This research was possible through funding from the University of Neuch&#226;tel. We would like to thank all the students that helped in improving this technique, namely our colleague Kevin Thievent. We would also like to thank the members of the Thomas Lab for making their laboratory available. We would like to thank Janet Teeple for her help with mosquito rearing. We would also like to thanks Loyal Hall in the laboratory of Pr. Tom Baker for his help in the preparation of the micro capillary glass tubes.

Enjeksiyon ve Diseksiyon 1. Tuzlu Çözüm <ol><li> PH = 6.8 de 1.3 mM NaCI, 0.5 mM KCI, 0.2 mM CaCl2 elde etmek üzere distile su NaCl, KCl, ve CaCl2 eklenerek tuz çözeltisi hazırlayın. </li><li> Şeffaf boncuk renk tuz çözeltisi için 99 mililitre% 0.1 metil yeşil çözelti 1 ml. Bu% 0.001 metil yeşil &quot;enjeksiyon çözüm&quot; dir. </li><li> Daha sonra, tuzlu su çözeltisi, 45 ml% 0.1 metil yeşil çözelti 5 ml. Bu% 0.01 metil yeşil &quot;diseksiyon çözüm&quot; Diseksiyon sırasında boncuk renklerini ve gözlem kolaylaştırmak için 10 kat daha fazla konsantre metil yeşil olduğunu. NOT: Gerekirse Filtre tampon sterilize edin. </li></ol> 2. Kılcal Hazırlık <ol><li> Isı çekme microcapillary cam tüpler küçük boncuklar (φ = 40 um) 42 biraz daha büyük bir çok ince bir ipucu elde etmek için. </li><li> Açık veCımbız ile ucu kırarak ya da ucu kapalı testere ile her kılcal djust. </li></ol> 3. Sivrisinek Yetiştirme <ol><li> 26 ± 1 ° C&#39;de arka A. gambiae, 70 ±% 5 bağıl nem ve 12:12 saat ışık: karanlık döngüsü. yetişkin kadın tutmak ve onlara% 10 şeker çözeltisi erişim sağlar. </li></ol> Kapiller 4. Boncuk Seçimleri <ol><li> 5 santimetrelik bir petri tabağına negatif yüklü boncuklar (çapı 40-120 um) ve 0.009 g dökün ve önceden enjeksiyon çözeltisi, 30 dakika içinde 5 mL. NOT: amaç boya kolay enjeksiyon için boncuk seçim yapmak için boncuk renk izin vermektir. onlar steril kavanoz gelmediği takdirde boncuk otoklavlayın. </li><li> Bir kılcal pipet ampul monte edilmiş bir kılcal kullanarak, görsel bir binoküler mikroskop altında tuzlu su çözeltisi 0.1 ul küçük boncuk seçin. NOT: Aynı anda birden fazla boncuk aşılamak zaman 0.1 ul toplam hacmi üç boncuk seçin.</li></ol> 5. Sivrisinek Taşıma ve Aşılama <ol><li> Bir böcek aspiratör kullanılarak, 50 ml bir tüp içinde, her sivrisinek yerleştirin. </li><li> ezilmiş buz (2-5 dk) tüp koyarak kısaca her sivrisinek Chill. </li><li> binoküler mikroskop altında sağ tarafta bir sivrisinek yerleştirin. </li><li> Bir stereomikroskop altında, sıkıca göğüs boşluğu manikür sol tarafında delip ardından sıvı ve boncuk enjekte ederek kılcal sivrisinek aşılamak. NOT: mümkün olduğunca sivrisinek olarak dik kılcal tutarak uçuş kaslarının zarar vermemeye özen gösterin. </li><li> kılcal çıkarın. </li><li> tüy entomoloji forseps kullanarak, ayrı ayrı sivrisinek örgü ile kaplı bir 180 ml&#39;lik plastik bir kap içinde her kadın yerleştirin; Onlara% 10 şeker çözeltisi erişim sağlar. NOT: sivrisinek şeker çözeltisi damla takılıyorum olabilir Dikkat, şeker çözeltisi ile gereklidir. </li></ol> 6. SivrisinekDiseksiyon ve Boncuk Fotoğrafçılık <ol><li> Sivrisinekler 24 saat aşılamadan sonra hayatta olup olmadığını kontrol edin. Eğer öyleyse, onların durumunu kontrol ve uçabiliyor sadece sivrisinek tutmak. </li><li> -20 ° C&#39;de bu sivrisinek dondurun. NOT: Gerekirse Onlar diseksiyon aylar önce birkaç gün muhafaza edilebilir. </li><li> 32X büyütmede bir binoküler mikroskop altında cımbız kullanarak sivrisineklerin kanatları kaldırın. saydam bant ile cam slaytlar üzerine kanatlarını sabitleyin. her sivrisinek için kullanılan kodunu yazın. </li><li> Bir tarayıcı cam slaytlar koyun ve 1.200 dpi onları tarayın. Not: kod her sivrisinek için okunabilir olup olmadığını kontrol edin. </li><li> cam bir mikroskop lamı üzerine ve diseksiyon çözeltisi içinde, baş ve karın toraks ayırmak için forseps kullanabilir. </li><li> boncuk almak için forseps ile toraks açın. NOT: Boncuk karın hareket etmiyor, birbirleri ile temas halinde değildir ve çoğu t serbestçe yüzenO Hemolimf. olmayan bir melanized boncuk bulmak zor olabileceğinden, forseps ile boncuk kırmak için değil dikkatli olun. Boncuklar rengi elde etmek için 1 ilâ 5 dakika bekleyin. </li><li> resmi çekmeden önce, bir cam mikroskop lamı üzerine diseksiyon çözümün bir damlacık boncuk aktarın. NOT: boncuk yıkamak gerek yok. </li><li> Bir kamera ile donatılmış bir mikroskop kullanarak, standart bir aydınlatma ayarı ve 400X büyütmede her boncuk bir dijital görüntü almak. NOT: kendi karşılaştırma yapabilmek için farklı resim arasındaki aydınlatma değiştirmeyin. </li></ol> 7. Resim Analizi: Boncuk melanization <ol><li> Görüntü analiz yazılımı açın. &quot;Dosyasında&quot; üzerine tıklayın ve ardından &quot;açık&quot; bir boncuk görüntüyü bulmak için. </li><li> Tıklayın &quot;analiz edin.&quot; Tıklayın &quot;set ölçümlerin.&quot; &quot;Set ölçümler&quot; penceresinde &quot;gri ortalama değer&quot; kutusunu işaretleyin. </li><li> daire seçim aracı simgesini tıklayın. boncuk çevresini seçin; kalırboncuk yan ve çevresinde aydınlık halo kaçının. </li><li> &quot;Analiz&quot; menüsünü tıklayın. Tıklayın &quot;tedbir.&quot; boncuk enine kesit alanının ortalama gri değer ve boyutunu elde edilir. <ol><li> &quot;- Gri değerini ortalama 256.&quot;: Her boncuk melanizm değerini elde etmek için, bir hesap makinesi veya hesap yazılımını kullanarak aşağıdaki işlemi gerçekleştirmek melanizm tedbir 0 tamamen beyaz bir görüntü temsil eden, 0 ile 256 arasında bir değerdir. </li></ol></li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+causes+and+consequences+of+immunopathology.">Evolutionary causes and consequences of immunopathology.</a> Annu Rev Ecol Evol S. 36, 373-397 (2005).</li><li>Best, A., Long, G., White, A., Boots, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+implications+of+immunopathology+for+parasite+evolution.">The implications of immunopathology for parasite evolution.</a> P Roy Soc B-Biol Sci. 279 (1741), 3234-3240 (2012).</li><li>Cator, L. J., George, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Manipulation&amp;#34;+without+the+parasite:+altered+feeding+behaviour+of+mosquitoes+is+not+dependent+on+infection+with+malaria+parasites.">34;Manipulation&amp;#34; without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites.</a> P Roy Soc B-Biol Sci. 280 (1763), 20130711 (2013).</li><li>Voordouw, M. J., Lambrechts, L., Koella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+maternal+effects+after+stimulation+of+the+melanization+response+in+the+yellow+fever+mosquito+Aedes+aegypti.">No maternal effects after stimulation of the melanization response in the yellow fever mosquito Aedes aegypti.</a> Oikos. 117 (8), 1269-1279 (2008).</li><li>Paskewitz, S., Riehle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Plasmodium+refractory+and+susceptible+strains+of+Anopheles+gambiae+to+inoculated+Sephadex+beads.">Response of Plasmodium refractory and susceptible strains of Anopheles gambiae to inoculated Sephadex beads.</a> Dev comp immunol. 18 (5), 369-375 (1994).</li></ol>

The authors declare that they have no competing financial interests.

Bu enjeksiyon tekniği teşvik ve sivrisinek melanizm tepkisini incelemek için yararlıdır. Örneğin, burada bağışıklık uyaranlar yükün etkisi incelenmiştir. Bu prosedürde kritik adım düzgün sivrisinek aşılamak etmektir. Uçuş kaslara veya sivrisinek kendisine herhangi bir aşırı hasar besleme gelen sivrisinek önleyebilir veya diseksiyonu önce öldürebilir. İkinci önemli bir adım onları öldürmeden onları dışarı vurmak için yeterince uzun buz üzerinde sivris...

Kendi manikür ya da orta bağırsak epiteli 4 kırmayı 3 - Böcekler parazit ve patojenler 1 karşı kendilerini korumak için bağışıklık yanıtları güveniyor. Sivrisinekler, bu tepkiler bakteriler 5, virüs 6, filarial nematod 7 ve sıtma parazitleri 1,8,9 karşı etkilidir. 12 - sivrisinekler, önemli bir immün yanıt melanin 10 yabancı parçacıkların kapsülleme olduğunu. 12 - Bu kapsülleme midgut veya sistem 10 dolaşan hemolimf meydana gelebilir. Bu melanizm yanıtı ön-fenoloksidaz sonucu 10 kaskad olduğu - 12 ve parazit ölümüne veya fagositoz yol açabilir. Hemocytes hücrelerin sayısı sınırlıdır yetişkin sivrisinek, olarak, melanizm Plasmodium parazitler veya filarial nematod 7 karşı gibi bir humoral tepki vardır.Diğer bazı böcekler, bu doğrudan 7 melanize için parazitin etrafında toplanıp hemocytes hücredir. Ayrıca melanin da yumurta üretimi ve manikür yaralar 7 şifa gibi birçok fizyolojik süreç için gereklidir. 18 - immün yanıtın uyarılması çeşitli tarım ve halk sağlığı modeli sistemlerinde 13 böcek bağışıklık incelemek için bir araç olarak kullanılır. 16,19 - konak-parazit etkileşimleri 14 çalışmak için Anopheles içinde gambiae sivrisinek, Afrika&#39;da sıtma büyük vektör kullanılır. Bu teknikler kendi örüntü tanıma reseptörleri (PRR) 2 ile parazit tespit böceklerin kapasitesine dayanmaktadır. Sivrisinekler ayrıca patojen ilişkili moleküler desenleri (PAMPS) olarak biyoloji ile müdahale diğer molekülleri tespit veya kolajen ve nükleik asitlerin serbest bırakılması nedeniyle kendi hasarlı hücreleri tespit edebilir. Sivrisinek bağışıklık hücresi23 - Bu hemocytes olarak s Tespit 20 kullanılmaktadır. Ana bağışıklık sinyal yolları vardır IMD, Toll, JAK / STAT 24 ve ribonükleik asit enterferans (RNAi) 25,26. Toll ve IMD yollar Hem melanizm yanıtını etkileyen ve pro-fenoloksidaz ile etkileşim 10 kaskad - 12. melanizm yanıtını uyarmak için kullanılan standart bir araç göğüs boşluğunun hemolimf içine küçük bir boncuk ile bir sivrisinek aşılama olduğunu. Melanin kapsülleme derecesi daha sonra sivrisinek diseksiyon ile boncuk aldıktan sonra 19 ölçülebilir. Birçok çalışmada, tek bir kordon sivrisinek 15,16,27 başına enjekte edilmiştir, ancak daha fazla boncuk enjekte melanizm yanıtı 19 sınırlarını incelemek için mümkündür. Bu boncuklar sivrisinek fizyolojisi rahatsızlık sınırlamak için bir enjeksiyon çözeltisi (serum fizyolojik) kullanılarak enjekte edilir vesivrisinek 15,16,27 kurutulması. Bir boya boncuk seçimi kolaylaştırmak için bu çözeltiye ilave edilir. Bu boncuk 15,16,27 almak için kullanılan disseksiyon çözeltisi için aynıdır. Patojenik olmayan uyaranlara böcekleri aşılamak avantajı immün yanıt üzerinde doğrudan etkisi odaklanmak yeteneğidir. 31 ya da bağışıklık kaçırma - 31 - 34 sebebiyle parazit patojenite 28 immünsupresyonun 29 hiçbir komplike etkileri vardır. Ayrıca, bu tür uzun ömürlü ya da doğurganlık gibi diğer yaşam öyküsü özellikleri üzerinde uyarıların sonuçları da ele alınabilir. Böylece, evrimsel ekoloji okuyan araştırmacılar bu tür araçları 2,35,36 gerektirebilir. Örneğin, bağışıklık meydan bombus arıları açlık altında kısaltılmış ömre sahiptir. Bağışıklık uyarılara ve dağıtımları Benzer olumsuz etkileri genellikle kısa sonuçlanan farklı omurgasız modellerin gözlenmiştirer ömrü ya da daha az üreme başarısı 13,27,37. Bu tür çalışmalar, değişen ortamlarda 2,4,38 gerçekleştirilebilir. Bağışıklık uyarıcı da İmmünopatolojiye 39,40 doğrudan odaklanan olanlara ilgi çekicidir. Bu protokol, melanizm yanıtı uyarmak ve doğrudan melanin miktarını ölçmek için sivrisinek ile boncukların aşılama dayanır. Bu, farklı deneysel ortamlarda melanizm yanıtı nicel ve nitel çalışma sağlar. Böyle bir araç, bu tür antibakteriyel yanıt olarak diğer bağışıklık tepkilerinin uyarılması için uzatılabilir bakteriler 41 ısı ile öldürdü. Aynı zamanda pek çok ekolojik ortamlarda yapılabilir.

<table><tbody><tr><td>Microcapillary glass tubes GB120TF-10</td><td>science-products.com</td><td>GB120TF-10</td><td>http://www.science-products.com/Products/CatalogG/Glass/Glass.html</td></tr><tr><td>Microcaps Capillary pipette bulb</td><td>Drumond</td><td>1-000-9000</td><td></td></tr><tr><td>negatively charged Sephadex CM C-25 beads</td><td>Sigma-Aldrich, Steinheim, Germany</td><td>C25120 SIGMA</td><td>need few to start</td></tr><tr><td>Methyl green</td><td>Sigma-Aldrich</td><td>323829 ALDRICH</td><td>need few to start</td></tr><tr><td>Software ImageJ</td><td>opensource</td><td></td><td>Version 1.47f7 or later</td></tr></tbody></table>

inoculating anopheles gambiae mosquitoes with beads to induce and measure the melanization immune response

immün yanıtın uyarılması etkinliğini ve bağışıklık mekanizmaları incelemek için omurgasız çalışmalarda ortak bir araçtır. parçacıklar, bağışıklık sistemi tarafından algılanır ve immün efektör üretimini teşvik edecek şekilde Bu uyarım, böceklerin içine patojenik olmayan partiküllerin enjekte dayanır. Biz sivrisinek Anopheles gambiae içinde melanizm tepkisinin uyarılması burada odaklanır. Melanin koyu bir tabaka ile yabancı partiküllerin ve parazitlerin kapsül içinde melanizm yanıt sonuçları. Bu yanıtı uyarmak için, sivrisinek microcapillary cam tüpler kullanılarak göğüs boşluğunda boncuk ile aşılanır. Daha sonra, 24 saat sonra, sivrisinekler boncuk almak için kesilir. tanenin melanization derecesi görüntü analizi yazılımı kullanılarak ölçülür. Boncuk parazitlerin patojenik etkileri yoktur, ya da kapasite kaçınmak veya immün yanıtı bastırmak için. Bu enjeksiyonlar measu bir yoludurBağışıklık etkinlik ve doğurganlık veya uzun ömürlü gibi diğer yaşam öyküsü özellikleri, immün uyarıların etkisiyle yeniden. Tam doğrudan konak-parazit etkileşimleri inceleyerek aynı değildir, ama bağışıklık ve evrimsel ekoloji incelemek için ilginç bir araçtır.

<html> Bazı boncuk diğerlerinden daha az melanin (Şekil 1) ile kaplandı sivrisineklerin aynı şekilde boncuklar melanize tüm yoktu. Gerçekten de, bazı boncuklar (Şekil 1), diğerleri tamamen karanlık oysa, çünkü melanization eksikliği mavi kalmıştır. Melanizm değeri (mavi ve unmelanized boncuğa karşılık), 0 ile 100 (koyu ve ağır melanized boncuğa karşılık gelir) (Şekil 2a ve 2b) arasında bir ...

Watch this Scientific Journal Video about Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response at JoVE.com

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

aşılanması<em&gt; Anofel gambiae</emMelanization Bağışıklık Tepkisi tetikleyebilecek ve Tedbir Boncuk&gt; ile Sivrisinekler

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

inoculating-anopheles-gambiae-mosquitoes-with-beads-to-induce-measure

Research

JoVE Journal

Immunology and Infection

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

7.9K Görüntüleme.  Université de Neuchâtel. Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

Video: Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

Bir, Enjeksiyon. stephensi Embriyolar Sıtma dayanıklı Sivrisinekler oluşturun

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

Biyoloji

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

Sıtma Anopheles Vektörlerinde SiteYe Yönelik φC31 Aracılı Entegrasyon ve Kaset Değişimi

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...

Genetik

Microinjection Method for Anopheles gambiae Embryos

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.

Microinjection Method for Anopheles gambiae Embryos

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

Anopheles gambiae Embriyoları için Mikroenjeksiyon Yöntemi

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Bir<em&gt; İn Vitro</em&gt; HIV Co-enfeksiyon Bağlamında Sıtma için Bağışıklık Yanıtları Ölçüm Modeli

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

İmmünoloji ve Enfeksiyon

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Canlı zayıflatılmış intradermal aşılama sonrasında Fare Deri ve akan lenf düğümü gelen Myeloid Hücre İzolasyonu<em&gt; Plasmodium</em&gt; Sporozoitler

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Manyetik Rezonans Görüntüleme Kullanarak Deneysel Serebral Sıtma Bir Modelinde Ödem Gelişimi ve Mikrovasküler Patolojinin İn Vivo Takibi

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Tıp

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

Anofellerde Plasmodium falciparum Enfeksiyonunun Tespiti için Standart Membran Besleme Testi

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Genetiği Değiştirilmiş Plasmodium berghei Sporozoitlerinin Üretilmesi

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

içine RNAi Tetik Teslimat<em&gt; Anofel gambiae</em&gt; Pupa

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

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