VK was funded by grants from the IUP Department of Biology and School of Graduate Studies and Research (Graduate Student Professional Development). CQD and TWS were supported by the IUP School of Graduate Studies and Research (Faculty Publication Costs/Incidental Research Expenses). We also thank members of the Diep laboratory for maintaining the zebrafish facility.

Bu protokol açıklanan prosedürler Pennsylvania Indiana Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. 1. Hasat Embriyolar <ol><li> 28.5 ° C, pH 7, 500-1,500 uS arasındaki iletkenlik zebrafish muhafaza ve 14 saat ışık ve 10 saat 24 ışıksız bir ışık / karanlık çevrimi. Böyle Tü, AB ya da Tü / AB melez olarak yabani tip suşlar kullanın. Farklı suşları kimyasal arıtma 25 farklı yanıt verebilir. </li><li> Bir çiftleşme tankına balık sistem su ilave edilerek hasat yumurta önceki gece çiftleşme için balık ayarlayın. tanka bir erkek ve dişi balık ekleyin ve bir bölücü ile iki balık ayırın. Her balık çifti 50-300 yumurta bir dizi üretecek. bu yeterli yumurta balık 30 çift kurmak, üretilecek sağlamak. Tipik olarak, Başbakan çiftleşme yaşta balık çiftlerinin yaklaşık% 50 (6-9 aylık) 200 çifti başına yumurta ve 3&#39;e kadar maksimum sonuçlanan yumurta üretecekBu deney için 000 yumurta. </li><li> ışığı yanar sonra ertesi sabah, çiftleşme başlatmak için bölücü kaldırın. Yumurtalar için her 15 dakikada bir çiftleşme tankları kontrol edin. </li><li> Balık yumurtalarını kez, bir çay süzgeci kullanarak tüm embriyolar hasat ve E3 tamponu (5 mM NaCl, 0.17 mM KCI, 0.33 mM CaCl2, 0.33 mM MgSO 4) ile büyük bir kap içine bunları birleştirmek. 1.5 hpf, kaldırmak ve bir mikroskop altında plastik bir transfer pipet ile döllenmemiş yumurta atmak. Döllenmiş yumurtalar 34 şeffaf ise döllenmemiş yumurtalar opak bulunmaktadır. </li><li> Her bir tedavi durumu için 50 mi E3 tamponu ihtiva eden bir 100 x 15 mm ebadındaki bir cam Petri kabı içine başına 50 embriyo. 33 yemekleri toplam 11 tedavi koşulları ve 3 tekerrürlü için gereklidir. </li></ol> 2. Şifalı Embriyolar <ol><li> 2 saat sonrası fertilizasyon (hpf) 35 bakımları yürütmek. 28.5 ° C embriyolar / larvaları inkübe ve 120 hpf larvaları inceleyin. lea gerçekleştirmekst İstatistiksel analiz için her tedavi durumun üç suret. </li><li> 2 HPF 3 Petri kapları (her biri 50 embriyo) için bir transfer pipetiyle E3 tamponunu çıkarın ve E3 tamponu içinde seyreltilmiş 300 mM 50 ml etanol ekleyin. etanol buharlaşmasını en aza indirmek için Parafilm ile Petri kapları örtün. <ol><li> 22 saat boyunca etanol embriyolar maruz devam edin. Sonra bir transfer pipet ile etanol çıkarın. E3 tampon 50 ml ekleyin ve etanol yıkamak için çanak birkaç kez girdap. Bir transfer pipet ile bu E3 tamponu çıkarın ve yıkama adım 2 kez daha tekrarlayın. </li></ol></li><li> 2 HPF 3 Petri kapları (her biri 50 embriyo) için bir transfer pipetiyle E3 tamponunu çıkarın ve yeni bir E3 tamponu 50 ilave edin. </li><li> 2 hpf 9 Petri kapları (her biri 50 embriyo) için bir transfer pipetiyle E3 tamponunu çıkarın ve E3 tamponu içinde çözülmüş 1.000 mg / l sodyum nitrit 50 ilave edin. Önceden kullanarak stok nitrit çözeltisinin konsantrasyonu onaylayındiyazotizasyonda modifikasyonu (USEPA Yöntem 354.1) spektrofotometrik yöntem 28. <ol><li> 46 saat 3 yemekler, 70 saat boyunca 3 yemekler ve 94 saat süreyle 3 yemekler açığa devam edin. günlük taze yapılmış nitrit çözeltisi ile nitrit çözeltisi değiştirin. </li><li> Her maruziyet süresinden sonra, bir transfer pipet ile Nitrit çıkarın. E3 tampon 50 ml ekleyin ve nitrit yıkamak için çanak birkaç kez girdap. Bir transfer pipet ile bu E3 tamponu çıkarın ve yıkama adım 2 kez daha tekrarlayın. </li></ol></li><li> 2 HPF 3 Petri kapları (her biri 50 embriyo) için bir transfer pipetiyle E3 tamponunu çıkarın ve 200 mg / L sodyum nitrit 50 ilave edin. 400, 600, 800 ile bu işlemi tekrarlayın ve sodyum nitrit 1,000 mg / L. <ol><li> 70 saat süre ile nitrite embriyolar maruz devam edin. günlük taze yapılmış nitrit çözeltisi ile nitrit çözeltisi değiştirin. </li><li> pozlama süresinden sonra, bir transfer pipet ile Nitrit çıkarın. E3 tampon 50 ml ekleyin ve birkaç çanak girdapzaman nitrit yıkayın. Bir transfer pipet ile bu E3 tamponu çıkarın ve yıkama adım 2 kez daha tekrarlayın. </li></ol></li><li> 2 HPF 3 Petri kapları (her biri 50 embriyo) için bir transfer pipetiyle E3 tamponunu çıkarın ve E3 tamponu içinde çözülmüş 1.000 mg / L sodyum nitrat 50 ml ekleyin. Kadmiyum indirgeme (USEPA Yöntem 353.3) spektrofotometrik 28 bir modifikasyonu kullanılarak, önceden hazır nitrat çözeltisi konsantrasyonunu kontrol edin. <ol><li> 70 saat boyunca nitrata embriyolar maruz devam edin. günlük taze yapılmış nitrat çözeltisi ile nitrat çözeltisi değiştirin. </li><li> pozlama süresinden sonra, bir transfer pipet ile nitratı temizler. E3 tampon 50 ml ekleyin ve nitrat yıkamak için çanak birkaç kez girdap. Bir transfer pipet ile bu E3 tamponu çıkarın ve yıkama adım 2 kez daha tekrarlayın. </li></ol></li><li> maruziyet her gün boyunca, bir stereomikroskopta kullanarak ölü embriyo / larvaların sayısını. Ölüm İşaretlerkalp atışı eksikliği ve kan dolaşımını, ya da gözlem 1 dakika sonra hareketlilik eksikliği sayılabilir. E3 tampon kirlenmesini azaltmak için bir transfer pipet ile ölü embriyolar / larva çıkarın. </li><li> Deneyler 120 hpf bittiğinde, larva euthanize. <ol><li> Bir transfer pipet ile E3 tamponu çıkarın. Daha sonra, (pH 7 tamponlanmış)% 0.2, MS-222, 50 ml ilave edilir ve 10 dakika boyunca bekleyin. </li><li> Bir transfer pipet ile MS-222 çıkarın. MS-222 yıkamak için E3 tampon ve girdap 50 ml ekleyin. </li><li> Bir transfer pipet ile E3 tamponu çıkarın ve larvaları düzeltmek için% 4 paraformaldehid (PFA) 20 ml ekleyin. çanak birkaç kez girdap. şişeyi doldurmak için yeterli PFA ile birlikte bir cam şişenin içine larva aktarmak için bir aktarma pipeti kullanarak. gece buzdolabında şişeleri (O / N) saklayın. </li></ol></li><li> Bir dijital kamera ile Stereoskop kullanarak sabit larva fotoğraf çekmek. 30X büyütme, ISO 200 ve 200 msn pozlama süresini kullanın. larvaları yönlendirmek böylece le anteriorft ve dorsal alanının üst etmektir. </li></ol>

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The authors have nothing to disclose.

Burada anlatılan yöntemi nitrit ve nitrat teratojenik potansiyelini değerlendirirken zebrafish faydasını ortaya koyar. diğer omurgalılar ile karşılaştırıldığında, Zebra balığı yüksek doğurganlığını, dış döllenme, optik şeffaflık ve hızlı bir gelişme içeren avantajlara sahip. (Örneğin casper zebrabalıkları 36 gibi) pigmentasyon eksikliği mevcut mutantlar da iç organların görünürlüğünü artırmak için yardımcı olur. Ayrıca, canlı balık 37 analizini ...

Teratojenez ağır vakalarda 1 kalıcı yapısal ve fonksiyonel anormallikler, büyüme geriliği, ya da düşük yapma yol açarak bir embriyo ya da ceninin normal gelişimini bozan bir süreçtir. Bu çeşitli şekillerde 2 embriyonik gelişim müdahale bazı doğal maddelerin (teratojenler), neden olabilir. insan fetal gelişim sırasında, radyasyon, enfeksiyöz ajanlar, toksik metaller ve organik kimyasallar gibi ortak teratojenler morfogenetik hatalar ile epicanthic kıvrımlar kusurları (üst göz kapağındaki deri kıvrımı) ve klinodaktili (kavisli parmak veya ayak) neden olduğu bildirilmiştir 1. teratogenez moleküler mekanizmasını anlamak tedavi ve korunma geliştirilmesine yönelik ilk adımdır. Böyle Afrika gibi bazı omurgalı modeller terat etkilenen moleküler yolları belirlemek için kullanılır olmuştur kurbağa (Xenopus laevis) ve zebrafish (Danio rerio) pençeliogens. Önceki çalışmalar epidemiyolojisi, toksikoloji ve teratogenez 3-7 için bir model olarak zebrafish kullandık. Scholz ve diğ. Çevresel toksisite değerlendirmesi için bir &quot;altın standart&quot; olarak kabul zebrafish. Bu 8 olarak ortaya çıkar araştırmacılar gelişimsel defekti görüntülemenizi sağlar zebrafish embriyo, şeffaflık, kısmen, kaynaklanmaktadır. İnsan genlerinin yaklaşık% 70&#39;i insan kusurları 9 çalışmak için Zebra balığı bir arzu omurgalı modeli yapma, zebrafish ortologlara var. Diğer çalışmalar bu ilişkiyi 12 desteği yok iken bazı epidemiyolojik çalışmalar çiftlik gıdalar ve su yaygın olarak mevcut olduğu nitrat ve nitrit, sürmüşlerdir, doğum kusurlarına veya spontan abortus 10,11 ile ilişkilidir. Nitrat (NO 3 -) ve nitrit (NO 2 -) toprak ve su doğal olarak bulunur. Bu bitkiler için bir azot kaynağı ve bir N bir parçasıdıritrogen döngüsü 13. Böyle nitrat yüksek gübre kullanımı çiftlikleri yeşil fasulye, havuç, kabak, ıspanak, ve pancar gibi yiyecekler ölçüde nitrat ve nitrit 7 seviyelerini yükseltmiştir. (Özellikle toprak ikinci tur 30) yüksek nitrat suda yüksek nitrat gıdalar ve balık ile beslenen ineklerden elde edilen süt nitrat ve nitrit 14 büyük miktarda tüketen insanlarda yol açabilir. Nitrat ve nitrit de yaygın dramatik insanlar 12 ile yutulur miktarı artar gıda muhafaza, kullanılmaktadır. Nitrat ve nitrit Optimal düzeyleri vasküler homeostaz ve fonksiyon, nörotransmisyon ve immünolojik konak savunma mekanizmaları 13-15 gibi fizyolojik süreçlerde önemli bir rol oynaması. Bununla birlikte, nitrat ve nitrit yüksek düzeyde maruz Özellikle bebeklerde ve çocuklarda 16, yan etkilere neden olabilir. Özeti Alınan nitrat mikroflorasının ve inci ağız boşluğunda nitrit da dönüştürüldüğübağırsak mikroflorası 17 e gastrointestinal sistem. Nitrat yeteneği 18 taşıyan kendi oksijen hemoglobin zarar, methemoglobin hemoglobin oksidasyonu ile mavi bebek sendromu için yüksek risk altında bebeklerin koyar. Bu, daha şiddetli vakalarda periferal dokulara uzanan cilt mavi renk elde edilir. Diğer belirtiler dokular sonuçların inhibe oksijenasyonu, en ağır koma ve ölüm 19,20 yol açar. Benzer semptomlar nitrat 21 daha yüksek konsantrasyonlarda bebeklerde ve yetişkinlerde görülür. Yükseltilmiş nedeniyle siyanoz, baş ağrısı nitrit zehirlenmesi sonuçlarına erişkinlerde methemoglobin düzeyleri, solunum bozuklukları, 31 ve ölüm nedeniyle hayati doku hipoksisi 32,33 ilgili komplikasyonlar tedavi edilmediği takdirde. daha yüksek seviyelerde yutulur nitrat, çeşitli sağlık komplikasyonlara neden olabilir. Çocukluk diyabet, tekrarlayan ishal ve tekrarlayan solunum yolu enfeksiyonlarıÇocuklarda yüksek nitrat alımı 11,17,22 ile bağlantılı olmuştur. nitrat yüksek düzeyde kronik maruz kalma idrar ve dalak kanaması ile ilişkilidir. Nitrat Akut yüksek doz maruz karın ağrısı, kas güçsüzlüğü, dışkı ve idrarda, bayılma kan ve ölüm 11 gibi tıbbi koşulları geniş bir yelpazede yol açabilir. Yüksek seviyelerde nitrata prenatal maruziyet nöral tüp ve kas-iskelet defekt 11 ile ilişkili olmuştur. Yeni bir rapor nitrit ile zebrafish embriyolar tedavi kesesi ödem, kraniofasial ve eksenel malformasyonlar sarisi yol açtı, ve mesane noninflation 5 yüzmek olduğunu gösterdi. Bu çalışmada, kendi teratojenik potansiyeli belirlemek için nitrat ve nitrit ile zebra balığı embriyolar tedavi etmek için bir yöntem ortaya koymaktadır. Embriyolar, farklı konsantrasyonlarda ve farklı zaman uzunlukları nitrit maruz bırakılmıştır. Bu kurulu bir teratojen 23 olduğu Etanol, bir pozitif kontrol olarak kullanıldı. Our yöntem nitrit yüksek konsantrasyonlarda ve uzun pozlama süreleri hem hayatta zarar ve şiddetli (brüt gelişimsel kusurlar) hafif (ödem) arasında değişen çeşitli fenotipleri yol açtığını gösterdi. Bu nedenle, zebra balığı doğrudan epidemiyolojik çalışmalar tamamlamak için embriyolar üzerinde nitrat ve nitrit potansiyel teratojenik etkileri keşfetmek için bir modeldir.

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			<td height="20" style="height:20px;width:191px;">Ethanol</td>
			<td style="width:185px;">Sigma-Aldrich</td>
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			<td height="20" style="height:20px;width:191px;">KIMAX glass Petri Dish</td>
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			<td style="width:104px;">89001-244</td>
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			<td height="20" style="height:20px;width:191px;">MS-222</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">E10521</td>
			<td></td>
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			<td height="40" style="height:40px;width:191px;">NitraVer 5 Nitrate Reagent</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">14034-46</td>
			<td></td>
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			<td height="20" style="height:20px;width:191px;">NitriVer 3 Nitrite Reagent</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">14065-99</td>
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			<td height="20" style="height:20px;width:191px;">Parafilm</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">3-374-10</td>
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			<td height="20" style="height:20px;width:191px;">Paraformaldehyde</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">158127</td>
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			<td height="20" style="height:20px;width:191px;">S6E stereomicroscope</td>
			<td style="width:185px;">Leica</td>
			<td style="width:104px;">10446294</td>
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			<td height="20" style="height:20px;width:191px;">Sodium nitrate</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">S343</td>
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			<td height="20" style="height:20px;width:191px;">Sodium nitrite</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">S347</td>
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			<td height="40" style="height:40px;width:191px;">Transfer pipets</td>
			<td style="width:185px;">Laboratory Products Sales</td>
			<td style="width:104px;">L320072</td>
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			<td height="20" style="height:20px;width:191px;">Glass vials</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">03-338B</td>
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zebrafish as a model to assess the teratogenic potential of nitrite

ortamda yüksek nitrat seviyeleri insanlardaki konjenital bozuklukların ve düşüklere neden olabilir. Tahminen bu bağırsak ve tükürük bakterilerin nitratın nitrite dönüştürülmesi kaynaklanmaktadır. fakir üreme sonuçlara yol açabilir Ancak, her ne diğer memeli çalışmalarda, yüksek nitrit düzeyleri, doğum kusurlarına neden yok. Böylece, nitrit teratojenik potansiyel açık değildir. Kolayca nitrit veya ilgi duyulan herhangi bir başka kimyasal teratojenik etkileri değerlendirmek için bir omurgalı bir model sistemi olması yararlı olacaktır. Burada, toksisite ve embriyonik kusurları için bileşiklerin taranması için zebrabalıkları (Danio rerio) kullanımını göstermektedir. Zebra balığı embriyolar onlara teratojenik çalışmalar için iyi bir model yapma, dışarıdan döllenmiş ve hızlı bir gelişme vardır. Biz olumsuz nitrite maruz kalma süresinin artması hayatta kalmasını etkilediğini göstermektedir. nitrat bunu yapmaz nitrit konsantrasyonunu arttırarak da olumsuz, hayatta kalma etkiler. Embriyoların tha içint nitrit poz hayatta, çeşitli kusurlar perikardial dahil oluşabilir ve mesane noninflation ve kraniofasial malformasyon yüzmek, sac ödem yolk. Sonuçlarımız zebra balığı nitrit teratojenik potansiyeli çalışmaları için uygun bir sistem olduğunu göstermektedir. Bu yaklaşım kolayca erken, omurgalı gelişimi üzerindeki etkileri, diğer kimyasal maddeler test etmek için uyarlanabilir.

22 saat süre ile, 300 mM etanol maruz önceki raporlarda 5,23,26 tutarlı sağkalım (veriler gösterilmemiştir), üzerinde herhangi bir etki. etanol, bilinen bir teratojen ve pozitif kontrol olarak hizmet gibi bu beklenir. Gözlenen fenotipleri perikardiyal ödem, yüzme kesesi noninflation (Şekil 1), kraniofasyal kusurları ve gelişimsel gecikme (veriler gösterilmemiştir) dahil. nitrit ile m...

Watch this Scientific Journal Video about Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite at JoVE.com

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

teratojen Pozlama doğum kusurlarına neden olabilir. Balığı kimyasal teratojenik potansiyeli belirlemek için faydalıdır. Biz maruz kalma farklı zamanlarda da nitrit değişik düzeylerde embriyoların açığa tarafından zebrabalıkları yararını göstermektedir. Biz nitrit toksik ve ağır gelişimsel bozukluklara neden olduğunu göstermektedir.

Bir Model Olarak Zebra balığı Nitrit teratojenik potansiyelini değerlendirmek için

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

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9.4K Görüntüleme.  Indiana University of Pennsylvania.  teratojen Pozlama doğum kusurlarına neden olabilir. Balığı kimyasal teratojenik potansiyeli belirlemek için faydalıdır. Biz maruz kalma farklı zamanlarda da nitrit değişik düzeylerde embriyoların açığa tarafından zebrabalıkları yararını göstermektedir. Biz nitrit toksik ve ağır gelişimsel bozukluklara neden olduğunu göstermektedir. 

Video: Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

İmmünoloji ve Enfeksiyon

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Bir Kök Hücre tabanlı Gen Terapisi Tümör Modeli olarak BLT Insanlaşmış Fare Kullanma

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

Böcek<em&gt; Galleria mellonella</em&gt; Bakteriyel Patogenez İnceleyecek Güçlü Enfeksiyon Model olarak

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Zebra balığı Doğuştan Bağışıklık Sağlık Bir Göstergesi Olarak Solunum Burst Tepki Kantitasyonu

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Kullanımı<em&gt; Galleria mellonella</em&gt; Bir Model Organizma olarak Eğitim için<em&gt; Legionella pneumophila</em&gt; Enfeksiyon

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

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 ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Bir Zebra balığı Larva Modeli Doğuştan Bağışıklık Tepki Non-invaziv Görüntüleme<em&gt; Streptococcus iniae</em&gt; Enfeksiyon

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Eğitim için bir genetik Yaklaşım olarak yardımcı T hücrelerinin retroviral İletimi Mekanizmaları kendi Farklılaşma ve Kontrol Fonksiyonunun

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Aşırı Ezme ve İnsanın Saflaştırılması<em&gt; Cis</em&gt; -prenıltransferaz in<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

Hamam böceği virülans, Patogenez ve uyuşturucu etkinliği araştırmak için alternatif bir Non-memeli hayvan Model tıslama Madagaskar

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...