The authors thank Dr. Lynda Partridge for the provision of advice and laboratory facilities.

1) Preparação de Linha de Células: <ol><li> O desenvolvimento da linha celular RBL-2H3.1 expressando α FcsRI eqüinos: <ol><li> Usando técnicas básicas de cultura de tecidos para linhas de células em monocamada, transfectar células RBL-2H3.1 parental usando o plasmídeo pEE6, que transporta o gene equina FceRIa (GenBank: Y18204.1) 28. Adicionar 2 ug -1 ul do DNA de plasmídeo a 0,8 ml de células a uma densidade de 1,2 x 10 7 células mL @ 1. Electroporate as células a 250 V, 960 uF utilizando um 0,4 centímetros electrocuvette depois incubar imediatamente em gelo durante 10 min. </li><li> Selecione as células transformadas utilizando meios contendo 0,4 g de sulfato de geneticina G418, em seguida, classificar as restantes células vivas através FACS por marcá-los com um anticorpo IgE fluorescente. Use o RBL-2H3.1 resultando expressar eqüinos linha celular FceRIa para a investigação 2, 3. </li></ol></li><li> Pré-ensaio de anticorpo de sensibilização: <ol><li&gt; Colheita da RBL-2H3.1 expressando células eqüinos FceRIa de um confluente placa de Petri. Lavar em seguida, re-suspender as células em meio de cultura a uma densidade celular de 5x10 5 células ml -1. </li><li> Adicionar a IgE de interesse para as células suspensas a uma concentração final de 1 ng mL-1, em seguida, a placa 100 ul de células numa placa de 96 poços em colunas 1-6 e incubar a 37 ° C + 5% de CO 2 + 90% humidade relativa durante 16 horas. Após o tempo de incubação, e antes de realizar o ensaio de libertação, verificar os poços sob um microscópio para o bem de confluência e aderência celular. </li></ol></li></ol> 2) Lançamento de Ensaio: <ol><li> Lavar as células: <ol><li> Tampão de libertação Quente (PIPES 25 mM, cloreto de sódio 120 mM, cloreto de potássio 5 mM, cloreto de magnésio 0,04 mM e 1 mM de cloreto de cálcio) a 37 ° C para permitir uma lavagem das células suavemente. </li><li> Lave as células sacudindo a placa para remover a mídia de células e adicionar 100 ml quente, 37 ° C, tampão de libertação. Repita duas vezes. </li></ol></li><li> Desafio Antígeno: <ol><li> Prepara-se uma diluição em série do antigénio (NIP-HSA ou DNP-HSA) de 0 ng ml -1, 0,1 ng ml -1, 1 ng ml-1, 10 ng mL-1, 100 ng ml -1, 1.000 ng ml -1, 10.000 ng ml-1 em tampão de liberação e quente a 37 ° C. </li><li> Após a segunda lavagem das células, os meios de descarte e substituído por 100 ul das soluções de antigénio. Certifique-se de que os poços de uma mesma fila (A1-6 por exemplo) têm a mesma concentração de antigénio adicionados a eles. </li><li> Configurar um controle negativo na linha A, adicionando 0 ng antígeno -1 ml. Adicionar o aumento da concentração de antigénio para baixo as linhas (BG), seguido de triton x-tampão (5% de Triton X-100) H em células de linha para lisar as células a serem utilizadas como um controlo positivo. Incubar a 37 ° C durante 20 minutos para permitir que as células de libertar os seus mediadores. </li></ol></li><li> Configurar controles bem individuais: <ol><li> Após a incubação, transferir 50 ul de sobrenadante de célula para a outra metade dos poços (placa de poços A1-6 A7-12, etc). Descartar as restantes 50 ul de sobrenadante e substituir com 50 ul de Triton-x tampão para permitir a medição da quantidade de mediadores libertados em cada cavidade nas colunas 7-12 como uma percentagem do total de mediadores no interior das células na coluna 1-6 . </li></ol></li><li> O substrato enzimático: </li><li> Adicionar 50 ul de substrato β-hexosaminidase (50 mM de 4-nitrofenilo N-acetil-β-D-glucosaminida preparado em DMSO diluídos até 2 mM, adicionando-o ao tampão de citrato ácido cítrico 0,2 M e 0,2 M de acetato de sódio, pH 4,5) a todos os poços para facilitar a conversão do substrato a 4-nitrofenol pela enzima β-hexosaminidase. Incubar as placas a 37 ° C durante 2 horas. </li></ol><img alt="Figura 2.4.1" fo:content-width="4in" src="/files/ftp_upload/52222/52222fig241.jpg" width = &quot;400&quot; /&gt; <ol start="5"><li> Encerrando a reação: <ol><li> Parar a reacção pela adição de 150 ul de tampão Tris (1 M Tris-HCl, pH 9) a cada poço como o elevado pH do tampão de pára a reacção e transforma o 4-nitrofenol em uma cor amarela. </li></ol></li><li> Lendo e analisando os resultados: <ol><li> Ler a placa utilizando uma placa espectrofotómetro a 405 nm para medir a absorvência da cor amarela. Calculado a percentagem de β-hexosaminidase libertada utilizando a seguinte fórmula: </li></ol></li></ol><img alt="Figura 2.6.1" fo:content-width="1in" src="/files/ftp_upload/52222/52222fig261.jpg" width="100" /><ol start="2"><li> Aplicar esta fórmula para cada poço, após o que é feita uma média para cada linha. A1 e A7 representam a localização dos poços da placa de 96 poços. Traçar uma curva de porcentagem de liberação β-hexosaminidase (o que corresponde a total liberação de mediador) açaíantígeno nst concentração 2, 3. </li></ol>

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The authors declare that they have no competing financial interests in this paper.

Em resumo, os resultados desta investigação mostraram que, quando as células RBL-2H3.1 expressando FceRIa eqüinos são sensibilizados com IgE eqüinos e desafiado por um antígeno, eles dão um pico de liberação mediador de 36,68% ± 4,88% do montante total de mediador dentro do As células, em comparação com o RBL-2H3.1 células parentais não expressam eqüinos FceRIa. Assim, este ensaio fornece uma ferramenta útil para investigar e estudar respostas alérgicas eqüinos in vit...

Alergia é conhecida há milênios. Um tratamento da asma foi descrita no texto médico egípcio antigo conhecido como o Papiro de Ebers (~ 1.550 aC) e discutido ervas remédios para tratá-la 7. Hoje alergia é classificada como uma resposta de hipersensibilidade do tipo I, em que o tipo de célula T helper 2 (TH 2) o braço do sistema imunitário dirige a produção de (IgE) anticorpos imunoglobulina E em resposta a antigénios ambientais chamados alérgenos. Estes são diversas substâncias que normalmente interagem com as células do sistema imunológico e estimular a síntese e secreção de citocinas pró-inflamatórias, incluindo a interleucina-4 e interleucina-13 8, 9, conforme se em partículas do fumo do cigarro ou do escape de diesel de partículas que aumentam a síntese de IgE 10. O aumento nas manifestações alérgicas em países industrializados nos últimos 50 anos, tem sido atribuída a uma combinação do efeito depoluentes ambientais e uma tendência para um ambiente mais higienizado, que combinam a mudar a resposta imunológica para um perfil predominou por TH duas citocinas, tal como proposto pelo &quot;Hipótese da Higiene&quot; 11. Como mencionado acima, os seres humanos não são os únicos mamíferos afetados por alergia. Notavelmente cavalos e cães também podem desenvolver reações alérgicas clássicos e um estudo de 12 mostrou que, como nos seres humanos, a alergia eqüina é atribuída a fatores genéticos e ambientais. Como consequência, esses animais apresentam bons modelos para o estudo da interação entre causas genéticas e ambientais de alergia, a sua progressão de sensibilização para a doença, e as possíveis estratégias de intervenção uma vez que as manifestações clínicas definidas em Em 1887, Stömmer foi a primeira pessoa a descrever a semelhança entre o ser humano e asma equina 13, o efeito da histamina sobre o sistema cardiovascular é equinamuito semelhante à dos seres humanos 14. Cavalos também são a pedra angular da indústria de corridas a cavalo, que vale US $ 72 bilhões, com um volume de apostas de US $ 115 bilhões por ano 15. A maioria dos cavalos de corrida contemporâneos são descendentes do pequeno número de cavalos árabes criados por Lady Anne Blunt de 1878 em diante. Cavalos de corrida modernos são comumente pura a escolha de habilidades de desempenho. Eles são propensas a doenças genéticas, uma das quais é a sua susceptibilidade para montar respostas alérgicas. Eles também têm mil vezes mais altos níveis séricos de IgE do que até mesmo os mais severamente alérgica seres humanos 16. Respostas alérgicas cavalo são geralmente se manifesta como hipersensibilidade picada de inseto (IBH) 17, 18. Resultados IBH na dermatite devido a picadas de insetos formam no gênero Culicoides. Outra forma de doença alérgica eqüina é obstruções das vias respiratórias recorrentes (RAO), isso se manifesta nos pulmões e vias respiratórias. É caracterizada por chiado e laboratóriorespiração ORed. RAO comumente ocorre em resposta a esporos, e altos níveis de IgE alérgeno-específicos foram registrados em cavalos que sofrem de RAO em um estudo de 19, embora outra investigação não confirmou esta 20. Estudos sobre alergia equina girado em torno da tentativa de controlar e neutralizar equina IgE através do desenvolvimento de anticorpos monoclonais anti-IgE equino (mAbs) 21, 22. Além disso, o estudo de 23 discute a produção dos domínios extracelulares de α o de elevada afinidade do receptor de Fc equina cadeia (FceRIa) receptor, na tentativa de detectar e quantificar soro eqüino IgE. Um estudo relacionado por Ledin 24 discute uma nova abordagem visando neutralizar IgE soro por priming do sistema imunológico através de um auto / não-auto imunógeno. Todos estes estudos, no entanto, faltava um ensaio eficaz para testar a segurança e eficácia dos seus protocolos. Neste artigo, vamos agora apresentar tal sys ensaiotempe aplicável ao estudo de estratégias de diagnóstico e terapêuticos relevantes para o sistema equina, onde libertação β-hexosaminidase, como um indicador de mediador de células desgranulação, foi avaliada em células RBL-2H3.1 expressam equina FceRIa. Este protocolo baseia-se em publicações anteriores 25, 4, 5, 2, 3 descrevem a engenharia de células RBL transfectadas com o gene que codifica o domínio de ligação do receptor de IgE de alta afinidade para IgE a partir de espécies diferentes. O protocolo explica como realizar um ensaio de libertação de β-hexosaminidase, cujos resultados são apresentados como a média ± desvio padrão de experiências em triplicado. O ensaio de liberação foi desenvolvido pela primeira vez por Siraganian e Gancho 25 para estudar alergia humano. O grupo de laboratório liderado pelo Dr. Reuben Siraganian também desenvolveu a linha de células RBL. Estas células RBL foram desenvolvidos para expressar o FceRIa humana e o protocolo foi publicada por 4. A peça finaldo ensaio de veio com o desenvolvimento do plasmídeo pSV no papel por Neuberger 26, que descreveu a produção de anticorpos IgE por uma clonagem do seu gene de cadeia pesada a jusante de um gene de rato para uma região variável de IgE que tem como alvo o hapteno 4-hidroxi-3 -nitro-fenacetilo (NP), o anticorpo quimérico resultante era totalmente funcional. A capacidade de desenvolver qualquer IgE direccionamento do mesmo hapteno, ao mesmo tempo, a clonagem do seu receptor na superfície das células RBL resultaram na padronização do ensaio tornando-se um protocolo útil para medir a desgranulação de basófilos. O ensaio tem prós e contras. As vantagens do ensaio é a sua adaptabilidade para ser utilizado em qualquer sistema de mamífero, o nosso laboratório tem, assim, usou-o para testar para a desgranulação nos sistemas humanos, caninos e equinos, e isso pode ser conseguido simplesmente através da síntese de IgE do organismo e a clonagem do seu receptor sobre a superfície das células RBL. Por outro lado,os contras do ensaio é que as células RBL são muito sensíveis a alterações térmicas, mecânicas e de PH, tornando-os dão uma variação de níveis de degranulação dentro do mesmo ensaio. É, portanto, altamente recomendável que os ensaios são sempre repetidas em triplicata e, em seguida, uma média é tomada a partir delas. Além disso, as células RBL tendem a deslocar para um fenótipo de não desprendimento, se forem deixados em cultura de tecidos durante tempos prolongados (&gt; 10 semanas) 27, tornando complicada a sua manutenção. Eles também são propensos a infecções por micoplasma, bactérias que não são visíveis a partir de um microscópio de luz e não modificam a morfologia da célula, mas iria alterar drasticamente os seus níveis de degranulação. Assim, são necessários testes de micoplasma regulares.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RBL-2H3.1 Expressing Equine Fc&#949;RI&#945;</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>Equine IgE anti NIP-HSA</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>96 Well Plate</td>
			<td>Sigma</td>
			<td>CLS3595</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Multi Channel Pipette</td>
			<td>Anachem</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Galaxy R</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4Hydroxy-5-iodo-3-nitrophenylacetic acid</td>
			<td>Cambridge Research Biochemicals</td>
			<td>N-1070-1</td>
			<td>NIP-OH was conjugated with Human Serum Albumin to make NIP-HSA in the lab</td>
		</tr>
		<tr>
			<td>Dinitrophenyl Conjugated to Human Serum Albumin</td>
			<td>Sigma</td>
			<td>A6661</td>
			<td>Abbreviated DNP-HSA</td>
		</tr>
		<tr>
			<td>Plate Spectrophotometer</td>
			<td>Anthos Labtec HT2</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipes</td>
			<td>Sigma</td>
			<td>P1851</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Triton x100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4-nitrophenyl N-acetyl-&#946;-D-glucosaminide</td>
			<td>Sigma</td>
			<td>N9376</td>
			<td>Stock solution called &#946;-hexosaminidase substrate was 50mM prepared in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma</td>
			<td>251275</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma</td>
			<td>S7670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

development of an in vitro model system for studying the interaction of equus caballus ige with its high-affinity receptor fc&#949;ri

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

As células RBL-2H3.1 parentais e as transfectadas com o gene do receptor de FceRIa equino foram sensibilizados primeiro com IgE de murganho anti DNP-HSA e desafiados com o antigénio DNP-HSA. Rato IgE se liga ao receptor endógeno de rato em ambas as linhas de células e, assim, actua como um controlo para testar a viabilidade de libertação de ambas as linhas celulares para libertar mediadores (Figura 1 A). Esta é uma verificação importante e deve ser levada a cabo rotineiramente desde cima prolon...

Watch this Scientific Journal Video about Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÎµRI at JoVE.com

Development of an &lt;em&gt;in vitro&lt;/em&gt; model system for studying the interaction of &lt;em&gt;Equus&lt;/em&gt; &lt;em&gt;caballus&lt;/em&gt; IgE with its high-affinity receptor Fc&amp;#949;RI

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Desenvolvimento de um<em&gt; In vitro</em&gt; Sistema modelo para estudar a interacção de<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE ao seu receptor de elevada afinidade FceRI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

development-an-vitro-model-system-for-studying-interaction-equus

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Immunology and Infection

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

12.3K visualizações.  King Abdulaziz University. The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine FcεRIα gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Vídeo: Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Desenvolvimento de um marcador negativo selecionáveis ​​para Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Imunologia e Infecção

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

In vitro a formação de biofilme em um slide Câmara de 8 poços

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Estabelecimento de um<em&gt; In vitro</em&gt; Sistema para estudar o comportamento intracelular de<em&gt; Candida glabrata</em&gt; Em humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel<em&gt; In vitro</em&gt; Modelo para estudar as interações entre a Whole Humano Sangue e endotélio

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Vitro Análise do mediada por Myd88 Cellular resposta imune a vírus do Nilo Ocidental Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Um<em&gt; In Vitro</em&gt; Modelo para medição de respostas imunes a Malária no Contexto do HIV co-infecção

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Estudar comunidades microbianas<em&gt; In Vivo</em&gt;: Um modelo de interação mediada por anfitrião entre<em&gt; Candida Albicans</em&gt; E<em&gt; Pseudomonas aeruginosa</em&gt; Nas Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

A<em&gt; In Vitro</em&gt; Modelo da barreira sangue-cérebro que usa espectroscopia de impedância: Um foco na interação celular T Cell-endotelial

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualizando os Efeitos de escarro no desenvolvimento de biofilmes usando um modelo Chambered lamela

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Desenvolvimento de um sistema de entrega de DNA Economical por &quot;Acufection&quot; e sua aplicação à Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...