Ringraziamo John Kelly e Martin Taylor (London School of Hygiene &amp; Tropical Medicine) per la fornitura di T. b. brucei GVR35-VSL-2 e il Dr. Andrea Zelmer (LSHTM) per un consiglio su imaging in vivo. Questo lavoro è stato sostenuto dalla Fondazione Bill e Melinda Gates Foundation globale Health Program (codice di autorizzazione OPPGH5337).

Etica Tutto il lavoro è stato realizzato sotto l&#39;approvazione del Regno Unito per la casa Ufficio Animali (procedure scientifiche) Act 1986 e la London School of Hygiene &amp; Tropical Medicine Animal Welfare and Ethics Review Board. ARRIVARE linee guida sono seguite in questo rapporto. 1. In Vivo Passaggio di Bioluminescent Trypanosoma brucei brucei <ol><li> Rimuovere uno stock crioconservati (stabilate definito) di T. b. brucei ceppo GVR35-VSL-2 (disponibile dal professor John Kelly presso la London School of Hygiene &amp; Tropical Medicine attraverso MTA) 9 da azoto liquido e permettono di equilibrare a temperatura ambiente. Diluire stabilate a 2 x 10 4 trypanosomes / ml in sterile tampone fosfato glucosio (PBS-G) pH 8,0 (1 L PBS + 15 g di glucosio) 10. Nota: T. b. brucei GVR35-VSL-2 non è contagioso per gli esseri umani e richiede SAPO licenza strutture per effettuare esperimenti. </li><li> inject 20-25 g femminile del mouse CD1 con 0,2 ml trypanosomes diluiti per via intraperitoneale (ip) con un ago da 25 G. Questo è il &quot;mouse donatore&quot;. Posizionare il mouse infetto in uno specifico patogeno libero (SPF) -cage e mangimi ad libitum. Nota: i topi CD1 sono un ceppo outbred e T. b. infezioni brucei GVR35 sono ben caratterizzati in questo ceppo. Tuttavia, questo protocollo è applicabile anche a ceppi inbred o geneticamente modificati, ma è importante notare che il colore della pelliccia possono influire sulla sensibilità dell&#39;imaging 6,9. </li><li> Monitorare parassitemia periferica dal prelievo di sangue. Posizionare il mouse in un dispositivo di immobilizzazione di plastica e prendere 5 ml di sangue dalla coda per via venosa con 21 G ago o venesection e mescolare 1/10 con cloruro di ammonio sterile 0,85% per la lisi dei globuli rossi e consentire una più facile conteggio. </li><li> Contare il sangue diluito in un Neubauer emocitometro. <ol><li> Caricare il sangue diluito in entrambe le camere. Conta l&#39;intero gr centraleid di una camera per dare n trypanosomes. Concentrazione di trypanosomes nel sangue = n x 10 4 trypanosomes / ml, regolare per qualsiasi fattore di diluizione e calcolare la media delle due camere. Nota: Dopo circa 5-7 giorni dopo l&#39;infezione, parassitemia periferica dovrebbe aumentare in misura sufficiente a svolgere una infezione ad una concentrazione di 2 x 10 4 tripanosomi / ml. </li></ol></li><li> Quando i livelli di trypanosome sono sufficienti per il numero di topi richiesti alta, passare al punto 1.6. Un livello parassitemia di 6 x 10 4 trypanosomes / ml in un topo donatore è sufficiente per infezione di 10 topi. Nota: Per le dimensioni statisticamente significative del gruppo, n = 6 per ogni gruppo di trattamento di topi sono necessari 8. Per gli altri modelli di infezione, calcoli di potenza dovrebbero essere applicati per determinare le dimensioni dei gruppi appropriati. </li><li> Topi posto in una scatola di calore (a 28-30 ° C per circa 5-10 minuti) per consentire le vene coda di dilatarsi.Per indurre anestesia terminale, somministrare 20 mg / kg ev pentobarbital con un ago 25 G. Conferma del mouse è anestetizzato premendo delicatamente i cuscinetti plantari per garantire che non vi è alcun riflesso di ritiro. </li><li> Eparinizzare un ago G 21 aliquotandolo 5 ml eparina a 5.000 USP unità / ml in un tubo Bijou e succhiare due volte dentro e fuori l&#39;ago di una siringa da 1 ml. </li><li> Prendere la eparinizzato 21 G ago e siringa da 1 ml, ed eseguire puntura cardiaca sul mouse passaggio anestetizzato inserendo l&#39;ago centralmente sotto la gabbia toracica (una goccia di sangue piscina nella base dell&#39;ago) e tirare delicatamente lo stantuffo in modo da non comprimere la camera del cuore. Raccogliere un minimo di 0,7 ml di sangue. </li><li> Diluire sangue infetto per produrre un inoculo di 2 x 10 4 trypanosomes / ml in PBS-G pH 8,0 come al punto 1.1. Nota: A questo punto, il sangue rimanente può essere crioconservati per produrre stabilate miscelando con 8% glicerolo, 20% inattivato al calore ca fetalesiero lf (hi-FCS) e il 72% di sangue infetto. Lentamente congelare a -80 ° C usando un lento congelamento contenitore congelamento per ottenere una velocità di raffreddamento di -1 ° C / min, prima del trasferimento a azoto liquido. </li></ol> 2. L&#39;infezione di topi sperimentale <ol><li> Posizionare CD1 topi di sesso femminile (~ 21-25 g) in una scatola di riscaldamento a delicatamente calda per dilatare le vene. Posizionare i topi riscaldati in un sistema di ritenuta di plastica e iniettare 0,2 ml inoculo diluito per via endovenosa con un ago G 25 nella vena della coda, pari a 4.000 trypanosomes per topo. Mettere pressione al sito di iniezione in seguito per fermare l&#39;emorragia. Nota: infezione intraperitoneale è un percorso valida di infezione ed è stato utilizzato in studi precedenti 10, ma abbiamo trovato questo meno riproducibile rispetto alla via endovenosa. </li><li> Randomize topi infetti e la gabbia in gruppi di 3 in gabbie SPF-ventilati. Come controllo, iniettare CD1 topi femmina con wild type parassita non bioluminescente, T. b. brucei GVR35 (n =3). </li><li> Monitorare l&#39;infezione attraverso il sangue Film Microscopia <ol><li> Spot 5 ml di sangue da prelievo venoso o salasso su un vetrino ed eseguire uno striscio di sangue (utilizzando un secondo vetrino spingere delicatamente la goccia di sangue in tutto il vetrino per produrre un film sottile). Lasciare le diapositive asciugare all&#39;aria. </li><li> Fissare lo striscio di sangue con il 100% di metanolo e macchia con Giemsa per 10 minuti prima di risciacquare con dH 2 O e lasciare asciugare all&#39;aria. Sotto obiettivo 100X contare il numero di tripanosomi in 10 campi di vista (FOV). Nota: le riprese sangue viene eseguita insieme a tutto l&#39;animale di imaging non invasivo più presto dopo l&#39;infezione di un giorno. Durante l&#39;elaborazione di un numero di animali, questo metodo di quantificazione tripanosoma è preferito rispetto conteggio emocitometro come i campioni possono essere conservati a temperatura ambiente e successivamente contati. </li></ol></li></ol> 3. bioluminescenza Imaging per tenere traccia delle infezioni Nota: Per monitorare l&#39;infezione, tutto animAl imaging non invasivo può essere utilizzato. <ol><li> Preparare una soluzione stock di D-luciferina, il substrato luciferasi di lucciola, in Dulbecco tampone fosfato (DPBS) a 30 mg / ml. Filtro sterilizzare e congelare in aliquote a -20 ° C. Nota: D-Luciferin è sensibile alla luce, quindi proteggerlo dalla luce avvolgendo aliquote in un foglio. Non più volte di gelo-disgelo luciferina in quanto può influenzare l&#39;attività 11. </li><li> Scaldare una aliquota di D-luciferina a temperatura ambiente. Rimuovere ogni mouse dalla gabbia (compresi topi di controllo infettate con nonbioluminescent ceppo wild type parassita) e iniettare 150 mg / kg di D-luciferina riscaldato (diluito in DPBS) per via intraperitoneale con un ago 25 G. Posizionare il mouse in una camera isofluorane per circa 5 minuti per indurre l&#39;anestesia con il 2% isofluorane / O 2 miscela a 2,5 L / min. Nota: I topi diventano immobile quando sono sotto anestesia. Per l&#39;imager qui utilizzato, 3 topi possono essere elaborati in un momento. Se i topi sono anesthetzato per più di 30 minuti, unguento oftalmico lacrima artificiale può essere applicato per prevenire gli occhi dei topi si secchi. </li><li> Per l&#39;imager qui descritto, aprire il software e inizializzare la macchina utilizzando il software secondo le istruzioni del produttore. Acquisire le immagini una volta fotocamera e la piastra riscaldata hanno raggiunto la temperatura. Nota: questo strumento non è di solito spento, consentendo alla macchina di eseguire calibrazioni di fondo durante la notte. Nel caso in cui deve essere riavviata o deve eseguire un controllo di calibrazione prima. </li><li> Mettere i topi anestetizzati nella termocamera (cfr 3,5). Utilizzare grandi separatori neri tra i topi per ridurre al minimo sanguinare attraverso di qualsiasi segnale bioluminescente luminoso. topi immagine utilizzando una serie di tempi di esposizione; 1, 3, 10, 30, 60 e 180 secondi, con medie discretizzazione, 1 f / stop e un filtro aperto, e il campo visivo E (12.5 x 12.5 cm 2). Nota: I topi di controllo infettati con il tipo selvatico parassiti GVR35 nonbioluminescent forniscono un backgrvalore di bioluminescenza ound. Da un esperimento di cinetica di luciferina (Figura 6), con questo modello, abbiamo trovato che i picchi di segnale bioluminescenza a 10 minuti dopo la somministrazione e l&#39;imaging dura circa 5 minuti per 3 topi. Prendete questo lasso di tempo in considerazione quando anestetizzare e di imaging i topi. </li><li> Per garantire l&#39;imaging approfondito dei topi, ruotare per ottenere una ventrale, dorsale e vista laterale. Mantenere la coerenza dell&#39;ordine di orientamento per l&#39;imaging tra animali sequenziali. </li><li> Dopo l&#39;imaging, permettono topi per recuperare da anestesia sotto osservazione prima di tornare a SPF-gabbia. </li><li> Continuare l&#39;imaging topi ogni 7 giorni fino al giorno 21 dopo l&#39;infezione. Ci dovrebbe essere un costante aumento del segnale bioluminescenza sull&#39;intera animale. </li><li> Al D21, immagini e striscio di sangue dei topi come descritto in precedenza, e la dose topi con 40 mg / kg per via intraperitoneale Diminazene aceturato. Questo farmaco cancellerà il parasitemia periferico, ma non attraverserà la barriera emato-Brain barriera, e permette quindi una migliore visualizzazione dell&#39;infezione CNS. Nota: Diminazene aceturato è un farmaco comunemente utilizzate per il trattamento di tripanosomiasi animale fase iniziale. </li><li> Continuare l&#39;imaging e il monitoraggio a D28 e D35. </li></ol> 4. Conferma CNS infezione <ol><li> Al D35, topi immagine sopra descritto (passi 3,1-3,4). </li><li> Dopo l&#39;imaging, permettono di recuperare i topi da topi anestesia e Exsanguinate come dettagliato nella fase 1,6-1,8, raccolta di sangue per ulteriori analisi. </li><li> Dopo puntura cardiaca, perfusione topi con 20 ml di PBS (opzionale: aggiungere 3 mg / ml di luciferina alla soluzione di perfusione) attraverso il cuore per cancellare il sangue dagli organi e di reintrodurre etichetta luciferina che potrebbero aver eliminato dalla regione del cervello durante l&#39;intervallo di tempo dal punto 4.1. </li><li> Rimuovere delicatamente il cervello, utilizzando forbici curve, tagliando dalla base attorno alla circonferenza del cranio e sollevamento fuori la parte superiore del cranio, per permettere al cervelloper essere delicatamente tirato fuori con le pinze curve. </li><li> Posizionare il cervello asportato su una sezione di plastica nera e pipettare 50 ml di 15 mg / ml luciferina sopra l&#39;organo prima di imaging, per garantire un&#39;adeguata luciferina è stato aggiunto al cervello asportato. Immagine utilizzando le impostazioni come sopra. Questo dimostrerà localizzazione dell&#39;infezione agli emisferi cerebrali. Nota: quantificazione valle parassitemia sul cervello asportato può essere effettuata da qPCR come precedentemente descritto 8. Altre applicazioni a valle alternative potrebbero includere l&#39;istologia o immunoistochimica per identificare i parassiti nel tessuto cerebrale dopo la fissazione. </li></ol> 5. La quantificazione della bioluminescenza Imaging Nota: bioluminescenza sia quantificabile secondo la regione di interesse (ROI) con il software di imaging e corretta per lo sfondo bioluminescenza. <ol><li> Utilizzando lo strumento ROI, creare un ROI di forma rettangolare per intero animale quantificazione unnd posizionarlo dell&#39;immagine mouse sopra per coprire punta del naso alla base della coda del topo. Utilizzare la stessa casella di dimensione per ogni animale e ogni punto del tempo. Osservando il ROI per il cervello, utilizzare una ROI circolare nello stesso modo, posizionandolo sopra la regione della testa del mouse nell&#39;immagine. </li><li> Se necessario, regolare le immagini a comparire sullo stesso spettro per una rappresentazione visiva di infezione. Nota: il software utilizzato qui genera una mappa di colori per ogni immagine regolata automaticamente per i conteggi minimo e massimo di fotoni. Per consentire un confronto visivo tra una serie di immagini, la scala di colore deve essere impostato gli stessi valori per tutte le immagini. <ol><li> Usando l&#39;immagine con il più alto bioluminescenza e la scheda &#39;Regolazione immagine&#39; sulla tavolozza degli strumenti, selezionare una scala logaritmica e un adeguato minima scala di colori e massima (da determinare empiricamente). Applicare questa scala a tutte le immagini di tutto l&#39;esperimento. </li></ol></li></ol>

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The authors have nothing to disclose.

Lo sviluppo di un T. bioluminescente b. brucei GVR35 ceppo permette la visualizzazione di una infezione tripanosoma dalla presto per la fase tardiva. Precedenti modelli di infezione erano in grado di rilevare la fase avanzata, quando i parassiti sono nel cervello, in tempo reale dalla microscopia striscio di sangue, e richiedevano l&#39;abbattimento e la rimozione dei cervelli dal topi infettati per determinare parassita fardello 12. La bioluminescenza riduce la variabilità inter-mouse come...

Human African trypanosomiasis (HAT), or sleeping sickness, is caused by the vector-borne protozoan parasites of the Trypanosoma brucei spp1. Estimated numbers of current cases is fewer than 7 thousand every year with almost 70 million people exposed to the risk of the parasite infection within the African continent. The disease, which is most often lethal if left untreated, comprises an early hemolymphatic stage where parasites are present in the blood, progressing to the late stage when parasites invade the central nervous system (CNS) and are no longer susceptible to treatment by early stage trypanosomal drugs2. The current drug therapies for late-stage HAT have both complex, prolonged, treatment regimens and severe adverse effects as well as reported resistance, therefore research into new drug therapies is imperative3,4. 
The study of late-stage human African trypanosomiasis (HAT) within traditional mouse models is lengthy and complex, with the removal of brain tissue being required to monitor parasitic burden5. The animal infective strain T. b. brucei is used as the study model of trypanosomiasis with the late stage appearing 21 days post infection (dpi). To monitor the wild type nonbioluminescent parasite infection in the mouse model, peripheral blood films or quantitative PCR are the only methods to determine parasite burden. For parasite burden in the brain, the mouse needs to be culled, brain excised and qPCR carried out on tissues, making it impossible to track parasites through multiple time points in the late stage infection. This results in the inability to follow real-time infections within the central nervous system (CNS). 
In vivo bioluminescence imaging (BLI) can provide highly sensitive, non-invasive detection of parasite dissemination and disease progression in a mouse model that can be followed in a single animal for the entirety of the experiment6. BLI is based on the emission of light in the visible spectrum produced by a luciferase-catalyzed reaction. The emitted photons are then detected by a charge coupled device (CCD) camera7. For this purpose, the pathogen is genetically modified to express a luciferase protein and the substrate, luciferin, is introduced at time points of interest by injection. The main advantage of this method is the ability to carry out longitudinal studies, in which the same animal can be imaged several times with minimal adverse effects. The acquired bioluminescence signal can be quantified, thus indicating the pathogen burden.
The optimization and validation of a red-shifted bioluminescent T. b. brucei has enabled the investigation of the late stage infection through non-invasive procedures, detecting parasites earlier than blood film microscopy and greatly reducing the time, cost and numbers of animals needed to study CNS infection and drug screening in late-stage trypanosomiasis8,9. In this protocol we demonstrate infection of mice with bioluminescent trypanosomes and how to then visualize the parasites in vivo for quantification of disease progression and CNS penetration.

<table><tbody><tr><td>PBS</td><td>Sigma, UK</td><td>P4417</td><td>tablets pH 7.4</td></tr><tr><td>Glucose</td><td>Sigma, UK</td><td>G8270</td><td>99.5% (molecular) grade</td></tr><tr><td>Ammonium chloride</td><td>Sigma, UK</td><td>A9434</td><td>99.5% (molecular) grade</td></tr><tr><td>Heparin (lithium salt)</td><td>Sigma, UK</td><td>H0878</td><td></td></tr><tr><td>Hi-FCS</td><td>Gibco, Life Technologies, UK</td><td>10500-064</td><td>500 ml</td></tr><tr><td>DPBS</td><td>Sigma, UK</td><td>D4031</td><td>Sterile filtered</td></tr><tr><td>Mr. Frosty</td><td>Nalgene, UK</td><td></td><td></td></tr><tr><td>Giemsa</td><td>Sigma, UK</td><td>G5637</td><td></td></tr><tr><td>D-Luciferin</td><td>Perkin Elmer, UK</td><td></td><td></td></tr><tr><td>Sigma, UK</td><td>115144-35-9</td><td></td><td></td></tr><tr><td>Diminazene aceturate</td><td>Sigma, UK</td><td>D7770</td><td>Analytical grade</td></tr><tr><td>IVIS Lumina II</td><td>Perkin Elmer, UK</td><td></td><td>other bioimagers available &lt;em&gt;e.g.&lt;/em&gt; from Bruker, Kodak</td></tr><tr><td>Living Image v. 4.2</td><td>Perkin Elmer, UK</td><td></td><td>proprietary software for Perkin Elmer IVIS instruments; other instruments may have their own</td></tr><tr><td>1 ml Syringe</td><td>Fisher Scientific, UK</td><td>10142104</td><td></td></tr><tr><td>20 ml Syringe</td><td>Fisher Scientific, UK</td><td>10743785</td><td></td></tr><tr><td>25 G Needles</td><td>Greiner Bio-one</td><td>N2516</td><td></td></tr><tr><td>21 G Needles</td><td>Greiner Bio-one</td><td>N2138</td><td></td></tr><tr><td>Twin-frosted microscope slide</td><td>VWR, UK</td><td>631-0117</td><td></td></tr><tr><td>1.5 ml Microcentrifuge tube</td><td>StarLab, UK</td><td>I1415-1000</td><td></td></tr><tr><td>7 ml Bijou tube</td><td>StarLab, UK</td><td>E1412-0710</td><td></td></tr><tr><td>Mouse restrainer</td><td>Sigma, UK</td><td>Z756903</td><td>our restrainer was made in-house, this is a similar model</td></tr></tbody></table>

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.

Questo protocollo dimostra come seguire la progressione della malattia dopo l&#39;infezione di topi con T. b. brucei, un modello per la tripanosomiasi africana umana. La figura 1 mostra la sequenza temporale del protocollo sperimentale, dimostrando il calendario per la fasi di trattamento e di imaging. La Figura 2 mostra un tipico campo di vista in uno striscio di sangue Giemsa-macchiate fisso utilizzato per quantificare parassitemia periferico,...

Watch this Scientific Journal Video about Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis at JoVE.com

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

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 ...

bioluminescence-imaging-to-detect-late-stage-infection-african

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.7K Views.  London School of Hygiene & Tropical Diseases. 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.

Video: Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

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

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

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

Imaging in vivo del transgenico parassiti Leishmania in un host live

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

Ricerca

Immunologia e infezioni

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

Completa dei costi e monitoraggio di laboratorio efficace di HIV / AIDS: un modello di ruolo africano

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

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 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Imaging batterica<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

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

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

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

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

In Vivo Imaging Systems (IVIS) il rilevamento di un neuro-invasiva Virus encephalitic

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

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Separazione dei<em&gt; Plasmodium falciparum</em&gt; Fine Stage-eritrociti infettati da mezzi magnetici

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Utilizzo di proteine ​​fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

Doppia bioluminescenza Imaging della progressione tumorale e dell'angiogenesi

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...

Medicina

Protocollo per il modello bioluminescente in vivo della valutazione dei farmaci per la malattia di Chagas

Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, expediting the development of new drugs and vaccines is necessary. However, this process is filled with difficulties such as highly complex parasite biology and disease pathogenesis and, as typical for neglected tropical diseases, comparatively limited funding for research and development. Thus, in vitro and in vivo study models that can sufficiently reproduce infection and disease key features while providing rational use of resources are essential for progressing research for these conditions. One example is the in vivo bioluminescence imaging (BLI) mouse model for Chagas disease, which provides highly sensitive detection of long wavelength light generated by Trypanosoma cruzi parasites expressing luciferase. Despite this technique becoming the standard approach for drug efficacy in vivo studies, research groups might still struggle to implement it due to a lack of proper practical training on equipment handling and application of quality control procedures, even when suitable BLI equipment is readily available. Considering this scenario, this protocol aims to guide from planning experiments to data acquisition and analysis, with details that facilitate the implementation of protocols in research groups with little or no experience with BLI, either for Chagas disease or for other infectious disease mouse models.

Autore in primo piano: Monitoraggio in tempo reale del carico di parassiti e della risposta dell'host

The protocol provided here describes the detailed steps to perform drug efficacy studies in a bioluminescent model of Trypanosoma cruzi infection, with a focus on data acquisition, analysis, and interpretation. Troubleshooting and quality control procedures to minimize technical issues are also provided.

Demistificazione dell'imaging in bioluminescenza in vivo di un modello murino della malattia di Chagas per studi sull'efficacia dei farmaci

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, ...

Bioluminescenza Imaging per rilevare fase avanzata di infezione di africani Tripanosomiasi

Riepilogo

Esplora Altri Video

Capitoli in questo video

Imaging in vivo del transgenico parassiti Leishmania in un host live

Completa dei costi e monitoraggio di laboratorio efficace di HIV / AIDS: un modello di ruolo africano

Imaging bioluminescenza di attività NADPH ossidasi in diversi modelli animali

Bioluminescent Imaging batterica

In Vivo Imaging Systems (IVIS) il rilevamento di un neuro-invasiva Virus encephalitic

Separazione dei

Utilizzo di proteine fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

Doppia bioluminescenza Imaging della progressione tumorale e dell'angiogenesi

Demistificazione dell'imaging in bioluminescenza in vivo di un modello murino della malattia di Chagas per studi sull'efficacia dei farmaci

Demistificazione dell'imaging in bioluminescenza in vivo di un modello murino della malattia di Chagas per studi sull'efficacia dei farmaci

Bioluminescenza Imaging per rilevare fase avanzata di infezione di africani Tripanosomiasi

Riepilogo

Esplora Altri Video

Capitoli in questo video

Imaging in vivo del transgenico parassiti Leishmania in un host live

Completa dei costi e monitoraggio di laboratorio efficace di HIV / AIDS: un modello di ruolo africano

Imaging bioluminescenza di attività NADPH ossidasi in diversi modelli animali

Bioluminescent Imaging batterica

In Vivo Imaging Systems (IVIS) il rilevamento di un neuro-invasiva Virus encephalitic

Separazione dei

Utilizzo di proteine ​​fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

Doppia bioluminescenza Imaging della progressione tumorale e dell'angiogenesi

Demistificazione dell'imaging in bioluminescenza in vivo di un modello murino della malattia di Chagas per studi sull'efficacia dei farmaci

Demistificazione dell'imaging in bioluminescenza in vivo di un modello murino della malattia di Chagas per studi sull'efficacia dei farmaci

Utilizzo di proteine fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano