This research was possible through funding from the University of Neuch&#226;tel. We would like to thank all the students that helped in improving this technique, namely our colleague Kevin Thievent. We would also like to thank the members of the Thomas Lab for making their laboratory available. We would like to thank Janet Teeple for her help with mosquito rearing. We would also like to thanks Loyal Hall in the laboratory of Pr. Tom Baker for his help in the preparation of the micro capillary glass tubes.

1. solução salina por injeção e Dissection <ol><li> Preparar a solução salina através da adição de NaCl, KCl, e CaCl 2 de água destilada para obter NaCl 1,3 mM, KCl 0,5 mM, e CaCl 2 0,2 mM, a pH = 6,8. </li><li> Adicionar 1 ml de solução verde 0,1% de metilo a 99 ml da solução de soro fisiológico para colorir as contas transparente. Esta é a &quot;solução injectável&quot; verde metil 0,001%. </li><li> Em seguida, adicionam-se 5 ml de solução a 0,1% verde de metilo a 45 ml de solução salina. Esta &quot;solução de dissecação&quot; verde metil 0,01% é 10 vezes mais concentrada em verde metil para facilitar a coloração das pérolas e sua observação durante a dissecção. NOTA: Filtro de esterilizar o tampão, se necessário. </li></ol> 2. Preparação Capilar <ol><li> Calor-pull tubos microcapilar de vidro para obter uma ponta muito fina ligeiramente maior do que as mais pequenas esferas (φ = 40 mm) 42. </li><li> Aberta e umdjust cada capilar, quebrando a ponta com uma pinça ou serrando a ponta fora. </li></ol> 3. Mosquito Empinar <ol><li> Traseira A. gambiae a 26 ± 1 ° C, 70 ± 5% de umidade relativa, e uma luz 12:12 hr: ciclo escuro. Mantenha as fêmeas adultas e dar-lhes acesso a uma solução de açúcar de 10%. </li></ol> 4. Seleções do grânulo com a Capilar <ol><li> Pour 0,009 g de grânulos carregados negativamente (40-120 um de diâmetro) numa placa de Petri 5 centímetros e adicionam-se 5 ml de solução de injecção de 30 minutos de antecedência. NOTA: O objetivo é permitir que o corante de cor as contas para fazer a seleção talão para a injeção mais fácil. Autoclave as contas, se eles não vêm em um frasco estéril. </li><li> Usando um tubo capilar montado em um bolbo de pipeta capilar, seleccionar visualmente o menor grânulo em 0,1 mL da solução salina sob um microscópio binocular. NOTA: Quando inocular várias esferas simultaneamente, selecione três contas a partir de um volume total de 0,1 mL.</li></ol> 5. Manuseamento Mosquito e inoculação <ol><li> Usando um aspirador de insectos, colocar cada mosquito em um tubo de 50 ml. </li><li> Relaxar cada mosquito brevemente, colocando o tubo em gelo triturado (2-5 min). </li><li> Coloque um mosquito em seu lado direito sob o microscópio binocular. </li><li> Sob um microscópio estereoscópico, inocular o mosquito com o capilar por perfuração firmemente através do lado esquerdo da cutícula cavidade torácica e depois injectar o líquido e o talão. NOTA: Tome cuidado para não danificar os músculos de vôo, segurando o capilar como perpendicularmente ao mosquito possível. </li><li> Remover o capilar. </li><li> Utilizando uma pinça entomologia pluma, coloque cada fêmea individualmente em um copo de plástico de 180 ml coberto com um mosquiteiro; dar-lhes acesso a uma solução de açúcar de 10%. NOTA: É necessário ter cuidado com a solução de açúcar, como o mosquito pode ficar preso em gotas da solução açucarada. </li></ol> 6. MosquitoDissecção e Fotografia Bead <ol><li> Verifique se os mosquitos estão vivos 24 horas após a inoculação. Se assim for, verificar a sua condição e manter apenas os mosquitos que são capazes de voar. </li><li> Congelar esses mosquitos a -20 ° C. NOTA: Eles podem ser mantidos vários dias a meses antes da dissecação, se necessário. </li><li> Remover asas dos mosquitos, usando uma pinça sob um microscópio binocular em 32X de ampliação. Fixar as asas em lâminas de vidro com fita adesiva transparente. Anote o código usado para cada mosquito individual. </li><li> Coloque as lâminas de vidro em um scanner e digitalizá-los a 1.200 dpi. NOTA: Verifique se o código é legível para cada mosquito individual. </li><li> Em uma lâmina de microscópio de vidro e na solução de dissecção, utilizar uma pinça para separar o tórax da cabeça e do abdómen. </li><li> Abra o tórax com uma pinça para recuperar as contas. NOTA: Beads que não se movem para o abdômen, não estão em contacto uns com os outros ea maioria deles flutuar livremente em tele hemolinfa. Tenha cuidado para não quebrar o cordão com a pinça, como um cordão não melanizadas pode ser difícil de encontrar. Espere por 1 a 5 min para grânulos para obter uma cor. </li><li> Transferir as contas em uma gota de solução de dissecção em uma lâmina de vidro, antes de tirar a foto. NOTA: Não há necessidade de lavar as contas. </li><li> Usando um microscópio equipado com uma câmera, dê uma imagem digital de cada conta com uma definição de iluminação padrão e aumento de 400X. NOTA: Não altere a iluminação entre as diferentes imagens para permitir a sua comparação. </li></ol> 7. Análise de Imagem: Bead melanizacao <ol><li> Abra o software de análise de imagem. Clique em &quot;Arquivo&quot; e depois em &quot;abrir&quot; para encontrar uma imagem de talão. </li><li> Clique em &quot;analisar&quot;. Clique em &quot;medições previstas.&quot; Marque a caixa &quot;significa valor de cinza&quot; na janela &quot;Definir medidas&quot;. </li><li> Clique no ícone do círculo ferramenta de seleção. Selecione o perímetro talão; permanecem emlado do talão e evitar o halo luminoso em torno dele. </li><li> Clique no menu &quot;analisar&quot;. Clique em &quot;medida&quot;. Obter o valor médio de cinzentos e o tamanho da área da secção transversal do cordão. <ol><li> Para obter o valor melanizacao de cada grânulo, realize a seguinte operação usando uma calculadora ou calculadora software: &quot;256 - valor médio de cinza.&quot; A medida melanização é um valor entre 0 e 256, com 0 representando uma imagem totalmente branco. </li></ol></li></ol>

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J., George, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Manipulation&amp;#34;+without+the+parasite:+altered+feeding+behaviour+of+mosquitoes+is+not+dependent+on+infection+with+malaria+parasites.">34;Manipulation&amp;#34; without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites.</a> P Roy Soc B-Biol Sci. 280 (1763), 20130711 (2013).</li><li>Voordouw, M. J., Lambrechts, L., Koella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+maternal+effects+after+stimulation+of+the+melanization+response+in+the+yellow+fever+mosquito+Aedes+aegypti.">No maternal effects after stimulation of the melanization response in the yellow fever mosquito Aedes aegypti.</a> Oikos. 117 (8), 1269-1279 (2008).</li><li>Paskewitz, S., Riehle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Plasmodium+refractory+and+susceptible+strains+of+Anopheles+gambiae+to+inoculated+Sephadex+beads.">Response of Plasmodium refractory and susceptible strains of Anopheles gambiae to inoculated Sephadex beads.</a> Dev comp immunol. 18 (5), 369-375 (1994).</li></ol>

The authors declare that they have no competing financial interests.

Esta técnica de injeção é útil para estimular e estudar a resposta melanizacao em mosquitos. Por exemplo, aqui nós estudamos o efeito da carga de estímulos imunológico. O passo crítico neste procedimento é adequada para inocular o mosquito. Qualquer dano excessivo aos músculos de vôo ou para o próprio mosquito pode impedir que o mosquito da alimentação ou podem matá-lo antes da dissecção. Um segundo passo fundamental é manter os mosquitos no gelo tempo suficiente para derr...

Insetos dependem de respostas imunes para se proteger contra parasitas e patógenos 1 - 3 que violam através da sua cutícula ou a sua epitélio do intestino médio 4. Em mosquitos, estas respostas são eficientes contra bactérias, vírus 5 6, nematóides filarial 7 e parasitas da malária 1,8,9. Em mosquitos, uma resposta imune chave é a encapsulação de partículas estranhas com melanina 10-12. Este encapsulamento pode ocorrer no intestino ou na hemolinfa sistema de circulação 10 - 12. Esta resposta melanização é o resultado de o pró-fenoloxidase cascata 10 - 12, e que pode conduzir à morte dos parasitas ou a sua fagocitose. Em mosquitos adultos, onde o número de células hemócitos é limitado, melanização é uma resposta humoral, como contra parasitas de Plasmodium ou nemátodos filariais 7.Em alguns outros insetos, é diretamente as células hemócitos que se reúnem em torno do parasita para melanize-los 7. Além disso, a melanina também é essencial para vários outros processos fisiológicos como a produção de ovos e feridas cutícula cura 7. A estimulação de respostas imunes é usado como uma ferramenta para o estudo da imunidade de insectos em vários sistemas de modelo de saúde pública agrícola e 13 - 18. É utilizado em Anopheles gambiae os mosquitos, o principal vetor de malária na África, para estudar as interações parasita-hospedeiro 14 - 16,19. Estas técnicas baseiam-se na capacidade de insectos para detectar parasitas com os seus receptores de reconhecimento de padrões (PRR) 2. Os mosquitos também pode detectar outras moléculas que interferem com a sua biologia, como padrões moleculares associados a patógenos (PAMPs), ou detectar suas próprias células danificadas devido à liberação de colágeno e ácidos nucleicos. A célula imune mosquitos tais como os hemócitos são utilizados para a detecção de 20-23. As principais vias de sinalização imunes são IMD, Toll, JAK / STAT 24, e interferência do ácido ribonucleico (RNAi) 25,26. Ambos Toll e vias IMD influenciar a resposta melanizacao e interagir com o pró-fenoloxidase cascata 10-12. A ferramenta de padrão usado para estimular a resposta melanização é a inoculação de um mosquito com um pequeno rebordo na hemolinfa da cavidade torácica. O grau de encapsulamento melanina pode então ser medida 19 depois de recuperar o cordão através da dissecação do mosquito. Na maioria dos estudos, apenas uma esfera foi injectada por mosquito 15,16,27, mas injectando mais grânulos é possível, a fim de estudar os limites da resposta melanização 19. Estes grânulos são injectados utilizando uma solução para injecção (soro fisiológico), para limitar a perturbação do mosquito e fisiologiaa dessecação do mosquito 15,16,27. Um corante é adicionado a esta solução para facilitar a selecção de grânulo. É o mesmo para a solução de dissecção usado para recuperar o grânulo 15,16,27. A vantagem de inoculação de insectos com estímulos não patogénico é a capacidade de focar sobre o efeito directo sobre a resposta imune. Não há efeitos que complicam devido ao parasita patogenicidade 28, imunossupressão 29-31, ou a evasão imune 31-34. Além disso, as consequências das estimulações de outras características da história de vida, tais como a longevidade ou fecundidade, pode também ser estudado. Assim, os pesquisadores que estudam ecologia evolutiva pode exigir tais ferramentas 2,35,36. Por exemplo, zangões desafiou-imuno têm uma vida útil encurtada sob fome. efeitos negativos semelhantes de estímulos imunitários e implantações foram observadas em diferentes modelos de invertebrados, resultando muitas vezes num curtoer a vida útil ou o sucesso reprodutivo menos 13,27,37. Tais estudos podem ser realizados em ambientes variados 2,4,38. Estimular a imunidade também é de interesse para aqueles focando diretamente imunopatologia 39,40. Este protocolo baseia-se na inoculação de grânulos com mosquitos para estimular a resposta melanização e directamente medem a quantidade de melanina. Isso permite estudo quantitativo e qualitativo da resposta melanizacao em diferentes configurações experimentais. Uma tal ferramenta pode ser estendido para a estimulação de outras respostas imunes, tais como a resposta antibacteriana a bactérias 41 aquecer-morto. Ele também pode ser realizado em muitos contextos ecológicos.

<table border="0" cellpadding="0" cellspacing="0" width="718">
	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:301px;">Microcapillary glass tubes GB120TF-10</td>
			<td style="width:177px;">science-products.com</td>
			<td style="width:84px;">GB120TF-10</td>
			<td style="width:156px;">http://www.science-products.com/Products/CatalogG/Glass/Glass.html</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:301px;">Microcaps Capillary pipette bulb</td>
			<td style="width:177px;">Drumond</td>
			<td style="width:84px;">1-000-9000</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:301px;">negatively charged Sephadex CM C-25 beads</td>
			<td style="width:177px;">Sigma-Aldrich, Steinheim, Germany</td>
			<td style="width:84px;">C25120 SIGMA</td>
			<td style="width:156px;">need few to start</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:301px;">Methyl green</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:84px;">323829 ALDRICH</td>
			<td style="width:156px;">need few to start</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:301px;">Software ImageJ</td>
			<td style="width:177px;">opensource</td>
			<td style="width:84px;"></td>
			<td style="width:156px;">Version 1.47f7 or later</td>
		</tr>
	</tbody>
</table>

inoculating anopheles gambiae mosquitoes with beads to induce and measure the melanization immune response

A estimulação de respostas imunes é uma ferramenta comum em estudos de invertebrados para examinar a eficácia e os mecanismos de imunidade. Esta estimulação é baseado em a injecção de partículas não patogénicas em insectos, como as partículas vão ser detectado pelo sistema imunológico e induzem a produção de efectores imunitários. Nós nos concentramos aqui na estimulação da resposta melanizacao no mosquito Anopheles gambiae. Os resultados de resposta melanizacao no encapsulamento de partículas estranhas e parasitas com uma camada escura de melanina. Para estimular essa resposta, os mosquitos são inoculados com contas na cavidade torácica, utilizando tubos microcapilar de vidro. Então, depois de 24 horas, os mosquitos são dissecados para recuperar as contas. O grau de melanização do grânulo é medida utilizando software de análise de imagem. Grânulos não têm os efeitos patogénicos de parasitas, ou a sua capacidade de evitar ou suprimir a resposta imunitária. Estas injecções são uma forma de measure eficácia imunológico e o impacto da estimulação do sistema imunológico em outros traços da história de vida, tais como fecundidade e longevidade. Ele não é exactamente o mesmo que estudando directamente interacções hospedeiro-parasita, mas é uma ferramenta interessante para o estudo da imunidade e da sua ecologia evolutiva.

<html> Os mosquitos que nem todos melanize os grânulos do mesmo modo, como algumas esferas foram cobertas com menos melanina do que outras (Figura 1). Na verdade, alguns grânulos permaneceu azul devido a uma falta de melanização, ao passo que outros foram completamente escuro (Figura 1). O valor melanização foi padronizado por interpolação linear para um valor entre 0 (o que corresponde a um cordão azul e unmelanized) e 100 (correspondendo a uma ...

Watch this Scientific Journal Video about Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response at JoVE.com

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

inoculando<em&gt; Anopheles gambiae</em&gt; Os mosquitos com grânulos para induzir e medir a melanizacao Resposta Imunológica

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

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Immunology and Infection

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

7.9K visualizações.  Université de Neuchâtel. Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

Vídeo: Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Medir a carga bacteriana e resposta imune em camundongos infectados com Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Imunologia e Infecção

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Uma Introdução à Vespas parasitas de<em&gt; Drosophila</em&gt; E Resposta Imune a antiparasitários

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagem intravital da Thymus rato usando 2-Photon Microscopia

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

Medição quantitativa da resposta imune e do sono em<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Quantificação da Resposta explosão respiratória como um indicador da imunitário inatas Saúde em Zebrafish

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Usando RNA-interferência para investigar a imunitário inatas de resposta em macrófagos

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

A utilização de orofaringe Intratraqueal Administração PAMP e Lavagem Broncoalveolar para avaliar a resposta imune do hospedeiro em camundongos

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Vitro Análise do mediada por Myd88 Cellular resposta imune a vírus do Nilo Ocidental Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Imagem de Intravital dois fotões de leucócitos durante a resposta imune em fígado de rato lipopolissacarídeo-tratados

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Um modelo do rato para avaliar a resposta imune inata à infecção de Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...