Name: bioluminescence imaging to detect late stage infection of african trypanosomiasis
Uploaded: 2016-05-18T14:00:00.000+00:00
Duration: 7 min 59 s
Description: Watch this Scientific Journal Video about Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis at JoVE.com

Agradecemos a John Kelly e Martin Taylor (London School of Hygiene &amp; Tropical Medicine) para a prestação de T. b. brucei GVR35-VSL-2 e Dr. Andrea Zelmer (LSHTM) para o conselho em imagem in vivo. Este trabalho foi financiado pela Fundação Programa de Saúde Global Bill e Melinda Gates (número de concessão OPPGH5337).

Ética Todo o trabalho foi realizado sob a aprovação do Reino Unido Escritório Animais (Scientific Procedures) Act 1986 e da London School of Hygiene &amp; Tropical Medicine Animal Welfare and Ethics Review Board. CHEGADA orientação são seguidos neste relatório. 1. In Vivo Passagem de Bioluminescent Trypanosoma brucei brucei <ol><li> Remover um estoque criopreservado (stabilate denominado) de T. b. brucei estirpe GVR35-VSL-2 (disponível a partir de Professor John Kelly na London School of Hygiene &amp; Tropical Medicine através MTA) 9 de azoto líquido e deixar equilibrar à temperatura ambiente. Diluir stabilate a 2 x 10 4 tripanossomas / ml em solução salina tamponada com fosfato de glicose (PBS-G) estéril de pH 8,0 (1 L PBS + glucose 15 g) 10. Nota: T. b. brucei GVR35-VSL-2 é não infecciosa para os seres humanos e requer SAPO licenciado instalações para a realização de experimentos. </li><li> inject de 20-25 g feminina rato CD1 com 0,2 ml diluído tripanossomas por via intraperitoneal (ip) com uma agulha 25 G. Este é o &quot;rato doador&quot;. Coloque o rato infectado em um Specific Pathogen Free (SPF) -cage e alimentação ad libitum. Nota: ratinhos CD1 são uma estirpe não consanguíneos e T. b. infecções brucei GVR35 estão bem caracterizados nesta estirpe. No entanto, este protocolo é igualmente aplicável às estirpes puras ou geneticamente modificados, mas é importante notar que a cor da pele pode ter impacto sobre a sensibilidade da imagem 6,9. </li><li> Monitorar parasitemia periférica por amostragem de sangue. Colocar o rato dentro de um limitador de plástico e levar de 5 ul de sangue a partir da cauda por punção venosa com 21 L de agulha ou flebotomia e misturar 1/10 com cloreto de amónio estéril de 0,85%, para lisar as células vermelhas do sangue e permitir a contagem mais fácil. </li><li> Contar o sangue diluído em um Neubauer hemocitômetro. <ol><li> Carregar o sangue diluído em ambas as câmaras. Contagem de toda a central de grID de uma câmara para dar N tripanossomas. Concentração de tripanossomas sanguíneos em = N x 10 4 / ml tripanossomas, ajustar para qualquer factor de diluição e calcular a média das duas câmaras. Nota: Após cerca de 5-7 dias após a infecção, parasitemia periférica deve subir suficientemente para levar a cabo uma infecção com uma concentração de 2 x 10 4 tripanossomas / ml. </li></ol></li><li> Quando os níveis de tripanossomas são altos o suficiente para o número de ratos necessários, vá para a etapa 1.6. Um nível de parasitémia de 6 x 10 4 tripanossomas / ml em um rato dador é suficiente para a infecção de 10 ratinhos. Nota: Para tamanhos estatisticamente significativas de grupo, n = 6 para cada grupo de tratamento de ratinhos são necessários 8. Para outros modelos de infecção, cálculos de alimentação teria de ser aplicado para determinar tamanhos de grupo apropriados. </li><li> Coloque ratos em uma caixa de calor (a 28-30 ° C durante cerca de 5-10 minutos) para permitir que as veias da cauda para dilatar.Para induzir a anestesia terminal, administrar 20 mg / kg iv pentobarbital com uma agulha 25 G. Confirmar rato é anestesiado, pressionando suavemente as patas para garantir que não haja reflexo de retirada. </li><li> Heparinizar uma agulha G 21, por alíquotas de 5 ml de heparina em 5.000 USP unidades / ml para um tubo de bijou e chupando duas vezes dentro e para fora da agulha ligada a uma seringa de 1 ml. </li><li> Leve o heparinizado 21 G agulha e seringa de 1 ml, e realizar punção cardíaca no rato passagem anestesiado pela inserção da agulha centralmente por baixo da caixa torácica (uma gota de sangue vai reunir na base da agulha) e puxe o êmbolo de modo a não entrar em colapso da câmara do coração. Recolha um mínimo de 0,7 ml de sangue. </li><li> Dilui-se o sangue infectado para produzir um inoculo de 2 x 10 4 tripanossomas / ml em PBS-G a pH 8,0 como passo 1.1 acima. Nota: Neste ponto, o sangue restante pode ser criopreservadas para produzir stabilate por mistura com 8% de glicerol, 20% de Ca fetal inactivado pelo calorsoro lf (oi-FCS) e sangue infectado de 72%. Lentamente congelar a -80 ° C por meio de um processo lento de congelação-congelação recipiente para atingir uma taxa de arrefecimento de 1 ° C / min, antes de transferir para azoto líquido. </li></ol> 2. infecção de camundongos Experimental <ol><li> Coloque CD1 camundongos fêmeas (~ 21-25 g) em uma caixa de aquecimento para gentilmente quente para dilatar veias. Colocar os ratinhos aquecidos em um sistema de retenção de plástico e injecção de 0,2 ml de inoculo diluiu-se por via intravenosa com uma agulha G 25 na veia da cauda, ​​o equivalente a 4.000 tripanossomas por ratinho. Coloque a pressão no local da injecção depois de deter a hemorragia. Nota: infecção intraperitoneal é um percurso válido de infecção e tem sido utilizado em estudos anteriores 10, mas verificou-se esta menos reprodutível do que a via intravenosa. </li><li> Randomize camundongos infectados e gaiola em grupos de 3 em gaiolas SPF-ventilados. Como controle, injetar camundongos fêmeas CD1 com tipo selvagem parasita não bioluminescente, T. b. brucei GVR35 (n =3). </li><li> Monitorar a infecção através do sangue Film Microscopia <ol><li> Spot 5 ul de sangue de punção venosa ou venesection sobre uma lâmina de vidro e efectuar um esfregaço de sangue (utilizando uma segunda lâmina de vidro empurrar suavemente a gota de sangue através da corrediça para produzir uma película fina). Deixar as lâminas secar ao ar. </li><li> Fixar o esfregaço de sangue com 100% de metanol e mancha com Giemsa durante 10 min antes de enxaguar com dH2O e deixar secar ao ar. No âmbito do objectivo 100X contar o número de tripanossomas em 10 campos de visão (FOV). Nota: as filmagens de sangue é realizada juntamente com toda animais imagem não invasivos mais cedo um dia após a infecção. Quando o processamento de um número de animais, este método de quantificação tripanossoma é preferível a contagem em hemocitómetro que as amostras podem ser armazenadas à temperatura ambiente e contadas mais tarde. </li></ol></li></ol> 3. A bioluminescência Imaging para Rastrear Infection Nota: Para controlar a infecção, anim todaal imagem não invasiva pode ser usado. <ol><li> Prepara-se uma solução stock de D-luciferina, o substrato da luciferase do pirilampo, em Dulbecco Salina Tamponada com Fosfato (DPBS) a 30 mg / ml. Filtro de esterilizar e congelar em alíquotas a -20 ° C. Nota: D-luciferina é sensível à luz, portanto, proteger da luz por envolvimento em folha de alíquotas. Não repetidamente congelamento-descongelamento luciferina, pois ele pode afetar a atividade 11. </li><li> Aquecer-se uma alíquota de D-luciferina à temperatura ambiente. Remover cada ratinho a partir da gaiola (incluindo ratinhos de controlo infectadas com o tipo selvagem estirpe nonbioluminescent parasita) e injectar 150 mg / kg de D-luciferina aquecido (diluídas em DPBS) por via intraperitoneal com uma agulha 25 G. Colocar os ratos dentro de uma câmara de isofluorano durante aproximadamente 5 min para induzir a anestesia com 2% de isoflurano / O2 mistura em 2,5 L / min. Nota: Os ratos tornam-se imóvel quando eles estão sob anestesia. Para o gerador de imagens usado aqui, 3 ratinhos pode ser processado de cada vez. Se os ratos são anesthetzado por mais de 30 min, pomada oftálmica lágrima artificial pode ser aplicada para evitar os olhos dos ratos de secar. </li><li> Para o gerador de imagens descrito aqui, abra o software e inicializar a máquina usando o software de acordo com as instruções do fabricante. Adquirir imagens depois da câmera e placa aquecida ter atingido a temperatura. Nota: Este instrumento não é normalmente desligado, permitindo que a máquina para realizar calibrações de fundo durante a noite. No caso em que ele precisa para reiniciar, ele deve executar uma verificação de calibração em primeiro lugar. </li><li> Coloque os ratos anestesiados para o gerador de imagens (ver 3.5). Use grandes separadores negros entre os ratos para minimizar a sangrar através de qualquer sinal bioluminescente brilhante. camundongos de imagem usando um conjunto de tempos de exposição; 1, 3, 10, 30, 60 e 180 segundos, com binning médio, 1 f / stop e um filtro aberto, eo campo de visão E (12,5 x 12,5 cm2). Nota: Os ratinhos de controlo infectadas com o tipo selvagem parasitas GVR35 nonbioluminescent fornecer um backgrvalor bioluminescência ound. A partir de uma experiência cinética de luciferina (Figura 6) com este modelo, verificou-se que os picos do sinal de bioluminescência em 10 minutos após a administração e de imagem demora aproximadamente 5 minutos para 3 ratinhos. Tome este prazo em consideração quando anestesiar e de imagem dos camundongos. </li><li> Para garantir a imagem completa dos ratos, gire para obter um ventral, dorsal e lateral. Manter a consistência da ordem de orientação para a imagem latente entre animais sequenciais. </li><li> Depois de imagem, permitem que os ratos se recuperar da anestesia sob observação antes de retornar a SPF-gaiola. </li><li> Continue imagiologia ratos a cada 7 dias até o dia 21 pós-infecção. Deveria haver um aumento constante no sinal de bioluminescência ao longo de todo o animal. </li><li> No D21, imagem e filme de sangue dos ratos, como descrito acima, e dose de ratos com 40 mg / kg diminazeno aceturate intraperitoneal. Esta droga irá limpar a parasitemia periférica, mas não vai atravessar a barreira sangue-braibarreira n, e, portanto, permite uma melhor visualização da infecção do SNC. Nota: Diminazena aceturate é uma droga bem estabelecida usados ​​para tratar em estágio inicial tripanossomíase animal. </li><li> Continue imagem e monitoramento em D28 e D35. </li></ol> 4. Confirmando CNS Infection <ol><li> No D35, os ratinhos da imagem, como especificado acima (passos 3,1-3,4). </li><li> Após a imagiologia, permitir que os ratinhos recuperar da anestesia e ratinhos desangrar como detalhado no passo 1,6-1,8, recolha de sangue para posterior análise. </li><li> Após a punção cardíaca, perfundir ratos com 20 ml de PBS (opcional: adicionar 3 mg / ml de luciferina a solução de perfusão) através do coração para limpar o sangue dos órgãos e para reintroduzir etiqueta luciferina que pode ter afastada da região do cérebro ao longo do intervalo de tempo a partir do passo 4.1. </li><li> Suavemente remover o cérebro, usando tesoura curva, cortando a partir da base à volta da circunferência do crânio, e levantando a parte superior do crânio, para permitir que o cérebropara ser suavemente retirado com pinças curvas. </li><li> Colocar o cérebro excisada para uma secção de plástico preto e pipeta 50 ul de 15 mg / ml de luciferina sobre o órgão antes da imagem, para garantir luciferina adequada foi adicionado à cerebral excisado. Imagem usando as configurações acima. Isto irá demonstrar a localização da infecção para os hemisférios cerebrais. Nota: A jusante quantificação da parasitemia no cérebro excisado pode ser levada a cabo por qPCR como anteriormente descrito 8. Outras aplicações a jusante alternativos podem incluir histologia ou imuno-histoquímica para identificar parasitas no tecido cerebral após a fixação. </li></ol> 5. A quantificação de bioluminescência de imagem Nota: A bioluminescência pode ser quantificada utilizando-se a região de interesse (ROI) com o software de imagem e corrigido para o fundo bioluminescência. <ol><li> Usando a ferramenta de ROI, criar um ROI rectangular para toda animais quantificação umand posicioná-lo sobre a imagem do mouse para cobrir ponta do nariz à base da cauda do rato. Usar a mesma caixa de tamanho para cada animal e cada ponto de tempo. Ao olhar para o ROI para o cérebro, use um ROI circular da mesma forma, posicionando-o sobre a região da cabeça do rato na imagem. </li><li> Se desejar, ajuste as imagens apareçam no mesmo espectro para uma representação visual da infecção. Nota: O software utilizado aqui gera um mapa de cores para cada imagem ajustada automaticamente para a contagem mínima e máxima de fótons. Para permitir a comparação visual entre uma série de imagens, a escala de cores deve ser ajustado para os mesmos valores para todas as imagens. <ol><li> Usando a imagem com a maior bioluminescência e na guia &#39;Ajuste de imagem &quot;na paleta de ferramentas, selecione uma escala logarítmica e um mínimo de escala de cores adequado e máximo (a ser determinado empiricamente). Aplicar esta escala a todas as imagens durante todo o experimento. </li></ol></li></ol>

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The authors have nothing to disclose.

O desenvolvimento de um T. bioluminescente b. brucei GVR35 estirpe permite a visualização de uma infecção por tripanossoma do início ao final de estágio. Modelos de infecção anteriores não foram capazes de detectar a fase tardia, quando os parasitas estão no cérebro, em tempo real, a partir de microscopia de esfregaço de sangue, e exigia o abate e remoção dos cérebros dos ratos infectados para determinar a carga parasitária 12. A bioluminescência reduz a variabilidade inter-...

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.

Este protocolo demonstra como seguir a progressão da doença após a infecção de camundongos com T. b. brucei, um modelo para a tripanossomíase humana Africano. A Figura 1 mostra a linha do tempo do protocolo experimental, demonstrando o calendário de tratamento e de imagem etapas. A Figura 2 mostra um típico campo de visão em um esfregaço de sangue Giemsa-manchadas fixa usada para quantificar parasitemia periférica, com tripanossomas e...

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.

Bioluminescência de imagem para detectar a infecção fase tardia Africano Tripanossomíase

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 visualizações.  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.

Vídeo: 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 de imagens de parasitas Leishmania transgênicas em um host ao vivo

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

Imunologia e Infecção

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.

Monitoramento Laboratório abrangente e Custo Efetivo de HIV / AIDS: um Modelo de Papel Africano

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

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

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

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

Imagem de bioluminescência de NADPH oxidase em diferentes modelos animais

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

Bioluminescent Bacterial Imaging In Vivo

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

Bioluminescent Bacterial Imaging In Vivo

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

Imagem bacteriana Bioluminescent<em&gt; Em Vivo</em

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

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

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

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

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

In Vivo Imaging Systems Detecção (IVIS) de um vírus de Neuro-invasiva encefalítico

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

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

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

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

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

Separação de<em&gt; Plasmodium falciparum</em&gt; Fase Tardia eritrócitos infectados por meio magnético

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

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

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

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

Utilizando Proteínas Fluorescentes para monitorar glicossomo Dynamics do Trypanosoma Africano

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

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

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

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

Imagem de bioluminescência dupla de progressão tumoral e angiogênese

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

Medicina

Protocolo para Avaliação de Medicamentos para Modelo Bioluminescente In Vivo da Doença de Chagas

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

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

Autor em destaque: monitoramento em tempo real da carga parasitária e da resposta do hospedeiro

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

Desmistificando imagens de bioluminescência in vivo de um modelo de camundongo com doença de Chagas para estudos de eficácia de medicamentos

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

Bioluminescência de imagem para detectar a infecção fase tardia Africano Tripanossomíase

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