Name: expression of exogenous antigens in the mycobacterium bovis bcg vaccine via non-genetic surface decoration with the avidin-biotin system
Uploaded: 2018-01-31T13:00:00
Description: Watch this Scientific Journal Video about Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System at JoVE.com

Biz Dr R Stokes BCG Pasteur zorlanma için ve A. Talal teknik destek için teşekkür ederiz. Ayrıca GenScript gen sentezi ile yardım için teşekkür.

Bütün hayvanlar hayvan bakımı ve kullanımı Komiteler British Columbia Üniversitesi tarafından onaylanmış protokoller uyarınca muhafaza. Deneyler hayvan bakımı ve kullanımı Komiteler tarafından onaylanmış ve hayvan bakım kuralları Kanada Konseyi göre gerçekleştirilen. Hayvan güvence refah A11-0247 sayıdır.
1. nesil Monomeric Avidin füzyon protein plazmid ifade
<ol><li>Alt monomeric avidin sıra12 pDEST17 plazmid 133 için karşılık gelen "C...

<ol>
	<li>Horwitz, M. A., Harth, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+vaccine+against+tuberculosis+affords+greater+survival+after+challenge+than+the+current+vaccine+in+the+guinea+pig+model+of+pulmonary+tuberculosis.">A new vaccine against tuberculosis affords greater survival after challenge than the current vaccine in the guinea pig model of pulmonary tuberculosis.</a> Infect. Immun. 71 (4), 1672-1679 (2003).</li><li>Pym, A. S., 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=Recombinant+BCG+exporting+ESAT-6+confers+enhanced+protection+against+tuberculosis.">Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis.</a> Nat Med. 9 (5), 548-553 (2003).</li><li>Grode, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+vaccine+efficacy+against+tuberculosis+of+recombinant+Mycobacterium+bovis+bacille+Calmette-Guerin+mutants+that+secrete+listeriolysin.">Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guerin mutants that secrete listeriolysin.</a> The J Clin Invest. 115 (9), 2472-2479 (2005).</li><li>Bastos, R. G., Borsuk, S., Seixas, F. K., Dellagostin, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+Mycobacterium+bovis+BCG.">Recombinant Mycobacterium bovis BCG.</a> Vaccine. 27 (47), 6495-6503 (2009).</li><li>Mederle, I., 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=Plasmidic+versus+insertional+cloning+of+heterologous+genes+in+Mycobacterium+bovis+BCG:+impact+on+in+vivo+antigen+persistence+and+immune+responses.">Plasmidic versus insertional cloning of heterologous genes in Mycobacterium bovis BCG: impact on in vivo antigen persistence and immune responses.</a> Infect Immun. 70 (1), 303-314 (2002).</li><li>Singh, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Approach+to+Cancer+Immunotherapy+Tumor+Cells+Decorated+with+CD80+Generate+Effective+Antitumor+Immunity+A+Novel+Approach+to+Cancer+Immunotherapy+Tumor+Cells+Decorated+with+CD80+Generate+Effective+Antitumor+Immunity.">A Novel Approach to Cancer Immunotherapy Tumor Cells Decorated with CD80 Generate Effective Antitumor Immunity A Novel Approach to Cancer Immunotherapy Tumor Cells Decorated with CD80 Generate Effective Antitumor Immunity.</a> Cancer Res. 63 (14), 4067-4073 (2003).</li><li>Oizumi, S., Strbo, N., Pahwa, S., Deyev, V., Podack, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+requirements+for+enhanced+antigen+cross-presentation+to+CD8+cytotoxic+T+lymphocytes.">Molecular and cellular requirements for enhanced antigen cross-presentation to CD8 cytotoxic T lymphocytes.</a> J Immunol. 179 (4), 2310-2317 (2007).</li><li>Sharif-Paghaleh, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+efficacy+of+dendritic+cell+vaccination+by+early+detection+of+(99m)+Tc-HMPAO-labelled+CD4(+)+T+cells.">Monitoring the efficacy of dendritic cell vaccination by early detection of (99m) Tc-HMPAO-labelled CD4(+) T cells.</a> Eur J Immunol. 44 (7), 2188-2191 (2014).</li><li>Sun, 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=A+broad-range+of+recombination+cloning+vectors+in+mycobacteria.">A broad-range of recombination cloning vectors in mycobacteria.</a> Plasmid. 62 (3), 158-165 (2009).</li><li>Laitinen, O. 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=Rational+design+of+an+active+avidin+monomer.">Rational design of an active avidin monomer.</a> J. Biol. Chem. 278 (6), 4010-4014 (2003).</li><li>Flynn, J. L., Chan, J., Triebold, K. J., Dalton, D. K., Stewart, T. A., Bloom, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+role+for+interferon+gamma+in+resistance+to+Mycobacterium+tuberculosis+infection.">An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection.</a> J Exp Med. 178 (6), 2249-2254 (1993).</li><li>Liao, T. Y. A., Lau, A., Joseph, S., Hytonen, V., Hmama, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+immunogenicity+of+the+Mycobacterium+bovis+BCG+vaccine+by+non-genetic+bacterial+surface+decoration+using+the+avidin-biotin+system.">Improving the immunogenicity of the Mycobacterium bovis BCG vaccine by non-genetic bacterial surface decoration using the avidin-biotin system.</a> PLoS ONE. 10 (12), e0145833 (2015).</li><li>Green, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avidin+and+streptavidin.">Avidin and streptavidin.</a> Methods Enzymol. 184, 51-67 (1990).</li><li>Howarth, M., Takao, K., Hayashi, Y., Ting, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+quantum+dots+to+surface+proteins+in+living+cells+with+biotin+ligase.">Targeting quantum dots to surface proteins in living cells with biotin ligase.</a> PNAS. 102 (21), 7583-7588 (2005).</li><li>Lesch, H. P., Kaikkonen, M. U., Pikkarainen, J. T., Yla-Herttuala, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avidin-biotin+technology+in+targeted+therapy.">Avidin-biotin technology in targeted therapy.</a> Expert Opin Drug Deliv. 7 (5), 551-564 (2010).</li><li>Paganelli, G., Bartolomei, M., Grana, C., Ferrari, M., Rocca, P., Chinol, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioimmunotherapy+of+brain+tumor.">Radioimmunotherapy of brain tumor.</a> Neurol Res. 28 (5), 518-522 (2006).</li><li>Cummings, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+immunogenicity+of+a+plant-produced+recombinant+monomer+hemagglutinin-based+influenza+vaccine+derived+from+influenza+A+(H1N1)pdm09+virus:+a+Phase+1+dose-escalation+study+in+healthy+adults.">Safety and immunogenicity of a plant-produced recombinant monomer hemagglutinin-based influenza vaccine derived from influenza A (H1N1)pdm09 virus: a Phase 1 dose-escalation study in healthy adults.</a> Vaccine. 32 (19), 2251-2259 (2014).</li><li>Kolotilin, I., 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=Plant-based+solutions+for+veterinary+immunotherapeutics+and+prophylactics.">Plant-based solutions for veterinary immunotherapeutics and prophylactics.</a> Vet Res. 45, 114-117 (2014).</li><li>Demonte, D., Drake, E. J., Lim, K. H., Gulick, A. M., Park, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-based+engineering+of+streptavidin+monomer+with+a+reduced+biotin+dissociation+rate.">Structure-based engineering of streptavidin monomer with a reduced biotin dissociation rate.</a> Proteins. 81 (9), 1621-1633 (2013).</li><li>Deghmane, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoamide+dehydrogenase+mediates+retention+of+coronin-1+on+BCG+vacuoles,+leading+to+arrest+in+phagosome+maturation.">Lipoamide dehydrogenase mediates retention of coronin-1 on BCG vacuoles, leading to arrest in phagosome maturation.</a> J Cell Sci. 120 (19), 3489-3489 (2007).</li><li>Soualhine, 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=Mycobacterium+bovis+bacillus+Calmette-Guerin+secreting+active+cathepsin+S+stimulates+expression+of+mature+MHC+class+II+molecules+and+antigen+presentation+in+human+macrophages.">Mycobacterium bovis bacillus Calmette-Guerin secreting active cathepsin S stimulates expression of mature MHC class II molecules and antigen presentation in human macrophages.</a> J Immunol. 179 (8), 5137-5145 (2007).</li><li>Bubb, M. O., Green, F., Conradie, J. D., Tchernyshev, B., Bayer, E. A., Wilchek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+antibodies+to+avidin+in+human+serum.">Natural antibodies to avidin in human serum.</a> ImmunolLett. 35 (3), 277-280 (1993).</li><li>Petronzelli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+use+of+avidin+is+not+hampered+by+antiavidin+antibodies+in+humans.">Therapeutic use of avidin is not hampered by antiavidin antibodies in humans.</a> Cancer Biother Radiopharm. 25 (5), 563-570 (2010).</li><li>Bigini, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+fate+of+avidin-nucleic+acid+nanoassemblies+as+multifunctional+diagnostic+tools.">In vivo fate of avidin-nucleic acid nanoassemblies as multifunctional diagnostic tools.</a> ACS Nano. 8 (1), 175-187 (2014).</li></ol>

BCG yüzeyinde belirli antijenleri veya bakteri'nın immünojenisite verimli bir şekilde geliştirmek için beklenen belirli fonksiyonel özellikler eklemek için hızlı ve etkili görüntü eksojen proteinlerin için genetik olmayan bir yaklaşım bu çalışmada bildirdi. BCG hücre yüzeyine kolayca biotinylated avidin füzyon protein ile anlık yüzey dekorasyon için olabilir gösterdi. Genetik dönüşümü ve olumlu klonlar yelpazesi gerektirir iken 2-3 ay zaman lag toplam yordam içinde 2 h, gerçekleştirilebi...

Çeşitli stratejileri geçerli TB aşı BCG protein adjuvan sistemleri, viral vektörlü teknolojileri, zayıflatılmış canlı M.tb suşları ve genetiği değiştirilmiş BCG suşları, ya genlerinin tanıtmak için de dahil olmak üzere, eski yerine koymak için önerilen yeterince enfeksiyon1 ya da Mtbsırasında gösterilir değil BCG antijenleri aşırı ifade-belirli antijenleri BCG2' mevcut değil. Genetik mühendisliği, ancak, birçok engeller Emanet, zaman alan işlem ve düşük verimlilik ifade vektörel çizimler4,5belirsiz düzey de dahil olmak üzere karşı karşıya. BCG iyileştirilmesi açısından, alternatif bir yaklaşım immünojenisite belirsiz genetik değişiklikler için gerek kalmadan geliştirmek için gereklidir.
Bu çalışmada, biotin tanınmış yüksek afinite avidin etkileşim dayanır BCG hücre yüzeyi ilgi rekombinant proteinlerin görüntü için yeni bir strateji tanıtmak. Bu yaklaşım hızlı ve tekrarlanabilir eki rekombinant avidin füzyon proteinlerin biotinylated maksimal iyileştirme onun etkinliğinin onun mükemmel emniyet koruyarak ulaşmak için BCG geniş işlemler kolaylaştırır BCG yüzeyi sağlar kayıt, kullanım yılda gözlenen.
Biotin için avidin ilgi son derece yüksektir (Kd 10−15 M =) ve bir kez kurulan, biotin avidin karmaşık çok kararlı ve yalnızca koşullar6denaturing altında bozulmuş. Ancak, bu tür bir gen transferi yöntemi alternatif olarak hizmet için etkileşim için uzun vadeli ama geri döndürülebilir ekran rekombinant proteinlerin gereklidir. Böylece, biz burada bir düşük benzeşme monomeric avidin tanıttı (Kd 10−7 M =) protein tersinir sürümü götürür yüzey hücreleri sunulması antijen içinde bir kez yutulur BCG dekore edilmiştir. Kavramının bir kanıtı olarak sağlamak için bu yöntem ovalbumin (OVA)7,8' den türetilmiş bir vekil antijen karşılık gelen bir monomeric avidin chimeric protein kullanarak test ettik. BCG hücre yüzeyine kolayca ve hızla monomeric avidin füzyon proteinler ile dekore edilebilir olduğunu ve bu bağlama BCG yüzeye istikrarlı ve bakteriyel büyüme ve hayatta kalma tespit değişiklikler olmadan tekrarlanabilir sonuçlar gösterdi. Ayrıca, monomeric avidin OVA (AviOVA) ile erimiş süslenmiş BCG BCG tarafından indüklenen için benzer bir bağışıklık yanıtı tetikleyebilir genetik olarak aynı antijen ifade bulduk vitro ve içinde vivo. Bu teknoloji bakteri yüzeyindeki ilgi proteinlerin geri döndürülebilir ekran bu nedenle geleneksel gen transferi yaklaşımlar etkili bir yerini ve BCG geniş işlemler ve daha fazla aşı uygulamaları için bir platform sağlayabilir geliştirme.

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expression of exogenous antigens in the mycobacterium bovis bcg vaccine via non-genetic surface decoration with the avidin-biotin system

Tüberküloz (TB) ciddi bir bulaşıcı hastalık ve biricik elde edilebilir aşı M. bovis bacillus Calmette-Guerin (BCG) çocuk şiddetli TB menenjit ve Dissemine TB bazı türlerinin karşı koruma için güvenli ve etkili ama başaramamak-e doğru korumak olduğunu Akciğer TB karşı hangi hastalığın en yaygın biçimidir. Şu anda BCG geliştirmek için umut verici stratejileri ya dönüştürmenin immunodominant M. tuberculosis (Mtb)kodlama genlerimizle itimat-belirli antijenleri ve/veya tamamlayıcı antijen teşvik etkenler kodlama genler ile hücreleri sunulması. Bu yaklaşımlara büyük sınırlamalar düşük verimlilik, düşük istikrar ve güvenliğini ifade vektörel çizimler belirsiz düzeyini içerir. Bu çalışmada, BCG uluslara bakteri, tercihan--dan dönüştürme yüzeyinde ilgi eksojen proteinler ile ilgili genlerin kodlama Plasmid'ler ile oluşur aşı geliştirme için alternatif bir yaklaşım sunuyoruz. İlk olarak, proteinler ilgi erime içinde standart E. coli monomeric avidin ile ifade edilir ifade sistemleri ve biotinylated BCG yüzeyine süslemek için kullandı. Hayvan deneyleri BCG yüzey yedek ovalbumin antijen ile dekore edilmiş kullanarak değiştirilmiş bakteri tamamen immunojenik ve belirli T hücre yanıt ikna yeteneğine sahip olduğunu göstermektedir. Tamamen, kesinlikle burada sunulan veri uluslara zahmetli geleneksel yaklaşım eksojen nükleik asitler ile değiştirir geçerli BCG aşısı yeniden şekillendirilmesi için bir roman ve verimli yöntem destekler.

Yukarıda açıklanan genel yordamlar ile fizibilite BCG yüzey biotinylation ve vekil antijen OVA ile dekorasyon incelenmiştir. Sonra değiştirilmiş BCG immünojenisite oldu test içinde vivo. Bakteriyel yüzey kolayca avidin chimeric antijenleri bakteriyel fenotipleri tespit herhangi bir değişiklik olmadan hızlı ekran için biotin ile etiketli oldu. Elde edilen değiştirilmiş BCG verimli antijen sunumu hücreleri tarafından yutulur ve OVA özgü bağışıklık yanıt...

Watch this Scientific Journal Video about Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System at JoVE.com

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Bakteriyel bir yüzey hızlı antijen görüntülemek için yeni bir teknik olan yüzey biotinylation proteinler füzyon monomeric avidin ile ilgi maruz takip içerir sunulur. BCG seçili antijenleri ile başarıyla yükleme yüzey dekorasyon geleneksel genetik yaklaşımlar yerine düşündüren onun immünojenisite geliştirir.

Eksojen antijenleri Mycobacterium bovis BCG aşısı Avidin-biotin sistemi ile sigara genetik yüzey dekorasyon ile ifade

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Research

JoVE Journal

Immunology and Infection

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

Mikobakteriler canlılık Tedbir Fonksiyonel Tam Kan Testi, Reporter Gen BCG veya M. Tb (BCG Takip edilen konu kullanarak Lüks / M. Tb Lüks)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Yenidoğan ve Erişkin Merkezi sinir sisteminin Kültüre Mikroglia

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

FDA onaylı Vero hücrelerinde Aşı Geliştirme için bir araya getiren Arenavirus üretimi

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

Bakülovirüs İfade Sistemi ile Novel H7N9 influenza virüs hemaglutinin Fonksiyonel rekombinant ve Neuraminidase Proteinlerin İfadesi

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

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

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

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 ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÃƒÅ½Ã‚ÂµRI

Bir Geliştirilmesi<em&gt; In vitro</emEtkileşimini incelemek için&gt; bir model sistem<em&gt; Equus</em&gt;<em&gt; Caballus</emYüksek afiniteli reseptör FcεRI&gt; ile IgE

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-GuÃƒÆ’Ã†'Ãƒâ€šÃ‚Â©rin

ile Enfeksiyon sırasında boşaltılması Lenf düğümleri içine Fare Cilt Dendritik Hücre Göç Eğitim Bir KAKE tabanlı Testi<em&gt; Mycobacterium bovis</em&gt; Basil Calmette-Guerin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Bir kullanma Lenfosit Ekstravazasyonunun analizi<em&gt; İn Vitro</em&gt; İnsan Kan-beyin bariyeri Model

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Membran yuvası arıtma için çapraz-sunudaki eksojen antijenleri endoplazmik retikulum ilişkili bozulma

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Mycobacterium tüberküloz ile muhabir enzim Floresans farelerde görüntüleme

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...