Gostaríamos de agradecer a Kerstin Thim (AstraZeneca, Lund, Suécia), Benita Dahlberg e Dr. Anders Eklund (Karolinska Institutet, em Estocolmo, Suécia), bem como Dr. Martin Stampfli (Universidade McMaster, Hamilton, ON, Canadá) para obter assistência hábil e conselhos de especialistas.

Os estudos com animais foram aprovados pelo comitê de ética bem-estar animal do Norte Estocolmo. Os procedimentos experimentais foram realizados em conformidade com a lei sueca. 1. Gerando um LPS Aerosol <ol><li> Dissolve-se 0,5 g purificado P. aeruginosa LPS em 50 ml de solução salina estéril com agitação suave e verificar dissolução. Dilui-se 1 ml de LPS dissolvidos em 9 ml de solução salina estéril, a uma concentração final de 1 mg / ml. Proteger da luz com papel alumínio e armazenar a -20 ° C. </li><li> Descongelar LPS solubilizados no escuro à temperatura ambiente e misture bem imediatamente antes da utilização. </li><li> Em um ventilado nível II capô biohazard, inserir uma entrada de vermelho em um nebulizador e ligue o nebulizador ao ar ambiente pressurizado através do tubo fornecido pelo (esquema de avaliação apresentando os dispositivos experimentais na Figura 1) fabricante. CUIDADO: equipamento de protecção individual adequado, incluindo uma meia peça facial Respirat reutilizávelou com filtros de partículas, óculos de proteção, luvas e vestuário de protecção deve ser utilizado durante o curso da exposição. </li></ol><img alt="Figura 1" src="/files/ftp_upload/51470/51470fig1highres.jpg" /> Figura 1:. Apresentação esquemática dos dispositivos experimentais utilizados para a geração de um aerossol A entrada do nebulizador é ligado a uma alimentação de ar. A saída do nebulizador é primeiro ligado a um medidor de fluxo através de um tubo de 15,9 milímetros e um filtro de ar, e de fornecimento de ar é ajustado para 5,0 L / m a 2 kbar pressão. A saída está próxima ligado a uma caixa de acrílico equipados com tampas removíveis e 5 furos mm para evitar aumento de pressão. <ol start="4"><li> Ligue a saída do nebulizador a um medidor de fluxo de massa através de um filtro de ar. Ligue o medidor de vazão em massa para uma alimentação eléctrica. </li><li> Ajuste o suprimento de ar para 5 L / min, com a pressão restante em 1,0-2,0 bar. </li><li> Remova o medidor de vazão em massa edesligue o suprimento de ar. </li><li> Ligue a saída do nebulizador para um tubo de 15,9 milímetros, o que se bifurca e conecta-se duas caixas de Plexiglas com as dimensões: 150 x 163 x 205 milímetros, equipados com tampas removíveis. Cada caixa deve ter um buraco de cinco milímetros no lado oposto da entrada, para evitar acúmulo de pressão. </li><li> Coloque até 5 ratos em cada caixa de acrílico e feche as tampas. </li><li> Abra o nebulizador e encher o inserto com pelo menos 4 ml de LPS dissolvidos em soro fisiológico estéril ou apenas veículo (solução salina estéril; o volume não deverá exceder 8 ml). Volte a ligar a entrada para o suprimento de ar. </li><li> Permitir que o aerossol para fluir para dentro das caixas de Plexiglas fechados durante 10 min. Monitorar continuamente os animais. Certifique-se o fornecimento de ar permanece firmemente fixado à entrada do nebulizador. </li><li> Desligue o fornecimento de ar. Com as pálpebras fechadas, deixe os animais permanecem nas caixas de acrílico para 2 min. </li><li> Abrir as tampas e para permitir que o aerossol para dispersar, e devolver os animais atele gaiolas. Se os animais aparecem molhados, coloque as gaiolas em uma almofada de aquecimento definido para fogo baixo, para evitar a hipotermia. </li><li> Monitorizar os animais continuamente durante os primeiros 30 minutos, e posteriormente, a cada 2 horas durante as primeiras 6 horas. Os animais devem apresentar padrão respiratório e da actividade normal durante e após o procedimento de nebulização. </li></ol> 2. Lavagem Broncoalveolar (BAL) <ol><li> No ponto final experimental, profundamente anestesiar os animais com isoflurano a efeito tal como recomendado pela equipe de veterinários da sua instituição. Aperte a pata traseira para verificar se há um reflexo de retirada para assegurar suficiente profundidade da anestesia para realizar cirurgia de grande porte. Spray para baixo a pele dos animais com etanol 70%. </li><li> Abra o abdômen com uma tesoura e cortar a aorta para desangrar o animal. Coloque um pedaço de tecido sobre o abdômen para absorver o sangue. </li><li> Follwing eutanásia, use um único corte ântero-posterior da tesoura paraexpor o tórax. Levante a caixa torácica pela ponta anterior do esterno e use a tesoura para perfurar o diafragma no ponto mais ventral, sem cortar qualquer lobo pulmonar. Abra a caixa torácica, fazendo dois cortes no sentido ântero-posterior (reuniões abaixo da mandíbula). </li><li> Delicadamente, puxe a caixa torácica com a pinça, e cortar a traqueia, abaixo da laringe. </li><li> Levante a traquéia com a pinça e remover os pulmões, cortando os ligamentos que ligam os lobos para a cavidade torácica, e puxando pelo tecido adiposo e cardíaca. </li><li> Insira um tubo de polietileno (diâmetro interno de 0,58 mm; diâmetro externo: 0,965 milímetros) na traquéia e fixar o tubo com uma corda de fio de seda. </li><li> Empate fora do multilobe (os quatro lobos do pulmão direito) com fio de seda. </li><li> Insira uma agulha de calibre 23 para dentro do tubo de polietileno e injetar lentamente 250 mL de gelada PBS estéril para o lobo único com uma seringa de 1 ml. </li><li> Tocar com cuidado os pulmões30 vezes e recolher o líquido através da seringa. Repetir o processo com 200 ul de PBS (aproximadamente 300 ul de PBS deve ser recuperado a partir do lobo único a partir do volume total de 450 ul injectados). </li><li> Mantenha o lavado bronco-alveolar (LBA) em gelo, ou enumerar as células do LBA imediatamente. Contar as células com um heamocytometer, utilizando uma solução de Turk para corar as células 11. Calcula-se o número total de células através da multiplicação do número de células com o factor de diluição da solução de coloração e o volume dentro do campo contado do heamocytometer. </li><li> Prepare a contagem diferencial de células a partir de células citocentrifugada como descrito noutro local 11. </li></ol> 3. Formalin fixação do tecido pulmonar para avaliação histológica <ol><li> Retire o multilobe e snap-freeze em gelo seco. Armazenar a -80 ° C. </li><li> Montar uma seringa de 60 ml com o êmbolo removido em um cavalete de suporte de metal. Encher a seringa com 10% de formalina paraa altura de 20 cm acima da bancada do laboratório, representando 20 cm de pressão constante. </li><li> Ligar-se a agulha de calibre 23, fixada à traqueia para a seringa de 60 ml através de um tubo de plástico com uma válvula para controlar o fluxo de formalina. </li><li> Insuflar o lobo pulmonar com formalina durante 5 min. Desligar a agulha e removê-lo em conjunto com o tubo de polietileno a partir da traqueia, enquanto em puxando o fio de seda para fechar a traqueia e manter a pressão no pulmão. </li><li> Submergir o lóbulo do pulmão em formalina e fixar durante 24 horas a 4 ° C. </li><li> Lavar o tecido fixado três vezes durante pelo menos 20 minutos com etanol a 70%, e desidratar a xileno através do seguinte esquema (1 h cada): <ul><li> 3x etanol a 70% </li><li> 3x etanol a 95% </li><li> 3x etanol a 100% </li><li> 3x xileno </li><li> 1x (líquido) parafina </li></ul></li><li> Incorporar tecido desidratado em parafina, cortadas em 4-5 mm seções, e mancha com HE para permitir a avaliação histológica.</li>
</ol>

<ol>
	<li>King, J. D., Kocincova, D., Westman, E. L., Lam, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Lipopolysaccharide+biosynthesis+in+Pseudomonas+aeruginosa.">Review: Lipopolysaccharide biosynthesis in Pseudomonas aeruginosa.</a> Innate Immun. 15, 261-312 (2009).</li><li>Grommes, J., Soehnlein, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+neutrophils+to+acute+lung+injury.">Contribution of neutrophils to acute lung injury.</a> Mol Med. 17, 293-307 (2011).</li><li>Pesci, A., 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=Inflammatory+cells+and+mediators+in+bronchial+lavage+of+patients+with+chronic+obstructive+pulmonary+disease.">Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease.</a> Eur Respir J. 12, 380-386 (1998).</li><li>Hoogerwerf, J. 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=Lung+Inflammation+Induced+by+Lipoteichoic+Acid+or+Lipopolysaccharide+in+Humans.">Lung Inflammation Induced by Lipoteichoic Acid or Lipopolysaccharide in Humans.</a> Am. J. Respir. Crit. Care Med. 178, 34-41 (2008).</li><li>Scheuchenzuber, W. J., Eskew, M. L., Zarkower, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+humoral+responses+to+Escherichia+coli+and+sheep+red+blood+cell+antigens+introduced+via+the+respiratory+tract.">Comparative humoral responses to Escherichia coli and sheep red blood cell antigens introduced via the respiratory tract.</a> Exp Lung Res. 13, 97-112 (1987).</li><li>Asti, C., 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=Lipopolysaccharide-induced+lung+injury+in+mice.+I.+Concomitant+evaluation+of+inflammatory+cells+and+haemorrhagic+lung+damage.">Lipopolysaccharide-induced lung injury in mice. I. Concomitant evaluation of inflammatory cells and haemorrhagic lung damage.</a> Pulm Pharmacol Ther. 13, 61-69 (2000).</li><li>Brand, P., 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=Total+deposition+of+therapeutic+particles+during+spontaneous+and+controlled+inhalations.">Total deposition of therapeutic particles during spontaneous and controlled inhalations.</a> Journal of pharmaceutical sciences. 89, 724-731 (2000).</li><li>Liu, F., Li, W., Pauluhn, J., Trubel, H., Wang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+acute+lung+injury+in+rats:+comparative+assessment+of+intratracheal+instillation+and+aerosol+inhalation.">Lipopolysaccharide-induced acute lung injury in rats: comparative assessment of intratracheal instillation and aerosol inhalation.</a> Toxicology. 304, 158-166 (2013).</li><li>Skerrett, S. 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=Role+of+the+type+1+TNF+receptor+in+lung+inflammation+after+inhalation+of+endotoxin+or+Pseudomonas+aeruginosa.">Role of the type 1 TNF receptor in lung inflammation after inhalation of endotoxin or Pseudomonas aeruginosa.</a> American Journal of Physiology - Lung Cellular and Molecular Physiology. 276, L715-L727 (1999).</li><li>Roos, A. B., 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=Lung+epithelial-C/EBPbeta+contributes+to+LPS-induced+inflammation+and+its+suppression+by+formoterol.">Lung epithelial-C/EBPbeta contributes to LPS-induced inflammation and its suppression by formoterol.</a> Biochem Biophys Res Commun. 423, 134-139 (2012).</li><li>Didon, L., 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=Lung+epithelial+CCAAT/enhancer-binding+protein-beta+is+necessary+for+the+integrity+of+inflammatory+responses+to+cigarette+smoke.">Lung epithelial CCAAT/enhancer-binding protein-beta is necessary for the integrity of inflammatory responses to cigarette smoke.</a> Am J Respir Crit Care Med. 184, 233-242 (2011).</li><li>Silverpil, E., 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=Negative+feedback+on+IL-23+exerted+by+IL-17A+during+pulmonary+inflammation.">Negative feedback on IL-23 exerted by IL-17A during pulmonary inflammation.</a> Innate Immunity. 19, 479-492 (2013).</li><li>Mercer, P. F., 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=Proteinase-Activated+Receptor-1,+CCL2+and+CCL7+Regulate+Acute+Neutrophilic+Lung+Inflammation.">Proteinase-Activated Receptor-1, CCL2 and CCL7 Regulate Acute Neutrophilic Lung Inflammation.</a> American Journal of Respiratory Cell and Molecular Biology. , (2013).</li><li>Korkmaz, B., Horwitz, M. S., Jenne, D. E., Gauthier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases. Pharmacological Reviews. 62, 726-759 (2010).</li><li>Bafadhel, M., 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=Acute+Exacerbations+of+COPD:+Identification+of+Biological+Clusters+and+Their+Biomarkers.">Acute Exacerbations of COPD: Identification of Biological Clusters and Their Biomarkers.</a> Am. J. Respir. Crit. Care Med. , 201104-200597 (2011).</li><li>Hurst, J. R., Perera, W. R., Wilkinson, T. M. A., Donaldson, G. C., Donaldson, G. C., Wedzicha, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+and+Upper+and+Lower+Airway+Inflammation+at+Exacerbation+of+Chronic+Obstructive+Pulmonary.">Systemic and Upper and Lower Airway Inflammation at Exacerbation of Chronic Obstructive Pulmonary.</a> Am. J. Respir. Crit. Care Med. 173, 71-78 (2006).</li><li>Rabe, K. F., 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=Global+Strategy+for+the+Diagnosis,+Management,+and+Prevention+of+Chronic+Obstructive+Pulmonary+Disease:+GOLD+Executive+Summary.">Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary.</a> Am. J. Respir. Crit. Care Med. 176, 532-555 (2007).</li></ol>

Abraham Roos recebeu uma taxa conferencista da Boehringer Ingelheim, bolsas de viagem de AstraZeneca R &amp; D e uma bolsa de pesquisa da AstraZeneca R &amp; D. Magnus Nord é um funcionário em tempo integral da AstraZeneca R &amp; D e tem recebido bolsas de pesquisa da AstraZeneca R &amp; D. Johan Grunewald e Tove Berg são co-pesquisadores em um projeto de pesquisa relacionado financiado pela AstraZeneca R &amp; D.

Em aerossol de LPS gera uma resposta inflamatória nas vias aéreas, caracterizada por neutrófilos na submucosa epitelial, espaços circundantes as vias aéreas de condução, bem como os espaços alveolares. Isto é, em conjunto com o aumento do conteúdo de proteína total na LBA, indicativo de perda de plasma, representativo da patologia da lesão pulmonar aguda. Como LPS induz uma inflamação estéril, a reacção é independente da resposta imune adaptativa, e existem limitações para a relevância para infecç?...

O lipopolissacárido (LPS) é um componente da parede celular de bactérias gram-negativas 1. Desafio ao LPS é um modelo bem documentado da lesão pulmonar aguda, uma síndroma caracterizada por inflamação e edema agudo neutrofílica 2. Além disso, a neutrofilia pulmonar é também uma característica da doença pulmonar obstrutiva crónica (DPOC) 3, e provocação com LPS em seres humanos tem sido utilizado para modelar as exacerbações da DPOC 4. Assim, modelos experimentais de exposição LPS são clinicamente relevantes e ferramentas valiosas para entender a patologia humana. O objetivo da administração pulmonar de LPS aerossol descritas aqui é gerar uma resposta inflamatória neutrofílica na condução e vias respiratórias, sem envolvimento sistêmico. Várias técnicas de provocação com LPS foram descritos anteriormente. Injecção intra-venosa do LPS é a via de administração mais utilizada. Embora esta técnica é facilmente acessível, tele dano primário é para o endotélio, com destruição secundário do epitélio pulmonar após a migração de neutrófilos para o pulmão. A administração intra-venosa também induz inflamação sistémica 2, o que pode complicar o quadro clínico em modelos animais. Inflamação sistémica está em contraste não observados com a administração intra-traqueal. Esta técnica, no entanto, é trabalhoso e requer anestésicos, bem como treinamento considerável 5, 6. Além disso, a deposição pulmonar por esta via de administração é dependente de respiração 7. Assim, a deposição pulmonar é afectada pela profundidade da anestesia necessários para a administração intra traqueal e variável deposição nas vias aéreas pode ser observada. Em contraste, a administração pulmonar com LPS requer o mínimo de formação de aerossol, e pode ser facilmente conseguido com um grande número de animais com pouca ou nenhuma variação entre indivíduos 5, 8.Um estudo recente confirmou que o fornecimento de aerossol é superior a via intra-traqueal em relação à deposição, e que doses mais pertinentes do LPS induzir inflamação neutrofílica com este modelo 8. Estudos anteriores demonstraram que a desafio aerossolizado Psuedomonas aeruginosa LPS gera uma resposta inflamatória significativa no lúmen das vias aéreas e do parênquima pulmonar, incluindo os espaços alveolares 9, 10. A inflamação é caracterizada pela predominância de neutrófilos e presença de edema pulmonar, e pode, assim, ser usado para endereçar patogénese da lesão pulmonar aguda e ganhar conhecimento adicional sobre os mecanismos que contribuem para a patologia da doença.

<table border="0" cellpadding="0" cellspacing="0" style="width:931px;" width="929">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Name of the material/equipment&#160;</td>
			<td style="width:244px;">Company</td>
			<td style="width:145px;">Catalog number</td>
			<td style="width:235px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:307px;">Purified Pseudomonas aeruginosa LPS&#160;</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Harmful. Recomended purification. LPS purified from other bactria may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Pari LC sprint star nebulizer</td>
			<td style="width:244px;">PARI Respiratory Equipment Inc.&#160;</td>
			<td style="width:145px;">023G1250</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">TSI mass flowmeter 4040</td>
			<td style="width:244px;">TSI</td>
			<td style="width:145px;">4040</td>
			<td style="width:235px;">Alternative product from supplier may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Saint-Gobain 15.9 mm Tygon tube</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">Z685704</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Plexiglas boxes with removable lids</td>
			<td style="width:244px;">Custom built</td>
			<td style="width:145px;">N/A</td>
			<td style="width:235px;">150 x 163 x 205 mm (a 2 mm hole on the side).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Half Facepiece Reusable Respirator</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">7503</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Advanced Particulate Filters (P100)&#160;</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">2291</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sissors</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">233-1104</td>
			<td style="width:235px;">Preferred scissors may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Forceps&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">232-1313</td>
			<td style="width:235px;">Preferred forceps may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Intramedic PE50 polyethylene tube</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">427411</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Ethicon 2-0 Perma-hand silk tread&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">95056-992</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">26 &#189;&#160; gage needle&#160;</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">1 mL BD slip-tip syringe, non-sterile</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=301025">301025</a></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">60 mL BD Luer-Lok syringe, non-sterile, polypropolene&#160;</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">301035</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fluka Hematoxylin-Eosin</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">3972</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">T&#252;rk&#39;s solution</td>
			<td style="width:244px;">Merck Millipore</td>
			<td style="width:145px;">109277</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Table top centrifuge</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative manufacturers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cytospin 4 cytocentrifuge</td>
			<td style="width:244px;">Thermo Scientific</td>
			<td style="width:145px;">A78300003</td>
			<td style="width:235px;">Alternative centrifuge can be used.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEMA-3 stat pack</td>
			<td style="width:244px;">Fisher Scientific</td>
			<td style="width:145px;">23-123-869</td>
			<td style="width:235px;">Alternative staining kits exists.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">HT501128&#160;</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
	</tbody>
</table>

a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide

A lesão pulmonar aguda (LPA) é uma doença grave, caracterizada por neutrofilia alveolar, com opções limitadas de tratamento e alta mortalidade. Modelos experimentais de ALI são fundamentais para melhorar a nossa compreensão da patogênese da doença. O lipopolissacárido (LPS) derivadas a partir de bactérias gram positivas induz inflamação neutrofílica nas vias aéreas e do parênquima pulmonar de ratos. Administração pulmonar eficaz de compostos, tais como LPS é, no entanto, difícil de alcançar. Na abordagem aqui descrita, a administração pulmonar em ratinhos é obtido por desafio com aerossol Pseudomonas aeruginosa LPS. LPS foi dissolvido em aerossol através de um nebulizador conectado ao ar comprimido. Os ratinhos foram expostos a um fluxo contínuo de aerossol de LPS numa caixa de plexiglas durante 10 minutos, seguido de 2 minutos após o condicionamento de aerossol foi descontinuado. Entubação traqueal e subsequente lavagem broncoalveolar, seguido por perfusão de formalina foi realizada próximo, o que permite a caracterização do p estérilinflamação ulmonar. Aerosolized LPS gera uma inflamação pulmonar caracterizada por neutrofilia alveolar, detectado no lavado bronco-alveolar e pela avaliação histológica. Esta técnica pode ser configurado com um custo pequeno, com poucos aparelhos, e exige treinamento e experiência mínima. O sistema de exposição pode, portanto, ser realizado rotineiramente em qualquer laboratório, com o potencial de melhorar a nossa compreensão da patologia pulmonar.

Desafio para aerossol P. aeruginosa LPS geralmente produz uma resposta inflamatória marcado no lúmen das vias aéreas e do espaço alveolar, caracterizado por uma predominância de neutrófilos em ambos os pontos de tempo precoces e tardios. Aerosolized LPS induz neutrophilia pulmonar C57BL / 6BY e BALB / c foram expostos a aerossol P. aeruginosa LPS ou veículo sozinho e neutrófilos foram enumerados no LBA. O número total de células no FLBA d...

Watch this Scientific Journal Video about A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide at JoVE.com

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Descreve-se um método para a indução de inflamação pulmonar neutrofílica desafio por aerossol para lipopolissacarídeo por nebulização, para modelar a lesão pulmonar aguda. Além disso, também são descritas as técnicas cirúrgicas básicas para isolamento pulmonar, intubação traqueal e lavado bronco-alveolar.

Um método para gerar Pulmonar Neutrofilia Usando Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

a-method-for-generating-pulmonary-neutrophilia-using-aerosolized

Research

JoVE Journal

Immunology and Infection

11.6K visualizações.  Karolinska Institutet.  Descreve-se um método para a indução de inflamação pulmonar neutrofílica desafio por aerossol para lipopolissacarídeo por nebulização, para modelar a lesão pulmonar aguda. Além disso, também são descritas as técnicas cirúrgicas básicas para isolamento pulmonar, intubação traqueal e lavado bronco-alveolar. 

Vídeo: A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

Um método para Etiquetagem Vascularização no embrião do rato

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Imunologia e Infecção

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population1, and almost two million people are killed by TB each year.2 Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3 The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2
Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in &gt; 95% of the MDR-TB isolates.4, 5 The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2
Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7 meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene.8 It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9 It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

Diagnóstico da tuberculose pulmonar com o teste Xpert MTB / RIF

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

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 ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Geração de vetores recombinantes Herpesvirus aviária com CRISPR/Cas9 Gene edição

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Co-imunoprecipitação ensaio para estudar interações funcionais entre receptores e enzimas

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Novo protocolo para a geração de células dendríticas imunogênicas fisiológicas

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Visualização da resistência bacteriana usando sondas de antibióticos fluorescentes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

Exposição à fumaça de cigarro em camundongos usando um sistema de inalação de corpo inteiro

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

Geração de Transgênicos e Nocautes em Espécies Strongyloides por Microinjeção

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

Microscopia de Fluorescência De Largura Intravital de Microcirculação Pulmonar em Lesão Pulmonar Aguda Experimental Usando um Sistema de Imagem Estabilizada a Vácuo

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...