Este trabalho foi financiado pelo National Institutes of Health (NIH) e do AI091759 de Crohn e Colite Fundação de William da América e Modell Shelby Família Research Award Foundation (a MN). Esquistossomo camundongos infectados foram fornecidos pelo Centro de Recursos NIAID esquistossomo (Contrato NIAID N01 A130026).

1. Purificação Schistosoma mansoni Ovos <ol><li> Infect suíça Webster-murganhos com cercarias esquistossomo, que é a fase infecciosa na vida Schistosoma 8 ciclo. Alternativamente, obter esquistossomo camundongos infectados a partir do NIAID Esquistossomose Resource Center ( <a href="http://www.schisto-resource.org/" target="_blank">http://www.schisto-resource.org/~~V</a> ). Deixar ratinhos durante 6-7 semanas após a infecção, através da qual ponto do tempo, os ovos são presente no fígado e pode ser recuperado como descrito abaixo (ver fig. 1). </li><li> Euthanize ratinhos com CO 2, e molhada com etanol a 70%. Coloque o mouse em volta. Use uma pinça e tesouras sem corte para cortar a pele e peritônio subjacente. O fígado Excise, localizado abaixo da caixa torácica e diafragma. </li><li> Finamente moída fígados e adicionar 20 mL por fígado de mistura de digestão, composto de colagenase / dispase (0,5 mg / mL), penicilina (100 U / mL) e estreptomicina (100 ug / mL) em PBS. Coloque mixture num tubo Falcon 50 mL e incubar durante a noite num agitador de 37 ° C. </li><li> Centrifugar mistura a 400 xg / 3 min / 20 ° C. Gentilmente deitar fora o excesso de líquido e tubo de enchimento com PBS. Centrifugar novamente e repita lavagem PBS. Após centrifugação passado, ressuspender o pellet em 25 ml de PBS. </li><li> Coe o sedimento ressuspenso através de um padrão de cozinha metálico filtro (50 mL usar uma seringa de êmbolo para esmagar os pedaços de fígado através de, num copo de vidro), seguido pela passagem da mistura através de uma peneira fina (utilizar seringa de 10 mL para empurrar mistura através da peneira fina para um copo de vidro segundo). </li><li> Coloque a mistura em uma tensa tubo Falcon 50 mL. Centrifugar a 400 xg / 5 min / 20 ° C. Cuidadosamente derramar sobrenadante sem perder pelota. Ressuspender o granulado em 3 ml de PBS. </li><li> Sobreposição misturar suavemente sobre uma Percoll 20%, 40 mL de gradiente de 8 mL de Percoll e 32 mL de sacarose 0,25 M (FW = 342,3 assim 8,56 g sucrose/100 mL H 2 0). </li><li> Centrifugar 10 min / 800 xg / 20 ° C. Pipetar fora de umd descartar camada superior gelatinosa de células do fígado. Remover ovo pellet no fundo com uma pipeta de transferência e colocado num tubo Falcon 15 mL. </li><li> Lavagem pelete 3X em 10 mL de PBS / 1 mM EDTA / EGTA 1 mM configurações de centrífuga usando: 3 min 30 / xg / 20 ° C. Ressuspender em 500 uL de PBS. </li><li> Sobreposição mistura sobre uma nova Percoll 25%, 10 mL de gradiente (2,5 mL de Percoll e 7,5 mL 0,25 M de sacarose), utilizando as definições de centrífuga: 10 min / 800 xg / 20 ° C. </li><li> Lavar 3 vezes em 10 mL de PBS com configurações de centrífuga: 3 min 30 / xg / 20 ° C. </li><li> Na última lavagem de 10 mL, remover 100 uL para contagem de ovos. Ovos resolver rapidamente, de modo que o tubo Falcon deve ser bem misturada antes de remover amostra. Dilui-se a amostra de 100 uL com 900 uL de PBS. 100 L da mistura deve ser contado como gotas sobre uma lâmina de microscópio utilizando um microscópio de dissecação (1:10 diluição). Dicas de grande calibre da pipeta deve ser usado. </li><li> Ressuspender ovos em 50.000 ovos / ml em PBS. Os ovos podem ser armazenados durante vários meses à temperatura de -80 ° C e descongelado uma vez ou TWICe para o uso. Antes da utilização, inspeccionar sob um microscópio para garantir que os ovos são ainda intacta (ver fig. 1). </li></ol> 2. Sensibilização intraperitoneal com S. Ovos mansoni: dia 0 <ol><li> Preparar ovos em 5000 eggs/100 PBS uL em um tubo de snapcap 5 mL. Para injecção, estimar ovos 50% mais que o necessário. </li><li> Carregar uma agulha de calibre 23 3/4inch e uma seringa de 1 mL com 100 uL de suspensão de ovo por ratinho. Ovos resolver, de modo agulha de carga com ovos imediatamente antes da utilização. </li><li> Seringa de rock para trás e para misturar os ovos antes de cada injeção, e injetar intraperitonealmente 100 uL de suspensão por mouse. </li></ol> 3. Retro-orbital Desafio intravenosa com S. Ovos mansoni: Dia 14 <ol><li> Prepare ovos, como acima a 5.000 uL eggs/100. </li><li> Anestesiar os ratos em câmara especializado com isoflurano (4%), ou por via intraperitoneal com xilazina (10 mg / kg) e cetamina (120 mg / kg). Profundidade da Anesthesia é determinada por beliscar de almofada da pata e garantir a ausência de resposta física. </li><li> Após carregamento da seringa de 1 ml e 23 agulha de calibre 3/4inch com ovos, utilize uma pinça para dobrar a agulha com um ângulo de 90 ° com o bisel voltado para baixo para o ângulo. Verifique se não há bolhas de ar. </li><li> Coloque o mouse de um lado. Retrair pele de modo olho vai sobressair, e injectar 100 uL para os vasos por trás do globo ocular em um ângulo de 45 ° para o nariz. </li><li> Retirar a agulha e aplicar uma ligeira pressão para controlar o sangramento. </li></ol> Todos estes procedimentos devem ser realizadas com cuidado, e ratinhos deve ser monitorizada até que eles se recuperar da anestesia. Estes protocolos foram aprovados pela Universidade da Pensilvânia Animal Care Institucional e Comitê de Uso (IACUC). 4. Colheita Experimental: Dia 22 <ol><li> Euthanize camundongos com CO 2. </li><li> Rato molhado com etanol 70%, e em lugar de volta. </li><li> Usando contundente forceps, segure o abdômen do rato e fazer uma pequena incisão na pele. Remova cuidadosamente a pele com uma tesoura e expor a caixa torácica e do peritônio. </li><li> Corte abrir o peritônio. Cortar o aorta abdominal para recuperar o sangue com uma pipeta de Pasteur. Guarde no gelo. </li><li> Mover o fígado para baixo para expor o diafragma. Use tesouras finas para cortar o diafragma. Cuidadosamente cortado e aberto da caixa torácica para expor o tecido pulmonar. </li><li> Recuperar os linfonodos parathymic que drenam o pulmão com uma pinça fina. Estes são dobrados por baixo da caixa torácica, em ambos os lados do timo 9. Armazenar sobre gelo em 1ml meios estéreis (DMEM suplementado com 10% inactivado pelo calor soro fetal de vitelo, 100 U / mL de penicilina, estreptomicina 100 ug / mL, 2 mM de L-glutamina, 50 mM 2-mercaptoetanol, todos disponíveis a partir de Invitrogen). </li><li> Para recuperar o BAL, e inflar os pulmões para análise histológica, intubação orotraqueal deve ser realizada. Expor a traqueia, movendo as glândulas salivares para o lado, e placing pinça fina sob a traquéia. Utilizando uma tesoura fina, cortar um furo no meio da traqueia, e inserir tubagem para dentro dos pulmões. Fixe tubulação, amarrando com fio cirúrgico. </li><li> Anexar PBS-enchido seringa de 1 mL para a tubagem e insuflar os pulmões com 1 ml de PBS. Cuidadosamente recuperar lavagem BAL com seringa. Guarde no gelo. </li><li> Anexar paraformaldeído a 4% (PFA) em PBS-seringa de 1 mL para a tubagem e inflar os pulmões. </li><li> Remova o tubo, e aperte o fio cirúrgico para evitar vazamento do PFA. </li><li> Dissecar cuidadosamente para fora do tecido pulmonar e colocar em um tubo Falcon de 50 ml com 5 ml PFA 4%. </li></ol> 5. Preparação de tecidos recuperados <ol><li> Soro: Depois do armazenamento em gelo durante 30 min a 2 h, para o sangue coagular, o soro é recuperado a partir do sangue por centrifugação (13000 xg / 10 min / 4 ° C). O soro é armazenado a -20 ° C e pode ser usado para os testes ELISA de citocinas ou S. mansoni ovo antigénio-específica isotipo IgG ELISAs <sup&gt; 6, 10. </li><li> Gânglios linfáticos: As suspensões de células únicas são preparados e as contagens de células usadas para avaliar a magnitude da resposta imune. Para examinar a polarização de células Th2, as células são re-estimuladas com S. mansoni antígeno de ovo por 72 horas. A coloração de citocinas Intracellar pode ser examinada por citometria de fluxo, e os sobrenadantes pode ser recuperado e armazenado a -20 ° C para o ELISA de citocinas Th2 (IL-4, IL-5, IL-13) 6. </li><li> BAL: As células são sedimentadas (400 xg / 5 min / 4 ° C). Fluido BAL é recuperado e armazenado a -20 ° C para análise de citocinas Th2 por ELISA. Contagens BAL células são usadas como uma leitura para a inflamação das vias aéreas. As preparações Citocentrífuga de 10.000-100.000 células (500 rpm / 5 min / 4 ° C), seguido por coloração Diffquik irá permitir a enumeração de infiltrado inflamatório 4, 6. Alternativamente, as células de BAL pode ser examinada por citometria de fluxo 6. </li><li> O tecido pulmonar: tecido de pulmão é stored durante a noite a 4 ° C. Tecido fixo é parafina, seccionados e montados em lâminas para análise histológica e de imunofluorescência. </li></ol> 6. Os resultados representativos Este protocolo detalhes todos os passos necessários para (i) preparação purificada ovos de Schistosoma mansoni para uso in vivo (Fig. 1), (ii) sensibilizar e desafiar camundongos com estes ovos e (iii) recuperar os órgãos para exame da resposta inflamatória pulmonar. Este modelo in vivo de forma reprodutível acciona uma resposta Th2 pulmão potente inflamatória. Isto é mostrado pela inflamação das vias aéreas e eosinofilia como visualizado por contagem de células BAL e preparações Citocentrífuga (Fig. 2). H e secções coradas E-pulmonares pode ser examinada por ovo induzida por granulomas (Fig. 3a), e coloração de imunofluorescência de secções de pulmão permite a visualização de macrófagos alternativamente activados e Th2 citocina induzida por genes, tais como resistência-estanho como molécula (RELM) α (Fig. 3b). O antigénio específico CD4 resposta de citocinas Th2 é examinado por ELISAs para IL-5, IL-4 e IL-13 de antigénio-estimuladas células dos nódulos linfáticos parathymic (Fig. 4). <img alt="A Figura 1" src="/files/ftp_upload/3905/3905fig1.jpg" /> Figura 1 Schistosoma mansoni modelo de injeção de ovo imagens representativas dos fígados granulomatosas, característicos de altas S. mansoni de ovos-cargas e de ovos de S. mansoni são mostrados -.. Tamanho médio (L) 120 mM por (W) 50 mM. <img alt="A Figura 2" src="/files/ftp_upload/3905/3905fig2.jpg" /> Figura 2. S. mansoni injeção ovo leva a inflamação das vias aéreas. A. LBA número de células (x10 5) de ingênuos (N) ou S. mansoni (Sm) ovo camundongos injetados. * P &lt;0,05. B. Preparação Citocentrífuga de células BAL. Mac, macrófagos; Eos, eosinófilos, linfa, linfócitos. Bar, 10 uM. <img alt="A Figura 3" src="/files/ftp_upload/3905/3905fig3.jpg" /> Figura 3. Visualização de granulomas pulmonares e macrófagos alternativamente ativados em torno do ovo de S. mansoni. A. Granuloma pulmonar em torno do ovo Sm (seta preta) foi visualizado em H &amp; E seções manchadas de pulmão. B. técnica de imunofluorescência para RELMα (verde), receptor de manose (vermelho) e DAPI (azul) revela, em alternativa, macrófagos ativados (seta branca) no granuloma ao redor do autofluorescente Sm ovo (seta preta). Bar, 50 um. <img alt="A Figura 4" src="/files/ftp_upload/3905/3905fig4.jpg" /> Figura 4. S. mansoni ovo antigénio específico resposta de citocinas Th2. Drenagem células linfáticas parathymic nó de ratinhos ingénuos (N) ou Sm ovo injectado foram estimuladas com Sm &lt;/ Em&gt; antigénio ovo durante 72 horas, seguido por ELISA dos sobrenadantes para IL-4, IL-5 e IL-13. Escala, ng / mL. * P &lt;0,05.

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Aqui, um modelo empregando ovos de Schistosoma mansoni para induzir a inflamação do pulmão do tipo 2 é descrito. Características deste modelo incluem a resposta Th2 potente imunológico, inflamação das vias aéreas e formação de granuloma pulmonar. Uma vez que estes parâmetros são dependentes de respostas de células T CD4 + Th2 11, este é um modelo útil para investigar os efeitos da proteína deleções ou específicos de linhagem específica sobre respostas de células Th2. Aqui, as le...

<table border="1"><tbody><tr><td> Reagente </td><td> Tamanho / Descrição </td><td> Companhia </td><td> Catalogo </td></tr><tr><td> Colagenase / dispase </td><td> 500 mg </td><td> Roche Applied Science </td><td> 11 097 113 001 </td></tr><tr><td> Diff Quik Hema 3 Stain </td><td> Empacotar </td><td> Fisher Scientific Co. LLC </td><td> 122-911 </td></tr><tr><td> DMEM </td><td> Líquido </td><td> Gibco </td><td> 11965 </td></tr><tr><td> Falcon de 50 mL </td><td> Empacotar </td><td> Falcão </td><td> 352070 </td></tr><tr><td> Intramedic Tubing PE </td><td> 0,58 milímetros </td><td> Becton Dickinson and Co. </td><td> 427410 </td></tr><tr><td> Isoflurano </td><td> 250 mL </td><td> Abade da Saúde Animal </td><td> 52600405 </td></tr><tr><td> Ketamina </td><td> 100 mg / mL </td><td> Fort Dodge, Saúde Animal </td&gt; <td> N / A </td></tr><tr><td> Anticorpo para o receptor de manose </td><td> Biotina </td><td> Abd Serotec </td><td> MCA2235B </td></tr><tr><td> Filtro metálico </td><td> Coador de cozinha padrão </td><td> Alvo </td><td> Laurentiis </td></tr><tr><td> Paraformaldeído </td><td> 16% </td><td> Electron Microsc. Ciências </td><td> 15710 </td></tr><tr><td> Penicilina / estreptomicina </td><td> 100x </td><td> Gibco </td><td> 15070 </td></tr><tr><td> Percoll </td><td> 1 litro </td><td> GE Healthcare Biosciences </td><td> 17 01 0891 </td></tr><tr><td> Anticorpo RELMα </td><td> 50 ug </td><td> Peprotech </td><td> 500-P214 </td></tr><tr><td> Fio cirúrgico </td><td> 2,0 USP 100 yds </td><td> Surgical Specialties Corp </td><td> SP118 </td></tr><tr><td> Agulha de seringa </td><td> 23g, 3/4 polegada </td><td> BD Vacutainer Lab. Med. </td><td>305143 </td></tr><tr><td> EUA Standard Testing Sieve </td><td> 150 æM, 0,0059 &quot; </td><td> WS Tyler, Inc. </td><td> ASTME-11, Spec. N º 100 </td></tr><tr><td> Xilazina </td><td> 20 mg / mL </td><td> Mordomo </td><td> N / A </td></tr></tbody></table>

using eggs from schistosoma mansoni as an in vivo model of helminth-induced lung inflammation

Parasitas Schistosoma são vermes de sangue que infectam cerca de 200 milhões de pessoas em todo o mundo 1. Na infecção crónica com Schistosoma, a patologia grave, incluindo fibrose hepática e esplenomegalia, é causada pela resposta imunitária aos ovos do parasita ao invés de o próprio parasita 2. Ovos do parasita induzir uma resposta Th2 caracterizada pela produção de IL-4, IL-5 e IL-13, a activação de macrófagos alternativa eo recrutamento de eosinófilos. Aqui, nós descrevemos injecção de ovos de Schistosoma mansoni como um modelo para examinar parasita respostas específicas de citocinas Th2 no pulmão e gânglios linfáticos de drenagem, a formação de granulomas pulmonares em torno do ovo, e inflamação das vias aéreas. Após sensibilização intraperitoneal e desafio intravenosa, S. ovos mansoni são transportados para o pulmão através das artérias pulmonares, onde são presos no interior do parênquima pulmonarpor granulomas compostos de linfócitos, eosinófilos e macrófagos alternativamente activados 3-6. Associadas com inflamação formação de granuloma, nos espaços bronco-alveolar, a expansão dos nodos linfáticos de drenagem e activação das células T CD4 pode ser observado. Aqui detalhamos o protocolo para o isolamento de ovos de Schistosoma mansoni a partir de fígados infectados (modificado de 7), sensibilizando e desafiando camundongos, e recuperando os órgãos (lavado bronco-alveolar (LBA), pulmão e linfonodos de drenagem) para análise. Nós também incluir histológico dados representativos e imunológicas e sugestões para análise imunológica adicional. Em geral, este método proporciona um modelo in vivo para investigar as respostas induzidas por helmintos imunológicas no pulmão, que é amplamente aplicável para o estudo da Th2 doenças inflamatórias, incluindo infecção helmíntica, doenças fibróticas, inflamação alérgica e asma. As vantagens deste modelo para o estudo da type inflamação 2 no pulmão incluem a reprodutibilidade de uma resposta Th2 potente inflamatória no pulmão e gânglios linfáticos de drenagem, a facilidade de avaliação da inflamação por exame histológico dos granulomas em torno do ovo, eo potencial para armazenamento a longo prazo do parasita ovos.

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Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Ovos de Schistosoma mansoni são potentes estimuladores do tipo T helper 2 (Th2) resposta imunológica, característica da infecção por parasita, asma e inflamação alérgica. Este protocolo utiliza S. mansoni injecção de ovo para gerar CD4 de resposta de citocinas induzida por Th2 inflamatória no pulmão, caracterizada por formação de granuloma pulmonar em torno do ovo, eosinofilia e macrófagos activação alternativa.

Usando Ovos de<em&gt; Schistosoma mansoni</em&gt; Como um<em&gt; Em vivo</em&gt; Modelo de Helmintos induzida por inflamação pulmonar

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

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Immunology and Infection

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

13.7K visualizações.  University of Pennsylvania. Ovos de Schistosoma mansoni são potentes estimuladores do tipo T helper 2 (Th2) resposta imunológica, característica da infecção por parasita, asma e inflamação alérgica. Este protocolo utiliza S. mansoni injecção de ovo para gerar CD4 de resposta de citocinas induzida por Th2 inflamatória no pulmão, caracterizada por formação de granuloma pulmonar em torno do ovo, eosinofilia e macrófagos activação alternativa.

Vídeo: Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Transformação e Cercarial In vitro Cultivo de Schistosoma mansoni Esquistossômulos

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Imunologia e Infecção

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 ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Indução de enxerto-versus-hospedeiro de Doenças e<em&gt; Em Vivo</em&gt; Monitoramento celular T Usando um modelo murino de MHC de correspondência

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

No método de imagem in vivo Distinguir inflamação aguda e crônica

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Um<em&gt; Ex vivo</em&gt; Modelo de um Ataque de T-Cell dirigiu-Oligodendrocyte em Agudos fatias de cérebro

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Propagação do vírus Influenza em ovos de galinha embrionados

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Um método otimizado para o isolamento e expansão de células invariante Natural Killer T de Rato Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

Sondas de rastreio óptico do romance bactérias específicas no tecido de pulmão humano Ex Vivo por Endomicroscopy Laser Confocal

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Avaliação do citotóxicos e efeitos imunomoduladores de substâncias no pulmão humano de precisão de corte fatias

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Isolamento e caracterização de vesículas extracelulares de adultos Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...