Este trabalho foi financiado pela Transregio Sonderforschungsbereich 19 (SFB/TR19, C2) da Deutsche Forschungsgemeinschaft (DFG), o Ministério Federal da Economia e Tecnologia projeto ZIM-KF 2727801MD0 e do Centro de Competência Inovação - respostas imunes humoral em Doenças Cardiovasculares ( ZIK CAMINHADA, BMBF FKZ 03Z2CN12) do Ministério Federal da Educação e Pesquisa, na Alemanha. Habitação e experimentos com animais foram realizados de acordo com as recomendações da Sociedade para Laboratory Animal Science (Gesellschaft für Versuchstierkunde, GV-SOLAS) e da Federação Europeia de Laboratório Animal Science Association (FELASA).

1. IgG isolamento de amostras de pacientes <ol><li> Prepare mini-colunas de imunoadsorção preenchendo 3 ml anti-IgG Sepharose por paciente 2 ml de EDTA plasma em um vazio coluna Econo Pac. IgG isolamento requer, pelo menos, 2 ml de plasma do paciente. </li><li> Colocar um filtro na parte superior da sefarose anti-IgG, cortou a ponta inferior da coluna e prima do filtro para atingir uma taxa de fluxo de cerca de 1 gota / seg. Deixe a solução de preservação correr para fora da coluna. </li><li> Lavar a coluna com 3 volumes de NaCl a 0,9% por volume de plasma. </li><li> Pipetar a plasma na coluna. Quando o plasma foi completamente imerso na sefarose anti-IgG, lava-se com o mesmo volume de NaCl 0,9%. </li><li> Pipeta de volume de plasma de uma glicina 0,2 M de HCl, pH 2,8 na coluna e deixar o imergir em sefarose. Adicionar outra quantidade de glicina HCl na coluna. Recolher o fluxo através de um tubo de 15 ml e ajustar o pH para 7,3 com 0,5 M de Tris-HCl, pH 8,0. </li><li> Para regenerar a coluna, Lava-se com 3 volumes de plasma glicina HCl. Após uma lavagem final com dois volumes de NaCl a 0,9%, a coluna pode ser utilizada para a amostra de plasma a seguir. Alternativamente, a coluna pode ser preenchida com uma solução de preservação e armazenado a 4-8 ° C durante até 4 semanas. </li><li> Para utilização em cardiomiócitos, a fracção de IgG recolhido terá de ser dialisada contra tampão experimental (EB). Para a preparação de EB, adicionar 117 mM NaCl, 2,8 mM KCl, 0,6 mM MgCl2, 1,2 mM de KH 2 PO 4, 1,2 mM CaCl2, 10 mM de HEPES e 20 mM de glucose a 1 L de água destilada num agitador magnético e ajustar o pH para 7,3. </li><li> Encher a fracção de IgG a um tubo de membrana de celulose de éster de diálise com um peso molecular de corte de 100 kDa. Colocar o tubo em 1 EB L e agitar lentamente com um agitador magnético, a 4 ° C. Depois de 8 horas, transferir o tubo de diálise em 3 L de EB fresco e dialisar para outro h 20 a 4 ° C. </li><li> Seguindo calor diálise, a amostra durante 30 min a 57 ° C num banho de água a inactivatfactores do complemento e. Determinar a concentração de IgG total e se preparar alíquotas contendo 330 ug de IgG de cada. Quando a adição da amostra para a medição de cardiomiócitos (passo 4.3), enchem-se as alíquotas de 1 ml com EB. Alíquotas não diluídas podem ser armazenadas a -20 ° C até à sua utilização. </li></ol> 2. Isolamento de cardiomiócitos de ratos <ol><li> Para a preparação de Ca 2 + sem tampão de isolamento (IB), adicionar 110 mM NaCl, 2,6 mM KCl, 1,2 mM de MgSO4, 1,2 mM de KH 2 PO 4, 20 mM de HEPES e 11 mM de glucose a 1 L de água destilada, utilizando um campo magnético agitador. Ajustar o pH a 7,4 à temperatura ambiente e filtrar através de um filtro de 0,2 uM topo frasco para esterilização. </li><li> Encher 80 ml de IB no reservatório superior de um sistema de controlo termostático Langendorff perfusão. Ajuste as configurações do termostato para manter uma temperatura de tampão de 37 ° C e arejar tampão com 95% de O2 / 5% de CO 2 durante 30 min. </li><li> Pesar cerca de 10.000 - 12.000 U de collagenase tipo II, 175 mg de ácido gordo livre de BSA e dissolver em 20 ml contendo IB 22,5 ul de uma solução 100 mM de CaCl stock 2. Controlar o pH do tampão de isolamento no reservatório superior e ajustar se necessário. </li><li> Anestesiar um rato Wistar de 175 - 200 g com 375 tiopental peso mg / kg de peso corporal. Adicionar 2500 UI de heparina à solução de tiopental. Após toracotomia do coração, a extirpar cuidadosamente com um arco aórtico intacta e transferi-lo para gelado de NaCl 0,9%. Limpar o coração a partir do sangue e do tecido circundante e da transferência de NaCl a 0,9% fresco. Expor a aorta, abra o redutor de fluxo do sistema de Langendorff para se obter uma velocidade de queda de 2-3/sec e anexar o coração à cânula. </li><li> Perfundir o coração por até 3 min, com uma taxa de fluxo de 1 gota / segundo para lavar o sangue restante. Começam a circular as IB no sistema Langendorff. Remover 20 mL de tampão a partir do reservatório superior, preencher a solução de colagenase e de recarga como tampão tanto quanto necessário para atingir 80 ml. Circular o c<html> solução ollagenase por outro 27 min. Taxa de fluxo deve ser controlado periodicamente e mantido a 1 gota / seg usando o redutor de fluxo. </li><li> Retire o coração da cânula, limpa a partir de átrios e aorta e pique em pedaços pequenos. Permitir que a solução de colagenase a correr para o reservatório inferior, a reduzir o volume a um máximo de 40 ml e ainda mais digerir os ventrículos picados por 10 - 15 min sob arejamento contínuo com 95% O% 2/5 CO 2. Em seguida, filtrar a suspensão de células através de uma gaze de 200 ^ M de tamanho de malha para um tubo de 50 ml. </li><li> Restabelecer o teor de Ca 2 + das células em 3 passos: centrifugar a suspensão de células a 43 xg durante 2 min à temperatura ambiente e Ressuspender as células em 10 ml IB contendo 200 ^ M de CaCl2. Centrifugar novamente e ressuspender as células em 10 ml de IB com 500 ^ M CaCl 2. Após uma centrifugação final, ressuspender em cardiomiócitos EB filtrada estéril (ver 1.8) a uma densidade de 50.000 células / ml. </li></ol>e_title &quot;&gt; 3. Fura-2 coloração dos cardiomiócitos <ol><li> Brasão uma lamela 4 bem compartimentado com 10 mg por laminina bem e espere até que esteja completamente seco. </li><li> Encha 1 ml da suspensão de cardiomiócitos em cada poço utilizando uma micropipeta com uma ponta de corte. Permitir que as células a aderir durante 1 hora à temperatura ambiente. </li><li> Diluir 10 uL de 1 mg / ml de Fura-2:00 em solução de 5 ml de EB. Manter a solução de coloração no escuro. </li><li> Tire tampão e solta células da lamela e pipeta 0,5 ml da solução de coloração em cada poço. Incubar as células durante 10 min sob agitação moderada. Subsequentemente, retire a solução de coloração, lavar as células uma vez com 1 ml de EB e, finalmente, encher EB 0,5 ml em cada poço. Evite luz direta durante o processo de coloração e sempre manter as células manchadas no escuro. </li></ol> 4. Gravação do Encurtamento celular e Ca 2 + Transients <ol><li> Coloque a lamela câmaras numa m fluorescência inversaicroscope cálcio ligado a um sistema de gravação de miócitos e contractilidade. Posicionar um eléctrodo feitos com um influxo e efluxo de tubo na primeira cavidade. Cardiomiócitos Superfuse com 1 EB ml / min e iniciar a estimulação eléctrica (20 V, 1 Hz, 5 ms de duração). Permitir que as células se adaptar à estimulação por cerca de 2 min. </li><li> Escolha uma haste intacta forma de cardiomiócitos com estrias claras e contração constante. Posicionar a célula no centro do campo visual e abrir o canal da câmara. Orientar a célula horizontalmente dentro da área de vídeo através da rotação do adaptador enquadramento célula e mover a platina do microscópio. </li><li> Ajuste a detecção de borda elementos de controle da área de vídeo (figura 2), abrir o canal de fluorescência e registro 10-15 contrações para obter a redução de células inicial e Ca 2 + transiente. </li><li> Fechar o canal de luz do microscópio a fim de evitar o branqueamento do corante fluorescente. Comutar o fluxo através da EB para a amostracontornar com uma válvula de 3-vias e cardiomiócitos superfuse com 1 ml de IgG paciente. Parar a bomba antes do ar atingir o bem. </li><li> Abrir o canal de luz e gravar outro 10-15 contrações para obter a resposta aguda de células. Pausar a gravação e fechar o canal de luz novamente. Repetir a gravação após 2 e 5 min. </li><li> Retire o eletrodo do poço. Limpe cuidadosamente os tubos com EB e continuar com o bem próxima da lamela câmaras. </li><li> Analisar o encurtamento celular e Ca 2 + transiente com o software IonWizard usando o &quot;Edge-Length/Length&quot; e &quot;2-fura numérico subtraído / Ratio&quot; janela, respectivamente. Calcula-se a percentagem de alteração a partir do encurtamento celular inicial e Ca 2 + transiente da altura do pico que se refere à linha de base (% de pico bl h) para a fase aguda, a 2 minutos e a resposta de 5 min. </li></ol>

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O método apresentado oferece um modo adequado, para detectar auto-anticorpos funcionalmente eficazes cardíacos em pacientes com DCM de origem incerta. Em comparação com os outros métodos, por exemplo, a detecção de anticorpos funcionais activos contra o β1-adrenérgico por seu impacto sobre os níveis de cAMP 6, o nosso método é independente de um epitopo específico. Claro que há outros epítopos ensaios independentes descritos na literatura, ou seja, a taxa de contagem de cardio...

<table border="1"><tbody><tr><td> Nome do reagente / equipamento </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Immunosorba solução de preservação </td><td> Fresenius Medical Care </td><td> 903609211 </td><td></td></tr><tr><td> A colagenase do tipo 2 </td><td> Sistema de célula </td><td> LS004176 </td><td></td></tr><tr><td> BSA, ácidos graxos livres </td><td> Sigma-Aldrich </td><td> A6003 </td><td></td></tr><tr><td> Laminina </td><td> Sigma-Aldrich </td><td> L2020 </td><td></td></tr><tr><td> Fura-02:00 </td><td> Sigma-Aldrich </td><td> F0888 </td><td> 1 mg / ml em DMSO </td></tr><tr><td> Econo Pac colunas de cromatografia </td><td> BioRad </td><td> 732-1010 </td><td></td></tr><tr><td> Spectra / Por Biotech éster de celulose Diálise Membrane </td><td> Spectrumlabs </td><td> 131417 </td><td></td></tr><tr><td> 4 bem Chambered lamela </td><td> Nunc </td><td> 155383 </td><td></td></tr><tr><td> O cálcio dos miócitos e Sistema de Gravação Contratilidade </td><td> IonOptix </td><td></td><td></td></tr><tr><td> IonWizard software de análise de 6,0 </td><td> IonOptix </td><td></td><td></td></tr></tbody></table>

measurement of antibody effects on cellular function of isolated cardiomyocytes

Cardiomiopatia dilatada (CMD) é uma das principais causas para a insuficiência cardíaca em adultos mais jovens 1. Embora predisposição genética e exposição a substâncias tóxicas são causas conhecidas para essa doença em cerca de um terço dos pacientes, a origem de DCM permanece amplamente claro. Num número substancial de pacientes, os auto-anticorpos contra epítopos cardíacos foram detectados e são suspeitos de desempenhar um papel crucial na iniciação e progressão da doença de 2,3. A importância dos anticorpos cardíacos é sublinhado por uma melhoria hemodinâmica observada em pacientes com CMD após a eliminação de auto-anticorpos por imunoadsorção 3-5. Uma variedade de antigénios específicos já foram identificados 2,3 e anticorpos contra estes objectivos podem ser detectados por imunoensaios. No entanto, estes ensaios não pode discriminar entre o estímulo (ou seja funcionalmente eficaz) e bloqueio de auto-anticorpos. Há cada vez mais evice que esta distinção é 6,7 crucial. Pode também considerar-se que os objectivos estabelecidos para um certo número de anticorpos cardiotrópicos ainda não identificado e, portanto, não pode ser detectada por imunoensaios. Assim, foi estabelecido um método para a detecção de anticorpos funcionalmente activos cardiotrópicos, independentemente do seu antigénio correspondente. O pano de fundo para o método é a elevada homologia normalmente observada para as regiões funcionais de proteínas cardíacas em mamíferos entre 8,9. Isto sugere que os anticorpos dirigidos contra antigénios cardíacos humanos reagem de forma cruzada com as células-alvo não-humanos, o que permite o teste de IgG de pacientes com CMD em cardiomiócitos de ratos adultos. O nosso método consiste em três passos: primeiro, a IgG é isolada a partir de plasma de paciente com Sepharose acoplada anti-IgG de anticorpos obtidas a partir de colunas de imunoadsorção (PlasmaSelect, Teterow, Alemanha). Em segundo lugar, os cardiomiócitos adultos são isolados através de perfusão com colagenase num aparelho de perfusão Langendorff usando aprotocolo modificado de trabalhos anteriores 10,11. Os cardiomiócitos obtidos estão ligados a esfregaços laminina revestidos septadas e coradas com Fura-2, um corante fluorescente de cálcio selectivo que pode ser facilmente levado para dentro da célula para observar o cálcio intracelular (Ca2 +) conteúdo 12. No último passo, o efeito de IgG do paciente sobre a redução das células e os transientes de Ca 2 + de cardiomiócitos de campo estimulado é monitorizada utilizando uma linha de cálcio comercial miócito e contractilidade sistema de monitorização (IonOptix, Milton, MA, EUA), ligado a um padrão inverso fluorescente microscópio.

Dois exemplos para a medição do inotropismo celular no campo estimulado cardiomiócitos adultos (Figura 2), utilizando um dos miócitos Ca 2 + e do sistema de gravação de contratilidade são dadas abaixo. Figura 3 dá uma impressão de uma medida de controlo, enquanto que na Figura 4, o efeito dos anticorpos cardiodepressive de um paciente com DCM é mostrado. Em ambos os exemplos, encurtamento celular inicial e Ca 2 +</s...

Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

O método apresentado oferece um modo para detectar auto-anticorpos funcionais eficazes cardiotrópicos no plasma de pacientes com cardiomiopatia dilatada, independentemente do antigénio específico, por meio da análise do impacto da imunoglobulina paciente isolado no encurtamento celular e os transientes de cálcio intracelular em cardiomiócitos de ratos isolados.

Medição dos Efeitos de anticorpos sobre a função celular de cardiomiócitos isolados

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

measurement-antibody-effects-on-cellular-function-isolated

Research

JoVE Journal

Immunology and Infection

11.8K visualizações.  University Medicine Greifswald. O método apresentado oferece um modo para detectar auto-anticorpos funcionais eficazes cardiotrópicos no plasma de pacientes com cardiomiopatia dilatada, independentemente do antigénio específico, por meio da análise do impacto da imunoglobulina paciente isolado no encurtamento celular e os transientes de cálcio intracelular em cardiomiócitos de ratos isolados.

Vídeo: Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Medição do Ca citosólico 2 + Em Isolated linfáticos contráteis

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

Imunologia e Infecção

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Um Ensaio de microplacas para avaliar os efeitos químicos de RBL-2H3 degranulação dos mastócitos: Efeitos do Triclosan, sem utilização de solventes orgânicos

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Métodos para a detecção quantitativa de anticorpos induzida pela ativação do complemento no Glóbulos Vermelhos

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Análise dos efeitos de células estromais no recrutamento de leucócitos de Fluxo

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Citometria de fluxo de partículas ligadas aos Bet v 1 alérgeno no PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

Identificação de um epítopo de célula B linear Monoclonal Antibody-péptido reconhecido Digitalização-assistida

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Teste de tolerância à glicose intraperitoneal, medição da função pulmonar e a fixação do pulmão para estudar o impacto da obesidade e metabolismo prejudicado em resultados pulmonares

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Avaliação dos efeitos de drogas baseados em anticorpo murino da medula óssea e ativação de macrófagos peritoneais

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Um método para a medição da função da glândula salivar em ratos

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

Construção do fago sintética exibida Fab biblioteca com diversidade sob medida

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...