Bu çalışma, Wellcome Trust Beate Kampmann (056.608, GRO77273) için kişisel burslar tarafından finanse edildi.

1. BCG lüks muhabiri mikobakteriler stok hazırlanması <ol><li> BCG büyümeye lux Middlebrook 7H9 broth içeren% 0.2 gliserol,% 0.05 arasında 80 ve% 10 ADC zenginleştirme orta-logaritmik faz 200rpm az sallayarak (servis plazmid pSMT1 ile dönüştürülmüş M. bovis BCG Montreal suşu). </li><li> Luminometre çift tüp (900μl PBS + 100μl BCG lüks kültür) 1ml steril PBS BCG lüks kültür 1:10 seyreltme hazırlayın. </li><li> N-desil aldehit (luciferin enzim substratı) içeren tüp makine sırtına yük ve kapağı sabitleniyor ve tüpler sağlamak. </li><li> Luminometreye yerleştirilen 3 boş emişli tüplerin her birine 100 ucl x 3 enjekte etmek için ana program üzerinden substrat ile Başbakan luminometre enjektör. </li><li> Luminometre 2 tüpler içine yükleyin ve her tüp içine substrat 100 ucl enjekte bir program aracılığıyla okuma ve 1 sn aralıklarla 20 saniye boyunca okuyun. </li><li> Stok 1x10 8 RLU / ml 2x10 8 RLU / ml (Bu büyüme 3-4 gün sürer) ulaştığında, 50ml şahin tüp kültür eşit hacimde steril% 30 gliserol ekleyin ve hafifçe karıştırın. </li><li> -80 Etiketli 2 ml vidalı kapaklı mikrotüp ve mağaza ° C içine kısım 1.5ml hacimleri </li></ol> 2. Belirlenmesi stok RLU / CFU korelasyon ve alikotları içeriği <ol><li> Havalandırmalı bir kap ile 200ml Erlenmeyer şişesinde% 10 ADC ek Middlebrook 7H9 kültür ortamı 15ml ekleyin. </li><li> % 20 Tween hygromycin ve 30μl 15μl ekleyin. </li><li> Güvenlik kabini, oda sıcaklığında (RT) dondurucu ve defrost BCG lüks bir şişe çıkarın . Orta flakon içeriğini ekleyin ve kapağını sıkın. </li><li> 37 200rpm az sallayarak ° C&#39;de 4 gün inkübe edin. </li><li> Her gün makyaj CFU belirlenmesi için seri 10-kat dilüsyonları (1:10, 1:100, 1:1000, 1:10,000 ve 1:100,000). </li><li> Paralel olarak aynı dilüsyonları kurmak ve lüminesans belirlemek için (1.2-1.6) çoğaltmak. </li><li> Middlebrook 7H11 agar (% 0.5 gliserol,% 10 OADC ek,% 20 Tween ve hygromycin, 1 ml 1 ml içeren sıvı bir ortamda 1 litre) 3 bölmesi iki tabak hazırlayın. </li><li> Sıvı bireysel kamara arasında eşit şekilde dağıtarak, ayrı serpme kullanarak her plaka bir segment üzerine her seyreltme 100μl, dışarı Plaka. </li><li> Parafilm her tabak, steril bir plastik torba yerine, otoklav bandı ile mühür Seal, ve 37 inkübe ° C, 2 hafta boyunca aşağı bakacak şekilde kapaklı. </li><li> CFU (2-3 hafta) görünene kadar düzenli olarak kontrol edin. </li><li> Koloniler saymak için, kuvöz ve bir koloni sayacı yer plakaları çıkarın. Yinelenen plakalardan her seyreltme için ortalama hesaplayın. </li><li> Her seyreltme için eşdeğer RLU ve CFU sayıları kullanarak RLU / CFU oranını hesaplayın. Bu oran, 3 ve 5 RLU / CFU arasında olmalıdır. </li></ol> 3. Tam kan inokülasyon için BCG lüks kültür hazırlanması <ol><li> Havalandırmalı bir kap ile 200ml Erlenmeyer şişesinde% 10 ADC ek Middlebrook 7H9 kültür ortamı 15ml ekleyin. </li><li> % 20 Tween hygromycin ve 30μl 15μl ekleyin. </li><li> Güvenlik kabini RT dondurucu ve defrost BCG lüks bir şişe çıkarın. Orta flakon içeriğini ekleyin ve kapağını sıkın. </li><li> 37 200rpm az sallayarak ° C 2 ila 4 gün inkübe edin. </li><li> Luminometre çift tüp (900μl PBS + 100μl BCG lüks kültür) 1ml steril PBS BCG lüks kültür 1:10 seyreltme hazırlayın. </li><li> Başbakan luminometre substrat ile (yukarıda açıklandığı gibi), 2 tüpler yavaşça girdap ve luminometre yüklemek ve okuma (yukarıda açıklandığı gibi) almak. </li><li> 7x10 6 RLU eşdeğer (monosit sayısı varsayarak, 2x10 yaklaşık 5 CFU / ml kan ve BCG oranı 1:1 monositler için bir inokulum verecek vermek için PBS içinde kültür sulandırmak için bu okuma kullanın. kan ml başına 4x10 için yaklaşık 5 2x10, 5 monositler) . </li></ol> 4. Tam kan hazırlanması <ol><li> 3 alın, koruyucu içermeyen heparin içeren bir tüp 5ml venöz kan. </li><li> 50ml şahin tüp kan transferi ve eşit miktarda glutamin ve 25mm Hepes (Herhangi bir kalem / strep) içeren RPMI 1640 ile seyreltilir . </li><li> 0hrs için 3 ve 96hrs için 3, gerekirse ek timepoints için ek tüp ayarlayın: Her zaman noktası için steril küçük ama tüpleri (toplam 6 tüpler içine üç nüsha olarak seyreltilmiş kan kısım 900μl </li><li> % 10 ADC takviyesi + 50μg/ml hygromycin (BCG lüks kültürü eskiden olduğu gibi aynı ortamda) Middlebrooks 7H9 kültür orta kısım 900μl büyümesini kontrol çoğaltın. </li><li> Her bir tüp kan seyreltilmiş BCG lux, 100μl ekleyin ve 2 büyüme kontrolütüpler. </li><li> İyice karıştırın ve 37 sallanan inkübatör kendi tarafında zaman 96hr noktası ve 2 büyümesini kontrol için 3 bijous ° C, 20 dev / dak (CO 2 gerekli değildir). </li></ol> 5. Ölçüm lüminesans (0hr ve 96hrs) <ol><li> 10 dakika 2000g 3 bijous santrifüjleyin. </li><li> , -80 Dondurmak için 2 ml vidalı kapaklı mikrotüpler pelet ve yeri rahatsız etmeden dikkatlice süpernatant 300μl kaldırmak ° C, daha sonraki sitokin analiz için. </li><li> Kaldırılır süpernatant hacmi yerine her küçük ama PBS 300μl ekleyin. </li><li> Her küçük ama içeriğini ilgili 50ml şahin tüp içine aspire edin ve her tüp 8 ml distile su ekleyin. 10 dakika için bir zamanlayıcı başlayın. Nb: Bu zamana duyarlı bir adım olduğunu ve bu kuluçka hücreleri ilk kez su ile temas ettiğinde itibaren, herhangi bir 10 dakika daha uzun olmamalıdır. </li><li> 2ml için distile su ve 5 saniye vorteks ile ilgili şahin tüpe devrilme önce her küçük ama durulayın. </li><li> 10 dakikalık inkübasyon sonunda, 10 dakika için 2000g şahin tüpler santrifüj. </li><li> Surfanios dezenfektan içine Durusu süpernatant. </li><li> Her pelet ve vorteks birkaç cam boncuk ekleyin. </li><li> 1ml steril PBS her tüp ve vorteks ekleyin. </li><li> Luminometre tüplerde, PBS (900μl PBS + 100μl örnek) ile yinelenen ve 96hr zaman noktası, her bir örnek için 01:10 dilüsyonları olun. Büyüme kontrollerin her biri için aynı dilüsyonları olun. </li><li> Yavaşça vorteks tüpleri ve luminometre yüklemek ve okuma (yukarıda açıklandığı gibi) almak Nb: aynı örnek triplicates arasında Okumalar, biri diğerinin% 15 içinde olmalıdır . </li></ol> 6. Temsilcisi sonuçları: Metabolik aktivite suboptimal olduğu gibi, yani önce örnekleri aşılamak için 48-72 saat içinde büyüdü, tam kan lüks deneyleri için büyüme logaritmik faz, bakteri kullanılması önemlidir dondurucu düz dışarı ya durumunda veya sabit faz . Önce büyüme değişkenliği önlemek için bir dizi deneyler ya da 48 veya 72 saat için standardize edilmelidir. Şekil 1 BCG lüks bir temsilcisi kültürünün büyüme eğrisi gösterir. Sabit faz ulaşana kadar iki katına çıkma süresi 24 saat. <img alt="Şekil 1" src="/files/ftp_upload/3332/3332fig1.jpg" /> Şekil 1: BCG Temsilcisi büyüme eğrisi lüks 7H9 orta ve RLU ve CFU korelasyon 96 saat içinde. RLU değerleri her zaman CFU ile korele olsa da, tutarlı bir enfeksiyon çokluğu garanti etmek için bir dizi deney boyunca aynı donmuş stoğu kullanmak için iyi bir neden (tek bir CFU ilgili RLU sayısı, stok bağlı olarak değişebilir. İçişleri Bakanlığı). Tablo 1, aşılama zamanı (T 0) ve 96 saat ham tarihten bir örnek gösterir . Büyüme oranları, formül T 96 / T 0 kullanılarak hesaplanır, ama başka zaman aralıkları içerisinde de ölçülebilir . Önemli hücre ölümü gerçekleşir gibi olur, ancak tavsiye, 96 saat ötesinde kültürlerin kullanmak için değil. <img alt="Tablo 1" src="/files/ftp_upload/3332/3332table1.jpg" /> Tablo 1, aşılama (T0) süresi ve 96 saate kadar (T96) ve 3 erişkin vericilerden hesaplanan büyüme oranları ham veri örneği . Mevcut antijen spesifik bellek yanıtları ve muhtemelen de nötrofil sayısı bağlı olarak, büyüme oranları, çocukların kan Şekil 2 ve HIV enfeksiyonu olmadan burada görüldüğü gibi, bireyler arasında değişir. Ortalama olarak, genç çocuklar yetişkinlere göre daha yüksek büyüme oranlarına sahip, Tüberkülin deri testi (TDT) + ve bireylerin TST-ve bireylerin daha düşük büyüme oranları ve HIV enfeksiyonlu hastalarda, CD4 T hücre popülasyonunun eksikliği nedeniyle yüksek büyüme oranlarına sahip mikobakteriler için hücresel immün yanıtları anahtar arabulucular biri. <img alt="Şekil 2" src="/files/ftp_upload/3332/3332fig2.jpg" /> Şekil 2 Inter-donör değişkenliği: altta yatan HIV durumuna bağlı olarak, hastaların bir dizi T 0&#39;a karşı 96 saat Büyüme oranları. Şekil 3, 12 aylık bir süre içinde ve kontrol deneyleri için tekrar tekrar kanadı tek bir donör iki kanadı 64 donörlerin sonuçları özetleyen bir süre içinde büyüme oranlarının Tekrarlanabilirlik gösterilmiştir. Değişkenlik potansiyel nedenleri gibi birçok biyoanalizlere gözlenen ana sitokinlerin düzeyleri içinde mikobakteriyel hassasiyete ya da değişkenlik değişiklikler olabilir. <img alt="Şekil 3" src="/files/ftp_upload/3332/3332fig3.jpg" /> Şekil 3. 12 ay içinde 12 kez (a) Tek donör 12 metreye kadar 2 kez (b) birden fazla (64) donörlerinonths.

<ol>
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Çıkar çatışması ilan etti.

Potansiyel olarak gelecek vaat eden aşı adayı ekrana uygulanır, tüberküloz aşısı araştırmaları bağlamında Bakterisit deneyleri, hayvan modellerinde de kurulmuştur. Ana immünojenite, okuma gibi çeşitli organların fonksiyonları bakteri yükünün azaltılması. M. enfekte insan bakterisidal testlerinin ilk nesil, tek başına makrofajlar kullanılarak dizayn edilmiştir tüberküloz. CFU, en az 3 haftalık bir süre sonra ana mikobakteriyel hayatta kalma o...

<table border="1"><tbody><tr><td> Reaktif </td><td> Şirket </td><td> Kedi yok </td></tr><tr><td> BCG aşısı lüks </td><td> Çeşitli </td><td> n / a </td></tr><tr><td> Middlebrook 7H11 agar </td><td> Becton Dickinson </td><td> 283810 </td></tr><tr><td> Middlebrook 7H9 broth </td><td> BD Biosciences </td><td> 271.310 (500g) </td></tr><tr><td> Middlebrook ADC zenginleştirme </td><td> BD Biosciences </td><td> 212352 </td></tr><tr><td> Middlebrook OADC takviyesi </td><td> Becton Dickinson </td><td> 212240 </td></tr><tr><td> Tween 80 </td><td> Sigma Aldrich </td><td> 274364 </td></tr><tr><td> Gliserin </td><td> Sigma Aldrich </td><td> G5150 </td></tr><tr><td> Hygromycin B </td><td> Roche </td><td> 10843555001 (20ml) </td></tr><tr><td> N-desil aldehit </td><td> Sigma </td><td> D7384-100G </td></tr><tr><td> Etanol (% 99,7) </td><td> VWR </td><td> 101077 </td></tr><tr><td> Steril PBS </td><td> House </td><td> n / a </td></tr><tr><td> L-Glutamin, 25mm Hepes RPMI-1640 </td><td> Sigma </td><td> R0883 </td></tr><tr><td> Steril distile Su </td><td> House </td><td> n / a </td></tr><tr><td colspan="3"> Ekipman </td></tr><tr><td> Tüp luminometre (enjektabl port zorunlu) </td><td> Berthold AutoLumat LB953 veya tek tüp luminometre Sirius </td><td></td></tr><tr><td> Luminometre tüpler, 12 x 75mm, pyrex çerçevesiz </td><td> VWR gelen Corning </td><td> 99445-12 </td></tr><tr><td> Bacalı kapakları Erlenmeyer şişeler, steril polikarbonat 250ml </td><td> Corning üzerinden Fisher Scientific </td><td> 2150329 </td></tr><tr><td> BD Vacutainer Kan Alma Tüpleri (koruyucu madde içermeyen heparin) </td><td> BD </td><td> 367676 </td></tr><tr><td> 50ml Falcon tüpleri, konik </td><td> BD </td><td> 352077 </td></tr><tr><td> 7ml STERILIN örnek polipropilen küçük ama borular </td><td> VWR </td><td> 99445-12 </td></tr><tr><td> STERILIN Evrensel konteynerler (30 ml) </td><td> Laboratuar Analiz LTD </td><td> 128C </td></tr><tr><td> Cam boncuklar 2mm çaplı </td><td> SigmaAldrich </td><td> Z143928 </td></tr><tr><td> 10ml pipet ve pipet çocuk </td><td> herhangi </td><td></td></tr><tr><td> 1000μl ve 200μl pipetler ve filtre ipuçları </td><td> herhangi </td><td></td></tr><tr><td colspan="3"> Genel laboratuvar ekipmanları: </td></tr><tr><td> Sınıf II güvenlik kabini kabin </td><td></td><td></td></tr><tr><td> Buzdolabı (4 ° C) </td><td></td><td></td></tr><tr><td> Santrifüj </td><td></td><td></td></tr><tr><td> Tüpler karıştırmak için Rocking çalkalayıcı platformu (180 0) </td><td></td><td></td></tr><tr><td> Orbital Çalkalayıcı inkübatör bakteri kültürleri büyüyen 37 ° C&#39;de </td><td></td><td></td></tr></tbody></table>

a functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged bcg or m.tb (bcg lux/m.tb lux)

Fonksiyonel testlerin uzun süre belirli bir aşı immünojenite ölçme önemli bir rol oynamıştır. Bu, geleneksel serum bakterisidal titreleri olarak ifade edilir. Çocukluk aşıları yanıt serum bakterisidal titreleri çalışmaları rutin olarak kullanılması, koruyucu bağışıklık yanıtlarını ve bu cut-off için seviyelerini geliştirmek ve cut-off doğrulamak sağlamıştır. Testler TB aşıları gibi efektör T hücre yanıtları şeklinde öncelikle hücre-aracılı bağışıklık neden olan aşıların rutin değerlendirme içine ileri atılmıştır. Hayvan modeli, belirli bir aşı adayı performans rutin standart bakterisidal tayinlerde değerlendirilir ve mevcut tüm roman TB aşı adayı 1 insan çalışmalarda faz önce bu adımı değerlendirilmesi maruz kalmış. Immünojenite ve bu nedenle yeni anti-TB aşıların koruyucu etkinlik olasılığını değerlendirme ideal bakterisidal testleri ölçümleri de dahil olmak üzere, insan modelleri benzer bir adım akıllıca değerlendirme geçmelidir. Potansiyel olarak gelecek vaat eden aşı adayı ekrana uygulanır, tüberküloz aşısı araştırmaları bağlamında Bakterisit deneyleri zaten iyi, hayvan modellerinde yılında kurulmuş olan. Ana immünojenite, okuma gibi çeşitli organların fonksiyonları bakteri yükünün azaltılması. Ancak, bugüne kadar yeni anti-TB aşıları için böyle testleri klinik çalışmalarda içine dahil edilmiştir. Mikobakterilerin insan makrofaj içinde öldürülmesi neden tam mekanizmaları hakkında belirsizlik hala var olmasına rağmen, mikobakteriler ile makrofaj ve T hücrelerinin etkileşimi açıkça gereklidir. Bu yazıda anlatılan testi onların göreceli bireysel katkı hakkında varsayımlar yapmadan tam kan mevcut diğer tüm hücresel ve humoral faktörler gibi temel hücresel bileşenleri çalışmaları sağlar bakterisidal testlerinin bir roman neslini temsil ediyor. Grubumuz tarafından açıklanan tam kan tahlil küçük hacimlerde kullanır ve zaten TB-endemik ayarları yetişkinler ve çocuklar çalışmalarda istihdam edilmiştir. Biz, BCG aşısı immünojenite gösterilen mikobakterilerin büyüme, HIV-pozitif hastalarda mikobakteriyel sağkalım üzerine in vitro anti-retroviral tedavi ve D vitamini etkisinin yanı sıra artmıştır. Burada metodoloji özetlemekte ve bu nispeten basit, düşük maliyetli ve sahada dostu bir model kullanarak tekrarlanabilirlik veri mevcut. Not: Tanımlar / Kısaltmalar BCG lux = M. bovis BCG, Montreal gerilme, mikobakteriyel GroEL (hsp60) promotör kontrolü altında, Vibrio harveyi luxAB genleri taşıyan servis plazmid pSMT1 dönüştürdü. CFU = Koloni Oluşturan Birim (mikobakteriyel canlılığının bir önlem).

Watch this Scientific Journal Video about A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux at JoVE.com

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

Mikobakterilerin numaralandırma için alternatif bir yaklaşım tarif<em&gt; In vitro</em, Muhabir-gen kullanan&gt; yerine koloni oluşturan birim (CFU) mikobakteriler etiketli. Organizmalar &quot;Survival&quot; gibi bir konak cevabı belirteçler tüberküloz bağlamda ev sahibi / patojen etkileşimleri çalışmaları için düşük maliyetli, çok yönlü ve fonksiyonel bir sistem, aynı anda ölçülür.

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 ...

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Research

JoVE Journal

Immunology and Infection

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

13.8K Görüntüleme.  Imperial College London. Mikobakterilerin numaralandırma için alternatif bir yaklaşım tarif In vitro yerine koloni oluşturan birim (CFU) mikobakteriler etiketli. Organizmalar "Survival" gibi bir konak cevabı belirteçler tüberküloz bağlamda ev sahibi / patojen etkileşimleri çalışmaları için düşük maliyetli, çok yönlü ve fonksiyonel bir sistem, aynı anda ölçülür.

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

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Bir Tam Kan fenotipleme Testi Kullanarak Çok Merkezli Klinik Araştırmalar için Binbaşı Periferik Kan Lökosit Populasyonlarının Numaralama

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

İmmünoloji ve Enfeksiyon

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Antijenler Kompakt Hyperbranched Polyglycerols Of Aşılama Membran By Fonksiyonel Kırmızı Kan Hücreleri Korumalı

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Kaplama Testi kullanarak niteliksel Pnömokok Bakteriyosinlerin Bakteriyel Üretim ve Hassasiyet Tedbir

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Basit Akım Sitometrisi Yöntemi Tüm Kan Monosit subpopulations için glikoz alımını ve Glikoz Transporter Anlatım Tedbir

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

Koruyucu etkinliği ve Pulmoner Bağışıklık Tepkisi Fare Subkutan ve Burun BCG İdaresi ardından

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Bir Monosit Tek tabakalı Testi Kullanımı Fcy reseptör aracılı fagositoz değerlendirin

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

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

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 protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

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

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

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 ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

En iyi duruma getirilmiş Interferon-gamma ELISpot tahlil ölçü birimi T hücre yanıtları sonrasi hipererjik eskiden şiling şimdi domuz modelinde

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

In Vitro Mikroakışkan Hastalık Modeli Gerçek Zamanlı Olarak Tam Kan-Endotel Etkileşimleri ve Kan Pıhtısı Dinamikleri Çalışma

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...