이 작품은 건강 연구의 캐나다 연구소 (MSH - 95368, 청소 - 89,773 및 CZ하는 걸레 - 106623)과 혁신에 대한 캐나다 재단에 의해 지원되었다. SCM은 위장 박사 교제의 CIHR / 캐나다 협회의받는 사람입니다. CZ는 CIHR 새로운 탐정이다.

1. Trichuris muris 달걀을 전파 <ol><li> (또는 유전자 취약 마우스 Trichuris muris 달걀의 새로운 배치를 생성하려면, 20-30 immunodeficient 생쥐를 감염 () 예 NOD.Cg - Prkdc scid Il2rg tm1Wjl / SzJ (NSG) 또는 129S6/SvEvTac-Rag2 tm1Fwa (RAG2 - - /) 예. 경구 gavage으로 약 300 Trichuris muris 달걀과 6~8주 된 AKR / J). </li><li> 32-35일 후 CO 2 질식하여 쥐를 희생. </li><li> 마우스의 복부 측면을 폭로하고 70 %의 에탄올과 함께 복부가 젖었어. </li><li> 사용 포셉은 복부 피부를 파악하고 가위를 사용하여 작은 절개를합니다. 복부 내용을 노출 피부를 잘라. </li><li> 이게 맹장과 근위 대장을 식별하고 그들을 제거합니다. </li><li> 500 U / ML 페니실린, 500 μg / ML 스트렙토 마이신 (P / S)와 Dulbecco의 수정 이글 중간 (DMEM)을 포함 10cm 배양 접시에있는 맹장과 근위 대장를 놓습니다. 루멘과 벌레를 노출 열어이게 맹장을 잘라. 인산염의 비커에 포셉 플레이스이게 맹장을 사용하면 호수 (PBS)를 버퍼 및 배설물을 제거 부드럽게 발라줍니다. </li><li> 페트리 접시에이게 맹장을 반환하고 부드럽게 벌레를 꺼내와 DMEM + P / S. 5 MLS를 포함하는 6 자 접시의 우물에 넣어 부드러운 곡선 포셉 (Roboz RS - 5047)을 사용하여 </li><li> 생쥐의 모든에 대해이 과정을 반복합니다. </li><li> 37 습도와 부화를 유지하기 위해 젖은 종이 타월 ° 4 시간을위한 C와 유리 섬유 복합 컨테이너 6 잘 접시를 넣어. </li><li> 4 시간은 부드러운 곡선 집게를 사용하여 웜을 제거하고 5mL DMEM을 포함하는 6 자 접시 중 2 개의 새 우물로 그들을 분할 후 + P / S. 15 ML 팔콘 튜브의 원래 잘 장소의 내용을 수확. 4 시간의 항원 (일반 단백질 수율 20 생쥐 10-20 MG 아르 PBS 투석 후 세포 restimulation에 사용할 수있는)과 resuspend로 3,000 RPM 및 제거하고 예비 뜨는에서 튜브를 회전 달걀이 포함된 autoclaved 수돗물에 펠릿합니다. </li><li> ° C 야간 37 위로 여섯 잘 접시를 넣어. 제거 및 웜 처분. 팔콘 튜브 5 분 3,000 rpm으로 회전에 우물의 내용을 수확. 하룻밤 항원 (일반 단백질 수율 20 생쥐 10-20 MG 아르 PBS 투석 후, Trichuris 자세 특정 항체 isotype의 ELISAs에 사용할 수있는)과 resuspend 달걀 함유 autoclaved 수돗물에서 펠렛을 같이 뜨는하고 예약을 제거합니다. </li><li> 4 시간과 야간 계란을 결합하고 75cm 2 플라스크에 전송 후 어떤 남은 웜을 제거하는 70μm 스트레이너를 통해 계란을 필터링합니다. 실온 (RT) 6 주 호일로 덮여 어두운 장소에 술병 보관하십시오. </li><li> autoclaved 수돗물에 달걀을 씻어 50 μl 50 가능한 계란에서 resuspend. 이렇게하려면 ~ 50ml 멸균 수돗물에 5 분 및 resuspend을 위해 3000 RPM의 계란을 원심 분리기. 유리 슬라이드 50 μl 계란과 장소를 제거합니다. 40x 배율을 사용하면 50 μl에 가능한 embryonated 달걀의 개수를 계산합니다. 50 μl 50 가능한 알을주고 달걀을 희석. 가능한 계란의 이미지에 대한 Kopper와 맨스필드 16 연구를 참조하시기 바랍니다. </li></ol> 2. Trichuris muris 달걀 (200 알 / 마우스)의 실험 생쥐의 급성 감염 <ol><li> 철저하게 resuspend 계란은 생쥐의 수를 치료 정도로 볼륨을 제거 X 2 (예 : 10 쥐 200 μl x를 받아 10 쥐 X 2 = 4 알을 MLS)와 14 ML 스냅 캡 튜브에서 그들을 놓으십시오. </li><li> 섞어 14 ML 스냅 캡 튜브를 반전. 적절한 크기의 먹이를 바늘 1 ML 주사기 (우리는 18 게이지 1 1 / 2 &quot;바늘을 사용 25g 무게 쥐)에 계란 200 μl를 가져와. 한손으로 아저씨에서 마우스와 다른 손을 사용은 경구 gavage으로 달걀을 관리 . </li><li> 21일 만요. </li></ol> 3. 실험 수확 <ol><li> 일 21 CO 2를 사용하여 쥐를 희생. </li><li> 심장 관통하여 생쥐에서 혈액을 수집이 아닌 heparinized 주사기에 4에서 저장 ° C. 그 다음날은 -20 ° C.에서 혈청 및 동결을 분리 혈청은 엘리사에 의해 Trichuris 특정 IgGs 및 IGE의 검출에 사용될 수 있습니다. </li><li> 마우스의 복부 측면을 폭로하고 70 %의 에탄올과 함께 복부가 젖었어. </li><li> 사용 포셉은 복부 피부를 파악하고 가위를 사용하여 작은 절개를합니다. 피부 및 복부 내용을 폭로 기본 멤브레인 컷. </li><li> 소장, 맹장 및 결장을 확인합니다. 포셉과 맹장를 잡고 복강 밖으로 당기십시오. 포셉 또 한 쌍의와 mesenteric 림프절 (mLNs)를 노출 오른쪽으로 작은 창자를 밀어. 미디어 2 MLS를 포함한 15 ML 팔콘의 내장과 장소로 절단되지 않도록주의되는 mLNs를 제거합니다. 원하는 경우 이러한 엘리사와 세포내 F로 시토킨 분석 72 시간을위한 처리 및 체외 (1 μg / ML αCD3/CD28 또는 50μg/ml 4h Trichuris 자세 24 잘 접시 1 ML에 4x10 6 / ML)에서 restimulated 수 있습니다낮은 cytometry. </li><li> 이게 맹장과 결장을 잘라 버릴거야. </li><li> 포셉을 사용하여 2 ML Axygen 튜브의 말초 콜론과 장소에서 배설물로 알약을 밖으로 밀어. -20 ° C.에 프리즈 방패 알약은 분자 β (RELM - β) resistin와 같은 표현을위한 서양 얼룩으로 분석하실 수 있습니다. </li><li> 이게 맹장 (~ 0.5 cm)의 끝부분을 제거합니다. 배설물이 집게와 맹장의 끝부분을 잡고 제거하고 PBS는 배양 접시에있는 3 ML의 주사기와 22 게이지 바늘을 사용하여 함께 플러시하려면 다음과 같이하십시오. 5 ML 팰컨 스냅 캡 튜브에 신선한 4 % paraformaldehyde에 놓습니다. 4 스토어 ° C. 이들은 파라핀 - 임베디드, sectioned 및 histological 분석을위한 슬라이드에 장착할 수 있습니다. </li><li> RNA에 대한 근위 대장 (~ 1cm)의 조각을 제거합니다. 500 μl RNAlater 2 ML Axygen 튜브에 샘플을 넣어. 4 스토어 ° C. 이 샘플은 qPCR에 의해 mRNA의 표현에 대해 분석하실 수 있습니다. </li><li> -20 ° C.에 표시 페트리 접시와 동결에이게 맹장의 나머지 부분을 플레이스 </li></ol> 4. 이게 맹장에서 Trichuris muris 벌레의 열거 <ol><li> ~ 5 ML 물이 들어있는 배양 접시에있는 냉장고와 장소에서이게 맹장을 제거합니다. </li><li> 가위를 사용하여 루멘을 폭로이게 맹장을 잘라 냈어. </li><li> 포셉과 맹장을 보유하고 적극적으로 대변의 대부분을 제거하는 맹장를 흔들. </li><li> 물을 포함하고 부드럽게 IECs에게 기본 조직을 긁어 부드러운 곡선 집게를 사용하여 다른 배양 접시에이게 맹장을 전송. </li><li> 물을 포함하는 새로운 배양 접시에이게 맹장을 전송하고 적극적으로 작은 조각으로 조직을 추출, 집게로 남아있는 조직을 다쳤어요. </li><li> 배양 접시의 모든 3에있는 벌레의 모든 통계를 해부 현미경을 사용합니다. 당신이 벌레 같은 벌레를 위해 접시에서 제거 찾아서 두 번 계산되지 않습니다. 웜이 손상된 경우 (예 : 반으로 깨진) 정확한 숫자는 파악에만 두꺼운 또는 얇은 끝이 계산합니다. </li></ol> 5. 대표 결과 Trichuris muris 감염 모델 연구가 기생충이 부담하고 체계적인 면역 반응뿐만 아니라, histologically 검사 말초이게 맹장 내의 염증 반응을 계량 할 수 있습니다. 수확이게 맹장의 루멘가 노출되면, Trichuris 벌레는 해부 현미경 (그림 1A - B)를 사용하여 열거 수 있습니다. Trichuris에 면책의 매개 변수는 항체 모성 동물 IgG1로 전환 isotype (Th2 타입 면역과 관련된), 그리고 민감한 동물 IgG2a (Th1 타입의 면역과 관련된) (그림 1C)을 포함합니다. 잔 세포 특정 단백질 resistin 같은 분자 β (RELM - β)의 표현은 Trichuris의 통관과 관련된과 서양 얼룩 분석 (그림 2)에 의해 감지입니다. 이게 맹장의 말초 부분은 고블렛 셀 반응 및 감염에 대한 응답으로 장의 염증의 범위 (그림 3)을 평가하기 위해주기적인 산성 - 쉬프 (PAS) 물들일 수 있습니다. <img src="/files/ftp_upload/2774/2774fig1.jpg" alt="그림 1"/> 그림 1. Trichuris muris에 면책의 부량. (A) 저항 (B6) 쥐가 하루 21 이후의 감염에 의해 Trichuris의 muris을 추방. 반대로, 민감한 (AKR / J) 생쥐는 만성 기생충 ​​설립에 결과 Trichuris에 감염된다. 각 막대는 평균 ± 그룹당 4-8의 SEM 동물을 나타냅니다. (B) 웜은 해부 현미경을 사용하여 계산됩니다. 톱 패널 격리 벌레를 보여줍니다, 하단 패널은 IECs과 대소변의 존재에 벌레를 보여줍니다. (C) Trichuris 특정 IgG2a의 titres는 배설 Trichuris의 항원 (O / N 자세, 5 μg / 코팅 접시에 희석 혈청 엘리사 (1시 2분 희석, 희석 1시 20분에서 시작하고 1:2560에 종료)에 의해 결정됩니다 ML). 민감한 AKR / J 마우스는 모성 B6 생쥐보다 Trichuris 특정 혈청 IgG2a의 특히 높은 수준의 수 있습니다. <img src="/files/ftp_upload/2774/2774fig2.jpg" alt="그림 2"/> 그림 2. Trichuris muris의 통관은 잔 세포 특정 단백질 RELM - β 7 표현과 연결됩니다. RELM - β 생산 Trichuris - 감염 방지 (B6)에서 배설물 여럿 발견하지만, 서양 얼룩 분석하여 쥐 (AKR / J) 감염될 수는 없습니다. 각 레인, (N) = 나이브 (TM) = 21 일 Trichuris 감염된 쥐 한 동물의 대표입니다. <img src="/files/ftp_upload/2774/2774fig3.jpg" alt="그림 3"/> 그림 3. 말초이게 맹장 다음 Trichuris 감염의 Histological 시험. 말초이게 맹장의 5-7 μm의 섹션은 PAS 물들다. 고블렛 셀 증식 및 점액 생산 방지 (B6)에서 분명하지만, Trichuris 감염 다음 쥐 (AKR / J) 취약하지 않습니다.

<ol>
	<li>Cliffe, L. J., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Trichuris+muris+system:+a+paradigm+of+resistance+and+susceptibility+to+intestinal+nematode+infection.">The Trichuris muris system: a paradigm of resistance and susceptibility to intestinal nematode infection.</a> Adv. Parasitol. 57, 255-307 (2004).</li><li>Else, K. J., Finkelman, F. D., Maliszewski, C. R., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine-mediated+regulation+of+chronic+intestinal+helminth+infection.">Cytokine-mediated regulation of chronic intestinal helminth infection.</a> J. Exp. Med. 179, 347-351 (1994).</li><li>Bancroft, A. J., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th1+and+Th2+cells+and+immunity+to+intestinal+helminths.">Th1 and Th2 cells and immunity to intestinal helminths.</a> Chem. Immunol. 71, 192-208 (1998).</li><li>Bancroft, A. J., McKenzie, A. N., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+IL-13+in+resistance+to+intestinal+nematode+infection.">A critical role for IL-13 in resistance to intestinal nematode infection.</a> J. Immunol. 160, 3453-3461 (1998).</li><li>Helmby, H., Takeda, K., Akira, S., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+(IL)-18+promotes+the+development+of+chronic+gastrointestinal+helminth+infection+by+downregulating+IL-13.">Interleukin (IL)-18 promotes the development of chronic gastrointestinal helminth infection by downregulating IL-13.</a> J. Exp. Med. 194, 355-364 (2001).</li><li>Owyang, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+25+regulates+type+2+cytokine-dependent+immunity+and+limits+chronic+inflammation+in+the+gastrointestinal+tract.">Interleukin 25 regulates type 2 cytokine-dependent immunity and limits chronic inflammation in the gastrointestinal tract.</a> J. Exp. Med. 203, 843-849 (2006).</li><li>Artis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RELM&#946;/FIZZ2+is+a+goblet+cell-specific+immune-effector+molecule+in+the+gastrointestinal+tract.">RELM&#946;/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract.</a> Proc. Natl. Acad. Sci. U. S. A. 101, 13596-13600 (2004).</li><li>Datta, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+genes+in+intestinal+tissue+that+are+regulated+after+infection+with+an+intestinal+nematode+parasite.">Identification of novel genes in intestinal tissue that are regulated after infection with an intestinal nematode parasite.</a> Infect. Immun. 73, 4025-4033 (2005).</li><li>Cliffe, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+intestinal+epithelial+cell+turnover:+a+new+mechanism+of+parasite+expulsion.">Accelerated intestinal epithelial cell turnover: a new mechanism of parasite expulsion.</a> Science. 308, 1463-1465 (2005).</li><li>Artis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+weapons+in+the+war+on+worms:+identification+of+putative+mechanisms+of+immune-mediated+expulsion+of+gastrointestinal+nematodes.">New weapons in the war on worms: identification of putative mechanisms of immune-mediated expulsion of gastrointestinal nematodes.</a> Int. J. Parasitol. 36, 723-733 (2006).</li><li>Finkelman, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+regulation+of+host+defense+against+parasitic+gastrointestinal+nematodes:+lessons+from+studies+with+rodent+models.">Cytokine regulation of host defense against parasitic gastrointestinal nematodes: lessons from studies with rodent models.</a> Annu. Rev. Immunol. 15, 505-533 (1997).</li><li>Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+helminth+infection:+immunopathology+and+resistance+during+intestinal+nematode+infection.">Enteric helminth infection: immunopathology and resistance during intestinal nematode infection.</a> Chem. Immunol. 66, 41-61 (1997).</li><li>Khan, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+intestinal+muscle+contraction+by+interleukin-9+(IL-9)+or+IL-9+neutralization:+correlation+with+worm+expulsion+in+murine+nematode+infections.">Modulation of intestinal muscle contraction by interleukin-9 (IL-9) or IL-9 neutralization: correlation with worm expulsion in murine nematode infections.</a> Infect. Immun. 71, 2430-2438 (2003).</li><li>Khan, W. I., Blennerhasset, P., Ma, C., Matthaei, K. I., Collins, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stat6+dependent+goblet+cell+hyperplasia+during+intestinal+nematode+infection.">Stat6 dependent goblet cell hyperplasia during intestinal nematode infection.</a> Parasite Immunol. 23, 39-42 (2001).</li><li>Akiho, H., Blennerhassett, P., Deng, Y., Collins, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+IL-4,+IL-13,+and+STAT6+in+inflammation-induced+hypercontractility+of+murine+smooth+muscle+cells.">Role of IL-4, IL-13, and STAT6 in inflammation-induced hypercontractility of murine smooth muscle cells.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 282, 226-2232 (2002).</li><li>Kopper, J. J., Mansfield, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+improved+methods+for+delivery+of+Trichuris+muris+to+the+laboratory+mouse.">Development of improved methods for delivery of Trichuris muris to the laboratory mouse.</a> Parasitol. Res. 107, 1103-1113 (2010).</li><li>Bancroft, A. J., Else, K. J., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-level+infection+with+Trichuris+muris+significantly+affects+the+polarization+of+the+CD4+response.">Low-level infection with Trichuris muris significantly affects the polarization of the CD4 response.</a> Eur. J. Immunol. 24, 3113-3118 (1994).</li></ol>

동물 실험은 브리티시 컬럼비아 대학교에서 정한 지침과 규정에 따라 수행되었다.

이 프로토콜은 자세한 조사가 필요에 따라 수정할 수있는 표준 고용량 급성 Trichuris muris 감염합니다. 예를 들어, 마우스가 희생될 수 있으며, 조직은 다른 일 수확. 생쥐가 성공적으로 가득 벌레 부담을 설립한다는 결정하기 위해 그들은 모두 쥐가 약 200 벌레의 부담을 가지고해야하는 시점에서, 하루 14 희생하실 수 있습니다. 생쥐도 감지하는 웜이 성숙에 도달하고 그 삶의 기간 동안 호스?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 동물 먹이 니들스 (18 × 1 ½ &quot;) </td><td> 포퍼 </td><td> 7912 </td><td></td></tr><tr><td> 부드러운 곡선 집게 </td><td> Roboz </td><td> RS - 5047 </td><td></td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11,965 </td><td></td></tr><tr><td> NOD.Cg - Prkdc scid Il2r gtm1Wjl / SzJ (NSG) </td><td> 잭슨 연구소 </td><td> 005557 </td><td> 이들은 우리가 사용 생쥐되며, 단, immunodeficient 마우스 또는 민감한 긴장이 작동합니다. </td></tr><tr><td> RNAlater </td><td> Qiagen </td><td> 76,104 </td><td></td></tr><tr><td> 2 ML 튜브 </td><td> Axygen </td><td> MCT - 200 - C </td><td></td></tr><tr><td> 15 ML 튜브 </td><td> 매 </td><td> 352096 </td><td></td></tr><tr><td> 6 웰 플레이트 </td><td> 매 </td><td> 353046 </td><td></td></tr><tr><td> Paraformaldehyde </td><td> 전자 현미경 과학 </td><td> 15,710 </td><td></td></tr><tr><td> α - RELMβ 항체 </td><td> PeproTech 부동산 </td><td> 0694270Rb </td><td></td></tr></tbody></table>

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

Trichuris muris은 생쥐의 자연 병원체이며 인간과 가축 1 감염 Trichuris의 수종에 생물학과 antigenically 비슷합니다. Infective 달걀이 구두 gavage, 말초 소장에서 부화에 의해 부여됩니다, 라인 -이게 맹장과 근위 결장과 성숙시 crypts이 웜은 환경 1에 달걀을 풀어 그 창자 상피 세포 (IECs)를 침공. 이 모델은 요소를 검토하는 강력한 도구입니다 컨트롤 CD4 + T는 헬퍼 (TH) 세포 활성화뿐 아니라 창자 상피의 변화. 이러한 C57BL / 6 BALB / C와 같은 저항 근친 직전에 발생하는 면역 반응이 Th2 편광 크린 시토킨 (IL - 4, IL - 5, IL - 13)과 Th1 - 관련 크린 시토킨 동안 (IL 웜의 퇴학이 특징입니다 -12, IL - 18, IFN - γ) 유전자 취약 AKR / J 마우스 2-6에서 만성 감염을 추진하고 있습니다. Th2 크린 시토킨 신속 IECs의 매출, 고블렛 셀 차별화, 모집 및 상피 투과성 및 웜 퇴학 7-15에 연루되어 모든있는 평활근의 수축에 변화를 포함한 창자 microenvironment의 생리 변화를 추진하고 있습니다. 여기 자세히 후속 실험에서 사용할 수 Trichuris muris 달걀을 전파를위한 프로토콜을. 우리는 또한 이후의 감염 분석을위한 제안 샘플 실험의 수확을 제공합니다. 전반적으로,이 프로토콜은 위장관뿐만 아니라 IECs의 면역 이펙터 기능에 목 proclivity에 관한 질문을 해결하기 위해 사용할 수있는 Trichuris muris 마우스 감염 모델을 수행할 수있는 기본 도구와 연구자를 제공할 것입니다.

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

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

Trichuris muris 감염은 모성 쥐가 보호 Th2 반응을 생성하고 민감한 쥐가 병적인 Th1 반응을 생성하는 Th2 면역의 창자 모델입니다.

Trichuris muris 감염 : 굿에서 2 형 내성 및 염증의 모델

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

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

Research

JoVE Journal

Immunology and Infection

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

16.2K 조회수.  University of British Columbia. Trichuris muris 감염은 모성 쥐가 보호 Th2 반응을 생성하고 민감한 쥐가 병적인 Th1 반응을 생성하는 Th2 면역의 창자 모델입니다.

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

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

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

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

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

의 캡슐 혈청 형<em&gt; 폐렴 구균</em&gt; 라텍스 응집에 의해

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

연구

면역학 및 감염병학

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

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

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

절연, 특성화 및 잇몸 면역 세포 네트워크의 기능 시험

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

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

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

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

호중구의 분리 및 분석 치료제에 림프종 세포 감도에서 자신의 역할을 확인하는 방법

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

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

적용 형광 공명 에너지 전달 (FRET)을하여 이펙터 전좌 효율을 검사합니다<em&gt; Coxiella burnetii의</em&gt; siRNA를 음소거시

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

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

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

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

마우스의 실험 세균성 폐렴의 유도 및 표현형에 대한 비 침습적 및 기술적 비 집약적 인 방법

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

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

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

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

정밀 절단 라이브 폐 수지상 세포를 시각화하기 위해 마우스 폐 슬라이스

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

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

형광 기반의 뉴 라미니다 아제 억제 분석은 항 바이러스제의 뉴 라미니다 아제 억제제 클래스에 인플루엔자 바이러스의 감수성을 평가하기 위해

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

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

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

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

선 동적인 장 질병 마우스 모델에서 창 자 염증 단백질 폐지를 신호 하는 맛의 효과

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

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

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

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

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

바이러스 성 감염 및 초파리 Melanogaster 상호 작용 호스트-바이러스의 분석의 설립

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

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

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

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

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

갤러리아 mellonella Waxworm 감염 모델 전파 칸디 다에 대 한

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