Biz T. sağlamak için John Kelly ve Martin Taylor (Hijyen ve Tropikal Tıp London School) teşekkür ederim b. brucei GVR35-VSL-2 ve in vivo görüntüleme tavsiye için Dr. Andrea Zelmer (LSHTM). Bu çalışma Bill ve Melinda Gates Vakfı Küresel Sağlık Programı (hibe sayısı OPPGH5337) tarafından desteklenmiştir.

ahlâk Tüm çalışmalar UK Home Office Animals onayı (Bilimsel Prosedürler) Yasası 1986 ve Hijyen ve Tropikal Tıp Hayvan Refahı ve Etik Değerlendirme Kurulu London School of altında yürütülmüştür. kılavuz ARRIVE bu raporda takip edilmektedir. Bioluminescent Trypanosoma brucei brucei Vivo Passage 1. <ol><li> T. bir Kriyoprezerve stok (adlandırılır stabilate) çıkarın b. brucei suşu sıvı azot 9 (MTA aracılığıyla Hijyen ve Tropikal Tıp London School Profesörü John Kelly temin edilebilir) GVR35-VSL-2 ve oda sıcaklığına gelmesini bekleyin. Steril fosfat tamponlu glukoz tuz (PBS-G), pH 8.0 (1 L PBS + 15 g glukoz) 10, 2 x 10 4 tripanozomlar / ml stabilate seyreltilir. Not: T. b. brucei GVR35-VSL-2 insanlarda bulaşıcı olmayan ve SAPO deneyleri yürütmek için imkanları lisanslı gerektirir. </li><li> enjvb 25 G iğne ile intraperitonal yoldan (İP) ile tripanozomlarına seyreltilmiş 0.2 ml 20-25 gram ağırlığında dişi CD1 fare. Bu &quot;donör fare&quot; dir. Belirli Patojen Free (SPF) -cage ve yem ad libitum içine enfekte fare yerleştirin. Not: CD1 fareler bir outbred zorlanma ve T. vardır b. brucei GVR35 enfeksiyonlar suşta iyi karakterize edilir. Ancak, bu protokol aynı zamanda doğal ya da genetiği değiştirilmiş suşları için geçerli olmakla birlikte, görüntüleme 6,9 hassasiyetine etkileyebilecek o kürk rengi dikkat etmek önemlidir. </li><li> kan numunesi alarak periferik parasitemia izleyin. plastik süzgeç içine fare koyun ve 21 G iğne veya venesection ile venepunkturdan tarafından kuyruk kan 5 ul almak ve kırmızı kan hücreleri parçalanması ve daha kolay sayım sağlamak için% 0.85 steril amonyum klorür ile 1/10 karıştırın. </li><li> Neubauer hemositometrede Seyreltilmiş Kan sayın. <ol><li> Her iki odalarına seyreltilmiş kan yükleyin. tüm merkez gr saymakbir odanın id n tripanozomlann vermek. Kan = n x 10 4 tripanozomlar / ml tripanozomlann konsantrasyonu, herhangi bir seyreltme faktörü ayarlamak ve iki odadan ortalamasını hesaplar. Not: Yaklaşık 5-7 gün sonra enfeksiyon, periferal parazitemi 2 x 10 4 tripanozomlar / ml&#39;lik bir konsantrasyonda bir enfeksiyon gerçekleştirmek için yeterli artacaktır. </li></ol></li><li> tripanosom seviyeleri 1.6 adıma geçmek, gerekli farelerin sayısı için yeterince yüksek olduğunda. Bir verici bir fare 6 x 10 4 tripanozomlar / ml&#39;lik bir parazit seviyesi 10 farelerin enfeksiyonu için yeterlidir. Not: istatistiksel olarak anlamlı bir grup boyutları için, n = 6 fare, her tedavi grubu için 8 ihtiyaç vardır. diğer enfeksiyon modeller için, güç hesapları uygun grup boyutlarını belirlemek için uygulanacak gerekir. </li><li> (Yaklaşık 5-10 dakika 28-30 ° C&#39;de) bir ısı kutusu içine yerleştirin fareler kuyruk damarları genişletmek için izin vermek.Terminal anestezi neden 20 mg / 25 G iğne ile kg pentobarbital iv yönetmek için. Onayla fare hafifçe hiçbir geri çekilme refleksi olmadığından emin olmak için, pedallar Sürüş Esnasında basılarak uyuşturulduktan. </li><li> Bir Bijou tüpüne 5.000 USP birim / ml&#39;de 5 mi heparin aliquoting ve bir 1 ml şırınga ucunda iğne üzerinden iki emerek, bir 21 G iğne Heparinize. </li><li> heparinize 21 G iğne ile 1 ml şırınga alın ve göğüs kafesi altında merkezi iğne sokarak anestezi geçiş fare üzerinde kalp ponksiyon gerçekleştirin (kan boncuk iğne tabanında havuz olacak) ve yavaşça pistonu geri çekin böylece kalbin odasına daraltmak için değil. kan 0.7 ml az toplayın. </li><li> Yukarıdaki adım 1.1 olarak PBS-G pH 8.0 içinde 2 x 10 4 tripanozomlar / ml&#39;lik bir aşının üretilmesi için enfekte olmuş kan seyreltilir. Not: Bu noktada, geri kalan kan% 8 gliserol,% 20 ısıyla inaktive edilmiş cenin ca karıştırılarak stabilate üretilmesi için dondurularak saklanabilirEğer serumu (hi-FCS) ve% 72 enfekte kan. Yavaş yavaş sıvı azot aktarmadan önce, C / dk ° -1 soğutma hızını elde etmek için bir kap dondurma yavaş dondurma kullanılarak -80 ° C &#39;de dondurma. </li></ol> Deneysel Fare 2. Enfeksiyon <ol><li> nazikçe ılık damarları genişletmek için bir ısıtma kutusu içine CD1 dişi fareler (~ 21-25 gr) yerleştirin. plastik kısıtlama içine ısıtılmış fareler yerleştirin ve fare başına 4.000 tripanozomlann eşdeğer kuyruk damar içine 25 G iğne ile damar 0.2 ml seyreltilmiş inokulum enjekte. Kanamayı durdurmak için daha sonra enjeksiyon yerinde basıncı yerleştirin. Karın içi enfeksiyon enfeksiyonun geçerli bir yol olduğunu ve daha önceki çalışmalarda 10 kullanılmıştır, ancak intravenöz yolla bu daha az tekrarlanabilir bulduk: edin. </li><li> SPF-havalandırmalı kafeslerde 3&#39;lü gruplar halinde enfekte fare ve kafesi Rastgele. Bir kontrol olarak, yabani tip olmayan biyolojik olarak ışık veren parazit, T ile CD1 dişi farelerin enjekte b. brucei GVR35 (n =3). </li><li> Kan Film Mikroskopi sayesinde Enfeksiyon Monitör <ol><li> Noktası 5 ul bir cam lam üzerine venepunkturdan ya venesection kan ve (hafifçe ince bir film oluşturmak için bir slayt üzerinde kan damlayı ikinci cam slayt kullanılarak) kan yayma gerçekleştirin. kuru hava slaytlar bırakın. </li><li> DH 2 O ile yıkamadan önce 10 dakika süreyle Giemsa ile% 100 metanol ve leke kan yayma düzeltmek ve kurumaya bırakın. 100X objektif altında görünümü (fov) 10 alanda tripanozomlann sayısını. Not: Kan çekim gibi erken bir gün sonrası enfeksiyon gibi bütün hayvan invaziv olmayan görüntüleme yanında yapılır. Hayvanlar, bir dizi işleme tabi tutarken numuneler, oda sıcaklığında saklanabilir ve daha sonra sayılabilir gibi, tripanosom kantitatif bu yöntem hemositometre sayımı tercih edilir. </li></ol></li></ol> 3. Biyoparlaklık Görüntüleme Enfeksiyon iz Not: enfeksiyon izlemek için, bütün animEl non-invaziv görüntüleme kullanılabilir. <ol><li> 30 mg / ml&#39;de Dulbecco Fosfat Tamponlu Salin (DPBS) D-lusiferin, ateşböceği lusiferaz alt-tabaka, bir stok çözelti hazırlayın. Filtre sterilize ve -20 ° C&#39;de parçalar halinde dondurma. Not: D-luciferase duyarlı ışık, bu nedenle folyo alikotları sararak ışıktan korumak. Bu aktiviteyi 11 etkileyebilir olarak art arda luciferase-çözülme donma yok. </li><li> Oda sıcaklığına kadar D-luciferase bir kısım ısıtın. (Nonbioluminescent vahşi tip parazit türü ile enfekte edilmiş kontrol farelerinde de dahil olmak üzere), kafesin her fare çıkarın ve intraperitonal 25 G iğne ile 150 mg / (DPBS içinde seyreltilmiş) ısıtıldı, D-luciferase kg enjekte edilir. / Dakika 2.5 L% 2 izofloran / O 2 karışımı ile anestezi sağlamak için yaklaşık 5 dakika boyunca, bir izofloran odasına fareler yerleştirin. Not: Onlar anestezi altında olduğunda Fareler hareketsiz hale gelir. Burada kullanılan kamera için, 3 fare, bir anda işlenebilir. farenin anesthet isedaha uzun bir süre 30 dakika yanıstarak, oftalmik merhem, yapay gözyaşı kurumasını farelerinin gözleri önlemek için uygulanabilir. </li><li> Burada anlatılan görüntüleyici için Yazılımı açmak ve üreticinin talimatlarına göre yazılımı kullanarak makineyi başlat. Kamera ve ısıtmalı plaka sıcaklığı ulaştığında görüntü kazanır. Not: Bu cihaz, genellikle bir gecede arka plan kalibrasyonları yapmak için makinenin sağlayan kapalı değil. yeniden başlatmak gerekiyor durumunda, öncelikle bir kalibrasyon kontrolü çalıştırmanız gerekir. </li><li> (3.5 bakınız) kamera içine anestezi fareler yerleştirin. herhangi bir parlak bioluminescent sinyal yoluyla kanama en aza indirmek için fareler arasında büyük siyah ayırıcılar kullanın. pozlama süreleri bir dizi kullanarak görüntü fareler; Görünüm E orta binning, 1 f / durdurma ve açık filtre ve alanla 1, 3, 10, 30, 60 ve 180 saniye, (12.5 x 12.5 cm 2). Not: vahşi tip nonbioluminescent GVR35 parazit ile enfekte kontrol fareleri bir backgr sağlamakound biyoparlaklık değer. Bu model ile, bir lusiferin kinetik deney (Şekil 6), biz 10 dakika biyoışıldama sinyal tepe Uygulamayı takip ve görüntüleme yaklaşık 3 fareler için 5 dakika sürer bulmuşlardır. anestezi ve fareler görüntüleme dikkate bu zaman ölçeği alın. </li><li> Farelerin tam görüntüleme sağlamak için, ventral, dorsal ve lateral görünüm elde etmek için döndürün. ardışık hayvanlar arasındaki görüntüleme için oryantasyon sırayla tutarlılığı korumak. </li><li> görüntüleme sonra fareler SPF-kafes dönmeden önce gözlem altında anestezi kurtarmak için izin verir. </li><li> gün 21 sonrası enfeksiyon kadar her 7 gün fareler görüntüleme devam edin. tüm hayvan üzerinde biyoparlaklık sinyali istikrarlı bir artış olmalıdır. </li><li> D21, görüntü ve kan filmi / kg diminazene aceturate intraperitonal 40 mg fareler, yukarıda tarif edilen ve bir doz olarak fareler. Bu ilaç periferik parasitemia silecektir ancak kan Brai geçmeyeceğimn bariyer ve bu nedenle MSS enfeksiyonu daha iyi görselleştirme sağlar. Not: Diminazene aceturate erken evre, hayvan trypanosomiasis tedavisi için kullanılan iyi bilinen bir ilaçtır. </li><li> D28 ve D35 de görüntüleme ve izleme devam edin. </li></ol> 4. MSS Enfeksiyon Onaylama <ol><li> D35 &#39;de, yukarıda açıklandığı gibi resim fareleri (3.1-3.4 adım). </li><li> görüntüleme sonra, daha fazla analiz için kan toplama aşamasında 1.6-1.8 detaylı olarak farenin anestezi ve asıl bulbus olfaktoryusları alındı ​​farelerden kurtarmak için izin verir. </li><li> kardiyak delme ardından, 20 ml PBS ile fareler serpmek (isteğe bağlı: 3 mg / perfüzyon çözüm luciferase ml ekleyin) organlara kan temizlemek ve zaman aralığındaki beyin bölgesi temizlenir olabilir luciferin etiketi reintroduce kalp yoluyla adım 4.1. </li><li> Yavaşça beyin izin, kavisli makas kullanılarak kafatası çevresi etrafında tabanından kesme ve kafatasının üst kısmı kapalı kaldırarak beyin kaldırmakhafifçe kavisli bir forseps ile kaldırılması gerekir. </li><li> Yeterli luciferase Kesilerek çıkarılan beynin eklendi sağlamak için, siyah plastik pipet önce görüntüleme organ üzerindeki 15 mg / ml lusiferin 50 ul bir bölümü üzerine Kesilerek çıkarılan beynin yerleştirin. Yukarıdaki ayarları kullanarak görüntü. Bu beyin hemisfer enfeksiyon lokalizasyonu gösterecektir. Not: Daha önce 8 açıklandığı gibi kesilmiştir beyin üzerinde parazitemi Mansap miktar qPCR yapılabilir. Diğer alternatif aşağı uygulamalar tespit sonucu beyin dokusunda parazitleri tespit etmek histoloji veya immünohistokimyasal içerebilir. </li></ol> Biyoparlaklık Görüntüleme 5. kantitasyonu Not: Biyoparlaklık görüntüleme yazılımı ile ilgi alanları (ROI) bölgesini kullanarak sayısal ve arka plan biyoparlaklık için düzeltilebilir. <ol><li> ROI aracını kullanarak, bütün hayvan kantitatif a için bir dikdörtgen ROI oluşturmaknd fare kuyruk tabanına burnunun ucunu kapatmak için fare resmin üzerine getirin. Her hayvan ve her zaman noktası için aynı boyutta kutusunu kullanın. beyin için ROI bakarken, görüntüdeki fare kafası bölge üzerinde konumlandırma, aynı şekilde dairesel ROI kullanın. </li><li> İstenirse, enfeksiyon görsel gösterimi için aynı spektrum görüntülenmesini ayarlayın. Not: Burada kullanılan yazılım otomatik olarak minimum ve maksimum foton sayıları için ayarlanmış her bir görüntü için bir renk haritası oluşturur. görüntülerin bir dizi arasındaki görsel karşılaştırma etkinleştirmek için, renk skalası, tüm görüntüler için aynı değerlere ayarlanmalıdır. <ol><li> En yüksek biyolüminesans ve araç paleti üzerinde &#39;Görüntü ayarlamak&#39; sekmesi ile görüntüyü kullanarak, logaritmik ölçek ve uygun bir renk skalası minimum ve maksimum (ampirik olarak tespit edilmesi) seçin. deney boyunca tüm görüntüleri bu ölçek uygulayın. </li></ol></li></ol>

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The authors have nothing to disclose.

Bir biyolojik olarak ışık veren T gelişimi b. brucei GVR35 suşu geç dönemde erken bir tripanozom enfeksiyon görselleştirme sağlar. Önceki enfeksiyon modelleri parazitler beyin olduğunda kan filmi mikroskopi gerçek zamanlı olarak, geç evre tespit koyamadık ve parazit yükünün 12 belirlemek için enfekte farelerin beyinleri seçilmesini ve çıkarılması gerekti. tek bir fare enfeksiyon bütünlükleri içinde ve herhangi bir aşamada gözlemlenebilir enfeksiyonun hızlı bi...

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 border="0" cellpadding="0" cellspacing="0" width="860">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">PBS</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">P4417</td>
			<td style="width:283px;">tablets pH 7.4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Glucose</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">G8270</td>
			<td style="width:283px;">99.5% (molecular) grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Ammonium chloride</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">A9434</td>
			<td style="width:283px;">99.5% (molecular) grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Heparin (lithium salt)</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">H0878</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:233px;">Hi-FCS</td>
			<td style="width:189px;">Gibco, Life Technologies, UK</td>
			<td style="width:155px;">10500-064</td>
			<td style="width:283px;">500 ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">DPBS</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">D4031</td>
			<td style="width:283px;">Sterile filtered</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Mr. Frosty</td>
			<td style="width:189px;">Nalgene, UK</td>
			<td style="width:155px;"></td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Giemsa</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">G5637</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="44" rowspan="2" style="height:44px;width:233px;">D-Luciferin</td>
			<td style="width:189px;">Perkin Elmer, UK</td>
			<td style="width:155px;"></td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:189px;">Sigma, UK</td>
			<td style="width:155px;">115144-35-9</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Diminazene aceturate</td>
			<td style="width:189px;">Sigma, UK</td>
			<td style="width:155px;">D7770</td>
			<td style="width:283px;">Analytical grade</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:233px;">IVIS Lumina II</td>
			<td style="width:189px;">Perkin Elmer, UK</td>
			<td style="width:155px;"></td>
			<td style="width:283px;">other bioimagers available e.g. from Bruker, Kodak</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:233px;">Living Image v. 4.2</td>
			<td style="width:189px;">Perkin Elmer, UK</td>
			<td style="width:155px;"></td>
			<td style="width:283px;">proprietary software for Perkin Elmer IVIS instruments; other instruments may have their own</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">1 ml syringe</td>
			<td style="width:189px;">Fisher Scientific, UK</td>
			<td>10142104</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">20 ml syringe</td>
			<td style="width:189px;">Fisher Scientific, UK</td>
			<td>10743785</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">25G Needles</td>
			<td style="width:189px;">Greiner Bio-one</td>
			<td>N2516</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">21G Needles</td>
			<td style="width:189px;">Greiner Bio-one</td>
			<td>N2138</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">Twin-frosted microscope slide</td>
			<td style="width:189px;">VWR, UK</td>
			<td>631-0117</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">1.5 ml microcentrifuge tube</td>
			<td style="width:189px;">StarLab, UK</td>
			<td>I1415-1000</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:233px;">7 ml Bijou tube</td>
			<td style="width:189px;">StarLab, UK</td>
			<td>E1412-0710</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:233px;">Mouse restrainer</td>
			<td style="width:189px;">Sigma, UK</td>
			<td>Z756903</td>
			<td style="width:283px;">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.

Bu protokol T. ile farelerin enfeksiyonu takiben hastalığın ilerlemesini takip etmek nasıl gösterir b. brucei, insan Afrika trypanosomiasis için bir model. Şekil 1 tedavi ve görüntüleme aşamaları için takvimi gösteren deneysel protokol çizelgesini gösterir. 2 periferik parasitemia ölçmek için kullanılan sabit bir Giemsa lekeli kan yayması bakış tipik bir alanı gösterir Şekil, tripanozomlar ve k...

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.

Biyoparlaklık Görüntüleme Afrika Trypanosomiasis Geç Evre Enfeksiyon Algılama için

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 Görüntüleme.  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

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.

HIV / AIDS Kapsamlı ve Maliyet Etkin Laboratuvar Kontrolü: Afrikalı bir Rol Modeli

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

İmmünoloji ve Enfeksiyon

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.

Farklı Hayvan Modelleri NADPH Oksidaz Aktivitesinin Biyoparlaklık Görüntüleme

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

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.

Ayrılması<em&gt; Plasmodium falciparum</emManyetik Yoluyla&gt; Geç Evre-enfekte Eritrositler

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

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D modelli Görüntüleme<em&gt; Citrobacter rodentium</emFarelerde&gt; Enfeksiyonlar

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In vivo Görüntüleme Yöntemi Akut ve Kronik İltihap ayırt

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

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.

Floresan Proteinleri kullanarak Afrika tripanosom içinde Glycosome Dynamics Monitör

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

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Fare Fetal Karaciğer Kültür Sistemi Terminali Eritropoiesis Erken ve Geç Dönemlerinde Hedef Gene İşlevleri teşrih etmek

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

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.

Çok ilaca dirençli için Antimikrobiyal Mavi Işık Tedavisinin Vivo İncelenmesinin Acinetobacter baumannii Biyoparlaklık ımaging&#39;i kullanarak Enfeksiyonlar Yanık

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

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Aşırı Ezme ve İnsanın Saflaştırılması<em&gt; Cis</em&gt; -prenıltransferaz in<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity.
Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal.
These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations.

Afrika, anyon-değiştiriciler (Diethylaminoethyl-selüloz sütunlar) tarafından kandan da dahil olmak üzere hücre dışı Tripanozomlar arıtma

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is ...