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><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.

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

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.

Canlı bir Host Transgenik Leishmania Parazitlerinin in vivo Imaging

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

İmmünoloji ve Enfeksiyon

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 ...

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 ...

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.

Bir Nöro-İnvazif encephalitic Virüs Vivo Görüntüleme Sistemleri (IVIS) Algılama

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.

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 ...

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.

Yüksek verimli Gen Etiketleme içinde<em&gt; Trypanosoma brucei</em

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

Tripanozomiyaz Tespiti ve Sınıflandırması için AI Programı

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.

Yazar Spotlight: Mikroskobik Görüntülerden Yapay Zeka Güdümlü Tripanozom Türlerinin Tespiti 

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.

Hibrit bir derin öğrenme modeli kullanarak tripanozom parazitlerinin üstün otomatik tanımlanması

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

Biyomühendislik

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.

Tümör Ilerlemesi ve anjiyogenezinin çift Bioluminescence görüntülenmesi

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

Biyoparlaklık Görüntüleme Afrika Trypanosomiasis Geç Evre Enfeksiyon Algılama için

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Canlı bir Host Transgenik Leishmania Parazitlerinin in vivo Imaging

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

Farklı Hayvan Modelleri NADPH Oksidaz Aktivitesinin Biyoparlaklık Görüntüleme

Bir Nöro-İnvazif encephalitic Virüs Vivo Görüntüleme Sistemleri (IVIS) Algılama

Floresan Proteinleri kullanarak Afrika tripanosom içinde Glycosome Dynamics Monitör

Yüksek verimli Gen Etiketleme içinde

Hibrit bir derin öğrenme modeli kullanarak tripanozom parazitlerinin üstün otomatik tanımlanması

Hibrit bir derin öğrenme modeli kullanarak tripanozom parazitlerinin üstün otomatik tanımlanması

Hibrit bir derin öğrenme modeli kullanarak tripanozom parazitlerinin üstün otomatik tanımlanması

Tümör Ilerlemesi ve anjiyogenezinin çift Bioluminescence görüntülenmesi