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/química , Conformação Proteica , Ratos Wistar , Soroalbumina Bovina/química
3.
J Immunoassay Immunochem ; 39(5): 471-484, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30188776

RESUMO

Hafnium(IV) oxide is a material with properties that can increase the sensitivity, durability, and reliability of biosensors made from silicon dioxide and other semiconductor materials due to its high dielectric constant, thermodynamic stability, and the simplicity with which it can be deposited. This work describes the use of this material in biosensors based on field-effect transistors to detect ions and DNA, in immunosensors to detect an antigen-antibody complex, its use as a contrast material in computed tomography scans and the possibility of using it in optic biosensors in the infrared region. Its low cost and versatility in the field of biosensors is underscored.


Assuntos
Técnicas Biossensoriais , Háfnio/química , Óxidos/química , Tomógrafos Computadorizados , Complexo Antígeno-Anticorpo/análise , Complexo Antígeno-Anticorpo/imunologia , DNA/análise , Humanos , Íons/análise
4.
Histopathology ; 72(4): 601-608, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28881045

RESUMO

AIMS: The technique used for classification of membranoproliferative glomerulonephritis (MPGN) has been changed from an electron microscopy-based to an immunofluorescence (IF)-based semiquantitative technique with immunoperoxidase (IP) staining as a backup option when IF is not possible. Since data on that matter is lacking, our aims were to study the interobserver variability, the correlation and the reclassification of MPGN based on these two techniques. METHODS AND RESULTS: We retrospectively analysed cases of type 1 MPGN. We repeated IF staining and performed IP staining for IgG, kappa, lambda, C3c and C4d in 35 renal biopsies, among which 19 biopsies had matched IP and IF samples. We observed substantial to near-perfect agreement among the seven observers for both IF and IP (W coefficients from 0.66 for IF lambda to 0.89 for IF C4d). Of the 19 cases with matched IP and IF samples, five (26%) turned out to have different diagnoses on IF and on IP. Also, the ability of C4d to discriminate immune complex-mediated glomerulonephritis (ICGN) from C3 glomerulopathy (C3G) was poor, with areas under the curve of 0.44 [95% confidence interval (CI) 0.24-0.63] and 0.66 (95% CI 0.50-0.81) for the receiver operating characteristic curves of IF and IP respectively. Limitations include the fact that no clinical data regarding complement activation were available. CONCLUSION: The diagnosis of ICGN versus C3GN depends on the immunochemical technique used. Also, the use of C4d failed to discriminate ICGN from C3G in our study. Further validation studies are required to avoid misdiagnosis based on kidney biopsy.


Assuntos
Imunofluorescência/métodos , Glomerulonefrite Membranoproliferativa/diagnóstico , Técnicas Imunoenzimáticas/métodos , Adolescente , Adulto , Idoso , Complexo Antígeno-Anticorpo/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
5.
Int J Rheum Dis ; 21(1): 194-199, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28762647

RESUMO

BACKGROUND: Lupus nephritis (LN) is a feared complication of systemic lupus erythematosus (SLE). Renal biopsy is valuable to assess disease severity and prognosis, but no histological data are available for Indigenous Australians (IA). We compared histopathology between IA and non-IA patients (NI) with LN in northern Australia and describe main outcomes. METHODS: Retrospective cohort study of all patients with biopsy evidence of LN at Royal Darwin Hospital over a 10-year period. Biopsies were classified by International Society of Nephrology criteria with clinical finding and vital status obtained from electronic health records. Data analyses used Australian Bureau of Statistics 2011 census population, nonparametric testing and lifetable estimates. RESULTS: The study cohort contained 42 patients (mean age 30 years,86% female and 74% IA). The estimated annual incidence of biopsy-proven LN was 7/100 000 for IA versus 0.7/100 000 for NI (P < 0.01). More IA patients had full-house immune complex deposition (79% vs. 21%, P < 0.05), but fewer IA patients had proliferative LN (classes III + IV) (42% vs. 72%) (P < 0.01). Five and 10-year patient (69% and 50%) and renal survival (87% and 53%) in IA were much worse than for NI patients. The reported causes of death were infections (38.6%), end-stage renal disease (23%), cardiovascular events (15.4%). CONCLUSION: Indigenous Australians more frequently have histological evidence of LN with a broader spectrum of immune complex deposition but less severe renal inflammation compared to non-Indigenous patients. The relative contribution of LN to reduced patient and renal survival for Indigenous Australians thus requires further study.


Assuntos
Rim/patologia , Nefrite Lúpica/etnologia , Nefrite Lúpica/patologia , Grupo com Ancestrais Oceânicos , Adulto , Complexo Antígeno-Anticorpo/análise , Biópsia , Causas de Morte , Feminino , Humanos , Incidência , Estimativa de Kaplan-Meier , Rim/imunologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/mortalidade , Masculino , Northern Territory/epidemiologia , Prognóstico , Estudos Retrospectivos , Fatores de Tempo
6.
Parasit Vectors ; 10(Suppl 2): 481, 2017 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-29143646

RESUMO

BACKGROUND: Antigen testing is routinely used to diagnose canine Dirofilaria immitis infections. Immune complex dissociation (ICD) methods, which were employed in the original heartworm antigen tests to release antigen that was bound by endogenous canine antibodies, were discontinued with improvements in assay reagents. The purpose of this study was to evaluate different ICD methods for detection of heartworm antigen by microtiter plate ELISA and assess the performance in samples from pet dogs. METHODS: The original PetChek® Heartworm Test (IDEXX Laboratories, Inc.) utilized pepsin at an acidic pH for ICD prior to antigen testing. Performance and characteristics of the pepsin ICD method were compared with those for heat treatment (with and without EDTA) and acid treatment. RESULTS: All four methods released complexed antigen in serum samples when tested using microtiter plate ELISA. Heat treatment required ≥600 µL of serum or plasma, whereas pepsin and acid methods needed only a 50-µL sample. Samples from 1115 dogs submitted to IDEXX Laboratories between 2014 and 2016 for investigation of discrepant heartworm results were evaluated with and without pepsin ICD using the PetChek Heartworm Test. Samples from 10% (n = 112) of the dogs were antigen positive with the ICD protocol only while 90% of the results remained unchanged. In a prospective study, antigen levels with and without ICD were evaluated for 12 dogs receiving pre-adulticide heartworm treatment with a macrocyclic lactone and doxycycline for 28 days. Serial samples revealed that three dogs had a reduction in detectable heartworm antigen within 4 weeks of initiating treatment. In these cases, heartworm antigen levels could be recovered with ICD. CONCLUSIONS: Heartworm antigen testing with ICD can be a valuable diagnostic tool for patients with discrepant results that have had intermittent use of a preventive, or have been treated with a macrocyclic lactone and doxycycline. Heartworm therapies may reduce antigen production and favor immune complexing in some dogs, resulting in false-negative results. Therefore, it is important to confirm positive heartworm antigen test results before initiating therapy.


Assuntos
Complexo Antígeno-Anticorpo/análise , Antígenos de Helmintos/análise , Dirofilaria immitis/isolamento & purificação , Dirofilariose/diagnóstico , Doenças do Cão/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Complexo Antígeno-Anticorpo/imunologia , Antígenos de Helmintos/imunologia , Dirofilaria immitis/efeitos dos fármacos , Dirofilaria immitis/imunologia , Dirofilariose/tratamento farmacológico , Dirofilariose/imunologia , Dirofilariose/parasitologia , Doenças do Cão/tratamento farmacológico , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Cães , Filaricidas/administração & dosagem , Lactonas/administração & dosagem , Estudos Prospectivos
7.
Bioanalysis ; 9(20): 1603-1615, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29072493

RESUMO

AIM: Development of drug-related surrogate positive controls for isotype anti-drug antibodies assay remains challenging. Efforts on antibody engineering or chemical crosslinking have been made. However, multiple epitope recognition, purity and stability are often of concern. To tackle these challenges, we used LC-SPDP/SMPB crosslinking to conjugate polyclonal anti-drug IgG and human immunoglobulin isotype (hIgE, hIgA, hIgM or hIgG4) with optimized conditions. RESULTS: The final product was a hybrid of anti-drug IgG cross-linked to human isotype immunoglobulin through stable thioether bond. The characteristics of the hybrids and their performance as assay positive controls were further evaluated. CONCLUSION: The results demonstrate that LC-SPDP/SMPB chemical crosslinking method is suitable for generating stable hybrids to be used as assay positive controls in isotype anti-drug antibodies assay.


Assuntos
Anticorpos/análise , Cromatografia em Gel , Isotipos de Imunoglobulinas/análise , Anticorpos/química , Anticorpos/metabolismo , Complexo Antígeno-Anticorpo/análise , Ensaio de Imunoadsorção Enzimática , Humanos , Isotipos de Imunoglobulinas/química , Maleimidas/química , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Sulfetos/química
8.
MAbs ; 9(4): 664-679, 2017 May/Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28387583

RESUMO

A number of studies have attempted to elucidate the binding mechanism between tumor necrosis factor (TNF) and clinically relevant antagonists. None of these studies, however, have been conducted as close as possible to physiologic conditions, and so the relationship between the size distribution of TNF-antagonist complexes and the antagonists' biological activity or adverse effects remains elusive. Here, we characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum and in phosphate-buffered saline (PBS). Fluorescence-detected sedimentation velocity analytical ultracentrifugation analyses revealed that adalimumab and infliximab formed a range of complexes with TNF, with the major complexes consisting of 3 molcules of the respective antagonist and one or 2 molcules of TNF. Considerably greater amounts of high-molecular-weight complexes were detected for infliximab in human serum. The emergence of peaks with higher sedimentation coefficients than the adalimumab monomer as a function of added human serum albumin (HSA) concentration in PBS suggested weak reversible interactions between HSA and immunoglobulins. Etanerept exclusively formed 1:1 complexes with TNF in PBS, and a small amount of complexes with higher stoichiometry was detected in human serum. Consistent with these biophysical characterizations, a reporter assay showed that adalimumab and infliximab, but not etanercept, exerted FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab. This study shows that assessing distribution profiles in serum will contribute to a more comprehensive understanding of the in vivo behavior of therapeutic proteins.


Assuntos
Adalimumab/análise , Complexo Antígeno-Anticorpo/análise , Fluorescência , Infliximab/análise , Fator de Necrose Tumoral alfa/análise , Adalimumab/química , Complexo Antígeno-Anticorpo/química , Humanos , Infliximab/química , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Fator de Necrose Tumoral alfa/química , Ultracentrifugação
9.
PLoS One ; 12(2): e0171447, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28199361

RESUMO

The innate immune system of insects responds to wounding and pathogens by mobilizing multiple pathways that provide both systemic and localized protection. Key localized responses in hemolymph include melanization, coagulation, and hemocyte encapsulation, which synergistically seal wounds and envelop and destroy pathogens. To be effective, these pathways require a targeted deposition of their components to provide protection without compromising the host. Extensive research has identified a large number of the effectors that comprise these responses, but questions remain regarding their post-translational processing, function, and targeting. Here, we used mass spectrometry to demonstrate the integration of pathogen recognition proteins, coagulants, and melanization components into stable, high-mass, multi-functional Immune Complexes (ICs) in Bombyx mori and Aedes aegypti. Essential proteins common to both include phenoloxidases, apolipophorins, serine protease homologs, and a serine protease that promotes hemocyte recruitment through cytokine activation. Pattern recognition proteins included C-type Lectins in B. mori, while A. aegypti contained a protein homologous to Plasmodium-resistant LRIM1 from Anopheles gambiae. We also found that the B. mori IC is stabilized by extensive transglutaminase-catalyzed cross-linking of multiple components. The melanization inhibitor Egf1.0, from the parasitoid wasp Microplitis demolitor, blocked inclusion of specific components into the IC and also inhibited transglutaminase activity. Our results show how coagulants, melanization components, and hemocytes can be recruited to a wound surface or pathogen, provide insight into the mechanism by which a parasitoid evades this immune response, and suggest that insects as diverse as Lepidoptera and Diptera utilize similar defensive mechanisms.


Assuntos
Aedes/metabolismo , Complexo Antígeno-Anticorpo/análise , Bombyx/metabolismo , Hemócitos/metabolismo , Proteínas de Insetos/metabolismo , Aedes/citologia , Aedes/crescimento & desenvolvimento , Aedes/imunologia , Animais , Complexo Antígeno-Anticorpo/metabolismo , Apolipoproteínas/análise , Apolipoproteínas/metabolismo , Bombyx/citologia , Bombyx/crescimento & desenvolvimento , Bombyx/imunologia , Cromatografia Líquida de Alta Pressão , Coagulantes/química , Coagulantes/metabolismo , Citocinas/metabolismo , Hemócitos/citologia , Proteínas de Insetos/análise , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia , Larva , Lectinas/análise , Lectinas/metabolismo , Melaninas/química , Melaninas/metabolismo , Peptídeo Hidrolases/análise , Peptídeo Hidrolases/metabolismo , Peptídeos/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas em Tandem , Transglutaminases/metabolismo
10.
Elife ; 52016 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-28035901

RESUMO

Despite the central role that antibodies play in the adaptive immune system and in biotechnology, much remains unknown about the quantitative relationship between an antibody's amino acid sequence and its antigen binding affinity. Here we describe a new experimental approach, called Tite-Seq, that is capable of measuring binding titration curves and corresponding affinities for thousands of variant antibodies in parallel. The measurement of titration curves eliminates the confounding effects of antibody expression and stability that arise in standard deep mutational scanning assays. We demonstrate Tite-Seq on the CDR1H and CDR3H regions of a well-studied scFv antibody. Our data shed light on the structural basis for antigen binding affinity and suggests a role for secondary CDR loops in establishing antibody stability. Tite-Seq fills a large gap in the ability to measure critical aspects of the adaptive immune system, and can be readily used for studying sequence-affinity landscapes in other protein systems.


Assuntos
Complexo Antígeno-Anticorpo/análise , Ensaios de Triagem em Larga Escala , Oligonucleotídeos/química , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/química , Sequência de Aminoácidos , Afinidade de Anticorpos , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Humanos , Cinética , Modelos Moleculares , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Relação Estrutura-Atividade
11.
Electrophoresis ; 37(15-16): 2163-9, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27066909

RESUMO

Herein, we have developed an in-capillary assay for simultaneous detection of the assembly and disassembly of the multivalent HA tag peptide and antibody. HA tag with hexahistidine at C terminus (YPYDVPDYAG4 H6 , termed YPYDH6 ) was conjugated with quantum dots (QDs) by metal-affinity force to form a multivalent HA tag (QD-YPYDH6 ). QD-YPYDH6 and monoclonal anti-HA antibody (anti-HA) were sequentially injected into the capillary. They were mixed and assembled inside the capillary. The reaction products were online discriminated and detected by fluorescence coupled capillary electrophoresis (CE-FL). For the in-capillary assay, the binding efficiency of the multivalent HA tag and antibody on was influenced by the molar ratio and injection time. Such novel assay could even give out the self-assembly kinetic constant of QDs and YPYDH6 as KD of 34.1 µM with n (binding cooperativeness) of 2.2 by Hill equation. More importantly, the simultaneous detection of the assembly and imidazole (Im) induced disassembly of the QD-YPYDH6 -anti-HA complex was achieved in a single in-capillary assay. Our study demonstrated a new method for the online detection of antigen-antibody interactions.


Assuntos
Complexo Antígeno-Anticorpo/análise , Eletroforese Capilar/métodos , Pontos Quânticos/química , Anticorpos Monoclonais , Fluorescência , Histidina/imunologia , Oligopeptídeos/imunologia
12.
AIDS Res Hum Retroviruses ; 32(8): 756-62, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26988426

RESUMO

BACKGROUND: Accurate methods for cross-sectional incidence estimation are needed for HIV surveillance and prevention research. We developed an avidity assay based on the fourth-generation Genetic Systems HIV Combo Ag/Ab EIA (Bio-Rad Combo assay) and evaluated its performance. MATERIALS AND METHODS: The Bio-Rad Combo assay was modified incubating samples with and without 0.025 M diethylamine (DEA). The avidity index (AI) was calculated as the ratio of the DEA-treated to untreated result for a specific sample. We analyzed 2,140 samples from 808 individuals from the United States with known duration of HIV infection. The mean duration of recent infection (MDRI) and the false-recent rate (FRR, fraction of samples from individuals known to be infected >2 years misclassified as recent) were calculated for AI cutoffs of 20%-90% for the avidity assay alone and in combination with a viral load assay (VL, limit of detection 400 copies/ml). Factors associated with misclassification of samples collected ≥2 years after infections were also evaluated. RESULTS: The MDRI for the Bio-Rad Combo Avidity assay ranged from 50 days using an AI cutoff of 20% to 276 days using an AI cutoff of 90%; the FRR ranged from 0% to 9%. When samples with a VL <400 copies/ml were classified as nonrecent, the FRRs were reduced approximately twofold and the MDRI estimates were reduced by ∼20%. An AI cutoff of 50% provided an MDRI of 135 days with an FRR of 2.1%. All samples from elite suppressors had an AI >80%. In adjusted analysis, viral suppression and low CD4 cell count were significantly associated with misclassification among individuals infected >2 years. CONCLUSIONS: This modified Bio-Rad Combo Avidity assay may be a useful tool for cross-sectional HIV incidence estimation. Further research is needed to evaluate use of this assay in combination with other assays to accurately estimate population-level HIV incidence.


Assuntos
Complexo Antígeno-Anticorpo/análise , Anticorpos Anti-HIV/sangue , Antígenos HIV/sangue , Infecções por HIV/diagnóstico , HIV-1/isolamento & purificação , Técnicas Imunoenzimáticas/normas , Adulto , Afinidade de Anticorpos , Complexo Antígeno-Anticorpo/biossíntese , Contagem de Linfócito CD4 , Estudos Transversais , Dietilaminas/química , Feminino , Anticorpos Anti-HIV/química , Antígenos HIV/química , Infecções por HIV/sangue , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Estados Unidos/epidemiologia , Carga Viral/imunologia
13.
Clin Exp Immunol ; 185(1): 72-80, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26953930

RESUMO

The aim of this study was to provide proof-of-concept for quantitative and qualitative label-free detection of immune complexes through myeloid cells with imaging surface plasmon resonance. Surface plasmon resonance imaging was first applied to monitor the binding of human sera from healthy and rheumatoid arthritis (RA) patients to immobilized citrullinated RA-specific peptide antigens, histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Next, the binding of monocytoid cell line U937 to the resulting immune complexes on the sensor surface was monitored. As control, binding of U937 was monitored to immunoglobulin (Ig)G subclasses simultaneously. Cell response results were compared to results of cyclic citrullinated peptide 2 (CCP2) enzyme-linked immunosorbent assay (ELISA), clinical RA diagnosis and antigen-specific antibody distribution of the samples. Human IgG3 triggered the most pronounced response, followed by IgG1 and IgG4, while IgG2 did not result in U937 cell binding. Serum samples obtained from RA patients resulted in a significantly increased cell response to VCP2 compared to healthy controls. The strength of cell response towards VCP2 immune complexes showed significant correlation with levels of antigen-specific IgA, IgG and IgG3. Cellular responses on VCP2 immune complexes showed significant association with both CCP2-based serological positivity and European League Against Rheumatism (EULAR) criteria-based clinical RA diagnosis. Immunoglobulin-triggered binding of monocytoid cells can be monitored using a label-free multiplex technology. Because these binding events are presumably initiated by Fc receptors, the system provides a tool for biological detection of autoantibodies with diagnostic value, here exemplified by anti-citrullinated antibodies. This provides added information to antibody levels, as interaction with Fc-receptor-expressing cells is also affected by post-translational modification of the immunoglobulins.


Assuntos
Complexo Antígeno-Anticorpo/análise , Artrite Reumatoide/diagnóstico , Autoanticorpos/metabolismo , Fragmentos de Peptídeos/sangue , Peptídeos Cíclicos/sangue , Proteínas Virais/sangue , Complexo Antígeno-Anticorpo/metabolismo , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Autoanticorpos/química , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas Imobilizadas/sangue , Proteínas Imobilizadas/química , Soros Imunes/química , Imunoglobulina G/sangue , Fragmentos de Peptídeos/química , Peptídeos Cíclicos/química , Ligação Proteica , Ressonância de Plasmônio de Superfície , Células U937 , Proteínas Virais/química
14.
J Diabetes Complications ; 30(4): 693-9, 2016 May-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26861948

RESUMO

BACKGROUND: Circulating immune complexes (IC) containing modified forms of LDL (mLDL) are strongly pro-inflammatory and when present in high levels are associated with the development of diabetic complications. OBJECTIVE: We investigated whether levels of oxidized LDL (oxLDL), malondialdehyde-LDL (MDA-LDL) and advanced glycation end products-LDL (AGE-LDL) as well as IgG and IgM antibodies reacting with MDA-lysine epitopes expressed by oxLDL and MDA-LDL isolated from circulating IC were associated with progression to macroalbuminuria in type 2 diabetes (VADT cohort). METHODS: Levels of mLDL in IC were measured in 905 patients, a median of two years after entry into the study. Participants were followed for an average of 3.7years for renal outcomes. Generalized logistic regression models were used to quantify the association of increased levels of biomarkers and development of abnormal albuminuria. Normal, persistent micro- (ACR ≥30), incident micro- (ACR ≥30) and incident macroalbuminuria (ACR ≥300) were the outcomes of interest. RESULTS AND CONCLUSIONS: Patients with macro (n=78) or non-persistent microalbuminuria (n=81) at baseline were excluded. Odds ratios for endpoints in relation to high versus low (defined using a median split) biomarker levels are found in Fig. 1. Our study demonstrates that high levels of AGE-LDL as well as of IgG antibodies (but not IgM antibodies) reacting with MDA-LDL lysine epitopes in circulating IC predict the development of macroalbuminuria in patients with type 2 diabetes. These data support the pathogenic role of modified LDL IgG antibodies but not the protective role of modified LDL IgM antibodies.


Assuntos
Albuminúria/etiologia , Complexo Antígeno-Anticorpo/análise , Autoanticorpos/análise , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/fisiopatologia , Produtos Finais de Glicação Avançada/sangue , Insuficiência Renal/fisiopatologia , Albuminúria/diagnóstico , Albuminúria/epidemiologia , Biomarcadores/sangue , Estudos de Coortes , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/imunologia , Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/epidemiologia , Nefropatias Diabéticas/imunologia , Progressão da Doença , Epitopos , Feminino , Seguimentos , Humanos , Imunoglobulina G/análise , Incidência , Lipoproteínas LDL/antagonistas & inibidores , Lipoproteínas LDL/sangue , Masculino , Malondialdeído/antagonistas & inibidores , Malondialdeído/sangue , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Insuficiência Renal/complicações , Insuficiência Renal/diagnóstico , Insuficiência Renal/epidemiologia , South Carolina/epidemiologia
15.
J Helminthol ; 90(6): 773-778, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26884092

RESUMO

Ocular lesions have been reported in patients with schistosomiasis; however, the problem with studying schistosomal infection of the human eye is that biopsies are almost impossible to take, and histopathological examination of suspicious lesions can only be undertaken post-mortem or after enucleation. This work aimed to study the possible effects and pathogenesis of schistosomiasis on the eye. This study involved 55 hamsters; five hamsters remained non-infected and the remaining 50 hamsters were infected with Schistosoma mansoni cercariae. Infected hamsters were sacrificed on weeks 8, 12, 16 and 20 post-infection (pi). Eye sections were prepared and stained for histopathological and immunohistochemical studies. Histopathological changes detected in hamsters infected after 16 and 20 weeks included looseness and oedema of the innermost retinal layers together with hyperplastic polypoid growth. Neither eggs nor granulomata were detected in eye sections throughout the experimental period. Deposition of S. mansoni antigen was revealed in 35% of infected hamsters. Later, on weeks 16 and 20 pi, moderate subepithelial conjuctival deposits and marked subchoroidal and scleral deposition were detected. In conclusion, the deposition of schistosomal antigen and immune complexes may play a pivotal role in the ocular changes that occur in schistosomiasis, even in the absence of detectable Schistosoma eggs. Schistosomiasis should be suspected in cases with unexplained ophthalmological findings, especially in endemic areas.


Assuntos
Oftalmopatias/patologia , Oftalmopatias/parasitologia , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomose mansoni/patologia , Esquistossomose mansoni/parasitologia , Animais , Complexo Antígeno-Anticorpo/análise , Antígenos de Helmintos/análise , Cricetinae , Modelos Animais de Doenças , Histocitoquímica , Imuno-Histoquímica , Microscopia , Fatores de Tempo
16.
ACS Nano ; 10(1): 1640-7, 2016 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-26690745

RESUMO

Point-of-care (POC) testing has the potential to enable rapid, low-cost, and large-scale screening. POC detection of a multiplexed biomarker panel can facilitate the early diagnosis of non-small cell lung cancer (NSCLC) and, thus, may allow for more timely surgical intervention for life-saving treatment. Herein, we report the nanoporous glass (NPG) integrated volumetric bar-chart chip (V-Chip) for POC detection of the three NSCLC biomarkers CEA, CYFRA 21-1, and SCCA, by the naked eye. The 3D nanostructures in the NPG membrane efficiently increase the number of binding sites for antibodies and decrease the diffusion distance between antibody and antigen, enabling the low detection limit and rapid analysis time of the NPG-V-Chip. We utilized the NPG-V-Chip to test the NSCLC biomarker panel and found that the limit of detection can reach 50 pg/mL (10-fold improvement over the original V-Chip), and the total assay time can be decreased from 4 to 0.5 h. We then detected CEA in 21 serum samples from patients with common cancers, and the on-chip results showed good correlation with the clinical results. We further assayed 10 lung cancer samples using the device and confirmed the results obtained using conventional ELISA methods. In summary, the NPG-V-Chip platform has the ability of multiplex, low detection limit, low cost, lack of need for accessory equipment, and rapid analysis time, which may render the V-Chip a useful platform for quantitative POC detection in resource-limited settings and personalized diagnostics.


Assuntos
Anticorpos Antineoplásicos/análise , Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Antígeno Carcinoembrionário/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Queratina-19/genética , Neoplasias Pulmonares/diagnóstico , Serpinas/genética , Complexo Antígeno-Anticorpo/análise , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Antígeno Carcinoembrionário/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Diagnóstico Precoce , Expressão Gênica , Vidro/química , Humanos , Queratina-19/metabolismo , Dispositivos Lab-On-A-Chip , Limite de Detecção , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Nanoestruturas/química , Sistemas Automatizados de Assistência Junto ao Leito , Porosidade , Serpinas/metabolismo
17.
J Biol Chem ; 291(3): 1368-86, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26582197

RESUMO

CD4(+) T-cells in systemic lupus erythematosus (SLE) patients show altered T-cell receptor signaling, which utilizes Fc-receptor γ-chain FcRγ-Syk. A role for FcγRIIIa activation from immune complex (IC) ligation and sublytic terminal complement complex (C5b-9) in CD4(+) T-cell responses is not investigated. In this study, we show that the ICs present in SLE patients by ligating to FcγRIIIa on CD4(+) T-cells phosphorylate Syk and provide a co-stimulatory signal to CD4(+) T-cells in the absence of CD28 signal. This led to the development of pathogenic IL-17A(+) and IFN-γ(high) CD4(+) T-cells in vitro. Cytokines IL-1ß, IL-6, TGF-ß1, and IL-23 were the only requirement for the development of both populations. SLE patients CD4(+) T-cells that expressed CD25, CD69, and CD98 bound to ICs showed pSyk and produced IFN-γ and IL-17A. This FcγRIIIa-mediated co-signal differentially up-regulated the expression of IFN pathway genes compared with CD28 co-signal. FcγRIIIa-pSyk up-regulated several toll-like receptor genes as well as the HMGB1 and MyD88 gene transcripts. ICs co-localized with these toll-like receptor pathway proteins. These results suggest a role for the FcγRIIIa-pSyk signal in modulating adaptive immune responses.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Ativação Linfocitária , Proteínas Tirosina Quinases/metabolismo , Receptores de IgG/metabolismo , Receptores Toll-Like/agonistas , Imunidade Adaptativa , Complexo Antígeno-Anticorpo/análise , Complexo Antígeno-Anticorpo/isolamento & purificação , Complexo Antígeno-Anticorpo/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Células Cultivadas , Complexo de Ataque à Membrana do Sistema Complemento/análise , Complexo de Ataque à Membrana do Sistema Complemento/isolamento & purificação , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Proteína HMGB1/agonistas , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/sangue , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fator 88 de Diferenciação Mieloide/agonistas , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/sangue , Receptores de IgG/sangue , Receptores de Interleucina-1/agonistas , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Quinase Syk , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Regulação para Cima
18.
J Immunol Methods ; 428: 58-68, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26643682

RESUMO

We herein established a new method to evaluate allergic responses in mice rapidly and easily with ethical improvement by reducing the number of animals used. A single intravenous injection of a mixture of anti-OVA monoclonal IgE and fluorescein-ovalbumin (FITC-OVA) induced the distinctive spotted distribution of FITC-OVA in skin, named "ASDIS (Anaphylaxis-dependent Spotted Distribution of a fluorescent-labeled Immune complex in Skin)", and this was easily detected by in vivo imaging. The parallel induction of hypothermia, scratching, serum histamine increases, and ASDIS as well as the inhibition of ASDIS by either the systemic administration of a histamine H1 receptor antagonist or mast cell-depleting antibody suggested that our method, which only required 15 min, induced these allergic responses including ASDIS. Relatively mild but significant ASDIS was induced also in mice with passive systemic anaphylaxis by the method, requiring 2 separate days. The painting of anti-histamines on the skin markedly reduced ASDIS in the painted area only, suggesting the potential of this model to simultaneously compare the anti-allergic effects of several candidate compounds with control drugs in the same mice. ASDIS was suggested to originate from extravasated FITC-OVA/OE-1 immune complexes from blood to skin tissues other than mast cells. Our new method has the advantages of rapidity, easy method, and lower animal numbers to evaluate anti-allergic compounds as well as the characteristics of the used antibody, antigen, labeling molecules, additives, and other formulations. Our model for inducing ASDIS may contribute to the development of anti-allergic drugs, especially those intended for application to the skin.


Assuntos
Alérgenos/imunologia , Anafilaxia/tratamento farmacológico , Antialérgicos/administração & dosagem , Antialérgicos/farmacologia , Complexo Antígeno-Anticorpo/análise , Ovalbumina/imunologia , Pele/efeitos dos fármacos , Administração Tópica , Alérgenos/administração & dosagem , Anafilaxia/imunologia , Animais , Antialérgicos/uso terapêutico , Complexo Antígeno-Anticorpo/imunologia , Fluorescência , Imunoglobulina E/administração & dosagem , Imunoglobulina E/imunologia , Injeções Intravenosas , Camundongos , Camundongos Pelados , Camundongos Endogâmicos DBA , Ovalbumina/administração & dosagem , Pele/imunologia
19.
J Biochem ; 158(5): 367-72, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26381536

RESUMO

The major function of major histocompatibility complex (MHC) class II molecules is the presentation of peptide antigens to helper T cells. However, when misfolded proteins are associated with MHC class II molecules in the endoplasmic reticulum, they are transported to the cell surface by MHC class II molecules without processing to peptides. Of note, misfolded proteins complexed with MHC class II molecules are specifically recognized by autoantibodies produced in patients with autoimmune diseases such as rheumatoid arthritis and antiphospholipid syndrome. Furthermore, autoantibody binding to misfolded proteins complexed with MHC class II molecules is associated with the susceptibility to autoimmune diseases conferred by each MHC class II allele. Therefore, misfolded proteins rescued from degradation by MHC class II molecules may be recognized as 'neo-self' antigens by the immune system and be involved in the pathogenicity of autoimmune diseases.


Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Autoanticorpos/metabolismo , Doenças Autoimunes/metabolismo , Genes MHC da Classe II , Modelos Biológicos , Deficiências na Proteostase/metabolismo , Animais , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/análise , Autoanticorpos/análise , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Predisposição Genética para Doença , Humanos , Polimorfismo Genético , Transporte Proteico , Proteólise , Deficiências na Proteostase/genética , Deficiências na Proteostase/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo
20.
Thromb Haemost ; 114(6): 1127-35, 2015 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-26245154

RESUMO

Development of inhibitory antibodies against factor VIII (FVIII) is a severe complication of replacement therapy in haemophilia A. Patients with inhibitors are treated with high FVIII doses in the context of immune tolerance therapy (ITT). Data from haemophilia A mouse model suggest that high FVIII concentrations prevent the formation of antibody secreting cells (ASCs) from memory B cells (MBCs) by inducing apoptosis. Fc gamma receptor 2B (CD32) is an important regulator of B cell function, mediating inhibitory signals after cross-linking with the B cell receptor. Here, the role of CD32 in the regulation of FVIII-specific MBCs was investigated using F8-/- and F8-/-CD32-/- knockout mice and monoclonal antibodies (mAbs). The initial immune response was similar between F8-/- and F8-/-CD32-/- mice, including concentration of anti-FVIII antibodies and number of FVIII-specific ASCs in spleen and bone marrow. In contrast, formation of ASCs from MBCs upon rhFVIII re-stimulation in vitro was abolished in F8-/-CD32-/- mice, whereas FVIII/anti-FVIII immune complexes significantly enhanced ASC formation in F8-/- mice. Inhibition of CD32 by mAbs or F(ab)2 fragments prevented ASC formation in a dose-dependent manner. Transfer of B cell-depleted splenocytes using CD45R (B220) depletion from CD32-competent mice did not restore ASC formation in F8-/-CD32-/- cells confirming that CD32 is required on B cells. We conclude that CD32 is a crucial regulator of FVIII-specific B cells and is required for the differentiation of MBCs into ASCs. Inhibition of CD32 could potentially improve the efficacy of FVIII in the context of ITT.


Assuntos
Subpopulações de Linfócitos B/imunologia , Fator VIII/imunologia , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Receptores de IgG/fisiologia , Transferência Adotiva , Animais , Anticorpos Heterófilos/biossíntese , Anticorpos Heterófilos/imunologia , Anticorpos Monoclonais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Complexo Antígeno-Anticorpo/análise , Apoptose , Medula Óssea/imunologia , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Fator VIII/genética , Fator VIII/farmacologia , Fator VIII/uso terapêutico , Hemofilia A/imunologia , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Knockout , Receptores de IgG/antagonistas & inibidores , Receptores de IgG/deficiência , Receptores de IgG/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Baço/imunologia
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