Bu çalışma, Kanada Sağlık Araştırma Enstitüsü (MSH-95.368, MOP-89.773 ve MOP-106.623 - CZ) ve Kanada Yenilik Vakfı tarafından desteklenmiştir. SCM, Gastroenteroloji doktora sonrası burs CIHR / Kanada Derneği alıcı. CZ CIHR Yeni Araştırmacı.

1. Trichuris muris yumurta Çoğaltım <ol><li> (Ya da genetik olarak yatkın fareler Trichuris muris yumurta yeni toplu üretmek için, 20-30 bağışık yetmezliği olan farelere enfekte () örneğin NOD.Cg-Prkdc SCID Il2rg tm1Wjl / SzJ (NSG) veya 129S6/SvEvTac-Rag2 tm1Fwa (RAG2 - /) örn. ağızdan sonda ile yaklaşık 300 Trichuris muris yumurta ile 6-8 hafta eski AKR / J). </li><li> CO 2 boğulma 32-35 gün sonra fareler kurban. </li><li> Fare ventral tarafında ortaya çıkarmak ve% 70 etanol ile karın ıslak. </li><li> Forseps kullanarak karın cilt kavramak ve makas kullanarak küçük bir kesi yapmak. Karın içeriğini ortaya çıkarmak için cildi kesin. </li><li> Çekum ve proksimal kalın bağırsak belirleyin ve bunları kaldırın. </li><li> Dulbecco&#39;nun Modifiye Eagle Orta (DMEM) 500 U / ml penisilin ve 500 mg / ml streptomisin (P / S) içeren bir 10 cm petri çekum ve proksimal kalın bağırsak yerleştirin. Lümen ve solucanlar ortaya çıkarmak için açık çekum kesin. Fosfat beher forseps yer çekum tamponlu salin (PBS) ve dışkı çıkarmak için hafifçe sallayın. </li><li> Petri çekum dönün ve hafifçe solucanlar çekin ve 6 plaka DMEM + P / S 5 ml içeren bir iyi bir yer onları yumuşak kavisli bir forseps (Roboz RS-5047) </li><li> Tüm fareler için bu işlemi tekrarlayın. </li><li> 37 nem ve inkübe korumak için nemli bir kağıt havlu ° C, 4 saat için bir Tupperware kap içinde 6 plaka koyun. </li><li> 4 saat yumuşak kavisli bir forseps kullanarak solucanları kaldırmak ve 5ml DMEM içeren 6 plaka 2 yeni kuyu bölebilirsiniz sonra + P / S 15 ml şahin tüp orijinal yeri iyi ve içeriğini Hasat. 4 saat antijen (tipik protein verimi 20 fareler başına 10-20 mg PBS diyaliz, sonra hücre restimulation için kullanılabilir) ve tekrar süspansiyon olarak 3000 rpm ve kaldır ve yedek süpernatant tüp Spin yumurta içeren pelet otoklavlanmış musluk suyu. </li><li> 6 plaka ° C gecede 37 geri koyun. Çıkarın ve solucanlar atmayın. Bir şahin tüp ve 3000 rpm&#39;de 5 dakika boyunca spin kuyuların içeriğini Hasat. Süpernatant ve rezerv gecede antijen (PBS diyaliz, tipik bir protein verimi 20 fareler başına 10-20 mg sonra Trichuris Ag spesifik antikor izotip ELISA&#39;larla için kullanılabilir) ve tekrar süspansiyon yumurta içeren otoklavlanmış musluk suyu pelet olarak çıkarın. </li><li> 4 saat ve gece boyunca yumurta birleştirin ve 75 cm 2 balona transfer daha sonra herhangi bir artık solucanları kaldırmak için 70μm süzgeç vasıtasıyla yumurta filtre . Şişesi, 6 hafta boyunca oda sıcaklığında (RT) folyo ile kaplanmış karanlık bir yerde tutun. </li><li> Otoklavlanmış musluk suyu yumurta yıkayın ve 50 ul 50 yaşayabilir yumurtaların tekrar süspansiyon. Bunu yapmak için, 3000 rpm&#39;de 5 dakika ~ 50ml steril musluk suyu ve tekrar süspansiyon yumurta santrifüj. Bir bardak slayt 50 ul yumurta ve yer çıkarın. 40x büyütme kullanarak 50 ul yaşayabilir embriyolu yumurta sayısını. 50 ul 50 yaşayabilir yumurtaların vermek için yumurta sulandırınız. Kopper ve Mansfield 16 tarafından yapılan çalışmada yaşayabilir yumurtaların görüntüler için bakın lütfen. </li></ol> 2. Trichuris muris yumurta (200 yumurta / fare) ile deneysel fareler Akut enfeksiyon <ol><li> Iyice tekrar süspansiyon yumurta ve farelerin sayısı tedavisi için yeterli hacmi kaldırmak X 2 (örneğin 10 fareler için 200 ul x alabilir 10 fareler x 2 = 4 yumurta ml) ve 14 ml ek bileşenini kap tüp koyun. </li><li> Karıştırmak için 14 ml ek bileşenini kap tüp ters çevirin. 1 ml şırınga uygun bir boyut besleme iğne (18 gauge 1 1 / 2 &quot;iğneler kullanılır 25 gram ağırlığında fare) içine yumurta 200 ul getirin. Fare ve diğer elinizi kullanarak bir el ense ağızdan sonda ile yumurta yönetmek . </li><li> 21 gün bekleyin. </li></ol> 3. Deneysel hasat <ol><li> Gün 21 CO 2 kullanarak fareleri kurban. </li><li> Kalp ponksiyon farelerden alınan kan toplayın, heparinize olmayan şırınga ve 4 mağaza ° C Ertesi gün, -20 ° C&#39;de, serum ve donma izole Serum Trichuris özel IgGs ve IgE tespiti için ELISA yöntemi ile kullanılabilir. </li><li> Fare ventral tarafında ortaya çıkarmak ve% 70 etanol ile karın ıslak. </li><li> Forseps kullanarak karın cilt kavramak ve makas kullanarak küçük bir kesi yapmak. Deri ve karın içeriğini ortaya çıkarmak için temel membran kesin. </li><li> Küçük bağırsak, çekum ve kolon tanımlayın. Forseps ile çekum alın ve karın boşluğundan dışarı doğru çekin. Forseps başka bir çift ile, mezenterik lenf düğümleri (mLNs) teşhir hakkı üzerinde ince bağırsak itin. Medya içeren 2 ml, 15 ml Falcon bağırsak ve yerine kesmek için dikkatli olmak mLNs çıkarın. İsterseniz bu ELISA ve hücre içi f sitokin analizi için 72 saat işlem ve in vitro (1 ml, 1 mcg / ml αCD3/CD28 veya 50μg/ml 4h Trichuris Ag 24 kuyucuğu 4x10 6 / ml) restimulated olabilirdüşük sitometri. </li><li> Çekum ve kolon kesin. </li><li> Forseps kullanarak, 2 ml Axygen tüp distal kolon ve yerden fekal pelet dışarı itmek. Freeze az -20 ° C. Fekal pelet molekülü-β (AHLAK-β) resistin gibi ifade western blot ile analiz edilebilir. </li><li> Çekum (~ 0.5 cm) ucu çıkarın. Dışkı forseps ile çekum ucu tutun kaldırın ve PBS bir petri 3 ml şırınga ve 22-gauge iğne ile yıkayın. Bir 5 ml Falcon ek kap tüp içinde taze% 4 paraformaldehid yerleştirin. 4 ° C saklayınız Bu parafine gömülü kesitli ve histolojik analiz için kızaklar üzerinde monte edilebilir. </li><li> RNA (~ 1 cm) proksimal kolonda bir parça kaldırın. 500 ul RNAlater 2 ml Axygen tüpe örnek koyun. 4 ° C saklayınız Bu örnekleri qPCR mRNA için analiz edilebilir. </li><li> Etiketli petri ve dondurucuda -20 ° C&#39;de çekum geri kalanı yerleştirin. </li></ol> 4. Çekum Trichuris muris solucanlar sayımı <ol><li> Çekum ~ 5 ml su içeren bir petri dondurucu ve yerden çıkarın. </li><li> Makas kullanarak, lümen maruz çekum kesilerek açılır. </li><li> Forseps ile çekum tutun ve şiddetle dışkı çoğunluğu kaldırmak için çekum sallamak. </li><li> Su içeren ve yavaşça IECS altta yatan doku kazıyın yumuşak kavisli bir forseps kullanarak başka bir petri çekum aktarın. </li><li> Su içeren yeni bir petri çekum aktarın ve şiddetle, küçük parçalar halinde doku müthiş, kalan doku forseps ile kazımak. </li><li> Tüm petri kutularının 3 solucanlar saymak için bir diseksiyon mikroskobu kullanın. Eğer bir solucan aynı solucan sağlamak için çanak çıkarın bulmak gibi iki kez sayılmaz. Solucanlar hasarlı ise (yani yarı yarıya kırık) doğru bir sayım almak için sadece kalın ya da ince uçları saymak. </li></ol> 5. Temsilcisi Sonuçlar Trichuris muris enfeksiyon modeli araştırmacılar parazit yükünü ve sistemik immün yanıtların yanı sıra, histolojik olarak incelemek için distal çekum içinde inflamatuvar yanıtı ölçmek için sağlar. Hasat çekum lümen maruz kaldığında, Trichuris solucanlar bir diseksiyon mikroskobu (Şekil 1A-B) ile numaralandırılan. Trichuris için bağışıklık parametreleri de antikor dayanıklı hayvanlar IgG1 için geçiş izotip (Th2 tip bağışıklık ile ilgili) ve duyarlı hayvanlarda IgG2a (Th1-tipi bağışıklık ile ilişkili) (Şekil 1C) içerir. Goblet hücre-spesifik protein resistin gibi molekülü-β (AHLAK-β) İfade Trichuris klerensi ile ilişkilidir ve Western blot analizi (Şekil 2) tarafından algılanamaz. Çekum distal kısmı goblet hücre yanıtları ve enfeksiyona yanıt olarak intestinal inflamasyon ölçüde (Şekil 3) değerlendirmek için periyodik asit-Schiff (PAS) ile boyandı. <img src="/files/ftp_upload/2774/2774fig1.jpg" alt="Şekil 1"/> Şekil 1. Trichuris muris bağışıklık Kantitasyonu. (A) Dayanıklı (B6) fareler gün 21 sonrası enfeksiyon Trichuris muris sınırdışı. Buna karşılık, duyarlı (AKR / J) farelerde kronik parazit kurulması, Trichuris ile enfekte olma. Her çubuk, ortalama ± SEM grup başına 4-8 hayvanlar temsil eder. (B) Worms diseksiyon mikroskobu kullanılarak sayılır. Üst panel, izolasyon solucanlar gösterir, alt panel IECS ve dışkı varlığı solucanlar gösterir. (C) Trichuris özel IgG2a titreleri boşaltım Trichuris antijen (O / N Ag, 5 mg / ile kaplı plakalar üzerinde seyreltilmiş serum ELISA (1:2 seyreltme 1:20 seyreltme başlayan ve biten 1:2560) tarafından belirlenir ml). Duyarlı AKR / J farelerin dayanıklı B6 farelere göre daha Trichuris-spesifik serum IgG2a özellikle yüksek seviyeleri var. <img src="/files/ftp_upload/2774/2774fig2.jpg" alt="Şekil 2"/> Şekil 2. Trichuris muris Gümrükleme goblet hücre-spesifik protein AHLAK-β 7 ifade ile ilişkilidir. AHLAK-β üretim Trichuris enfekte dayanıklı (B6) fekal pelet saptandı, ancak western blot analizi ile fareler (AKR / J) duyarlı değildir. Her şerit, (N) = Naive (Tm) = 21 gün Trichuris bulaşmış farelerin tek bir hayvan temsilcisidir. <img src="/files/ftp_upload/2774/2774fig3.jpg" alt="Şekil 3"/> Şekil 3. Distal çekum aşağıdaki Trichuris enfeksiyon histolojik incelenmesi. Distal çekum 5-7 mikron bölümlerini PAS ile boyandı. Goblet hücre hiperplazisi ve mukus üretimi dayanıklı (B6) belirgindir, ancak Trichuris enfeksiyon fareler (AKR / J) duyarlı değildir .

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British Columbia Üniversitesi tarafından belirlenen kurallara ve yönetmeliklere uygun olarak hayvanlar üzerinde deneyler yapıldı.

Bu protokol, araştırmacı tarafından gerekli olarak modifiye edilebilir bir standart yüksek doz akut Trichuris muris enfeksiyon. Örneğin, fare kurban edilebilir ve dokuların farklı günlerde hasat. Farelerin tam solucan yükü başarıyla kurduk belirlemek için hangi noktada tüm fareler yaklaşık 200 solucanlar bir yük taşımak, 14. günde kurban edilebilir. Fareler de 32 gün için tespit edilen solucan olgunluğa erişmiş olacak ve ömrünün süresi için ev sahibi ile kalacaktı enfekte olabil...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> Hayvan Besleme İğneler (18 x 1 ½ &quot;) </td><td> Çıtçıt </td><td> 7912 </td><td></td></tr><tr><td> Pürüzsüz eğimli Forseps </td><td> Roboz </td><td> RS-5047 </td><td></td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11965 </td><td></td></tr><tr><td> NOD.Cg Prkdc SCID Il2r gtm1Wjl / SzJ (NSG) </td><td> Jackson Laboratuvarları </td><td> 005557 </td><td> Bunlar kullanılan fareler, ancak, herhangi bir bağışık yetmezliği olan farelere veya zorlanma duyarlı çalışması gerektiğini. </td></tr><tr><td> RNAlater </td><td> Qiagen </td><td> 76104 </td><td></td></tr><tr><td> 2 ml tüpler </td><td> Axygen </td><td> MCT-200-C </td><td></td></tr><tr><td> 15 ml tüpler </td><td> Şahin </td><td> 352096 </td><td></td></tr><tr><td> 6 kuyucuğu </td><td> Şahin </td><td> 353046 </td><td></td></tr><tr><td> Paraformaldehit </td><td> Elektron Mikroskopi Bilimi </td><td> 15710 </td><td></td></tr><tr><td> α-RELMβ antikor </td><td> PeproTech Inc. </td><td> 0694270Rb </td><td></td></tr></tbody></table>

trichuris muris infection: a model of type 2 immunity and inflammation in the gut

Trichuris muris farelerin doğal bir patojen ve enfekte insanlar ve hayvancılık 1 Trichuris türleri biyolojik ve antijenik olarak benzer . Enfektif yumurta ağızdan sonda ile distal ince bağırsakta ambar tarafından verilen, çizgi çekum ve proksimal kolon ve olgunlaşma üzerine kript solucanlar ortamı içine 1 yumurta serbest intestinal epitel hücrelerinde (IECS) işgal. Bu model faktörleri incelemek için güçlü bir araç olduğunu kontrol CD4 + T helper (Th) hücre aktivasyonu yanı sıra bağırsak epitel değişiklikleri. C57BL / 6 ve BALB / c gibi dayanıklı kendilenmiş suşları, ortaya çıkan immün yanıt, polarize Th2 sitokinlerin (IL-4, IL-5 ve IL-13) ve ilişkili Th1 sitokinlerin iken (IL solucanlar sürülmesi ile karakterizedir -12, IL-18, IFN-γ) kronik enfeksiyonlar, genetik olarak yatkın AKR / J fareler 2-6 teşvik . Th2 sitokinleri hızlı IECS ciro, goblet hücre farklılaşması, işe alma ve epitelyal permeabilite ve düz kas kasılması, solucan sınırdışı 7-15 suçlanmıştır hangi değişiklikler dahil olmak üzere bağırsak mikroçevresinin fizyolojik değişiklikler teşvik eder. Burada detay Trichuris muris yumurta sonraki deneylerde kullanılan propaganda için bir protokol. Ayrıca enfeksiyon sonrası analizi için önerileri ile örnek bir deneysel hasat sağlar. Genel olarak, bu protokol, gastrointestinal sistem gibi IECS immün efektör fonksiyonları Th eğilimine ilişkin soruları yanıtlamak için kullanılabilecek bir Trichuris muris fare enfeksiyon modeli gerçekleştirmek için temel araçları araştırmacılar sağlayacaktır .

Watch this Scientific Journal Video about Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut at JoVE.com

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris enfeksiyon dayanıklı farelerde koruyucu bir Th2 yanıtı oluşturmak ve duyarlı fareler patolojik bir Th1 yanıtı Th2 dokunulmazlık bağırsak modelidir.

Trichuris muris Enfeksiyon: Gut Tip 2 Bağışıklık ve İnflamasyon Bir Model

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

trichuris-muris-infection-model-type-2-immunity-inflammation

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Immunology and Infection

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

16.2K Görüntüleme.  University of British Columbia. Trichuris muris enfeksiyon dayanıklı farelerde koruyucu bir Th2 yanıtı oluşturmak ve duyarlı fareler patolojik bir Th1 yanıtı Th2 dokunulmazlık bağırsak modelidir.

Video: Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Kapsüler Serotipleme<em&gt; Streptococcus pneumoniae</em&gt; Lateks Aglutinasyon tarafından

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

İmmünoloji ve Enfeksiyon

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

İzolasyon, Karakterizasyonu ve Dişeti Bağışıklık Hücre Ağı Fonksiyonel Sınav

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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 ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Farelerde Deneysel Bakteriyel Pnömoni İndüksiyonunun ve fenotipleme bir Non-invaziv ve Teknik Olmayan yoğun Yöntem

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Hassas kesimli Canlı Pulmoner Dendritik Hücreler Visualize&#39;a Fare Akciğer Dilimleri

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

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 ...

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the &#945;-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

Protein Halifeliğin bağırsak iltihabı bir iltihabi bağırsak hastalığı fare modeli üzerinde sinyal tat etkileri

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Viral enfeksiyon ve analiz Drosophila Melanogaster ana bilgisayar virüs etkileşimin kurulması

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

Galleria mellonella Waxworm enfeksiyon modeli Dissemine kandidiyazis için

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...