Yazarlar Therwa Hamza, John E. Tidwell, Nina Clovis, ve deneysel yardım ve redaksiyon için Suzanne Smith Suzanne Smith teşekkürler. Yazarlar ayrıca, onun desteği ve Mikrobiyoloji, İmmünoloji ve West Virginia Üniversitesi&#39;nde Hücre Biyoloji Bölümü biyolojik güvenlik laboratuar kullanımı için bakteriyel klinik izolat ve John B. Barnett, Doktora sağlamak için John Thomas, Doktora ederim. Yazarlar Osteosentez ve Travma Bakım Vakfı ve Ulusal Bilim Vakfı (# 1003907) mali destek için minnettarım. Mikroskop deneyleri ve görüntü analizi de Meryem Babb Randolph Kanser Merkezi ve NIH hibe P20 RR016440 tarafından kısmen desteklenen Batı Virginia Üniversitesi Görüntüleme Tesis içinde yapılmıştır. Kan Hayvan kullanımı Batı Virginia Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onaylanmıştır çizer. Tüm deneyler ilgili tüm guidelin uygun idam edildies, düzenlemeler ve düzenleyici kurumlar.

1. Hazırlanması ve PRP Etkinleştirme 1.1 Kan beraberlik <ol><li> Izofluran inhalasyonu (indüksiyon için O 2% 2 ve idamesi için% 1) ile tavşan uyuştur. </li><li> 20 ml&#39;lik bir enjektöre 2 ml 0.129 M tri-sodyum sitrat (bir anticoagulent çözüm) çizin. Tri-sodyum sitrat solüsyonu 50 ml damıtılmış H2O içinde 1.897 g tri-sodyum sitrat çözücü ve bir 0.22 um filtre ile steril filtre edilerek hazırlanır. </li><li> % 70 etanol kullanılarak tavşan kulak sterilize. </li><li> Bir kelebek iğne (25 g) ile şırınga aracılığıyla bağlı tavşan kulak damarından (örneğin, 5 ml) ile kan çizin. </li><li> Nazik çalkalama ile tri-sodyum sitrat çözeltisi ile kan karıştırılır. Kan ve tri-sodyum sitrat çözeltisi hacmi oranı 09:01 olduğu. </li></ol> 1,2 TZP hazırlama (Şekil 1) <ol><li> 50 ml plastik santrifüj tüpüne antikoagüle kan aktarın. 10 ul bir alikotu atınhemocytometry kullanarak bazal trombosit sayısını belirlemek için kan. </li><li> Bir rotor ile bir santrifüj içinde oda sıcaklığında 10 dakika (RT) için 300 x g&#39;da kan santrifüjleyin. Düşük hızlanma ve fren hızı (Şekil 1) ayarlayın. </li><li> Santrifüjlemeden sonra, kan üç tabakaya ayrılmıştır. Alt tabaka esas olarak kırmızı kan hücreleri, orta tabaka (genellikle &quot;buffy coat&quot; olarak anılacaktır) konsantre trombosit ve lökositlerin oluşur ve üst tabaka esas olarak kan sıvı bileşeni olan plazma, ve trombosit (almaktadır Şekil 1). Bir hücre kültürü kaputu santrifüj tüpüne dikkatle taşınması; katmanları rahatsız etmeyin. Bir 1 ml plastik pipet kullanarak 15 ml plastik tüp içine plazma tüm, buffy coat ve 2-3 mm kalınlığında kırmızı kan hücre tabakası aktarın. </li><li> RT 15 dakika boyunca 3.000 xg&#39;de aktarılan örnek ikinci kez santrifüjleyin. Üst tabaka (süpernatant) trombosit ile zayıf plazmanın (PPP), bir kabul edilird, yeni bir tüpe aktarılmıştır. </li><li> PPP 2.0 x 10 6 trombosit / ul (hemocytometry tarafından belirlenen) elde etmek için kullanılıyorsa, geri kalan kan örneği içinde, trombosit toplama ayarlayarak PRP elde edilir. </li></ol> 1,3 TZP aktivasyon <ol><li> 1000 IU / ml çalışma konsantrasyonu için 5 ml% 10 kalsiyum klorür ile 5000 IU Sığır trombini eriterek PRP etkinleştirme çözeltisi hazırlayın. </li><li> PRP ve PPP için aktivasyon çözümü ekleyin ve sürekli PRP ve PPP-jeller oluşturmak için pipetleme çözüm karıştırmak. PRP veya PPP için aktivasyon çözelti hacmi oranı 1:4 olduğunu. </li></ol> 2. Öldür Eğrisi Assay kullanma TZP vitro Antimikrobiyal Testi (Şekil 2) olarak <ol><li> Steril bir inokülasyon döngü kullanarak, S. çeşitli koloniler ekleyin plastik bir tüp içinde Mueller Hinton broth (MHB) 5 ml içine gecede plaka kültüründen aureus. Girdap kısaca ve daha sonra 37 2 saat için örnek inkübe ° C Sonraki, Bakteri ortamın optik yoğunluk bir spektrofotometre kullanılarak belirlendi ve ~ 1 &#39;e eşit bir optik yoğunluk&#39; e ayarlandı x 10 8 CFU / önceden belirlenmiş standart bir eğri esas ml. </li><li> 1 x 10 6 CFU / mL elde etmek ve buz üzerinde mikroorganizma yerleştirmek için PBS kullanarak 100x seyreltme yapın. </li><li> Kurma ve steril, tek kullanımlık 5 ml yuvarlak tabanlı polistiren tüpler etiket, ve her bir tüp içinde 2 ml değerindeki bir son hacim için Tablo 1 &#39;de, aşağıdaki gruplar içindeki hazırlanır. </li><li> Etkinleştirme (jel oluşumu) için trombin çözeltisi ile muamele edildi polistiren tüpler için ilk PRP, PPP veya PBS, ekleyin. Daha sonra, MHB ekleyin ve sonra S. aureus, mikroorganizma (1 x 10 6 CFU / ml), 1 x 10 5 CFU / ml &#39;lik nihai konsantrasyon elde edildi. </li><li> ° C 150 rpm&#39;de orbital ajitasyon ile 37 inkübe edin. </li><li> Önceden belirlenmiş zaman noktalarında (örneğin 0, 1 ve 2 saat) At, tekrar pipetleme (Bu yoluyla her tüp çözümleri mixbakteri TZP jelden içinde sıkışıp olabilir beri adım) önemlidir. , Örnek 10 ul al steril% 0.9 salin ile seri seyreltin ve Triptik Soy Agar (TSA,% 5 koyun kanı ile) CFU sayımı için plaka üzerine her seyreltme 100 ul kısım Pipeti. </li><li> 37 gecede Kültür agar plakaları ° C, daha sonra saymak ve plaka koloniler kaydedin. Konu x-ekseni ve CFU / y-ekseni üzerinde ml zaman (saat) bir ölçek üzerinde logarithimic veriler. </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>

Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Trombosit zengin plazma giderek şifa destekleyici özellikleri 15-17 nedeniyle klinik uygulamalar için kullanılır olmuştur. Bu çalışmada, TZP enfeksiyonun önlenmesi için yeni bir yaklaşım olarak sunuldu. PRP MRSA, MSSA, Grup A Streptokok, ve Neisseria gonorrhoeae karşı güçlü antimikrobiyal özelliklere sahip olduğu bulunmuştur. Konvansiyonel antibiyotik tedavilere kıyasla PRP önemli avantajları, enfeksiyonun önlenmesi için şunlardır: (1) Cari antibiyotik tedavileri ...

İmplant ile ilişkili enfeksiyonu önemli bir klinik komplikasyondur. Staphylococcus aureus (S. aureus) implant ile ilişkili enfeksiyonlar en sık izole edilen mikroorganizmalar biridir. Bu implantların yüzeyleri kaplar ve antibiyotiğe dirençli enfeksiyon 1,2 varabiliyor biyofilm üretme kapasitesine sahiptir. Implant ilişkili enfeksiyon tedavisi sık tekrarlanan debridman ve uzun süreli parenteral antibiyotik tedavisi için uzun süreli yatış gerektirir. Antibiyotik dirençli durumlarda, implantın çıkartılması gerekli olabilir. Bakterilerin antibiyotik yükselen direniş aynı zamanda Hastalık Kontrol ve Önleme (CDC) Merkezleri tarafından anılır olmuştur &quot;dünyanın en acil sağlık sorunlarından biridir.&quot; Zaman içinde, yeni ve etkili antimikrobiyal tedavilerin geliştirilmesi olmaksızın, çoklu ilaca dirençli patojenler geleneksel antibiyotiklerle tedavi edilemez olacağı mümkündür. Implant ilişkili Önlenmesienfeksiyon önemlidir ve yeni yaklaşımlar profilaktik ajanlar ya da bu gibi enfeksiyonların önlenmesi için gereklidir. Trombosit zengin plazma (TZP), kemik ve kemik grefti iyileşmesi 3-5 ile yardımcı olabilir 30&#39;dan fazla büyüme faktörleri içeren otolog kan konsantrasyonu. Kemik rejenerasyonu ve yumuşak doku olgunlaşmasını artırmak için PRP uygulama giderek çünkü trombositler tarafından yayımlanan çeşitli büyüme faktörleri, yüksek konsantrasyonu kliniklerinde rapor edilmiştir. PRP Çeşitli özellikleri PRP de antimikrobiyal özellikleri 6-9 olabileceğini gösteriyor. PRP trombosit çok sayıda, lökositlerin yüksek bir konsantrasyonda (bakterilere ve mantarlara karşı host defense etkilere sahip olabilir), ve birden fazla antimikrobiyal peptitler 7,8,10 içerir. Kardiyak cerrahi hastalarında büyük bir kohort bir çalışmada, bu ortaya çıktı bu yaranın kapanması anlaml sırasında TZP-jel intraoperatif kullanımınıicantly yüzeyel ve derin sternum enfeksiyonu 11 insidansı azalmıştır. Bu nedenlerle ve gözlemler için, PRP, onun iyi çalışılmış şifa arttırıcı özellikleri yanında, antimikrobiyal özelliklere sahip olduğu varsayıldı. Enfeksiyonu önlemek için TZP kullanmanın potansiyel avantajları şunlardır: (i) PRP konvansiyonel antibiyotik tedavilere kıyasla direnç indükleme olasılığı düşüktür. PRP şifa destekleyici özellikleri patojenler ve insan hücreleri implant yüzeyleri 12 yarış olarak böylece enfeksiyon oran azaltarak bakteriyel eki önlemek için sızdırmazlık sağlayabilir, (ii) PRP de enfeksiyon önleme üzerinde sinerjik bir etkiye sahip olabilir iyileşmesine katkıda özelliklere sahiptir , 13. (Iii) PRP doğal biyouyumlu ve güvenli ve bulaşıcı hastalıkların riski içermez. Bizim uzun vadeli hedefi implant ile ilişkili enfeksiyonu önlemek için yeni bir yaklaşım olarak PRP kullanmaktırons. Bu çalışmanın amacı, in vitro antimikrobiyal özelliklerde PRP incelemek için, bir iki defa santrifüj yaklaşım kullanılarak PRP hazırlamak için, ve bu gibi antimikrobiyal özelliklerini değerlendirmek için protokoller tanımlanmasıdır.

<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

İmplant ile ilişkili enfeksiyon antibiyotik direnci, hayvanlar ve insanlar arasındaki antibiyotik dirençli bakteri iletim ve enfeksiyonların tedavisinde yüksek maliyeti artan nedeniyle daha fazla ve sağlık sektöründe daha zorlu dünya haline geliyor. Bu çalışmada, trombosit açısından zengin plazma (PRP) potansiyel antimikrobiyal özellikleri esas implant ile ilişkili enfeksiyonu önlemede etkili olabilir, yeni bir strateji ifşa. Şifa teşvik etmek için onun iyi araştırılan özellikleri nedeniyle, PRP (biyolojik bir ürün) giderek ortopedik ameliyatlar, periodontal ve oral cerrahi, maksillofasiyal cerrahi, plastik cerrahi, spor hekimliği, vb dahil olmak üzere klinik uygulamalar için kullanılır olmuştur PRP implant ile ilişkili enfeksiyonların önlenmesinde konvansiyonel antibiyotik tedavileri için gelişmiş bir alternatif olabilir. PRP kullanımı geleneksel antibiyotik tedavisi si ile karşılaştırıldığında avantajlı olabilirPRP antibiyotik direnci indüklediği ve PRP antimikrobiyal ve şifa teşvik edici özelliklerine enfeksiyon önleme üzerinde sinerjik bir etkiye sahip olabilir olasılığı düşüktür nce. Biliyorsunuz patojenler ve insan hücreleri implant yüzeyleri için yarış olduğu bilinen ve teşvik şifa PRP özelliklerini böylece enfeksiyon oran azaltarak insan hücresi eki artırabilirsiniz. Buna ek olarak, doğal olarak PRP biyo-uyumlu, güvenli ve bulaşıcı hastalıkların risk içermez. Bizim araştırma için, yaygın ortopedik enfeksiyonları bulunan ve PRP bu bakterilere karşı in vitro antimikrobiyal özellikleri olup olmadığını incelenerek çeşitli klinik suşların seçtiniz. Biz aynı trombosit konsantrasyonu tüm örnekler için elde edilmesine olanak tanıyan iki santrifüj yaklaşım kullanarak PRP hazırladık. Biz tutarlı antimikrobiyal bulgulara ulaşılmıştır ve PRP Methi gibi bakterilere karşı in vitro antimikrobiyal özellikleri güçlü olduğunu buldukcillin duyarlı ve metisiline dirençli Staphylococcus aureus, Grup A Streptokok, ve Neisseria gonorrhoeae. Bu nedenle, TZP kullanımı enfeksiyonu önlemek ve implant ile ilişkili enfeksiyonların masraflı post-operatif tedavi ihtiyacını azaltmak için potansiyeli olabilir.

PRP tekrarlanabilir bir kez santrifüj yaklaşım (Şekil 1) kullanılarak hazırlanmıştır. PRP metisiline dirençli S. karşı in vitro antimikrobiyal özellikleri (CFU kadar 100 kat azalma) güçlü sunmak bulundu aureus (MRSA) (Şekil 3), dünya çapında yaygın olan hastanelerde 14 bulunur. Benzer şekilde, TZP metisiline duyarlı S. karşı güçlü antimikrobiyal özellikleri vardır aureus (MSSA), Grup A Str...

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

İmplant ile ilişkili enfeksiyonu önemli bir klinik komplikasyondur. Bu çalışma, implant ile ilişkili enfeksiyonların önlenmesi için trombosit açısından zengin plazma (PRP) kullanarak bir yaklaşım açıklar, sabit trombosit konsantrasyonu ile PRP hazırlama protokolü sunar ve PRP yeni tespit antimikrobiyal özellikleri ve bu antimikrobiyal özelliklerinin incelenmesi için ilgili protokoller raporları<em&gt; In vitro.</em

Hazırlama ve: Enfeksiyon Önleme Yeni Bir Yaklaşım olarak PRP<em&gt; In vitro</emPRP&gt; Antimikrobiyal Özellikleri

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 Görüntüleme.  West Virginia University. İmplant ile ilişkili enfeksiyonu önemli bir klinik komplikasyondur. Bu çalışma, implant ile ilişkili enfeksiyonların önlenmesi için trombosit açısından zengin plazma (PRP) kullanarak bir yaklaşım açıklar, sabit trombosit konsantrasyonu ile PRP hazırlama protokolü sunar ve PRP yeni tespit antimikrobiyal özellikleri ve bu antimikrobiyal özelliklerinin incelenmesi için ilgili protokoller raporları In vitro.

Video: 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

Fare T-hücrelerinin, T-hücre reseptörleri retroviral İletimi

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 ...

İmmünoloji ve Enfeksiyon

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.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

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.

Basit ve Sağlam In vivo Ve In vitro</emVirüs Meclisi Öğrenmenin> Yaklaşım

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.

Karşı Antibiyotik Etkinlik Test Yapay Balgam Orta Kullanımı<em&gt; Pseudomonas aeruginosa</em&gt; Kistik Fibrozis Akciğer Daha İlgili Koşullar

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 Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Bir<em&gt; In vitro</emİnsan Makrofaj Farklılaşma ve Polarizasyon heterojenliği Eğitim için&gt; Model

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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.

Kullanımı<em&gt; Galleria mellonella</em&gt; Bir Model Organizma olarak Eğitim için<em&gt; Legionella pneumophila</em&gt; Enfeksiyon

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.

Membran Kutup, Membran Dürüstlük ve Hücre içi İyon Konsantrasyonlarında İzleme değişiklikler<em&gt; Streptococcus pneumoniae</em&gt; Floresan boyalar kullanarak

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.

Batı Nil Virüsü Mutant Soy Enfeksiyon MyD88-aracılı Hücresel Bağışıklık Tepki Vitro Analizi

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.

Basit ve Etkin Yaklaşım Mutant Vaccinia Virüs Vektörler Construct

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.

Eğitim için bir genetik Yaklaşım olarak yardımcı T hücrelerinin retroviral İletimi Mekanizmaları kendi Farklılaşma ve Kontrol Fonksiyonunun

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