Agradecemos Lee Tuddenham (IBMC, em Estrasburgo) para amplificar e titulação MCMV-Luc e Thomas Baumert (INSERM U748, Estrasburgo) para permissão de uso do biotério do Instituto de Virologia. Apoio financeiro do INSERM, Université de Strasbourg ea Agence Nationale de la Recherche (ANR-08-MIEN-005-01) é reconhecido. A participação inicial de Sonia Beroud e Laetitia Lelieur durante seu projeto de Mestre também é reconhecido.

1. Preparação da suspensão virai <ol><li> Obter a tensão Smith de MCMV expressando luciferase (MCMV-Luc 15) do laboratório de Ulrich Koszninowski. Neste recombinante, o gene da luciferase é inserido no locus IE2 do genoma do MCMV. </li><li> Para amplificar MCMV-Luc, infectar uma linha celular estromal de medula óssea de murino (M2-10B4, ATCC # CRL-1972) com MCMV em diferentes multiplicidade de infecção (MOI, com 0,001 a 1) 16. Por isso, adicionar o vírus a células cultivadas em meio isento de soro a 37 ° C. Após uma hora, remover o sobrenadante e substituir com meio DMEM suplementado com soro de vitela fetal a 10% (FCS). Cinco dias mais tarde, a colheita do meio de cultura e armazenamento de alíquotas a -80 ° C até à sua utilização. </li><li> Titulação da suspensão virai por ensaio de placas. <ol><li> Dividir células 3T3 (ATCC # CRL-1658) numa placa de 24 poços a 40.000 células / poço em 1 ml de DMEM suplementado com FCS a 10% contendo penicilina (200 UI / ml) e steptomycin (200 mg / mL) E incubar durante 24 horas a 37 ° C. </li><li> Preparar diluições em série (10 -1 a 10 -8) de uma aliquota de suspensão de MCMV em DMEM e infectar as células-alvo (3T3) com 200 ul de diluição após a remoção de meio de cultura celular. Incubar 1 hora a 37 ° C. Adicionar 1 ml de Topo Medium (DMEM 3% de FCS, 2% (v / v), gentamicina, 200 UI / ml de penicilina, 200 mg / ml de estreptomicina, 8 g / L de carboximetil celulose) e incubar 5 dias a 37 ° C. </li><li> Fixar as células por adição de 200 ul de 10% de formaldeído (diluição de formaldeído com água deve ser realizado numa câmara de químico para evitar inalações tóxicas) a cada poço, e a incubação de 6 horas à temperatura ambiente. Remova o meio de cultura por pipetagem + fixador para fora e adiciona-se 200 ul de solução de coloração de células (1% de violeta de cristal (w / v) em etanol a 20%). Antes do uso, diluir uma parte com 9 partes de água Milli-Q. Incubar 2 min à temperatura ambiente, lavar a placa 3-4x por imersão em água, deixar secar e contar o número de placas com um binóculo. </li></ol></li> &lt;li&gt; Diluir MCMV-Luc em DMEM para obter 50 PFU em 50 ul e deixar em gelo até à sua utilização. Usando inóculos virais mais elevados induz letalidade antes dos neonatos chegar a 2-3 semanas de idade. Em nossa experiência, a 500 PFUs induziu letalidade em todos os recém-nascidos de 5 a 7 dias após a injecção. </li></ol> 2. A injeção de recém-nascidos <ol><li> Quando recém-nascidos (de 4 a 20 ratos hr de idade) estão disponíveis, manipular o lixo e as fezes da gaiola com luvas antes de manusear cuidadosamente os filhotes para evitar a contaminação com odores &quot;estrangeiros&quot; que gostaria de sublinhar a mãe e induzir a rejeição dos filhotes . </li><li> Realizar uma injecção intraperitoneal usando uma seringa de insulina (29 g) como mostrado na Figura 1A. Segure o recém-nascido com a pele dorsal. Com o lado ventral para cima, introduzir a agulha sob a pata dianteira e empurre-o suavemente por via subcutânea para atingir a cavidade peritoneal (um pouco abaixo do umbigo). Atualmente, injetar 1% de azul de metileno diluído em DPBS e monitorar a sobrevivência de practice antes de executar a técnica de infecções virais. Desde azul de metileno é claramente visível durante o processo de injeção, recomendamos realizar esta injeção &quot;treinamento&quot; em primeiro lugar para garantir a entrega correta da suspensão viral na cavidade intraperitoneal. </li><li> Eliminar as pequenas gotas de sangue que possam surgir e colocar o recém-nascido de volta na gaiola. </li></ol> 3. In vivo de imagens <ol><li> No dia 7, lidar com os recém-nascidos com os mesmos cuidados, como mencionado anteriormente. Injectar intraperitonealmente uma mistura de anestésicos (4 mg / ml de cetamina, 0,8 mg / ml de xilazina) e luciferina (0,15 mg / g de peso corporal) em 50 ul em cada animal. O peso médio corporal dos animais é de 1-2 g e a dose de anestésicos por animal é: 40 mg de cetamina, xilazina 8 ug Espera 12-15 min, até que a luciferina atinge o seu máximo de emissão. Isto tem de ser determinada de antemão e depende do fornecedor luciferina e do sistema de imagem utilizada. </li><li> Enquanto os filhotes são anesthetized, marcá-los para identificação futura colheita e um pequeno pedaço de tecido que pode ser então utilizada para a futura genotipagem se os animais mutantes são utilizados. </li><li> Coloque as crias no sistema de aquisição de imagem e executar da luz emitida por todo o corpo. Sete dias após a infecção, o vírus foi replicado em várias cópias em órgãos-alvo, tais como o fígado, rins, pulmões e glândulas salivares e, portanto, a duração da exposição deve ser curta. No nosso caso, realizar a aquisição de 10 a 20 segundos (Figura 1B). A plataforma na câmara de aquisição do dispositivo de imagem necessita de ser aquecido para evitar arrefecimento excessivo da temperatura do corpo dos animais anestesiados. Além disso, é útil para obter imagens de baixa resolução de vários animais dentro de um ciclo de aquisição e, em seguida, subir a plataforma mais próxima da lente da câmera, a fim de se concentrar em uma parte de um animal particular ou um órgão isolado após dissecação. O software deve permitir mudanças rápidasdos parâmetros (tempo de exposição, mostra a distância / lente, sensibilidade), quantificação de regiões definidas de interesse e futuro exportação de imagens instantâneas. </li><li> Para adquirir o sinal de luz a partir da cabeça apenas cobrir o corpo dos neonatos deitado lateralmente com um papel espesso e escuro que vai mascarar os fotões emitidos ao nível dos órgãos em que ocorre alta replicação viral (fígado, pulmões, glândulas salivares) e realizar aquisição de 5 a 10 min (Figura 1C). Realizar aquisição do lado oposto do animal, nas mesmas condições. </li><li> Reintroduzir os filhotes na gaiola e colocar uma lâmpada de aquecimento na parte superior para evitar resfriamento excessivo dos neonatos anestesiados. Regularmente monitorar a recuperação pós-anestésica. Com a dose indicada, a anestesia actualmente dura 30 min. </li><li> Repita o mesmo procedimento nos dias 9, 11 e 14. Nos dias 11 e 14, alterar a dose de anestésicos (6 mg / ml de cetamina, 1,2 mg / ml de xilazina). </li></ol>

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Os autores declaram não competindo interesse financeiro. Declaração de ética Os animais foram mantidos em condições livres de patógenos na instalação do Institut d&#39;Imunologia et d&#39;Hématologie cuidados com os animais. Manipulação de ratos e procedimentos experimentais foram realizados de acordo com a lei francesa para a Protecção dos Animais de Laboratório. O procedimento foi aprovado pelo serviço veterinário de la Préfecture du Bas-Rhin (França) com o número de autorização A-67-345.

Usando a tecnologia de imagem vivo para monitorar MCMV-Luc disseminação de ratos recém-nascidos, pudemos observar propagação viral para o cérebro de animais mutantes, ao contrário do tipo selvagem. Além disso dissecação do animal e imagiologia ex vivo do cérebro confirmou a presença do vírus luminescente no sistema nervoso central. Além disso, também realizada imuno-histoquímica (não mostrado) em secções finas de cérebro com um anticorpo específico para a proteína E1 MCMV...

Citomegalovírus Humano (HCMV/HHV-5) é um membro da família β-herpesvírus. HCMV é altamente prevalente, patógeno oportunista que é geralmente adquirida durante a infância como uma infecção assintomática 1. Tal como todos os vírus de herpes, o HCMV persiste durante toda a vida do hospedeiro, cujo sistema imunitário bem controla a replicação viral. Episódios de reativação viral ocorrem principalmente em indivíduos imunocomprometidos, como pacientes transplantados que recebem medicamentos para evitar a rejeição do enxerto 2. Nos adultos, o HCMV também foi ligada a glioblastomas 3. Além disso, o HCMV é um patógeno importante para recém-nascidos com imunidade imaturo 4-6. A infecção primária no desenvolvimento do feto ou recém-nascido pode ter consequências graves. Infecção por HCMV é a causa infecciosa mais comum de defeitos congênitos e doenças da infância em países desenvolvidos. Estima-se que a incidência de infecção neonatal HCMV afecta 0,5-1% of todos os nascidos vivos, entre os quais 5-10% vão sofrer de sintomas graves, tais como microcefalia ou hipoplasia. Além disso, 10% das crianças infectadas com infecção viral subclínica, mais tarde, desenvolver sequelas levando a retardo mental, perda, defeitos visuais ou convulsão e epilepsia 7,8 audição. Ao contrário de outros herpesvírus humanos, tais como herpes simplex 1 (HSV-1/HHV-1) que podem ser inoculadas aos ratos via diferentes vias de injecção 9, a replicação do citomegalovírus é específico da espécie. Este recurso investigações de HCMV patogênese que são realizadas em diferentes modelos animais severamente prejudicada (rato, porquinho da índia, macaco rhesus) e seus respectivos CMVs específica do host genuínos. Todos os CMVs apresentam semelhanças significativas no tamanho do genoma e organização, tropismo tecidual e regulação da expressão gênica. Eles também induzir patologias semelhantes nas suas respectivas hospedeiro. Apesar da diversidade genômica serinterpolação de HCMV e rato citomegalovírus (MCMV) (50% das ORFs presentes no vírus humano estão identificadas no CMV de murino), o modelo de rato demonstrou recentemente a ser vantajoso, principalmente porque as estirpes mutantes podem ser testados quanto à sua capacidade para controlar viral replicação in vivo. Isto levou a uma tela genética que permitiu uma estimativa do número de genes do rato expressos na fase adulta, que compõem a &quot;resistome&quot; para este vírus 10. No total, isto indica que os ratinhos infectados com MCMV representam um modelo atractivo para o estudo das interacções hospedeiro-vírus em adultos. A exploração de infecção congênita por CMV é mais complexa porque as diferenças na organização camada da placenta entre humanos e camundongos prejudicar a transmissão da mãe para o feto da infecção viral em camundongos. Recentemente, a injeção direta de MCMV na placenta no dia 12,5 de gestação permitiu infecção cerebral de ratos recém-nascidos que levaram à deterioração 11 de audição. No entanto, a maioria investigations agora usam injeção intraperitoneal de 4-20 neonatos hr de idade para proporcionar a disseminação viral sistêmica potencialmente levando à infecção cerebral hematogênica, um modelo que é mais relevante do que a de uma injeção intracraniana. Este protocolo fornecido conhecimentos importantes sobre patogénese CMV e, mais particularmente, foi demonstrado que a infecção MCMV de recém-nascidos resulta em replicação virai em células neuronais e gliais localizados nos focos inflamatórios, que são infiltrados com células mononucleares como macrófagos 12. Este relatório também descrito morfogénese alterada do cerebelo acompanhado com proliferação diminuída granular neuronal e da migração e indução de múltiplos genes do interferão-estimuladas. O papel essencial das células T CD8 + para o controlo de MCMV no sistema nervoso central também foi avaliado pelo mesmo grupo 13. Um aspecto importante a considerar quando se analisa o efeito patológico de um micróbio é a dinâmica do infectaríons. No caso do MCMV, é particularmente crucial para explorar e quantificar a progressão da disseminação do vírus no cérebro em desenvolvimento de modo a compreender e prever a magnitude das futuras lesões neurobiológicos. Tradicionalmente, a quantificação da progressão de uma infecção normal requer o sacrifício dos animais infectados titule o patógeno em tecidos, tais como o cérebro, que são de outro modo inacessíveis. Este tipo de protocolo é agora desafiado por necessária melhoria do bem-estar animal e os 3Rs (Reduzir, Refine, Replace) Princípios 14. Usando em tecnologias de imagem in vivo pode permitir uma redução drástica do número de animais que são necessárias em experiências de infecção in vivo. Aqui, vamos relatar e descrever a análise tempo-curso de disseminação viral para o cérebro sobre intraperitoneal MCMV-Luc injeção para recém-nascidos de rato. Usando os mesmos animais, nós rastreados e monitorizados in vivo os locais de intensa viral de replicação durante um período de 2 semanas.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Fisher Scientific</td>
			<td>W3523A</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Methylene blue</td>
			<td>Sigma Aldrich</td>
			<td>319112</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Insulin needles</td>
			<td>VWR</td>
			<td>613-4897</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ketamine</td>
			<td>CentraVet</td>
			<td>Ket 201</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Xylazine/Vetranal</td>
			<td>Sigma Aldrich</td>
			<td>46995</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DPBS</td>
			<td>DUTSCHER</td>
			<td>P0436500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Luciferin</td>
			<td>Caliper</td>
			<td>760504</td>
			<td></td>
		 </tr>
		 <tr>
			<td>gentamycin</td>
			<td>Sigma Aldrich</td>
			<td>G1272</td>
			<td></td>
		 </tr>
		 <tr>
			<td>penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15070</td>
			<td></td>
		 </tr>
		 <tr>
			<td>carboxymethylcellulose</td>
			<td>Sigma Aldrich</td>
			<td>C4888</td>
			<td></td>
		 </tr>
		 <tr>
			<td>formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		 </tr>
		 <tr>
			<td>crystal violet</td>
			<td>Sigma Aldrich</td>
			<td>C3886</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>IVIS 50</td>
			<td>Caliper/Perkin Elmer</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

use of in vivo imaging to monitor the progression of experimental mouse cytomegalovirus infection in neonates

Citomegalovírus humano (HCMV ou HHV-5) é um agente patogénico de risco de vida em indivíduos imunocomprometidos. Após a infecção congênita ou neonatal, o vírus pode infectar e se replicar no cérebro em desenvolvimento, o que pode induzir a danos neurológicos graves, incluindo a surdez e retardo mental. Apesar do potencial de gravidade dos sintomas, as opções terapêuticas são limitados pela falta de uma vacina e a ausência de uma terapia antiviral específica. Por outro lado, uma descrição precisa dos acontecimentos moleculares que ocorrem durante a infecção do sistema nervoso central (SNC), uma vez que ainda falta observações principalmente derivam da autópsia de crianças infectadas. Vários modelos de animais, tais como macaco rhesus CMV, foram desenvolvidos e forneceu importantes insights patogénese CMV no SNC. No entanto, apesar da sua proximidade evolutiva com seres humanos, este modelo foi limitada pelo procedimento de inoculação intracraniana utilizada para infectar os animais e contrasistently induzir infecção do SNC. Além disso, considerações éticas têm promovido o desenvolvimento de modelos alternativos, entre os quais a infecção neonatal de ratos recém-nascidos com citomegalovírus rato (MCMV) foi recentemente levado a avanços significativos. Por exemplo, foi reportado que a injecção intraperitoneal de MCMV para Balb / c recém-nascidos resulta em infecção de neurónios e células gliais em áreas específicas do cérebro. Estas descobertas sugerem que a inoculação experimental de ratinhos pode recapitular os défices induzidos pela infecção por HCMV em crianças. No entanto, uma análise dinâmica de infecção MCMV de neonatos é difícil de realizar, porque metodologia clássica requer o sacrifício de um número significativo de animais em diferentes pontos de tempo para analisar a carga viral e / ou os parâmetros de auto-imunes. Para contornar esse obstáculo e permitir futuras investigações dos raros animais mutantes, foram aplicados in vivo tecnologia de imagem para realizar uma análise tempo-curso do viral difusão no cérebro após injecção periférica de um MCMV recombinante expressando a luciferase C57Bl / 6 neonatos.

Uma experiência representativa está ilustrada na Figura 1. Após a injecção intraperitoneal de 50 PFU de MCMV-Luc (painel A mostra uma injecção semelhante realizada com azul de metileno para visualizar o percurso da agulha subcutânea), recém-nascidos, foram anestesiados e receberam simultaneamente 0,3 mg de substrato de luciferase (luciferina, Caliper). Quinze minutos mais tarde, os animais foram colocados lado ventral para cima na câmara de aquisição do IVIS 50 (In Vivo Imaging System, calib...

Watch this Scientific Journal Video about Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates at JoVE.com

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

O citomegalovírus (HCMV) A infecção humana de recém-nascidos representa uma importante causa de retardo mental, mas os eventos moleculares que levam à induzida por vírus patogênese ainda são pouco compreendidos. Para investigar a dinâmica da infecção cerebral, adaptamos todo-animais<em&gt; In vivo</em&gt; Imagiologia para realizar a análise tempo-curso de recém-nascidos infectados com um vírus recombinante luciferase.

Utilização de<em&gt; In vivo</em&gt; Imagem para monitorar a progressão da infecção pelo citomegalovírus Experimental Rato em recém-nascidos

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

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Research

JoVE Journal

Immunology and Infection

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

14.8K visualizações.  Université de Strasbourg. O citomegalovírus (HCMV) A infecção humana de recém-nascidos representa uma importante causa de retardo mental, mas os eventos moleculares que levam à induzida por vírus patogênese ainda são pouco compreendidos. Para investigar a dinâmica da infecção cerebral, adaptamos todo-animais In vivo Imagiologia para realizar a análise tempo-curso de recém-nascidos infectados com um vírus recombinante luciferase.

Vídeo: Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

In vivo de imagens de parasitas Leishmania transgênicas em um host ao vivo

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Imunologia e Infecção

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Não-invasiva por imagem do Homing leucócitos e Migração In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

O Uso de diacetato carboxifluoresceína Succinimidyl Ester (CFSE) para monitorar a proliferação de linfócitos

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso de meio de escarro Artificial para testar a eficácia antibiótica contra<em&gt; Pseudomonas aeruginosa</em&gt; Em condições mais relevantes para o Pulmão Fibrose Cística

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

In Vivo Imaging Systems Detecção (IVIS) de um vírus de Neuro-invasiva encefalítico

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

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 ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

O Uso de fluorescentes alvo Matrizes para a Avaliação de respostas de células T<em&gt; In vivo</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Imagem latente da produção in situ da gama do interferon no spleen do rato depois da infecção dos monocytogenes de Listeria

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

Um ensaio ex vivo para estudar candida albicans Hórgênese Háfal no Trato Gastrointestinal

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Infecção recorrente do trato urinário de Escherichia coli desencadeada por gardnerella vaginalis exposição bexiga em camundongos

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...