Bu proje kısmen Ulusal Enstitüsü alerji ve enfeksiyon hastalıkları gelen federal fonları ile finanse edilmiştir, Ulusal Sağlık enstitüleri, bölümü sağlık ve insan Hizmetleri, CEIRS altında P01AI097092-04S1 (P.E.L.) sözleşme.

Bu el yazması önceden oluşturulmuş deneyler aşağıdaki Icahn School of Medicine'nın kurumsal kodu etik ve düzenlemeler yapıldı.
1. grip virüsü Hemagglutinin yolu ile Transfection ya da enfeksiyon ifade
Transfection:
<ol><li>Plaka insan embriyonik böbrek (HEK 293T) hücre yoğunluğu 2 x 104 hücreleri/iyi bir beyaz doku kültürü ', 96-şey plaka tedavi ve 37 °C kuluçka (%5 CO2ile) 4 h için oturmak izin.</li>
	<li>Transfect 100 içeren hücreleri ng DNA viral hemagglutinin ve transfection reaktif 50 μL toplam hacmi bir kuyu başına 0.2 μL için kodlama. Not: Her ikisi de 1 Opti-en az temel medya (MEM) indirimli Serum orta x seyreltilmiş Plazmid ve transfectant reaktif.</li>
	<li>Tabak 18 h için % 5 CO2 bir 37 °C kuluçka kuluçka sonra transfection medyayı çıkarın.</li>
</ol>2. enfeksiyon
<ol><li>2 x 104 hücreleri/iyi bir beyaz doku kültürü'yoğunluğu plaka A549 veya Madin Darby köpek böbrek hücreleri 96-şey plaka tedavi ve en az 18 h 37 °C kuluçka %5 CO2için büyür.</li>
	<li>1 seyreltik virüs MEM x ve hücreleri enfeksiyon 3, 1 veya 1 h 37 °C kuluçka %5 CO2için 0,33 çokluğu, bulaştırmak. Toplam hacim 100 μL eşit olmalıdır. Not: 10 x MEM MEM çalışma stok virüs seyreltme yukarıda belirtilen için x 1 yapmak için su ile seyreltilmiş.</li>
	<li>Enfeksiyon sonra inoculum kaldırın ve 1 100 μL ekleyin MEM tripsin yokluğunda plaka içine x.</li>
	<li>18-24 h 37 °C kuluçka %5 CO2ile enfekte hücreleri büyümek.</li>
</ol>3. FcRg/NFAT-aracılı etkinleştirme monoklonal antikorlar veya Sera Luciferase etkinliğinin değerlendirilmesi
<ol><li>İlk olarak, transfected veya enfekte hücre büyüme ortamı tahlil medya (1 %4 (vol/vol) ile desteklenmiş x RPMI 1640 düşük IgG fetal sığır sera) 25 µL ile değiştirin.</li>
	<li>Ayrı bir 96-şey tabak içinde dört kat dilutions 30 μg/tahlil medya kullanarak mL başlayan ilgi antikorların gerçekleştirin. Dilutions triplicates içinde gerçekleştirmek ve bir örnek plaka düzen şekil 1A' gösterilmektedir. Bir arka plan kontrol (tahlil arabellek yalnız) yanı sıra antikor (antikor kontrol) dışlayan bir denetim eklemeyi unutmayın. Not: her iki 'yok antikor' için ve arka plan kontrolleri, tahlil arabelleği antikor yerine 25 µL ekleyin.</li>
	<li>Seri olarak seyreltilmiş antikorların 25 μL adım 3.2 için karşılık gelen kuyu transfected hücreleri veya enfekte hücreleri tabağa aktarın.</li>
	<li>Plaka (%5 CO2ile) bir 37 °C kuluçka 15dk için kuluçkaya.</li>
	<li>Fare FcgRIV veya insan FcgRIIIa (hücre/iyi 75.000) ifade uygun değiştirilmiş efektör Jurkat hücrenin 25 μL ekleyin. Her biri için Toplam hacim de 75 µL olmalı ve antikor başlangıç son konsantrasyonu 10 µg / (şekil 1B) mL'dir. Not: arka plan denetimi için tahlil arabelleği efektör hücreler yerine 25 µL ekleyin. Antikor-bağımlı Hücre-aracılı fagositoz incelemek için değiştirilmiş bir Jurkat hücre insan FcgRIIa ifade kullanılabilir.</li>
	<li>Plaka bir 37 ° C kuluçka %5 CO2 ile 6 h için kuluçkaya.</li>
	<li>Tezcan ve sıcak luciferase substrat arabellek 37 °C su kullanmadan önce yaklaşık 1 h için. Not: luciferase substrat tampon yapmak için liyofilize luciferase tahlil substrat sağlanan tahlil arabelleği yeniden oluşturma. Sulandırılan substrat arabellek-30 °C-10 °C ilâ altı hafta boyunca saklanabilir.</li>
	<li>75 μL/iyi luciferase substrat arabelleği arka plan denetimi dışında bütün wells ekleyin.</li>
	<li>Bir luminometer üzerinde okuyun.</li>
	<li>Kat indüksiyon ile aşağıdaki denklemi hesaplamak: kat indüksiyon (Induced değeri arka plan değeri) = / (mAb değeri arka plan değeri).</li>
</ol>

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Yazarlar hiçbir çıkar çatışmaları bildirin.

Burada açıklanan yöntemi bir HA özel Monoklonal antikor fare FcγRIV meşgul yeteneğini ölçmek kullanıcı sağlar. Hedef antijen, grip virüsü hemagglutinin, hücre yüzeyinde enfeksiyon sonra virüs veya plazmid DNA transfection tarafından ifade edilir. Tahlil daha fazla diğer FcγRs (insan veya fare) ile birlikte farklı yüzey-viral proteinlerin kullanmaya müsait. Ayrıca, biz sera örnekleri antikorlar poliklonal bir bağlamda Fc efektör fonksiyonları (veri) gösterilmez, ikna yeteneği değerlendirmek ...

Mevsimsel grip aşısı tarafından sağlanan bağışıklık hemagglutinin reseptör bağlama sitenin hedef antikor varlığı ölçen hemaglütinasyon inhibisyon (hı) tahlil1ile, geleneksel olarak değerlendirildi. Bu HI-aktif antikorlar bağışıklık sterilize görüşmek ama genellikle onların genişliğini koruma, grip virüsü select bir kaç suşları için dokunulmazlık sağlayan dar. Yalıtım ve hemagglutinin (HA) tanımak geniş reaktif monoklonal antikorlar karakterizasyonu evrensel grip aşısı geliştirilmesi ulaşmak içinde2,3,4, önermek 5 , 6 , 7 , 8. bir evrensel grip aşısı önemli amaçlarından biri güçlü antikor yanıtı grip virüsü9,10,11,12 korunmuş bölgelerde doğru ikna etmek için , 13 , 14 , 15 , 16. geniş reaktif antikorları nötralize ve sigara nötralize antikorlar17,18, oluşan bu korunmuş epitopları tanımak için Fc-FcγR etkileşim gerektirecek şekilde gösterilmiş olan en uygun koruma vivo içinde ve vurgulamak Fc-aracılı bağışıklık grip virüsü19,20genel bağışıklık yanıtı için katkı.
Koruma ilişkilendirir enfeksiyon bağışıklık değerlendirirken açısından büyük önem taşıyor. Bu ölçümler bilim adamları ve klinisyenler aşı etkinliği, verilerin önemi ve tedavinin gidişatı tahmin etmek izin verir. Grip virüs bulaşmasına karşı koruma tek kurulan ilişkili hemaglütinasyon inhibisyon tahlil olduğunu. 1:40 titresi hastalık1,21 -riski % 50 azalma ile ilişkili ve küresel HA başında bulunan site Bağlayıcısı reseptör hedefleyerek Aglutinasyon inhibe antikor varlığını yansıtır. Ancak, ayrıca T-hücre yanıt-e doğru-ebilmek var olmak daha iyi ilişkili grip virüs hastalık Yaşlılarda karşı koruma dikkat edilmelidir. İlginçtir, bir rapora neuraminidase inhibisyon aktivitesi dokunulmazlık grip22daha iyi bir tahmin olabileceğini düşündürmektedir. Neyse ki, nötralizasyon veya neuraminidase inhibisyon deneyleri HA sapı veya neuraminidase özel antikorlar ölçebilirsiniz gibi tipik vitro deneyleri. Bu geleneksel vitro deneyleri çoğu ancak, sadece hesaba katmak antikor antijen bağlayıcı bölgenin fonksiyonu ve Fc bölge rolü ölçüyor musunuz. Ayrıca, Fc reseptör nişan vivo içinde yolu ile korumak sigara nötralize antikorlar katkısını değildir tespit edilen17,18. Fc-aracılı bağışıklık antikor aracılı hücre bağımlı sitotoksisite (CCDA) gibi neden antikorlar tarafından tanınan koruma ölçmek için sağlam vitro tahlil gereklidir.
Aşağıda açıklanan yöntemi fare monoklonal antikorlar Fc-aracılı işlevleri kullanılarak bir etkinleştirme ifade genetiği değiştirilmiş Jurkat hücre kültürünü ikna yeteneği değerlendiriyor antikorundan 1 FcR, FcγRIV yazın. FcγR antikor angajman Bu Tetikleyicileri nükleer faktör aktive T-Hücre-aracılı luciferase faaliyet hücre içi sinyal transduces. Tahlil yalıtım ve birincil efektör hücrelerin kültür ve harekete geçirmek-in Fc-aracılı efektör fonksiyonları19,23,24 algılamak için Akış Sitometresi kullanımını gerektiren geleneksel teknikleri üzerinde çeşitli avantajları vardır ,25,26. İlk olarak, burada açıklanan protokol kolayca farklı viral hedefler, FcγRs ve antikor (insan ve fare) farklı türlerden adapte edilebilir. İkinci olarak, değiştirilmiş bir Jurkat hücre kullanımı satır ışıldama ölçme bir plaka okuyucu kullanarak kolayca çözümlenebilir bir geniş format tahlil için bir FcγR bir luciferase muhabir gen aktive T-hücre (NFAT) nükleer faktör altında ile verir ifade. Burada, geleneksel harekete geçirmek birincil efektör hücrelerin NFAT-luciferase indüksiyon antikor angajman bir FcγR Jurkat hücre yüzeyi üzerine yerine kullanılır. Bu 96-şey plaka biçimi tahlil triplicates yedi dilutions-geleneksel yöntemlerle hantal olabilir örnekleri bir dizi ile en çok dört farklı örnekleri ölçümü için izin verir. Deney tasarımı ve veri yorumlanması etkileyebilir bu tahlil ile dikkate alınacak ek noktalar vardır. Viral hedef antikor tanıma amacıyla hücre yüzeyinde ifade gerekir. Bu etkisini azaltmak için hedef antijen (örneğin bir iç viral protein) doğrudan bir plaka yüzeyinde kaplı olması. Ancak, bu henüz ciddi bir şekilde test edilmemiştir. Ayrıca, değiştirilmiş Jurkat hücre kültürünü FcγR etkinleştirme yalnızca bir türü ifade eder ve tüm reseptörleri fizyolojik bir bağlamda birincil hücre satırlarını hızlı iken herhangi bir inhibitör FcγRs ifade değil.
Biz daha önce bu tahlil epitope özgüllük poliklonal bir bağlamda Fc-aracılı efektör fonksiyonları düzenlenmesinde önemli bir rol oynar ve antikor bağımlı Hücre-aracılı yanıt en iyi aktivasyonu iki noktası gerektirir göstermek için kullanılan 27,28başvurun. Burada, biz yeteneği meşgul ve FcγRIV aktive antikorundan etkinleştirmek için SAP özgü monoklonal antikorların değerlendiriyor bir yöntemi açıklanmaktadır. Özel durumlar29,30olmakla birlikte, çok sayıda geniş reaktif antikorlar hemagglutinin antigenically korunmuş sapı bölgenin hedef ve böylece evrensel bir grip gelişiminde önemli bir rol oynamaktadır aşı.

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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">A549 cells&#160;</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CCL-185</td>
			<td style="width:353px;">Adenocarcinomic human alveolar basal epithelial cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">HEK 293T cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CRL-3216</td>
			<td>Human embryonic kidney cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Lipofectamine 2000</td>
			<td style="width:141px;">Life Technologies&#160;</td>
			<td style="width:184px;">11668019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Opti-MEM Reduced Serum Medium</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">51985-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">White tissue culture treated 96-well plate</td>
			<td>Corning, Inc.&#160;</td>
			<td>3917</td>
			<td>Assay plates</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:243px;">10X MEM</td>
			<td style="width:141px;">Gibco</td>
			<td style="width:184px;">11430-030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing murine FcgRIV</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">M1201</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing human FcgRIIIa</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G7010</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing human FcgRIIa</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G9901</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luminometer</td>
			<td>BioTek</td>
			<td>Synergy H1 Multi-Mode reader</td>
			<td>Luminescence plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Bio-Glo Luciferase Assay System</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G7940</td>
			<td>Contains luciferase assay buffer and luciferase assay substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Madin Darby canine kidney cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CCL-34</td>
			<td>Canine kidney cells</td>
		</tr>
	</tbody>
</table>

a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies

Antikorlar karşı antijen bağlayıcı etki alanları ve Fc-bölgeler ile viral patojenler doğuştan gelen ve edinilmiş bağışıklık yanıtı kaplin çok önemli bir rol oynamaktadır. Burada, biz nasıl belgili tanımlık harekete geçirmek ölçmek için tarif Fc efektör fonksiyonları monoklonal antikorlar grip virüsü hemagglutinin bir etkinleştirme ifade genetiği Jurkat hücre kültürünü kullanımı ile hedefleme tarafından 1 Fc yazın-FcγR. bu yöntemi kullanarak katkı immünglobulin tarafından haiz belirli Fc-FcγR etkileşimlerin bir vitro tahlil kullanarak belirlenebilir.

Biz bu gösterdi bir sapı özgü mAb, 6F12, ama değil kafa özgü kafa özgü mAb, PY102, CD107a ve interferon γ19etkinleşmiş tarafından birincil doğal katil hücreler etkinleştirmek başardı. Bir SAP özgü antikor birincil insan NK hücreleri aktive yeteneği modellemek için bir SAP özgü mAb, 6F12, sağlam tarafından fare FcγRIV ifade değiştirilmiş Jurkat hücre etkinleştirebilirsiniz göstermek ~ 14-fold. Öte yandan, kafa özgü mAb, PY102 ve kontrol IgG değil (<strong class...

Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Fc-aracılı efektör fonksiyonları aktivasyonu antikorlar tarafından hedefleyen grip virüsü hemagglutinin ölçmek için bir yöntem açıklanmaktadır. Bu tahlil de Fc-aracılı bağışıklık ikna etmek için monoklonal antikorlar veya poliklonal sera diğer viral yüzey glikoproteinlerin hedefleme yeteneklerini değerlendirmek için adapte edilebilir.

Grip Hemagglutinin özel antikorlar tarafından indüklenen Fc-aracılı efektör fonksiyonları değerlendirmek için bir yöntem

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

a-method-to-assess-fc-mediated-effector-functions-induced-influenza

Research

JoVE Journal

Immunology and Infection

7.6K Görüntüleme.  Icahn School of Medicine at Mount Sinai. Fc-aracılı efektör fonksiyonları aktivasyonu antikorlar tarafından hedefleyen grip virüsü hemagglutinin ölçmek için bir yöntem açıklanmaktadır. Bu tahlil de Fc-aracılı bağışıklık ikna etmek için monoklonal antikorlar veya poliklonal sera diğer viral yüzey glikoproteinlerin hedefleme yeteneklerini değerlendirmek için adapte edilebilir.

Video: A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Sitotoksik T Lenfositler Beta Hücre Ölümü aracılığı ile değerlendirin yöntemleri

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

İmmünoloji ve Enfeksiyon

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Tüm Influenza Kantitatif Analiz Basit Slot Blot Tahliller Evrensel Antikorlar kullanarak Viral Hemagglutinins ve nöraminidaz yazın

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Gribi için yüksek verimlilik Tespit Yöntemi

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

Vitro Testi HIV-spesifik CD4 T hücre efektör işlevi üzerinde immünoregülatör yolları etkisini değerlendirmek için

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Tüp bebek Patojen Kaynaklı Nötrofil Trans-epitel Göçünü Değerlendirmek için Coculture Tahlili

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

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

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

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 Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Gradient Santrifüj tarafından İnfluenza A Virüsü Kapsidlerin Vitro Demontaj içinde

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Uygulanması Floresan Rezonans Enerji Transferi (FRET) tarafından Efektör Translokasyon Verimliliği incelemek için<em&gt; Coxiella burnetii</em&gt; SiRNA susturma sırasında

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

Floresans tabanlı Nöraminidaz İnhibisyonu Tahlili antivirallerin nöraminidaz inhibitörü Class Nezle Virüslerinden eğilimi değerlendirilebilir

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Grip virüsü monoklonal antikorları nötralize kaçış türevleri üretimi

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...