Este trabalho foi financiado por bolsas pessoal do Wellcome Trust para Beate Kampmann (056.608, GRO77273).

1. Preparação de BCG estoque repórter lux micobactérias <ol><li> Crescer BCG lux (M. bovis BCG cepa Montreal transformado com o plasmídeo pSMT1 shuttle) com agitação a 200 rpm até meados de logarítmica fase em Middlebrook 7H9 caldo contendo 0,2% de glicerol, 0,05% entre 80 e 10% de enriquecimento ADC. </li><li> Prepare 1 ml de uma diluição de 1:10 de BCG cultura lux em PBS estéril em um tubo de luminômetro (900μl de PBS + 100μl BCG cultura lux) em duplicata. </li><li> Carga tubo contendo N-decyl aldeído (substrato da enzima luciferase) na parte traseira da máquina e garantir a tampa é presa e tubos no local. </li><li> Primeiro luminômetro injector com substrato através do programa principal, definido para injectar 100 ucl x 3 em cada um dos três tubos de priming vazias colocadas no luminômetro. </li><li> Carregar os dois tubos no luminômetro e fazer leituras através de um programa conjunto de injectar 100 ucl de substrato em cada tubo e ler por 20 segundos em intervalos de 1 seg. </li><li> Quando o estoque atingiu 1x10 8 RLU / ml a 2x10 8 RLU / ml (isso leva cerca de 3-4 dias de crescimento), adicionar igual volume de glicerol 30% estéril para a cultura em um tubo falcon de 50ml e misture delicadamente. </li><li> Alíquota de 1,5 ml em volumes rotulados microtubo tampa de rosca 2 ml e armazenar a -80 ° C. </li></ol> 2. Determinar ações RLU / UFC correlação e conteúdo de alíquotas <ol><li> Adicionar 15ml de Middlebrook 7H9 meio de cultura com 10% de suplemento ADC para um balão Erlenmeyer 200ml com tampa ventilada. </li><li> Adicionar 15μl de higromicina e 30μl de Tween 20%. </li><li> Remover um frasco de BCG lux do freezer e descongele em temperatura ambiente (RT) no gabinete de segurança. Adicionar o conteúdo do frasco para o meio e apertar a tampa. </li><li> Incube com agitação a 200 rpm a 37 ° C por 4 dias. </li><li> Em cada dia, tornar-se série de 10 diluições (1:10, 1:100, 1:1000, 1:10.000 e 1:100.000) para determinação UFC. </li><li> Configurar as diluições mesmo em paralelo e duplicar para determinar luminescência (ver parte 1,2-1,6). </li><li> Prepare dois 3 compartimentos placas de ágar Middlebrook 7H11 (1 l de meio líquido contendo glicerol 0,5%, 10% suplemento OADC, 1 ml de Tween 20% e 1 ml de higromicina). </li><li> Placa fora 100μl de cada diluição em um segmento de cada placa usando propagadores separadas, distribuindo o líquido igualmente através da câmara individual. </li><li> Selar cada placa com parafilme, coloque em um saco plástico estéril, vedação com fita autoclave, e incubar a 37 ° C por 2 semanas com tampa virada para baixo. </li><li> Inspecionar regularmente até aparecer UFC (2-3 semanas). </li><li> Para contar colônias, retire as placas da incubadora e coloque em um contador de colônias. Calcular a média para cada diluição das placas duplicadas. </li><li> Calcular a relação RLU / UFC usando RLU equivalentes e contagem de CFU para cada diluição. A relação deve estar entre 3 e 5 RLU / UFC. </li></ol> 3. Preparação de BCG cultura lux para inoculação em sangue total <ol><li> Adicionar 15ml de Middlebrook 7H9 meio de cultura com 10% de suplemento ADC para um balão Erlenmeyer 200ml com tampa ventilada. </li><li> Adicionar 15μl de higromicina e 30μl de Tween 20%. </li><li> Remover um frasco de BCG lux do freezer e descongele em temperatura ambiente no gabinete de segurança. Adicionar o conteúdo do frasco para o meio e apertar a tampa. </li><li> Incube com agitação a 200 rpm a 37 ° C durante 2 a 4 dias. </li><li> Prepare 1 ml de uma diluição de 1:10 de BCG cultura lux em PBS estéril em um tubo de luminômetro (900μl de PBS + 100μl BCG cultura lux) em duplicata. </li><li> Luminômetro privilegiada, com substrato (como descrito acima), gentilmente vortex os 2 tubos e carregar no luminômetro e fazer leituras (como descrito acima). </li><li> Use esta leitura para diluir até a cultura em PBS para dar o equivalente a 7x10 6 RLU (isso vai dar um inóculo de cerca de 2x10 5 de sangue UFC / ml e um rácio de BCG para os monócitos de cerca de 1:1, assumindo uma contagem de monócitos de cerca de 2x10 5 a 4x10 5 monócitos por ml de sangue). </li></ol> 4. Preparação de sangue total <ol><li> Levar de 3 a 5 ml de sangue venoso em tubo contendo heparina sem conservantes. </li><li> Transferir o sangue para um tubo falcon de 50ml e diluir com um volume igual de RPMI 1640 contendo HEPES glutamina e 25mm (sem pen / strep). </li><li> Alíquota de 900μl de sangue diluída em triplicata em tubos estéreis bijou para cada ponto de tempo (6 tubos no total:. 3 para 0hrs e 3 para 96h Configure tubos adicionais para timepoints adicionais, se necessário. </li><li> Alíquota de 900μl Middlebrooks meio de cultura 7H9 com 10% ADC suplemento + 50μg/ml higromicina (mesmo meio utilizado para a cultura da BCG lux) em duplicata para os controles de crescimento. </li><li> Adicionar 100μl do BCG diluído lux a cada tubo de sangue, e para o controle de duas crescimentotubos. </li><li> Misture bem e coloque os 3 bijous 96h para o ponto do tempo e os 2 controla o crescimento do seu lado na incubadora de balanço a 37 ° C, 20 rev / min (CO 2 não obrigatório). </li></ol> 5. Luminescência de medição (em 0hr e 96h) <ol><li> Centrifugar a 3 bijous em 2000g por 10 min. </li><li> Remova cuidadosamente 300 ul do sobrenadante sem perturbar o sedimento e coloque em 2ml tampa de rosca microtubos para congelar a -80 ° C para análise de citocinas subseqüentes. </li><li> Adicionar 300 ul de PBS a cada bijou para substituir o volume de sobrenadante removido. </li><li> Aspirar o conteúdo de cada bijou para a correspondente tubo falcon de 50ml e adicionar água destilada a cada tubo de 8ml. Iniciar um temporizador de 10 min. NB: Este é um passo sensíveis ao tempo, e esta incubação não deve ser qualquer mais de 10 min, começando a partir de quando as primeiras células entram em contato com a água. </li><li> Enxaguar cada bijou com 2 ml de água destilada e vortex por 5 segundos antes de derrubar para dentro do tubo falcon correspondente. </li><li> No final da incubação de 10 minutos, centrifugar os tubos falcon de 2000g por 10 minutos. </li><li> Decantar o sobrenadante em desinfetante Surfanios. </li><li> Adicionar algumas esferas de vidro a cada pellet e vortex. </li><li> Adicionar 1 ml de PBS estéril para cada tubo e vortex. </li><li> Em tubos luminômetro, fazer diluições 1:10 de cada amostra em duplicata com PBS (amostra + 900μl PBS 100μl) e no tempo 96h-ponto. Fazer as diluições mesmo para cada um dos controles de crescimento. </li><li> Suavemente os tubos vortex e carregar no luminômetro e fazer leituras (como descrito acima) Nb: Leituras entre triplicatas de uma mesma amostra deve ser de 15% uma da outra. </li></ol> 6. Resultados representativos: É importante o uso de bactérias na fase logarítmica de crescimento para os ensaios de sangue total lux, ou seja, cresceu mais de 48-72 horas antes da inoculação das amostras, como atividade metabólica é suboptimal se qualquer direto para fora do freezer ou em fase estacionária. O crescimento, antes deve ser padronizado para uma série de experimentos a 48 ou 72 horas para evitar a variabilidade. A Figura 1 mostra a curva de crescimento de uma cultura representativa da BCG lux. O tempo de duplicação é de cerca de 24 horas até fase estacionária é alcançado. <img alt="Figura 1" src="/files/ftp_upload/3332/3332fig1.jpg" /> Figura 1. Curva de crescimento Representante do BCG lux mais de 96 horas em meio 7H9 e correlação de RLU e UFC. Embora os valores de RLU sempre se correlacionam com UFC, o número de RLU correspondente a um único UFC pode variar dependendo do estoque, que é por isso que é boa prática usar o mesmo estoque congelado durante uma série de experimentos para garantir uma multiplicidade consistente de infecção ( MOI). A Tabela 1 mostra um exemplo de data-primas no momento da inoculação (T 0) e em 96 horas. Os rácios de crescimento são calculadas usando a fórmula T 96 / T 0, mas outros intervalos de tempo, claro, também pode ser medido. É, no entanto, não aconselhável a utilização de culturas para além das 96 horas, como a morte celular significativa ocorra. <img alt="Tabela 1" src="/files/ftp_upload/3332/3332table1.jpg" /> Tabela 1. Exemplo de dados brutos em tempo de inoculação (T0) e em 96 horas (T96) e rácios de crescimento calculados a partir de 3 dadores adultos. Dependendo do antígeno específico disponível respostas de memória ea contagem de neutrófilos, possivelmente, também, os rácios de crescimento variam entre indivíduos, como mostrado aqui na Figura 2 no sangue de crianças com e sem infecção pelo HIV. Em média, as crianças têm maiores taxas de crescimento do que os adultos, teste tuberculínico (TST) + ve indivíduos têm relações um crescimento inferior ao TST ve-indivíduos, e pacientes infectados pelo HIV tem rácios de crescimento devido à deficiência da população de células CD4 T, um dos principais mediadores da resposta imune celular de micobactérias. <img alt="Figura 2" src="/files/ftp_upload/3332/3332fig2.jpg" /> Figura 2 Inter-doador variabilidade:. Índices de crescimento de T 0 versus 96 horas de um conjunto de pacientes, dependendo do estado HIV subjacente. Reprodutibilidade dos índices de crescimento ao longo de um período de tempo é mostrado na Figura 3, que resume os resultados de 64 doadores sangrou duas vezes durante um período de 12 meses e de um único doador sangrou várias vezes para experimentos de controle. Causas potenciais de variabilidade podem ser mudanças na sensibilização por micobactérias ou variabilidade dentro de níveis de citocinas host, como observado em bioensaios de muitos. <img alt="Figura 3" src="/files/ftp_upload/3332/3332fig3.jpg" /> Figura 3. (A) único doador em 12 ocasiões em 12 meses. (B) múltiplas (64) doadores em 2 ocasiões com mais de 12 meses.

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Ensaios bactericida no contexto da pesquisa vacina contra a tuberculose estão bem estabelecidos em modelos animais, onde são aplicados para a tela vacinas candidatas potencialmente promissoras. Redução da carga bacteriana em várias funções órgãos como a principal leitura de imunogenicidade. A primeira geração de humanos ensaios bactericida foram projetados usando macrófagos sozinho, infectados com M. tuberculose. CFU serviu como a principal leitura de sobrevivência de m...

<table border="1"><tbody><tr><td> Reagente </td><td> Companhia </td><td> Cat Não </td></tr><tr><td> BCG lux </td><td> Vários </td><td> n / a </td></tr><tr><td> Middlebrook 7H11 agar </td><td> Becton Dickinson </td><td> 283810 </td></tr><tr><td> Middlebrook 7H9 caldo </td><td> BD Biosciences </td><td> 271310 (500g) </td></tr><tr><td> Middlebrook enriquecimento ADC </td><td> BD Biosciences </td><td> 212352 </td></tr><tr><td> Middlebrook suplemento OADC </td><td> Becton Dickinson </td><td> 212240 </td></tr><tr><td> Tween 80 </td><td> Sigma Aldrich </td><td> 274364 </td></tr><tr><td> Glicerina </td><td> Sigma Aldrich </td><td> G5150 </td></tr><tr><td> Higromicina B </td><td> Roche </td><td> 10843555001 (20ml) </td></tr><tr><td> N-decyl aldeído </td><td> Sigma </td><td> D7384-100G </td></tr><tr><td> Etanol (&gt; 99,7%) </td><td> VWR </td><td> 101077 </td></tr><tr><td> PBS estéril </td><td> Em Casa </td><td> n / a </td></tr><tr><td> RPMI-1640 com HEPES, L-Glutamina 25mM </td><td> Sigma </td><td> R0883 </td></tr><tr><td> Água destilada estéril </td><td> Em Casa </td><td> n / a </td></tr><tr><td colspan="3"> Equipamento </td></tr><tr><td> Luminômetro tubo (porta injetável obrigatório) </td><td> Berthold AutoLumat LB953 ou single-tubo luminômetro Sirius </td><td></td></tr><tr><td> Tubos luminômetro, 12 x 75mm, pirex sem aro </td><td> Corning da VWR </td><td> 99445-12 </td></tr><tr><td> Erlenmeyers com tampas ventiladas, estéril policarbonato 250ml </td><td> Corning via Fisher Scientific </td><td> 2150329 </td></tr><tr><td> BD Vacutainer Sangue Coleta Tubes (isenta de conservantes heparina) </td><td> BD </td><td> 367676 </td></tr><tr><td> Falcon 50ml tubos, cônico </td><td> BD </td><td> 352077 </td></tr><tr><td> 7ml espécime STERILIN polipropileno bijou tubos </td><td> VWR </td><td> 99445-12 </td></tr><tr><td> STERILIN Universal recipientes (30 ml) </td><td> Laboratório de Análises LTD </td><td> 128C </td></tr><tr><td> Contas de vidro 2mm de diâmetro </td><td> SigmaAldrich </td><td> Z143928 </td></tr><tr><td> 10ml pipetas e um menino de pipeta </td><td> qualquer </td><td></td></tr><tr><td> 1000μl e 200μl pipetas e pontas com filtro </td><td> qualquer </td><td></td></tr><tr><td colspan="3"> Equipamento geral de laboratório: </td></tr><tr><td> Classe II de segurança gabinete gabinete </td><td></td><td></td></tr><tr><td> Geladeira (4 ° C) </td><td></td><td></td></tr><tr><td> Centrifugador </td><td></td><td></td></tr><tr><td> Rocking shaker plataforma (180 0) para misturar os tubos </td><td></td><td></td></tr><tr><td> Orbital Shaker incubadora a 37 ° C para crescer culturas bacterianas </td><td></td><td></td></tr></tbody></table>

a functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged bcg or m.tb (bcg lux/m.tb lux)

Ensaios funcionais há muito tempo desempenhou um papel fundamental na medição da imunogenicidade de uma vacina dada. Este é convencionalmente expressa como soro títulos bactericida. Estudos de soro títulos bactericida em resposta a vacinas infantis nos permitiram desenvolver e validar níveis de corte para respostas imunes protetoras e tal cut-offs estão em uso rotineiro. Sem tais ensaios foram levadas adiante na avaliação de rotina de vacinas que induzem principalmente a imunidade mediada por células em forma de respostas de células T efetoras, tais como vacinas contra a tuberculose. No modelo animal, o desempenho de um candidato a vacina dada é rotineiramente avaliada em ensaios padronizados bactericida, e todos os atuais romance TB vacina candidatos foram submetidos a esta etapa na sua avaliação antes da fase 1 de testes em humanos. A avaliação da imunogenicidade e, portanto, a probabilidade de eficácia protectora do romance de vacinas anti-TB deve idealmente submetidos a uma avaliação por etapas semelhantes nos modelos humanos agora, incluindo medidas em ensaios bactericida. Ensaios bactericida no contexto da pesquisa vacina contra a tuberculose já estão bem estabelecidos em modelos animais, onde eles são aplicados a tela de vacinas candidatas potencialmente promissoras. Redução da carga bacteriana em várias funções órgãos como a principal leitura de imunogenicidade. No entanto, nenhum de tais ensaios foram incorporados ensaios clínicos para novos medicamentos anti-TB vacinas até o momento. Embora ainda haja incerteza sobre os mecanismos exatos que levam à morte de micobactérias no interior dos macrófagos humanos, a interação de macrófagos e células T com micobactérias é claramente necessária. O ensaio descrito neste trabalho representa uma geração nova de ensaios bactericida que permite estudos de tal chave componentes celulares com todos os outros fatores celular e humoral presentes no sangue inteiro sem fazer suposições sobre sua contribuição relativa individual. O ensaio descrito pelo nosso grupo utiliza pequenos volumes de sangue total e já foi empregada em estudos com adultos e crianças em TB endêmica configurações. Nós mostramos imunogenicidade da vacina BCG, aumento do crescimento de micobactérias em pacientes HIV-positivos, bem como o efeito da terapia anti-retroviral e vitamina D sobre a sobrevivência de micobactérias in vitro. Aqui resumimos a metodologia e apresentar os nossos dados de reprodutibilidade usando este modelo relativamente simples, de baixo custo e de campo-friendly. Nota: Definições / Abreviaturas BCG lux = M. bovis BCG, cepa Montreal, transformada com pSMT1 shuttle plasmídeo carregando a genes de Vibrio harveyi luxAB, sob o controle do GroEL micobactérias (Hsp60) promotor. UFC = Unidade Colony Forming (uma medida de viabilidade de micobactérias).

Watch this Scientific Journal Video about A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux at JoVE.com

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

Descrevemos uma abordagem alternativa para a enumeração de micobactérias<em&gt; In vitro</em&gt;, Que usa repórter gene-tagged micobactérias em vez de unidades formadoras de colônia (UFC). &quot;Survival&quot; de organismos, bem como anfitrião-resposta marcadores são medidos simultaneamente, proporcionando um sistema de baixo custo, versátil e funcional para os estudos de host / patógeno interações no contexto da tuberculose.

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 ...

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Immunology and Infection

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

13.8K visualizações.  Imperial College London. Descrevemos uma abordagem alternativa para a enumeração de micobactérias In vitro, Que usa repórter gene-tagged micobactérias em vez de unidades formadoras de colônia (UFC). "Survival" de organismos, bem como anfitrião-resposta marcadores são medidos simultaneamente, proporcionando um sistema de baixo custo, versátil e funcional para os estudos de host / patógeno interações no contexto da tuberculose.

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

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 ...

Imunologia e Infecção

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

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<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">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

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 ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Antígenos funcionais protegidos glóbulos vermelhos pela membrana enxerto de compactos Poligliceróis hiper

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Baseada em células de Citometria de Fluxo Ensaio medir citotóxicos Atividade

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Usando a sobreposição de ensaio para medir qualitativamente produção bacteriana e sensibilidade para pneumocócica Bacteriocinas

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

A Fluxo Simples Citometria método para medir glicose Captação e expressão de transportadores de glicose para monócitos subpopulações em sangue total

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

A eficácia de protecção e Pulmonar resposta imune Seguindo subcutânea e intranasal BCG Administração em Ratinhos

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

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 ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Expressão de antígenos exógenos a Mycobacterium bovis BCG através da decoração de superfície Non-genética com o sistema de avidina-biotina

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

Ensaio de ELISpot otimizado de Interferon-gama às respostas de célula T medida no modelo de porquinho da Índia após a vacinação

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...