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...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco, Rockville, MD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>4-MUNANA</td>
<td>&nbsp;</td>
<td>Biosynth International Inc., Itasca, IL, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum</td>
<td>&nbsp;</td>
<td>HyClone, Logan, Utah, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DakoCytomation, Fluorescent Mounting Media</td>
<td>&nbsp;</td>
<td>DAKO North America, Inc., Carpintenia, CA</td>
<td>S3023</td>
<td>15 mL</td>
</tr>
<tr>
<td>Tamiflu (Oseltamivir Phosphate)</td>
<td>&nbsp;</td>
<td>Hoffman La Roche</td>
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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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinação de adesivos de fita baseada em amostragem e de fluorescência In situ Hibridação para detecção rápida de Salmonella On Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Imunologia e Infecção

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

Basófilos Teste de ativação de Investigação de IgE-mediada Mecanismos Hipersensibilidade a Drogas

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Imagem quantitativa da linhagem específica Toll-like receptor-mediados sinalização em monócitos e células dendríticas a partir de pequenas amostras de sangue humano

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

A saliva, glândula salivar, e Coleta de hemolinfa de carrapatos Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Métodos para a detecção quantitativa de anticorpos induzida pela ativação do complemento no Glóbulos Vermelhos

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

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.

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

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 ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolamento de Murino macrófagos peritoniais de Cumprir a análise de expressão gênica Após Toll-like receptors Estimulação

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolamento e Activação de Linfócitos murinos

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Citometria de fluxo e assassinas naturais celular Lytic Actividade em sangue total humano

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.