Questo lavoro è stato finanziato dal NIH RO1 AI079253 e Department of Veterans Affairs.

1. Modelli animali <ol><li> Mouse: utilizzare p47 phox-/ - topi e per età e sesso-abbinato topi C57BL6/DBA. Ottenere l&#39;approvazione per gli esperimenti di cura degli animali e del Comitato Istituzionale uso. </li><li> Anestesia: Utilizzare un sistema continuo di amministrazione isoflurano per indurre l&#39;anestesia. Il sistema vaporizzatore (VetEquip) è riempito con isoflurano (2-3%). Verificare che i topi sono completamente anestetizzati osservando la respirazione, il movimento, e riflesso corneale in risposta a stimoli esterni. </li><li> Le procedure chirurgiche: Scrub l&#39;area chirurgica (da banco) con il 70% di etanolo. Indossare guanti sterili e un abito pulito chirurgico e la maschera. Preparare i topi dal clipping i capelli e applicando betadine alternata con etanolo 70% per l&#39;area in cui l&#39;incisione viene effettuata. Eseguire un intervento chirurgico su una superficie sterile e usare strumenti sterili. Monitor topi dopo l&#39;intervento fino a quando sveglio e muoversi liberamente. </li><li> Intratracheale zymosan amministrazione <ol><li> Somministrare anestesiacome descritto in 1.2. </li><li> Disinfettare area chirurgica (collo) con Betadine e 70% di etanolo ed esporre la trachea da dissezione chirurgica. </li><li> Pierce trachea con un ago di calibro 27 e iniettare zymosan (St. Louis, MO) ad una dose di 1 mg / g con un 0,5 ug / ul di soluzione (volume totale di 50 microlitri per un mouse 25 g) sciolto in PBS sterile. </li><li> Chiudi collo topi avvolti con 5-0 punti di sutura in seta sterili in condizioni asettiche. </li><li> Immagine dopo 4 ore e 24 ore. </li></ol></li><li> Legatura del cieco e puntura <ol><li> Somministrare anestesia, come descritto al punto 1.2. </li><li> Clip di capelli nella zona di intervento (addome) e disinfettare con betadine e il 70% di etanolo. </li><li> Eseguire una laparotomia mediana e identificare il cieco. </li><li> Legare il distale 50% del cieco esposta con sutura di seta 4-0 e cieco puntura distale alla legatura con un passaggio di un ago da 21 gauge. </li><li> Chiudere l&#39;incisione con 4-0 punti di sutura in seta sterili. </li><li> Immagine dopo 4 ore e24 ore. </li></ol></li><li> Orale tetracloruro di carbonio (CCl 4) somministrazione <ol><li> Somministrare anestesia, come descritto al punto 1.2. </li><li> Somministrare CCl 4 (2 mg / g, St. Louis, MO), disciolto in olio di mais mediante sonda gastrica. La procedura di CCl4 somministrazione deve essere effettuata in un&#39;area designata conformemente IBC / EHS politiche. </li><li> Immagine dopo 4 ore e 24 ore. </li></ol></li></ol> 2. Acquisizione di un&#39;immagine Bioluminescent <ol><li> Inizializzare il IVIS 200 (Xenogen Corporation, Alameda, CA) Imaging System, impostato in modalità luminescenza, e attendere che il charge-coupled device (CCD) per bloccare la temperatura. </li><li> Selezionare il tempo di esposizione, binning (media) e f / stop (8) le impostazioni. </li><li> Topi di luogo in posizione supina nella camera di imaging su un palco riscaldato per mantenere la temperatura corporea normale. Somministrare anestesia, come descritto al punto 1.2 con cono di naso, mentre i topi sono nella camera di IVIS imaging. </li><li> Amministrare L-012<html> (20 ug / g) disciolto in PBS sterile (10 pg / pl) per via endovenosa con retro-orbitale iniezione ad ogni tempo selezionato per l&#39;imaging. </li><li> Cattura di immagini che iniziano a 2 minuti dopo l&#39;iniezione L-012. Si utilizzano in genere un 40 secondi di esposizione. </li></ol> 3. Analisi dei dati con regione di interesse <ol><li> Analizzare i dati utilizzando un software immagine vivente V.4.2 (Xenogen Corporation, Alameda, CA). </li><li> Aprite l&#39;immagine e selezionare la misurazione della Regione di strumenti di interesse. </li><li> Selezionare la regione di interesse forma e dimensioni sul / o torace e addome dopo sovrapponendo una pseudo-colore immagine digitale (che rappresenta rilevazione dei fotoni) per un immagine fotografica del mouse. </li><li> Quantificare l&#39;intensità del segnale (flusso di fotoni) dalla regione di interesse. </li></ol> 4. Statistica <ol><li> Utilizzare pacchetto software di analisi statistica effettuare ANOVA a due vie con Bonferroni post-test in punti temporali individuali. </li></ol>TLE &quot;&gt; 5. rappresentativi Risultati Zymosan è un pro-infiammatoria cellulare prodotto lieviti ed un potente attivatore di ossidasi NADPH 3. Abbiamo precedentemente dimostrato che zymosan intratracheale induce una più robusta infiammazione polmonare neutrofila e pro-infiammatorie la produzione di citochine in p47 phox-/ - rispetto al tipo selvatico topo 1. Qui, il nostro obiettivo è stato quello di confrontare generazione di ROS nei polmoni di tipo selvatico e p47 phox-/ - topi dopo sfida zymosan. A 4 ore dopo la somministrazione intratracheale zymosan, abbiamo osservato un significativo aumento emissione di fotoni sul petto nei topi di tipo selvatico rispetto al basale nonché un aumento della emissione di fotoni in rispetto al tipo selvatico p47 phox-/ - topi a 4 ore e 24 ore. Al contrario, i segnali in bioluminescenza p47 phox-/ - mice è rimasto a livello basale a 4 ore dopo il trattamento zymosan (Fig. 1 AB). Così, NADPH ossidasi sembra be la principale fonte di generazione di ROS nei polmoni in seguito alla somministrazione zymosan. Quindi, abbiamo valutato produzione di ROS nel CLP-indotta modello di sepsi. La sepsi è una minaccia per la vita associata con la sindrome d&#39;organo lesioni, fra cui danno polmonare acuto (ALI) e sindrome da distress respiratorio (ARDS) 4, 5. ROS-mediata pregiudizio è stato considerato un fattore importante di guida indotta da sepsi disfunzione multi-organo. Abbiamo trovato che i ROS sono aumentate significativamente sul petto (4 ore) e sopra l&#39;addome (24 ore) in seguito CLP nei topi di tipo selvatico rispetto al basale. Tuttavia, i livelli di ROS in p47 phox-/ - mice erano simili ai livelli basali in entrambi i punti di tempo (Fig. 2 AC). Questi risultati dimostrano che la produzione di ROS nel CLP-indotta modello di sepsi mouse è NADPH ossidasi-dipendente. Infine, abbiamo usato l&#39;imaging bioluminescente per rilevare la produzione di ROS in un 4-indotto modalità di danno epatico CCll. CCl 4 provoca necrosi epatocellulare, ed è stato utilizzato per modellare entrambe le lesioni acute del fegato e fibrosi epatica 6. In contrasto con i modelli precedentemente descritti di infiammazione e lesioni, abbiamo scoperto che la produzione di ROS nell&#39;addome era modesto (circa il 35%) è aumentato rispetto al basale, in p47 phox-/ - topi a 4 ore dopo la somministrazione CCl 4. Tuttavia, l&#39;entità di CCl 4-ROS indotta generazione era significativamente maggiore nei topi di tipo selvatico che in p47 phox-/ - mice (Fig.3 AB). Questi dati suggeriscono che sia la generazione p47 phox contenente NADPH ossidasi-dipendente e indipendente dal ROS si verificano in CCl 4 acuta indotta da danno epatico. <img alt="Figura 1" src="/files/ftp_upload/3925/3925fig1.jpg" /> Figura 1. Produzione di ROS nei polmoni dopo il trattamento zymosan. A) Rappresentante bioluminescence l&#39;imaging di produzione di ROS. Notare che, oltre al petto, luminescenza è rilevabile sull&#39;addome in entrambi di tipo selvatico e p47 phox-/ - topi e luminescenza addominale è invariata dal trattamento zymosan. B) conta di fotoni dal petto di tipo selvatico e p47 phox-/ - topi dopo una singola iniezione intratracheale (IT) di zymosan. L&#39;imaging bioluminescenza è stata eseguita al basale, 4 h, e 24 h. Emissione di luce dalla regione di interesse sul petto è stato identificato utilizzando l&#39;indicato pseudo-colore scala. I risultati sono presentati come media ± SE, n = 6-9 per gruppo, * = p &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/3925/3925fig1large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img alt="Figura 2" src="/files/ftp_upload/3925/3925fig2.jpg" /> Figura 2. Rilevamento ROS dopo CLP. A) l&#39;imaging bioluminescenza Rappresentante di produzione di ROS, BConta fotoni) dal petto, e C) conta di fotoni dall&#39;addome di tipo selvatico e p47 phox-/ - mice al basale, 4 ore, e 24 ore dopo CLP. I risultati sono presentati come media ± SE, n = 4-5 per gruppo, * = p &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/3925/3925fig2large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img alt="Figura 3" src="/files/ftp_upload/3925/3925fig3.jpg" /> Figura 3. Produzione di ROS in addome dopo CCl 4 indotta da danno epatico. A) l&#39;imaging bioluminescenza Rappresentante di produzione di ROS e B) conta di fotoni dall&#39;addome di tipo selvatico e p47 phox-/ - mice al basale, 4 ore, e 24 ore dopo somministrazione orale CCl 4 trattamento. I risultati sono presentati come media ± SE;. N = 4-5 per gruppo, * = p &lt;0,05 <a href="http://www.jove.com/files/ftp_upload/3925/3925fig3large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> </a&gt;.

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Nessun conflitto di interessi dichiarati.

Misurazione &quot;tempo reale&quot; di specie reattive dell&#39;ossigeno (ROS) in animali viventi può essere ottenuto utilizzando sonde fluorescenti e chemiluminescenza. Mentre sonde fluorescenti soffrono avente deboli segnale-rumore 12 rapporti, la tecnica di imaging descritta è più sensibile per la rilevazione di emissione di luce dopo una reazione chimica di ROS con il luminol substrato a base di L-012. Come tutte le tecniche di imaging bioluminescenti, questa metodologia è limitata dalla lunghezza d&#...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> L-012 </td><td> Wako Chemicals USA, Inc. </td><td> 120-04891 </td><td></td></tr><tr><td> Zymosan </td><td> Sigma, St. Louis, MO </td><td> Z4250 </td><td></td></tr><tr><td> tetracloruro di carbonio </td><td> Sigma, St. Louis, MO </td><td> 289116 </td><td></td></tr></tbody></table>

bioluminescence imaging of nadph oxidase activity in different animal models

NADPH ossidasi è un enzima critico che media difesa dell&#39;ospite antibatterica e antimicotica. Oltre al suo ruolo nella difesa dell&#39;ospite antimicrobica, NADPH ossidasi ha funzioni critiche di segnalazione che modulano la risposta infiammatoria 1. Pertanto, lo sviluppo di un metodo per misurare in &quot;tempo reale&quot; la cinetica di NADPH ossidasi derivata generazione di ROS dovrebbe essere un valido strumento di ricerca per comprendere i meccanismi pertinenti per ospitare difesa, infiammazione e lesioni. Malattia granulomatosa cronica (CGD) è una malattia ereditaria del NADPH ossidasi caratterizzato da gravi infezioni e infiammazioni eccessivo. L&#39;attivazione della NADPH ossidasi dei fagociti richiede traslocazione delle sue subunità citosoliche (p47 phox, p67 phox e p40 phox) e Rac ad una membrana legato flavocytochrome (composto da un phox gp91 e p22 phox eterodimero). Perditadi funzione mutazioni in uno qualsiasi di questi risultati NADPH ossidasi componenti in CGD. Come per i pazienti con CGD, gp91 phox-carente topo e p47 phox-carenza topi hanno difettoso fagocita attività della NADPH ossidasi e di accoglienza alterata difesa 13, 14. Oltre a fagociti, che contengono i componenti sopra descritti NADPH ossidasi, una varietà di altri tipi di cellule esprimono diverse isoforme di ossidasi NADPH. Qui, descriviamo un metodo per quantificare la produzione di ROS nei topi di vita e di delineare il contributo della NADPH ossidasi di generazione di ROS in modelli di infiammazione e lesioni. Questo metodo si basa su ROS reagire con L-012 (un analogo del luminol) per emettere luminescenza che è registrato da un dispositivo ad accoppiamento di carica (CCD). Nella descrizione originale della L-012 probe, L-012-dipendente chemiluminescenza è stata completamente abolita dal superossido dismutasi, indicando che il principale ROS rilevato in questa reazione era superossido di anione 15. Studi successivi hanno dimostrato che la L-012 in grado di rilevare altri radicali liberi, comprese le specie reattive dell&#39;azoto 16, 17. Kielland et al. 17 hanno dimostrato che l&#39;applicazione topica del forbolo miristato acetato, un potente attivatore di NADPH ossidasi, portato in NADPH ossidasi-dipendente generazione di ROS che potrebbe essere rilevata nel topo, utilizzando la sonda luminescente L-012. In questo modello, hanno mostrato che la luminescenza L-012-dipendente è stata abolita nel p47 phox-carente topo. Abbiamo confrontato generazione di ROS nei topi di tipo selvatico e NADPH ossidasi-carente p47 phox-/ - mice 2 nei seguenti tre modelli: 1) somministrazione intratracheale di zymosan, un pro-infiammatoria della parete cellulare fungina prodotto derivato che può attivare NADPH ossidasi; 2) legatura del cieco e la puntura (CLP), un modello di sepsi intra-addominale secondaria infiammazione polmonare acuta e del pregiudizio, e 3) tetracloruro di carbonio per via orale(CCl 4), un modello di ROS-dipendente danno epatico. Questi modelli sono stati specificamente selezionati per valutare ossidasi NADPH-dipendente generazione di ROS nel contesto di infiammazione non infettiva, sepsi polimicrobica e tossina danno indotto organo, rispettivamente. Confrontando bioluminescenza nei topi di tipo selvatico di p47 phox-/ - mice ci permette di delineare il contributo specifico di ROS generato da p47 phox contenente NADPH ossidasi al segnale bioluminescente in questi modelli. Risultati di imaging bioluminescenza che hanno dimostrato un aumento dei livelli di ROS nei topi di tipo selvatico rispetto al p47 phox-/ - topi ha indicato che NADPH ossidasi è la principale fonte di generazione di ROS in risposta a stimoli infiammatori. Questo metodo fornisce un approccio minimamente invasivo per il monitoraggio &quot;in tempo reale&quot; di generazione di ROS durante l&#39;infiammazione in vivo.

Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH ossidasi è la principale fonte di specie reattive dell&#39;ossigeno (ROS) in fagociti. A causa della natura effimera dei ROS, è difficile misurare e monitorare i livelli di ROS negli animali viventi. Un metodo minimamente invasivo per la quantificazione seriale di ROS in topi viventi è descritto.

Imaging bioluminescenza di attività NADPH ossidasi in diversi modelli animali

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

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Immunology and Infection

16.1K Views.  Vanderbilt University School of Medicine. NADPH ossidasi è la principale fonte di specie reattive dell'ossigeno (ROS) in fagociti. A causa della natura effimera dei ROS, è difficile misurare e monitorare i livelli di ROS negli animali viventi. Un metodo minimamente invasivo per la quantificazione seriale di ROS in topi viventi è descritto.

Video: Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

La diagnosi di Ecto-e endoparassiti in ratti e topi di laboratorio

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

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Immunologia e infezioni

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Saliva, le ghiandole salivari, e Collection emolinfa dalle zecche Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Long Term intravitale Multiphoton Microscopia Imaging di cellule immunitarie in sani e malati di fegato Uso Mice CXCR6.Gfp Reporter

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Isolamento e Trapianto di diversi invecchiato murini del timo innesti.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Bioluminescenza Imaging per rilevare fase avanzata di infezione di africani Tripanosomiasi

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Flusso analisi di citometria di Natural Killer cellulare Lytic attività nel Sangue umano intero

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

In Vivo Investigation of Antimicrobial Blue Light Therapy per la multi-resistente baumannii Acinetobacter Masterizzare Infezioni Utilizzando bioluminescenza Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

Non invasivo<em&gt; In Vivo</em&gt; Fluorescenza ottica di imaging dell&#39;attività MMP infiammatoria utilizzando un agente fluorescente di imaging attivo

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Immagini dal vivo di antifungini attività da Human neutrofili e monociti primaria in risposta a<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

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 ...