우리는 Trichopria drosophilae, 유전자 변형 뜬 종자를위한 교수 토니의 IP, 그리고 안티 Spätzle 항체에 대해 교수 칼 하시 모토위한 교수 토드 Schlenke에 감사를드립니다. 이 프레 젠 테이션에 대한 그들의 공헌에 대한 연구소의 현재와 과거 회원을 감사드립니다. NIH (S06 GM08168, 대두 41399-009, 그리고 G12-RR03060), USDA (NRI / USDA CSREES 2006-03817하고 2009-35302-05277) 및 PSC-CUNY에서이 작품은 다음과 같은 보조금에 의해 지원되었다.

<ol>
	<li>Schlenke, T. A., Morales, J., Govind, S., Clark, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+infection+strategies+in+generalist+and+specialist+wasp+parasitoids+of+Drosophila+melanogaster.">Contrasting infection strategies in generalist and specialist wasp parasitoids of Drosophila melanogaster.</a> PLoS Pathog. 3, 1486-1501 (2007).</li><li>Chiu, H., Morales, J., Govind, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+immuno-electron+microscopy+localization+of+p40,+a+protein+component+of+immunosuppressive+virus-like+particles+from+Leptopilina+heterotoma,+a+virulent+parasitoid+wasp+of+Drosophila.">Identification and immuno-electron microscopy localization of p40, a protein component of immunosuppressive virus-like particles from Leptopilina heterotoma, a virulent parasitoid wasp of Drosophila.</a> J. Gen. Virol. 87, 461-470 (2006).</li><li>Gueguen, G., Rajwani, R., Paddibhatla, I., Morales, J., Govind, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VLPs+of+Leptopilina+boulardi+share+biogenesis+and+overall+stellate+morphology+with+VLPs+of+the+heterotoma+clade.">VLPs of Leptopilina boulardi share biogenesis and overall stellate morphology with VLPs of the heterotoma clade.</a> Virus Res. 160, 159-165 (2011).</li><li>Paddibhatla, I., Lee, M. J., Kalamarz, M. E., Ferrarese, R., Govind, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+sumoylation+in+systemic+inflammation+and+immune+homeostasis+in+Drosophila+larvae.">Role for sumoylation in systemic inflammation and immune homeostasis in Drosophila larvae.</a> PLoS Pathog. 6, e1001234 (2010).</li><li>Sorrentino, R. P., Carton, Y., Govind, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+immune+response+to+parasite+infection+in+the+Drosophila+lymph+gland+is+developmentally+regulated.">Cellular immune response to parasite infection in the Drosophila lymph gland is developmentally regulated.</a> Dev. Biol. , 243-265 (2002).</li><li>Sorrentino, R. P., Melk, J. P., Govind, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+contributions+of+dorsal+group+and+JAK-Stat92E+pathway+genes+to+larval+hemocyte+concentration+and+the+egg+encapsulation+response+in+Drosophila.">Genetic analysis of contributions of dorsal group and JAK-Stat92E pathway genes to larval hemocyte concentration and the egg encapsulation response in Drosophila.</a> Genetics. 166, 1343-1356 (2004).</li><li>Jung, S. H., Evans, C. J., Uemura, C., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+lymph+gland+as+a+developmental+model+of+hematopoiesis.">The Drosophila lymph gland as a developmental model of hematopoiesis.</a> Development. 132, 2521-2533 (2005).</li><li>Krzemien, J., Crozatier, M., Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+the+Drosophila+larval+hematopoietic+organ,+hemocyte+homeostasis+and+the+dedicated+cellular+immune+response+to+parasitism.">Ontogeny of the Drosophila larval hematopoietic organ, hemocyte homeostasis and the dedicated cellular immune response to parasitism.</a> Int. J. Dev. Biol. 54, 1117-1125 (2010).</li><li>Martinez-Agosto, J. A., Mikkola, H. K., Hartenstein, V., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hematopoietic+stem+cell+and+its+niche:+a+comparative+view.">The hematopoietic stem cell and its niche: a comparative view.</a> Genes Dev. 21, 3044-3060 (2007).</li><li>Lemaitre, B., Hoffmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+defense+of+Drosophila+melanogaster.">The host defense of Drosophila melanogaster.</a> Annu. Rev. Immunol. 25, 697-743 (2007).</li><li>Schlegel, A., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+"lower"+organisms:+what+worms,+flies,+and+zebrafish+can+teach+us+about+human+energy+metabolism.">Lessons from "lower" organisms: what worms, flies, and zebrafish can teach us about human energy metabolism.</a> PLoS Genet. 3, e199 (2007).</li><li>Ferrandon, D., Jung, A. C., Criqui, M., Lemaitre, B., Uttenweiler-Joseph, S., Michaut, L., Reichhart, J., Hoffmann, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+drosomycin-GFP+reporter+transgene+reveals+a+local+immune+response+in+Drosophila+that+is+not+dependent+on+the+Toll+pathway.">A drosomycin-GFP reporter transgene reveals a local immune response in Drosophila that is not dependent on the Toll pathway.</a> EMBO J. 17, 1217-1227 (1998).</li><li>Bruckner, K., Kockel, L., Duchek, P., Luque, C. M., Rorth, P., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PDGF/VEGF+receptor+controls+blood+cell+survival+in+Drosophila.">The PDGF/VEGF receptor controls blood cell survival in Drosophila.</a> Dev. Cell. 7, 73-84 (2004).</li><li>Tokusumi, T., Shoue, D. A., Tokusumi, Y., Stoller, J. R., Schulz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+hemocyte-specific+enhancer-reporter+transgenes+for+the+analysis+of+hematopoiesis+in+Drosophila.">New hemocyte-specific enhancer-reporter transgenes for the analysis of hematopoiesis in Drosophila.</a> Genesis. 47, 771-774 (2009).</li><li>Tokusumi, T., Sorrentino, R. P., Russell, M., Ferrarese, R., Govind, S., Schulz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+lamellocyte+transcriptional+enhancer+located+within+the+misshapen+gene+of+Drosophila+melanogaster.">Characterization of a lamellocyte transcriptional enhancer located within the misshapen gene of Drosophila melanogaster.</a> PLoS One. 4, e6429 (2009).</li><li>Morales, J., Chiu, H., Oo, T., Plaza, R., Hoskins, S., Govind, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+structure,+and+immune-suppressive+effects+of+virus-like+particles+of+a+Drosophila+parasitoid,+Leptopilina+victoriae.">Biogenesis, structure, and immune-suppressive effects of virus-like particles of a Drosophila parasitoid, Leptopilina victoriae.</a> J. Insect Physiol. 51, 181-195 (2005).</li><li>Bettencourt, R., Asha, H., Dearolf, C., Ip, Y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolymph-dependent+and+-independent+responses+in+Drosophila+immune+tissue.">Hemolymph-dependent and -independent responses in Drosophila immune tissue.</a> J. Cell Biochem. 92, 849-863 (2004).</li><li>Melk, J. P., Govind, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+analysis+of+Ganaspis+xanthopoda,+a+larval+parasitoid+of+Drosophila+melanogaster.">Developmental analysis of Ganaspis xanthopoda, a larval parasitoid of Drosophila melanogaster.</a> J. Exp. Biol. 202, 1885-1896 (1999).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

Drosophila의 기생 말벌에 대한 관심은 전체 genomes를 해독하기위한 분자 기술은 효율적이고 비용 효과가로 서징있다. 그러나 그들의 매우 잘 공부 호스트에 상대적으로, 말벌 생물학의 많은 매혹적인 측면이 모호한 상태로 유지됩니다. 이러한 범위, 면역 억제, superparasitism, 그리고 행동을 호스팅과 관련된 문제가 포함됩니다. 이 프레 젠 테이션의 초점은 파리의 면역 조직에서 감염의 효과를 ?...

<table border="1"><tbody><tr><td> 자료 </td><td> 유형 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 곤충의 문화 유지를위한 자료 </td><td></td><td></td><td></td></tr><tr><td> 누룩 </td><td> 활성 건조 </td><td> 피셔 과학 </td><td> S802453 </td></tr><tr><td> 음식 들아 </td><td> 옥수수 식사, 설탕 </td><td></td><td> 표준 제조법 </td></tr><tr><td> 꿀 </td><td> 클로버 </td><td> 네덜란드 골드 </td><td></td></tr><tr><td> 튜브 </td><td> 폴리 프로필렌 셸 튜브 (좁은) </td><td> 피셔 과학 </td><td> AS514 </td></tr><tr><td> 유리병에 폐쇄 </td><td> 코튼 플러그 </td><td> 피셔 과학 </td><td> AS212 </td></tr><tr><td> 유리병에 폐쇄 </td><td> 버즈 플러그 </td><tD&gt; Genesee 과학 </td><td> AS273 </td></tr><tr><td> 냉장 인큐베이터 </td><td> 정밀 815 </td><td> 써모 과학 </td><td> 3721 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 샘플 준비를위한 자료 </td><td></td><td></td><td></td></tr><tr><td> CO 2 탱크 </td><td> 뼈 건조 학년 </td><td> TW 스미스 </td><td> UN1013 </td></tr><tr><td> 주걱 </td><td> 마이크로 주걱 (14 ㎝) </td><td> 피셔 과학 </td><td> 21-401-15 </td></tr><tr><td> Pyrex 명소 테스트 플레이트 </td><td> 9 - 잘 해부 판 85mm X 100mm </td><td> 토마스 과학 </td><td> 7812G17 </td></tr><tr><td> 파스퇴르 Pipettes </td><td> 소다 석회 </td><td> J &amp; H Berge </td><td> 71-5200-05 </td></tr><tr><td> 집게 </td><td> 스타일 # 5 </td><td> 시그마 </td><td> T-4662 </td></tr><tr><td> 에탄올 </td><td> 190 증거의 USP </td><td> 피셔 과학 </td><td> 04-355-221 </td></tr><tr><td> 포름 알데히드 </td><td> 37% W / w </td><td> 피셔 과학 </td><td> F79-1 </td></tr><tr><td> 이차 항체 Cy3 AffiniPure 당나귀 안티 토끼 IgG (H + L) </td><td> 1시 50분 여진 546 nm의; 배출 565 nm의 </td><td> 잭슨 Immuno 리서치 연구소 주식 회사 </td><td> 711-165-152 </td></tr><tr><td> Antifade (N-프로필 gallate) </td><td> 1X PBS에 50 % 글리세롤 4 μg / ML </td><td> MP의 Biomedicals </td><td> 10274790 </td></tr><tr><td> 글리세린 </td><td></td><td> 피셔 과학 </td><td> G33-1 </td></tr><tr><td> Hoechst 33258 </td><td> 0.2 μg / ML 여기 352 nm의; 배출 461 nm의 </td><td> 분자 프로브 </td><td> H-1398 </td></tr><tr><td> Rhodamine phalloidin </td><td> 200 개가 / ML (6.6μm의) 여진 540 nm의; 배출 565 nm의 </td><td> 분자 프로브 </td><td> R415 </td></tr><tr><td> 알렉사 형석 488 phalloidin </td><td> 300 단위 / ML 여기 495 nm의; 배출 518 nm의 </td><td> 분자 프로브 </td><td> A12379 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Disposables </td><td></td><td></td><td></td></tr><tr><td> 병을 씻으십시오 </td><td> Fisherbrand </td><td> 피셔 과학 </td><td> 03-409-22A </td></tr><tr><td> Kimwipes </td><td> 킴벌리 클라크 </td><td> 피셔 과학 </td><td> 06-666A </td></tr><tr><td> 종이 타월 </td><td> 하나 플라이 C-공간 </td><td> 깃 </td><td> 901-7CFTB2400 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 현미경 사용법 </td><td> 및nbsp; </td><td></td><td></td></tr><tr><td> Leica의 stereomicroscope </td><td> MZFLIII </td><td> 제국 이미징 시스템즈 </td><td> 10446208 </td></tr><tr><td> 자이스 혈구 Stereomicroscope </td><td> Stemi 1000 또는 2000-C </td><td> 칼 자이스 혈구 </td><td> 000000-1006-126 </td></tr><tr><td> 광원 - LED </td><td> 거위 목처럼 생긴 것의 조명기 </td><td> 피셔 과학 </td><td> 12563501 </td></tr><tr><td> 무대 </td><td> 플레이트로 전송 라이트 박스 </td><td> 칼 자이스 혈구 </td><td> 455,137,000 </td></tr><tr><td> 자이스 혈구 레이저 공촛점 현미경 스캐닝 </td><td> LSM 510 </td><td> 칼 자이스 혈구 </td><td></td></tr><tr><td> 자이스 혈구 복합 현미경 </td><td> 직립 Axioplan이 </td><td> 칼 자이스 혈구 </td><td></td></tr></tbody></table><table border="1"><tbody><tr><td> 말벌의 변종 </td><td> 스트레인 날기의 </td></tr><tr><td> Leptopilina victoriae 16 </td><td> yw </td></tr><tr><td> Leptopilina boulardi 17 1 </td><td> UAS-GFP-지느러미 17 </td></tr><tr><td> Leptopilina heterotoma이 </td><td> SerpentHemoGal4 13 </td></tr><tr><td> Leptopilina heterotoma 14 1 </td><td> MSNF9-moCherry 14 </td></tr><tr><td> Trichopria drosophilae </td><td> MSNF-GFP 15 </td></tr><tr><td> Ganaspis xanthopoda 18 </td><td> yw 뱀의-Gal4 UAS GFP-Dorsal/Basc 4 </td></tr><tr><td></td><td> yw; Drosomycin-GFP/CyO Y + 12 </td></tr></tbody></table>

an introduction to parasitic wasps of drosophila and the antiparasite immune response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

Watch this Scientific Journal Video about An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response at JoVE.com

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (기생) 말벌을 포함한 여러 곤충들의 천적의 주요 클래스를 구성<em&gt; Drosophila melanogaster</em&gt;. 우리는 이러한 기생충을 전파하는 기법을 소개합니다<em&gt; Drosophila</em&gt; SPP. 과 면역 조직에 미치는 영향을 분석하는 방법을 보여줍니다<em&gt; Drosophila</em&gt; 애벌레.

의 기생충 말벌에 대한 소개<em&gt; Drosophila</em&gt; 그리고 Antiparasite 면역 반응

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

an-introduction-to-parasitic-wasps-drosophila-antiparasite-immune

Research

JoVE Journal

Immunology and Infection

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

18.5K 조회수.  The City College of New York, CUNY. Parasitoid (기생) 말벌을 포함한 여러 곤충들의 천적의 주요 클래스를 구성 Drosophila melanogaster. 우리는 이러한 기생충을 전파하는 기법을 소개합니다 Drosophila SPP. 과 면역 조직에 미치는 영향을 분석하는 방법을 보여줍니다 Drosophila 애벌레.

비디오: An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2 - 광자 현미경을 사용하여 마우스 Thymus의 Intravital 영상

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

연구

면역학 및 감염병학

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

면역 반응과 수면에서의 양적 측정<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

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.

제브라 피쉬의 선천성 면역 건강의 지표로 호흡 버스트 응답의 정량화

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

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

를 사용하여 폐렴 구균 식민지에서 생균제의 효과를 조사<em&gt; 체외</em&gt; 준수의 분석

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

마우스 대 식세포의 타고난 면역 반응을 조사하기 위해 RNA 간섭을 사용하여

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

마우스의 호스트 면역 반응을 평가하기 위해 구인두 기관 내 PAMP 관리 및 폐포의 활용

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

웨스트 나일 바이러스 돌연변이 균주의 감염에에서 MyD88 매개 세포 면역 반응의 생체 외 분석

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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.

의 Zebrafish의 애벌레 모델의 선천성 면역 반응의 비 침습적 영상<em&gt; 연쇄상 구균 iniae</em&gt; 감염

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

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

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is based on the injection of non-pathogenic particles into insects, as the particles will be detected by the immune system and will induce the production of immune effectors. We focus here on the stimulation of the melanization response in the mosquito Anopheles gambiae. The melanization response results in the encapsulation of foreign particles and parasites with a dark layer of melanin. To stimulate this response, mosquitoes are inoculated with beads in the thoracic cavity using microcapillary glass tubes. Then, after 24 hr, the mosquitoes are dissected to retrieve the beads. The degree of melanization of the bead is measured using image analysis software. Beads do not have the pathogenic effects of parasites, or their capacity to evade or suppress the immune response. These injections are a way to measure immune efficacy and the impact of immune stimulations on other life history traits, such as fecundity or longevity. It is not exactly the same as directly studying host-parasite interactions, but it is an interesting tool to study immunity and its evolutionary ecology.

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.

접종<em&gt; 아노 펠 레스 gambiae</emMelanization 면역 반응을 유도하고 측정하는 비즈&gt; 모기

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

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

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

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

Lipopolysaccharide 취급 마우스 간에 면역 반응 동안 백혈구의 2 광자 Intravital 이미징

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