Este trabalho foi financiado pelo Fundo de Ciência Austríaco (FWF) conceder P18820-B13. Declaração de ética: O estudo foi aprovado (PLUS_Ethik_090514) pelo Comitê de Ética de experimentos envolvendo seres humanos e / ou Animais da Universidade de Salzburg cumprimento das Declaraction de Helsínquia revista em 1983 e todos os pacientes participantes deram seu consentimento informado.

1. Preparação de conjugados de medicamentos <ol><li> Dissolver 10 mg DF em 1 ml dH 2 O em um tubo de reação de 1,5 ml. No uso de prosseguir 95 mL desta solução DF. Conjugar PP para HSA dissolver 30,4 mg de derivados PP (304 g / mol) em 500 mL de hidróxido de sódio 0,3 M (NaOH). </li><li> Adicionar 430,6 mL dH 2 O e 100 l 0,5 M 2 - (N-morfolino) etanosulfónico (MES) pH 6,5-95 mL de DF dissolvido. Adicionar 33,1 mL dH 2 O e 140 mL de 2 M MES pH 2,9 para a amostra PP. </li><li> Adicionar 57,5 mL de uma recém-preparados N-etil-N &#39;- (3-dimetilaminopropil) carbodiimida cloridrato de solução-mãe (EDC) dissolvido em dH 2 O com uma concentração de 100 mg / ml de amostra DF, ou 10 mL de solução EDC a amostra PP. Vortex as amostras para um minuto. </li><li> Adicionar 16,9 ml de solução HSA com uma concentração de 118 mg / ml para cada amostra. </li><li> Incubar as amostras por 2 horas em temperatura ambiente, agitando a 300 rounds / min. </li><li> Diálise do DF e amostras de PP 3 vezes contra 2 litros de tampão fosfato-salina pH (PBS) 7,4 por várias horas cada. Executar todas as etapas diálise a 4 ° C. </li><li> Centrifugar a amostra de 10 minutos a 14.000 xg e recolher o sobrenadante contendo DF ou PP conjugado com HSA. </li><li> Verifique sobrenadante para as proteínas através da realização de um SDS-PAGE, utilizando géis de 12%. Carga de cerca de 12 microgramas de HSA conjugado em um slot de gel. </li></ol> 2. Determinar a taxa de acoplamento de conjugados de medicamentos (Figura 2) <ol><li> Para a análise da taxa de acoplamento do DF e conjugados PP por HPSEC usar um de sódio 100 mM de tampão fosfato pH 6,5 contendo NaCl 150 mM e azida sódica 0,05%. Use um x 7.8 300 milímetros TSK-Gel-G2000 coluna SWXL. </li><li> Realizar a detecção de sinais usando um RI-online acoplada e um sistema de detector UV. </li><li> Preparar uma amostra HSA padrão com uma concentração de 1 mg / ml em dH 2 O. Para a calibração de detector injetar 50 ul HSA solução padrão. </li><li> Calcular o RI RI k constante ea UV UV k constante para o detector. Use a área de RI fórmulas = RI k * [(dn / dc) HSA * c (HSA)] e área de UV UV = k * [(dA / dc) HSA * c (HSA)] com (dn / dc) HSA = 0,185 e (dA / dc) HSA = 0,518 5. </li><li> Prepare DF e padrões PP derivados para HPSEC que 5, 10 e 30 nmol quantidades de ambos os padrões podem ser facilmente injetado. </li><li> Calcule os valores médios de (dn / dc) de drogas e (dA / dc) de drogas para ambos os padrões. Para DF, a (dn / dc) de 0,235 ± 0,010 e um (dA / dc) de 39,000 ± 0,493 foi determinado. Para PP, a (dn / dc) de 0,328 ± 0,016 e um de (dA / dc) 53,155 ± 2,464 foi determinado. </li><li> Injetar conjugados de medicamentos em HPSEC e determinar o seu RI e UV áreas dos picos. </li><li> Calcular a concentração de DF, derivado PP, e HSA, resolvendo as duas equações área RI RI = k * [(dn / dc) de drogas * c (droga) + (dn / dc) HSA * c (HSA)] UV área e = k UV * [(dA / dc) de drogas * c (droga) + (dA / dc) HSA * c (HSA)] para as incógnitas. </li></ol> 3. BAT usando conjugados de medicamentos <ol><li> Prepare sete taças citometria de fluxo e rotulá-las dependendo do seu conteúdo: controle sem manchas, controle negativo, controle positivo, e conjugados de drogas. </li><li> Pipetar 50 ul de B-CCR-STB buffer de estimulação (Flow2 CAST, Bühlmann Laboratories, Schönenbuch, CH) nos frascos reservados para o controle imaculada e negativos. Como controle positivo, use 50 ul de uma solução anti-FcεRI fornecido pelo kit CAST Flow2. Adicionar as quantidades apropriadas de solução do conjugado aos frascos reservado para a conjugados de medicamentos através da criação de uma série diluição de 1:10 de 0,02 -20 mg / ml DF conjugado e PP conjugada (concentração final no ensaio de um volume de 200 mL). Estes experimentos de titulação deve ser realizada para determinar a concentração de activação de basófilos ideal para cada amostra. </li><li> Adicionar 100 mL de buffer estimulação a cada tubo e sangue de 50 mL de EDTA paciente todo. Misture as amostras com cuidado. </li><li> Pipetar 20 l Flow2 reagente coloração CAST contendo anticorpos anti-CCR3 marcado com ficoeritrina (PE) e anticorpos anti-CD63 marcado com isotiocianato de fluoresceína (FITC) a cada tubo e misture novamente. Não pipetar reagentes coloração na amostra imaculada! </li><li> Incubar as amostras por 45 minutos em banho-maria a 37 ° C. </li><li> Reação de parada no gelo por 5 minutos. </li><li> Lisar eritrócitos pela adição de 2,0 ml de reagente de lise CAST Flow2 a cada frasco e vortex suavemente. Incubar as amostras no escuro por 10 minutos em temperatura ambiente. </li><li> Centrifugar amostras por 5 minutos a 500 xg eo sobrenadante cuidadosamente decantar. </li><li> Ressuspender as células em 500 mL de tampão de lavagem Flow2 CAST e armazenar em gelo até citometria de fluxo. </li><li> S Ajuste o citômetro de fluxo de tensãoettings para dispersão de frente e de lado (FSC, SSC), enquanto a aquisição da amostra imaculada. </li><li> Porta das células como linfócitos previsto da parcela SSC-FSC para a trama SSC-PE. Basófilos são identificados por um PE-anticorpo marcado anti-CCR3 como CCR3 oi SSC lo e porta-los para o enredo FITC-PE. O anticorpo anti-CD63 detectar basófilos ativados é marcado com FITC. </li><li> Definir o corte de no máximo 2,5% de basófilos CD63 como oi no controle negativo. Uma amostra é considerada positiva para a ativação de basófilos se&gt; 5% são basófilos CD63 oi eo índice médio de fluorescência (IMF) da amostra dividido pelo MFI do controle negativo superior a 2 (Figura 3). </li></ol> 4. Resultados representativos: Este experimento avalia BAT como uma ferramenta útil para detectar IgE-dependente hipersensibilidade a drogas. Conjugados de proteína de AINEs são usados ​​para a ativação de basófilos. Por HPSEC com RI e do estado de agregação de detecção UV, o grau de acoplamento, e de rendimento efetivo dos conjugados são determinados. A Figura 4 mostra como, 43,5% dos conjugados DF permaneceu monomérica com um grau de acoplamento de 5,0 DF / HSA. Para os agregados um grau de acoplamento de 9,5 foi determinado. O conjugado propyphenazone mostrou consistem em monômeros 68,5% com um grau de acoplamento de 21,2. O grau de acoplamento dos agregados foi de 27,9. No total, o grau de acoplamento determinada de conjugados DF foi de 7,6 DF e que de conjugados de PP foi de 23,2. BAT realizada com conjugados em condições otimizadas (concentração de conjugados, tempo de estimulação) permitiu a investigação da IgE-dependente reações de hipersensibilidade em AINE. Conforme mostrado na Figura 5, apenas o conjugado PP foi capaz de desencadear a activação de basófilos visualizado por uma upregulation na expressão de CD63 de superfície: 20,6% de basófilos foram ativados caracterizada por uma mudança de log-in intensidade de fluorescência. A IFM aumentou por um fator de 4,9 de 386 (controle negativo) para 1893. Em contraste, usando o conjugado DF apenas 3,1% dos basófilos foram ativadas que estava abaixo dos 5% de corte. Além disso, o IFM aumentou apenas por um fator 1,1 (limite de 2.0) a partir de 197 (controle negativo) para 210. Validade do ensaio é dado por (i) &lt;activação de basófilos 5% no controle negativo, (ii) uma mudança de log-in intensidade de fluorescência de uma subpopulação basófilos, e (iii)&gt; 50% basófilos ativados (controle positivo). <img alt="Figura 1" src="/files/ftp_upload/3263/3263fig1.jpg" /> Figura 1. Acoplamento de derivados DF e PP para HSA. Depois de dissolver a droga, a água, MES buffer, e EDC (reagente de acoplamento) são adicionados. As amostras são agitadas durante 1 minuto antes HSA é pipetado para as soluções de drogas. Dentro de duas horas agitando à temperatura ambiente as drogas covalentemente se ligam a HSA. Monômeros, bem como agregados podem resultar. <img alt="Figura 2" src="/files/ftp_upload/3263/3263fig2.jpg" /> Figura 2. Cálculo de graus de acoplamento. Em primeiro lugar, as constantes k RI e UV k são determinados. Portanto, HSA padrão com concentração conhecida é injetada no sistema HPSEC. (Dn / dc) e (dA / dc) de HSA são dados os valores de 0,185 e 0,518, respectivamente 5. As áreas dos picos são usados ​​para calcular as constantes. Segundo, para cada três padrões de drogas são injetadas para determinar droga e da concentração específicos e áreas de RI UV. Uma vez que o RI k e constantes UV k são conhecidos a partir das etapas acima, os derivados de drogas específicas (dn / dc) e (dA / dc) pode ser calculado. Terceiro, drogas HSA conjugados são injetadas em HPSEC. As áreas dos picos resultantes são usados ​​para calcular as concentrações de drogas e por HSA de pico, resolvendo as duas equações com duas variáveis ​​desconhecidas. <img alt="Figura 3" src="/files/ftp_upload/3263/3263fig3.jpg" /> Figura 3. Gating Basófilo. Células previsto como linfócitos são dispersão lado gated relação ao dispersor de frente (SSC-enredo FSC). Os basófilos são identificados como CCR3 oi SSC lo a partir dos linfócitos gated (SSC-CCR3-PE enredo). Basófilos são analisados ​​para ativação (CD63 oi) na parcela CD63-FITC-CCR3-PE. O controle negativo é usado para definir o corte de no máximo 2,5% restantes basófilos CD63 oi. <img alt="Figura 4" src="/files/ftp_upload/3263/3263fig4.jpg" /> Figura 4. Graus de acoplamento Determinado de conjugados de medicamentos. RI e os sinais UV mostram dois picos representando agregados (eluição com um volume de retenção baixa) e monômeros (eluição com um volume de retenção de altura) de drogas HSA conjugados. Percentagens de monômeros e agregados (determinado a partir de sinais de RI) e seus graus de acoplamento são retratados (n = 1). <img alt="Figura 5" src="/files/ftp_upload/3263/3263fig5.jpg" /> Figura 5. Basófilo Activatiem teste. Resultados da droga conjugada ativado amostras, controles negativos e controles positivos são mostrados na CD63-FITC-CCR3-PE parcelas (n = 1).

<ol>
	<li>Himly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgE-mediated+immediate-type+hypersensitivity+to+the+pyrazolone+drug+propyphenazone.">IgE-mediated immediate-type hypersensitivity to the pyrazolone drug propyphenazone.</a> J Allergy Clin Immunol. 111, 882-888 (2003).</li><li>Demoly, P., Pichler, W., Pirmohamed, M., Romano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+questions+in+Allergy:+1--drug+allergy/hypersensitivity.">Important questions in Allergy: 1--drug allergy/hypersensitivity.</a> Allergy. 63, 616-619 (2008).</li><li>Ebo, D. G., Hagendorens, M. M., Bridts, C. H., Schuerwegh, A. J., Clerck, L. S., Stevens, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+in+vitro+activated+basophils,+specific+IgE+and+skin+tests+in+the+diagnosis+of+pollen-associated+food+allergy.">Flow cytometric analysis of in vitro activated basophils, specific IgE and skin tests in the diagnosis of pollen-associated food allergy.</a> Cytometry Part B (Clinical Cytometry). 64B, (2005).</li><li>Harrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diclofenac+hypersensitivity:+antibody+responses+to+the+parent+drug+and+relevant+metabolites.">Diclofenac hypersensitivity: antibody responses to the parent drug and relevant metabolites.</a> PLoS One. 5, e13707-e13707 (2010).</li><li>Wen, J., Arakawa, T., Philo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-exclusion+chromatography+with+on-line+light-scattering,+absorbance,+and+refractive+index+detectors+for+studying+proteins+and+their+interactions.">Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions.</a> Anal Biochem. 240, 155-166 (1996).</li><li>De Weck, A. L., Sanz, M. L., Gamboa, P. M., Aberer, W., Bienvenu, J., Blanca, M., Demoly, P., Ebo, D. G., Mayorga, L., Monneret, G., Sainte Laudy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+tests+based+on+human+basophils:+more+potentials+and+perspectives+than+pitfalls.+II.+Technical+issues.">Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls. II. Technical issues.</a> J Investig Allergol Clin Immunol. 18, 143-155 (2008).</li><li>Ebo, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-assisted+allergy+diagnosis:+current+applications+and+future+perspectives.">Flow-assisted allergy diagnosis: current applications and future perspectives.</a> Allergy. 61, 1028-1039 (2006).</li><li>Poulsen, L. K., Quan, S., Kragh, C., Platzer, M. H., Skov, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basophil+granulocyte+in+allergic+reactions:+experimental+models+and+their+use+for+the+identification+of+drugs+with+effects+or+side+effects+on+basophils.">The basophil granulocyte in allergic reactions: experimental models and their use for the identification of drugs with effects or side effects on basophils.</a> Curr Med Chem - Anti-inflammatory &#38; Anti-Allergy Agents. 3, 167-180 (2004).</li><li>Sanz, M. L., Gamboa, P., de Weck, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+combined+test+with+flowcytometric+basophil+activation+and+determination+of+sulfidoleukotrienes+is+useful+for+in+vitro+diagnosis+of+hypersensitivity+to+aspirin+and+other+nonsteroidal+anti-inflammatory+drugs.">A new combined test with flowcytometric basophil activation and determination of sulfidoleukotrienes is useful for in vitro diagnosis of hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs.</a> Int Arch Allergy Immunol. 136, 58-72 (2005).</li></ol>

Não há conflitos de interesse declarados.

BAT é um bem estabelecida, embora ainda não utilizado rotineiramente método para diagnóstico de IgE mediada doença alérgica 6,7. Para hipersensibilidade a drogas, no entanto, sua aplicabilidade é comprometida, como compostos de baixo peso molecular não são capazes de receptores IgE crosslink, um pré-requisito para a ativação de basófilos 8. Portanto, as drogas sob investigação precisam ser covalentemente acoplado a proteínas de transporte adequado (por exemplo HSA). Importante, o grau de acoplamento (ie número de moléculas de droga por proteína transportadora) precisa ser controlada para garantir a atividade imunológica (ie receptor IgE cross-linking) de conjugados. Teoricamente, dois haptenos por molécula transportadora deve ser suficiente para o receptor de IgE de ligação cruzada e apenas cinco moléculas DF por HSA foram mostrados para desencadear a liberação de mediador em um ensaio baseado em células 4. Ambos os conjugados, PP-HSA e DF-HSA, foram determinados para mostrar um grau suficientemente elevado de acoplamento (HPSEC) e ser imunologicamente ativas tornando-os reagentes adequados para uso em BAT. Outro problema pode ser que os metabolitos de drogas pode ter um papel, como um metabólito em vez do fármaco pode causar a reação de hipersensibilidade. No caso do DF, essa possibilidade tem sido avaliada em detalhes anteriormente usando cinco principais Fase I metabólitos e uma variante de ligação 4. Fatores importantes para dominar a técnica descrita incluem a qualidade da amostra de sangue (&lt;12 horas desde a coleta de sangue, número de basófilos detectado&gt; 500) e otimização das condições de estimulação (dose, tempo). Além disso, os chamados não-respondedores, que nem sequer reagem com o controle positivo (anticorpos dirigidos contra o receptor IgE) têm de ser identificados. Portanto, critérios de validação tem que incluir um anticorpo anti-FcεRI como controle positivo. Uma vantagem importante do uso de conjugados de medicamentos é o fato de que eles parecem não tóxicos, em contraste com as drogas pura, como mostra a DF-HSA conjugado por co-estimulação com anticorpo anti-FcεRI na BAT 4. Pure DF, em contraste, exibe efeitos citotóxicos em uma concentração de 1,25 mg / ml causando problemas com a interpretabilidade da BAT 9. Geralmente, testes para citotoxicidade potencial é fortemente recomendado para cada conjugado droga recém-produzidos. Aqui, nós mostramos o potencial do PP-HSA conjugado usado em BAT para revelar hipersensibilidade mediadas por IgE. Em contraste, DF hipersensibilidade não está associada com IgE demonstrado pela falta de ativação de basófilos em resposta a DF-HSA conjugado. Importante, em caso de incerteza sobre um mecanismo IgE mediada, conjugados têm de ser avaliados para a atividade imunológica por ensaio in vitro em sistemas baseados em células como tem sido demonstrado por DF 4. Usando a configuração descrita de conjugar baixo peso molecular drogas covalentemente a proteína transportadora adequada moléculas uma série de reações de hipersensibilidade à droga, incluindo antibióticos, outros AINEs, radiocontrast mídia, relaxantes musculares, anestésicos, etc pode ser investigado por um envolvimento de uma mediada por IgE mecanismo. Assim, BAT pode servir como um complemento para os métodos existentes para o diagnóstico (teste cutâneo, o teste de provocação oral).

<table border="1"><tbody><tr><th> Nome do reagente / dispositivo </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> 2 - (N-Morfolino) etanosulfónico </td><td> Sigma </td><td> M3671 </td><td> - </td></tr><tr><td> 7,8 x 300 mm TSK Gel -G2000SWXL coluna </td><td> Tosoh Bioscience </td><td> 08540 </td><td> - </td></tr><tr><td> BD FACSCanto II </td><td> Becton Dickinson </td><td> 338962 </td><td> - </td></tr><tr><td> Centrífuga de bancada </td><td> Sigma </td><td> - </td><td> - </td></tr><tr><td> Diclofenaco de sódio </td><td> Sigma </td><td> D6899 </td><td> - </td></tr><tr><td> EDTA-Vacutainer </td><td> Becton Dickinson </td><td> 368589 </td><td> - </td></tr><tr><td> Flow2 Kit CAST </td><td> Bühlmann Laboratories </td><td> FK-CCR </td><td> Kit CAST eficazes 14 de junho de 2011 Flow2 agora é CAST Fluxo </td></tr><tr><td> HP1100 sistema HPLC </td><td> Hewlett Packard </td><td> - </td><td> - </td></tr><tr><td> Albumina de soro humano </td><td> Sigma </td><td> A9511 </td><td> - </td></tr><tr><td> Centrifugadora de laboratório </td><td> Eppendorf </td><td> - </td><td> - </td></tr><tr><td> N-etil-N&#39;-(3-dimetilaminopropil) cloridrato de carbodiimida (EDC) </td><td> Sigma </td><td> E6383 </td><td> - </td></tr><tr><td> Comprimidos PBS </td><td> AppliChem </td><td> A9199 </td><td> - </td></tr><tr><td> Derivados Propyphenazone </td><td> Departamento de Biologia Molecular, Universidade de Salzburg, Áustria </td><td> - </td><td> - </td></tr><tr><td> Sacudidor </td><td> Eppendorf </td><td> - </td><td> - </td></tr><tr><td> Azida de sódio </td><td> Sigma </td><td> S8032 </td><td> - </td></tr><tr><td> Cloreto de sódio (NaCl) </td><td> Sigma </td><td> S1679 </td><td> - </td></tr><tr><td> Bicarbonato de sódio </td><td> Sigma </td><td> 90421C </td><td> - </td></tr><tr><td> Hidróxido de sódio </td><td> Sigma </td><td> S5881 </td><td> - </td></tr><tr><td> Fosfato de sódio </td><td> Sigma </td><td> 342483 </td><td> - </td></tr><tr><td> Conjunto detector triplo </td><td> Viscotek </td><td> TDA302 </td><td> - </td></tr><tr><td> BioVortex pessoal V-1 mais </td><td> Peqlab </td><td> 90-V-1 </td><td> - </td></tr><tr><td> Banho-maria 1012 </td><td> GFL </td><td> 1012 </td><td> - </td></tr></tbody></table>

basophil activation test for investigation of ige-mediated mechanisms in drug hypersensitivity

Reações de hipersensibilidade contra os não-esteróides anti-inflamatórios não esteróides (AINE), como propyphenazone (PP) e diclofenaco (DF) pode se manifestar como Tipo I-como reações alérgicas 1. Na prática clínica, o diagnóstico de hipersensibilidade a drogas é realizado principalmente pelo histórico do paciente, como testes de pele não é de confiança e testes de provocação oral tem risco de vida riscos para o paciente 2. Daí, a evidência de uma pathomechanism IgE mediada subjacente é difícil de obter. Aqui, apresentamos um método in vitro baseado no uso de basófilos humano, derivados de drogas pacientes hipersensíveis que imita a reação alérgica efetoras in vivo. Como basófilos de drogas pacientes alérgicos transportar moléculas de IgE específica para a droga culpado, eles se tornam ativados durante a IgE crosslinking receptor e liberar alérgica moléculas efetoras. A ativação de basófilos pode ser monitorado pela determinação da expressão de CD63 upregulation de superfície por citometria de fluxo 3. No caso de drogas baixo peso molecular, conjugados são projetados para permitir IgE crosslinking receptor em basófilos. Como mostrado na Figura 1, dois representantes de AINEs, PP e DF, são covalentemente ligado à albumina sérica humana (HSA), através de um grupo carboxila reagem com o grupo primário amino de resíduos de lisina. DF transporta um grupo carboxila intrínseca e, portanto, pode ser usado diretamente 4, enquanto um grupo carboxila contendo derivados do PP teve de ser sintetizada organochemically antes do estudo 1. O grau de acoplamento dos compostos de baixo peso molecular da molécula de proteína transportadora e sua distribuição espacial é importante para garantir crosslinking de duas moléculas de receptor IgE. O protocolo aqui descrito aplica-se cromatografia de exclusão de alto desempenho de tamanho (HPSEC) equipado com um índice de refração seqüencial (RI) e ultra-violeta (UV) sistema de detecção para a determinação do grau de acoplamento. Como a metodologia descrita pode ser aplicada para outras drogas, o teste de activação de basófilos (BAT) tem o potencial para ser utilizado para a determinação de IgE mediada por mecanismos de hipersensibilidade a drogas. Aqui, nós determinamos como PP hipersensibilidade mediadas por IgE e hipersensibilidade DF como não-IgE mediada pela BAT.

Watch this Scientific Journal Video about Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity at JoVE.com

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Basófilos teste de ativação é uma potente ferramenta para a detecção de IgE-dependente alergias<em&gt; In vitro</em&gt;. Aqui, um protocolo otimizado para a ativação de basófilos teste é utilizado para investigar hipersensibilidade a drogas. Um método para a produção eficiente de drogas covalente de proteínas e seus conjugados de caracterização físico-química é descrito.

Basófilos Teste de ativação de Investigação de IgE-mediada Mecanismos Hipersensibilidade a Drogas

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

basophil-activation-test-for-investigation-ige-mediated-mechanisms

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Immunology and Infection

Unit 1

Adverse Drug Effects and Chemical Toxicity

SCIENTISTS IN ACTION

33.3K visualizações.  University of Salzburg. Basófilos teste de ativação é uma potente ferramenta para a detecção de IgE-dependente alergias In vitro. Aqui, um protocolo otimizado para a ativação de basófilos teste é utilizado para investigar hipersensibilidade a drogas. Um método para a produção eficiente de drogas covalente de proteínas e seus conjugados de caracterização físico-química é descrito.

Vídeo: Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modelo murino de CD40-ativação de células B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Imunologia e Infecção

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Facilitar Drug Discovery: um ensaio automatizado de alta Inflamação conteúdo em Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Trans-vivo Atraso Tipo Ensaio Hipersensibilidade para o Antigen Regulamento Específico

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Ativação e Mensuração de NLRP3 inflammasome Atividade Usando IL-1β em células dendríticas humanas Monocyte derivados

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Estática Adesão Ensaio para o Estudo da integrina Ativação em Linfócitos T

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

De alto rendimento quantitativo em tempo real de RT-PCR Ensaio para Determinar Perfis de Expressão do tipo I e III do interferão subtipos

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

In Vivo Investigation of Antimicrobial Therapy luz azul para-multirresistente Acinetobacter baumannii queimadura Infecções Usando bioluminescência Imagiologia

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Indução de Hepatite Autoimune Induzida por Drogas em CAMUNDONGOS BALB/c para o Estudo de Seus Mecanismos Patogênicos

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

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

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

Teste in vitro ELISA para avaliar a potência da vacina antirrábica

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

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.