Este estudo foi apoiado por Max McGee National Research Center for Juvenile Diabetesat Medical College of Wisconsin e infantil Research Institute of Wisconsin. Os financiadores não tiveram nenhum papel no desenho do estudo, coleta de dados e análise, ou preparação do manuscrito.

1. Antes de configurar supressão de um ensaio, um precisa de contas de revestimento tosylactivated com anti-CD3 humano (clone UCHT1, 1μg/ml concentração final) para a estimulação das células e, depois, verificar se as contas são eficientemente revestido através da criação de um ensaio de proliferação in vitro utilizando humana células T <ol><li> Tomar 1ml de M-450 contas tosylactivated do frasco original, realizada em suporte magnético e segure até que todas as contas tenham aderido ao lado do tubo. Remova o tubo de buffer enquanto ainda está no suporte magnético. Leve tubo de suporte magnético e adicionar 1 ml de buffer1, coloque o tubo no suporte magnético novamente e remover o buffer1 enquanto tubo está no suporte magnético; contas ressuspender em 1 ml de buffer1 e adicionar 40μl de anti-CD3 humano, agite a 37 ° C por 15 minutos, adicionar 0,1% w / v BSA e continuar agitando para o próximo 16 horas. </li><li> Lave as contas em buffer2 duas vezes durante 5 minutos a 2-8 ° C e de vez em buffer3 por 5 minutos a 2-8 ° C usando suporte magnético, como explicado acima; remover o tampão e ressuspender as contas em 1ml de buffer2; as contas são concentração de 4x10 8 ml / final e pronto para uso. </li><li> Alíquota de 50.000 e 25.000 PBMC / poço, em triplicata em placa de 96 poços e adicione número variável de CD3-revestido contas (4x10 8 contas / ml, CD3 1μg/ml, por exemplo 1, 2, 3, 4, 5 esferas / células ), a fim de determinar o número ideal de contas por célula. Após 72 horas de cultura, adicionar 1μCi [3 H] timidina e continuar incubando a 37 ° C para os próximos 16 horas. Células colheita em chapa de colheita Multiscreen (Millipore), adicione líquido de cintilação e ler contagens por minuto (cpm) / poço usando Top Conde NXT (Packard, CT). Use as contas / razão entre células quando CPMs são acima de 5000, mas menos de 15 mil para evitar a superestimulação de Tregs, que pode perder função supressiva. Normalmente, a razão de 3 esferas / células estimula tanto responder e células Treg em todos os grupos sujeitos testados até agora 1-4. </li></ol> 2. PBMC isolamento do sangue total de doadores saudáveis ​​ou de leukopacks humano ou buffy coat (BC), geralmente tiradas de voluntários saudáveis ​​e disponível gratuitamente a partir de centros de transfusão de sangue locais (Figura 1) <ol><li> Diluir o BC (~ 50ml) 1:06 com PBS (adicione 250ml). Agora não há volume total de 300ml. Lentamente camada de 25ml de BC diluída em cima de 15ml Ficoll-Paque PLUS adicionado 50ml tubos Falcon, sem perturbar as camadas. Centrifugar a 800xg (1400 rpm em uma centrífuga Sorvall com rotor girando balde SH-3000) por 30 minutos a 4 ° C, com freios desligado. </li><li> Recolher com cuidado a camada de PBMC (fase intermediária) e transferi-lo para uma nova 50ml tubos Falcon. Lavar PBMC preenchendo os tubos de até 50ml com DPBS. Coletar 2 pellets de células em um tubo. Centrifugar a 400xg por 10 min a 4 ° C. Repita o passo de lavagem duas vezes, cada vez que combina dois pellets de células em um tubo único. Combine todos os pellets de células em um tubo de 50ml único. </li><li> Contagem de PBMC usando Trypan teste de exclusão Azul. Fazer uma diluição de 1:10 em Trypan Blue adicionando 20μl de PBMC de 180μl de Trypan Blue mancha. Misture bem e leve 20μl a contar em hemocitômetro imediatamente. Contar números de todas as células sem mácula e apenas azul manchado localizados em dois blocos quadrados cada um contendo 16 quadrados menores. Tomar a média dos dois números e multiplicar por 10. Divida este número por 100 para obter o número de PBMC / ml. Porcentagem de células viáveis ​​calcular como [1 - (número de células azuis / número de células total) x100]. Proceder se a viabilidade é ≥ 95%. </li></ol> 3. MACS tipo pré-CD4 células T <ol><li> Antes de prosseguir, a transferência de 1 ml de PBMC em um novo tubo, adicionar 4ml de mídia para preparar células de irradiação com 5000rad-estes serão células apresentadoras de antígenos (APC). </li><li> Centrifugar o resto das células em 250xg em PBS/2mM tampão BSA EDTA/0.5% por 10 min a 4 ° C. Deitar fora o sobrenadante de células, e ressuspender em 4ml de tampão BSA PBS/2mM EDTA/0.5%. </li><li> Adicionar 200μl de microesferas de MACS anti-CD4 e incubar a 4 ° C por 20 minutos. </li><li> Lavagem adicionando 40 ml de tampão BSA PBS/2mM EDTA/0.5% e centrifugar a 250xg de 10min a 4 ° C. Deitar fora o sobrenadante e ressuspender em 8ml de desgaseificado à temperatura ambiente, PBS/2mM EDTA/0.5% tampão BSA. </li><li> Filtro-separada de suspensão da célula usando filtros pré-separação antes de carregá-los em uma coluna de LS. </li><li> Dividir a suspensão de células e 4ml camada de cada coluna LS mais calibrada (com 3 ml de tampão BSA deggassed PBS/2mM EDTA/0.5%). LS coluna é fixa no separador MidiMACS. Antes de suspensão celular se esgota, ou fluxo contínuo re-run ou adicionar 3 ml de temperatura ambiente deggassed PBS/2mM EDTA/0.5% tampão BSA e deixar que o tampão de corrida completamente. Adicione mais buffer até que ele sai claro. </li><li> Pipetar 5 ml tampão deggassed na coluna LS, LS remover Coluna do separador MidiMACS e coloque em um tubo de nova coleção estéril 15ml deixar ~ 1,0 ml por rodar. Coloque o êmbolo na coluna e empurre lentamente o resto dode volume para fora. </li><li> Faça o mesmo com as duas colunas LS e combinar as duas CD4 + frações. Adicione até 50ml células PBS e contar. Rendimento esperado é de até 5x10 8 células. Centrifugar a 400xg por 10 minutos e ressuspender as células bem em 2ml PBS/2mM tampão BSA EDTA/0.5%. </li></ol> 4. Fluorescente ativado Celular classificação de isolamento (FACS) (Figura 2) <ol><li> Faça um coquetel de anticorpos para marcadores de CD para os marcadores de células seguintes superfície (manter protegido da luz): 20 l de anti-humano CD8-FITC (clone RPA-T8), 20 l de anti-CD14 humano-FITC (clone M5E2; LPS receptor), 20 l de anti-CD32 humano-FITC (clone FLI8.26; FcγR do tipo II) e 6 mL de anti-humano CD116-FITC (clone M5D12; GM-CSFRα cadeia) e, alternativamente, adicionar anti 40μl -humanas CD4-APCCy7 (clone RPA-T4). </li><li> Tome 5μl de suspensão de células de 2ml e mancha com 2μl de cocktail mancha, que é um tubo para determinar o limiar (Fluorocromo Minus One-FMO) 5. </li><li> Adicionar 50 ul de anti-CD25 humano-PE (clone M-A251; IL-2Rα) para o coquetel de mancha, e adicione o cocktail de suspensão de células e incubar a 4 ° C 30 minutos. Lavar as células em tampão PBS, centrifugue a 400xg por 10 minutos e ressuspender as células a uma concentração de células de 10 7 / ml. </li><li> Prepare células imaculada e células ou grânulos corados com fluorocromo único para usar como controle de compensação para separação de células em FACS Aria (BD Biosciences, San Jose, NJ). </li><li> Aquisição de células no tubo FMO que permitirá ao usuário definir o limite para a classificação de células CD25 + T (Figura 2a). Definir uma porta em torno FITC-negativos para excluir as células-FITC células positivas compreendendo monócitos, macrófagos e todos os outros CD4 + T-não-células (Figura 2b). </li><li> Em um lote separado, desenhar portas para CD25, CD25low e T-CD25high células Tregs (top 1% das células que expressam o maior número de CD25) (Figura 2c). Subpopulações de células normalmente apresentam alta pureza (Figura 2d, 2e e 2f). </li><li> Centrifugar tubos de coleta com células em 400xg por 10 minutos e mantê-los no gelo até placas. </li></ol> 5. Criar cultura de células em placa de 96 poços (esquema anexado como Table1) em 200μl/well <ol><li> 50μl alíquota de CD3-revestido contas (1 mg / ml), calculado a ser três esferas / resposta celular em um poço, ressuspenso em media completo com 10% de soro AB humanos agrupados em U bottom-placas de 96 poços. (Por exemplo, para fazer media com 2ml CD3-revestido, tomar 7.5μl do estoque de CD3-revestido contas-final 4x10 concentração 8 ml / e diluir em 2 ml de mídia, cada 50μl conterá 75.000 beads-3beads/cell). </li><li> Diluir APC irradiada para 5x10 concentração celular 5 ml / e adicionar 50μl (conterá 2,5 x 10 4 células) em cada poço com estimulação previamente adicionados, incluindo poços rotulados como &quot;Tregs só&quot;, &quot;A APC só&quot; e &quot;media apenas&quot; na Tabela 1. </li><li> Adicionar 2,5 x 10 4 / CD4CD25 bem-T ou células CD4CD25low em triplicatas seguinte projeto na Tabela 1. </li><li> Adicionar Tregs para co-culturas (linha B, Tabela 1) na proporção 01:10 (2.500 células Treg) e poços rotulados como &quot;Tregs only&quot; e incubar a placa a 37 ° C em incubadora de CO 2 com 5% de CO 2 , na umidade saturada por 72 horas. </li><li> Pulso de poços com 1μCi [3 H] timidina e continuar incubação a 37 ° C para o próximo 16 horas. </li></ol> 6. Colheita e contando <ol><li> Células colheita na placa colheita multiscreen usando Packard filtermate colheitadeira ou sistema alternativo. </li><li> Adicionar líquido de cintilação (Microscint 20), tampa colheita placa com a cobertura de plástico transparente, em preparação para a etapa final. </li><li> Leia contagens por minuto (cpm) / poço usando Top Conde NXT (Packard, CT) ou sistema alternativo. </li></ol> 7. Porcentagem de supressão de computação <ol><li> Como as células foram cultivadas em triplicatas, a média é calculada para cada condição. Se o coeficiente de variação é&gt; 30%, o outlier é eliminado e apenas cpm a partir de dois poços são em média. Porcentagem de supressão é obtida pelo cálculo [(sc) / s] x 100%, onde s = cpm em cultura única e c = cpm em co-cultura. </li><li> Como naïve (CD25) e in vivo ativado (CD25low) células T efetoras são banhados como células T responder, a diferença na capacidade de Tregs para suprimir a cada um desses subconjuntos é capturado e usado como um indicador de prognóstico potencial funcional baseada na premissa de que células ativadas são mais difíceis de suprimir. </li></ol> 8. Resultados representativos: Grande variabilidade nos métodos utilizados e os resultados derivados do ensaio in vitro em supressão humana nos levou a realizar um estudo abrangente das condições que influenciam o ensaio 1. Temos desenvolvido um ensaio que os testes não só a função Treg, mas também a sua pureza, considerando a baixa relação entre Tregs: Teffs (1:10), que determinou anteriormente 6. Além disso, diferem em Tregs ªeir capacidade de suprimir com sucesso ingênua e vivo em células T ativadas, mesmo em indivíduos saudáveis, como mostrado na Figura 3 e em nossos estudos anteriores 2,4, que se torna mais proeminente se o equilíbrio imunológico está comprometido, como em indivíduos em risco de desenvolver DM1 . O ensaio funcionou muito bem no estudo onde comparou a função de supressão de recursos naturais (nTregs), induzível (iTregs) e em nTregs vitro expandido, permitindo-nos comparar a sua função entre o controle saudável, de início recente (RO) e DM1 de longa data (LS ) indivíduos DM1. Concluiu-se que assuntos RO DM1 tinham melhor capacidade de gerar funcional tanto iTregs e nTregs expandiu em comparação com indivíduos controle LS DM1 e saudável 7. Assim, este ensaio pode ser usado como uma excelente ferramenta para o reconhecimento tanto do Estado uma precoce e tardia de desequilíbrio imunológico. Esquema de apresentação esquemática uma das etapas envolvidas no ensaio in vitro em supressão <img src="/files/ftp_upload/3071/3071fig1.jpg" alt="Figura 1"/> Figura 1. Etapas do ensaio in vitro em supressão apresentados com fotografias <img src="/files/ftp_upload/3071/3071fig2.jpg" alt="Figura 2"/> Figura 2. Estratégia Gating FACS em isolamento celular. a) CD25 limite + foi ajustada de acordo com Fluorocromo Minus One (FMO), b) as células foram fechadas como FITC-negativos, c) FITC-negativos células foram novamente fechados e recolhidos como CD + CD25-, CD + CD25low e CD + CD25high ( Tregs) mostrada com porcentagens, d) FACS classificadas Tregs após a triagem, e) FACS classificadas CD + CD25low após a triagem, e f) FACS CD4 + CD25-classificadas células T após triagem <img src="/files/ftp_upload/3071/3071fig3.jpg" alt="Figura 3"/> Figura 3. Os resultados representativos de indivíduos saudáveis ​​a) Os resultados representativos de contagens por minuto (cpm) de indivíduos saudáveis ​​apresentado como único para todas as culturas de células subconjuntos envolvidos (naïve-CD25-, in vivo ativado-CD25low, células apresentadoras de antígenos-APC e células T reguladoras-Treg), bem como co-culturas de células de resposta T (CD25 ou CD25low) e Tregs. b) Percentagem de supressão de cada CD25 e células T CD25low responder por Tregs autólogo é apresentado para controle de indivíduos saudáveis ​​(n = 4). Supressão foi calculada como [(sc) / s] x 100%, onde s = cpm em cultura única e c = cpm em co-cultura. Embora pequena diferença na capacidade de Tregs para suprimir as células T responder foi notado, não foi significativa (teste t pareado p = 0,08). C) Apresentado são cpm em indivíduos de risco para cada cultura única, incluindo-CD25 e CD25low como responder células T, bem como a APC e Tregs, e co-culturas, onde cada resposta subconjunto de células T é semeado com Tregs Percentagem (CD25-/Tregs e CD25low/Tregs). d) de supressão de cada CD25 e CD25low células T responder por autólogo Tregs é apresentado para em indivíduos de risco (n = 4). A diferença na capacidade de Tregs para suprimir CD25 versus CD25low células T responder foi significativa (teste t pareado p = 0,04). Tabela 1. Esquemática criação de ensaio in vitro em supressão <table border="1"><tbody><tr><td></td><td> 03/01 </td><td> 06/04 </td><td> 09/07 </td><td> 12/10 </td></tr><tr><td> A </td><td> CD4CD25- </td><td> CD4CD25low </td><td> somente mídia </td><td> somente mídia </td></tr><tr><td> B </td><td> CD4CD25 / Tregs </td><td> CD4CD25low / Tregs </td><td> Tregs apenas </td><td> APC só </td></tr><tr><td> C </td><td></td><td></td><td></td><td></td></tr><tr><td> D </td><td></td><td></td><td></td><td></td></tr><tr><td> E </td><td></td><td></td><td></td><td></td></tr><tr><td> F </td><td></td><td></td><td></td><td></td></tr><tr><td> G </td><td></td><td></td><td></td><td></td></tr><tr><td> H </td><td></td><td></td><td></td><td></td></tr></tbody></table>

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Como a única característica única de Tregs, função supressiva deverá ser testado de forma confiável e de maneira uniforme entre os indivíduos em diferentes fases de desenvolvimento da doença dentro da mesma e entre diferentes estudos. Oferecemos os detalhes do ensaio de supressão desenvolvido em nosso laboratório como a nossa contribuição para a padronização deste ensaio. Em nosso estudo otimização, nós determinamos que a estimulação de células T com anti-CD3 humano revestido contas (UCHT1 clone, na...

<table border="1"><tbody><tr><th> Nome do reagente ou instrumento </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Ficoll-Paque PLUS </td><td> Amersham Pharmacia Biotech </td><td> 17-1440-03 </td><td></td></tr><tr><td> DPBS-1X </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> Trypan Blue </td><td> Invitrogen </td><td> 15250-061 </td><td></td></tr><tr><td> anti-CD4 microesferas </td><td> Miltenyi </td><td> 130-045-101 </td><td></td></tr><tr><td> Pré-separação filtros </td><td> Miltenyi </td><td> 130-041-407 </td><td></td></tr><tr><td> Coluna LS </td><td> Miltenyi </td><td> 130-042-401 </td><td></td></tr><tr><td> EDTA </td><td> Invitrogen </td><td> 15575-020 </td><td></td></tr><tr><td> BSA </td><td> Sigma-Aldrich </td><td> B4287 </td><td></td></tr><tr><td> Anti-humano CD4-APCCy7 (clone RPA-T4) </td><td> BD PharMingen </td><td> 557852 </td><td></td></tr><tr><td> Anti-humano CD25-PE (clone M-A251; IL-2Rα) </td><td> BD PharMingen </td><td> 555432 </td><td></td></tr><tr><td> Anti-humano CD8-FITC (clone RPA-T8) </td><td> BD PharMingen </td><td> 555366 </td><td></td></tr><tr><td> Anti-humano CD14-FITC (clone M5E2; receptor LPS) </td><td> BD PharMingen </td><td> 555397 </td><td></td></tr><tr><td> Anti-humano CD32-FITC (clone FLI8.26; FcγR do tipo II) </td><td> BD PharMingen </td><td> 555448 </td><td></td></tr><tr><td> Anti-humano CD116-FITC (clone M5D12; GM-CSFRα cadeia) </td><td> BD PharMingen </td><td> 554532 </td><td></td></tr><tr><td> Dynalbeads M-450 tosylactivated </td><td> Invitrogen </td><td> 140-13 </td><td></td></tr><tr><td> Anti-CD3 humano </td><td> Ancell </td><td> 144-024 </td><td></td></tr><tr><td> Buffer1 </td><td> Caseiro </td><td></td><td> 0,1 M Na 2 B 4 O 7 pH7.6 </td></tr><tr><td> Buffer2 </td><td> Caseiro </td><td></td><td> PBS/2mM BSA EDTA / 0,1% pH7.4 </td></tr><tr><td> Buffer3 </td><td> Caseiro </td><td></td><td> 0.2M Tris/0.1% BSA pH8.5 </td></tr><tr><td> RPMI completo media </td><td> Caseiro </td><td></td><td> RPMI 1640 media 2 mM L-glutamina 5 mM HEPES 100 U / mg / ml peni / strept 0,5 mM piruvato de sódio </td></tr><tr><td> [3 H] timidina </td><td> Perkin Elmer </td><td> NET027Z005MC </td><td></td></tr><tr><td> humanos agrupados soro AB </td><td> Atlanta Biologicals </td><td> S40110 </td><td></td></tr><tr><td> Placa de colheita Multiscreen </td><td> Millipore </td><td> MAHFC1H60 </td><td></td></tr><tr><td> Microscint 20 </td><td> Perkin Elmer </td><td> 6013621 </td><td></td></tr></tbody></table>

human in vitro suppression as screening tool for the recognition of an early state of immune imbalance

Células T reguladoras (Tregs) são mediadores críticos de tolerância imune a auto-antígenos. Além disso, eles são reguladores fundamentais da resposta imune após uma infecção. Apesar dos esforços para identificar marcador de superfície única sobre Tregs, a única característica única é a sua capacidade para suprimir a proliferação e função das células T efectoras. Embora seja claro que só em ensaios in vitro podem ser utilizados na avaliação da função Treg humano, este torna-se problemático quando se avalia os resultados de estudos transversais, onde as células saudáveis ​​e células isoladas de indivíduos com doenças auto-imunes (como Diabetes Tipo 1-DM1) precisam a serem comparados. Há uma grande variabilidade entre laboratórios no número e tipo de células T responder, a natureza e força de estimulação, Treg: rácios de resposta eo número e tipo de células apresentadoras de antígenos (APC) usado em humanos em testes de supressão vitro. Esta variabilidade dificulta a comparação entre os estudos que medem a função Treg difícil. O campo precisa de um ensaio de Treg supressão padronizados que vai funcionar bem com ambos os indivíduos saudáveis ​​e aqueles com doenças auto-imunes. Nós desenvolvemos um ensaio in vitro em supressão que mostra muito pouco da variabilidade intra-ensaio na estimulação de células T isolados de voluntários saudáveis ​​em comparação a indivíduos com base destruição auto-imune do pâncreas β-células. O objetivo principal desta peça é descrever um ensaio in vitro que permite a supressão humana comparação entre grupos de sujeitos diferentes. Além disso, este ensaio tem o potencial para delinear uma pequena perda em função nTreg e antecipar ainda mais a perda de, no futuro, assim, identificar temas que poderiam se beneficiar da terapia com imunomoduladores preventiva 1. Abaixo, fornecemos descrição detalhada das etapas envolvidas neste procedimento. Esperamos contribuir para a padronização do ensaio in vitro em supressão usado para medir a função Treg. Além disso, oferecemos este ensaio como uma ferramenta para reconhecer um estado inicial de desequilíbrio imunológico e um biomarcador em potencial funcional para DM1.

Watch this Scientific Journal Video about Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance at JoVE.com

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs são potentes supressores do sistema imune. Há uma falta de marcadores de superfície única para defini-los, portanto, definições de Tregs são principalmente funcionais. Aqui nós descrevemos um otimizado<em&gt; In vitro</em&gt; Teste capaz de identificar o desequilíbrio imunológico em indivíduos em risco de desenvolver DM1.

Humano In Vitro Supressão como ferramenta de triagem para o reconhecimento de um Estado precoce de desequilíbrio imunológico

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

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JoVE Journal

Immunology and Infection

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

14.4K visualizações.  Medical College of Wisconsin. Tregs são potentes supressores do sistema imune. Há uma falta de marcadores de superfície única para defini-los, portanto, definições de Tregs são principalmente funcionais. Aqui nós descrevemos um otimizado In vitro Teste capaz de identificar o desequilíbrio imunológico em indivíduos em risco de desenvolver DM1.

Vídeo: Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Imunologia e Infecção

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Quantificação da Resposta explosão respiratória como um indicador da imunitário inatas Saúde em Zebrafish

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Uma<em&gt; In vitro</em&gt; Modelo para estudar respostas imunes de sangue periférico humano células mononucleares para Vírus Respiratório Sincicial Humano Infecção

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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 ...

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. ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Estabelecimento de um programa de configuração de alta capacidade para a triagem de pequenas moléculas que modulam c-di-GMP Sinalização<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

Determinar a supressão imune Sistema contra CNS Proteção para farmacológicos intervenções na desmielinização auto-imune

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Análise de Linfócitos extravasamento Usando um<em&gt; In Vitro</em&gt; Modelo do Sangue-Cérebro humano Barreira

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Análise de 18FDG PET-CT Imaging como uma ferramenta para estudar infecção por Mycobacterium tuberculosis e tratamento em primatas não-humanos

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...