Wir danken John Kelly und Martin Taylor (London School of Hygiene &amp; Tropical Medicine) für T. Bereitstellung b. brucei GVR35-VSL-2 und Dr. Andrea Zelmer (LSHTM) für die Beratung über in - vivo - Bildgebung. Diese Arbeit wurde von der Bill and Melinda Gates Foundation Global Health Program (Grantnummer OPPGH5337) unterstützt.

Ethik Alle Arbeiten wurden im Rahmen der Genehmigung der britischen Home Office Tiere durchgeführt (Scientific Procedures) Act 1986 und der London School of Hygiene &amp; Tropical Medicine Tierschutz und Ethik Review Board. ANKOMMEN Leitlinie werden in diesem Bericht folgt. 1. In - vivo - Passage von Biolumineszenz Trypanosoma brucei brucei <ol><li> Entfernen eines kryokonservierten Lager (genannt Stabilat) von T. b. brucei Stamm GVR35-VSL-2 (erhältlich von Professor John Kelly an der London School of Hygiene &amp; Tropical Medicine durch MTA) 9 aus flüssigem Stickstoff und auf Raumtemperatur äquilibrieren. Verdünnte Stabilat bis 2 x 10 4 Trypanosomen / ml in steriler Phosphat - gepufferter Glukosesalzlösung (PBS-G) pH 8,0 (1 l PBS + 15 g Glucose) 10. Anmerkung: T. b. brucei GVR35-VSL-2 ist nicht infektiös für den Menschen und erfordert SAPO Einrichtungen lizenziert Experimente durchzuführen. </li><li> inject 20-25 g weiblichen CD1-Maus mit 0,2 ml Trypanosomen über die intraperitoneale Injektion (ip) mit einer 25 G-Nadel verdünnt. Dies ist der &quot;Spendermaus&quot;. Legen Sie die infizierte Maus in eine spezifische pathogenfreie (SPF) -cage und Futter ad libitum. Hinweis: CD1 - Mäuse sind ein Auszucht Stamm und T. b. brucei GVR35 - Infektionen sind in diesem Stamm gut charakterisiert. Jedoch ist dieses Protokoll auch für angeboren oder genetisch veränderte Stämme, aber es ist wichtig , dass die Fellfarbe zu beachten , kann auf die Empfindlichkeit der Bildgebung 6,9 auswirken. </li><li> Überwachen peripheren Parasitämie durch Blutentnahme. Platzieren Sie die Maus in eine Plastik Verzögerer und nehmen 5 ul Blut aus dem Schwanz von venepuncture mit 21 G-Nadel oder Aderlässe und mischen 1/10 mit 0,85% sterilem Ammoniumchlorid roten Blutkörperchen zu lysieren und einfacher Zählung ermöglichen. </li><li> Zählen Sie die verdünntes Blut in einer Neubauer-Zählkammer. <ol><li> Legen Sie das verdünnte Blut in beiden Kammern. Zählen Sie die gesamte zentrale grID von einer Kammer zu geben n Trypanosomen. Konzentration von Trypanosomen in Blut = n x 10 4 Trypanosomen / ml, für jede Verdünnungsfaktor einzustellen und die Mittel der beiden Kammern berechnen. Hinweis: Nach ca. 5-7 Tage nach der Infektion sollte peripheren Parasitämie steigen ausreichend um eine Infektion bei einer Konzentration von 2 x 10 4 Trypanosomen / ml durchzuführen. </li></ol></li><li> Wenn Trypanosomen ist hoch genug für die Anzahl der Mäuse erforderlich, bewegen sich auf 1,6 zu treten. Ein Parasitämie Niveau von 6 x 10 4 Trypanosomen / ml in einer Spendermaus ist für die Infektion von 10 Mäusen ausreichend. Hinweis: Für die statistisch signifikante Gruppengrößen, n = 6 für jede Behandlungsgruppe von Mäusen werden 8 benötigt. Für andere Modelle der Infektion, müssten Leistungsberechnungen angewandt werden, um geeignete Gruppengrößen zu bestimmen. </li><li> Ort Mäuse in einem Wärmefeld (bei 28-30 ° C für etwa 5-10 min) die Schwanzvenen zu erlauben, zu dilatieren.Zur Auslösung einer Klemme Narkose verabreichen 20 mg / kg Pentobarbital iv mit einer 25 G-Nadel. Bestätigen Sie die Maus wird durch sanftes Drücken der Fußballen narkotisiert, um sicherzustellen, gibt es keine Rückzugsreflex ist. </li><li> Heparinisierung eine 21 G Nadel durch Aliquotieren 5 ml Heparin bei 5000 USP-Einheiten / ml in einem Bijou Rohr und Saugen zweimal in die und aus der Nadel in eine 1 ml Spritze befestigt. </li><li> Nehmen Sie die heparinisierte 21 G Nadel und 1-ml-Spritze und führen Herzpunktur auf der narkotisierten Passage Maus durch die Nadel mittig unter dem Brustkorb eingefügt (eine Perle von Blut wird in der Basis des Nadel Pool) und ziehen Sie vorsichtig zurück auf den Kolben um nicht die Kammer des Herzens zu kollabieren. Sammeln Sie ein Minimum von 0,7 ml Blut. </li><li> Verdünnte infiziertem Blut ein Inokulum von 2 x 10 4 Trypanosomen / ml in PBS-G pH 8,0 als Schritt 1.1 oben zu erzeugen. Hinweis: An dieser Stelle kann das restliche Blut kryokonserviert werden Stabilat herzustellen, indem man mit 8% Glycerin mischen, 20% hitzeinaktiviertem fötalen calf Serum (hallo-FCS) und 72% infiziertem Blut. Langsam bei -80 ° C einfrieren ein langsam einfrieren Behälter mit einer Kühlrate von 1 ° C / min zu erreichen, bevor sie mit flüssigem Stickstoff übertragen. </li></ol> 2. Infektion von Versuchsmäusen <ol><li> Platzieren CD1 weibliche Mäuse (~ 21-25 g) in einen Heizkasten sanft warmen Venen zu dilatieren. Legen Sie die erwärmte Mäuse in eine Plastikstütze und injizieren 0,2 ml verdünnte Inokulum intravenös mit einer 25 G-Nadel in die Schwanzvene, das entspricht 4000 Trypanosomen pro Maus. Platzieren Druck an der Injektionsstelle danach die Blutung einzudämmen. Hinweis: Die intraperitoneale Infektion eine gültige Route der Infektion und wurde in früheren Studien verwendet worden , 10, aber wir haben festgestellt , diese weniger reproduzierbar als intravenöser Applikation. </li><li> Randomize infizierten Mäusen und Käfig in Gruppen von 3 in SPF-belüftete Käfige. Als Kontrolle injizieren CD1 weibliche Mäuse mit Wildtyp nicht Biolumineszenz - Parasiten, T. b. brucei GVR35 (n =3). </li><li> Überwachen Sie die Infektion durch Blut Film Mikroskopie <ol><li> Spot 5 ul Blut aus venepuncture oder Aderlässe auf einen Glasträger und führen Sie einen Blutabstrich (mit einem zweiten Glasträger vorsichtig die Tropfen Blut schieben über die Rutsche einen dünnen Film zu erzeugen). Lassen Sie Folien an der Luft trocknen. </li><li> Befestigen Sie den Blutabstrich mit 100% Methanol und Fleck mit Giemsa für 10 Minuten , bevor sie mit dH 2 O gespült und an der Luft trocknen lassen . Unter 100X Ziel zählen die Anzahl der Trypanosomen in 10 Bereichen (FOV). Hinweis: Blutdreharbeiten wird neben ganzen Tier nicht-invasive Bildgebung bereits einen Tag nach der Infektion durchgeführt. Wenn eine Anzahl von Tieren Verarbeitung dieses Verfahren der Trypanosomen Quantifizierung über Hämozytometer Zählen bevorzugt, da die Proben können später bei Raumtemperatur und gezählt gespeichert werden. </li></ol></li></ol> 3. Biolumineszenz Imaging-Infektion zu verfolgen Hinweis: Um die Infektion zu überwachen, ganze animal nichtinvasive Bildgebung verwendet werden. <ol><li> Vorbereitung einer Stammlösung von D-Luciferin, das Glühwürmchen-Luciferase-Substrat, in Dulbeccos phosphatgepufferter Salzlösung (DPBS) bei 30 mg / ml. Filter sterilisieren und in Aliquots bei -20 ° C einfrieren. Hinweis: D-Luciferin Licht empfindlich ist, daher schützen sie vor Licht von Aliquoten in Folie gewickelt wird. Nicht wiederholt Frost-Tau - Luciferin , wie es 11 - Aktivität beeinflussen können. </li><li> Wärmen eines Aliquots von D-Luciferin auf Raumtemperatur. Entfernen Sie jede Maus aus dem Käfig (einschließlich Kontrollmäuse infiziert mit nonbioluminescent Stamm Wildtyp-Parasiten) und injizieren 150 mg / kg des erwärmten D-Luciferin (verdünnt in DPBS) intraperitoneal mit einer 25 G-Nadel. Legen Sie die Mäuse in eine Isofluran Kammer für ca. 5 min zu induzieren Anästhesie mit 2% Isofluran / O 2 Mix bei 2,5 l / min. Hinweis: Die Mäuse werden unbeweglich, wenn sie unter Narkose sind. Für den Bildaufnehmer hier verwendet, 3 Mäuse können gleichzeitig verarbeitet werden. Wenn Mäuse sind anesthetized länger als 30 min, können ophthalmische künstliche Tränen Salbe angewendet werden, um die Augen der Mäuse verhindert das Austrocknen. </li><li> Verwendung der Software gemäß den Anweisungen des Herstellers für den Imager hier beschrieben, die Software öffnen und die Maschine zu initialisieren. Erwerben Sie Bilder einmal Kamera und beheizte Plattentemperatur erreicht haben. Hinweis: Dieses Gerät nicht in der Regel ausgeschaltet ist, so dass die Maschine über Nacht Hintergrund Kalibrierungen durchzuführen. Für den Fall, dass es neu gestartet werden muss, es muss eine Überprüfung der Kalibrierung laufen zuerst. </li><li> Legen Sie die narkotisierten Mäusen in den Imager (siehe 3.5). Verwenden Sie große schwarze Trennzeichen zwischen den Mäusen zu minimieren bluten durch jedes helle Biolumineszenz-Signal. Bild Mäuse eine Reihe von Belichtungszeiten; 1, 3, 10, 30, 60 und 180 Sekunden, mit mittlerer Binning, 1 f / stop und einem offenen Filter und Sichtfeld E (12,5 x 12,5 cm 2). Hinweis: Die Kontrollmäuse infiziert mit den Wildtyp-nonbioluminescent GVR35 Parasiten bieten eine background Biolumineszenz Wert. Aus einem Luciferin Kinetik Experiment (Abbildung 6) mit diesem Modell haben wir die Biolumineszenz Signalspitzen bei 10 Minuten nach der Verabreichung und Imaging dauert ca. 5 min für 3 Mäuse gefunden. Nehmen Sie diesen Zeitplan in Betracht, wenn anesthetizing und Abbilden der Mäuse. </li><li> Um sicherzustellen, gründliche Abbildung der Mäuse, drehen einen ventralen, dorsalen und Seitenansicht zu erhalten. Pflegen Sie die Konsistenz in der Reihenfolge der Orientierung für die Bildgebung zwischen aufeinanderfolgenden Tieren. </li><li> Nach der Bebilderung erlauben Mäuse aus der Narkose unter Beobachtung zu erholen, bevor zu SPF-Käfig zurück. </li><li> Weiter Mäuse Bildgebung alle 7 Tage bis zum Tag 21 nach der Infektion. Es sollte eine stetige Zunahme der Biolumineszenz-Signal über das gesamte Tier sein. </li><li> Bei D21, Bild und Blut Film die Mäuse wie oben beschrieben, und der Dosis-Mäuse mit 40 mg / kg Diminazen aceturat intraperitoneal. Dieses Medikament wird die periphere Parasitämie klar, aber das Blut-brai nicht überquerenn-Schranke und ermöglicht somit eine bessere Visualisierung der ZNS-Infektion. Hinweis: Diminazen aceturat ist ein gut etabliertes Medikament zur Frühstadium nagana behandeln. </li><li> Weiter Bildgebung und Überwachung bei D28 und D35. </li></ol> 4. Bestätigen ZNS-Infektion <ol><li> Bei D35, Bild Mäuse wie oben beschrieben (Schritte 3,1-3,4). </li><li> Nach der Bebilderung erlauben Mäuse aus der Narkose und exsanguinate Mäuse in so detailliert Schritt 1,6-1,8, das Sammeln zur weiteren Analyse Blut zu erholen. </li><li> Nach Herzpunktur, perfuse Mäuse, die mit 20 ml PBS (optional: add 3 mg / ml Luciferin zu Perfusionslösung) über das Herz das Blut aus den Organen zu löschen und Luciferin Etikett wieder einführen, die aus der Region des Gehirns über das Zeitintervall gelöscht haben könnte aus Schritt 4.1. </li><li> Vorsichtig in das Gehirn zu entfernen, die durch gekrümmte Schere, die um den Umfang des Schädels von der Basisschneiden und das Oberteil des Schädels Abhebens, das Gehirn zu ermöglichen,sanft herausgehoben mit einer gebogenen Pinzette werden. </li><li> Legen Sie das ausgeschnittene Gehirn auf einen Abschnitt aus schwarzem Kunststoff und Pipette 50 ul 15 mg / ml Luciferin über das Organ vor der Bildgebung, um sicherzustellen, ausreichende Luciferin hat ausgeschnittenen Gehirn hinzugefügt. Bild die Einstellungen wie oben. Dies wird die Lokalisierung der Infektion zu den Gehirnhälften demonstrieren. Hinweis: Downstream Quantifizierung von Parasitämie am exzidiert Gehirn kann durch qPCR , durchgeführt werden , wie zuvor 8 beschrieben. Andere alternative Downstream-Anwendungen könnte Histologie oder Immunhistochemie umfassen Parasiten im Hirngewebe folgenden Fixierung zu identifizieren. </li></ol> 5. Die Quantifizierung der Biolumineszenz-Bildgebung Hinweis: Die Biolumineszenz quantifiziert werden können, die Region von Interesse (ROI) mit der Bildverarbeitungssoftware und für die Hintergrund Biolumineszenz korrigiert. <ol><li> den ROI-Tool verwenden, erstellen Sie einen rechteckigen ROI für ganze Tier Quantifizierung einnd positionieren sie über die Maus Bild der Nasenspitze zur Basis des Schwanzes der Maus zu decken. Verwenden Sie die gleiche Größe Box für jedes Tier und jeden Zeitpunkt. Bei der Betrachtung der ROI für das Gehirn suchen, verwenden Sie einen kreisförmigen ROI in der gleichen Art und Weise, sie über die Maus Kopfbereich in die Bildposition. </li><li> Falls gewünscht, einstellen Bilder auf dem gleichen Spektrum für eine visuelle Darstellung der Infektion zu erscheinen. Hinweis: Die Software, die hier erzeugt eine Farbkarte für jedes Bild automatisch angepasst für minimale und maximale Photonenzählungen. Um einen visuellen Vergleich zwischen einer Reihe von Bildern zu ermöglichen, die Farbskala muss für alle Bilder auf die gleichen Werte eingestellt werden. <ol><li> Mit dem Bild mit der höchsten Biolumineszenz und der &quot;Bild anpassen&quot; Registerkarte auf der Werkzeugpalette, wählen Sie eine logarithmische Skala und eine entsprechende Farbskala Minimum und Maximum (empirisch ermittelt werden). Wenden Sie diese Skala auf alle Bilder während des gesamten Experiments. </li></ol></li></ol>

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The authors have nothing to disclose.

Die Entwicklung eines biolumineszenten T. b. brucei GVR35 Stamm ermöglicht die Visualisierung eines Trypanosomeninfektion von den frühen bis späten Stadium. Zurück Infektionsmodellen konnten die späten Stadium zu erkennen, wenn Parasiten im Gehirn, in Echtzeit aus dem Blut Film Mikroskopie sind und erforderlich , um die Keulung und Beseitigung von Gehirnen von der infizierten Mäuse Parasitenlast 12 zu bestimmen. Die Biolumineszenz reduziert inter Maus Variabilität als eine einzige Maus kann ü...

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.

Dieses Protokoll zeigt , wie Krankheitsprogression nach einer Infektion von Mäusen mit T. folgen b. brucei, ein Modell für die afrikanische Trypanosomiasis. 1 , die die Zeitleiste des Versuchsprotokoll zeigt, den Zeitplan für die Behandlung und Imaging - Schritte zeigt, Fig . 2 eine typische Sichtfeld in einer festen Giemsa-gefärbten Blutausstrich zeigt , verwendet periphere Parasitämie quantitativ zu bestimmen, mi...

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.

Biolumineszenz-Bildgebung zur Erkennung von Late Stage-Infektion der afrikanischen 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 ...

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

Research

JoVE Journal

Immunology and Infection

7.7K Aufrufe.  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.

Serie: 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.

In-vivo-Imaging von transgenen Leishmania Parasiten in einer Live-Host

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

Forschung

Immunologie und Infektion

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.

Umfassende &amp; Kostengünstig Laboratory Überwachung von HIV / AIDS: eine afrikanische 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 ...

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.

Biolumineszenz Imaging von NADPH-Oxidase-Aktivität in verschiedenen Tiermodellen

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

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) Nachweis eines Neuro-Invasive encephalitische Virus

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

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.

Mit fluoreszierenden Proteinen zu Glycosome Dynamics in der afrikanischen Trypanosomen auf Monitor

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

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Hochdurchsatz-Gene Tagging in<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

KI-Programm zum Nachweis und zur Klassifizierung von Trypanosomiasis

Superior Auto-Identification of Trypanosome Parasites by Using a Hybrid Deep-Learning Model

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of hotspot areas under active surveillance is a fundamental procedure for controlling disease transmission. Microscopic examination is a commonly used diagnostic method. It is, nevertheless, primarily reliant on skilled and experienced personnel. To address this issue, an artificial intelligence (AI) program was introduced that makes use of a hybrid deep learning technique of object identification and object classification neural network backbones on the in-house low-code AI platform (CiRA CORE). The program can identify and classify the protozoan trypanosome species, namely Trypanosoma cruzi, T. brucei, and T. evansi, from oil-immersion microscopic images. The AI program utilizes pattern recognition to observe and analyze multiple protozoa within a single blood sample and highlights the nucleus and kinetoplast of each parasite as specific characteristic features using an attention map.
To assess the AI program&#39;s performance, two unique modules are created that provide a variety of statistical measures such as accuracy, recall, specificity, precision, F1 score, misclassification rate, receiver operating characteristics (ROC) curves, and precision versus recall (PR) curves. The assessment findings show that the AI algorithm is effective at identifying and categorizing parasites. By delivering a speedy, automated, and accurate screening tool, this technology has the potential to transform disease surveillance and control. It could also assist local officials in making more informed decisions on disease transmission-blocking strategies.

Autor im Rampenlicht: KI-gesteuerter Nachweis von Trypanosomenspezies aus mikroskopischen Bildern 

Worldwide medical blood parasites were automatically screened using simple steps on a low-code AI platform. The prospective diagnosis of blood films was improved by using an object detection and classification method in a hybrid deep learning model. The collaboration of active monitoring and well-trained models helps to identify hotspots of trypanosome transmission.

Überlegene automatische Identifizierung von Trypanosomenparasiten durch Verwendung eines hybriden Deep-Learning-Modells

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of ...

Biotechnik

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.

Dual Biolumineszenz-Bildgebung der Tumorprogression und Angiogenese

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

Biolumineszenz-Bildgebung zur Erkennung von Late Stage-Infektion der afrikanischen Trypanosomiasis

Zusammenfassung

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Umfassende & Kostengünstig Laboratory Überwachung von HIV / AIDS: eine afrikanische Role Model

Biolumineszenz Imaging von NADPH-Oxidase-Aktivität in verschiedenen Tiermodellen

In Vivo Imaging Systems (IVIS) Nachweis eines Neuro-Invasive encephalitische Virus

Mit fluoreszierenden Proteinen zu Glycosome Dynamics in der afrikanischen Trypanosomen auf Monitor

Hochdurchsatz-Gene Tagging in

Überlegene automatische Identifizierung von Trypanosomenparasiten durch Verwendung eines hybriden Deep-Learning-Modells

Überlegene automatische Identifizierung von Trypanosomenparasiten durch Verwendung eines hybriden Deep-Learning-Modells

Überlegene automatische Identifizierung von Trypanosomenparasiten durch Verwendung eines hybriden Deep-Learning-Modells

Dual Biolumineszenz-Bildgebung der Tumorprogression und Angiogenese