Esta pesquisa foi financiada pelo NIH concede AI062732 K08, K12 HD001401 e 1UL1RR024139-02 e uma criança Grant Pesquisa em Saúde da Fundação Charles H. capa para SB-M. e pela Fundação de Pesquisa da Universidade Estadual de Nova York em Stony Brook.

1. EBV preparação de <ol><li> Subcultura crescimento exponencial B95-8 células (ATCC # CRL 1612, 13) a 3 x 10 5 células / ml em um frasco de 75 centímetros de cultura de tecido usando a técnica de dois estéril. As células são cultivadas em RPMI 1640 completo contendo inativado pelo calor 10% de soro fetal bovino (FBS), Penicilina / Estreptomicina em 100U/ml e 100 mg / ml, respectivamente, e anfotericina B, 0,5 mg / ml a 37 ° C na presença de 5% de CO 2. </li><li> Quarenta e oito horas depois, as células ressuspender em fresco RPMI completo 1640 em 1 x 10 6 células / ml. Para induzir a produção de vírus, estimulam as células com acetato de forbol tetradecanoyl 20ng/mL (TPA) por 1 hora em um padrão de incubadora de CO 2. Lave as células três vezes com RPMI 1640 para remover TPA. </li><li> Ressuspender as células do volume original de RPMI completo 1640 (de 1,2) e colocar o balão na incubadora de CO 2 por 96 horas. Este método tem sido mostrado para produzir títulos elevados de vírus infeccioso partigos 6. </li><li> Centrifugar a 600 x g por 10 min a 4 ° C para separar EBV contendo sobrenadante de cultura de células. Sobrenadante do filtro através de um filtro de 0,45 micron, alíquota, e armazenar a -70 ° C por mais de um ano. Uma alternativa é obter o sobrenadante de B95-8 células contendo EBV da ATCC (VR-1492) e uso na diluição recomendada pelo ATCC. </li></ol> 2. Isolamento de células mononucleares do sangue periférico (PBMC) <ol><li> Draw 10 ml de sangue do dador para uma seringa heparinizada ou um tubo de sangue heparinizado. Diluir 20 ml de sangue com PBS em temperatura ambiente em um tubo de 50 ml. </li><li> Underlay sangue diluído com 15 Ficoll Hypaque ml de meio de separação de linfócitos. Centrifugar a 225 x g sem freio em temperatura ambiente por 30 minutos. </li><li> Remover buffy coat e transferência para um novo tubo cônico de 50 ml. Aumentar o volume para 50 ml com PBS e girar a 600 xg à temperatura ambiente por 10 minutos. </li><li> Pour fora o sobrenadante. Lave as células pela ressuspensão pellet em 50 ml de PBS e centrifugação a 600 x g à temperatura ambiente por 10 minutos. Lavar duas vezes mais de uma maneira similar. </li><li> Ressuspender as células lavadas em 1 ml de RPMI completo. Use 5 mL de células para preparar uma diluição 1 / 10 em Trypan azul. Contagem de células vivas usando um hemocitômetro. </li><li> Ajustar o volume usando RPMI completo em um frasco de 25 centímetros de cultura de tecido 2 para obter uma concentração de células de 2 x 10 6 / ml. </li></ol> 3. Infecção com EBV <ol><li> Adicionar FK506 (AG Scientific) para a suspensão de células de 2,6 para uma concentração final de 20 nM. Frasco lugar na incubadora de CO 2 a 37 ° C durante uma hora. </li><li> Descongelar rapidamente uma alíquota de EBV. Remover balão de incubadora e adicione EBV para as células em 10/01 diluição. Tipicamente, esta diluição oferece uma MOI de 50-100. Frasco para misturar e coloque-o na posição vertical em incubadora de CO 2 a 37 ° C. </li></ol> ª notana Etapa 4, realizada para prever o resultado bem-sucedido, é uma etapa opcional. Passo 4 Se é para ser realizado, você vai precisar para incubar um frasco adicional de não-infectados PBMC de 2 x 10 6 células / ml como controle. 4. Identificação da população de células em proliferação (etapa opcional) <ol><li> Prepare FACS buffer: PBS + 5% de SFB. Armazenar a 4 ° C. </li><li> Tornar o anticorpo seguinte mistura em 50 mL de volume para cada coloração de células no Passo 4.6. Diluições devem ser feitas na FACS tampão contendo 1mg/ml de rato IgG (Sigma) para inibir a ligação não específica de anticorpos. <ol class="lroman"><li> single-coradas com PE de anticorpos anti-CD23 conjugado (1 / 50 de diluição) </li><li> single-coradas com FITC anticorpo anti-CD58 conjugado (1 / 50 de diluição) </li><li> single-coradas com PE-Cy5 anticorpo anti-CD19 conjugado (1 / 50 de diluição) </li><li> triple-coloração para CD23, CD58, e CD19 (1 / 50 cada diluição) </li><li> single-coradas com conjugado PE controle isotipoanticorpos combinados para CD23-PE (na mesma concentração de anticorpos anti-CD23) </li><li> single-coradas com anticorpo controle conjugado FITC isotipo combinados para CD58-FITC (na mesma concentração de anticorpos anti-CD58) </li><li> single-coradas com PE-Cy5 anticorpo controle isotipo conjugados combinados para CD19-PE-Cy5 (na mesma concentração de anticorpos anti-CD19) </li><li> triple-coradas com os três anticorpos isotipo controle. </li></ol> Armazenar temporariamente as misturas a 4 ° C no escuro. </li><li> Em três dias pós-exposição ao EBV, gentilmente frasco agite para obter uma suspensão celular mais uniforme. Remover 2ml de balão para um tubo Eppendorf de 2ml. Tubo giratório em uma microcentrífuga a 3000 rpm em temperatura ambiente por 3 minutos. </li><li> Aspirar o sobrenadante e ressuspender as células em tampão FACS, 5 x 10 6 a 1 x 10 7 células / ml. </li><li> Remover oito alíquotas 50μl em tubos Eppendorf individuais ou uma placa de 96 poços V-fundo. Tubos de rotação ou a placa a 1500 x gpor 3 minutos. </li><li> Descartar o sobrenadante e os tubos vortex / placa. Ressuspender as células em cada tubo / poço em 50 mL de tampão FACS contendo diluições apropriadas preparadas em 4.2. </li><li> Incubar os tubos / placa de gelo no escuro por 30 minutos. Células Centrifugar a 3000 rpm por 3 minutos, retire o sobrenadante e adicionar 200 mL de tampão FACS. Centrifugar as células novamente e remover o sobrenadante para completar a lavagem. Repita mais duas lavagens. </li><li> Ressuspender as células em 200 mL de buffer FACS e aquisição de dados usando um citômetro de fluxo, a aquisição de 20 mil eventos / tubo. </li><li> Analisar os dados usando WinMDI (freeware para FACS análise de dados no PC). Portão em células vivas usando perfis de dispersão para a frente e lateral. Então, as células portão ao vivo para a expressão de CD19 + (marcador de células B) depois de comparar com as células coradas com anticorpos isotipo controle pareado para anticorpos anti-CD19. Plot CD19 + B ao vivo células com intensidade de fluorescência de CD23 em eixo-x e intensidade de fluorescência de CD58 em eixo y. Determine CD23 + e CD58 + células, comparando a EBV-expostos de células marcadas com anticorpos isotipo controle pareado. Presença de CD23 + CD58 oi células EBV-expostos cultura (Figura 2C) prevê crescimento bem sucedida de EBV-infectados crescimento das células transformadas. Em contraste, CD23 + células CD58 oi não surgem quando as células não estão expostos a EBV (Figura 2A) ou exposto a uma cepa não imortalizando de EBV (Figura 2B). CD23 + células CD58 oi têm sido experimentalmente demonstrado submeter-se a proliferação e, posteriormente, estabelecer LCL (14). </li></ol> 5. Expansão e criopreservação de LCL <ol><li> Visualização de células por microscopia de luz: Por uma semana após a infecção EBV, grupos de células são visíveis por microscopia de luz. A Figura 3 mostra um exemplo dos primeiros aglomerados microscópicos (Figura 3B) num balão. </li><li> Como o tempo passa, tornam-se maiores aglomerados microscópicos de tal forma que aglomerados são visíveismacroscopicamente no frasco. Figura 3C mostra os clusters maior de células em LCL estabelecidos pela microscopia de luz. </li><li> Alimentando células: o dobro do volume de meio de cultura no frasco de cultura no dia 12. Posteriormente, expandir a cultura, aumentando seu volume de 2-3 vezes usando RPMI completo. </li><li> Periodicidade das células de alimentação deve ser determinado com base na taxa de crescimento celular. Quando o meio de cultura torna-se amarelo, geralmente é hora de substituir a mídia como acima. Normalmente, isso ocorre uma vez por semana. No entanto, algumas linhagens de células podem precisar ser alimentados mais ou menos freqüência. Expandir a cultura de 75-100 ml ao longo das próximas semanas. </li><li> Criopreservação: Quando o congelamento até as células, as células de centrifugação a 600 x g por 10 minutos em temperatura ambiente. Remover o sobrenadante e ressuspender pellet celular em meio de muito frio contendo 90% FBS e dimetilsulfóxido 10% no 1 x 10 7 células / ml. Coloque o tubo em nitrogênio líquido. </li></ol> 6. Resultados representativos: Resultado bem-sucedido é previsto pela presença de CD23 + CD58 oi células. Cerca de 2-3% de células vivas no dia 04/03 após a exposição ao EBV deve ter este perfil (Figura 2C). Outras sub-populações de CD23 +, CD23 -, CD58 +, e CD58 - células observadas após a exposição ao EBV mostram proliferação mínima 14. Un células infectadas (Figura 2A) e células expostas a EBV de HH514-16 células (Figura 2B), abrigando uma cepa não imortalizando de EBV, não demonstram CD23 CD58 + células oi. Você também pode visualizar as células por microscopia óptica para monitorar êxito: Como mencionado anteriormente, dentro de uma semana após a infecção EBV, pequenos aglomerados de células no frasco são visíveis por microscopia de luz (Figura 3B). Enquanto o tempo progride e as células se desenvolvem em LCL, clusters microscópicas tornam-se maiores (Figura 3C) de tal forma que aglomerados são visíveis macroscopicamente no frasco. Figura 1. Fluxo de Trabalho para a geração e criopreservação de linhagens de células linfoblastóides. Sangue periférico é centrifugada por um gradiente de Ficoll. PBMC presentes no revestimento buffy de um gradiente estabelecidas estão expostos a FK506 seguido por adição de EBV. EBV-expuseram as células são cultivadas a 37 ° C na presença de 5% de CO 2 para estabelecer e, posteriormente, expandir LCL para criopreservação. <img alt="Figura 2" src="/files/ftp_upload/3321/3321fig2.jpg" /> Figura 2. Identificação de um sub-população de células B deverá submeter-se a proliferação após a exposição ao EBV. Un-infectados PBMC (A) ou PBMC expostos a EBV derivadas de células HH514-16 (B) ou de células B95-8 (C) na presença de FK506 foram colhidas no dia 3. Células foram marcadas com fluorocromo-anticorpos dirigidos contra CD19, CD23, CD58 e. Depois de chaveamento em células vivass, un-infectados (A) e EBV-exposed (B e C) CD19 + células B foram examinados para a expressão de CD23 e CD58. A sub-população de células B expressando níveis de CD58 e alta de CD23 (CD23 oi CD58 +), observada apenas nas células expostas à transformação competentes EBV (derivado de B95-8 células), é descrito. <img alt="Figura 3" src="/files/ftp_upload/3321/3321fig3.jpg" /> Figura 3. Visualization dos ninhos de células em células infectadas pelo EBV-. PBMC foram tratados com FK506 e infectados com EBV. Un-infectados (A) e EBV-exposed (B) as células foram examinados uma semana após a infecção por microscopia de contraste de fase (aumento de 10x). Cinco semanas de idade linfoblastóides linha celular é mostrado na C.

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O método descrito neste trabalho gera LCL de doadores de sangue periférico com imortalização rápida e os tempos de criopreservação. Através do uso de FK506, uma célula T imunossupressoras, e altos títulos de vírus infecciosos que somos capazes de promover a proliferação de células infectadas pelo EBV B a partir de células mononucleares do sangue periférico. Estas intervenções fazem o método descrito mais eficiente, resultando em rápida expansão das células para os experimentos subseqüentes. Tradicionalmente, a transformação de crescimento tem sido monitorado através da visualização de aglomerados de células por microscopia de luz cerca de uma semana após a exposição ao EBV 6, 15. No entanto, o agrupamento de células não é um indicador específico de EBV-mediada transformação de crescimento. Temos demonstrado anteriormente identificação consistente da população de proliferação celular através de citometria de fluxo 14, fornecendo um método preciso e específico para determinar sucesso tão cedo quanto três dias à réer a exposição de células B para EBV.

<table border="1"><tbody><tr><td></td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Anfotericina B </td><td> Fisher Bioreagents </td><td> BP 928-250 </td></tr><tr><td> Dimetilsulfóxido </td><td> Sigma </td><td> D 2650 </td></tr><tr><td> Soro fetal bovino, inativado pelo calor </td><td> Sigma </td><td> F 4135 </td></tr><tr><td> Ficoll Hypaque media separação de linfócitos </td><td> Mediatech </td><td> 25-072 </td></tr><tr><td> FK506 </td><td> AG Scientific </td><td> F 1030 </td></tr><tr><td> IgG de soro de rato </td><td> Sigma </td><td> I 8765 </td></tr><tr><td> Rato anti-CD23 humano conjugado com PE </td><td> BD PharMingen </td><td> 555711 </td></tr><tr><td> Isotipo IgG1 rato conjugado com PE </td><td> BD PharMingen </td><td> 554680 </td></tr><tr><td> Rato anti-CD58 humano conjugado com FITC </td><td> Pierce Thermo Scientific </td><td> MA1-82159 </td></tr><tr><td> Isotipo IgG1 rato conjugado com FITC </td><td> BD PharMingen </td><td> 550616 </td></tr><tr><td> Rato anti-CD19 humano conjugado com PE-Cy5 </td><td> BD PharMingen </td><td> 555414 </td></tr><tr><td> Isotipo IgG1 rato conjugado com PE-Cy5 </td><td> Dako </td><td> X 0955 </td></tr><tr><td> Penicilina / estreptomicina </td><td> Gibco </td><td> 15140-122 </td></tr><tr><td> RPMI 1640 media </td><td> Sigma </td><td> R 8758 </td></tr><tr><td> Acetato de forbol Tetradecanoyl (TPA) </td><td> Calbiochem </td><td> 407952 </td></tr><tr><td> FACS Buffer (Phosphate Buffered Saline 1X + 5% de soro fetal bovino) </td><td></td><td></td></tr></tbody></table>

establishment of epstein-barr virus growth-transformed lymphoblastoid cell lines

Infecção de células B com vírus Epstein-Barr (EBV) leva à imortalização proliferação e subseqüentes, resultando no estabelecimento de linhas de células linfoblastóides (LCL) in vitro. Desde LCL estão latentes infectadas com EBV, eles fornecem um sistema modelo para investigar a latência e EBV-vírus impulsionado a proliferação de células B e tumorigênese 1. LCL têm sido utilizados para apresentar antígenos em uma variedade de testes imunológicos 2, 3. Além disso, LCL pode ser usado para gerar anticorpos monoclonais humanos 4, 5 e fornecer uma fonte potencialmente ilimitada quando o acesso a materiais biológicos primária é limitada 6, 7. Uma variedade de métodos têm sido descritos para gerar LCL. Métodos anteriores incluíram o uso de mitógenos, como fitohemaglutinina, lipopolissacarídeo 8 e 9 pokeweed mitógeno para aumentar a eficiência da EBV-mediada imortalização. Mais recentemente, outros autores utilizaram o immunosuppragentes essive como a ciclosporina A para inibir a morte celular mediada de células T infectadas B 7, 10-12. O período de tempo considerável da infecção EBV para o estabelecimento de linhas celulares impulsiona a necessidade de métodos mais rápidos e mais confiáveis ​​para EBV-driven de transformação do crescimento de células B. Usando uma combinação de alta título EBV e um agente imunossupressor, somos capazes de infectar consistentemente, transformar e gerar a partir de células LCL B no sangue periférico. Este método utiliza uma pequena quantidade de células mononucleares do sangue periférico, que são infectados em clusters in vitro de células pode ser demonstrada. A presença de CD23 com EBV, na presença de FK506, uma célula T imunossupressoras. Tradicionalmente, o crescimento de células B proliferam é monitorado através da visualização de aglomerados de células microscópicas cerca de uma semana após a infecção com EBV. Aglomerados de LCL pode ser visto a olho nu depois de várias semanas. Nós descrevemos um ensaio para determinar cedo se EBV mediada gCRESCIMENTO transformação for bem sucedida, mesmo antes aglomerados microscópico das células pode ser demonstrada. A presença de CD23 + células CD58 oi observados logo aos três dias pós-infecção indica um resultado positivo.

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Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Nós descrevemos um método para gerar linhas B transformada célula usando vírus Epstein-Barr. Nós também ilustrar um ensaio de romance que pode identificar as células B destinados a sofrer transformações logo aos três dias após a infecção.

Estabelecimento de Epstein-Barr Virus Crescimento transformada linhagens de células linfoblastóides

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

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Immunology and Infection

39.8K visualizações.  State University of New York at Stony Brook. Nós descrevemos um método para gerar linhas B transformada célula usando vírus Epstein-Barr. Nós também ilustrar um ensaio de romance que pode identificar as células B destinados a sofrer transformações logo aos três dias após a infecção.

Vídeo: Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

Derivação de Tímico linfoma de células T Linhas de Atm - / -</sup E<emP53> - / -</sup Ratos

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

Imunologia e Infecção

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Expandindo linfócitos T citotóxicos do sangue do cordão umbilical que citomegalovírus Target, vírus de Epstein-Barr, e Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

De células vivas de Alphaherpes Vírus Transporte anterógrada e Spread

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Avaliação da eficácia e toxicidade de RNAs Segmentação HIV-1 Produção para uso em Gene ou Quimioterapia

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

Um glóbulo vermelho simples método de lise para o estabelecimento de linhas celulares linfoblastóides B

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Um método simples e eficiente para estabelecer linhas de células T de pacientes com infecção crônica ativa vírus Epstein - Barr e NK

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identificação de codificação e Classes de RNA não-codificante expressa em suínos, sangue total

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Estabelecimento de infecção Viral e análise da interação vírus-hospedeiro em Drosophila Melanogaster.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Teste in vitro ELISA para avaliar a potência da vacina antirrábica

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...