Agradecemos Jessica Jones, Erica Clark, Constance Ducar, Donna Smith, Roy Lewis, Lily Apedaile, Joanne Wiesner, Devin Adams, Corey McBain e Stephen Voght para a sua assistência no desenvolvimento deste método manuscrito, e vídeo. Este trabalho foi financiado pela Fundação Bill e Melinda Gates CAVD concessão 38.645 (MJM) e do Instituto Nacional de Saúde concede UM1 AI068618 e U01 AI069481 (MJM). EA-N. é suportado pelo NIH Grant T32 AI007140. Agradecemos a confiança James B. Pendleton Charitable por sua doação de equipamentos generoso.

Nota: Para proteger os anticorpos fluoróforo conjugados da luz, executar todas as etapas de um gabinete de segurança bio-com a luz. 1. Anticorpo Preparação Painel de coloração <ol><li> O painel de coloração de anticorpos podem ser encontrados na Tabela 1. Concentração de anticorpos deve ser definido por meio de titulação com sangue total e no uso do equipamento de citometria de fluxo e mesmo procedimentos que serão utilizados para adquirir as amostras coradas fenotípicos. </li><li> Uma vez que os títulos de coloração apropriados são determinados, combinar todos os anticorpos para uma mistura única de um tubo de bloqueio-cap. Adicionar tampão de fluxo de lavagem (PBS de Dulbecco, com 2% de calor de soro bovino fetal inactivado), para perfazer um volume total de 100 ul. Dimensionar a mistura para o número de amostras a serem corados. Esta mistura pode ser armazenada a 4 ° C durante até oito semanas. </li></ol> 2. Coloração <ol><li> Se o sangue recolhido é para ser usado para outros fins em addição para este ensaio, defina uma alíquota de lado enquanto os mais sensíveis ao tempo procedimentos são realizados no sangue restante. A alíquota pode ser armazenado à temperatura ambiente durante até 4 horas após a punção venosa sem perda significativa de células. </li><li> Verificar se existe um grânulo intacto pellet no fundo do tubo e Trucount rótulo do tubo para identificar a amostra que está sendo manchadas. Em ensaios clínicos da HVTN, o Laboratório de Sistema de Gerenciamento de Dados (Frontier Ciência e Tecnologia Research Foundation; Amherst, Nova Iorque) é usado para rotular e rastrear amostras coradas. </li><li> Anote os números de lote e datas de vencimento de todos os reagentes. Anote o número de contagem Trucount tubo talão fornecido pelo fabricante no saco de tubos, certifique-se que o número de lote no saco corresponde ao número de lote no tubo. </li><li> Usar a reversão de pipetagem de precisão pipetar 100 uL de sangue total para dentro do tubo Trucount, logo acima do retentor metálico. Evitar manchas de sangue para o lado do tubo. </li><li&gt; Usando técnica (para a frente) regular pipetagem, pipeta 100 uL do painel misto anticorpos (ver Tabela 1) para dentro do tubo Trucount. Tapar o tubo e vórtice a baixa velocidade durante cerca de 15 segundos para misturar. Inspeccionar visualmente o tubo para assegurar que o sedimento do grânulo é completamente dissolvida. </li><li> Incubar o tubo Trucount durante 15 minutos à temperatura ambiente (15-30 ° C), no escuro. </li><li> Se necessário, diluem-se uma alíquota de 10 × solução de lise FACS para 1X utilizando dih 2 O. Adicionar 900 ul de 1 × solução de lise FACS para o tubo. </li><li> Tapar o tubo e agitar no vortex cuidadosamente a baixa velocidade durante cerca de 15 segundos para misturar. Empurre a tampa para baixo firmemente em posição de bloqueio no tubo e vedação com filme de laboratório. </li><li> Armazenar o tubo a -65 ° C a -95 ° C até que a amostra está pronta para a transferência para o laboratório de análise central ou para análise em casa. As amostras são estáveis, nesta fase, durante pelo menos quatro semanas. Se o transporte ou ensaio imediatamente, este passo pode ser omitido. </li></ol> 3. Expedição Nota: As instruções a seguir utilizam um sistema de transporte isolado de Inc., Saf-T-Pak projetado especificamente para transporte de categoria B isentos substâncias biológicas de acordo com a International Air Transport Association e (IATA) regulamentos. Se analisar as amostras no mesmo local que a coloração ocorreu, vá para a seção 4. <ol><li> As amostras podem ser fornecidas imediatamente após a coloração ou depois de terem sido congeladas a -65 ° C a -95 ° C. Enrole cada tubo completamente em papel alumínio e coloque na caixa de espécime manchado. Coloque a caixa de espécime manchada dentro de um saco estanque-poli com material absorvente. </li><li> Colocar o saco à prova de fugas e de poli-conteúdo dentro de um saco Tyvek e vedação com o ar o menos possível no saco. </li><li> Coloque o pacote de amostra (amostra dentro de embalagem secundária) dentro da caixa interna marrom. </li><li> Coloque o interior marrom bboi dentro do peito de isopor, prendendo-o no recuo para evitar deslocamento. </li><li> Encha o peito de isopor com gelo seco (cerca de 8 kg) e coloque a tampa firmemente no peito. </li><li> Segurança fita da caixa de transporte e navio como substância biológica, Categoria B (UN3373) com o gelo seco adequada (UN1845) marcações; seguir IATA PI-650 instruções. </li><li> Após a recepção, as amostras são armazenadas a -65 ° C a -95 ° C até serem analisadas. </li></ol> 4. Descongelamento e Análise por Citometria de Fluxo <ol><li> Retirar a amostra corada do congelador a descongelar à temperatura ambiente, no escuro, antes de recolher no citómetro de fluxo. Se a recolha de dados sobre mais do que uma amostra, padronizar o processo para todos os tubos por escalonamento descongelamento para que os tubos não estão sentados à temperatura ambiente durante mais de 1 hora cada. </li><li> As amostras devem ser obtidas utilizando um citómetro de fluxo de quatro a laser equipado com filtros adequados, tais como o LSR BDII. Utilize calibração citómetro padrão e métodos de compensação de fluorescência para coleta de dados 4. </li></ol> Nota: Não defina limites de dispersão de dispersão para a frente ou de lado durante a coleta de 5. Contas Trucount podem cair abaixo a configuração do limite mais baixo possível para estes parâmetros, causando um subconjunto de contas não devem ser contabilizados durante a análise. Se exigido pelo instrumento, para definir um limite, defina o menor possível Am Ciano limiar de canal. Porque leucócitos CD45 + coradas com o painel será Am Ciano positivo, e os grânulos Trucount também fluorescem no Ciano Am canal, este deve permitir a todos os dados relevantes para ser adequadamente recolhidos. <ol start="3"><li> Vortex do tubo de amostra durante 5 segundos antes de o colocar no citómetro de fluxo. Contas Trucount fluorescência altamente em muitos canais. Portão as contas durante a coleta, encontrando a população que é altamente positivo para o duplo-PE Cy5 e APC (o colo doisrs que mais facilmente distinguir os grânulos das células pode variar, dependendo de instrumentação). Selecione a porta talão como seu portão parar, e registro de dados até pelo menos 20.000 contas são adquiridos. </li><li> Analisar os dados utilizando software apropriado, tal como FlowJo (Treestar; Ashland, OR). Figura 1 mostra o esquema de propagação utilizado para a análise de diferentes populações de leucócitos a partir de um desenho representativo de controlo do sangue. </li></ol> 5. Cálculos Trucount <ol><li> Cada tubo contém uma Trucount pellet liofilizado de partículas fluorescentes. Após a adição de líquido para o tubo e agitação em vórtex, as pérolas devem ser distribuídos igualmente ao longo da amostra. O número de pérolas na pelete varia ligeiramente, o número de lote e podem ser encontrados sobre o saco de armazenamento para os tubos. </li><li> Portão das pérolas Trucount e populações de células, como mostrado na Figura 1 para determinar as contagens de eventos para cada população. Comparando o número de eventos in o portão do talão, o número total de pérolas originalmente no tubo lhe permitirá determinar a proporção da amostra recolhida, a qual pode então ser usada para determinar a concentração absoluta (isto é, células / uL) para cada população. A equação que se segue pode ser utilizado para este fim: concentração celular (# de células / uL de sangue total) = [# eventos / população (eventos # / # grânulo grânulos totais em pellet)] / 100 uL. </li></ol> 6. Resultados representativos <img src="/files/ftp_upload/4302/4302fig1.jpg" alt="Figura 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/4302/4302fig1highres.jpg" /> Figura esquema Gating 1. Utilizado para a análise de grandes populações de leucócitos que mostram dados representativos de um voluntário saudável. A) grânulos Trucount (i) são fechado e excluído a partir de células. Granulócitos (ii) são delineadas e lympohcytes e monócitos são divididos em 3 popluations: CD14linfócitos negativos (iii), todas as células CD14 negativas (iv), e não os linfócitos (V). B) CD14 linfócitos negativos são gated para distinguir células T CD4 + (VI), CD8 + T (vii), células B (VIII), CD56 brilhantes células NK (ix), células CD56 dim NK (x), e CD56 negativas células NK (xi). C) Todos células CD14 negativas são gated para distinguir mielóide (xii) e (xiii células plasmocitóide) dendríticas. D) Não são lympocytes fechado para distinguir não-clássicas (xiv), intermediários (xv) e clássica (xvi) monócitos. <a href="http://www.jove.com/files/ftp_upload/4302/4302fig1large.jpg" target="_blank">Clique aqui para ver maior figura</a> . Mais subconjuntos de células específicas, que não são mostradas na figura 1 (por exemplo, as células NKT ou neutrófilos) também pode ser distinguido através do painel são apresentadas, e o esquema de gating pode ser ampliado ou modificado para satisfazer as necessidades específicas de estudo. Gating passos certos mostrados são exclusivas para este método. Da nota particular, um portão de inclusão eportão exclusão são desenhados em torno das esferas de Trucount e colocado em cima da outra, de um a porta para as pérolas de contagem, e um para excluir os grânulos a partir da análise celular (Figura 1A). Além disso, como os linfócitos, monócitos, granulócitos e não são tão facilmente distinguidos no sangue total como o são em células mononucleares do sangue periférico por dispersão frontal e lateral de dispersão, gating estas células CD45 usando expressão dispersão lateral e é muitas vezes necessário (Figura 1A). Granulócitos contaminantes (círculos) que não podem ser separados a partir de linfócitos e monócitos CD45 e usando dispersão lateral são distinguíveis em algumas parcelas, pela sua expressão de alto CD16 (Figura 1C e Figura 1D). O número de granulócitos contaminantes é normalmente pequeno, e que não interfiram com a propagação de células de monócitos e células NK.

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Neste relatório, apresentamos um método baseado em talão para enumerar populações de leucócitos no sangue total fresco por citometria de fluxo e cobrir os parâmetros necessários para a sua utilização em um ensaio clínico multicêntrico com a análise da amostra centralizado. Este método baseia-se e otimiza o BD protocolo Trucount e permite seu uso confiável em um ambiente de estudo clínico multicêntrico. O método de coloração é simples e leva cerca de 45 minutos para ser realizado, tornando viável pa...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Trucount absolutos Tubos Contando </td><td> BD Biosciences </td><td> 340334 </td></tr><tr><td> FACS 10X de Lise Solução </td><td> BD Biosciences </td><td> 349202 </td></tr><tr><td> Categoria B &amp; Sistema de envio Isento, Duplas </td><td> Saf-T-Pak </td><td> STP-320 </td></tr><tr><td> CD45 anticorpo monoclonal AmCyan </td><td> BD Biosciences </td><td> 339192 </td></tr><tr><td> CD3 anticorpo monoclonal FITC </td><td> BD Biosciences </td><td> 349201 </td></tr><tr><td> CD8 PerCP-Cy 5,5 de anticorpo monoclonal </td><td> BD Biosciences </td><td> 341051 </td></tr><tr><td> CD4 Alexa Fluor 700 Antibo monoclonaldy </td><td> BD Biosciences </td><td> 557922 </td></tr><tr><td> HLA-DR anticorpo monoclonal ECD </td><td> Beckman Coulter </td><td> IM3636 </td></tr><tr><td> CD14 anticorpo monoclonal V450 </td><td> BD Biosciences </td><td> 560349 </td></tr><tr><td> CD19 anticorpo monoclonal PE </td><td> BD Biosciences </td><td> 555413 </td></tr><tr><td> CD16 anticorpo monoclonal APC-H7 </td><td> BD Biosciences </td><td> 560195 </td></tr><tr><td> CD56-PE Cy7 anticorpo monoclonal </td><td> BD Biosciences </td><td> 335791 </td></tr><tr><td> CD11c APC anticorpo monoclonal </td><td> BD Biosciences </td><td> 559877 </td></tr><tr><td> CD123 anticorpo monoclonal PE-Cy5 </td><td> BD Biosciences </td><td> 551065 </td></tr></tbody></table>

enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay

Criopreservação de leucócitos do sangue periférico é amplamente utilizado para preservação de células para a avaliação da resposta imune, em ensaios clínicos, e oferece muitas vantagens para facilidade e normalização das avaliações imunológicos, mas os efeitos prejudiciais deste processo têm sido observados em alguns subconjuntos de células, tais como granulócitos, células B , células dendríticas e 1-3. Ensaio leucócitos frescos dá uma visão mais precisa do estado in vivo das células, mas é muitas vezes difícil de realizar, no contexto de grandes estudos clínicos. Ensaios com células frescas são dependentes de compromissos voluntários e prazos e, se demorado, sua aplicação pode ser impraticável devido às horas de trabalho necessárias do pessoal de laboratório. Além disso, quando os ensaios são realizados em centros múltiplos, laboratórios com os recursos e treinamento necessários para realizar os ensaios não podem estar localizados em proximidade suficiente para locais clínicos. Para resolver estas questões, temos develvolvido um 11-cor do painel de coloração de anticorpo que pode ser utilizado com tubos de Trucount (Becton Dickinson, San Jose, CA) para o fenótipo e enumerar as principais populações de leucócitos no sangue periférico, produzindo mais robusto informação específica do tipo de célula do que os ensaios, tais como um hemograma completo (CBC) ou ensaios com comercialmente disponíveis painéis projetados para tubos Trucount que mancham por apenas alguns poucos tipos de células. O processo de coloração é simples, requer apenas 100 l de sangue total fresco, e leva cerca de 45 minutos, tornando-se viável para o padrão de processamento de sangue laboratórios para executar. É adaptado do tubo Trucount BD ficha técnica ( <a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">versão 8/2010</a> ). O cocktail de anticorpos de coloração pode ser preparada antecipadamente a granel em um laboratório de ensaio central e enviado para o laboratório de processamento no local. Tubos coradas podem ser fixadase congelada para a expedição para o laboratório central para análise multicolor análise de citometria de fluxo. Os dados gerados a partir deste painel de coloração pode ser usado para controlar as alterações nas concentrações de leucócitos ao longo do tempo em relação à intervenção e pode ser facilmente desenvolvidas para avaliar os estados de activação de tipos específicos de células de interesse. Neste relatório, nós demonstrar o procedimento usado pelos técnicos de processamento de sangue de laboratório para realização da coloração de sangue total fresco e os passos para analisar estas amostras coradas em um laboratório de ensaio central de apoio de um ensaio clínico multicêntrico. Os detalhes do procedimento de vídeo, uma vez que é realizada no contexto de um ensaio clínico draw sangue no HIV Vaccine Trials Network (HVTN).

Watch this Scientific Journal Video about Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay at JoVE.com

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Neste relatório, demonstrar os passos coloração e análise de um ensaio de fenotipagem em sangue total fresco para enumerar grandes inata e adaptativa populações de leucócitos. Ressaltamos considerações para executar estes procedimentos, no contexto de um ensaio clínico multicêntrico.

Enumeração dos principais Populações de Leucócitos do Sangue Periférico de ensaios clínicos multicêntricos usando um ensaio fenotipagem Sangue

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

enumeration-major-peripheral-blood-leukocyte-populations-for

Research

JoVE Journal

Immunology and Infection

14.9K visualizações.  Fred Hutchinson Cancer Research Center. Neste relatório, demonstrar os passos coloração e análise de um ensaio de fenotipagem em sangue total fresco para enumerar grandes inata e adaptativa populações de leucócitos. Ressaltamos considerações para executar estes procedimentos, no contexto de um ensaio clínico multicêntrico.

Vídeo: Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

Esgotamento das populações de células específicas de Esgotamento Complemento

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Imunologia e Infecção

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Purificação da população de células específicas de separação de células de fluorescência Ativado (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Expansão, Purificação e Avaliação Funcional de Células NK do sangue periférico

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Expansão do sangue periférico humano γδ células T usando zoledronato

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Um Ensaio de sangue Funcional inteiro para medir a viabilidade de micobactérias, utilizando-Reporter Gene Tagged BCG ou M. Tb (BCG Lux / M. Tb Lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

Avaliação rápida e específica da atividade da peroxidase de halogenação em amostras de sangue enriquecido por leucócitos

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Uso de um monócito monocamada Ensaio para Avaliar a fagocitose mediada pelo receptor Fcy

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran aumenta a eficiência de Lentivirus transdução das células NK primário murino e humano

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Análise de célula única do imunofenótipo e produção de citocinas no sangue periférico através de citometria de massa

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...