Todos os espectros de RMN utilizadas como exemplos ilustrativos são derivados de um estudo publicado anteriormente 7, que foi apoiada financeiramente pela Nestlé.

1. Colonização do germe-free animais e coleta de amostras <ol><li> Remover livre de germes animais de isoladores e abrigá-los em uma sala de criação em gaiolas convencionais equipados com filtro na frente dos animais convencionais, que servirão como controles (Figura 1). </li><li> Meia mistura do lixo (3 dias) retirado da gaiola de controle convencional, com a ninhada dos animais germ-free. Mantenha sempre 1 / 3 do lixo sujos convencionais cada vez que é necessário renová-lo, a fim de manter um nível de bactérias (mantê-lo pelo menos por 3 dias). </li><li> Coletar a urina em um microtubo 1,5 mL manipulando o mouse sobre o tubo e ajudar a micção por massagem suave do intestino. Snap-congelar imediatamente em nitrogênio líquido. Armazenar pelo menos a -40 ° C até a análise de RMN. Um volume mínimo de 20 mL é necessário para a aquisição com uma sonda de RMN de 5 mm, mas é recomendado o uso de 30 mL para melhorar a qualidade do perfil metabólico. </li><li> Os animais devem be sacrificados sem o uso de qualquer anestésico para evitar ressonâncias de RMN de confusão devido ao metabolismo hepático de compostos anestésico (por exemplo, use deslocamento cervical seguido de confirmação de morte por sangria) </li></ol> 2. Recomendação para coleta de biópsia hepática <ol><li> Não use nenhum produto que contenha álcool para evitar a contaminação. Lavar as ferramentas usando uma solução de água ou soro fisiológico. </li><li> Não perfurar vesícula biliar. Em caso de vazamento de bile, lave o tecido imediatamente com água ou soro fisiológico. </li><li> Recolher biópsias hepáticas (cerca de 15-50 mg) do lóbulo esquerdo, como mostrado na Figura 2. Para biópsias reprodutível, recolher de forma consistente no centro do lobo esquerdo evitando as áreas periféricas onde o tecido é mais fina. </li><li> Snap biópsias congelar em nitrogênio líquido e armazená-los imediatamente a -80 ° C até a análise de RMN. </li></ol> 3. RMN 1 H aquisição de urina microvolume </p&gt; <ol><li> Prepare 0.2M de sódio em solução tampão fosfato D2O (99,8%), pH 7,4 contendo 1 mM 3 - (trimetilsilil) do ácido propiônico d-4 (TSP). </li><li> Misturar 30 mL de urina com 30 mL de tampão fosfato de sódio. </li><li> Transferir 50 mL de solução mista em 1,7 milímetros do tubo de RMN capilar (Figura 3) (2) usando uma seringa de vidro 50 mL equipado com uma agulha de metal (OD 0,5 mm). Ter cuidado para evitar bolhas. </li><li> Coloque o adaptador capilar (Figura 3) (3) no topo da amostra de urina contendo capilar e colocá-lo em um microtubo de RMN de 2,5 mm para 5 milímetros sonda de RMN (Figura 3) (1). Use esta combinação de tubos como um regular tubo de RMN de 5 mm para aquisição espectral. </li><li> Use a haste de extração (Figura 3) (4) para remover capilar de 2,5 mm por tubos de RMN aparafusar o adaptador capilar com cuidado para retirá-la. </li></ol> . 4 1 H RMN HR MAS de tecido de biópsia hepática: a preparação da amostra <ol><li> MAS rotor componentes e ferramentas sãodescrito na Figura 4. </li><li> Inserir biópsia (cerca de 15-50 mg) em rotor de zircônio (Figura 4) (1) e preencher o resto do volume com pura D 2 O NMR para bloqueio. Tenha cuidado para não fazer quaisquer bolhas porque isso iria alterar a qualidade dos calços subsequente e qualidade de aquisição de dados. </li><li> Inserir 50 espaçador Teflon mL (Figura 4) (2) com o parafuso cilíndrico (Figura 4) (5). Desapertar-lo e calibrá-lo usando o medidor de profundidade do lado curto (Figura 4) (8). Nesta etapa, é importante prestar uma atenção específica para a amostra porque parte dela pode vazar pelo buraco espaçador. Se este for o caso, então parte da biópsia é destruído e peso da amostra não é mais confiável. Assim, é necessário começar de novo desde o início da preparação da amostra. </li><li> Coloque o pino thead (Figura 4) (3) e aperte-o suavemente com a chave de fenda (Figura 4) (6). Secar toda a água residual com um pedaço de tecido. </li><li> Coloque a tampa (Figura 4) (4) nasuperior do rotor e inseri-lo no embalador rotor (Figura 4) (6). Pressione com firmeza até que a tampa está no lugar. Não deve haver qualquer espaço deixado entre o rotor e sua tampa. </li><li> Marca a metade da parte inferior do rotor usando uma caneta preta para permitir a detecção óptica taxa de spin. </li><li> Coloque o rotor dentro do espectrômetro de RMN e começar a girar em 5 kHz. Adquirir um espectro de RMN H utilizando seqüência de pulsos CPMG 13 de acordo com as orientações do fabricante. </li><li> Use α ressonância glucose anomérico em 5,22 ppm (doublet) para calibrar espectros de RMN. </li><li> Para descompactar o rotor, proceder por remover a tampa usando o removedor cap (Figura 4) (9). Desapertar thead pin e remover espaçador de teflon com o parafuso cilíndrico. Lavar bem com água e detergente. </li></ol> 5. Resultados representante Atividade microbiana do intestino pode ser monitorado usando perfil metabólico urinário. Um grande número de micróbios urináriaco-metabólitos identificáveis ​​por 1 H RMN têm sido descritos na literatura 7,14-17. Estes microbiana co-metabólitos são particularmente úteis para monitorar o processo de colonização como eles fornecem uma maneira rápida e não-invasivo para estimar quando o ecossistema recém-criada é estável. Figura 5A ilustra claramente o aparecimento de micróbios do intestino co-metabólitos sobre o processo de colonização. Esta figura mostra um perfil metabólico urinário obtidas mediante o procedimento descrito no Passo 2 para um animal colonizadas 20 dias usando o procedimento descrito no Passo 1. Este animal não excretar qualquer sulfato de indoxil e quantidades muito pequenas de phenylacetylglycine (PAG) e sulfato de p-cresol no estado livre de germes (dia 0-azul). Como a colonização avança, estes três marcadores do metabolismo de proteínas pela microbiota intestinal aumentam consideravelmente para chegar a um equilíbrio no dia 20 (vermelho). Isto é particularmente fácil de monitorar, por um grupo de animais, como ilustrado na Figura 5B usando o PAGressonância. Este diagrama foi obtido pela integração da área sob a ressonâncias destaque em cinza na Figura 5A (δ 7,40-7,43), correspondendo a uma ressonância específica (trio) de PAG para um grupo de sete animais. 1 H Alta Resolução Angle Spinning Magia (MAS HR) espectroscopia de RMN é uma técnica não destrutiva que permite aquisições rápida e reprodutível de perfis metabólicos de qualquer tipo de biópsia 18. Neste protocolo, utilizamos esta técnica poderosa para obter um perfil metabólico hepática de 2 ratos antes (azul) e após (vermelho) colonização (Figura 6). Esta figura ilustra bem a informação que pode ser derivado de uma MAS RMN baseado em perfil metabólico. Numerosos aminoácidos, assim como metabólitos derivados do metabolismo energético, como a glicose, glicogênio, lactato, triglicérides, (D)-3-hidroxibutirato e nicotinurate pode ser visualizado. Estes perfis também contêm informações relevantes ao estresse oxidativo (ascórbico ou seja, umcid, glutationa), o metabolismo de nucleotídeos (ou seja, inosina, uridina) e metabolismo metilamina (ie colina, Trimetilamina-N-óxido). Neste exemplo, é muito claro que o livre de germes do mouse exibe quase nenhum glicogênio e quantidades muito baixas de glicose e triglicérides como foi publicado anteriormente 7. <img src="/files/ftp_upload/3642/3642fig1.jpg" alt="Figura 1."/> Figura 1. Visão geral do protocolo de colonização. Animais livres de germes e convencionais são alojados em gaiolas equipadas com filtros de lado a lado e suas ninhadas são trocadas para permitir a colonização progressiva da microbiota intestinal convencional (1). Atividade microbiana do intestino é monitorado usando um perfil metabólico H NMR-based (2-3). Metabolismo hepático é avaliado por um MAS RMN HR H-base de perfis metabólicos (4-5). <img src="/files/ftp_upload/3642/3642fig2.jpg" alt="Figura 2."/> Figura 2. Rato vivoanatomia r. O fígado é apresentado como o lado plano do órgão enfrenta a mesa. Para biópsias reprodutível, é aconselhável sempre coletar amostras do centro do lobo esquerdo como indicado pelo retângulo tracejado. <img src="/files/ftp_upload/3642/3642fig3.jpg" alt="Figura 3."/> Figura 3 1,7 milímetros kit NMR capilar para trabalhar com microvolumes-chave:.. 1: 2,5 microtubo NMR mm, 2: 1,7 mm tubo de RMN capilar, 3: adaptador capilar, 4: Extração rod. <img src="/files/ftp_upload/3642/3642fig4.jpg" alt="Figura 4."/> . MAS Figura 4 equipamentos rotor-chave:. 1: rotor MAS, 2: 50 espaçador Teflon mL, 3: thead pinos, 4: cap, 5: parafuso cilíndrico, 6: chave de fenda, 7: rotor embalador, 8: medidor de profundidade. <img src="/files/ftp_upload/3642/3642fig5.jpg" alt="Figura 5."/> Figura 5. Evolução dos perfis metabólicos urinários durante colonization. <ol class="ualpha"><li> Zoom na região aromática do espectro entre 6,8-7,8 ppm, onde co-metabólitos microbianos podem ser visualizados. Espectros de RMN 1 H foram obtidos a partir de um único indivíduo no dia 0 (azul), 4 (verde), 15 (laranja) e 20 (vermelho) pós-colonização. A zona cinzenta corresponde à área que foi integrado para fazer o diagrama em B. chave: 1-MeHistamine: 1-methylhistamine; indoxil-S: sulfato de indoxil;:; Seus p-Cresol-S Histidina: sulfato de p-Cresol; PAG : Phenylacetylglycine. </li><li> Concentração PAG média durante a colonização (n = 7). Teste t de Student foi utilizado para comparar a diferença na concentração de PAG em vários pontos temporais: a: p &lt;0,05 em relação ao dia 0; b: p &lt;0,01 em relação ao dia 10. </li></ol><img src="/files/ftp_upload/3642/3642fig6.jpg" alt="Figura 6."/> Figura 6. Típicos 600 MHz 1 H HR espectros de RMN MAS de biópsias hepáticas derivadas de livre de germes (azul) e ex-germ-free (red) camundongos. Prótons em negrito são responsáveis ​​pela ressonância triglicérides-chave:. 3-HB: 3-hidroxibutirato, GSH: glutationa reduzida, TGs: Triglicerídeos, TMAO: Trimetilamina-N-óxido.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+partial+least+squares+models+and+related+chemometric+approaches+in+metabonomics+and+metabolic+phenotyping.">The evolution of partial least squares models and related chemometric approaches in metabonomics and metabolic phenotyping.</a> Journal of Chemometrics. 24, 636-649 (2010).</li><li>Merrifield, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metabolic+system-wide+characterisation+of+the+pig:+a+model+for+human+physiology.">A metabolic system-wide characterisation of the pig: a model for human physiology.</a> Mol. Biosyst. , (2011).</li><li>Tugnoli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+human+gastric+mucosa+by+HR-MAS+magnetic+resonance+spectroscopy.">Molecular characterization of human gastric mucosa by HR-MAS magnetic resonance spectroscopy.</a> International Journal of Molecular Medicine. 14, 1065-1071 (2004).</li><li>Sitter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+HR+MAS+MR+spectroscopic+profiles+of+breast+cancer+tissue+with+clinical+parameters.">Comparison of HR MAS MR spectroscopic profiles of breast cancer tissue with clinical parameters.</a> NMR Biomed. 19, 30-40 (2006).</li><li>Beckonert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+profiling,+metabolomic+and+metabonomic+procedures+for+NMR+spectroscopy+of+urine,+plasma,+serum+and+tissue+extracts.">Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts.</a> Nat. Protoc. 2, 2692-2703 (2007).</li></ol>

Não temos nada a revelar.

Neste protocolo, descrevemos um procedimento colonização progressiva em um ambiente aberto para investigar o impacto da microbiota intestinal no metabolismo hepático avaliado por 1 H RMN HR MAS perfil da biópsia intacta. Vários métodos de colonização têm sido descritos na literatura. Os métodos mais comuns para colonizar animais com microbiota definida são gavagem oral ou água contaminada 19,20. Inoculação de fezes também pode ser usado como descrito anteriormente 21. O m?...

Tabela de reagentes e equipamentos específicos: <table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Microtubo 2,5 milímetros </td><td> Nova Era </td><td> NE-H5/2.5-V-Br </td><td></td></tr><tr><td> 1,7 milímetros tubo capilar </td><td> Sigma-Aldrich </td><td> NORS175001 </td><td></td></tr><tr><td> Adaptador capilar </td><td> Nova Era </td><td> NE-325-5/1.7 </td><td></td></tr><tr><td> Extração da haste </td><td> Nova Era </td><td> NE-341-5 </td><td></td></tr><tr><td> HR-MAS BL4 rotor com 50 mL kit espaçador esférica Teflon </td><td> Bruker </td><td> HZ07213 </td><td></td></tr><tr><td> Kit de ferramentas para 50 inserções mL </td><td> Bruker </td><td> B2950 </td><td></td></tr><tr><td> Antecedência III 600MHz NMR </td><td> Bruker </td><td></td><td></td></tr><tr><td> 1 H RMN HR MAS sonda sólida </td><td> Bruker </td><td></td><td></td></tr><tr><td> Óxido de deutério 99,9% </td><td> Sigma-Aldrich </td><td> 530867-1L </td><td></td></tr><tr><td> 3 - (trimetilsilil) propiônico ácido-d 4 (TSP) </td><td> Sigma-Aldrich </td><td> 269913 </td><td></td></tr></tbody></table>

assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1h nmr spectroscopy

É bem conhecido que as bactérias do intestino contribuir significativamente para a homeostase host, fornecendo uma gama de benefícios como a proteção imunológica e síntese de vitaminas. Eles também fornecem o host com uma quantidade considerável de nutrientes, fazendo com que este ecossistema um órgão metabólicas essenciais. No contexto da crescente evidência da relação entre a flora intestinal e síndrome metabólica, a compreensão da interação metabólica entre o anfitrião e sua microbiota intestinal está se tornando um desafio importante da biologia moderna. 1-4 Colonização (também referida como processo de normalização) designa o estabelecimento de micro-organismos em um animal livre de germes ex. Embora seja um processo natural que ocorre no nascimento, é também utilizado em adulto livre de germes animais para controlar o ecossistema intestinal floral e ainda determinar o seu impacto sobre o metabolismo hospedeiro. Um procedimento comum para controlar o processo de colonização é a utilização do método de gavagem com um single ou uma mistura de micro-organismos. Este método resulta em uma colonização muito rápido e apresenta a desvantagem de ser extremamente estressante 5. Portanto, é útil para minimizar o estresse e obter um processo mais lento de colonização para observar gradualmente o impacto da criação de bactérias sobre o metabolismo hospedeiro. Neste manuscrito, nós descrevemos um procedimento para avaliar a modificação do metabolismo hepático durante um processo de colonização gradual utilizando uma técnica não destrutiva de perfil metabólico. Propomos para monitorar a colonização microbiana do intestino através da avaliação da atividade metabólica microbiana do intestino refletida pela excreção urinária de microbiana co-metabólitos por um perfil H RMN baseado metabólica. Isto permite uma apreciação da estabilidade da atividade microbiana do intestino, além do estabelecimento estável do ecossistema microbiano do intestino geralmente avaliado pelo monitoramento bactérias fecais por DGGE (eletroforese em gel de gradiente de desnaturação). 6 Acolonização ocorre em um ambiente convencional aberta e é iniciada por uma maca, suja de animais convencionais, que servirá como controle. Roedores são animais coprófagos, isso garante uma colonização homogênea como descrito anteriormente. 7 Perfil metabólico hepático é medido diretamente a partir de uma biópsia do fígado intacto utilizando 1 H Angle Magia Alta Resolução Spinning espectroscopia de RMN. Esta técnica semi-quantitativa oferece uma forma rápida de avaliar, sem danificar a estrutura da célula, os principais metabolitos, tais como triglicérides, glicose e glicogênio, a fim de estimar mais a complexa interação entre o processo de colonização e do metabolismo hepático 7-10. Este método também pode ser aplicado a qualquer tecido biópsia 11,12.

Watch this Scientific Journal Video about Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy at JoVE.com

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

Um procedimento de colonização progressiva é descrito para melhor avaliar seu impacto sobre o metabolismo hepático host. Colonização é monitorado não invasiva através da avaliação da excreção urinária de microbiana co-metabólitos por RMN baseado em perfil metabólico, enquanto o metabolismo hepático é avaliado pela Alta Resolução Magia Angle Spinning (MAS HR) NMR perfil da biópsia intacta.

Avaliar as alterações metabólicas hepáticas Durante a colonização progressiva de Germ-free Mouse 1 H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

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Immunology and Infection

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

13.0K visualizações.  The University of Reading. Um procedimento de colonização progressiva é descrito para melhor avaliar seu impacto sobre o metabolismo hepático host. Colonização é monitorado não invasiva através da avaliação da excreção urinária de microbiana co-metabólitos por RMN baseado em perfil metabólico, enquanto o metabolismo hepático é avaliado pela Alta Resolução Magia Angle Spinning (MAS HR) NMR perfil da biópsia intacta.

Vídeo: Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

Quantificação da colonização fúngica, esporogênese, e produção de micotoxinas Utilizando Bioensaios Kernel

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Imunologia e Infecção

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

Colonização de Euprymna scolopes Squid por Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Detecção de MicroRNAs em Microglia por PCR em tempo real no SNC normal e durante Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caracterização de Respostas Inflamatórias Durante Colonização Intranasal com Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Avaliar a atividade anti-fúngica isolada de macrófagos alveolares por microscopia confocal

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Avaliação da invasão bacteriana de células cardíacas na cultura e colonização cardíaca em camundongos infectados usando listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Colheita seletiva de Marginating-hepática Leucócitos

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Um modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonização Vaginal

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Difusão molecular em membranas plasmáticas de linfócitos primários medida por fluorescência Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Deteção de colonização Enterohemorrhagic Escherichia Coli murino Host pelo sistema não-invasivo na Vivo bioluminescência

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...