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1.
Int Immunopharmacol ; 71: 285-294, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30928648

RESUMO

Lectins are proteins/glycoproteins of non-immune origin, which interact specifically and non-covalently with the carbohydrate moieties of respective receptors on the cell surface. In this study, a novel 65.2-kDa tetrameric lectin (AHL) was purified from Artocarpus hypargyreus Hance (A. hypargyreus) by affinity chromatography on a galactose-sepharose column. It is a glycoprotein with carbohydrate content of 6.91%. Its maximum haemagglutinating activity was maintained after incubation at a temperature range of 20-40 °C and pH range of 5.0-9.0. AHL-induced haemagglutination of erythrocytes was inhibited strongly by carbohydrates, such as methyl-galactose, methyl-mannose, and N-acetyl-d-galactosamine, indicating the existence of more than one carbohydrate binding sites in the AHL molecule. The AHL activity was gradually lost in the presence of urea and completely lost when being treated with ethylenediaminetetraacetic acid (EDTA). The immunomodulatory activity of AHL was assessed using human peripheral lymphocytes and rat peritoneal macrophages. AHL triggered proliferation and activation of human T lymphocytes and induced the release of Th1 cytokines, including IFN-γ, TNF-α and IL-6. Furthermore, AHL significantly stimulated the production of nitric oxide (NO) and pro-inflammatory cytokines, including TNF-α and IL-12, in rat peritoneal macrophages. However, AHL did not enhance proliferation of B cell-enriched rat splenocytes. Taken together, in this study, a novel immunomodulatory lectin was purified from A. hypargyreus and was found to be capable of inducing a Th1-type immune response, and thus, it may have potential immunoregulatory application in response to infections, immune diseases and cancer.


Assuntos
Macrófagos Peritoneais/imunologia , Lectinas de Plantas/imunologia , Linfócitos T/imunologia , Animais , Artocarpus/imunologia , Células Cultivadas , Cromatografia de Afinidade , Citocinas , Humanos , Ativação Linfocitária , Masculino , Estrutura Molecular , Óxido Nítrico/metabolismo , Fagocitose , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Ratos , Ratos Wistar
2.
Int Immunopharmacol ; 66: 1-12, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30415189

RESUMO

Pinellia pedatisecta, a widely used herb in Chinese medicine, has proinflammatory toxicity related to its Pinellia pedatisecta lectin (PPL), but the mechanism is still unknown. However, for safer use, it is necessary to clarify its proinflammatory mechanism. Herein, we studied the mechanism in RAW264.7 cells. PPL decreased the mitochondrial membrane potential (MMP) and increased the outflow of calcium, accompanied by the overproduction of reactive oxygen species (ROS), which resulted in the activation of the MAPK and NF-κB pathways and the release of IL-1ß. The maturation of IL-1ß relied on caspase-1 p20, the active caspase-1, as demonstrated by adding caspase-1 inhibitor. While caspase-1 was associated with the activation of the NLRP3 inflammasome, we further found that the stimulation of PPL also contributed to the activation. In addition, TXNIP was downregulated, whereas NLRP3/caspase-1 p20/ASC was upregulated, and there was binding of TXNIP with NLRP3. There was also binding of NLRP3 with ASC and caspase-1. Further, we found that N-acetylcysteine (NAC), an ROS scavenger, could inhibit the PPL-stimulated activation of these pathways and the release of IL-1ß. Moreover, PPL led to cell pyroptosis with pyknotic nuclei and plasma membrane rupture, which could be inhibited by NAC. All of these findings demonstrated an important role of ROS in the inflammation caused by PPL. Taken together, our data provide new mechanistic insights into the possible endogenous signaling pathways involved in the inflammation of RAW264.7 cells, stimulated by PPL.


Assuntos
Inflamação/metabolismo , Macrófagos/imunologia , Pinellia/imunologia , Lectinas de Plantas/imunologia , Piroptose/imunologia , Animais , Caspase 1/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Potencial da Membrana Mitocondrial/imunologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
3.
Methods Mol Biol ; 1911: 421-432, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30593642

RESUMO

Enzyme-linked immunosorbent assays (ELISAs) enable rapid detection and quantitation of antibodies in samples. Such assays can be highly sensitive and can be performed in most laboratories with basic equipment. Although detecting binding antibodies to the surface proteins of most pathogens by ELISA is not always indicative of antibody function, i.e., neutralizing activity of antibodies, the results can be used as a first step toward more in-depth analysis of antibody responses. Here we describe a method that can be used to standardize ELISAs for the detection of HCV envelope antibodies across laboratories and provide adaptations of the method to further characterize antibody responses in serum samples.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/isolamento & purificação , Hepatite C/imunologia , Lectinas de Ligação a Manose/imunologia , Lectinas de Plantas/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Cricetulus , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Hepacivirus/metabolismo , Anticorpos Anti-Hepatite C/imunologia , Humanos , Testes de Neutralização/instrumentação , Testes de Neutralização/métodos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia
4.
Sci Rep ; 8(1): 16292, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389954

RESUMO

Bacterial surfaces are decorated with carbohydrate structures that may serve as ligands for host receptors. Based on their ability to recognize specific sugar epitopes, plant lectins are extensively used for bacteria typing. We previously observed that the galactose-specific agglutinins from Ricinus communis (RCA) and Viscum album (VAA) exhibited differential binding to nontypeable Haemophilus influenzae (NTHi) clinical isolates, their binding being distinctly affected by truncation of the lipooligosaccharide (LOS). Here, we examined their binding to the structurally similar LOS molecules isolated from strains NTHi375 and RdKW20, using microarray binding assays, saturation transfer difference NMR, and molecular dynamics simulations. RCA bound the LOSRdKW20 glycoform displaying terminal Galß(1,4)Glcß, whereas VAA recognized the Galα(1,4)Galß(1,4)Glcß epitope in LOSNTHi375 but not in LOSRdKW20, unveiling a different presentation. Binding assays to whole bacterial cells were consistent with LOSNTHi375 serving as ligand for VAA, and also suggested recognition of the glycoprotein HMW1. Regarding RCA, comparable binding to NTHi375 and RdKW20 cells was observed. Interestingly, an increase in LOSNTHi375 abundance or expression of HMW1 in RdKW20 impaired RCA binding. Overall, the results revealed that, besides the LOS, other carbohydrate structures on the bacterial surface serve as lectin ligands, and highlighted the impact of the specific display of cell surface components on lectin binding.


Assuntos
Antígenos de Bactérias/metabolismo , Técnicas de Tipagem Bacteriana/métodos , Haemophilus influenzae/imunologia , Lipopolissacarídeos/metabolismo , Lectinas de Plantas/metabolismo , Antígenos de Bactérias/imunologia , Bioensaio/métodos , Galactose/metabolismo , Haemophilus influenzae/classificação , Haemophilus influenzae/metabolismo , Lipopolissacarídeos/imunologia , Análise em Microsséries/métodos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Lectinas de Plantas/imunologia
5.
Eur Ann Allergy Clin Immunol ; 50(6): 243-253, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30039691

RESUMO

Summary: Dietary lectins play a major role in the activation of mast cells / basophils by bridging cell surface IgE glycans to release histamine and other mediators. In the present study, the effect of mannose / glucose-specific banana lectin (BanLec) on the activation of mast cells / basophils from non-atopic and atopic subjects has been investigated. BanLec was purified from banana pulp in a yield of 7 mg/kg. Leukocytes isolated from heparinized blood of non-atopic / atopic subjects were used for quantitation of the released histamine. Approximately 28.2% of the atopics (n = 117) was positive by skin prick test (SPT) to purified BanLec (100 µg/mL concentration), and all the non-atopics (n = 20) were negative. Maximal release of histamine was seen at 2 µg of BanLec. In percent histamine release, an increase of 35-40% is observed in case of atopics (n = 7) compared to non-atopics (n = 5), and the histamine release from atopic and non-atopic subjects correlates fairly well with the total serum IgE levels (R2 = 0.817). BanLec also induces release of histamine (26.7%) from mast cells present in rat peritoneal exudate cells. BanLec can significantly activate and degranulate mast cells and basophils by cross-linking the trimannosidic core mannose of IgE glycans in atopic population as compared to non-atopic population; the activation is marginal in the case of non-atopics.


Assuntos
Basófilos/imunologia , Liberação de Histamina/imunologia , Mastócitos/imunologia , Musa/imunologia , Lectinas de Plantas/imunologia , Proteínas de Vegetais Comestíveis/imunologia , Adulto , Alérgenos/imunologia , Animais , Feminino , Histamina/sangue , Histamina/metabolismo , Humanos , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Musa/química , Ratos Wistar , Testes Cutâneos , Adulto Jovem
6.
Int J Mol Sci ; 19(3)2018 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-29510511

RESUMO

The plant cell wall shows a great diversity regarding its chemical composition, which may vary significantly even during different developmental stages. In this study, we analysed the distribution of several cell wall epitopes in embryos of Brachypodium distachyon (Brachypodium). We also described the variations in the nucleus shape and the number of nucleoli that occurred in some embryo cells. The use of transmission electron microscopy, and histological and immunolocalisation techniques permitted the distribution of selected arabinogalactan proteins, extensins, pectins, and hemicelluloses on the embryo surface, internal cell compartments, and in the context of the cell wall ultrastructure to be demonstrated. We revealed that the majority of arabinogalactan proteins and extensins were distributed on the cell surface and that pectins were the main component of the seed coat and other parts, such as the mesocotyl cell walls and the radicula. Hemicelluloses were localised in the cell wall and outside of the radicula protodermis, respectively. The specific arrangement of those components may indicate their significance during embryo development and seed germination, thus suggesting the importance of their protective functions. Despite the differences in the cell wall composition, we found that some of the antibodies can be used as markers to identify specific cells and the parts of the developing Brachypodium embryo.


Assuntos
Brachypodium/imunologia , Parede Celular/imunologia , Epitopos/imunologia , Sementes/imunologia , Brachypodium/crescimento & desenvolvimento , Brachypodium/ultraestrutura , Núcleo Celular/imunologia , Núcleo Celular/ultraestrutura , Parede Celular/ultraestrutura , Citoplasma/imunologia , Citoplasma/ultraestrutura , Lectinas de Plantas/imunologia , Sementes/ultraestrutura
7.
Glycoconj J ; 35(2): 205-216, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29374812

RESUMO

The carbohydrate specificities of Dioclea grandiflora lectins DGL-I1 and DGL-II, and Galactia lindenii lectin II (GLL-II) were explored by use of remodeled glycoproteins as well as by the lectin hemagglutinating activity against erythrocytes from various species with different glycomic profiles. The three lectins exhibited differences in glycan binding specificity but also showed overlapping recognition of some glycotopes (i.e. Tα glycotope for the three lectins; IIß glycotope for DGL-II and GLL-II lectins); in many cases the interaction with distinct glycotopes was influenced by the structural context, i.e., by the neighbouring sugar residues. Our data complement and expand the existing knowledge about the binding specificity of these three Diocleae lectins, and taken together with results of previous studies, allow us to suggest a functional map of the carbohydrate recognition which illustrate the impact of modification of basic glycotopes enhancing, permiting, or inhibiting their recognition by each lectin.


Assuntos
Dioclea/química , Lectinas de Plantas/imunologia , Especificidade de Anticorpos , Epitopos/química , Epitopos/imunologia , Hemaglutinação , Humanos , Lectinas de Plantas/química , Polissacarídeos/química , Polissacarídeos/imunologia
8.
Expert Rev Proteomics ; 15(2): 183-190, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29265940

RESUMO

INTRODUCTION: Serum proteins are generally glycosylated and solubilized, and are thus present as glycoproteins. The glycan structure of glycoproteins reflects cell differentiation status; glycan structures generated by diseased cells are distinguishable from those produced by healthy cells. Proteins may therefore serve as markers of tissues that secrete them. Several strategies for the identification of novel serum biomarkers using a combination of glycoscience-based technologies have been recently proposed. The selection of lectins for use as probes for identification of altered glycan structures represents a critical step. Areas covered: This review describes the identification of Wisteria floribunda agglutinin (WFA) as a probe that recognizes the altered glycan structure of glycoproteins secreted by diseased cells. WFA may be employed as a probe for several diseases, e.g., liver fibrosis, liver cirrhosis, prostate cancer, ovarian cancer, and IgA nephropathy. The advantage of employing WFA as a serum biomarker probe is that only very small amounts of WFA-positive glycoproteins are present in serum; therefore, WFA background in serum is very low. Expert commentary: Based on the findings to date, several WFA-positive serum biomarkers may be measured without pre-purification of target glycoproteins, indicating their utility as serum biomarkers in patients with various diseases.


Assuntos
Glomerulonefrite por IGA/sangue , Cirrose Hepática/sangue , Lectinas de Plantas/imunologia , Receptores de N-Acetilglucosamina/imunologia , Biomarcadores/sangue , Glicômica/métodos , Humanos
9.
Structure ; 25(11): 1667-1678.e4, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-28988747

RESUMO

High-quality reagents to study and detect glycans with high specificity for research and clinical applications are severely lacking. Here, we structurally and functionally characterize several variable lymphocyte receptor (VLR)-based antibodies from lampreys immunized with O erythrocytes that specifically recognize the blood group H-trisaccharide type II antigen. Glycan microarray analysis and biophysical data reveal that these VLRs exhibit greater specificity for H-trisaccharide compared with the plant lectin UEA-1, which is widely used in blood typing. Among these antibodies, O13 exhibits superior specificity for H-trisaccharide, the basis for which is revealed by comparative analysis of high-resolution VLR:glycan crystal structures. Using a structure-guided approach, we designed an O13 mutant with further enhanced specificity for H-trisaccharide. These insights into glycan recognition by VLRs suggest that lampreys can produce highly specific glycan antibodies, and are a valuable resource for the production of next-generation glycan reagents for biological and biomedical research and as diagnostics and therapeutics.


Assuntos
Anticorpos Monoclonais/química , Antígenos de Grupos Sanguíneos/análise , Lampreias/imunologia , Polissacarídeos/química , Receptores de Antígenos de Linfócitos T/química , Trissacarídeos/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Sítios de Ligação , Antígenos de Grupos Sanguíneos/imunologia , Tipagem e Reações Cruzadas Sanguíneas/métodos , Cristalografia por Raios X , Eritrócitos/química , Eritrócitos/imunologia , Humanos , Imunização , Modelos Moleculares , Lectinas de Plantas/química , Lectinas de Plantas/imunologia , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Trissacarídeos/imunologia , Trissacarídeos/metabolismo
10.
J Immunol Res ; 2017: 2471627, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28634588

RESUMO

Lectins are carbohydrate-binding proteins with various biological activities, such as antitumor and immunomodulatory effects. Although lectins have various biological activities, they are still limited by cytotoxicity in normal cells. To overcome this problem, we used the noncytotoxic part of Korean mistletoe lectin B-chain (KML-B) to induce maturation of dendritic cells (DCs). A previous study reported that KML-B induces DC maturation by triggering TLR-4, including expression of costimulatory molecules (CD40, CD80, and CD86), MHC II, and secretion of cytokines in DCs. Additionally, matured DCs by KML-B induced T helper (Th) cell activation and differentiation toward Th1 cells. However, the interaction of KML-B-treated DCs with CD8+ T cells is still poorly understood. In this study, we confirmed the ability of matured DCs by KML-B to stimulate cytotoxic T cells using OT-1 mouse-derived CD8+ T cells. KML-B induced MHC I expression in DCs, stimulation of CD8+ T cell activation and proliferation, and IFN-γ secretion. Moreover, tumor sizes were reduced by KML-B treatment during vaccination of OVA257-264-pulsed DCs. Here, we confirmed induction of CD8+ T cell activation and the antitumor effect of KML-B treatment in DCs.


Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Imunoterapia/métodos , Neoplasias Experimentais/terapia , Lectinas de Plantas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/imunologia , Antígenos CD8/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Interferon gama/metabolismo , Ativação Linfocitária , Medicina Tradicional Coreana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/imunologia , Ovalbumina/imunologia , Carga Tumoral , Viscum album/imunologia
11.
Clin Exp Immunol ; 189(1): 21-35, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28268243

RESUMO

Neovascularization and jeopardized immunity has been critically emphasized for the establishment of malignant progression. Lectins are the diverse class of carbohydrate interacting proteins, having great potential as immunopotentiating and anti-cancer agents. The present investigation sought to demonstrate the anti-proliferative activity of Dolichos lablab lectin (DLL) encompassing immunomodulatory attributes. DLL specific to glucose and mannose carbohydrate moieties has been purified to homogeneity from the common dietary legume D. lablab. Results elucidated that DLL agglutinated blood cells non-specifically and displayed striking mitogenicity to human and murine lymphocytes in vitro with interleukin (IL)-2 production. The DLL-conditioned medium exerted cytotoxicity towards malignant cells and neoangiogenesis in vitro. Similarly, in-vivo anti-tumour investigation of DLL elucidated the regressed proliferation of ascitic and solid tumour cells, which was paralleled with blockade of tumour neovasculature. DLL-treated mice showed an up-regulated immunoregulatory cytokine IL-2 in contrast to severely declined levels in control mice. Mechanistic validation revealed that DLL has abrogated the microvessel formation by weakening the proangiogenic signals, specifically nuclear factor kappa B (NF-κB), hypoxia inducible factor 1α (HIF-1 α), matrix metalloproteinase (MMP)-2 and 9 and vascular endothelial growth factor (VEGF) in malignant cells leading to tumour regression. In summary, it is evident that the dietary lectin DLL potentially dampens the malignant establishment by mitigating neoangiogenesis and immune shutdown. For the first time, to our knowledge, this study illustrates the critical role of DLL as an immunostimulatory and anti-angiogenic molecule in cancer therapeutics.


Assuntos
Mitógenos/farmacologia , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Lectinas de Plantas/administração & dosagem , Lectinas de Plantas/farmacologia , Células A549 , Aglutinação , Animais , Aorta/efeitos dos fármacos , Técnicas de Cultura de Células , Membrana Corioalantoide/efeitos dos fármacos , Córnea/irrigação sanguínea , Córnea/efeitos dos fármacos , Dissacarídeos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imunomodulação , Interleucina-2/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mitógenos/imunologia , Lectinas de Plantas/imunologia , Ratos , Ratos Wistar , Sementes/química , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
Toxicon ; 127: 122-129, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28088476

RESUMO

Abrin, a type II ribosome inactivating protein from the Abrus precatorius plant, is extremely toxic. It has been shown to be 75 times more potent than its infamous sister toxin, ricin and their potential use in bio-warfare is a cause of major concern. Although several vaccine candidates are under clinical trials for ricin, none are available against abrin. The present study proposes a chimeric protein, comprising of 1-123 amino acids taken from the A chain of abrin and 124-175 amino acids from Abrus precatorius agglutinin A chain, as a vaccine candidate against abrin intoxication. The design was based on the inclusion of the immunogenic region of the full length protein and the minimal essential folding domains required for inducing neutralizing antibody response. The chimera also contains the epitope for the only two neutralizing antibodies; D6F10 and A7C4, reported against abrin till now. Active immunization with the chimera protected all the mice challenged with 45 X LD50 of abrin. Also, passive transfer of antibodies raised against the chimera rescued all mice challenged with 50 X LD50 of toxin. Hence the chimeric protein appears to be a promising vaccine candidate against abrin induced lethality.


Assuntos
Abrina/toxicidade , Abrus/química , Aglutininas/imunologia , Lectinas de Plantas/imunologia , Intoxicação por Plantas/prevenção & controle , Proteínas Recombinantes de Fusão/imunologia , Abrina/genética , Abrus/imunologia , Abrus/envenenamento , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Epitopos , Feminino , Humanos , Células Jurkat , Camundongos Endogâmicos BALB C , Lectinas de Plantas/genética , Intoxicação por Plantas/imunologia , Conformação Proteica , Coelhos , Proteínas Recombinantes de Fusão/genética , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
13.
J Immunol ; 198(5): 2082-2092, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28087670

RESUMO

Plant-derived dietary lectins have been reported to be involved in the pathogenesis of several inflammatory diseases, including inflammatory bowel disease, diabetes, rheumatoid arthritis, and celiac disease, but little is known about the molecular mechanisms underlying lectin-induced inflammation. In this study, we showed that plant lectins can induce caspase-1 activation and IL-1ß secretion via the NLRP3 inflammasome. Lectins were internalized and subsequently escaped from the lysosome and then translocated to the endoplasmic reticulum. Endoplasmic reticulum-loaded plant lectins then triggered Ca2+ release and mitochondrial damage, and inhibition of Ca2+ release and mitochondrial reactive oxygen species by chemical inhibitors significantly suppressed NLRP3 inflammasome activation. In vivo, plant lectin-induced inflammation and tissue damage also depended on the NLRP3 inflammasome. Our findings indicate that plant lectins can act as an exogenous "danger signal" that can activate the NLRP3 inflammasome and suggest that dietary lectins might promote inflammatory diseases via the NLRP3 inflammasome.


Assuntos
Doenças Autoimunes/imunologia , Canavalia/imunologia , Inflamassomos/metabolismo , Inflamação/imunologia , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Lectinas de Plantas/metabolismo , Animais , Sinalização do Cálcio , Caspase 1/metabolismo , Dieta , Retículo Endoplasmático/metabolismo , Humanos , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Lectinas de Plantas/imunologia , Espécies Reativas de Oxigênio/metabolismo
14.
Mucosal Immunol ; 10(5): 1122-1132, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28098245

RESUMO

Early and midgestational decidua of mice genetically ablated for expression of the natural killer (NK) cell natural cytotoxicity receptor (NCR; Ncr1Gfp/Gfp mice) shows restricted angiogenesis and atypically small uterine (u)NK cells. We hypothesized that NCR1 inactivation disturbs maturation and angiokine production by uterine natural killer (uNK) cells. Using histological and morphometric approaches, we observed that Ncr1Gfp/Gfp but not control C57BL/6 (B6) implantation sites sustain immature, non-granulated uNK cells into midpregnancy. Mouse uNK cells can be subclassified by their reactivity with Dolichos biflorus agglutinin (DBA) lectin; DBA+ uNK cells with greater Ncr1 expression were investigated. DBA+ uNK cells from Ncr1Gfp/Gfp mice show delayed maturation as indicated by shorter diameters and fewer cytoplasmic granules. Granules in mature Ncr1Gfp/Gfp uNK cells are ultrastructurally abnormal and abundance of granule-associated proteins (perforin, granzyme) and of cytoplasmic proteins (vascular endothelial growth factor; placental growth factor) differs from controls. Leukocyte-leukocyte conjugate formation in gestation day 6.5 and 8.5 intact Ncr1Gfp/Gfp decidua was less frequent than in B6; however, this difference involved leukocytes other than DBA+ uNK cells. These studies strongly support roles for NCR1 and its ligands in normal pregnancy promotion.


Assuntos
Antígenos Ly/metabolismo , Decídua/metabolismo , Células Matadoras Naturais/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Gravidez/imunologia , Útero/patologia , Animais , Antígenos Ly/genética , Diferenciação Celular , Células Cultivadas , Citotoxicidade Imunológica , Decídua/patologia , Feminino , Linfocinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Lectinas de Plantas/imunologia
15.
Methods Mol Biol ; 1389: 167-76, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27460244

RESUMO

Imaging flow cytometry is an emerging imaging technology that combines features of both conventional flow cytometry and fluorescence microscopy allowing quantification of the imaging parameters. The analysis of protein posttranslational modifications by glycosylation using imaging flow cytometry constitutes an important bioimaging tool in the glycobiology field. This technique allows quantification of the glycan fluorescence intensity, co-localization with proteins, and evaluation of the membrane/cytoplasmic expression. In this chapter we provide the guidelines to analyze glycan expression, particularly the ß1,6 GlcNAc branched N-glycans, on the membrane of intestinal T cells from inflammatory bowel disease patients.


Assuntos
Colite Ulcerativa/patologia , Citometria de Fluxo/métodos , Microscopia de Fluorescência/métodos , Linfócitos T/metabolismo , Linfócitos T/patologia , Colite Ulcerativa/imunologia , Glicosilação , Humanos , Citometria por Imagem , Intestinos/imunologia , Intestinos/patologia , Lectinas de Plantas/imunologia , Polissacarídeos/análise , Polissacarídeos/metabolismo
16.
J Exp Med ; 213(8): 1441-58, 2016 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-27401343

RESUMO

Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire(+) mTECs) is unclear. Here, we describe novel embryonic precursors of Aire(+) mTECs. We found the candidate precursors of Aire(+) mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire(+) mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire(+) mTECs and efficiently suppressed the onset of autoimmunity induced by Aire(+) mTEC deficiency. Mechanistically, pMECs differentiated into Aire(+) mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-ß receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire(+) mTECs.


Assuntos
Diferenciação Celular/imunologia , Células Epiteliais/imunologia , Regulação da Expressão Gênica/imunologia , Células-Tronco Embrionárias Murinas/imunologia , Timo/imunologia , Fatores de Transcrição/imunologia , Animais , Diferenciação Celular/genética , Células Epiteliais/citologia , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Lectinas de Plantas/genética , Lectinas de Plantas/imunologia , Timo/citologia , Fatores de Transcrição/genética
17.
BMC Immunol ; 17(1): 22, 2016 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-27377926

RESUMO

BACKGROUND: Mast cells are hematopoietically derived cells that play a role in inflammatory processes such as allergy, as well as in the immune response against pathogens by the selective and rapid release of preformed and lipid mediators, and the delayed release of cytokines. The native homotetrameric lectin ArtinM, a D-mannose binding lectin purified from Artocarpus heterophyllus seeds, is one of several lectins that are able to activate mast cells. Besides activating mast cells, ArtinM has been shown to affect several biological responses, including immunomodulation and acceleration of wound healing. Because of the potential pharmacological application of ArtinM, a recombinant ArtinM (rArtinM) was produced in Escherichia coli. The current study evaluated the ability of rArtinM to induce mast cell degranulation and activation. RESULTS: The glycan binding specificity of rArtinM was similar to that of jArtinM. rArtinM, via its CRD, was able to degranulate, releasing ß-hexosaminidase and TNF-α, and to promote morphological changes on the mast cell surface. Moreover, rArtinM induced the release of the newly-synthesized mediator, IL-4. rArtinM does not have a co-stimulatory effect on the FcεRI degranulation via. The IgE-dependent mast cell activation triggered by rArtinM seems to be dependent on NFkB activation. CONCLUSIONS: The lectin rArtinM has the ability to activate and degranulate mast cells via their CRDs. The present study indicates that rArtinM is a suitable substitute for the native form, jArtinM, and that rArtinM may serve as an important and reliable pharmacological agent.


Assuntos
Mastócitos/imunologia , Lectinas de Plantas/imunologia , Proteínas Recombinantes/imunologia , Animais , Artocarpus/imunologia , Degranulação Celular , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Histamina/metabolismo , Imunoglobulina E/metabolismo , Imunomodulação , Interleucina-4/metabolismo , Manose/metabolismo , NF-kappa B/metabolismo , Lectinas de Plantas/isolamento & purificação , Ligação Proteica , Ratos , Proteínas Recombinantes/isolamento & purificação , beta-N-Acetil-Hexosaminidases/metabolismo
18.
Scand J Gastroenterol ; 51(3): 344-53, 2016 03.
Artigo em Inglês | MEDLINE | ID: mdl-26340708

RESUMO

OBJECTIVE: As data on the effectiveness of tumor markers in detecting hepatocellular carcinoma (HCC) in cirrhotic patients are limited, we investigated the diagnostic accuracy of alpha-fetoprotein (AFP), protein induced by vitamin K absence or antagonist-II (PIVKA-II), and Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3). MATERIAL AND METHODS: This retrospective study enrolled 361 cirrhotic patients with HCC, and 276 cirrhotic patients without HCC occurrence. RESULTS: Most patients were men (n = 431, 67.7%); the median age was 57.0 years. The main etiology of chronic liver disease was chronic hepatitis B (n = 467, 73.3%). The sensitivity and specificity of combined three biomarkers was 87.0 and 60.1% in overall HCC, and 75.7 and 60.1% in early HCC, respectively (cutoff: 20 ng/mL for AFP, 40 mAU/mL for PIVKA-II, and 5% for AFP-L3). The area under the receiver operating characteristic curve (AUROC) for HCC diagnosis was 0.765 (95% confidence interval [CI], 0.728-0.801) for AFP; 0.823 (95% CI, 0.791-0.854) for PIVKA-II; and 0.755 (95% CI, 0.718-0.792) for AFP-L3. The AUROC for early HCC diagnosis was 0.754 (95% CI, 0.691-0.816) for AFP, 0.701 (95% CI, 0.630-0.771) for PIVKA-II, and 0.670 (95% CI, 0.596-0.744) for AFP-L3. Combining the three tumor markers increased the AUROC to 0.877 (95% CI, 0.851-0.903) for HCC diagnosis, and 0.773 (95% CI, 0.704-0.841) for early HCC diagnosis. CONCLUSION: Diagnostic accuracy improved upon combining the AFP, PIVKA-II, and AFP-L3 tumor markers compared to each marker alone in detecting HCC and early HCC in cirrhotic patients.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Idoso , Área Sob a Curva , Biomarcadores/sangue , Biomarcadores Tumorais/imunologia , Carcinoma Hepatocelular/virologia , Feminino , Hepatite B Crônica/complicações , Hepatite C Crônica/complicações , Humanos , Cirrose Hepática/virologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Lectinas de Plantas/imunologia , Isoformas de Proteínas/sangue , Precursores de Proteínas/sangue , Protrombina , Curva ROC , Estudos Retrospectivos , alfa-Fetoproteínas/imunologia , alfa-Fetoproteínas/metabolismo
19.
Biosens Bioelectron ; 78: 111-117, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26599480

RESUMO

Ricin is one of the most toxic plant toxins known. Its accessibility and relative ease of preparation makes it a potential agent for criminal or bio-terrorist attacks. Detection of ricin from unknown samples requires differentiation of ricin from the highly homologous Ricinus communis agglutinin which is currently not feasible using immunological methods. Here we have developed a simple and sensitive surface plasmon resonance (SPR) sensing system for rapid differentiation between ricin and agglutinin done in real time. Both lectins were quantified in a sandwich immunoassay-like setting by capturing with a cross-reactive antibody (R109) binding to both proteins while differentiating by injection of a ricin-specific antibody (R18) in a subsequent enhancement step. The SPR-assay was reproducible and sensitive for different R. communis cultivars, showing no false positive results when other lectins were tested. Quantification and differentiation of both molecules was also demonstrated from a crude castor bean extract and complex matrices. For the first time, we have demonstrated how the closely related lectins can be discerned and quantified in a single assay based on immunological methods. This novel approach delivers crucial information regarding the composition, purity, concentration, and toxicity of suspicious samples containing ricin in less than 30 minutes. Furthermore, we show how enhancement injections during SPR-measurements can be used to determine the ratio of two related proteins independently of the actual protein concentration by comparing normalized enhancement response levels.


Assuntos
Técnicas Biossensoriais/métodos , Lectinas de Plantas/isolamento & purificação , Ricina/isolamento & purificação , Anticorpos/química , Anticorpos/imunologia , Humanos , Imunoensaio , Lectinas de Plantas/imunologia , Ricina/imunologia , Ressonância de Plasmônio de Superfície
20.
Toxins (Basel) ; 7(12): 4906-34, 2015 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-26703723

RESUMO

Ricinus communis intoxications have been known for centuries and were attributed to the toxic protein ricin. Due to its toxicity, availability, ease of preparation, and the lack of medical countermeasures, ricin attracted interest as a potential biological warfare agent. While different technologies for ricin analysis have been established, hardly any universally agreed-upon "gold standards" are available. Expert laboratories currently use differently purified in-house materials, making any comparison of accuracy and sensitivity of different methods nearly impossible. Technically challenging is the discrimination of ricin from R. communis agglutinin (RCA120), a less toxic but highly homologous protein also contained in R. communis. Here, we established both highly pure ricin and RCA120 reference materials which were extensively characterized by gel electrophoresis, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI MS/MS), and matrix-assisted laser desorption ionization-time of flight approaches as well as immunological and functional techniques. Purity reached >97% for ricin and >99% for RCA120. Different isoforms of ricin and RCA120 were identified unambiguously and distinguished by LC-ESI MS/MS. In terms of function, a real-time cytotoxicity assay showed that ricin is approximately 300-fold more toxic than RCA120. The highly pure ricin and RCA120 reference materials were used to conduct an international proficiency test.


Assuntos
Lectinas de Plantas/análise , Ricina/análise , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Semente de Rícino , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Ensaio de Proficiência Laboratorial/normas , Lectinas de Plantas/química , Lectinas de Plantas/imunologia , Lectinas de Plantas/toxicidade , Padrões de Referência , Ricina/química , Ricina/imunologia , Ricina/toxicidade , Sementes/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Células Vero
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