Somos gratos ao laboratório Martin para discussões úteis. Esta publicação é apoiada por uma bolsa do Instituto Canadense de Pesquisa em Saúde (MOP-89783). AM é apoiado por um presidente de Pesquisa do Canadá Nível II Prêmio.

Etapa I: Isolamento de células B do baço via de enriquecimento magnética <ol><li> Camundongos experimentais devem ser envelhecidos 8-12 semanas para garantir a plena maturação do sistema imunológico. </li><li> Euthanize o mouse por deslocamento cervical e mergulhar em etanol 70%. Remoção cirúrgica do baço por fazer uma incisão através da pele e do músculo do hipocôndrio esquerdo do abdômen e corte-o em três pedaços grandes em tampão fosfato salino (PBS). Assegurar que todas as ferramentas e os reagentes são estéreis. </li><li> Usando o final de borracha de uma seringa de 1 mL êmbolo, gentilmente amasse o baço através de um filtro de células 70 mM em PBS. Certifique-se de usar um movimento de empurrar ao invés de um movimento de moagem como a moagem pode levar à ruptura de maior (ou seja, proliferação) das células. </li><li> Girar as células, não superior a 350 g por 5 minutos e ressuspender o sedimento em PBS. Contar as células ressuspenso utilizando um hemocitômetro. </li><li> Prepare uma suspensão de 1x10 8 células / mL em PBS com soro de ratos normais 5% em um tubo estéril de poliestireno 5 mL com uma tampa (volume máximo de 2 mL por tubo). </li><li> Adicionar 50 mL de EasySep Seleção rato Negative B cocktail de Enriquecimento de células para cada mL de células. Pipeta a mistura, os tubos de tampa e coloque na geladeira por 15 minutos. </li><li> Adicionar 100 mL EasySep Cocktail Seleção Biotina para cada mL de células. Pipeta a mistura, os tubos de tampa e coloque na geladeira por 15 minutos. </li><li> Adicionar 100 mL de EasySep nanopartículas magnéticas (garantir nanopartículas são bem misturados ea solução é homogênea) para cada mL de células. Pipeta a mistura, os tubos de tampa e coloque na geladeira por 5 minutos. </li><li> A solução de cima para 2,5 ml com PBS e colocar em um ímã EasySep por 5 minutos. Inverter o ímã e tubo e despeje rapidamente em um tubo de poliestireno novo. Não agite o tubo como células negativamente selecionados serão magneticamente ligado às paredes do tubo. </li><li> Para aumentar ainda mais a pureza da suspensão de células, o tubo pode ser reinserido no ímã para um adicional de 5 minutos e colocada em um novo tubo. Isto pode reduzir a recuperação das células em geral. </li><li> B pureza celular pode ser avaliado por análise de citometria de fluxo de marcadores de superfície de células B. Após o enriquecimento, nós sempre ver uma pureza&gt; 95% B220 células positivas. </li></ol> Etapa II: A coloração celular CFSE <ol><li> Volte a suspender as células isoladas em PBS suplementado com calorosa 0,1% de soro albumina bovina a uma concentração final de 1 x 10 6 células por ml. </li><li> Prepare uma solução estoque de 5mM CFSE (diluída em dimetil sulfóxido estéril) e adicione 2μL para cada mL de células no tubo. A concentração final de 10μM é ideal para a coloração de células B primário. </li><li> Incubar a 37 ° C por 10 minutos no escuro (note: CFSE é sensível à luz e deve ser mantido no escuro, quando possível). </li><li> Saciar a mancha, adicionando um volume igual de soro bovino para suas células e incubar no gelo por 5 minutos. </li><li> Lave as células, completando-se os tubos com meio de cultura (veja a etapa III para a receita) e centrifugação a 350 g. Note-se que pelo menos 5 vezes o volume equivalente de mídia deve ser adicionado para neutralizar a viscosidade do soro bovino e para as células a produzir uma pelota. </li><li> Lave as células duas vezes mais em meio de cultura. As células estão agora prontos para a estimulação. </li></ol> Etapa III: estimulação celular com LPS <ol><li> Prepare o seu in vitro meio de cultura celular B, completando RPMI 1640 com soro bovino fetal 10% e 50 mM β-mercaptoetanol /. </li><li> Prepare seu meio de estimulação, adicionando LPS numa concentração final de 50μg/mL. ML alíquota do meio de 125 a estimulação em cada poço de uma placa de cultura de fundo chato de 96 poços de tecido. </li><li> Prepare suas células CFSE manchada de B em meio de cultura sem LPS na concentração final de 3,2 x 10 6 células / mL. Adicionar 125 mL da suspensão de células para os poços contendo o seu meio de estimulação e misture delicadamente por pipetagem. </li><li> Cada poço contém agora 4 x 10 5 células em uma concentração de LPS final de 25 mcg / mL. Isto proporciona ótimos níveis de proliferação celular e mudança de classe, embora as alterações a estes valores podem ser feitas como desejado. </li><li> Incubar as células a 37 ° C e 5% CO 2 por 72 a 96 horas. Após 48 horas, clusters de grandes células B proliferam será claramente visível ao microscópio. </li></ol> Etapa IV: análise de citometria de fluxo de mudança de classe <ol><li> Quando estiver pronto para olhar para mudança de classe, remover as células de suas condições de cultura e lavá-los duas vezes em 1 mL de tampão de marcação (PBS com 2% de bovinos soro bovino). </li><li> Para evitar que a coloração não específica de anticorpos de suas células, as células pellet girando-os em 350 g e incubaçãote-los em 100μL de coloração de buffer com soro de rato normal e 5% de 1μg FcBlock per106 células por 15 minutos no gelo. Bloqueio de celular deve ser realizada no gelo. </li><li> Sem lavar as células, adicionar 1μg por 10 6 células de um anti-rato fluorescente etiquetado anticorpos IgG3 e permitir que as células a mancha por 30 minutos no gelo. </li><li> Lave as células duas vezes por centrifugação e ressuspender em 200-300 mL de tampão de coloração. As células estão agora prontos para a avaliação por citometria de fluxo. </li><li> CFSE é otimamente animado a 492 nm (por laser de argônio-ion) e emite a 517 nm. O espectro de excitação e emissão do anticorpo IgG3 é dependente de sua tintura conjugado fluorescente. </li></ol> Resultados representante Após o enriquecimento magnético, a suspensão celular deve ser semelhante a uma população homogênea de pequenas células indistinguíveis circular (Figura 1A). Após 48-72 horas de estimulação, núcleos isolados de células em proliferação alargada será claramente visível (Figura 1B). Uma grande parte das células não-proliferação aparecerá pequena e granular como eles sofrem apoptose. A RSE pode ser normalmente detectado nos níveis mais altos entre 72 e 96 horas após a estimulação antes da maioria das células começam a morrer 7. Nesta fase, as células devem ter sido submetidos a uma série de divisões celulares com células ligado aparecer na população mais tarde células filhas. Um gráfico de fluxo representante citometria de diluição CFSE e expressão de superfície de IgG3 após 96 horas de estimulação LPS pode ser visto na Figura 2. Tabela 1. Isotype comutação cocktails. Nota: Os valores de concentração de estimulantes são apenas recomendações. Titulações podem ser necessárias. <table border="1"><tbody><tr><td> Isotype desejado </td><td> Cocktail estimulação </td></tr><tr><td> IgG1and IgE </td><td> LPS (25μg/mL) e IL-4 (10 ng / mL) </td></tr><tr><td> IgG1and IgE </td><td> Anti-CD40 (10μg/mL) e IL-4 (10 ng / mL) </td></tr><tr><td> IgG2a </td><td> LPS (25μg/mL) e IFN-γ (10 ng / mL) </td></tr><tr><td> IgG2b e IgG3 </td><td> LPS (25μg/mL) </td></tr><tr><td> IgA </td><td> LPS (25μg/mL), TGF-β (2 ng / mL) e IL-5 (1,5 ng / mL) </td></tr></tbody></table><img src="/files/ftp_upload/2130/2130fig1.jpg" alt="Figura 1" /> Figura 1. Isolamento e LPS estimulação das células B do baço. (A) magneticamente enriquecido células B esplênicas antes da estimulação LPS. (B) 48 horas após a estimulação com 25 mg / mL de LPS. As células em crescimento são vistos como clusters em um fundo de células apoptóticas e explodir. <img src="/files/ftp_upload/2130/2130fig2.jpg" alt="Figura 2" /> Figura 2. Proliferação e recombinação mudança de classe após 96 horas. (A) diluição CFSE para LPS estimulou as células após 96 horas de cultura. Cada pico independente representa uma diluição de fluorescência CFSE correspondente a uma divisão celular independente. (B) a expressão IgG3 mostrando o surgimento de uma população IgG3 positivo após várias rodadas de divisão celular.

<ol>
	<li>Hodgkin, P. D., Lee, J. H., Lyons, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+differentiation+and+isotype+switching+is+related+to+division+cycle+number.">B cell differentiation and isotype switching is related to division cycle number.</a> J Exp Med. 184, 277-281 (1996).</li><li>McCall, M. N., Hodgkin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switch+recombination+and+germ-line+transcription+are+division-regulated+events+in+B+lymphocytes.">Switch recombination and germ-line transcription are division-regulated events in B lymphocytes.</a> Biochim Biophys Acta. 1447, 43-50 (1999).</li><li>Manis, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1+links+DNA+damage-response+pathways+to+immunoglobulin+heavy+chain+class-switch+recombination.">53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination.</a> Nat Immunol. 5, 481-487 (2004).</li><li>de Yebenes, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-181b+negatively+regulates+activation-induced+cytidine+deaminase+in+B+cells.">miR-181b negatively regulates activation-induced cytidine deaminase in B cells.</a> J Exp Med. 205, 2199-2206 (2008).</li><li>McBride, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+class+switch+recombination+and+somatic+mutation+by+AID+phosphorylation.">Regulation of class switch recombination and somatic mutation by AID phosphorylation.</a> J Exp Med. 205, 2585-2594 (2008).</li><li>Ramachandran, 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+RNF8/RNF168+ubiquitin+ligase+cascade+facilitates+class+switch+recombination.">The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination.</a> Proc Natl Acad Sci U S A. 107, 809-8014 (2010).</li><li>Zaheen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+constrains+germinal+center+size+by+rendering+B+cells+susceptible+to+apoptosis.">AID constrains germinal center size by rendering B cells susceptible to apoptosis.</a> Blood. 114, 547-554 (2009).</li><li>Chaudhuri, J., Alt, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class-switch+recombination:+interplay+of+transcription,+DNA+deamination+and+DNA+repair.">Class-switch recombination: interplay of transcription, DNA deamination and DNA repair.</a> Nat Rev Immunol. 4, 541-552 (2004).</li><li>Kaminski, D. A., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli+that+enhance+IgA+class+switching+increase+histone+3+acetylation+at+S+alpha,+but+poorly+stimulate+sequential+switching+from+IgG2b.">Stimuli that enhance IgA class switching increase histone 3 acetylation at S alpha, but poorly stimulate sequential switching from IgG2b.</a> Eur J Immunol. 37, 240-251 (2007).</li><li>Kaminski, D. A., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+class+switching+differs+among+SJL,+C57BL/6+and+129+mice.">Antibody class switching differs among SJL, C57BL/6 and 129 mice.</a> Int Immunol. 19, 545-556 (2007).</li></ol>

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O protocolo descrito acima fornece um ensaio padrão para analisar a expressão de AID e função durante a mudança de classe de células primárias de B murinos. Este protocolo usa LPS bacteriano para induzir a mudança de IgM para IgG3, apesar de modificações para a mídia estimulação pode ser feita para induzir a mudança para outros isotipos (resumidos na Tabela 1 8, 9). Temos notado que, por razões ainda desconhecidas, soro fetal bovino pode afetar o nível de classe de...

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<td>Phosphate Buffered Saline</td>
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<td>Gibco</td>
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<td>Stemcell Technologies</td>
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<td>Becton Dickinson Falcon</td>
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<td>Bovine Serum Albumin</td>
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<td>Rat Anti-Mouse IgG3</td>
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<td>Becton Dickinson Pharmingen</td>
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induction and assessment of class switch recombination in purified murine b cells

Imunidade humoral é o ramo do sistema imunológico mantido por células B e mediada através da secreção de anticorpos. Após a ativação de células B, o locus da imunoglobulina sofre uma série de modificações genéticas para alterar a capacidade de ligação e função efetora de anticorpos secretados. Este processo é destacado por um evento de recombinação do genoma conhecido como recombinação classe switch (CSR), no qual o padrão isotipo de anticorpos IgM é substituído por um de IgG, IgA ou IgE. Cada isotipo possui diferentes funções efetoras tornando CSR crucial para a manutenção da imunidade. Diversificação do lócus imunoglobulina é mediada pela enzima citidina desaminase ativação induzida (AID). Um vídeo esquemáticos descrevendo esse processo em detalhes está disponível online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). Atividade da AID e na via de RSE são comumente estudados na avaliação da função das células B e imunidade humoral em camundongos. O protocolo apresentado neste relatório apresenta um método de isolamento de células B do baço de camundongos e estimulação subseqüente com lipopolissacarídeo bacteriano (LPS) para induzir a mudança para classe IgG3 (para isotipos outro anticorpo ver Tabela 1). Além disso, a coloração fluorescente de células corante carboxifluoresceína succinimidyl éster (CFSE) é usado para monitorar a divisão celular de células estimuladas, um processo crucial para a mudança isotipo 1, 2. A regulamentação da AID e do mecanismo pelo qual ocorre RSE ainda não estão claros e, portanto, em ensaios in vitro interruptor classe fornecem um método confiável para testar esses processos em vários modelos de mouse. Estes testes têm sido utilizados anteriormente no contexto da deficiência do gene usando camundongos knockout 3. Além disso, in vitro comutação de células B pode ser precedida por transdução viral para modular a expressão gênica por RNA knockdown ou expressão do transgene 4-6. Os dados desses tipos de experimentos têm impactado a nossa compreensão da atividade AID, a resolução da reação de RSE, e mediada por anticorpos de imunidade no mouse.

Watch this Scientific Journal Video about Induction and Assessment of Class Switch Recombination in Purified Murine B Cells at JoVE.com

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Após a exposição de antígenos, subpopulações de células ativadas B passam por um processo conhecido como recombinação mudança de classe (CSR) para produzir isotipos de anticorpos com diferentes funções efetoras. O protocolo apresentado neste relatório explica como a RSE pode ser induzido e analisados<em&gt; In vitro</em&gt; Para efeitos de estudo B a função celular.

Indução e Avaliação de recombinação mudança de classe em purificada Células B murino

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

induction-assessment-class-switch-recombination-purified-murine-b

Research

JoVE Journal

Immunology and Infection

18.0K visualizações.  University of Toronto. Após a exposição de antígenos, subpopulações de células ativadas B passam por um processo conhecido como recombinação mudança de classe (CSR) para produzir isotipos de anticorpos com diferentes funções efetoras. O protocolo apresentado neste relatório explica como a RSE pode ser induzido e analisados In vitro Para efeitos de estudo B a função celular.

Vídeo: Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

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

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

A identificação de DNA Mutações em Purificada-Tronco Hematopoéticas / células progenitoras

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Detecção de True IgE-expressando Rato B células da linhagem

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Indução de Murino inflamação intestinal por transferência adotiva de Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; Células T em imunodeficientes Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

Avaliação das propriedades imunomoduladora de mesenquimais humanas Células-Tronco (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Um modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonização Vaginal

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

Genoma-largo mediada por análise de inibidor HDAC modulação de microRNAs e mRNAs em B células induzidas a recombinação de DNA de classe-interruptor sofrem e diferenciação de células plasmáticas

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

In Vitro e In Vivo de avaliação da atividade supressiva, T, B e células mieloides e respostas Humoral de pacientes transplantados

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Análise de cisalhamento induzida pelo fluxo de migração de murino Marginal Zone B células In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.