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1.
Cell Physiol Biochem ; 53(1): 229-241, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31302949

RESUMO

BACKGROUND/AIMS: Circulating or extracellular histones (EHs) in the bloodstream act as a damage-associated-molecular-pattern (DAMP) agent that plays a critical role in the pathogenesis of many diseases such as sepsis and sterile inflammation. To date, not much information is available to describe the mechanistic relationship between human erythrocytes and the cytotoxicity of EHs, the protein members from a highly conserved histone family across species. The present study explored this key question with a hypothesis that EHs induce eryptosis. METHODS: Freshly isolated human red blood cells (RBCs) from healthy donors were treated with EHs or agents for positive controls in a physiological buffer for 3 or 24 h. After treatments, flow cytometry was employed to quantify surface phosphatidylserine (PS) exposure from annexin-V-RFP binding, cell shrinkage from flow cytometric forward scatter (FSC) analysis, Ca2+ rise by fluo-4, reactive oxygen species (ROS) production by H2DCFDA, and caspase-3 activation by FAM-DEVD-FMK measurement. Hemolysis and membarne permeabilization were estimated respectively from hemoglobin release into supernatant and calcein leakage from RBC ghosts. RESULTS: With positive controls for validation, EHs in the pathophsyiological range were found to accumulate annexin-V binding on cell surface, decrease FSC, upregulate ROS production, elevate Ca2+ influx and increase caspase-3 activity in a 3-h incubation. Of note, no RBC hemolysis and no calcein release from ghosts were obtained after EHs treatment for 24 h. Interestingly, external Ca2+ was not a prerequisite for the EHs-mediated ROS production and PS externalization. Also, the eryptotic hallmarks in the apoptotic RBCs were partially blocked by heparin and antibody (Ab) against Toll-like receptor 2 (TLR2). CONCLUSION: EHs act as a DAMP agent in the human RBCs that induces eryptosis. The cytotoxic effect is rapid as the hallmarks of eryptosis such as cell shrinkage, surface PS exposure, [Ca2+]i rise, ROS production and caspase-3 activation can be seen 3 h after treatment in a dose-dependent manner. The EHs' cytotoxic effects could be blocked by heparin and the Ab against TLR2.


Assuntos
Eriptose/efeitos dos fármacos , Histonas/farmacologia , Anticorpos/imunologia , Anticorpos/farmacologia , Cálcio/metabolismo , Caspase 3/metabolismo , Células Cultivadas , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Heparina/farmacologia , Humanos , Espécies Reativas de Oxigênio/metabolismo , Receptor 2 Toll-Like/imunologia
2.
Talanta ; 202: 111-122, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171159

RESUMO

A new dual-modality immunosensor based on molecularly imprinted polymer (MIP) and a nanostructured biosensing layer has fabricated for the simultaneous detection of two important markers including prostate-specific antigen (PSA) and myoglobin (Myo) in human serum and urine samples. In the first step, 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP) was self-assembled on a gold screen printed electrode (SPE). Then, the target proteins were attached covalently to the DSP-SPE. The imprinted cocktail polymer ((MIP(PSA, Myo)-SPE)) was synthesized at the SPE surface using acrylamide as monomer, N,N'-methylenebisacrylamide as a crosslinker, and PSA and Myo as the templates, respectively. The MIP-SPE was specific for the impedimetric sensing of PSA and Myo. After that, a nanocomposite (NCP) was synthesized based on the decorated magnetite nanoparticles with multi-walled carbon nanotube, graphene oxide and specific antibody for PSA (Ab). Then, NCP incubated with (MIP(PSA, Myo)-SPE. The modified electrodes and synthesized nanoparticles were characterized using electrochemical impedance spectroscopy, dynamic light scattering, surface plasmon resonance and scanning electron microscopy. The limits of detections were found to be 5.4 pg mL-1 and 0.83 ng mL-1 with the linear dynamic ranges of 0.01-100 and 1-20000 ng mL-1 for PSA and Myo, respectively. The ability of proposed biosensor to detect PSA and Myo simultaneously with high sensitivity and specificity offers a powerful opportunity for the new generation of biosensors. This dual-analyte specific receptors-based device is highly desired for the integration with lab-on-chip kits to measure a wide panel of biomarkers present at ultralow levels during early stages of diseases progress.


Assuntos
Biomarcadores Tumorais/análise , Técnicas Biossensoriais , Técnicas Eletroquímicas , Mioglobina/análise , Polímeros/química , Antígeno Prostático Específico/análise , Neoplasias da Próstata/diagnóstico , Anticorpos/imunologia , Reações Antígeno-Anticorpo , Biomarcadores Tumorais/imunologia , Eletrodos , Humanos , Masculino , Impressão Molecular , Mioglobina/imunologia , Nanopartículas/química , Antígeno Prostático Específico/imunologia , Neoplasias da Próstata/imunologia
3.
Expert Opin Ther Pat ; 29(7): 481-485, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31216214

RESUMO

Introduction: OX40 is checkpoint inhibitor in cancer that coordinates the downregulation of the proliferation of antigen-specific lymphocytes. There is a great need to discover and develop new therapies focused on inhibiting the action of OX40 and consequently improving the immune response in the various types of cancer. Authors of patent US2018256711A1 propose a method to eradicate cancer that utilizes anti-OX40 agonist antibody in combination with anti-PD-L1 antagonist antibody. Areas covered: Patent US2018256711A1 describes a method of cancer combinatorial treatment consisting of the utilization of a pharmaceutical cocktail containing anti-OX40 and an anti-PD-L1 antibody. Expert opinion: The results of the clinical trials only support trials regarding the tolerability of combinatorial therapy, even when the objectives of determining the safety and pharmacokinetics of the treatment are proposed.


Assuntos
Antígeno B7-H1/antagonistas & inibidores , Neoplasias/terapia , Receptores OX40/agonistas , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/patologia , Patentes como Assunto , Resultado do Tratamento
4.
Folia Histochem Cytobiol ; 57(2): 94-100, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31237344

RESUMO

INTRODUCTION: A reduced number of interstitial Cajal-like cells (ICLCs) in the gallbladder have been proposed to play a role in the pathogenesis of cholelithiasis. Therefore, this prospective study was conducted to investigate the relationship between gallbladder contractility and the number of gallbladder ICLCs in patients with cholelithiasis. MATERIAL AND METHODS: Patients admitted to the Department of Hepatobiliary Surgery for cholecystectomy were divided into the cholelithiasis (n = 18) and non-cholelithiasis (n = 8) groups based on their clinical data. Patients' clinical data were collected on admission, and B-mode ultrasonography was performed to assess their gallbladder contractility. The resected gallbladder specimens were fixed, paraffin sections mounted on slides, and the immunofluorescence staining with the anti-human CD-117 and anti-human tryptase antibodies was performed to identify ICLSs and mast cells, respectively. The number of ICLCs was counted in 10 high-power fields (HPFs) randomly. RESULTS: Independent sample t-tests revealed differences between the cholelithiasis and non-cholelithiasis groups in the number of ICLCs (mean ± standard deviation: 88.61 ± 28.22 vs. 115.89 ± 27.87 per HPFs, P = 0.032) and gallbladder contractility (43.94% ± 18.50% vs. 61.00% ± 20.50%, P = 0.046). Pearson and Spearman cor-relation analyses revealed no significant correlation between the number of ICLCs and gallbladder contractility. CONCLUSION: The results suggest that the number of gallbladder ICLCs in the wall of the gallbladder of patients with or without cholelithiasis is not a decisive factor affecting gallbladder contractility.


Assuntos
Colelitíase/fisiopatologia , Esvaziamento da Vesícula Biliar/fisiologia , Vesícula Biliar/citologia , Vesícula Biliar/fisiologia , Telócitos/citologia , Adulto , Idoso , Animais , Anticorpos/imunologia , Contagem de Células , Colelitíase/patologia , Feminino , Vesícula Biliar/patologia , Cabras , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Estudos Prospectivos , Proteínas Proto-Oncogênicas c-kit/imunologia , Coelhos , Telócitos/patologia , Triptases/imunologia
5.
Anal Bioanal Chem ; 411(20): 5159-5174, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161323

RESUMO

Horseradish peroxidase (HRP) conjugated gluten-specific antibodies (G12, R5, 2D4, MIoBS, and Skerritt), from nine commercial gluten ELISA test kits, previously utilized in the development of a multiplex competitive ELISA for the detection of fermented-hydrolyzed gluten, were utilized in western blot analyses of 59 fermented-hydrolyzed foods from four food groups (beer, soy-based sauces, vinegar, and sourdough bread). The protein/peptide profiles generated by the nine gluten-specific antibodies varied in size distribution and intensity dependent on the type of food, with minor differences between related products. Cluster analysis of the estimated gluten concentration values (based on western blot band intensities relative to intact gluten standards at 2.5 µg/mL and 100 µg/mL) and that of the relative response of the nine gluten-specific antibodies to different gluten proteins/peptides, distinguished among the different categories of fermented-hydrolyzed foods; comparable to what was observed in the multiplex competitive ELISA. Further, unlike the competitive ELISA, the western blot analyses distinguished between the presence of antigenic proteinaceous materials and false positives due to the presence of binding inhibitors (as observed with four soy-based sauces and one vinegar). Limitations of western blot analysis often include lower sensitivity than the comparable competitive ELISA and problems quantitating gluten-derived peptides and proteins. As a result, western blot analysis provides an orthogonal approach that can be used to both confirm the multiplex competitive ELISA while also providing additional insight into the protein/peptide profile of fermented-hydrolyzed foods. Graphical abstract.


Assuntos
Western Blotting , Ensaio de Imunoadsorção Enzimática/métodos , Fermentação , Glutens/imunologia , Anticorpos/imunologia , Análise de Alimentos , Hidrólise
6.
Food Chem ; 297: 125006, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253309

RESUMO

Muscle foods, particularly fish products are highly exposed to oxidative stress during processing and storage, resulting in oxidative modification of proteins. Protein carbonyls content has been used as one of the measures of oxidative stress. Generally, the resulting carbonylated proteins (CPs) have so far been labeled with 2,4-dinitrophenyl (DNP) hydrazine and detected with anti-DNP antibody. However, the applicability of this method to food samples is limited by its high price, time-consuming procedure and possibility to perform the measurements just on soluble protein fractions. We developed a simpler, faster and cheaper method to assess CP level in muscle foods, including both soluble and insoluble protein fractions, which is based on a direct reaction of protein carbonyls with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). The paper describes a novel technique to label both soluble and insoluble carbonylated proteins with CHH and determine carbonyl content by fluorescence microscopy assay which correlates (R = 0.911) with conventional ELISA method.


Assuntos
Fluorometria/métodos , Proteínas Musculares/análise , Animais , Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática , Peixes/metabolismo , Congelamento , Microscopia de Fluorescência , Proteínas Musculares/química , Estresse Oxidativo , Fenil-Hidrazinas/química , Fenil-Hidrazinas/imunologia , Carbonilação Proteica , Reprodutibilidade dos Testes
7.
Chem Biol Interact ; 308: 317-322, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31170385

RESUMO

Acetylcholinesterase (AChE) hydrolyzes acetylcholine at cholinergic synapses, and which has various isoforms of AChE, i.e. AChER, AChEH and AChET, deriving from single gene. AChEH exists as a glycophosphatidylinositol (GPI)-linked dimer (G2), presents mainly in plasma membrane of mammalian erythrocyte. Transgenic mice with ACHE gene depletion were employed here to investigate the possible role of AChE in blood cell formation. ACHE knock-out mice were found to suffer normocytic anemia. In erythrocyte of ACHE-/- mice, the amount of hemoglobin, especially α-globin, was found to be markedly reduced. In addition, the number of erythrocyte and hematocrit of ACHE-/- mice were significantly lowered. To probe the role of AChE isoforms in erythroid differentiation, erythroblast-like cells (TF-1) over-expressed with different AChE isoforms were induced to differentiate by erythropoietin (EPO): this differentiation induced the expression of each AChE isoform. Only in the TF-1 cells over-expressed with AChEH, the EPO-induced transcriptions and protein expressions of α- and ß-globins could be significantly enhanced, which therefore suggested that AChEH might regulate the responsiveness of TF-1 cells to EPO. The alternation of EPO-induced downstream signaling might be accounted by association of AChE with EPO receptor in cell surface. The findings indicated the significance of AChE in erythroblast maturation, which provided an insight in elucidating possible mechanisms in regulating erythropoiesis.


Assuntos
Acetilcolinesterase/metabolismo , Receptores da Eritropoetina/metabolismo , Acetilcolinesterase/química , Acetilcolinesterase/imunologia , Animais , Anticorpos/imunologia , Diferenciação Celular , Linhagem Celular , Dimerização , Eritroblastos/citologia , Eritroblastos/metabolismo , Eritropoetina/farmacologia , Expressão Gênica/efeitos dos fármacos , Hemoglobinas/metabolismo , Humanos , Camundongos , Camundongos Knockout , Receptores da Eritropoetina/imunologia
8.
Scand J Immunol ; 90(3): e12795, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31148206

RESUMO

Antigen-specific molecules of the immune system, namely antibodies, the membrane immunoglobulins (mIgs) of B cells and T cell receptors (TcRs), can all signal their interaction with antigen. There are different mechanisms by which this signalling could occur. These mechanisms can be divided into two general categories: allosteric and non-allosteric. In allosteric mechanisms, the monovalent binding of the antigen to the receptor triggers a conformational change at the binding site that is propagated to an invariant part of the receptor, a change recognized by a sensing unit. We argue allosteric mechanisms are implausible. Non-allosteric mechanisms depend on steric effects due to the antigen's size and/or multivalency. We consider two non-allosteric mechanisms by which the mIg of B cells has been envisaged to signal its interaction with antigen: the popular cross-linking model and the dissociation activation model. We argue, on the basis of both experimental observations and physiological considerations, that the dissociation activation model, developed by Reth and his colleagues, is uniquely plausible.


Assuntos
Anticorpos/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Sistema Imunitário/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Humanos , Receptores de Antígenos de Linfócitos T/imunologia
9.
Eur J Histochem ; 63(2)2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31189296

RESUMO

The Kölliker's organ is a transient epithelial structure during cochlea development that gradually degenerates and disappears at postnatal 12-14 days (P12-14). While apoptosis has been shown to play an essential role in the degeneration of the Kölliker's organ, the role of another programmed cell death, autophagy, remains unclear. In our study, autophagy markers including microtubule associated protein light chain 3-II (LC3-II), sequestosome 1 (SQSTM1/p62) and Beclin1 were detected in the supporting cells of the Kölliker's organ through immunohistochemistry staining. In addition, Western blot and real-time PCR revealed a gradually decreased expression of LC3-II and an increased expression of p62 during early postnatal development. Compared to apoptosis markers that peaks between P7 and P10, autophagy flux peaked earlier at P1 and decreased from P1 to P14. By transmission electron microscopy, we observed representative autophagosome and autolysosome that packaged various organelles in the supporting cells of the Kölliker's organ. During the degeneration, these organelles were digested via autophagy well ahead of the cellular apoptosis. These results suggest that autophagy plays an important role in transition and degeneration of the Kölliker's organ prior to apoptosis during the early postnatal development.


Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Cóclea/embriologia , Cóclea/metabolismo , Animais , Anticorpos/imunologia , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Proteína Beclina-1/metabolismo , Caspase 3/genética , Caspase 3/imunologia , Caspase 3/metabolismo , Cóclea/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Imuno-Histoquímica/métodos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/imunologia , Proteína Sequestossoma-1/metabolismo , Fatores de Tempo
10.
Talanta ; 201: 217-225, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122414

RESUMO

This study describes, for the first time, the development of two platforms of competitive fluorescent immunoassays for bioanalysis of crizotinib (CZT), a potent drug used for the treatment of non-small cell lung cancer (NSCLC). These platforms were microwell-based heterogeneous fluoroimmunoassay (FIA) and a kinetic exclusion assay (KinExA) with KinExA™ 3200 immunosensor. Both FIA and KinExA were developed using same reagents; mouse anti-CZT antibody and a capturing reagent of CZT conjugated with bovine serum albumin (CZT-BSA). In the FIA, the CZT-BSA coated onto the microwells of the assay plate was present simultaneously in the assay mixture (CZT and its antibody). In the KinExA, the antibody was allowed to pre-equilibrate with CZT, and then the incubation mixture was rapidly passed through a microcolumn containing CZT-BSA coated onto polymethyl methacrylate (PMMA) beads. The analytical performances of both assays were comparatively evaluated in terms of assay working range, limit of detection, precision profile, and accuracy. The results revealed that KinExA yielded higher sensitivity and better precision than FIA; whereas, both assays had comparable accuracies. Both FIA and KinExA were superior to all the existing chromatographic methods for CZT in terms of the assay sensitivity, convenience, analysis throughputs. The proposed FIA and KinExA are anticipated to effectively contribute to the therapeutic drug monitoring (TDM) of CZT in clinical settings.


Assuntos
Antineoplásicos/sangue , Crizotinibe/sangue , Fluorimunoensaio/métodos , Animais , Anticorpos/imunologia , Antineoplásicos/química , Antineoplásicos/imunologia , Calibragem , Carcinoma Pulmonar de Células não Pequenas/sangue , Bovinos , Crizotinibe/química , Crizotinibe/imunologia , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Cabras , Humanos , Limite de Detecção , Neoplasias Pulmonares/sangue , Camundongos , Polimetil Metacrilato/química , Soroalbumina Bovina/química
11.
Talanta ; 201: 245-252, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122419

RESUMO

In this study, the coupling of magnetic enrichment of bacteria from real samples with rapid surface enhanced Raman spectroscopy (SERS) detection was reported. The selective isolation and enrichment for the model bacteria Escherichia coli (E. coli) was performed using E. coli (primary) antibody bound-magnetic gold (Fe3O4@Au) nanoparticles. Following isolation and enrichment, the rennet enzyme was used to cleave of casein modified Fe3O4/Au-PEI nanoparticles from primary antibody-bound bacteria to prevent the nanoparticle aggregation and provide the movement of bacteria on nitrocellulose membrane. In the first part of the study, optimization studies were carried out namely; the amounts of gold nanoparticles (AuNPs), polyethyleneimine coated magnetic gold (Fe3O4/Au-PEI) nanoparticles, casein and rennet enzyme. The SERS signals of DTNB (5,5'-Dithiobis(2-nitrobenzoic acid)) molecule were collected on the test line and a calibration curve was plotted by using signal intensities. The correlation between the concentration of E. coli and SERS signal was found to be linear within the range of 101-107 cfu/mL (R2 = 0.984, LOD = 0.52 cfu/mL and LOQ = 1.57 cfu/mL). The selectivity of the paper-based lateral flow immunoassay (LFIA) was examined with Bacillus subtilis (B. subtilis), Micrococcus luteus (M. luteus), Salmonella enteritidis (S. enteritidis) which did not produce any significant response compared with E. coli measurement. Finally, the developed paper-based LFIA was tested with urine and milk samples. The obtained SERS results were compared with a plate counting method results which were in a good accordance. The developed method was found as rapid and sensitive to E. coli with a total analysis time of less than 60 min.


Assuntos
Bactérias/isolamento & purificação , Técnicas de Tipagem Bacteriana/métodos , Nanopartículas de Magnetita/química , Leite/microbiologia , Papel , Urina/microbiologia , Animais , Anticorpos/imunologia , Bacillus subtilis/isolamento & purificação , Caseínas/imunologia , Quimosina/química , Escherichia coli/isolamento & purificação , Ouro/química , Limite de Detecção , Micrococcus luteus/isolamento & purificação , Salmonella enteritidis/isolamento & purificação
12.
Virchows Arch ; 475(1): 67-76, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31127385

RESUMO

With the approval of pembrolizumab for first- and second-line treatment of PD-L1+ non-small cell lung cancer (NSCLC), PD-L1 testing by immunohistochemistry (IHC) has become a necessity. However, the DAKO autostainer ASL48 for the FDA approved DAKO 22C3 pharmDx assay is not broadly available in Switzerland and other parts of Europe. The primary goal of this study was to cross-validate the 22C3 anti-PD-L1 antibody on Benchmark Ultra (VBMU) and Leica Bond (LBO) immunostainers. IHC protocols were developed for 22C3 on both platforms with the 22C3phDx using ASL48 as reference. A tissue microarray (TMA) was constructed from 23 NSCLC specimens with a range of PD-L1 staining results. Empty TMA sections and the 22C3 antibody were distributed to 16 participants for staining on VBMU (8 centers) and/or LBO (12 centers) using the centrally developed protocols. Additionally the performance of the Ventana SP263 assay was tested in five centers. IHC scoring was performed centrally. Categorical PD-L1 staining (0-49% vs. 50-100%) did not significantly differ between centers using VBMU, whereas data from LBO were highly variable (p < 0.001). The SP263 assay was well concordant with 22C3 on VBMU and with 22C3 pharmDx. PD-L1 IHC using a standardized 22C3 protocol on VBMU provides satisfactory results in most centers. The SP263 assay is confirmed as a valid alternative to 22C3 pharmDx. 22C3 PD-L1 IHC on LBO shows major staining variability between centers, highlighting the need for local validation and adjustment of protocols.


Assuntos
Anticorpos/imunologia , Antígeno B7-H1/análise , Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/química , Imuno-Histoquímica/métodos , Neoplasias Pulmonares/química , Automação Laboratorial , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Imuno-Histoquímica/instrumentação , Imuno-Histoquímica/normas , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Variações Dependentes do Observador , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Suíça , Análise Serial de Tecidos
13.
Vet Immunol Immunopathol ; 211: 38-43, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31084892

RESUMO

Natural antibodies (NAb) are antibodies that can bind to a particular antigen without any apparent antigenic stimulation. In this paper, a careful analysis has been carried out on NAb levels in goat kid serum; possible correlations with the total immunoglobulin (tot-Ig) levels and specific antibody (SpAb) response were considered. Twenty randomly chosen kids were submitted to a first blood sampling (day 0). After 60 and 100 days, new blood samplings were carried out in the same animals. On day 0, after blood collection, all animals were immunized with a commercial vaccine; the immunization was repeated 30 days apart. Some exogenous antigens were tested to verify their immunoreactivity to NAb. Among them, the synthetic hapten 2,4,6-trinitrophenyl (TNP) conjugated with bovine serum albumin, resulted as the antigen with the higher immunoreactivity to NAb. Tot-Ig levels increased over time (p < 0.001). On the contrary, NAb levels, both IgG- and IgM-isotypes, significantly decreased during the experimental period (p < 0.001 and <0.05, respectively). Linear regression analyses showed a high correlation between IgM-NAb and tot-IgM levels (p < 0.001) at all the evaluated sampling times. However, a significant correlation between IgG-NAb and IgM-NAb was found only at the 1st (p < 0.01) and at the 2nd sampling (p < 0.05). No significant correlations were found between SpAb response and the other assessed humoral immune parameters. The obtained results are discussed in the light of the possible use of NAb assessment for the evaluation of the immune system activity in goat.


Assuntos
Imunidade Adaptativa/imunologia , Anticorpos/imunologia , Cabras/imunologia , Imunoglobulinas/imunologia , Animais , Anticorpos/sangue , Formação de Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Cabras/sangue , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Imunoglobulinas/sangue , Masculino
14.
Anal Chim Acta ; 1067: 48-55, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31047148

RESUMO

Immunoassay is a powerful technique to identify and quantify biological molecules, which base on the specificity and selectivity of antigen-antibody interaction. Impedance-based immunosensor has recently shown a great potential to provide rapid and label-free detections. However, the conventional impedance-based immunosensors rely on dedicated electrochemical measurement interface which involves expensive fabrication procedures such as gold deposition and photolithography. In this work, we propose an ultra-low-cost and high processing efficiency platform for impedance-based immunosensing. With effortless operations of direct-laser-writing, an impedance-based immunoassay can be fabricated within 5 min in standard laboratories. The as-fabricated devices have shown great stability and a high device-to-device uniformity. In order to further validate impedance sensing system's performance, finite element analysis and impedance equivalent model analysis were performed. The measured data was consistent with the simulation results. With the standard gold electrodes surface bio-functionalization procedures, the disposable immunoassay can detect anti-IgG down to 10 ng/ml.


Assuntos
Anticorpos/análise , Impedância Elétrica , Imunoensaio , Lasers , Impressão , Anticorpos/imunologia , Eletrodos , Ouro/química , Imunoensaio/economia , Imunoglobulina G/imunologia , Propriedades de Superfície
15.
Nat Commun ; 10(1): 2109, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068594

RESUMO

Nanopore sensors detect individual species passing through a nanoscale pore. This experimental paradigm suffers from long analysis times at low analyte concentration and non-specific signals in complex media. These limit effectiveness of nanopore sensors for quantitative analysis. Here, we address these challenges using antibody-modified magnetic nanoparticles ((anti-PSA)-MNPs) that diffuse at zero magnetic field to capture the analyte, prostate-specific antigen (PSA). The (anti-PSA)-MNPs are magnetically driven to block an array of nanopores rather than translocate through the nanopore. Specificity is obtained by modifying nanopores with anti-PSA antibodies such that PSA molecules captured by (anti-PSA)-MNPs form an immunosandwich in the nanopore. Reversing the magnetic field removes (anti-PSA)-MNPs that have not captured PSA, limiting non-specific effects. The combined features allow detecting PSA in whole blood with a 0.8 fM detection limit. Our 'magnetic nanoparticle, nanopore blockade' concept points towards a strategy to improving nanopore biosensors for quantitative analysis of various protein and nucleic acid species.


Assuntos
Anticorpos/química , Técnicas Biossensoriais/instrumentação , Nanopartículas de Magnetita/química , Nanoporos , Anticorpos/imunologia , Técnicas Biossensoriais/métodos , Calicreínas/análise , Calicreínas/imunologia , Limite de Detecção , Membranas Artificiais , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/imunologia , Compostos de Silício/química , Fatores de Tempo
16.
Analyst ; 144(12): 3716-3720, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31134993

RESUMO

A photothermal immune-imaging assay was innovatively designed for the visual quantitative detection of cancer biomarkers by coupling CuxS nanocrystals with a portable infrared thermal imager on a smartphone. The rolling circle amplification (RCA) technique was used for the formation of a CuxS nanocrystal concatemer, thus opening up new territories in immunoassay development.


Assuntos
Biomarcadores Tumorais/análise , Imunoensaio/métodos , Nanocompostos/química , Antígeno Prostático Específico/análise , Smartphone , Anticorpos/imunologia , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Biomarcadores Tumorais/imunologia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Cobre/química , DNA/química , DNA/genética , Humanos , Imunoensaio/instrumentação , Raios Infravermelhos , Masculino , Nanocompostos/efeitos da radiação , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Antígeno Prostático Específico/imunologia , Sulfetos/química , Temperatura Ambiente
17.
Fish Shellfish Immunol ; 90: 349-362, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31067499

RESUMO

The myxozoan parasite Enteromyxum leei causes chronic enteritis in gilthead sea bream (GSB, Sparus aurata) leading to intestinal dysfunction. Two trials were performed in which GSB that had survived a previous infection with E. leei (SUR), and naïve GSB (NAI), were exposed to water effluent containing parasite stages. Humoral factors (total IgM and IgT, specific anti-E. leei IgM, total serum peroxidases), histopathology and gene expression were analysed. Results showed that SUR maintained high levels of specific anti-E. leei IgM (up to 16 months), expressed high levels of immunoglobulins at the intestinal mucosa, particularly the soluble forms, and were resistant to re-infection. Their acquired-type response was complemented by other immune effectors locally and systemically, like cell cytotoxicity (high granzyme A expression), complement activity (high c3 and fucolectin expression), and serum peroxidases. In contrast to NAI, SUR displayed a post-inflammatory phenotype in the intestine and head kidney, characteristic of inflammation resolution (low il1ß, high il10 and low hsp90α expression).


Assuntos
Imunidade Adaptativa , Doenças dos Peixes/imunologia , Imunidade Inata , Myxozoa/fisiologia , Doenças Parasitárias em Animais/imunologia , Dourada/imunologia , Animais , Anticorpos/imunologia , Proteínas de Peixes/imunologia , Imunoglobulinas/imunologia , Inflamação/imunologia , Inflamação/veterinária , Membrana Mucosa/imunologia
18.
Parasitol Res ; 118(6): 1919-1926, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31069534

RESUMO

In our previous study, proteomics analyses of host cells infected with Eimeria tenella sporozoites coupled with isobaric tags for relative and absolute quantitation, identified several host proteins related to Eimeria invasion. In this study, A 458-bp Gallus gallus fatty acid-binding protein 4 (FABP4) gene was cloned and subcloned to pET-28c(+) vector to construct the prokaryotic recombinant expression plasmid pET-28c(+)-FABP4. The 18.5 kDa recombinant FABP4 protein (rFABP4) was expressed and identified by western blotting. Expression of FABP4 in E. tenella sporozoite-infected DF-1 cells was downregulated significantly than in non-infected cells detected by western blotting and immunohistochemistry. The antibody inhibition assay showed that antibodies against FABP4 at 50, 100, 200, 300, and 400 µg/mL had no significant effect on sporozoite invasion. BMS-309403 and transforming growth factor-ß3 (TGF-ß3) was used to inhibit and improve the expression of FABP4 in DF-1 cells, respectively, and their effect on the sporozoite invasion of cells was detected by flow cytometry. Sporozoite invasion rate in the BMS-309403-treated group was not significantly affected; however, the invasion rate in the TGF-ß3-treated group declined significantly. These results show that host FABP4 plays a negative role in Eimeria invasion. However, further studies are needed to elucidate the exact mechanism of how FABP4 negatively regulates Eimeria invasion.


Assuntos
Galinhas/parasitologia , Coccidiose/veterinária , Eimeria tenella/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Regulação da Expressão Gênica/genética , Esporozoítos/metabolismo , Animais , Anticorpos/imunologia , Western Blotting , Linhagem Celular , Coccidiose/parasitologia , Regulação para Baixo , Eimeria tenella/genética , Eimeria tenella/imunologia , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/imunologia , Coelhos/parasitologia , Fator de Crescimento Transformador beta3/farmacologia
19.
J Agric Food Chem ; 67(24): 6874-6883, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31144502

RESUMO

We prepared a specific adsorptive nanocarrier for pesticide due to its challenge to cleanup and low detoxification in the treatment after intake, whether intentional or by mistake. We modified the plastic antibody (molecularly imprinted polymer (MIP)) on the surface of persistent luminescence nanoparticle (La3Ga5GeO14: Cr3+, Zn2+, LGGO) as the specific adsorptive nanocarrier for toxic molecules and realized the nanocarrier was widely distributed for absorbing pesticide and real-time in vivo bioimaging. We used LGGO as the core and trichlorphon as the template to prepare the plastic antibody nanocarrier. After in vivo bioimaging and biodistribution of mice, LGGO@MIP could be distributed evenly in the gastrointestinal tract, circulated in the blood for a long time, and finally excreted to achieve the adsorption and removal of pesticide in the body. The LGGO@MIP nanocarrier prepared in this study opens a new way for the treatment of poisoning.


Assuntos
Anticorpos/química , Nanotecnologia/métodos , Praguicidas/metabolismo , Plásticos/química , Imagem Corporal Total/métodos , Adsorção , Animais , Anticorpos/imunologia , Cinética , Luminescência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Impressão Molecular , Nanopartículas/química , Nanotecnologia/instrumentação , Praguicidas/química , Plásticos/metabolismo , Polímeros/síntese química , Polímeros/química , Triclorfon/química , Triclorfon/metabolismo , Imagem Corporal Total/instrumentação
20.
Neurochem Res ; 44(7): 1736-1744, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31037609

RESUMO

Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Neurons differentiated from hiPSCs may be promising tools to develop novel treatment methods for various neurological diseases. However, the detailed process underlying functional maturation of hiPSC-derived neurons remains poorly understood. Here, we analyze the developmental architecture of hiPSC-derived cortical neurons, iCell GlutaNeurons, focusing on the primary cilium, a single sensory organelle that protrudes from the surface of most growth-arrested vertebrate cells. To characterize the neuronal cilia, cells were cultured for various periods and evaluated immunohistochemically by co-staining with antibodies against ciliary markers Arl13b and MAP2. Primary cilia were detected in neurons within days, and their prevalence and length increased with increasing days in culture. Treatment with the mood stabilizer lithium led to primary cilia length elongation, while treatment with the orexigenic neuropeptide melanin-concentrating hormone caused cilia length shortening in iCell GlutaNeurons. The present findings suggest that iCell GlutaNeurons develop neuronal primary cilia together with the signaling machinery for regulation of cilia length. Our approach to the primary cilium as a cellular antenna can be useful for both assessment of neuronal maturation and validation of pharmaceutical agents in hiPSC-derived neurons.


Assuntos
Cílios/metabolismo , Cílios/ultraestrutura , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Fatores de Ribosilação do ADP/imunologia , Adenilil Ciclases/imunologia , Animais , Anticorpos/imunologia , Linhagem Celular , Cílios/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Humanos , Hormônios Hipotalâmicos/farmacologia , Imuno-Histoquímica , Lítio/farmacologia , Melaninas/farmacologia , Proteínas Associadas aos Microtúbulos/imunologia , Neurogênese/fisiologia , Neurônios/efeitos dos fármacos , Hormônios Hipofisários/farmacologia , Ratos Wistar , Receptores de Somatostatina/imunologia
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