Os autores agradecem Therwa Hamza, John E. Tidwell, Nina Clovis, e Suzanne Smith para assistência experimental e Suzanne Smith para revisão. Os autores também agradecer a John Thomas, PhD pela prestação dos isolados bacterianos clínicos e John B. Barnett, PhD por seu apoio e do uso do laboratório de segurança biológica no Departamento de Microbiologia, Imunologia e Biologia Celular da Universidade de West Virginia. Os autores reconhecem o apoio financeiro da osteossíntese e Trauma Care Foundation e National Science Foundation (# 1003907). Experimentos de microscopia e análise de imagem também foram realizadas na Universidade de West Virginia Facilidade Imaging, que é apoiado em parte pela Babb Mary Randolph Cancer Center e concessão de NIH P20 RR016440. O uso de animais de sangue desenha foram aprovados pela Universidade de West Virginia Animal Care Institucional e Comitê de uso. Todos os experimentos foram executados em conformidade com todas as Guidelin relevantees, regulamentos e agências reguladoras.

1. Preparação e ativação do PRP 1,1 coleta de sangue <ol><li> Anestesiar coelho por inalação de isoflurano (2% em O 2 para a indução e 1% para manutenção). </li><li> Desenhar 2 ml 0,129 M citrato de tri-sódio (uma solução anticoagulent) para uma seringa de 20 ml. A solução de citrato de tri-sódio é preparada por dissolução de 1,897 g de citrato de tri-sódio em 50 ml de H2O destilada e de filtragem com um filtro de 0,22 ^ M estéril. </li><li> Esterilizar a orelha de coelho utilizando etanol a 70%. </li><li> A obtenção de sangue (por exemplo, 5 ml) a partir da veia da orelha de coelho através de uma agulha de borboleta (25 G) ligada à seringa. </li><li> Misturar o sangue com a solução de citrato de tri-sódio, por agitação suave. A proporção de volume de sangue e de tri-sódio de solução de citrato é de 9:1. </li></ol> 1,2 preparação PRP (Figura 1) <ol><li> Transferir o sangue anticoagulado a um tubo de centrífuga de 50 ml em plástico. Tomar uma aliquota de 10 ulde sangue para determinar a contagem de plaquetas de base usando hemocytometry. </li><li> Centrifuga-se o sangue a 300 xg durante 10 min à temperatura ambiente (TA) numa centrífuga com rotor swing-out. Definir a velocidade de aceleração e de travagem para baixo (Figura 1). </li><li> Após a centrifugação, o sangue é separado em três camadas. A camada inferior é de glóbulos vermelhos, principalmente, a camada média (geralmente referido como o &quot;buffy coat&quot;) é composta por concentrado de plaquetas e leucócitos, e a camada superior é, principalmente, o plasma, o qual é o componente líquido do sangue e plaquetas ( Figura 1). Cuidadosamente transportar o tubo de centrífuga de um capuz de cultura de células, não perturbar as camadas. Transferir todo o plasma, buffy coat, e 2-3 mm de espessura da camada de células vermelhas do sangue para um tubo de plástico de 15 ml usando uma pipeta de plástico de 1 ml. </li><li> Centrifugar a amostra transferida uma segunda vez a 3000 xg durante 15 min à TA. A camada superior (sobrenadante) é considerado plasma pobre em plaquetas (PPP) ad é transferida para um novo tubo. </li><li> Obter PRP, ajustando a concentração de plaquetas na amostra de sangue restante utilizando PPP para se obter 2,0 x 10 6 plaquetas / ul (determinado por hemocytometry). </li></ol> 1,3 ativação PRP <ol><li> Preparar PRP solução de activação através da dissolução de 5,000 UI de trombina bovina, com 5 ml de cloreto de cálcio a 10% para a concentração de trabalho de 1000 IU / ml. </li><li> Adicionar a solução de activação para PRP e PPP, e misturar a solução por pipetagens repetidas para formar PRP e PPP-gels. A proporção em volume da solução de activação de PRP ou de PPP é 1:4. </li></ol> 2. In vitro Teste Antimicrobiano de PRP Usando Kills Ensaio de curva (Figura 2) <ol><li> Usando um loop de inoculação estéril, adicionar várias colónias de S. aureus a partir de uma cultura da placa durante a noite em 5 ml de caldo Mueller Hinton (MHB) num tubo de plástico. Vortex brevemente e então incubar a amostra durante 2 horas a 37 ° C. Próximo, A densidade óptica dos meios de bactérias foi determinada utilizando um espectrofotómetro e ajustadas para uma densidade óptica igual a ~ 1 x 10 8 UFC / ml com base na curva padrão pré-determinado. </li><li> Prepare uma diluição de 100 vezes usando PBS para se obter 1 x 10 6 CFU / ml e colocar os inóculos em gelo. </li><li> Configurar e rotular estéreis descartáveis ​​de 5 ml, os tubos de fundo redondo de poliestireno, e preparar os seguintes grupos de amostras como indicado na Tabela 1, para um volume final de 2 ml em cada tubo. </li><li> Adicionar PRP, PPP, ou PBS em primeiro lugar aos tubos de poliestireno, seguida da solução de trombina para a activação (formação do gel). Em seguida, adicione MHB e, em seguida, o S. inóculos aureus (1 x 10 6 CFU / ml) para se obter a concentração final de 1 x 10 5 CFU / ml. </li><li> Incubar os tubos a 37 ° C com agitação orbital a 150 rpm. </li><li> Em pontos de tempo pré-determinados (por exemplo, 0, 1 e 2 h), misturar as soluções de cada um dos tubos através de pipetagem de repetição (istopasso é importante uma vez que as bactérias podem ser presos no interior do gel PRP). Tomar 10 ul de amostra, dilua serialmente com solução salina estéril a 0,9%, e pipetar uma alíquota de 100 ul de cada diluição sobre um meio de ágar de soja tríptico (TSA, com sangue de ovelha a 5%) para a contagem de CFU placa. </li><li> Cultura das placas de agar durante a noite a 37 ° C, em seguida, contar e registar as colónias da placa. Os dados do gráfico em escala logarítmica, com o tempo (hr) no eixo x e CFU / ml no eixo y. </li></ol>

<ol>
	<li>Gristina, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial-centered+infection:+microbial+adhesion+versus+tissue+integration.">Biomaterial-centered infection: microbial adhesion versus tissue integration.</a> Science. 237, 1588-1595 (1987).</li><li>Gristina, A. G., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adherence+to+biomaterials+and+tissue.+The+significance+of+its+role+in+clinical+sepsis.">Bacterial adherence to biomaterials and tissue. The significance of its role in clinical sepsis.</a> J. Bone Joint Surg. Am. 67, 264-273 (1985).</li><li>Everts, P. 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=Reviewing+the+structural+features+of+autologous+platelet-leukocyte+gel+and+suggestions+for+use+in+surgery.">Reviewing the structural features of autologous platelet-leukocyte gel and suggestions for use in surgery.</a> Eur. Surg. Res. 39, 199-207 (2007).</li><li>Marx, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma+(PRP):+what+is+PRP+and+what+is+not+PRP.">Platelet-rich plasma (PRP): what is PRP and what is not PRP.</a> Implant. Dent. 10, 225-228 (2001).</li><li>Toscano, N., Holtzclaw, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+considerations+in+the+use+of+platelet-rich+plasma.">Surgical considerations in the use of platelet-rich plasma.</a> Compend. Contin. Educ. Dent. 29, 182-185 (2008).</li><li>Cieslik-Bielecka, A., Gazdzik, T. S., Bielecki, T. M., Cieslik, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+the+platelet-rich+gel+has+antimicrobial+activity?.">Why the platelet-rich gel has antimicrobial activity?.</a> Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 103, 303-306 (2007).</li><li>Yeaman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+platelets+in+antimicrobial+host+defense.">The role of platelets in antimicrobial host defense.</a> Clin. Infect. Dis. 25, 951-970 (1997).</li><li>Tang, Y. Q., Yeaman, M. R., Selsted, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+from+human+platelets.">Antimicrobial peptides from human platelets.</a> Infect Immun. 70, 6524-6533 (2002).</li><li>El-Sharkawy, H., 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=Platelet-rich+plasma:+growth+factors+and+pro-+and+anti-inflammatory+properties.">Platelet-rich plasma: growth factors and pro- and anti-inflammatory properties.</a> J. Periodontol. 78, 661-669 (2007).</li><li>Krijgsveld, 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=Thrombocidins,+microbicidal+proteins+from+human+blood+platelets,+are+C-terminal+deletion+products+of+CXC+chemokines.">Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines.</a> J. Biol. Chem. 275, 20374-20381 (2000).</li><li>Trowbridge, C. 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=Use+of+platelet+gel+and+its+effects+on+infection+in+cardiac+surgery.">Use of platelet gel and its effects on infection in cardiac surgery.</a> J. Extra Corpor. Technol. 37, 381-386 (2005).</li><li>Gristina, A. G., Naylor, P., Myrvik, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+from+biomaterials+and+implants:+a+race+for+the+surface.">Infections from biomaterials and implants: a race for the surface.</a> Med. Prog. Technol. 14, 205-224 (1988).</li><li>Subbiahdoss, G., Kuijer, R., Grijpma, D. W., vander Mei, H. C., Busscher, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biofilm+growth+vs.+tissue+integration:+"the+race+for+the+surface"+experimentally+studied.">Microbial biofilm growth vs. tissue integration: "the race for the surface" experimentally studied.</a> Acta Biomater. 5, 1399-1404 (2009).</li><li>Klevens, R. 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=Invasive+methicillin-resistant+Staphylococcus+aureus+infections+in+the+United+States.">Invasive methicillin-resistant Staphylococcus aureus infections in the United States.</a> JAMA. 298, 1763-1771 (2007).</li><li>Foster, T. E., Puskas, B. L., Mandelbaum, B. R., Gerhardt, M. B., Rodeo, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma:+from+basic+science+to+clinical+applications.">Platelet-rich plasma: from basic science to clinical applications.</a> Am. J. Sports Med. 37, 2259-2272 (2009).</li><li>Carlson, N. E., Roach, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma:+clinical+applications+in+dentistry.">Platelet-rich plasma: clinical applications in dentistry.</a> J. Am. Dent. Assoc. 133, 1383-1386 (2002).</li><li>Man, D., Plosker, H., Winland-Brown, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+autologous+platelet-rich+plasma+(platelet+gel)+and+autologous+platelet-poor+plasma+(fibrin+glue)+in+cosmetic+surgery.">The use of autologous platelet-rich plasma (platelet gel) and autologous platelet-poor plasma (fibrin glue) in cosmetic surgery.</a> Plast. Reconstr. Surg. 107, 229-237 (2001).</li><li>Fridkin, S. K., 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=Epidemiological+and+microbiological+characterization+of+infections+caused+by+Staphylococcus+aureus+with+reduced+susceptibility+to+vancomycin,+United+States,+1997-2001.">Epidemiological and microbiological characterization of infections caused by Staphylococcus aureus with reduced susceptibility to vancomycin, United States, 1997-2001.</a> Clin. Infect. Dis. 36, 429-439 (1997).</li><li>Jackson, C. R., Fedorka-Cray, P. J., Davis, J. A., Barrett, J. B., Frye, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence,+species+distribution+and+antimicrobial+resistance+of+enterococci+isolated+from+dogs+and+cats+in+the+United+States.">Prevalence, species distribution and antimicrobial resistance of enterococci isolated from dogs and cats in the United States.</a> J. Appl. Microbiol. 107, 1269-1278 (2009).</li><li>Murray, C. K., 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=Recovery+of+multidrug-resistant+bacteria+from+combat+personnel+evacuated+from+Iraq+and+Afghanistan+at+a+single+military+treatment+facility.">Recovery of multidrug-resistant bacteria from combat personnel evacuated from Iraq and Afghanistan at a single military treatment facility.</a> Mil. Med. 174, 598-604 (2009).</li><li>Durr, M., Peschel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+meet+defensins:+the+merging+concepts+of+chemoattractants+and+antimicrobial+peptides+in+host+defense.">Chemokines meet defensins: the merging concepts of chemoattractants and antimicrobial peptides in host defense.</a> Infect Immun. 70, 6515-6517 (2002).</li><li>Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+antibiotics.">Peptide antibiotics.</a> Lancet. 349, 418-422 (1997).</li><li>Dohan Ehrenfest, D. M., Rasmusson, L., Albrektsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+platelet+concentrates:+from+pure+platelet-rich+plasma+(P-PRP)+to+leucocyte-+and+platelet-rich+fibrin+(L-PRF).">Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF).</a> Trends Biotechnol. 27, 158-167 (2009).</li><li>Kalen, A., Wahlstrom, O., Linder, C. H., Magnusson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+content+of+bone+morphogenetic+proteins+in+platelets+varies+greatly+between+different+platelet+donors.">The content of bone morphogenetic proteins in platelets varies greatly between different platelet donors.</a> Biochem. Biophys. Res. Commun. 375, 261-264 (2008).</li><li>Weibrich, G., Kleis, W. K., Hafner, G., Hitzler, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factor+levels+in+platelet-rich+plasma+and+correlations+with+donor+age,+sex,+and+platelet+count.">Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count.</a> J. Craniomaxillofac. Surg. 30, 97-102 (2002).</li><li>Mazzucco, L., Balbo, V., Cattana, E., Guaschino, R., Borzini, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+every+PRP-gel+is+born+equal.+Evaluation+of+growth+factor+availability+for+tissues+through+four+PRP-gel+preparations:+Fibrinet,+RegenPRP-Kit,+Plateltex+and+one+manual+procedure.">Not every PRP-gel is born equal. Evaluation of growth factor availability for tissues through four PRP-gel preparations: Fibrinet, RegenPRP-Kit, Plateltex and one manual procedure.</a> Vox Sang. 97, 110-118 (2009).</li><li>Lei, H., Gui, L., Xiao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+anticoagulants+on+the+quality+and+biological+efficacy+of+platelet-rich+plasma.">The effect of anticoagulants on the quality and biological efficacy of platelet-rich plasma.</a> Clin. Biochem. 42, 1452-1460 (2009).</li><li>Redler, L. H., Thompson, S. A., Hsu, S. H., Ahmad, C. S., Levine, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma+therapy:+a+systematic+literature+review+and+evidence+for+clinical+use.">Platelet-rich plasma therapy: a systematic literature review and evidence for clinical use.</a> Phys. Sportsmed. 39, 42-51 (2011).</li><li>Whitman, D. H., Berry, R. L., Green, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+gel:+an+autologous+alternative+to+fibrin+glue+with+applications+in+oral+and+maxillofacial+surgery.">Platelet gel: an autologous alternative to fibrin glue with applications in oral and maxillofacial surgery.</a> J. Oral Maxillofac. Surg. 55, 1294-1299 (1997).</li><li>Anitua, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+rich+in+growth+factors:+preliminary+results+of+use+in+the+preparation+of+future+sites+for+implants.">Plasma rich in growth factors: preliminary results of use in the preparation of future sites for implants.</a> Int. J. Oral Maxillofac. Implants. 14, 529-535 (1999).</li><li>Whitman, D. H., Berry, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+improving+the+handling+of+particulate+cancellous+bone+and+marrow+grafts+using+platelet+gel.">A technique for improving the handling of particulate cancellous bone and marrow grafts using platelet gel.</a> J. Oral. Maxillofac. Surg. 56, 1217-1218 (1998).</li><li>Currie, L. J., Sharpe, J. R., Martin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fibrin+glue+in+skin+grafts+and+tissue-engineered+skin+replacements:+a+review.">The use of fibrin glue in skin grafts and tissue-engineered skin replacements: a review.</a> Plast. Reconstr. Surg. 108, 1713-1726 (2001).</li><li>Nikulin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+calcium,+thrombin+and+nucleotides+(ADP,+cAMP,+cGMP)+on+blood+platelet+glycolysis+and+energy+metabolism.">Effect of calcium, thrombin and nucleotides (ADP, cAMP, cGMP) on blood platelet glycolysis and energy metabolism.</a> Farmakol. Toksikol. 43, 585-590 (1980).</li><li>Hantgan, R. R., Taylor, R. G., Lewis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+interact+with+fibrin+only+after+activation.">Platelets interact with fibrin only after activation.</a> Blood. 65, 1299-1311 (1985).</li><li>Hantgan, R., Fowler, W., Erickson, H., Hermans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+assembly:+a+comparison+of+electron+microscopic+and+light+scattering+results.">Fibrin assembly: a comparison of electron microscopic and light scattering results.</a> Thromb. Haemost. 44, 119-124 (1980).</li><li>Li, B., Jiang, B., Boyce, B. M., Lindsey, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+polypeptide+nanoscale+coatings+incorporating+IL-12+for+the+prevention+of+biomedical+device-associated+infections.">Multilayer polypeptide nanoscale coatings incorporating IL-12 for the prevention of biomedical device-associated infections.</a> Biomaterials. 30, 2552-2558 (2009).</li><li>Li, B., Jiang, B., Dietz, M. J., Smith, E. S., Clovis, N. B., Rao, K. M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+local+MCP-1+and+IL-12+nanocoatings+for+infection+prevention+in+open+fractures.">Evaluation of local MCP-1 and IL-12 nanocoatings for infection prevention in open fractures.</a> J. Orthop. Res. 28, 48-54 (2010).</li><li>Boyce, B. M., Lindsey, B. A., Clovis, N. B., Smith, E. S., Hobbs, G. R., Hubbard, D. F., Emery, S. E., Barnett, J. B., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additive+effects+of+exogenous+IL-12+supplementation+and+antibiotic+treatment+in+infection+prophylaxis.">Additive effects of exogenous IL-12 supplementation and antibiotic treatment in infection prophylaxis.</a> J. Orthop. Res. 30 (2), 196-202 (2012).</li></ol>

Os autores declaram que não têm interesses conflitantes financeiros.

Plasma rico em plaquetas tem sido cada vez mais utilizado para aplicações clínicas, devido às suas propriedades promotoras de cura 15-17. No presente estudo, o PRP foi apresentado como uma nova abordagem para a prevenção de infecção. PRP foi encontrado com fortes propriedades antimicrobianas contra MRSA, MSSA, Grupo A Streptococcus e Neisseria gonorrhoeae. As principais vantagens do PRP, em comparação com os convencionais tratamentos com antibióticos, para a prevenção de infecç...

Implante infecção associada é uma complicação clínica significativa. Staphylococcus aureus (S. aureus) é um dos microorganismos mais comuns isoladas de implante de infecções associadas. Ele é capaz de produzir um biofilme que cobre as superfícies dos implantes, podendo levar a resistente a antibióticos 1,2 a infecção. Tratamento de implante infecção associada freqüentemente requer hospitalização a longo prazo para desbridamentos repetidos e terapia parenteral prolongada de antibióticos. Em casos resistentes aos antibióticos, a remoção do implante pode ser necessária. A resistência crescente das bactérias aos antibióticos também foi referido pelos Centros de Controle e Prevenção de Doenças (CDC) como &quot;um dos problemas de saúde do mundo mais prementes.&quot; Com o tempo, sem o desenvolvimento de novos e eficazes tratamentos antimicrobianos, é possível que vários fármacos patógenos resistentes será tratável com antibióticos convencionais. Prevenção de implante associadainfecção, é importante e novos agentes profiláticos ou abordagens são necessários para a prevenção de tais infecções. Plasma rico em plaquetas (PRP) é uma concentração de sangue autólogo que contém mais de 30 fatores de crescimento que podem ajudar com osso e enxerto ósseo cura 3-5. A aplicação de PRP para aumentar a regeneração óssea e maturação dos tecidos moles tem sido cada vez relatada em clínicas devido à sua elevada concentração de vários factores de crescimento libertados pelas plaquetas. Várias características do PRP indicam que o PRP pode também ter propriedades antimicrobianas 6-9. PRP contém um grande número de plaquetas, uma elevada concentração de leucócitos (os quais podem possuir hospedeiro-defesa acções contra bactérias e fungos), e vários peptídeos antimicrobianos 7,8,10. Em um estudo recente de um grande grupo de pacientes cirúrgicos cardíacos, foi revelado que o uso intra-operatório de gel de PRP durante encerramento de feridas signifvamente diminuiu a incidência de infecção do esterno superficial e profunda 11. Por estas razões e observações, a hipótese de que o PRP, além de suas bem estudadas cura propriedades promotoras, tem propriedades antimicrobianas. As vantagens potenciais da utilização de PRP para prevenir a infecção pode incluir: (i) O PRP é menos provável que induza a resistência em comparação com os convencionais tratamentos com antibióticos. (Ii) PRP também tem propriedades que promovem a cura e que podem ter um efeito sinérgico na prevenção de infecção; curando-promovendo PRP de propriedades podem proporcionar uma vedação para impedir a fixação bacteriana, reduzindo assim as probabilidades de infecção por agentes patogénicos e células humanos estão correndo para superfícies de implante 12 , 13. (Iii) o PRP é inerentemente biocompatível e seguro e livre do risco de doenças transmissíveis. Nosso objetivo de longo prazo é usar o PRP como uma nova abordagem para evitar implante associada infections. O objectivo deste estudo foi o de preparar PRP utilizando uma abordagem de centrifugação, duas vezes, para examinar PRP de propriedades in vitro anti-microbianos, e para descrever os protocolos para a avaliação de tais propriedades antimicrobianas.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bovine thrombin</td>
 <td>King Pharmaceuticals, Inc </td>
 <td>60793-215-05</td>
 <td>Thrombin (bovine origin)</td>
 </tr>
 <tr>
 <td>Calcium chloride</td>
 <td>King Pharmaceuticals, Inc</td>
 <td>60793-215-05</td>
 <td>10% calcium chloride</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>E7023</td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Baxter</td>
 <td>1001936060</td>
 <td></td>
 </tr>
 <tr>
 <td>Mueller Hinton broth</td>
 <td>Becton, Dickinson and Company</td>
 <td>275710</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline</td>
 <td>Sigma-Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <td>Tri-sodium citrate</td>
 <td>Sigma-Aldrich</td>
 <td>W302600</td>
 <td></td>
 </tr>
 <tr>
 <td>Tryptic soy agar</td>
 <td>Fisher Scientific</td>
 <td>R01202</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Kendro Laboratory Products</td>
 <td>750043077</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe filter</td>
 <td>Millipore</td>
 <td>SLGP033RS</td>
 <td></td>
 </tr>
 </tbody>
</table>

prp as a new approach to prevent infection: preparation and in vitro antimicrobial properties of prp

Implante infecção associada está a tornar-se cada vez mais difícil para a indústria de cuidados de saúde em todo o mundo, devido ao aumento de resistência a antibióticos, a transmissão de bactérias resistentes aos antibióticos entre animais e seres humanos, e os elevados custos de tratamento de infecções. No presente estudo, nós divulgamos uma nova estratégia que pode ser eficaz na prevenção de infecções associadas implante com base nas propriedades antimicrobianas potenciais de plasma rico em plaquetas (PRP). Devido às suas propriedades bem estudadas para promover a cura, PRP (um produto biológico) tem sido cada vez mais utilizado para aplicações clínicas, incluindo cirurgias ortopédicas, cirurgias periodontais e oral, cirurgias maxilo-faciais, cirurgias plásticas, medicina esportiva, etc PRP poderia ser uma alternativa avançada para tratamentos com antibióticos convencionais na prevenção de infecções associadas implante. A utilização de PRP pode ser vantajosa em comparação com os tratamentos convencionais antibiótico since PRP é menos provável que induza a resistência aos antibióticos e antimicrobianos de PRP e curando-propriedades promotoras podem ter um efeito sinérgico na prevenção de infecções. É bem conhecido que organismos patogénicos e células humanos estão correndo para superfícies de implantes, e as propriedades de cura de PRP de promoção pode melhorar a ligação de células humanas, reduzindo assim as probabilidades de infecção. Além disso, o PRP é inerentemente biocompatível e seguro e livre do risco de doenças transmissíveis. Para o nosso estudo, nós selecionamos várias clínicas estirpes de bactérias que são comumente encontrados em infecções ortopédicas e analisou se o PRP tem propriedades in vitro antimicrobianos contra estas bactérias. Nós preparamos PRP utilizando uma abordagem de centrifugação duas vezes o que permite que a mesma concentração de plaquetas a ser obtidos para todas as amostras. Nós conseguimos consistentes resultados antimicrobianos e descobriram que o PRP tem fortes propriedades in vitro antimicrobianos contra bactérias como methicillin sensíveis e resistentes à meticilina Staphylococcus aureus, Streptococcus do grupo A, e Neisseria gonorrhoeae. Portanto, a utilização de PRP pode ter o potencial para prevenir a infecção e para reduzir a necessidade de um tratamento pós-operatório de implante dispendioso infecções associadas.

PRP é reprodutivelmente preparados usando uma abordagem de centrifugação duas vezes (Figura 1). PRP é encontrado para apresentar forte (até 100 vezes a redução em UFCs) propriedades in vitro antimicrobianas contra resistente à meticilina S. aureus (MRSA) (Figura 3), que é vulgarmente encontrado em hospitais em todo o mundo 14. Da mesma forma, o PRP tem fortes propriedades antimicrobianas contra meticilina sensível S. aureus (MSSA)...

Watch this Scientific Journal Video about PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP at JoVE.com

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implante infecção associada é uma complicação clínica significativa. Este estudo descreve uma abordagem utilizando plasma rico em plaquetas (PRP) para evitar as infecções associadas implante, apresenta o protocolo para a preparação de PRP com a concentração de plaquetas constante, e relata as propriedades antimicrobianas recentemente identificados de PRP e protocolos relacionados para examinar tais propriedades antimicrobianas<em&gt; In vitro.</em

PRP como uma nova abordagem para prevenir a infecção: Preparação e<em&gt; In vitro</em&gt; Propriedades antimicrobianas do PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

prp-as-new-approach-to-prevent-infection-preparation-vitro

Research

JoVE Journal

Immunology and Infection

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

21.2K visualizações.  West Virginia University. Implante infecção associada é uma complicação clínica significativa. Este estudo descreve uma abordagem utilizando plasma rico em plaquetas (PRP) para evitar as infecções associadas implante, apresenta o protocolo para a preparação de PRP com a concentração de plaquetas constante, e relata as propriedades antimicrobianas recentemente identificados de PRP e protocolos relacionados para examinar tais propriedades antimicrobianas In vitro.

Vídeo: PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transdução retroviral de receptor de células T no rato células T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Imunologia e Infecção

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

Simples e robusto In vivo E In vitro Abordagem para o Estudo Assembléia Virus

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso de meio de escarro Artificial para testar a eficácia antibiótica contra<em&gt; Pseudomonas aeruginosa</em&gt; Em condições mais relevantes para o Pulmão Fibrose Cística

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Uma<em&gt; In vitro</em&gt; Modelo para estudar respostas imunes de sangue periférico humano células mononucleares para Vírus Respiratório Sincicial Humano Infecção

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Utilização de<em&gt; Galleria mellonella</em&gt; Como um organismo modelo para estudar<em&gt; Legionella pneumophila</em&gt; Infecção

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Acompanhamento da evolução das Membrane Polaridade, integridade de membrana e intracelulares Ion concentrações no<em&gt; Streptococcus pneumoniae</em&gt; Usando corantes fluorescentes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

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 ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Uma abordagem simples e eficiente para construir vectores de vírus mutante Vaccinia

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Transdução retroviral de células T helper como uma abordagem genética para estudar mecanismos que controlam a sua diferenciação e função

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...