Biz usta yardım için Kerstin Thim (AstraZeneca&#39;yı, Lund, İsveç), Benita Dahlberg ve Dr. Anders Eklund (Karolinska Enstitüsünde, Stockholm, İsveç) yanı sıra Dr. Martin STAMPFLI (McMaster Üniversitesi, Hamilton, ON, Kanada) teşekkür etmek istiyorum ve uzman tavsiyesi.

Hayvan çalışmaları Kuzey Stockholm hayvan refahı etik kurulu tarafından onaylandı. Deneysel işlemler İsveç hukuka uygun olarak yapılmıştır. 1. Bir LPS aerosol üretimi <ol><li> S. saflaştırılarak 0.5 g çözülür Hafifçe çalkalama ile 50 ml steril tuzlu su içinde aeruginosa LPS ve çözünme edin. 1 mg / ml&#39;lik nihai bir konsantrasyona kadar, 9 ml steril tuzlu su içinde LPS çözülmüş 1 ml seyreltin. -20 ° C&#39;de alüminyum folyo ve mağaza ile ışıktan koruyun. </li><li> Oda sıcaklığında, karanlıkta, çözünür LPS çözülme ve kullanım öncesinde de hemen karıştırın. </li><li> Bir havalandırılmış seviye II biohazard kaput, bir püskürtücü içine kırmızı bir giriş eklemek, ve üretici (Şekil 1 deneysel cihazlar sunan yorum şeması) tarafından sağlanan boru aracılığıyla basınçlı oda havasına nebulizeri bağlayın. DİKKAT: Uygun kişisel koruyucu ekipman, yarım yüz parça yeniden Respirat dahilveya partikül filtreleri, gözlük, eldiven ve koruyucu giysiler ile maruz kalma sırasında kullanılmalıdır. </li></ol><img alt="Şekil 1," src="/files/ftp_upload/51470/51470fig1highres.jpg" /> Şekil 1:. Bir aerosolün üretilmesi için kullanılan deneysel cihazların şematik sunumu püskürtücü giriş hava kaynağına bağlanır. nebulizer çıkış önce 15,9 mm boru ve bir hava filtresi ve hava beslemesi ile bir akış ölçere bağlı olan 2 kbar basınçta 5.0 L / m ayarlanır. çıkış çıkarılabilir kapakları ve basınç birikmesini önlemek için 5 mm&#39;lik delikleri ile donatılmış bir pleksiglas kutu bağlı yanındadır. <ol start="4"><li> Bir hava filtresi aracılığıyla bir kitle debi ölçer için nebulizatör çıkışını bağlayın. Bir elektrik kaynağına kitle debi ölçer bağlayın. </li><li> 1.0-2.0 barda kalan basınç, 5 L / dk hava kaynağını ayarlayın. </li><li> Kitle debimetre çıkarın veHava kaynağını ayırın. </li><li> 150 x 163 x 205 mm, çıkarılabilir kapak ile donatılmış: ikiye bölen ve boyutları ile iki pleksiglas kutular bağlanan bir 15,9 mm tüp için nebulizatör çıkışını bağlayın. Her kutu basınç birikmesini önlemek için giriş karşı tarafında bulunan 5 mm&#39;lik bir delik, olmalıdır. </li><li> Her pleksiglas kutuya 5 fareler kadar yerleştirin ve kapaklarını kapatın. </li><li> Nebülizatör açın ve sadece steril tuzlu su ya da aracı madde içinde eritilebilir, en az 4 mi LPS ile ekleme doldurun (steril tuzlu su, cilt 8 ml geçmemelidir). Hava kaynağına giriş yeniden bağlayın. </li><li> Aerosol 10 dakika kapalı pleksiglas kutulara akmasına izin verin. Sürekli hayvanları izleyin. Emin hava besleme nebulizatör girişine sıkıca güvenli kalır emin olun. </li><li> Hava kaynağını ayırın. Kapakları kapalı, hayvanlar 2 dakika pleksiglas kutulara kalsın. </li><li> Kapaklarını açın ve aerosol dağıtmak için izin ve t hayvanların dönmekO kafesleri. Hayvanlar ıslak görünüyorsa, hipotermi önlemek için, düşük ısı ayarlı bir ısıtma yastığı kafesleri yerleştirin. </li><li> İlk 30 dakika boyunca sürekli hayvanları izlemek, ve ilk 6 saat sonra her 2 saatte. Hayvanlar sırasında ve nebulization prosedürü sonra normal solunum paterni ve aktivite göstermelidir. </li></ol> 2. Bronkoalveoler Lavaj (BAL) <ol><li> Kurumunuzun veteriner personeli tarafından tavsiye edilen deneysel uç noktasında, derin etkisi izofluran ile hayvanları uyutmak. Majör cerrahi yapmak için anestezi yeterli derinlik sağlamak için bir geri çekilme refleksi kontrol etmek için arka pençe sıkıştırın. % 70 etanol ile hayvanların kürk aşağı püskürtün. </li><li> Makas kullanırken karın açın ve hayvan asıl bulbus olfaktoryusları için aort sever. Kan emmek için karın üzerine doku parçası yerleştirin. </li><li> Ötenazi follwing, makas tek ön-arka kesim kullanıntoraks maruz. Sternum ön ucu ile göğüs kafesi yukarı kaldırın ve herhangi bir akciğer lobunun içine kesmeden, en ventral noktada diyaframı delmek için makas kullanın. Ön-arka yönde iki kesim (çene altındaki toplantı) yaparak göğüs kafesi açın. </li><li> Yavaşça forseps kullanarak göğüs kafesi dışında çekin ve gırtlak altında trakea kesti. </li><li> Forseps ile trakea yukarı kaldırın ve göğüs boşluğuna lobu bağlayan ligament kesme ve hafifçe adipoz ve kardiyak doku tarafından çekerek akciğerleri kaldırmak. </li><li> Bir polietilen tüp (: 0.58 mm; dış çap: iç çapı 0.965 mm) takın trakea içine ve ipek iplik bir dize ile tüp sabitleyin. </li><li> Ipek iplik ile multilobe (sağ akciğerin dört lobu) kapalı kravat. </li><li> Polietilen boru içine bir 23 gog iğne yerleştirin ve yavaşça 1 ml şırınga ile bir lob içine buz soğukluğunda steril PBS 250 ul enjekte edilir. </li><li> Dikkatle akciğerleri dokunun30 kat ve şırınga yoluyla sıvının toplanması. 200 ul PBS ile işlemi tekrarlayın (yaklaşık 300 ul PBS 450 ul toplam enjekte birimden tek lob kurtarıldı olmalıdır). </li><li> Buz üzerinde BAL sıvısında (BAL) tutun, ya da hemen BAL hücreleri numaralandırmak. Hücreleri 11 leke Türk çözümünü kullanarak, bir heamocytometer hücreleri sayın. Boyama çözeltisi seyreltme faktörü ve heamocytometer ve sayılan alanında hacmi ile hücrelerin sayısı çarpılarak toplam hücre sayısını hesaplayın. </li><li> Başka bir yerde 11 açıklandığı gibi sitosantrifüjden geçirilidi hücrelerden farklı hücre sayımları hazırlayın. </li></ol> Histolojik Değerlendirme Akciğer Doku 3. Formol Fixation <ol><li> Multilobe çıkarın ve ek-freeze kuru buz üzerinde. -80 ° C&#39;de saklayın. </li><li> Bir metal destek standı kaldırıldı piston ile 60 ml şırınga monte edin. % 10 formalin şırınga doldurunSabit basınç 20 cm temsil laboratuvar tezgah üstünde 20 cm, yüksekliği. </li><li> Formalin akışını kontrol etmek için bir valf ile bir plastik tüp ile 60 ml şırınga trakea sabitlenmiş 23 gauge iğne bağlayın. </li><li> 5 dakika boyunca formalin ile akciğer lobunun insuflasyon. İğneyi çıkarın ve ipek iplik çekerek trakea kapatmak ve akciğer basıncı korumak için ise trakea polietilen tüp ile birlikte çıkarın. </li><li> Formalin akciğer lobu Batmak ve 4 ° C&#39;de 24 saat saptamak. </li><li> % 70 etanol ile en az 20 dakika boyunca sabit bir dokuya üç kez yıkayın ve aşağıdaki rejimi (1 saat her biri) ile ksilen dihidrat: <ul><li> 3x% 70 etanol </li><li> 3x% 95 etanol </li><li> 3x% 100 etanol </li><li> 3x ksilen </li><li> 1x (sıvı) parafin </li></ul></li><li> Hematoksilin ve eosin ile Embed susuz 4-5 mikron bölümleri kesilmiş parafin doku, ve leke histolojik değerlendirme için izin verir.</li>
</ol>

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İbrahim Roos Boehringer Ingelheim, AstraZeneca Ar-Ge ve AstraZeneca Ar-Ge bir araştırma bursu seyahat hibelerinden öğretim ücreti aldı. Magnus Nord AstraZeneca Ar-Ge tam zamanlı çalışan ve AstraZeneca Ar-Ge araştırma hibe aldı. Johan Grunewald ve Tove Berg AstraZeneca Ar-Ge tarafından finanse alakasız bir araştırma projesi üzerinde birlikte araştırmacılar vardır.

Aerosol haline LPS epitel alt mukozada nötrofiller tarafından karakterize edilen solunum yolları enflamatuar bir tepki üretir, iletken hava yolları, hem de alveoler boşluk çevreleyen alanlarda. Bu arada plazma sızıntı, akut akciğer hasarı patoloji temsilcisi göstergesidir BAL&#39;da artan toplam protein içeriği ile vardır. LPS steril iltihabı teşvik olarak, reaksiyon adaptif bağışıklık tepkisinin bağımsızdır ve bakteriyel enfeksiyonlara karşı ilgisi sınırlamalar vardır. teknik, bununla bi...

Lipopolisakarid (LPS), gram negatif bakterilerin 1 &#39;in bir hücre duvarı bileşeni. LPS meydan akut akciğer hasarı, akut nötrofilik inflamasyon ve ödem 2 ile karakterize bir sendrom iyi belgelenmiş bir modeldir. Buna ek olarak, pulmoner nötrofilinin da KOAH alevlenme 4 modellemek için kullanılır olmuştur kronik obstrüktif akciğer hastalığı (KOAH) 3, ve insanlarda LPS bir özelliğidir. Böylece, LPS maruz deneysel modeller klinik insan patoloji anlamak için ilgili ve değerli araçlardır. Burada tarif edilen aerosol haline LPS&#39;nin pulmoner yoldan amacı, sistemik katılımı olmadan iletken ve solunum yollarının bir nötrofilik inflamatuar yanıtı elde etmektir. LPS meydan okumasının çeşitli teknikleri, daha önce tarif edilmiştir. LPS&#39;nin damardan zerk edilerek verilme yolu, en yaygın olarak kullanılan bir yoldur. Bu teknik, kolayca erişilebilir, ancak, tO birincil hasar akciğere nötrofil göç aşağıdaki pulmoner epitel ikincil yıkımı ile, endotel olduğunu. İçi venöz yönetimi de hayvan modellerinde klinik tabloyu komplike edebilir sistemik inflamasyon 2, uyarmaktadır. Sistemik inflamasyon içi trakeal için elde edilmemiştir tersidir. Bu teknik, bununla birlikte, emek yoğun ve anestezi hem de önemli bir eğitim 5, 6 gerektirir. Bundan başka, bu uygulama yoluyla pulmoner birikimi nefes 7 bağlıdır. Bu nedenle, pulmoner birikimi solunum yollarında içi trakeal yönetim ve değişken yerleştirme gözlenebilir için gerekli anestezinin derinliği ile etkilenir. Bunun aksine, aerosol haline getirilmiş LPS ile pulmoner uygulama minimum eğitim gerektirir ve kolayca küçük hayvanların çok sayıda ya da bireyler 5, 8 arasında bir varyasyon gerçekleştirilebilir.Yeni bir çalışmada, solunum hacmi birikimi ile ilgili içi trakeal yönlendirmek için üstün olduğunu, ve LPS&#39;nin daha fazla dozları bu modele 8 nötrofilik enflamasyonu doğrulamaktadır. Aerosol Pseudomonas aeruginosa LPS alveoller 9, 10 de dahil olmak üzere solunum yolu lümeni ve akciğer parankimi, belirgin bir inflamatuar yanıt oluşturur Önceki çalışmalar meydan göstermiştir. inflamasyon nötrofil ve pulmoner ödem varlığı, bir üstünlük kurmasıyla karakterize edilir ve bu nedenle, akut akciğer hasarı patogenezini ele almak ve hastalık patolojisine katkıda mekanizmaların daha ayrıntılı bilgi edinmek için kullanılabilir.

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			<td height="22" style="height:22px;width:307px;">Name of the material/equipment&#160;</td>
			<td style="width:244px;">Company</td>
			<td style="width:145px;">Catalog number</td>
			<td style="width:235px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:307px;">Purified Pseudomonas aeruginosa LPS&#160;</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Harmful. Recomended purification. LPS purified from other bactria may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Pari LC sprint star nebulizer</td>
			<td style="width:244px;">PARI Respiratory Equipment Inc.&#160;</td>
			<td style="width:145px;">023G1250</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">TSI mass flowmeter 4040</td>
			<td style="width:244px;">TSI</td>
			<td style="width:145px;">4040</td>
			<td style="width:235px;">Alternative product from supplier may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Saint-Gobain 15.9 mm Tygon tube</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">Z685704</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Plexiglas boxes with removable lids</td>
			<td style="width:244px;">Custom built</td>
			<td style="width:145px;">N/A</td>
			<td style="width:235px;">150 x 163 x 205 mm (a 2 mm hole on the side).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Half Facepiece Reusable Respirator</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">7503</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">3M Advanced Particulate Filters (P100)&#160;</td>
			<td style="width:244px;">3M</td>
			<td style="width:145px;">2291</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sissors</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">233-1104</td>
			<td style="width:235px;">Preferred scissors may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Forceps&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">232-1313</td>
			<td style="width:235px;">Preferred forceps may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Intramedic PE50 polyethylene tube</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">427411</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Ethicon 2-0 Perma-hand silk tread&#160;</td>
			<td style="width:244px;">VWR</td>
			<td style="width:145px;">95056-992</td>
			<td style="width:235px;">Recomended brand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">26 &#189;&#160; gage needle&#160;</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">1 mL BD slip-tip syringe, non-sterile</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=301025">301025</a></td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">60 mL BD Luer-Lok syringe, non-sterile, polypropolene&#160;</td>
			<td style="width:244px;">BD</td>
			<td style="width:145px;">301035</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fluka Hematoxylin-Eosin</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">3972</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">T&#252;rk&#39;s solution</td>
			<td style="width:244px;">Merck Millipore</td>
			<td style="width:145px;">109277</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Table top centrifuge</td>
			<td style="width:244px;"></td>
			<td style="width:145px;"></td>
			<td style="width:235px;">Alternative manufacturers exist.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cytospin 4 cytocentrifuge</td>
			<td style="width:244px;">Thermo Scientific</td>
			<td style="width:145px;">A78300003</td>
			<td style="width:235px;">Alternative centrifuge can be used.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEMA-3 stat pack</td>
			<td style="width:244px;">Fisher Scientific</td>
			<td style="width:145px;">23-123-869</td>
			<td style="width:235px;">Alternative staining kits exists.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:244px;">Sigma-Aldrich</td>
			<td style="width:145px;">HT501128&#160;</td>
			<td style="width:235px;">Alternative suppliers exist.</td>
		</tr>
	</tbody>
</table>

a method for generating pulmonary neutrophilia using aerosolized lipopolysaccharide

Akut akciğer hasarı (ALI) tedavi seçenekleri kısıtlı ve yüksek mortalite ile alveol nötrofilisi ile karakterize ciddi bir hastalıktır. ALI Deneysel modeller hastalığın patogenezinde anlayışımızı arttırmak anahtardır. Gram pozitif bakterilere türetilen Lipopolisakarid (LPS) nötrofilik hava yollarında inflamasyon ve farelerin akciğer parankimi neden olur. LPS gibi bileşiklerin etkin pulmoner şevki, Bununla birlikte, elde etmek zordur. Burada tarif edilen bir yaklaşımda, fareler pulmoner uygulama, aerosol, Pseudomonas aeruginosa, LPS meydan okumasının elde edilir. Çözünmüş LPS basınçlı hava bağlı bir püskürtücü ile aerosol edildi. Fareler, aerosol kesildikten sonra 2 dakika havalandırma ve ardından, 10 dakika boyunca bir pleksiglas kutuya LPS aerosol sürekli bir akışına maruz bırakılmıştır. Formalin perfüzyon ardından entübasyon ve sonraki bronkoalveoler lavaj, sonraki steril p karakterizasyonu için olanak sağlayan, yapıldıulmonary inflamasyon. Aerosolize LPS bronkoal- lavaj ve histolojik değerlendirilmesi ile tespit alveol nötrofilisi ile karakterize bir akciğer iltihabı, üretir. Bu teknik birkaç aletleri ile küçük bir maliyetle kurmak ve minimal eğitim ve uzmanlık gerektirir. pozlama sistemi böylece rutin akciğer patolojisi anlayışımızı geliştirmek için potansiyele sahip, herhangi bir laboratuvarda yapılabilir.

Aerosollaştınlmış P. Mücadelesi aeruginosa LPS genellikle erken ve geç zaman noktalarında hem de nötrofillerin bir üstünlüğü ile karakterize solunum yolu lümeni ve alveollerde, belirgin bir inflamatuar yanıt verir. Aerosol haline LPS pulmoner nötrofiliyi indükler C57BL / 6BY ve BALB / c fareleri, aerosol P. maruz bırakıldı aeruginosa LPS veya tek başına araç ve nötrofiller BAL&#39;da sayıldı. C57BL taşı...

Watch this Scientific Journal Video about A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide at JoVE.com

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Biz akut akciğer hasarı modeli, nebülizasyonu tarafından aerosolleşmiş lipopolisakkaride meydan tarafından nötrofilik akciğer inflamasyonu uyarılması için bir yöntem açıklanmaktadır. Buna ek olarak, akciğer izolasyonu, entübasyon ve bronkoalveoler lavaj temel cerrahi teknikler de tarif edilmiştir.

Aerosolize Lipopolisakarid kullanma Yaratma Pulmoner nötrofilisi için bir yöntem

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

a-method-for-generating-pulmonary-neutrophilia-using-aerosolized

Research

JoVE Journal

Immunology and Infection

11.6K Görüntüleme.  Karolinska Institutet.  Biz akut akciğer hasarı modeli, nebülizasyonu tarafından aerosolleşmiş lipopolisakkaride meydan tarafından nötrofilik akciğer inflamasyonu uyarılması için bir yöntem açıklanmaktadır. Buna ek olarak, akciğer izolasyonu, entübasyon ve bronkoalveoler lavaj temel cerrahi teknikler de tarif edilmiştir. 

Video: A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

Embriyonik Farelerde damarları Etiketleme Bir Yöntem

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

İmmünoloji ve Enfeksiyon

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population1, and almost two million people are killed by TB each year.2 Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3 The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2
Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in &gt; 95% of the MDR-TB isolates.4, 5 The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2
Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7 meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene.8 It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9 It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

Xpert MTB / RIF Testi ile Akciğer Tüberkülozu Tanısı

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

Peptid: Epitop-spesifik T-hücrelerinin MHC tetramer bazlı Zenginleştirme

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Çekal ligasyon ve Farelerde Autophagy incelemek için bir model olarak Sepsis Patamaya kaynaklı

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Fare Dendritik Hücre Alt Grupları İzolasyonunda bir Verimli ve Yüksek Verim Yöntem

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

İki fotonlu Lipopolysaccharide tedavi fare karaciğer bağışıklık yanıtında sırasında lökositlerin Intravital görüntüleme

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Reseptörleri ve enzimler arasındaki işlevsel etkileşimler çalışmak için Co-immunoprecipitation tahlil

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Fizyoloji Immunogenic dendritik hücreler oluşturma için yeni protokol

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Floresan Antibiyotik Probları Kullanılarak Bakteriyel Direncin Görüntülenmesi

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

Tüm Vücut Soluma Sistemi Kullanan Farelerde Sigara Dumanına Maruz Kalma

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...