Estamos muito gratos a K. Nakagaki, H. Dr. Ishii, Dr. K. Suzuki, Yamagata A., K. Oofusa por suas valiosas contribuições.

1. Produção e purificação de sGMRα <ol><li> Amplificar cDNA de sGMRα da biblioteca de cDNA placenta humana por reação em cadeia da polimerase (PCR). </li><li> Adicionar 50 base de 5&#39;-UTR seqüência de final poliedrina baculovírus e 27 RGS base de codificação Sua tag-de-final 3 &#39;do fragmento amplificado pela PCR por outro PCR. </li><li> Inserir o produto amplificado de PCR em um pMSG1.1MG plasmídeo. </li><li> Injetar o psGMR/M1.1MG construir com o vetor helper pHA3PIG em ovos de bicho-da-tensão pnd-w1. </li><li> Traseira do chocados G0 larvas às traças a 25 ° C. Tela de embriões obtidos por meio de cruzamentos G1 entre irmãos ou com pnd-w1 para a expressão MGFP nos olhos para obter transgênicos bichos tendo o gene sGMRα. </li><li> Extrato recombinante sGMRα dos casulos de bicho-da-transgênicos com tampão fosfato contendo 500mM NaCl e mexa a 4 ° C por 24h. </li><li> Centrífuga de amostra em 20.000 g por 15 min para remover um precipitados. </li><li> Aplicar o sobrenadante para uma coluna de níquel afinidade. </li><li> Lavar a coluna com 10mM imidazol, NaCl 500mM e fosfato de sódio 20mM, pH6.3, e eluir com um gradiente linear de imidazol de 10mm a 250mm. Piscina as frações contendo o sGMRα purificado e diálise contra tampão fosfato (pH7.4) (PBS). </li></ol> 2. Biotinilação de GM-CSF recombinante <ol><li> Remover preparação sorbitol por diálise contra PBS. </li><li> Adicionar 1 ml de solução de sódio 20mM frio meta-periodato a 4 ° C para a solução em gelo. </li><li> Incubar a amostra por 30min no gelo no escuro. </li><li> Adicionar glicerol para a solução em uma concentração final de 15mM. </li><li> Diálise a solução contra tampão acetato de sódio 100mM (pH5.5) </li><li> Adicionar biotina hidrazida para a solução em uma concentração final de 5mM e continuamente agitar a solução para 2h em temperatura ambiente. </li><li> Remover o material não reagiu por diálise contra PBS, pH7.4. </li></ol> 3. Uma célula-livre GM-CSF receptor-ligand-binding ensaio <ol><li> Placa de um casaco de microtitulação com 50μl de anticorpo monoclonal anti-polyHistidine overnight a 4 ° C. </li><li> Lavar cinco vezes com 500μl de PBST. </li><li> Adicionar 100μl de uma solução de bloqueio e incubar por 3h a 4 ° C. </li><li> Lavar cinco vezes com 500μl de PBST. </li><li> Adicionar 50μl de sGMRα na solução de bloqueio e incubar overnight a 4 ° C. </li><li> Lavar cinco vezes com 500μl de PBST. </li><li> Adicionar 25μl das amostras contendo GM-CSF auto-anticorpos e, em seguida, 25μl de biotinilado GM-CSF. Incubar por 1h a 4 ° C para formar sGMRα-GM-CSF complexo. </li><li> Lavar cinco vezes com 500μl de PBST. </li><li> Adicionar 50μl de 0.4mm fosfatase alcalina estreptavidina para uma hora a 4 ° C para detectar a biotinilado GM-CSF ligado sGMRα. </li><li> Lavar cinco vezes com 500μl de PBST. </li><li> Adicionar 50μl de um substrato quimioluminescente para a solução e incubados por 1h em temperatura ambiente. </li><li> Usando um leitor de placas de quimioluminescência, detectar a atividade quimioluminescência. </li></ol> 4. Resultados representativos: O primeiro passo para a GM-CSF via de transdução de sinal receptor é a ligação do GM-CSF a GM-CSF alpha receptor na superfície da célula. Porque a GM-CSF auto-anticorpos se liga especificamente GM-CSF e bloquear a sua ligação ao receptor in vitro 16,17, temos a hipótese de que os auto-anticorpos inibem esta primeira reação diretamente ligação a GM-CSF. Como descrito anteriormente (Ref. 15/09), foi aplicado transgênicos tecnologia-da-seda para obter uma grande quantidade de sGMRα recombinante com alta pureza. Quando o bicho derivados sGMRα recombinante foi carregado no SDS-PAGE em condições não-condições redutoras, o sGMRα mostrou tanto monomérica (45 kDa) e dimérica (90 kDa) formas, enquanto que apenas a forma monomérica foi detectado em condições redutoras (Figura 1B) , indicando que o sGMRα recombinante foi uma mistura de monômeros e dissulfeto-linked dimmers 18. Utilizando o sistema de células livres (Fig. 2A), avaliou-se a inibição da GM-CSF ligação a sGMRα pela GM-CSF auto-anticorpos (Fig. 2B e Figura. 2C). Estes métodos foram descritos na referência 19 por Urano et al. A inibição do crescimento foi estreitamente relacionado com a inibição de ligação (r = 0,988, p = 0,002) em várias concentrações dos auto-anticorpos GM-CSF (Fig. 3A) 19. Da mesma forma, a inibição de ligação foi significativamente correlacionada com a inibição do crescimento. A inibição de ligação aumentou de forma dose-dependente pela GM-CSF auto-anticorpos (Fig. 3B) 19. Estes dois parâmetros para as frações de soro IgG de diferentes pacientes foram correlacionados entre si (r = 0,589 p = 0,006, fig. 3C). Nem a inibição de ligação, nem a inibição do crescimento correlacionado com os valores de Kd, e assim, ambos os parâmetros não foram afetados pela afinidade de ligação 19. Consequentemente, reprodutibilidade de dados entre vinculativo e gCRESCIMENTO inibição obtidos através três experimentos independentes em três diferentes amostras foi avaliada. O coeficiente de variação indicou que o sistema livre de células foi mais excelente do que o de bioensaio (tabela 1). <img src="/files/ftp_upload/2742/2742fig1.jpg" alt="Figura 1" /> Figura 1. Fluxograma do procedimento para a produção de transgênicos sGMRα usando bichos. A) As estruturas dos vetores de transformação 9-11. B) SDS-PAGE e coloração Coomassie Brilliant Blue-do sGMRα purificada sob redutores e não redutores condições. sGMRα, solúvel GM-CSF receptor alfa; MGFP, monstro proteína verde fluorescente; BmNPVpol5&#39;-UTR, 5&#39;-não traduzida seqüência região de Bombyx mori poliedrose nuclear vírus poliedrina; polyA SV40, SV40 seqüência de sinal polyA; P 3xP3, 3xP3 promotor; P SER1, SER1 promotor; FIBL polyA, fibroína L de cadeia seqüência de sinal polyA; hr3, Bombyx mori vírus da poliedrose nuclear hr3 enhancer. <img src="/files/ftp_upload/2742/2742fig2.jpg" alt="Figura 2" /> Figura 2. Esquema do sistema de ensaio livre de células. A) Um ensaio de ligação competitiva utilizando sGMRα produzido pelo bicho da seda. B) Efeito de anticorpos neutralizantes e não-neutralizantes na inibição de ligação por celular sem sistema. C) A diferença de inibição de ligação entre várias concentrações de anticorpos neutralizantes e não-anticorpos neutralizantes. GM-CSF, estimulante de colônias de granulócitos e macrófagos fator; sGMRα, solúvel GM-CSF receptor alfa; Sua, RGS-His-tag, AP, fosfatase alcalina. <img src="/files/ftp_upload/2742/2742fig3.jpg" alt="Figura 3" /> Figura 3. GM-CSF inibição de ligação para sGMRα por efeito de GM-CSF anticorpos policlonais ou frações de soro IgG de pacientes com PAP auto-imunes. A) Relação entre a inibição de ligação e inibição do crescimento por GM-CSF auto-anticorpos. B) inibição Binding em várias concentrações de GM-CSF auto-anticorpos. C) Relação entre IC 50 por cento para a inibição da ligação e inibição do crescimento por cento em frações de soro IgG 19. IC 50, 50% da concentração inibitória; GM-CSF, granulócitos e macrófagos fator estimulante de colônia. <table border="1"><tbody><tr><td> Amostra </td><td> A </td><td> B </td><td> C </td></tr><tr><td colspan="4"> Inter-ensaio </td></tr><tr><td> # Das determinações </td><td> 3 </td><td> 3 </td><td> 3 </td></tr><tr><td> Valor médio (% de inibição de ligação) </td><td> 61,6 </td><td> 63,8 </td><td> 69,7 </td></tr><tr><td> Valor médio (inibição do crescimento%) </td><td> 21,0 </td><td> 73,4 </td><td> 82,8 </td></tr><tr><td> Coeficiente de variação (%) (% de inibição de ligação) </td><td> 6 </td><td> 5,6 </td><td> 8,3 </td></tr><tr><td> Coeficiente de variação (%) (inibição do crescimento%) </td><td> 64,4 </td><td> 18,3 </td><td> 10,4 </td></tr></tbody></table> Ambos os ensaios foram realizados em 5 ng / ml de GM-CSF e uma concentração igual a GM-CSF auto-anticorpos. Tabela 1. Comparação de coeficiente de variação entre vinculativo por cento e as inibições de crescimento obtidos através de três experimentos independentes.

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O ensaio livre de células estimou a capacidade de neutralização de GM-CSF auto-anticorpos com excelente reprodutibilidade e rapidez. A inibição de ligação por auto-anticorpos GM-CSF ou frações de soro do paciente IgG foi avaliada por este ensaio. Os dados mostraram uma correlação entre a inibição de ligação do ensaio livre de células ea inibição do crescimento de um bioensaio utilizando células TF-1, respectivamente. O bioensaio tem sido amplamente utilizada, mas abrigava dificuldades na comparação ...

a cell free assay system estimating the neutralizing capacity of gm-csf antibody using recombinant soluble gm-csf receptor

FUNDOS: Anteriormente, demonstramos que a capacidade de neutralizar, mas não a concentração de GM-CSF auto-anticorpos foi correlacionada com a severidade da doença em pacientes com proteinose alveolar pulmonar auto-imunes (PAP) 1-3. Como revogação da GM-CSF bioatividade no pulmão é a causa mais provável para PAP auto-imunes 4,5, é promissora para medir a capacidade de neutralização de GM-CSF auto-anticorpos para avaliar a gravidade da doença em cada paciente com PAP. Até agora, a capacidade de neutralização de GM-CSF auto-anticorpos foi avaliada através da avaliação da inibição do crescimento de células de medula óssea humana ou TF-1 células estimuladas com GM-CSF 6-8. No sistema de bioensaio, no entanto, muitas vezes é problemático para obter dados fiáveis, bem como comparar os dados de diferentes laboratórios, devido a dificuldades técnicas na manutenção das células em uma condição constante. OBJETIVO: Para imitar GM-CSF ligação ao GM-CSF receptor na superfície celular utilizando células livres receptor-ensaio de ligação. MÉTODOS: Transgênicos bicho-da-tecnologia foi aplicada para a obtenção de uma grande quantidade de recombinante solúvel GM-CSF receptor alfa (sGMRα) com alta pureza 9-13. O sGMRα recombinante foi contido nas camadas sericin hidrofílica de fios de seda sem ser fundida ao proteínas da seda, e assim, podemos facilmente extrair os casulos na pureza bom com soluções aquosas neutras 14,15. Felizmente, as estruturas de oligossacarídeos, que são críticos para a ligação com a GM-CSF, são mais semelhantes às estruturas de sGMRα humana do que aquelas produzidas por outros insetos ou leveduras. RESULTADOS: O sistema de ensaio livre de células usando sGMRα rendeu os dados com alta plasticidade e confiabilidade. GM-CSF ligação a sGMRα foi dose-dependente inibida por policlonal GM-CSF auto-anticorpos de forma semelhante ao bioensaio usando TF-1 células, indicando que o nosso novo livre de células do sistema de ensaio utilizando sGMRα é mais útil para a medição da atividade neutralizante de GM-CSF auto-anticorpos que o sistema de bioensaio utilizando TF-1 célula ou células humanas de medula óssea. CONCLUSÕES: Nós estabelecemos um ensaio livre de células quantificar a capacidade de neutralização da GM-CSF auto-anticorpos.

Watch this Scientific Journal Video about A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor at JoVE.com

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Nós projetamos um ensaio de receptor de células livres vinculativas, a fim de estimar a ligação de granulócitos e macrófagos fator estimulante de colônia (GM-CSF) para os receptores. Ele nos permite avaliar inibição competitiva do biotinilado GM-CSF ligação a solúvel GM-CSF receptor alfa por GM-CSF auto-anticorpos com excelente reprodutibilidade.

A célula do sistema de Ensaio grátis Estimar a capacidade de neutralização de GM-CSF recombinante usando anticorpo receptor GM-CSF Solúvel

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

a-cell-free-assay-system-estimating-neutralizing-capacity-gm-csf

Research

JoVE Journal

Immunology and Infection

12.6K visualizações.  Niigata University Medical and Dental Hospital. Nós projetamos um ensaio de receptor de células livres vinculativas, a fim de estimar a ligação de granulócitos e macrófagos fator estimulante de colônia (GM-CSF) para os receptores. Ele nos permite avaliar inibição competitiva do biotinilado GM-CSF ligação a solúvel GM-CSF receptor alfa por GM-CSF auto-anticorpos com excelente reprodutibilidade.

Vídeo: A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Bioenergética e os oxidativo estourou: Protocolos para o Isolamento e Avaliação de leucócitos humanos e plaquetas

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Imunologia e Infecção

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Selecção de alta Scalable Rendimento de bibliotecas de anticorpos em fagos sintética exibida-

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Geração e identificação de GM-CSF macrófagos derivados e dendríticas células alveolares do tipo de ratinho de Medula Óssea

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Avaliando Retinal microglia fagocítica Função<em&gt; In Vivo</em&gt; Usando um ensaio baseado em citometria de fluxo

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murino Linfócitos Rotulagem por<sup&gt; 64</sup&gt; Cu-Antibody Receptor Segmentação para<em&gt; In Vivo</em&gt; Tráfico celular por PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Geração de variantes de fuga de neutralizar os anticorpos monoclonais de vírus da gripe

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Medição da gripe neutralizando Antibody Responses to A(H3N2) vírus em soros humanos por Microneutralization ensaios usando as células MDCK-SIAT1

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Um novo sistema alimentador-livre para produção em massa de murino Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

In Vitro Diferenciação de Mouse Granulócito-macrófago-colônia-estimulando Factor (GM-CSF)-produzindo pilhas de T Helper (THGM)

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.