Os autores gostariam de agradecer a Amanda Tardo, Dr. Mostafa Bouljihad, Stephanie Feely, Carol Coyne, Dr. Erin Ribka, Dr. Didier e Pete Dorothy Kuebler por sua assistência técnica. Este trabalho foi financiado pelo NIH concede R01-DK076653 e 3R01-DK076653-02S1 para KS e da subvenção de funcionamento base do TNPRC RR000164.

1. Coleta de amostra de biópsia de pele <ol><li> Antes de procedimento de biópsia de pele, anestesiar animais por via intramuscular com 2,5 mg / kg de cloridrato de tiletamina e cloridrato de zolazepam mistura telazol (Fort Dodge Saúde Animal, Fort Dodge, IA). Monitorar os animais desde a administração do anestésico até decúbito e depois remover do seu gabinete. </li><li> Remover os pêlos da área de interesse da pele utilizando uma de Ouro Oster A5 Clipper Single Speed ​​Veterinária com uma lâmina de tamanho 40 (Oster Professional Products, McMinnville, TN) e preparar assepticamente com alternância matagal betadine e álcool. Garantir uma cortina fenestrado estéril sobre o local da biópsia selecionado. </li><li> Usando uma técnica asséptica, coloque um 4,0 milímetros Miltex Soco instrumento de biópsia cutânea (Miltex, York, PA) contra a pele enquanto gira o instrumento de 180 graus no sentido horário e anti-horário com leve pressão até que a biópsia transectos através das camadas dérmicas na ti subcutâneassue. Retirar a amostra da biópsia e agarrar a parte transeccionados da pele com uma pinça e livre do tecido subcutâneo. </li><li> Fechar o defeito da pele com fio de náilon 3-0 ligado a uma agulha 3 / 8 círculo de corte (Ethilon, Ethicon, Johnson &amp; Johnson Medical Limited, Berkshire, UK) em um padrão cruzado. Dar a todos os animais de cloridrato de buprenorfina 0,01 mg / kg (Hospira, Lake Forest, IL) por via intramuscular para analgesia pós-operatória. </li></ol> 2. Processamento das amostras <ol><li> Trabalhar com amostras de biópsia da pele de dermatite crônica e controles saudáveis ​​macacos rhesus. Obter 2-3 (4 mm de diâmetro) biópsia amostras de cada animal. </li><li> Fix primeira amostra em formol de zinco (Z-fix, Anatech Ltd., Battle Creek, MI) por 24 horas, lave em água por 30 min, lave brevemente em etanol 70%, e coloque em ASP300 processador de tecido Leica (Leica Microsystems Inc. , Buffalo Grove, KS) onde o tecido é desidratado com graus ascendentes de 70%, 80%, 95% e 100% de etanol 48min cada (Fisher Scientific, Pittsburgh, PA), seguido por duas mudanças de xileno (Fisher). </li><li> Incorporar em media de parafina (Fisher) para armazenamento de longo prazo na temperatura ambiente. Coloque a-20oC por 20 min antes do corte. Prepare 6 seções M usando um micrótomo rotativo (HM325, Microm International, Waldorf, Alemanha). Seções lugar em slides cobrado (Fisher) e ar seco a 60 º C durante a noite. Mancha com hematoxilina e eosina (H &amp; E) método padrão (descritas abaixo). </li><li> Fix segunda amostra em paraformaldeído 2% (USB Corp, Cleveland, OH) por 30 min em temperatura ambiente, lavar três vezes em tampão fosfato salino (PBS, Gibco-Invitrogen, Carlsbad, CA), lugar em sacarose 30% (Fisher) para 4 horas, e incorporar em outubro média de congelamento (Sakura Finetek, Torrence, CA). Mantenha a-80oC por 20 min antes do corte. Prepare 15 seções M utilizando o criostato (HM560, Thermo Scientific, Kalamazoo, MI). </li></ol> 3. Coloração H &amp; E <ol><li> Deparaffinize incorporarseções ded por três mudanças de xilenos (Fisher) e hidratar através etanóis classificados: três alterações de 100%, duas mudanças de 95%, uma mudança de 80% e água destilada, 2 min cada um. </li><li> Mancha com hematoxilina (Richard Allan-Científico, Kalamazoo, MI) por um min e siga por uma lavagem rápida em água corrente. </li><li> Contracoloração com eosina (Sigma) por 6 min. </li><li> Desidratar os tecidos manchados por 95% e etanol absoluto, duas mudas de 2 min cada um e em seguida, desmarque em três mudanças de xilenos, 3 min cada. </li><li> Monte lamela usando Meio de Montagem Cytoseal (Richard Allan-Científica) sobre a H &amp; E-seções manchada do tecido. </li></ol> 4. Avaliação histopatológica Inspecionar H &amp; E-corados biópsia da pele sob um microscópio de luz usando a 4 - aumento 40x e microscópio Leica com um SPOT introspecção câmera digital (Digital Instruments Inc., Michigan, EUA). 5. Imunofluorescência staining <ol><li> Trabalhar com seções outubro-embedded da pele para a coloração de imunofluorescência. Antes da coloração, de molho por 20 min em PBS contendo 0,2% de peixes de pele de gelatina (FSG, Sigma) com 0,1% Triton X-100 (Sigma) para remover a outubro e de permeabilizar o tecido. </li><li> Depois de lavar os slides, incube com 10% de soro normal de cabra (NGS, Sigma), diluído em PBS-FSG por 1 hora, à temperatura ambiente, em uma câmara de corrediça umidificado. </li><li> Para visualizar a deposição de IgA e tTG2 anticorpos, incubar slides com anti-rhesus IgA e tTG2 anticorpos primários, diluídos em 10% NGS-PBS-FSG por 1 hora. </li><li> Lavar com X-100 PBS-FSG-Triton (Sigma) por 10 min, seguido por PBS-FSG por 10 min. </li><li> Incube com secundário, isotipo de anticorpos específicos marcados com fluorocromos Alexa Fluor 488 (verde), 568 (vermelho) ou 633 (azul) (Molecular Probes-Invitrogen) na diluição 1:1.000. Use o corante ToPro-3 para visualizar o DNA nuclear. Use os 10% NGS-PBS-FSG como diluente de anticorpo. </li><li> Use a solução anti-quenching (Sigma) para montar as seções de tecido manchado. </li></ol> 6. Confocal imagem Realizar imagens com uma Leica TCS SP2 microscópio de varredura confocal a laser Verdadeiro (Leica, Wetzlar, Alemanha) equipado com três lasers (seis linhas disponíveis para a excitação): um laser de argônio-krypton a 488 nm (verde), um laser de 568 nm krypton ( vermelho) e um laser de hélio-neon a 633 nm (azul), que se estendem do visível para o lado vermelho do espectro. Registro de contraste de interferência diferencial (DIC) de imagens para avaliação das organizações não-rotulados arquitetura do tecido. 7. Resultados representante Um exemplo de reconhecimento positivo da ETG por ambos tTG2 e IgA é mostrado na figura. 3A. Consistentes com a situação em pacientes humanos e devido à prevalência muito menor de DH-como a dermatite de CD-like sensibilidade ao glúten também em macacos rhesus, a maioria das amostras de biópsia da pele são esperados para show a reação positiva com anticorpos tTG2 mas sem a presença de IgA + + tTG2 dupla células positivas (Fig. 4). Nestes casos DH-independentes, incluindo os casos de dermatite de origem microbiana, vai revelar alguns single-células positivas + IgA espalhados sob o epitélio, mas não sobreposição espectral entre as tTG2 e IgA-rotulados marcadores (Figs. 3B e 4). Portanto, é importante manter a ordem dos procedimentos de teste sugerido, começando com o exame clínico (Fig. 1), seguido de avaliação histopatológica (Fig. 2) e apenas se não houver dermatite causador da doença, outros agentes mais comuns podem ser identificados, confocal exame de microscopia de biópsias de pele manchada para tTG2 e IgA é garantido. Avaliações clínicas e / ou histopatológico por si só não são suficientes para o diagnóstico positivo desta condição. Nos casos em duplo positivo (tTG2 + IgA +) células são identificadas em torno da área de papilas dérmicas, DH-como o diagnóstico de dermatite pode ser emitido. Em tais casos, retirada do glúten da dietae administração de dieta livre de glúten é recomendado para aliviar os sintomas da dermatite. <img src="/files/ftp_upload/3154/3154fig1.jpg" alt="Figura 1" /> Figura 1. Região inguinal de EM96 macaco antes (A) e após (B) a retirada do glúten da dieta durante 5 semanas. Reintrodução do glúten da dieta coincidiu com o reaparecimento de dermatite (C). <img src="/files/ftp_upload/3154/3154fig2.jpg" alt="Figura 2" /> Figura 2. Coloração HE de EM96 biópsias de pele macaco coletados antes (A) e após (B) a retirada do glúten da dieta por nove semanas. A: Epitélio é engrossado (7-15 camadas de células epiteliais), com uma grossa crosta superficial de células hyperkeratotic infiltradas com neutrofilos degenerando. Neutrófilos aparecem também no interior do epitélio e são misturados com números moderada de linfócitos, células plasmáticas e histiócitos em torno do anexos na derme superior. B: Alguns hyperkeratosis ainda está presente, espessura epitelial é normalizada (4-7 camadas) com infiltração mínima linfoplasmocitário na derme superior. C: pele abdominal normal a partir do FR56 macaco, sem infiltração linfoplasmocitário, está incluído para comparação. Ampliação de 20x. <img src="/files/ftp_upload/3154/3154fig3.jpg" alt="Figura 3" /> Figura 3. Imagens de microscopia confocal de EM96 biópsias de pele macaco coletados antes (A) e após (B) a retirada do glúten da dieta por nove semanas. A: coloração IgA (verde), tTG2 (vermelho) e ToPro-3 marcador de DNA nuclear (azul). Co-localização de IgA e tTG2 aparece em amarelo ao redor da área de papilas dérmicas. B: amostra de pele tomada após 9 semanas de dieta sem glúten não mostra IgA dentro do epitélio, enquanto alguns IgA subepitelial ainda está presente (verde), tTG2 (vermelho) e ToPro-3 marcador de DNA nuclear (azul). <img src="/files/ftp_upload/3154/3154fig4.jpg" alt="Figura 4" /> Figura 4. </strong&gt; As imagens de microscopia confocal de GI24 biópsias de pele rhesus coletados enquanto animal foi exibindo os sintomas da dermatite (DH-independentes) crônica. A coloração IgA é a verde, em vermelho e tTG2 ToPro-3 marcador de DNA nuclear é em azul. As células + tTG2 estão presentes dentro do epitélio, alguns IgA + células abaixo do epitélio, enquanto que há sobreposição espectral extensa entre os marcadores de vermelho e verde é visto. <table border="1"><tbody><tr><td> Tag animais </td><td> Sexo [M / F] </td><td> Idade [Anos] </td><td> Os sintomas clínicos </td><td> Plasma tTG2 anticorpos </td><td> Dieta [1,2] * </td></tr><tr><td> HJ71 </td><td> F </td><td> 1,3 </td><td> Saudável </td><td> - </td><td> 1 </td></tr><tr><td> HD13 </td><td> M </td><td> 1,4 </td><td> Diarréia, saudável ocasional </td><td> + </td><td> 1 </td></tr><tr><td> FR56 </td><td> M </td><td> 5,4 </td><td> Diarréia crônica idiopática </td><td> - </td><td> 1 </td></tr><tr><td> EM96 </td><td> F </td><td> 5,0 </td><td> Dermatite crônica </td><td> + / - (Limítrofe) </td><td> 1 e 2 </td></tr><tr><td> GI24 </td><td> F </td><td> 4,6 </td><td> Dermatite crônica </td><td> - </td><td> 1 </td></tr></tbody></table> Tabela 1. TNPRC macacos rhesus utilizado para a coleta de biópsias de pele * A dieta 1 - regular macaco Chow (glúten contendo); Dieta 2 - dieta sem glúten

<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>

Não há conflitos de interesse declarados.

Nosso recém-criado modelo de primatas não-humanos da sensibilidade ao glúten foi recentemente utilizado para estudar as mudanças da permeabilidade intestinal desencadeada pelo glúten na dieta 5,6. Plasma tTG2 anticorpos, assim como intestinal tTG2 + + depósitos de IgA foram detectados em alguns macacos glúten-sensível. Desde que identificado recentemente incidentes raros de dermatite idiopática em colônia de macacos rhesus, onde nenhuma etiologia (infecciosas / metabólica) usual pôde ser estabelec...

<table border="1"><tbody><tr><td> Nome do reagente ou equipamento </td><td> Companhia </td><td> Número Catálogo / Código </td><td> Nota </td></tr><tr><td> IgA anti-humana-FITC * </td><td> Sigma, St. Louis, MO </td><td> F-5259 </td><td> 1:100 diluição de trabalho </td></tr><tr><td> Anti-Human tTG2 </td><td> Thermo Scientific, Kalamazoo, MI </td><td> MS-300-P1ABX </td><td> 1:100 diluição de trabalho </td></tr><tr><td> Anti-IgG1 ** </td><td> Invitrogen, Carlsbad, CA </td><td> A-21124 </td><td> 1:1.000 diluição de trabalho </td></tr><tr><td> ToPro-3 DNA *** sonda </td><td> Invitrogen, Carlsbad, CA </td><td> T-3605 </td><td> 1:1.000 diluição de trabalho </td></tr><tr><td> Zinco formalina (Z-fix) </td><td> Anatech Ltd., Battle Creek, MI </td><td> 171 </td><td></td></tr><tr&gt; <td> Congelamento outubro Médio </td><td> Sakura Finetek, Torrence, CA </td><td> 4583 </td><td></td></tr><tr><td> Cytoseal Montagem Médio </td><td> Richard Allan-Científico, Kalamazoo, MI </td><td> 8312-4 </td><td></td></tr><tr><td> Anti-Quenching Solução </td><td> Sigma, St. Louis, MO </td><td> P 3130 </td><td></td></tr><tr><td> Ethilon (Sutura Nylon) </td><td> Ethicon, Johnson &amp; Johnson, Berkshire, Reino Unido </td><td> 663G </td><td></td></tr><tr><td> Miltex Soco instrumento de biópsia cutânea </td><td> Miltex, York, PA </td><td> NA </td><td></td></tr><tr><td> Oster A5 Ouro Clipper Vet Único de velocidade com uma lâmina de tamanho 40 </td><td> Oster Produtos, McMinnville, TN </td><td> 78005-050 </td><td></td></tr><tr><td> Leica Processor Tissue </td><td> Leica Microsystems Inc., Buffalo Grove, KS </td><td> ASP300 </td><td></td></tr><tr><td> Micrótomo rotativo </td><td> Microm International, Waldorf, na Alemanha </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> Microscópio confocal </td><td> Leica, Wetzlar, Alemanha </td><td> TCS SP2 </td><td></td></tr></tbody></table> * Isotiocianato de fluoresceína ** Anticorpo secundário (Alexa Fluor 568-conjugados) usado após tTG2 anticorpo primário ** Dye

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

Transglutaminase tecidular 2 (tTG2) é uma enzima digestiva intestinal que deamidates já parcialmente digeridos da dieta do glúten peptídeos gliadina por exemplo. Em indivíduos geneticamente predispostos, tTG2 desencadeia respostas auto-imunes que se caracterizam pela produção de anticorpos e tTG2 sua deposição direta em 1,2 parede do intestino delgado. A presença de tais anticorpos constitui uma das características principais da doença celíaca (CD). Transglutaminase epidérmica (ETG) é outro membro da família transglutaminase, que também pode funcionar como auto-antígeno em uma pequena minoria de pacientes com DC. Nestes casos relativamente raros, ETG desencadeia uma reação auto-imune (erupção cutânea) clinicamente conhecida como dermatite herpetiforme (DH). Embora o mecanismo exato da patogênese da DH CD e não é bem compreendida, sabe-se que tTG2 e ETG epítopos antigênicos partes que podem ser reconhecidas por anticorpos de soro de pacientes de CD e DH 3,4. (Macaca mulatta) com dermatite (Tabela 1, fig. 1 e 2) foi usado para estudar os tecidos afetados. Em um animal (EM96) uma sobreposição espectral de IgA e tTG2 anticorpos (Fig. 3) foi demonstrada. A presença de duplo-positivos tTG2 + + células IgA foi focada na epiderme profunda, em torno da papila dérmica. Isto é consistente com lesões descritas em pacientes DH 3. Quando EM96 foi colocado em uma dieta livre de glúten, a dermatite, bem como tTG2 + IgA + depósitos desapareceu e já não eram detectáveis ​​(Figs. 1-3). Dermatite reapareceu no entanto, com base na re-introdução do glúten na dieta EM96 (não mostrado). Em macacos, incluindo animais com dermatite independentes, o tTG2 + + depósitos de IgA não foram detectadas. Dieta sem glúten-dependente remissão de dermatite em EM96 juntamente com presença de IgA + + tTG2 células em sua pele sugerem uma auto-imunes, como a DH-mecanismo para o desenvolvimento dessa condição. Este é o primeiro relatório de DH-como a dermatite em qualquer primata não-humano.

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

Dermatite herpetiforme (DH) é uma condição inflamatória crônica caracterizada por uma reação auto-imune entre IgA e transglutaminase epidérmica (ETG). DH se desenvolve em uma parcela muito pequena dos pacientes glúten-sensíveis e / ou doença celíaca. Os resultados deste estudo indicam que a DH também pode se desenvolver em um hospedeiro macaco rhesus com sintomas da dermatite idiopática.

Reconhecimento da transglutaminase epidérmica por 2 Anticorpos IgA transglutaminase tecidular e em um caso raro de Rhesus Dermatite

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 visualizações.  Tulane National Primate Research Center. Dermatite herpetiforme (DH) é uma condição inflamatória crônica caracterizada por uma reação auto-imune entre IgA e transglutaminase epidérmica (ETG). DH se desenvolve em uma parcela muito pequena dos pacientes glúten-sensíveis e / ou doença celíaca. Os resultados deste estudo indicam que a DH também pode se desenvolver em um hospedeiro macaco rhesus com sintomas da dermatite idiopática.

Vídeo: 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.

Fluxo multicolor de Citometria de Análises de resposta imune celular em 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 ...

Imunologia e Infecção

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.

Tecnologia IgY: Extração de anticorpos de gema de ovo de galinha por polietileno glicol (PEG) Precipitação

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.

Transformação e Cercarial In vitro Cultivo de Schistosoma mansoni Esquistossômulos

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.

Identificação de patógenos bacterianos raras, seqüência do gene 16rRNA e 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.

A ligação do anticorpo especificidade para Kappa (VK) Humanos (IgM) Anticorpos contendo Cadeia Luz: Acid polissiálico (PSA) Anexado a MACN como um estudo 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.

Geração de anticorpos monoclonais murinos pelo hibridoma Tecnologia

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.

Análise de células T CD8 específicas do vírus da imunodeficiência símia<sup&gt; +</sup&gt; Células-T em Rhesus Macaques por Peptide-MHC-I tetrâmero coloração

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.

Detecção e enriquecimento da Rare células B antígeno-específica para a análise do fenótipo e função

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.

Efeitos da exposição de formol para um modelo do rato da dermatite atópica, induzido pela capsaicina Neonatal tratamento

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.

Um método para avaliar funções efetoras mediada por Fc induzidas por anticorpos específicos de hemaglutinina de gripe

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