Tutti gli spettri NMR utilizzati come esempi illustrativi sono derivati ​​da uno studio precedentemente pubblicato con il 7 che è stato sostenuto finanziariamente dalla Nestlé.

1. Colonizzazione di germi senza animali e la raccolta dei campioni <ol><li> Rimuovere sterile animali da isolatori e la casa in una stanza di allevamento in gabbie tradizionali dotati di filtro davanti alla animali convenzionali, che servirà come controlli (Figura 1). </li><li> Mescolare mezzo della cucciolata (3 giorni) preso dalla gabbia di controllo convenzionale con la lettiera del germe senza animali. Tenere sempre 1 / 3 della lettiera sporca convenzionale ogni volta che è necessario rinnovarlo al fine di mantenere un livello di batteri (tenerlo almeno per 3 giorni). </li><li> Raccogliere l&#39;urina in un microtubo 1,5 ml gestendo il mouse sopra il tubo e aiutare minzione da massaggiare delicatamente l&#39;intestino. Snap-congelare immediatamente in azoto liquido. Memorizzare almeno a -40 ° C fino al momento dell&#39;analisi NMR. Un volume minimo di 20 l è necessario per l&#39;acquisizione con una sonda NMR 5 millimetri, ma si consiglia di utilizzare 30 microlitri per migliorare la qualità dei profili metabolici. </li><li> Gli animali devono be l&#39;eutanasia, senza uso di anestetico per evitare risonanze NMR confusione a causa del metabolismo epatico di composti anestetici (per esempio, utilizzare dislocazione cervicale seguito da conferma della morte per dissanguamento) </li></ol> 2. Raccomandazione per la raccolta della biopsia epatica <ol><li> Non utilizzare alcun prodotto contenente alcol per evitare la contaminazione. Lavare gli attrezzi con acqua o soluzione salina. </li><li> Non perforare cistifellea. In caso di perdita di bile, lavare tessuto immediatamente con acqua o soluzione salina. </li><li> Raccogliere biopsie epatiche (circa 15-50 mg) dal lobo sinistro come mostrato nella Figura 2. Per le biopsie riproducibile, raccogliere costantemente al centro del lobo sinistro evitando le zone periferiche dove il tessuto è più sottile. </li><li> Snap biopsie congelare in azoto liquido immediatamente e conservarli a -80 ° C fino al momento dell&#39;analisi NMR. </li></ol> 3. Acquisizione 1 H NMR di urina microvolumi </p&gt; <ol><li> Preparare una soluzione di sodio fosfato 0,2 M tampone in D2O (99,8%), pH 7,4 contenente 1 mM 3 - (trimetilsilil) propionico-d 4 (TSP). </li><li> Mescolare 30 ml di urina con 30 ml di tampone fosfato di sodio. </li><li> Trasferire 50 ml di soluzione mista in tubo di 1,7 millimetri NMR capillare (Figura 3 (2)) usando una siringa da 50 microlitri di vetro dotato di un ago di metallo (diametro 0,5 mm). Attenzione ad evitare le bolle. </li><li> Montare l&#39;adattatore capillare (Figura 3 (3)) sulla parte superiore del campione di urina contenente capillare e mettetelo in un microtubo NMR 2,5 mm per 5 millimetri sonda NMR (Figura 3 (1)). Utilizzare questa combinazione di tubi come un normale tubo NMR 5 mm per l&#39;acquisizione spettrale. </li><li> Utilizzare l&#39;asta di estrazione (Figura 3 (4)) per rimuovere capillare da 2,5 mm Tubo NMR avvitando l&#39;adattatore capillare delicatamente per estrarlo. </li></ol> . 4 1 H NMR HR MAS di tessuto biopsia epatica: la preparazione del campione <ol><li> MAS di componenti di rotori e gli strumenti sonodescritto nella Figura 4. </li><li> Inserire biopsia (circa 15-50 mg) in zirconio rotore (Figura 4 (1)) e riempire il resto del volume con puro D 2 O per NMR di blocco. Fare attenzione a non fare le bolle, perché questo altererebbe la qualità della spessoramento successive e la qualità di acquisizione dei dati. </li><li> Inserire 50 microlitri distanziatore in teflon (figura 4 (2)), utilizzando la vite cilindrica (figura 4 (5)). Svitare e calibrare usando il misuratore di profondità sul lato corto (figura 4 (8)). A questo punto, è importante prestare una specifica attenzione al campione perché una parte di esso può fuoriuscire attraverso il foro distanziatore. Se questo è il caso, allora parte della biopsia viene distrutta e il peso del campione non è più affidabile. E &#39;quindi necessario ricominciare dall&#39;inizio la preparazione del campione. </li><li> Posizionare il perno thead (Figura 4 (3)) e avvitare delicatamente con il cacciavite (Figura 4 (6)). Asciugare l&#39;acqua residua con un pezzo di tessuto. </li><li> Posizionare il tappo (figura 4 (4)) alsuperiore del rotore e di inserirlo nel packer rotore (Figura 4 (6)). Premere con decisione fino a quando il tappo è a posto. Non ci dovrebbe essere alcuno spazio lasciato tra il rotore e il suo tappo. </li><li> Segna la metà del fondo del rotore con una penna nera marker per consentire il rilevamento ottico velocità di rotazione. </li><li> Posizionare il rotore all&#39;interno dello spettrometro NMR e iniziano a girare a 5 kHz. Acquisire 1 H NMR utilizzando CPMG sequenza di impulsi 13 secondo le linee guida del produttore. </li><li> Utilizzare la risonanza glucosio anomerico α a 5,22 ppm (doppietta) per calibrare gli spettri NMR. </li><li> Per decomprimere il rotore, procedere togliendo il tappo utilizzando il dispositivo di rimozione tappo (figura 4 (9)). Svitare thead pin e rimuovere distanziatore in Teflon con la vite cilindrica. Lavare accuratamente con acqua e detersivo. </li></ol> 5. Rappresentante Risultati Attività microbica intestinale può essere monitorato tramite il profiling metabolico urinario. Un gran numero di microrganismi urinarico-metaboliti identificabili da 1 H NMR sono stati descritti in letteratura 7,14-17. Questi co-metaboliti microbici sono particolarmente utili per monitorare il processo di colonizzazione in quanto forniscono un modo rapido e non invasivo per valutare quando l&#39;ecosistema di nuova costituzione è stabile. 5A figura illustra chiaramente l&#39;aspetto del microbica intestinale co-metaboliti sul processo di colonizzazione. Questa figura mostra un profilo metabolico urinario ottenute seguendo la procedura descritta al punto 2 per un animale colonizzata 20 giorni utilizzando procedura descritta al punto 1. Questo animale non espellere qualsiasi solfato indossile e quantità molto piccola di phenylacetylglycine (PAG) e p-cresolo solfato presso il germe senza stato (giorno 0-blu). Come colonizzazione avanza, questi 3 marcatori del metabolismo delle proteine ​​dalla flora intestinale aumenta notevolmente per raggiungere un equilibrio al giorno 20 (rosso). Ciò è particolarmente facile da monitorare per un gruppo di animali, come illustrato in Figura 5B con il PAGrisonanza. Questo diagramma è stato ottenuto integrando l&#39;area sotto le risonanze evidenziate in grigio nella Figura 5A (δ 7,40-7,43), corrispondente ad una risonanza specifica (tripletta) di PAG per un gruppo di 7 animali. 1 H ad alta risoluzione Magia Angle Spinning (MAS HR) spettroscopia NMR è una tecnica non distruttiva che permette acquisizioni rapido e riproducibile di profili metabolici di ogni tipo di biopsia 18. In questo protocollo, abbiamo usato questa tecnica potente per ottenere un profilo metabolico epatico di 2 topi prima (blu) e dopo (rosso) colonizzazione (Figura 6). Questa figura illustra bene le informazioni che possono essere derivati ​​da un MAS NMR basati su profilo metabolico. Numerosi aminoacidi e metaboliti derivati ​​da metabolismo energetico come il glucosio, il glicogeno, lattato, trigliceridi, (D)-3-idrossibutirrato e nicotinurate possono essere visualizzati. Questi profili contengono anche informazioni utili per lo stress ossidativo (cioè un ascorbicocid, glutatione), il metabolismo dei nucleotidi (cioè inosina, uridina) e il metabolismo metilammina (cioè colina, Trimetilammina-N-ossido). In questo esempio, è molto chiaro che la priva di germi del mouse viene visualizzata quasi glicogeno e gli importi molto bassi di glucosio e trigliceridi, come è stato precedentemente pubblicato 7. <img src="/files/ftp_upload/3642/3642fig1.jpg" alt="Figura 1."/> Figura 1. Panoramica del protocollo di colonizzazione. Animali germ-free e convenzionali sono alloggiati in gabbie dotate di filtri fianco a fianco e le loro cucciolate vengono scambiati per consentire progressiva colonizzazione dalla flora intestinale convenzionale (1). Attività microbica intestinale è controllata mediante 1 H NMR del profilo metabolico-based (2-3). Il metabolismo epatico è valutata da 1 H HR MAS NMR basati su profili metabolici (4-5). <img src="/files/ftp_upload/3642/3642fig2.jpg" alt="Figura 2."/> Figura 2. Topo vivor anatomia. Il fegato viene visualizzato come il lato piatto dell&#39;organo di fronte al tavolo. Per le biopsie riproducibile, si consiglia di raccogliere campioni da sempre al centro del lobo sinistro, come indicato dal rettangolo tratteggiato. <img src="/files/ftp_upload/3642/3642fig3.jpg" alt="Figura 3."/> Figura 3 1.7 mm Kit NMR capillare di lavorare con microvolumi chiave:.. 1: 2,5 microtubo NMR mm, 2: 1,7 mm Tubo NMR capillare, 3: adattatore capillare, 4: asta di estrazione. <img src="/files/ftp_upload/3642/3642fig4.jpg" alt="Figura 4."/> . Figura 4 MAS attrezzature rotore chiave: 1.: Rotore MAS, 2: 50 microlitri distanziatore in Teflon, 3: THEAD pin, 4: cappuccio, 5: vite cilindrica, 6: cacciavite, 7: rotore packer, 8: profondimetro. <img src="/files/ftp_upload/3642/3642fig5.jpg" alt="Figura 5."/> Figura 5. Evoluzione dei profili metabolici urinari durante colonization. <ol class="ualpha"><li> Zoom sulla regione aromatica degli spettri tra 6,8-7,8 ppm in cui microbica co-metaboliti possono essere visualizzati. Spettri 1 H NMR sono stati ricavati da un singolo individuo, al giorno 0 (blu), 4 (verde), 15 (arancione) e 20 (rosso) post-colonizzazione. La zona grigia corrisponde alla zona che è stata integrata per rendere il diagramma in B. chiave: 1-MeHistamine: 1-methylhistamine; indossile-S: solfato indossile;:; suo p-cresolo, S Istidina: solfato di p-cresolo; PAG : Phenylacetylglycine. </li><li> PAG concentrazione media durante la colonizzazione (n = 7). Student t-test è stato utilizzato per confrontare la differenza di concentrazione PAG a vari time-punti: a: p &lt;rispetto al giorno 0 0.05; b: p &lt;0,01 rispetto al giorno 10. </li></ol><img src="/files/ftp_upload/3642/3642fig6.jpg" alt="Figura 6."/> Figura 6. Tipiche 600 MHz 1 H HR spettri NMR MAS di biopsie epatiche derivate da privo di germi (blu) ed ex-priva di germi (rid) i topi. Protoni in grassetto sono responsabili per la risonanza trigliceridi chiave:. 3-MP: 3-idrossibutirrato, GSH: glutatione ridotto, TG: trigliceridi, TMAO: Trimetilammina-N-ossido.

<ol>
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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>

Non abbiamo nulla da rivelare.

In questo protocollo, abbiamo descritto una procedura di progressiva colonizzazione in un ambiente aperto di approfondire l&#39;impatto della flora intestinale sul metabolismo epatico valutate di 1 profiling H NMR HR MAS di intatto biopsia. Vari metodi di colonizzazione sono state descritte in letteratura. I metodi più comuni per colonizzare gli animali con un microbiota definiti sono sonda gastrica o l&#39;acqua potabile contaminata 19,20. Inoculazione fecale può essere utilizzato anche come des...

Tabella di reagenti e attrezzature specifiche: <table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> 2,5 millimetri microprovette </td><td> New Era </td><td> NE-H5/2.5-V-Br </td><td></td></tr><tr><td> 1,7 millimetri tubo capillare </td><td> Sigma-Aldrich </td><td> NORS175001 </td><td></td></tr><tr><td> Adattatore capillare </td><td> New Era </td><td> NE-325-5/1.7 </td><td></td></tr><tr><td> Estrazione asta </td><td> New Era </td><td> NE-341-5 </td><td></td></tr><tr><td> HR-MAS BL4 rotore con 50 microlitri sferica Teflon spacer kit </td><td> Bruker </td><td> HZ07213 </td><td></td></tr><tr><td> Kit di strumenti per 50 inserti microlitri </td><td> Bruker </td><td> B2950 </td><td></td></tr><tr><td> Advance III 600MHz NMR </td><td> Bruker </td><td></td><td></td></tr><tr><td> 1 H HR MAS NMR solido sonda </td><td> Bruker </td><td></td><td></td></tr><tr><td> Ossido di deuterio 99,9% </td><td> Sigma-Aldrich </td><td> 530867-1L </td><td></td></tr><tr><td> 3 - (trimetilsilil) propionico acido-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

E &#39;noto che i batteri intestinali contribuiscono significativamente alla omeostasi host, fornendo una serie di benefici come la protezione immunitaria e la sintesi della vitamina. Essi hanno inoltre fornire l&#39;host con una notevole quantità di sostanze nutritive, facendo di questo ecosistema un organo metabolico essenziale. Nel contesto di una crescente evidenza del legame tra la flora intestinale e la sindrome metabolica, comprendere l&#39;interazione metabolica tra l&#39;host e la sua flora intestinale sta diventando una sfida importante della biologia moderna. 1-4 Colonizzazione (noto anche come processo di normalizzazione), designa la creazione di micro-organismi in un antico animale privo di germi. Mentre è un processo naturale che si verificano al momento della nascita, è utilizzato anche in adulti privo di germi animali per controllare l&#39;ecosistema intestinale floreali e ulteriormente determinare il suo impatto sul metabolismo host. Una procedura comune per controllare il processo di colonizzazione è quello di utilizzare il metodo gavage con un singlE o una miscela di microrganismi. Questo metodo comporta una colonizzazione molto veloce e presenta lo svantaggio di essere estremamente stressante 5. E &#39;quindi utile per ridurre al minimo lo stress e per ottenere un processo di colonizzazione lento ad osservare gradualmente l&#39;impatto di stabilimento sul metabolismo batterico ospite. In questo manoscritto, si descrive una procedura per valutare la modifica del metabolismo epatico durante un processo di colonizzazione graduale utilizzando una tecnica non distruttiva profili metabolici. Noi proponiamo di monitorare la colonizzazione microbica intestinale, mediante la valutazione dell&#39;attività microbica intestinale metabolica riflette l&#39;escrezione urinaria di metaboliti microbici co-profilatura di 1 H NMR basati metabolica. Questo consente un apprezzamento della stabilità dell&#39;attività microbica intestinale al di là della sede stabile l&#39;ecosistema microbico intestinale solitamente valutata attraverso il monitoraggio dei batteri fecali da DGGE (gel elettroforesi denaturante gradiente). 6 Ilcolonizzazione si svolge in un ambiente convenzionale aperta ed è iniziata da una cucciolata sporca sporca da animali convenzionali, che servirà come controlli. Roditori sono animali coprofagi, questo garantisce una colonizzazione omogenea come descritto in precedenza 7. Profiling metabolico epatico viene misurato direttamente da una biopsia del fegato intatto con 1 H ad alta risoluzione Magic Angle Spinning spettroscopia NMR. Questo semi-quantitativa tecnica offre un modo rapido per valutare, senza danneggiare la struttura delle cellule, i metaboliti importanti come trigliceridi, glucosio e glicogeno al fine di ulteriormente valutare la complessa interazione tra il processo di colonizzazione e il metabolismo epatico 7-10. Questo metodo può essere applicato a qualsiasi tessuto bioptico 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

Una procedura di progressiva colonizzazione è descritto per valutare ulteriormente il suo impatto sul metabolismo epatico ospitante. La colonizzazione è monitorato non invasivo, valutando l&#39;escrezione urinaria di co-metaboliti microbici da NMR basati su profili metabolici, mentre il metabolismo epatico viene valutata ad alta risoluzione Angle Spinning Magic (MAS HR) profilazione NMR di intatto biopsia.

Valutare alterazioni metaboliche epatiche durante la colonizzazione progressiva di Germe senza mouse da 1 H spettroscopia NMR

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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Research

JoVE Journal

Immunology and Infection

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

13.0K Views.  The University of Reading. Una procedura di progressiva colonizzazione è descritto per valutare ulteriormente il suo impatto sul metabolismo epatico ospitante. La colonizzazione è monitorato non invasivo, valutando l'escrezione urinaria di co-metaboliti microbici da NMR basati su profili metabolici, mentre il metabolismo epatico viene valutata ad alta risoluzione Angle Spinning Magic (MAS HR) profilazione NMR di intatto biopsia.

Video: 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.

Quantificazione di colonizzazione fungina, sporogenesi, e la produzione di micotossine Uso Bioassays 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 ...

Ricerca

Immunologia e infezioni

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.

La colonizzazione delle Euprymna scolopes Squid da 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.

Rilevamento dei microRNA nella microglia da Real-time PCR in CNS normale e durante neuroinfiammazione

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.

Caratterizzazione delle risposte infiammatorie durante la colonizzazione intranasale con streptococco 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.

Valutare l&#39;attività anti-fungina isolato Alveolar macrofagi dal Microscopia confocale

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.

Valutazione dell'invasione batterica delle cellule cardiache in coltura e colonizzazione cardiaca nei topi infetti utilizzando 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.

La raccolta selettiva di Marginating-epatiche leucociti

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.

Un modello murino di Gruppo B<em&gt; Streptococcus</em&gt; Colonizzazione vaginale

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.

Diffusione molecolare nel plasma membrane dei linfociti primari misurati da Fluorescence 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.

Rilevamento della colonizzazione di Escherichia Coli enteroemorragica in murino Host di sistema Non invasivo In Vivo bioluminescenza

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