Los autores desean agradecer a Amanda Tardo, el Dr. Mostafa Bouljihad, Feely Stephanie, Coyne Carol, la Dra. Erin Ribka, el Dr. Pete Didier y Kuebler Dorothy por su asistencia técnica. Este trabajo fue apoyado por subvenciones del NIH R01-DK076653 y 3R01-DK076653-02S1 para KS y la subvención de funcionamiento de la base de TNPRC RR000164.

1. La biopsia de piel la recogida de muestras <ol><li> Antes de la biopsia de piel, anestesiar a los animales por vía intramuscular con 2,5 mg / kg de clorhidrato de tiletamina y zolazepam mezcla de clorhidrato de Telazol (Fort Dodge Animal Health, Fort Dodge, IA). Monitor de los animales de la administración de anestesia hasta postración y retire de su gabinete. </li><li> Eliminar el vello de la piel del área de interés utilizando un Oster A5 de Oro Clipper velocidad Veterinaria individual con un tamaño de 40 cuchillas (Oster Professional Products, McMinnville, TN) y preparar asépticamente con alternancia de matorral Betadine y alcohol. Asegurar un paño fenestrado estéril sobre el sitio de la biopsia seleccionado. </li><li> Usando una técnica estéril, coloque un 4,0 mm Miltex instrumento perforación cutánea Biopsia (Miltex, York, PA) contra la piel mientras se gira el instrumento 180 grados en sentido horario y antihorario con una ligera presión hasta que la biopsia por punción de transectos a través de la dermis en el ti subcutáneaCUESTIÓN. Retire la muestra de la biopsia y agarre la parte seccionada de la piel con pinzas y sin el tejido subcutáneo. </li><li> Cierre el defecto de la piel con sutura de nylon 3-0 unido a una aguja 3 / 8 círculo de corte (Ethilon, Ethicon, Johnson &amp; Johnson Medical Limited, Berkshire, Reino Unido) en un patrón cruzado. Dar a todos los animales de 0,01 mg / kg de hidrocloruro de buprenorfina (Hospira, Lake Forest, IL) por vía intramuscular para la analgesia postoperatoria. </li></ol> 2. Procesamiento de la muestra <ol><li> Trabajar con muestras de biopsia de la piel dermatitis crónica y controles sanos macacos rhesus. La obtención de dos a tres (4 mm de diámetro) las muestras de biopsia de cada animal. </li><li> Fix primera muestra en formol zinc (Z-fix, Anatech Ltd., Battle Creek, MI) durante 24 horas, lave en agua durante 30 minutos, lave brevemente en etanol al 70%, y el lugar en ASP300 procesador de tejidos Leica (Leica Microsystems Inc. , Buffalo Grove, IL), donde los tejidos se deshidratan con los grados de ascenso de 70%, 80%, 95% y 100% de etanol 48minutos cada uno (Fisher Scientific, Pittsburgh, PA), seguido por dos cambios de xileno (Fisher). </li><li> Insertar en los medios de parafina (Fisher) para almacenamiento a largo plazo a temperatura ambiente. Lugar a -20 º C durante 20 minutos antes de cortar. Prepare 6 secciones micras con un micrótomo rotatorio (HM325, Microm Internacional, Waldorf, Alemania). Se colocan en las diapositivas cargada (Fisher) y aire seco a 60 º C durante la noche. Tinción con hematoxilina y eosina (H &amp; E) método estándar (descrito más adelante). </li><li> Fix segunda muestra en paraformaldehído al 2% (USB Corp, Cleveland, OH) durante 30 min a temperatura ambiente, lavar tres veces en tampón fosfato salino (PBS, Gibco-Invitrogen, Carlsbad, CA), lugar en el 30% de sacarosa (Fisher) para 4 horas, e incluir en el medio de congelación octubre (Sakura Finetek, Torrence, California). Mantener a los 80 ° C durante 20 minutos antes de cortar. Prepare 15 secciones micras utilizando el criostato (HM560, Thermo Scientific, Kalamazoo, MI). </li></ol> 3. H &amp; E tinción <ol><li> Desparafinar incrustarsecciones ded a través de tres cambios de xileno (Fisher) y rehidratar a través de etanoles clasificados: tres cambios de 100%, dos cambios de 95%, un cambio del 80% y agua destilada, a 2 minutos cada uno. </li><li> Tinción con hematoxilina (Richard Allan-Científico, Kalamazoo, MI) durante un minuto y seguir por un breve lavado en agua corriente del grifo. </li><li> Contratinción con eosina (Sigma) durante 6 minutos. </li><li> Deshidratar los tejidos teñidos a través del 95% y etanol absoluto, dos cambios de 2 minutos cada uno y claro en tres cambios de xileno, a 3 minutos cada uno. </li><li> Monte cubre objetos usando Medio Cytoseal de montaje (Richard Allan-científico) en el H &amp; E-manchado secciones de tejido. </li></ol> 4. Evaluación histopatológica Inspeccione H &amp; E-manchado secciones biopsia de la piel bajo un microscopio óptico con el 4 - 40 aumentos y un microscopio Leica con un punto penetración de cámaras digitales (Digital Instruments Inc., Michigan, EE.UU.). 5. Inmunofluorescencia staiNing <ol><li> Trabajar con secciones de piel octubre embebido para la tinción de inmunofluorescencia. Antes de la tinción, en remojo durante 20 min en PBS que contenía 0,2% de gelatina de piel de pescado (FSG, Sigma) con 0,1% Triton X-100 (Sigma) para eliminar los PTU y para permeabilizar el tejido. </li><li> Después de lavar las diapositivas, se incuban con 10% de suero normal de cabra (NGS, Sigma), diluido en PBS-FSG durante 1 hora a temperatura ambiente, en una cámara de diapositivas humidificado. </li><li> Para poder visualizar el depósito de IgA y anticuerpos tTG2, Incubar los portaobjetos con anti-rhesus IgA y anticuerpos primarios tTG2, diluido en un 10% NGS-PBS-FSG durante 1 hora. </li><li> Lavar con PBS-FSG-Triton X-100 (Sigma) durante 10 minutos, seguido por PBS-FSG durante 10 minutos. </li><li> Se incuba con el secundario, isotipo de los anticuerpos específicos marcados con fluorocromo Alexa Fluor 488 (verde), 568 (rojo) o 633 (azul) (Molecular Sondas-Invitrogen) a 1:1000 dilución. El uso del tinte ToPro-3 para visualizar el ADN nuclear. Utilizar el 10% de NGS-PBS-FSG como diluyente para anticuerpos. </li><li> Use la solución anti-temple (Sigma) para montar las secciones de tejidos teñidos. </li></ol> 6. Microscopía confocal Realizar imágenes con una Leica TCS SP2 verdadero microscopio de escaneo láser confocal (Leica, Wetzlar, Alemania) equipado con tres láseres (seis líneas de excitación está disponible): un láser de argón, kriptón a 488 nm (verde), un láser de criptón a 568 nm ( rojo) y un láser de helio-neón de 633 nm (azul), que abarcan desde el visible hasta el otro lado rojo del espectro. Registro de contraste de interferencia diferencial (DIC) imágenes para la evaluación de los no etiquetados arquitectura del tejido. 7. Resultados representante Un ejemplo de un reconocimiento positivo de la ETG por tanto tTG2 e IgA se muestra en la fig. 3A. De acuerdo con la situación en pacientes humanos, y debido a la prevalencia mucho más baja de DH-como dermatitis de CD-como la sensibilidad al gluten también en los macacos rhesus, la mayoría de las muestras de biopsia de piel se espera que show la reacción positiva con anticuerpos tTG2 pero sin la presencia de tTG2 + IgA + doble células positivas (Fig. 4). Estos casos DH-relacionados, incluyendo casos de dermatitis de origen microbiano, se revelan algunas células de un solo positivo IgA + esparcidos por debajo del epitelio, pero no solapamiento espectral entre las tTG2 e IgA marcados con marcadores (Fig. 3B y 4). Por lo tanto, es importante para mantener el orden de los procedimientos de prueba sugerido, a partir de la exploración clínica (Fig. 1), seguida de evaluación histopatológica (Fig. 2) y sólo si no que causan dermatitis y otras más comunes agentes pueden ser identificados confocal, examen microscópico de biopsias de piel manchada para tTG2 e IgA se justifica. Las evaluaciones clínicas y / o histopatológicos no son suficientes para el diagnóstico positivo de esta condición. En los casos en doble positivo (+ tTG2 IgA +) las células se identifican en el área de las papilas dérmicas, DH-como el diagnóstico dermatitis pueden ser emitidas. En tales casos, la retirada del gluten de la dietay la administración de la dieta sin gluten se recomienda con el fin de aliviar los síntomas de la dermatitis. <img src="/files/ftp_upload/3154/3154fig1.jpg" alt="Figura 1" /> Figura 1. Región inguinal de EM96 macaco antes (A) y después (B) la retirada del gluten de la dieta durante 5 semanas. La reintroducción del gluten de la dieta coincidió con la reaparición de la dermatitis (C). <img src="/files/ftp_upload/3154/3154fig2.jpg" alt="Figura 2" /> Figura 2. HE tinción de las biopsias de piel EM96 macacos recogidos antes (A) y después (B) la retirada del gluten de la dieta durante 9 semanas. R: El epitelio se espese (7-15 capas de células epiteliales), con una gruesa capa superficial de células hiperqueratósicas infiltrado de neutrófilos en degeneración. Los neutrófilos aparecen también en el interior del epitelio y se mezclan con un número moderado de linfocitos, células plasmáticas e histiocitos alrededor de los anexos en la dermis superior. B: Algunos hyperkeratosis todavía está presente, el espesor del epitelio está normalizado (4-7 capas) con mínima infiltración linfoplasmocitario en la dermis superior. C: La piel normal abdominal de FR56 macacos, sin infiltración linfoplasmocitaria, se incluye para la comparación. Magnificación de 20x. <img src="/files/ftp_upload/3154/3154fig3.jpg" alt="Figura 3" /> Figura 3. Imágenes de microscopía confocal de EM96 biopsias de piel macacos recogidos antes (A) y después (B) la retirada del gluten de la dieta durante 9 semanas. A: tinción IgA (verde), tTG2 (rojo) y ToPro-3 de marcadores de ADN nuclear (azul). Co-localización de IgA y tTG2 aparece en amarillo en la zona de papilae dérmica. B: muestra de piel tomada después de 9 semanas de dieta sin gluten no muestra IgA en el epitelio, mientras que algunos subepitelial de IgA está presente (verde), tTG2 (rojo) y ToPro-3 de marcadores de ADN nuclear (azul). <img src="/files/ftp_upload/3154/3154fig4.jpg" alt="Figura 4" /> Figura 4. </strong&gt; imágenes de microscopía confocal de GI24 biopsias de piel macaco rhesus recogidos, mientras que los animales se presentan los síntomas de las enfermedades crónicas (DH-relación) dermatitis. La tinción de IgA es de color verde, de rojo y tTG2 ToPro-3 de marcadores de ADN nuclear es de color azul. Las células + tTG2 están presentes en el interior del epitelio, algunas células IgA + por debajo del epitelio, mientras que no se superponen amplio espectro entre los marcadores de color rojo y verde que se ve. <table border="1"><tbody><tr><td> Animal Etiqueta </td><td> Sexo [M / F] </td><td> Edad [Años] </td><td> Los síntomas clínicos </td><td> Plasma tTG2 anticuerpos </td><td> Dieta [1,2] * </td></tr><tr><td> HJ71 </td><td> F </td><td> 1.3 </td><td> Saludable </td><td> - </td><td> 1 </td></tr><tr><td> HD13 </td><td> M </td><td> 1.4 </td><td> Diarrea sanos, ocasionales </td><td> + </td><td> 1 </td></tr><tr><td> FR56 </td><td> M </td><td> 5.4 </td><td> Diarrea crónica idiopática </td><td> - </td><td> 1 </td></tr><tr><td> EM96 </td><td> F </td><td> 5.0 </td><td> Dermatitis crónica </td><td> + / - (Borderline) </td><td> 1 y 2 </td></tr><tr><td> GI24 </td><td> F </td><td> 4.6 </td><td> Dermatitis crónica </td><td> - </td><td> 1 </td></tr></tbody></table> Tabla 1. TNPRC macacos rhesus utilizados para la recogida de biopsias de piel * Dieta 1 - regular comida mono (con gluten), la dieta 2 - dieta libre de gluten

<ol>
	<li>Diosdado, B., Wijmenga, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+the+adaptive,+innate+and+regulatory+immune+responses+in+the+intestinal+mucosa+of+celiac+disease+patients.">Molecular mechanisms of the adaptive, innate and regulatory immune responses in the intestinal mucosa of celiac disease patients.</a> Expert. Rev. Mol. Diagn. 5 (5), 681-700 (2005).</li><li>Schuppan, D., Dennis, M. D., Kelly, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celiac+disease:+epidemiology,+pathogenesis,+diagnosis,+and+nutritional+management.">Celiac disease: epidemiology, pathogenesis, diagnosis, and nutritional management.</a> Nutr. Clin. Care. 8 (2), 54-69 (2005).</li><li>Sardy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+transglutaminase+(TGase+3)+is+the+autoantigen+of+dermatitis+herpetiformis.">Epidermal transglutaminase (TGase 3) is the autoantigen of dermatitis herpetiformis.</a> J. Exp. Med. 195 (6), 747-757 (2002).</li><li>Rose, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+against+epidermal+transglutaminase+are+a+sensitive+diagnostic+marker+in+patients+with+dermatitis+herpetiformis+on+a+normal+or+gluten-free+diet.">Autoantibodies against epidermal transglutaminase are a sensitive diagnostic marker in patients with dermatitis herpetiformis on a normal or gluten-free diet.</a> J. Am. Acad. Dermatol. 61 (1), 39-43 (2009).</li><li>Bethune, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+transport+and+enzymatic+detoxification+of+gluten+in+gluten-sensitive+rhesus+macaques.">Transepithelial transport and enzymatic detoxification of gluten in gluten-sensitive rhesus macaques.</a> PLoS ONE. 3 (3), e1857-e1857 (2008).</li><li>Mazumdar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+transepithelial+passage+of+the+immunogenic+33-residue+peptide+from+&#x03B1;-2+gliadin+in+gluten-sensitive+macaques.">Visualization of transepithelial passage of the immunogenic 33-residue peptide from &#x03B1;-2 gliadin in gluten-sensitive macaques.</a> PLoS ONE. 5 (4), e10228-e10228 (2010).</li><li>Schuppan, D., Junker, Y., Barisani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celiac+disease:+from+pathogenesis+to+novel+therapies.">Celiac disease: from pathogenesis to novel therapies.</a> Gastroenterol. 137 (6), 1912-1933 (2009).</li><li>Hunt, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+identified+genetic+risk+variants+for+celiac+disease+related+to+the+immune+response.">Newly identified genetic risk variants for celiac disease related to the immune response.</a> Nature Genet. 40 (4), 395-402 (2008).</li><li>Abadie, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+genetic+and+immunological+insights+into+a+model+of+celiac+disease+pathogenesis.">Integration of genetic and immunological insights into a model of celiac disease pathogenesis.</a> Annu. Rev. Immunol. 29, 493-525 (2011).</li></ol>

No hay conflictos de interés declarado.

Nuestra reciente creación no humana modelo de primates de la sensibilidad al gluten se ha utilizado recientemente para estudiar los cambios de permeabilidad intestinal provocada por gluten de la dieta 5,6. Plasma tTG2 anticuerpos, así como intestinal tTG2 + + depósitos de IgA se detectaron en algunos macacos sensibilidad al gluten. Ya que recientemente identificado incidentes raros de dermatitis idiopática en la colonia de macacos rhesus, que no se pudo habitual (infecciosas / metabólicas) etiología est...

<table border="1"><tbody><tr><td> Nombre del reactivo o equipo </td><td> Empresa </td><td> Número de catálogo / Código de producto </td><td> Nota </td></tr><tr><td> Anti-IgA humana-FITC * </td><td> Sigma, St. Louis, MO </td><td> F-5259 </td><td> 1:100 dilución de trabajo </td></tr><tr><td> Anti-Human tTG2 </td><td> Thermo Scientific, Kalamazoo, MI </td><td> MS-300-P1ABX </td><td> 1:100 dilución de trabajo </td></tr><tr><td> Anti-ratón IgG1 ** </td><td> Invitrogen, Carlsbad, CA </td><td> A-21124 </td><td> 1:1000 dilución de trabajo </td></tr><tr><td> ToPro-3 *** sonda de ADN </td><td> Invitrogen, Carlsbad, CA </td><td> T-3605 </td><td> 1:1000 dilución de trabajo </td></tr><tr><td> Zinc formalina (Z-fix) </td><td> Anatech Ltd., Battle Creek, MI </td><td> 171 </td><td></td></tr><tr&gt; <td> Octubre de congelación Medio </td><td> Sakura Finetek, Torrence, CA </td><td> 4583 </td><td></td></tr><tr><td> Cytoseal montaje Medio </td><td> Richard Allan-Científico, Kalamazoo, MI </td><td> 8312-4 </td><td></td></tr><tr><td> Anti-enfriamiento Solución </td><td> Sigma, St. Louis, MO </td><td> P 3130 </td><td></td></tr><tr><td> Ethilon (Sutura de nylon) </td><td> Ethicon, Johnson &amp; Johnson, Berkshire, Reino Unido </td><td> 663G </td><td></td></tr><tr><td> Miltex sacador cutáneo instrumento de biopsia </td><td> Miltex, York, PA </td><td> NA </td><td></td></tr><tr><td> Oster A5 de oro Single Speed ​​Clipper veterinario con un tamaño de 40 cuchillas </td><td> Productos Oster, McMinnville, TN </td><td> 78005-050 </td><td></td></tr><tr><td> Leica procesador de tejidos </td><td> Leica Microsystems Inc., Buffalo Grove, IL </td><td> ASP300 </td><td></td></tr><tr><td> Micrótomo rotatorio </td><td> Microm Internacional, Waldorf, Alemania </td><td> HM325 </td><td></td></tr><tr><td> Criostato </td><td> Thermo Scientific, Kalamazoo, MI </td><td> HM560 </td><td></td></tr><tr><td> Microscopio Confocal </td><td> Leica, Wetzlar, Alemania </td><td> TCS SP2 </td><td></td></tr></tbody></table> * Isotiocianato de fluoresceína ** Secundaria (Alexa Fluor 568-conjugado) anticuerpo utilizado después de tTG2 anticuerpo primario ** Tinte

recognition of epidermal transglutaminase by iga and tissue transglutaminase 2 antibodies in a rare case of rhesus dermatitis

Transglutaminasa tisular 2 (tTG2) es una enzima digestiva intestinal que deamidates ya parcialmente digerido la dieta gluten péptidos de la gliadina por ejemplo. En individuos genéticamente predispuestos, tTG2 desencadena la respuesta autoinmune que se caracteriza por la producción de anticuerpos y tTG2 su deposición directa en 1,2 pequeña pared intestinal. La presencia de anticuerpos de ese tipo constituye una de las características principales de la enfermedad celiaca (EC). Transglutaminasa epidérmica (ETG) es otro miembro de la familia de la transglutaminasa, que también puede funcionar como un autoantígeno en una pequeña minoría de los pacientes con EC. En estos casos relativamente raros, ETG desencadena una reacción auto inmune (una erupción de la piel) clínicamente se conoce como dermatitis herpetiforme (DH). Aunque el mecanismo exacto de la patogenia de CD y DH no se entiende bien, se sabe que tTG2 y ETG epítopos antigénicos compartir que pueden ser reconocidos por los anticuerpos de suero de los pacientes de CD y DH 3,4. (Macaca mulatta) con dermatitis (tabla 1, fig. 1 y 2) se utilizó para estudiar los tejidos afectados. En un animal (EM96) una superposición espectral de IgA y anticuerpos tTG2 (Fig. 3) se demostró. La presencia de doble positivas tTG2 + IgA + células se centró en la epidermis de profundidad, alrededor de las papilas dérmicas. Esto es consistente con las lesiones descritas en pacientes con DH 3. Cuando EM96 fue colocado en una dieta libre de gluten, dermatitis, así como tTG2 + IgA + depósitos desaparecido y ya no eran detectables (Fig. 1-3). Dermatitis reapareció sin embargo, sobre la base de re-introducción del gluten de la dieta en el EM96 (no mostrado). En los macacos otros como animales no relacionados con la dermatitis, la tTG2 + IgA + depósitos no fueron detectados. Dieta libre de gluten-dependiente de la remisión de la dermatitis en el EM96 junto con la presencia de tTG2 + IgA + células en la piel indican una enfermedad autoinmune, como DH-mecanismo para el desarrollo de esta condición. Este es el primer informe de DH-como la dermatitis en los primates no humanos.

Watch this Scientific Journal Video about Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis at JoVE.com

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

La dermatitis herpetiforme (DH) es una enfermedad inflamatoria crónica caracterizada por una reacción autoinmune entre IgA y la epidermis transglutaminasa (ETG). DH se desarrolla en una porción muy pequeña de los pacientes con sensibilidad al gluten y / o enfermedad celíaca. Los resultados de este estudio indican que DH también puede desarrollarse en una gran mono rhesus con síntomas de la dermatitis idiopática.

Reconocimiento de la transglutaminasa epidérmica por IgA y anticuerpos transglutaminasa tisular 2 en un raro caso de Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

recognition-epidermal-transglutaminase-iga-tissue-transglutaminase-2

Research

JoVE Journal

Immunology and Infection

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

24.4K vistas.  Tulane National Primate Research Center. La dermatitis herpetiforme (DH) es una enfermedad inflamatoria crónica caracterizada por una reacción autoinmune entre IgA y la epidermis transglutaminasa (ETG). DH se desarrolla en una porción muy pequeña de los pacientes con sensibilidad al gluten y / o enfermedad celíaca. Los resultados de este estudio indican que DH también puede desarrollarse en una gran mono rhesus con síntomas de la dermatitis idiopática.

Video: Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

Multicolor análisis de citometría de flujo de la respuesta inmune celular en macacos rhesus

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Investigación

Inmunología e Infección

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

Tecnología IgY: Extracción de anticuerpos de pollo de yema de huevo por el polietilenglicol (PEG) Precipitación

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Transformación y cercarias In vitro El cultivo de la Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

La identificación de patógenos bacterianos raras por 16S rRNA gen secuenciación y MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

Antibody Binding Specificity for Kappa (V&amp;#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

Anticuerpo especificidad de unión de Kappa (VK) Humanos (IgM) de luz que contiene la cadena-: Ácido polisiálico (PSA) unido a NCAM como un estudio de caso

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Generación de anticuerpos monoclonales murino por el hibridoma Tecnología

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Análisis de Inmunodeficiencia CD8 Simian Virus-específica<sup&gt; +</sup&gt; Células T en macacos rhesus por el péptido-MHC-I de tetrámeros La tinción

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Detección y enriquecimiento de Rare células B específicas de antígeno para el análisis del fenotipo y la función

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

Efectos de la exposición de formaldehído a un modelo de rata de la Dermatitis atópica inducida por el tratamiento Neonatal de capsaicina

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Un método para evaluar las funciones efectoras mediadas por Fc inducidas por los anticuerpos específicos de la hemaglutinina de la gripe

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...