Apoio parcial por doações para MRS é de Ciências Naturais e Pesquisa de Engenharia Council of Canada (NSERC), o Harry Botterell Foundation for Neuroscience Research, ARC, eo Garfield Research Kelly Cardiovascular e Fundo de Desenvolvimento. SRA é um receptor da Rainha do Prêmio de Pesquisa da Universidade e do Robert J. Wilson Fellowship. PJ é um receptor de Prêmio de Pós-Graduação da rainha e do Robert J. Wilson Fellowship. AG e SA são os destinatários do Prêmio de Pós-Graduação da Rainha. SF foi o ganhador da Bolsa de Pós-Graduação em Ontario Ciência e Tecnologia (OGSST). AG é o destinatário da Rainha Franklin Bolsa de Pós-Graduação Bracken. SA é o destinatário da Rainha RS Bolsa de Pós-Graduação McLaughlin.

1. Ressuscitando macrófagos Congelados <ol><li> Antes de ressuscitar células congeladas do freezer -80 ° C, é preciso preparar meio de cultura usando estéril filtrado Médio Modified Dulbecco Águia (DMEM) suplementado com 10% soro fetal bovino (FBS) e 5 mg / mL de plasmocin. Plasmocin é uma solução de antibiótico utilizado em nossa pesquisa para eliminar e evitar a contaminação por micoplasma em culturas de células. </li><li> Para um frasco de células congeladas, acrescenta um 4 mL, 1 mL do meio de cultura para um frasco de 25 centímetros cultura 2 células em uma capa de contenção de risco biológico estéril. </li><li> Quando o frasco de células congeladas são retiradas do freezer -80 ° C, eles são rapidamente descongeladas pelo aquecimento mão no capô estéril, que leva cerca de 2-3 min. </li><li> Assim que as células são descongeladas, eles são rapidamente transferidos para o balão contendo o meio condicionado utilizando uma pipeta de 1 mL estéril. </li><li> O frasco contendo as células são colocadas em uma cultura de tecidos incubadora a 37 ° C e 5% CO 2. </li><li> As células marcada para o crescimento e adesão em uma base diária até que se tornem 75% a 90% confluentes usando um microscópio invertido. </li><li> Durante a incubação de cultura de células, 12 mm lâminas de vidro circular são esterilizados, cobrindo-os em etanol 70% em um grande prato de vidro petri por 1 hr no gabinete de contenção de risco biológico, o álcool é posteriormente removido e armazenado para reusage, e as laterais de vidro molhado são deixados expostos ao fluxo de ar filtrado estéril no gabinete sob um UV por 24 horas ou até que estejam secos. </li></ol> 2. Células chapeamento para o ensaio sialidase <ol><li> Antes do plaqueamento das células, uma única lâmina de vidro estéril circular é cuidadosamente colocado em um poço de uma BD Falcon 24 poços de fundo plano com tampa da cultura de tecidos placa de poliestireno tratada usando uma curva completa sterilized flamejante, 4 &quot;, serrilhada, de aço inoxidável fórceps . Para um frasco de células em confluência de 75%, normalmente os usamos 12 poços da placa de cultura. </li><li> Para as células de macrófagos aderentes, o meio DMEM condicionado é removido do frasco utilizando uma pipeta de 5 mL estéreis no gabinete de contenção estéril. Lave as células aderentes uma vez com Tris buffered 1x estéril salina pH 7,4 para remover qualquer meio de soro contendo, e adicionar 1 mL de cálcio médio livre de fosfato (CMF) solução salina tamponada, pH 7,4, o suficiente para cobrir as células. As células são colocadas na incubadora a 37 ° C por aproximadamente 10 min ou até que as células começam a decolar no frasco. O frasco pode ser balançado suavemente ou agitado para soltar qualquer resquício de células aderentes. </li><li> Para a 1 mL de células em suspensão no frasco, adicionar 3 mL de DMEM meio condicionado. Tomar 0,5 ml de células em suspensão e esterilizados transferiu-os para a placa de tecido cultivado contendo os 12 milímetros lâminas de vidro circular. Aproximadamente, 0,5 x 10 6 células são transferidas para a lâminas de vidro circular. </li><li> O tecido células de cultura contendo placa são incubados a 37 ° C em 5% CO 2 por 24 horas, a fim de permitir que as células aderem à lâminas de vidro circular. </li></ol> 3. Ensaio sialidase 1. Tornando o controle <ol><li> Coloque uma lâmina de microscópio (fosco: uma final, 1slide, 25 x 75 x 1 mm) no gabinete de contenção ou de bancada de laboratório e adicione, misturando em ordem seqüencial os seguintes compostos: 1 mL DAKO + 1 mL de 4-Muñana e por último + 3 mL de Tris buffered saline pH 7,4 (TBS). </li><li> Depois de remover a mídia de um poço que contém as células, levante cuidadosamente a lâmina de vidro circular com os 4 fórceps &quot;para o canto do bem. Retire cuidadosamente a lâmina de vidro com a pinça e coloque o slide com as células virada para a solução de mistura em a lâmina de microscópio. </li><li> Imediatamente, tomar o slide microcope a um microscópio epi-fluorescente que foi definida para o filtro UV e tem uma câmera digital para capturar as imagens fluorescentes. </li><li> O foco do microscópio em contraste de fase até que você possa ver as células e então mudar para o modo fluorescente. Tirar fotos das células sob luz fluorescente de 1 min, 2 min e 3 min. Tire uma foto única das células sob contraste de fase. </li></ol> 2. Fazendo o teste positivo <ol><li> Identificar os compostos a seguir na ordem seqüencial em outra lâmina de microscópio: 1 mL DAKO + 1 mL de 4-Muñana mL + 2 de LPS + 1 mL de TBS. </li><li> Repita os passos 3.1.2 a 3.1.4 acima. </li></ol> 3. Fazendo o teste positivo juntamente com inibidor da neuraminidase Tamiflu <ol><li> Identificar os seguintes compostos na seguinte ordem seqüencial em outra lâmina de microscópio: 1μL DAKO + 1μL de 4-Muñana + 2 mL de LPS + 1 mL de Tamiflu. </li><li> Concentrações Tamiflu são pré-preparados em 1x Tris buffered saline pH 7,4; a concentração final dos compostos em contato com as células vivas na lâmina de vidro circular terá um fator de diluição de 5. </li><li> Repita os passos 3.1.2 a 3.1.4 acima. </li></ol> 4. Determinação da Concentração de Inibidores necessárias para inibir 50% da Atividade sialidase (IC 50) <ol><li> Para quantificar o fluorescente em torno das células, as imagens são analisadas utilizando ImageJ 1.38x com o plugin para medir analisar RBG. Ter 50 leituras aleatórias da fluorescência em torno das células e calcular a média da fluorescência total. </li><li> O IC 50 é gerada a partir de um gráfico da concentração de inibidor convertidos em unidades de log (por exemplo, log ng / mL) no eixo x, contra a fluorescência média no eixo y por meio de regressão não-linear. </li></ol> 5. Segredos para o Sucesso <ol><li> Certifique-se que as células em cultura são a contaminação livre de micoplasma. Nós usam rotineiramente Plasmocin em meio de cultura para controlar isso. </li><li> O substrato artificial neuraminidase, 4-Muñana, devem ser utilizados preparados na hora. O substrato preparado pode ser usado dentro de uma semana para o ensaio sialidase. </li><li> Se a linha de célula tem um expressão de receptores de baixo na superfície da célula, pode-se usar uma dose mais elevada do ligante para a estimulação. </li><li> Temos também que as células experientes várias passagens por mais de 6 vezes pode perder seu fenótipo do receptor. Recentemente, as células ressuscitado deve ser cultivado. </li></ol> 6. Resultados representante Ver protocolo de animação do ensaio sialidase com resultados representante no anexo <a href="http://www.jove.com/files/ftp_upload/2142/szewczuk_JoVE_sialidase_assay.pptx">arquivo Powerpoint</a> .

<ol>
	<li>Amith, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu1+desialylation+of+sialyl+alpha-2,3-linked+beta-galactosyl+residues+of+TOLL-like+receptor+4+is+essential+for+receptor+activation+and+cellular+signaling.">Neu1 desialylation of sialyl alpha-2,3-linked beta-galactosyl residues of TOLL-like receptor 4 is essential for receptor activation and cellular signaling.</a> Cell Signal. 22, 314-324 (2010).</li><li>Amith, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+pathogen+molecule-induced+Toll-like+receptor+activation+and+cell+function+on+Neu1+sialidase.">Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase.</a> Glycoconjugate Journal. 26, 1197-1212 (2009).</li><li>Woronowicz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+neurotrophic+factor+activation+of+Trk+tyrosine+kinase+receptors+on+cellular+sialidase.">Dependence of neurotrophic factor activation of Trk tyrosine kinase receptors on cellular sialidase.</a> Glycobiology. 17, 10-24 (2007).</li><li>Jayanth, P., Amith, S. R., Gee, K., Szewczuk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu1+Sialidase+and+Matrix+Metalloproteinase-9+Cross-talk+is+Essential+for+Neurotrophin+Activation+of+Trk+Receptors+and+Cellular+Signaling.">Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk is Essential for Neurotrophin Activation of Trk Receptors and Cellular Signaling.</a> Cell. Signal. 22, 1193-1205 (2010).</li><li>Finlay, T. M., Jayanth, P., Amith, S. R., Glimour, A., Guzzo, C., Gee, K., Beyaert, R., Szewczuk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymoquinone+from+nutraceutical+black+cumin+oil+activates+Neu4+sialidase+in+live+macrophage,+dendritic,+and+normal+and+type+I+sialidosis+human+fibroblast+cells+via+GPCR+G&#945;i+proteins+and+matrix+metalloproteinase-9.">Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR G&#945;i proteins and matrix metalloproteinase-9.</a> Glycoconj J. 27, 329-348 (2010).</li><li>Seyrantepe, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Phagocytosis+in+Macrophages+by+Neuraminidase+1.">Regulation of Phagocytosis in Macrophages by Neuraminidase 1.</a> Journal of Biological Chemistry. 285, 206-215 (2010).</li></ol>

SRA e PJ contribuíram igualmente como primeiro autor.

Utilizando o teste recentemente desenvolvido para detectar atividade de sialidase em células macrófago vivo 2, usamos essa tecnologia para detectar atividade de sialidase em ligante induzida por atividade sialidase em viver BMC-2 macrófagos de uma forma dependente da dose, bem como em viver DC-2.4 células dendríticas , HEK-TLR4/MD2, HEK293, SP1 nas células mamárias adenocarcinoma, WT humanos e 1140F01 e WG0544 tipo I fibroblastos sialidose. Inibidores da neuraminidase, como o Tamiflu (fosfato de oselta...

<table><tbody><tr><td>DMEM</td><td>GIBCO, by Life Technologies</td><td></td><td></td></tr><tr><td>4-MUNANA</td><td>Biosynth International, Inc</td><td></td><td></td></tr><tr><td>Fetal calf serum</td><td>Hyclone</td><td></td><td></td></tr><tr><td>DakoCytomation, Fluorescent Mounting Media</td><td>Dako</td><td>S3023</td><td>15 mL</td></tr><tr><td>Tamiflu (Oseltamivir Phosphate)</td><td>Hoffmann-La Roche AG</td><td></td><td></td></tr></tbody></table>

detection of  neu1 sialidase activity in regulating toll-like receptor activation

Mamíferos Toll-like receptors (TLRs) são uma família de receptores que reconhecem patógenos padrões moleculares associados. Não são apenas os TLRs sensores crucial da microbiana (por exemplo, vírus, bactérias e parasitas) infecções, eles também desempenham um papel importante na fisiopatologia de doenças infecciosas, doenças inflamatórias e, possivelmente, em doenças auto-imunes. Assim, a intensidade ea duração das respostas TLR contra as doenças infecciosas devem ser rigorosamente controladas. Segue-se que o entendimento a integridade estrutural dos receptores de sensor, suas interações ligante e componentes de sinalização é essencial para a proteção imunológica subseqüentes. Seria também oferecem oportunidades importantes para a modificação de doenças através da manipulação sensor. Embora as vias de sinalização de sensores de TLR estão bem caracterizadas, os parâmetros de controle de interações entre os sensores e seus ligantes ainda permanecem mal definidos. Recentemente, identificou um novo mecanismo de ativação do TLR por seu ligante natural, que não foi observado anteriormente 1,2. Ele sugere que a ativação induzida por ligante TLR é rigidamente controlado por Neu1 ativação sialidase. Temos também informou que Neu1 regule receptores neurotrofina como TRKA TrkB e 3, que envolvem Neu1 e matriz metaloproteinase-9 (MMP-9) cruzada no complexo com os receptores 4. O ensaio sialidase foi inicialmente usado para encontrar um romance ligante thymoquinone, na ativação de Neu4 sialidase na superfície celular de macrófagos, células dendríticas e células fibroblasto via GPCR Gαi proteínas e MMP-9 5. Para os receptores TLR, nossos dados indicam que Neu1 sialidase já está em complexos com receptores TLR-2, -3 e -4, e é induzida mediante ligação do ligando a qualquer receptor. Ativado Neu1 hidrolisa sialidase sialil α-2 ,3-β-ligados galactosyl resíduos distante ligação do ligando para remover impedimento estérico a TLR-4 dimerização, MyD88/TLR4 recrutamento complexo, NFkB ativação e respostas pró-inflamatórias da célula. Em um relatório de colaboração, sialidase Neu1 foi mostrado para regular a fagocitose em macrófagos 6. Tomados em conjunto, o ensaio sialidase nos forneceu insights poderosos para os mecanismos moleculares do ligante induzida por ativação do receptor. Embora a relação exata entre Neu1 sialidase e ativação de TLR, receptores Trk ainda não foi completamente elucidado, isso representaria uma nova abordagem ou pioneira de vias de regulação celular.

Watch this Scientific Journal Video about Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation at JoVE.com

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

O ensaio sialidase é uma abordagem simples técnica que irá elucidar mecanismo molecular romance (s) de sensores de TLR de infecções microbianas e envolvimento em doenças inflamatórias ao nível do receptor na superfície celular de macrófagos ao vivo.

Detecção de Atividade Neu1 sialidase em Regulação Toll-like receptor de ativação

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

detection-neu1-sialidase-activity-regulating-toll-like-receptor

Research

JoVE Journal

Immunology and Infection

11.5K visualizações.  Queen's University - Kingston, Ontario. O ensaio sialidase é uma abordagem simples técnica que irá elucidar mecanismo molecular romance (s) de sensores de TLR de infecções microbianas e envolvimento em doenças inflamatórias ao nível do receptor na superfície celular de macrófagos ao vivo.

Vídeo: Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Ativação e Mensuração de NLRP3 inflammasome Atividade Usando IL-1β em células dendríticas humanas Monocyte derivados

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Imunologia e Infecção

A Pipeline to Investigate the Structures and Signaling Pathways of Sphingosine 1-Phosphate Receptors

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of sphingolipids in the cell membrane, is one of the best-characterized LPLs that regulates a variety of cellular physiological responses via signaling pathways mediated by sphingosine 1-phosphate receptors (S1PRs). This implicated that the S1P-S1PRs signaling system is a remarkable potential therapeutic target for disorders, including multiple sclerosis (MS), autoimmune disorders, cancer, inflammation, and even COVID-19. S1PRs, a small subset of the class A G-protein coupled receptor (GPCR) family, are composed of five subtypes: S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5. The lack of detailed structural information, however, impedes the drug discovery targeting S1PRs. Here, we applied the cryo-electron microscopy method to solve the structure of the S1P-S1PRs complex, and elucidated the mechanism of activation, selective drug recognition, and G-protein coupling by using cell-based functional assays. Other lysophospholipid receptors (LPLRs) and GPCRs can also be studied using this strategy.

S1P exerts its diverse physiological effects through the S1P receptors (S1PRs) subfamily. Here, a pipeline is described to expound on the structures and function of S1PRs.

Um pipeline para investigar as estruturas e caminhos de sinalização de receptores sphingosine 1-fosfato

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of ...

Bioquímica

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Glycoengineering metabólica do ácido siálico usando N-acil-modificado Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Bioengenharia

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

Um ensaio celular do repórter do macrófago para examinar a sinalização nf-kB/AP-1 mediada pelo receptor em macrófagos em camadas de proteína adsorbed em superfícies poliméricas

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Triagem de Nanopartículas Bioativas em Células Imunes Fagocíticas para Inibidores da Sinalização de Receptores Toll-like

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Medicina

DCs derivadas de monócitos dessialilados de PBMCs isoladas usando tratamento com sialidase

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

Autor em destaque: Maturação de células dendríticas usando tratamento enzimático baseado em sialidases da superfície celular 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

Geração de Células Dendríticas Derivadas de Monócitos com Fenótipos Sialilados Diferentes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Determinação de ácidos sialic em amostras de fígado e leite de tipo selvagem e<em&gt; CMAH</em&gt; Ratinhos de eliminação.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Biologia

Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells downstream of pattern recognition receptors. Best studied are the Dectin-1 and Dectin-2 members of the C-type lectin-like receptor family that induce a SYK- and CARD9-dependent signaling cascade leading to NF-&#954;B activation, in a MALT1-dependent manner. By contrast, Toll-like receptors (TLR), such as TLR-4, propagate NF-&#954;B activation but signal via an MYD88/IRAK-dependent cascade. Nonetheless, whether MALT1 might contribute to TLR-4 signaling has remained unclear. Recent evidence with MLT-827, a potent and selective inhibitor of MALT1 paracaspase activity, indicates that TNF- production downstream of TLR-4 in human myeloid cells is independent of MALT1, as opposed to TNF- production downstream of Dectin-1, which is MALT1 dependent. Here, we addressed the selective involvement of MALT1 in pattern recognition sensing further, using a variety of human and mouse cellular preparations, and stimulation of Dectin-1, MINCLE or TLR-4 pathways. We also provided additional insights by exploring cytokines beyond TNF-, and by comparing MLT-827 to a SYK inhibitor (Cpd11) and to an IKK inhibitor (AFN700). Collectively, the data provided further evidence for the MALT1-dependency of C-type lectin-like receptor &#8212;signaling by contrast to TLR-signaling.

MALT1 regulates innate immunity but how this occurs remains ill-defined. We used the selective MALT1 paracaspase inhibitor MLT-827 to unravel the contribution of MALT1 to innate signaling downstream of Toll-like or C-type lectin-like receptors, demonstrating that MALT1 regulates the production of myeloid cytokines, and downstream of C-type lectin-like receptors, selectively.

Mieloide inata sinalização via regulamento por MALT1 Paracaspase atividade

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells ...

Detecção de Atividade Neu1 sialidase em Regulação Toll-like receptor de ativação

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Geração de Células Dendríticas Derivadas de Monócitos com Fenótipos Sialilados Diferentes

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Determinação de ácidos sialic em amostras de fígado e leite de tipo selvagem e

Mieloide inata sinalização via regulamento por MALT1 Paracaspase atividade