Agradecemos a Hye Sook-Oh, Joanne Morello e Alan Collmer, Departamento de Fitopatologia e Biologia Vegetal-Microbe, Cornell University, pelas valiosas discussões. Agradecemos a Jesse Munkvold para comentários sobre o artigo. O financiamento foi fornecido pelo National Science Foundation Programa Genoma Plant, número prêmio DBI-0605059 (GBM).

Parte 1: O crescimento das plantas e manutenção Nicotiana benthamiana plantas utilizada no ensaio deve ser de cerca de 7 semanas de idade. Eles devem ser aparadas pelo menos 4-5 dias antes do ensaio para remover todos os ramos axilares e flores. É uma boa idéia para remover ramos axilares logo depois que eles surgem de forma a tornar as plantas mais facilmente gerenciáveis. Parte 2: cultura bacteriana DIA 1: <ol><li> Placas ou culturas de todas as estirpes utilizadas no ensaio são iniciadas ao mesmo tempo. Para a Pseudomonas spp. que incluem dois indutores e 3 cepas desafio, bactérias raia em placas KBM contendo os antibióticos adequados de estoques congelados glicerol (Tabela 2). Uma pequena quantidade de bactérias deve ser espalhado no centro do prato. Incubar as placas a 30 ° C por cerca de 18-24 horas. Para Agrobacterium, iniciar uma cultura líquida em duas LB ml de uma placa nova e crescer durante a noite a 250 rpm, 30 ° C. </li></ol> 2 º DIA: <ol start="2"><li> Por Pseudomonas, adicione 150-200 mL de KBM líquido para as bactérias e espalhe-os sobre a placa inteira usando um espalhador de vidro estéril. Coloque o prato de volta na incubadora e deixá-lo crescer por mais 20-24 horas. Deve haver um gramado bacteriana na placa no dia seguinte, indicando um bom crescimento. Para Agrobacterium, iniciar uma cultura secundária em cerca de 50-10 ml LB com 20 mM acetosyringone e deixá-lo crescer durante a noite para 20 horas a 250 rpm, 30 ° C. </li></ol> Parte 3: Estações Preparação para o ensaio 2 º DIA: <ol><li> Um dia antes do ensaio as plantas devem ser movido para um quarto em 22-24 ° C, luz ~ 35-40% de umidade relativa e constante. </li><li> Mark círculos em folhas que são pelo menos 1,5 cm de diâmetro com um marcador preto grosso. Dois a quatro círculos podem ser marcados por folha. Os círculos devem ser bem espaçadas e de preferência não atravessar uma grande veia. Escolha bem-expandida folhas. Evitar que as folhas velhas ou folhas que são difíceis ou grossa ao toque e evitar a marcação círculos nas partes inferior da folha. </li></ol> Círculos marcando um dia antes do experimento não é essencial para o protocolo. No entanto, ele economiza tempo se houver um grande número de plantas que estão sendo analisadas, e faz com que seja mais fácil de conseguir o sincronismo preciso entre a indução da PTI e inoculações desafio. Parte 4: Indução da PTI DIA 3: <ol><li> Colher as células a partir da placa em 10 mM MgCl 2 para P. fluorescens e P. putida. Para Agrobacterium, centrifugar a cultura para coletar células e ressuspender em 10 mM MgCl 2 + 10 mM MES pH 5,6. Repetir a centrifugação e ressuspender em solução de 2-MES MgCl. Medir a densidade óptica (DO) das células a 600 nm. Ajustar o ODS para os valores finais necessários conforme descrito na Tabela 2. Pseudomonas deve ser finalmente ressuspenso em 10 mM MgCl 2 e Agrobacterium na MgCl 2-MES solução. Um volume total de 25 ml é suficiente para inocular cerca de 40-50 pontos. </li><li> Infiltrar no PTI indutores para os círculos pré-marcadas nas folhas utilizando uma seringa de 1 ml sem agulha. Anote a ordem em que as plantas foram infiltradas eo tempo exato em que a infiltração foi iniciada. </li></ol> Parte 5: inoculação Desafio <ol><li> Prepare P. syringae pv tomate. DC3000, Pst DC3000 Δ hopQ1-1 e Ps pv. tabaci para o desafio, tal como descrito na parte 4.1 e Tabela 2. </li><li> Sete horas após a infiltração indutor, executar a infiltração desafio na mesma ordem de plantas como a indução foi feito. Geralmente, um ponto na periferia do círculo primeira inoculação pode ser usado como o centro do círculo segunda inoculação. Se há pontos múltiplos em uma folha, em seguida, certifique-se as infiltrações não se sobrepõem. </li><li> Diluições placa de série de todas as culturas utilizadas no ensaio de KBM ou LB placas contendo os antibióticos apropriados para determinar o número exato UFC. </li></ol> Parte 6: A pontuação para repartição do PTI Dia 5: <ol><li> Olhe para o surgimento de morte celular por ETI os pontos que foram desafiados com Pst DC3000. Morte celular dentro da área de sobreposição de infiltração indica uma repartição dos PTI e deve ser marcado como um fenótipo positivo. </li></ol> Dia 7: <ol start="2"><li> Olhe para o surgimento de morte celular devido à doença nos locais que foram desafiados com Pst DC3000 Δ hopQ1-1 ou pv Ps. Tabaci </em&gt;. Pontuação para um fenótipo positivo da mesma maneira como descrito na parte 6.1. </li></ol> Quando as plantas controle (que não deve ser comprometida por PTI) começam a mostrar a morte celular na área de sobreposição de infiltração, parar de fazer quaisquer observações adicionais. Parte 7: resultados Representante A Figura 1 mostra o resultado de um ensaio quando P. fluorescens foi usado como indutor e DC3000 Pst como o desafio. <img src="/files/ftp_upload/1442/fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1. P. fluorescens foi infiltrada em N. benthamiana folhas (círculo preto) para induzir a PTI e 7 horas depois, o local foi desafiado com Pst DC3000 (círculo branco). Plantas silenciadas para FLS2 mostrou um colapso da PTI na região onde P. fluorescens foi infiltrada (A), como visto por morte celular. Plantas controle que não foram silenciadas não mostrou a morte celular na área de sobreposição devido à indução da PTI (B). Setas vermelhas indicam ausência ou presença de morte celular na área de sobreposição de infiltração. Fotografias foram tiradas dois dias após a infiltrações. Por favor, <a href="https://www.jove.com/files/ftp_upload/1442/fig1.jpg">clique aqui</a> para ver uma versão ampliada da figura 1. <table border="1"><tbody><tr><td> Indutor PTI </td><td> Morte celular provocando desafio </td><td> Natureza da morte celular </td><td> Código </td></tr><tr><td> Pseudomonas fluorescens 55 </td><td> Pseudomonas syringae pv tomato (Pst). DC3000 6 </td><td> ETI </td><td> Pf / DC </td></tr><tr><td> P. putida KT2440 </td><td> Pst DC3000 </td><td> ETI </td><td> Pp / DC </td></tr><tr><td> P. fluorescens 55 </td><td> Pst DC3000 Δ hopQ1-1 6 </td><td> Doença </td><td> Pf/Q1-1 </td></tr><tr><td> Agrobacterium tumefaciens GV2260 </td><td> P. syringae pv. tabaci 11528R </td><td> Doença </td><td> Agro / ptab </td></tr></tbody></table> Tabela 1: Combinações de PTI induzir e morte celular provocando micróbios usados ​​no ensaio para a morte celular PTI. <table border="1"><tbody><tr><td> Estirpe bacteriana </td><td> Meio de seleção </td><td> Finais OD usado em experimento </td><td> CFU correspondente / ml </td></tr><tr><td> P. fluorescens 55 </td><td> KBM Ampicilina (100 ug / ml) </td><td> 0,5 </td><td> 1 x 10 9 </td></tr><tr><td> P. putida KT2440 </td><td> KBM Ampicilina (100 ug / ml) </td><td> 0,5 </td><td> 1 x 10 8 </td></tr><tr><td> A. tumefaciens GV2260 </td><td> LB Rifampicina (100 ug / ml) </td><td> 0,5 </td><td> 5 x 10 8 </td></tr><tr><td> Pst DC3000 </td><td> KBM Rifampicina (100 ug / ml) </td><td> 0,02 </td><td> 2 x 10 7 </td></tr><tr><td> Pst DC3000 Δ hopQ1-1 </td><td> KBM Rifampicina (100 ug / ml) </td><td> 100 de diluição de 0,1 vezes </td><td> 1 x 10 6 </td></tr><tr><td> P. syringae pv. tabaci 11528R </td><td> KBM Rifampicina (100 ug / ml) </td><td> 100 de diluição de 0,1 vezes </td><td> 1 x 10 6 </td></tr></tbody></table> Tabela 2: Condições de cultura e níveis de inóculo utilizado no ensaio. O OD e níveis correspondentes UFC pode variar entre diferentes marcas de espectrofotômetros.

<ol>
	<li>Abramovitch, R. B., Anderson, J. C., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+elicitation+and+evasion+of+plant+innate+immunity.">Bacterial elicitation and evasion of plant innate immunity.</a> Nat Rev Mol Cell Biol. 7 (8), 601-60 (2006).</li><li>Jones, J. D., Dangl, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plant+immune+system.">The plant immune system.</a> Nature. 444 (7117), 323-323 (2006).</li><li>Zipfel, C., Robatzek, S., Navarro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+disease+resistance+in+Arabidopsis+through+flagellin+perception.">Bacterial disease resistance in Arabidopsis through flagellin perception.</a> Nature. 428 (6984), 764-764 (2004).</li><li>Oh, H. S., Collmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+resistance+against+bacteria+in+Nicotiana+benthamiana+leaves+is+accompanied+by+reduced+vascular+staining+and+suppressed+by+multiple+Pseudomonas+syringae+type+III+secretion+system+effector+proteins.">Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins.</a> Plant J. 44 (2), 348-348 (2005).</li><li>Goodin, M. M., Zaitlin, D., Naidu, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotiana+benthamiana:+its+history+and+future+as+a+model+for+plant-pathogen+interactions.">Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.</a> Mol Plant Microbe Interact. 21 (8), 1015-1015 (2008).</li><li>Wei, C. F., Kvitko, B. H., Shimizu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pseudomonas+syringae+pv.+tomato+DC3000+mutant+lacking+the+type+III+effector+HopQ1-1+is+able+to+cause+disease+in+the+model+plant+Nicotiana+benthamiana.">A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana.</a> Plant J. 51 (1), 32-32 (2007).</li></ol>

A célula de ensaio de morte podem ser usadas para determinar o envolvimento de um gene no PTI. Por exemplo, perda de função mutantes para um gene de interesse pode ser testado com este ensaio. Das 4 combinações de indutor e inoculações desafio dada na Tabela 1, é possível que apenas uma combinação pode resultar em um fenótipo em seu fundo de plantas. Temos observado que as plantas silenciadas para FLS2 mostrar uma avaria do PTI na Pf / DC e Pf/Q1-1 combinações de indutor e inoculações desafio (Tabela 1). É essencial que as condições ambientais para o ensaio são o mais próximo possível às descritas na parte 3.1. A baixa umidade provoca a morte celular para continuar rápido demais, tornando o ensaio difícil de marcar. Se as condições descritas no nosso protocolo não funcionam em seu laboratório, em seguida, tentar alterar o nível de indutor ou inoculação desafio. Outro parâmetro que pode ser modificado é o intervalo de tempo entre indução e desafio, embora tenhamos encontrado que as lacunas de tempo mais curto fez com que o ensaio de quebrar muito rapidamente em plantas controle. Recomendamos que, pelo menos, 4-5 plantas ser testado em um experimento e um fenótipo positivo deve ser repetido em pelo menos 3-4 experimentos independentes.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP300-100</td>
<td>Prepare as a1M stock, adjust pH and filter sterilize. Store at room temperature. </td>
</tr>
<tr>
<td>Life Science UV/Vis Spectrophotometer</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>DU 730</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

assay for pathogen-associated molecular pattern (pamp)-triggered immunity (pti) in plants

Para perceber patógenos potenciais em seu ambiente, as plantas usam receptores de reconhecimento de padrões (PRRs) presentes nas membranas seu plasma. PRRs reconhecer recursos microbianos conservada chamado patógeno-padrões moleculares associados (PAMPs) e isso leva à detecção de PAMP disparado imunidade (PTI), o que efetivamente previne a colonização de tecidos vegetais por patógenos não 1,2. O sistema mais bem estudado no PTI é a via de três FLS2-dependente. FLS2 reconhece a flg22 PAMP que é um componente dos flagelos bacterianos. Patógenos de sucesso possuem fatores de virulência ou efetores que pode suprimir PTI e permitir que o patógeno para causar a doença 1. Algumas plantas, por sua vez possuem genes de resistência que detectam efetores ou sua atividade, o que leva a efetoras disparado imunidade (ETI) 2. Descrevemos uma célula de ensaio da morte-based para PTI modificado a partir Ah, e Collmer 4. O ensaio foi padronizado em N. benthamiana, que está sendo usado cada vez mais como um sistema modelo para o estudo da planta-patógeno interações 5. PTI é induzida pela infiltração de uma cepa não-patogênica bacteriana em folhas. Sete horas depois, uma cepa bacteriana que causa a doença tanto ou que ativa ETI é infiltrado em uma área de sobreposição da zona de infiltração original. PTI induzida pela infiltração primeiro é capaz de retardar ou prevenir o aparecimento de morte celular, devido à infiltração segundo desafio. Por outro lado, a aparência de morte celular na área de sobreposição de inoculação indica uma repartição dos PTI. Quatro combinações diferentes de indutores do PTI e inoculações desafio foram padronizados (Tabela 1). O ensaio foi testado em não-silenciadas N. benthamiana plantas que serviram como controle e plantas silenciadas para FLS2 que estava previsto para ser comprometida em sua capacidade de desenvolver PTI.

Watch this Scientific Journal Video about Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants at JoVE.com

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

Uma célula de ensaio de morte com base em plantas para PTI Nicotiana benthamiana é descrito.

Ensaio para Pathogen-Associated Padrão Molecular (PAMP) Triggered-Imunidade (PTI) em Plantas

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

assay-for-pathogen-associated-molecular-pattern-pamp-triggered

Research

JoVE Journal

Immunology and Infection

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

27.0K visualizações.  Boyce Thompson Institute for Plant Research. Uma célula de ensaio de morte com base em plantas para PTI Nicotiana benthamiana é descrito.

Vídeo: Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Seleção de Plasmodium falciparum Parasitas para Cytoadhesion para células do cérebro humano endotelial

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Imunologia e Infecção

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Investigando os efeitos dos probióticos sobre pneumocócica Colonização Usando um<em&gt; In Vitro</em&gt; Adesão Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Um fluxo de Adesão Ensaio para o Estudo de Recrutamento de leucócitos para hepáticas humanas senoidais endotélio sob condições de estresse de cisalhamento

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

High Yield purificação<em&gt; Plasmodium falciparum</em&gt; Merozoítos Para uso em opsonizantes Anticorpo Ensaios

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

-Fluorescência baseado Neuraminidase Ensaio de Inibição Para avaliar a susceptibilidade do vírus influenza A de neuraminidase Inibidor de Classe Antivirais

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Modelos de mouse da infecção por Helicobacter e patologias gástricas

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Estabelecimento de infecção Viral e análise da interação vírus-hospedeiro em Drosophila Melanogaster.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Imagem latente confocal de RNA Double-Stranded e receptores de reconhecimento padrão na infecção pelo vírus de RNA de sentido negativo

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;