저자는 유럽위원회 (European Commission) 일곱 번째 프레임 워크 프로그램 (PIOF-GA-2009-255466)와 아일랜드 보건 연구위원회 (HRA_POR/2010/138)에서이 원고에 관련된 지원을 인정하고 싶습니다. 럭스 태그 E. 대장균 박사 코르 막 Gahan, 대학 대학 코르크에서 일종의 선물이었다.

1. 종양 유도 <ol><li> 일상적인 종양 유도를 들어, 혈청이없는 배지 200 μl에 떠있는 셀의 최소 tumorigenic 선량은 무료로 감염의 측면 6~8주 세 여성 Balb / C 또는 athymic MF1-nu/nu 마우스로 (SC) 피하 주입 된 N 21 게이지 주사기 바늘을 사용하여 = 6 (할렌, 옥스퍼드 셔, 영국) (1 X 10 6 4T1 세포). 혈구와 Trypan 파랑 염료 제외 (Gibco)를 사용 시각적 개수에 의해 결정으로 접종에 사용되는 세포의 생존 능력은보다 큰 95%했다. </li><li> 종양 설립 후, 종양은 성장하고 발전 할 수 있었고, 2 회 모니터링했다. 종양 볼륨이 종양의 가장 긴 직경이며, B는 직경을에 가장 긴 직경 수직 공식 V = (AB 2) Π / 6에 따라 계산되었습니다. </li></ol> 2. 세균 준비 <ol><li> 이 protoco에 사용 된 세균의 변형난 E.했다 대장균 K-12 MG1655, 비 단백질 독소 표현 세균에 의해 감지 할 수 있도록 그 luxABCDE 인코딩 플라스미드을 품고, 스트레인을 BLI. E. 통합 luxABCDE를 포함하는 대장균 MG1655은 37 aerobically 성장했습니다 ° LB 매체에서 C (시그마 - 알드리치, 아일랜드) 300 μg / ML 에리스로 마이신 (엠)와 보충. MG1655의 발광 생물 파생는 플라스미드 p16S 룩스 제정 P 도움말 luxABCDE 오페론 1 포함 된을 사용하여 만들어졌습니다. </li><li> 마우스로 관리를위한 준비, 문화는 중간 로그 단계 (600 nm의에서 광학 밀도)에 200 rpm으로 흔드는에서 37 ° C에서 LB 매체에 incubated되었습니다. 박테리아는 (6,000 × g로 5 분)을 원심 분리하여 수확 PBS (시그마)로 세척하고, PBS 1 희석 된 × 10 7 식민지 형성 단위 (CFU) / IV 관리를위한 ML, 또는 gavage 1 × 10 10. </li></ol> 3. 세균 Administra기 <ol><li> 종양 볼륨에서 약 100mm 3에 도달하면 마우스가 무작위로 실험 그룹으로 나누어되었다. 정맥 주사 관리를 들어, 절제된 마우스 각 28G 주사기 바늘을 사용하여 측면 꼬리 정맥에 직접 주입, 100 μl에 10 6 세포를 받았다. 각 inoculum의 가능한 개수는 회고 도금에 의해 결정되었다. </li><li> GIT의 식민지 연구를 들어, 10 9 박테리아 세포는 구두 세 번 일에 gavage에 마우스 당 100 μl으로 시행되었다. 기존의 공생 박테리아 수준은 이전의 구두 gavage 1 개시 7 일 동안 마우스 식수 5 MG / ML 스트렙토 마이신의 추가에 의해 공급 이전에 감소했다. </li></ol> 4. BioLuminescence 영상 <ol><li> 생체 BLI 영상의 2D는 IVIS100 (캘리퍼)를 사용하여 수행되었습니다. 정의 시간 포인트 후 세균 관리에서, 쥐 캘리퍼스의 XGI-8 가스 마취를 사용하여 anesthetized했다3 % Isofluorane, 그리고 전체 - 신체 이미지 분석 시스템은 고감도에서 2-5 분 동안 IVIS 100 시스템에 수행되었다. </li><li> 3D 영상의 경우, anesthetized 마우스는 등쪽의 이미징을위한 광학 이미징 시스템 (IVIS 스펙트럼, 캘리퍼스)의 내부에 마우스 이미징 셔틀에 배치되었습니다. 3D 광학 재건을위한 세균 루시 페라 신호의 이미지를 취득하려면 500-580 nm의에 이르기까지 방출 필터 파장은 소음 비율에 신호를 극대화 할 수 필터 당 3-4 분 빈 16 수집 시간으로 사용되었습니다. 이 이미지 수집 과정의 일환으로, 구조화 된 빛이 이미지는 높이 맵을 정의하기 위해 얻은 것입니다. 이지도는 입력 확산 광 단층 촬영 영상 (DLIT) 음수가 아닌 최소 제곱 최적화 2를 사용 3D 광학 이미지를 형성하는 데 사용 된 reconstructions 알고리즘했다. </li><li> 이미지 분석 : 관심 지역을 파악하고 생활 이미지 소프트웨어 (캘리퍼)를 사용하여 정량 하였다. </li></ol> 5. 대표하는결과 본 연구에서는 비 병원성 세균 공생 E.coli K-12 MG1655 luxABCDE 오페론을 표현하는 IV는 SC 4T1의 이종 이식 종양을 간직한 생쥐에 투여했다. 세균 룩스 신호는 마우스의 종양에 구체적으로 발견 된 후 IV-행정 (그림 2). 샘플 마우스에서 박테리아의 문화 복구 가능한 박테리아 숫자와 감지 빛의 양 (그림 3) 사이의 선형 관계의 존재를 확인합니다. GIT에 구두 - 관리 공생 박테리아의 생체 이미징에서 또한 3D BLI를 사용하여 달성된다. <img src="/files/ftp_upload/4318/4318fig1.jpg" alt="그림 1" fo:content-width="4in" fo:src="/files/ftp_upload/4318/4318fig1highres.jpg"/> 1 그림. 프로토콜 타임 라인. 피하 종양은 마우스에서 유도하고, 박테리아는 종양 개발 (100mm 3)에 따라 관리됩니다. 라이브 쥐 BLI 인스턴트 메신저 아르여러 시간 지점 게시물 세균 관리 (화살표가 일반적인 시간을 표시)에 세. <img src="/files/ftp_upload/4318/4318fig2.jpg" alt="그림 2" /> 그림 2. E.의 관리 종양 베어링 마우스에 대장균 MG1655 luxABCDE. 피하 4T1 종양은 MF1 뉴 / 뉴 마우스와 E.에서 유도 된 대장균 MG1655 luxABCDE는 종양 개발에 실시. 각 동물은 측면 꼬리 정맥에 직접 주사 10 6 세포를 받았습니다. 마우스는 샘플 희생 마우스 종양에서 가능한 박테리아의 후속 복구 (CFU)로 연구 기간 동안 4 개의 시간대 포인트 (검은 점 Z-축 및 이미지) (막대 그래프)에서 이미징되었다. 세균 번호와 특히 종양에 시간 (대표 마우스 시점에 따라 그림)을 통해 관찰 된 플라스미드 유전자 발현에 향상시킬 수 있습니다. <a href="http://www.jove.com/files/ftp_upload/4318/4318fig2large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> .<img src="/files/ftp_upload/4318/4318fig3.jpg" alt="그림 3" /> 그림 3. Intratumoral 세균 번호와 Bioluminescence 관계. 가능한 박테리아는 종양의는 다양한 시간 포인트 이후 IV 관리에서 BLI에 다음 종양에서 예 생체 박테리아 문화가 열거되었다. 생체 bioluminesce 단위를 기준으로 세균의 숫자 값 (CFU)를 로그하는 것은 그래프로 나타납니다. 세균 수와 세균 bioluminescence 신호 사이의 강력한 상관 관계가 관찰 R 2 = 0.9717 1. <img src="/files/ftp_upload/4318/4318fig4.jpg" alt="그림 4" /> 4 그림. 손쥐 위장관의 3D IVIS 이미지 E.하여 식민지 대장균 MG1655은. 마우스의 GIT는 E.의 10 9 CFU의 구두 행정부에 의해 개척했고 세 번 일 대장균. 식민지 마우스의 3D 단층 촬영에서 샘플 격리 된 이미지가 표시됩니다.3D 이미지는 해부학 등록을 제공하는 해골의 디지털 마우스 아틀라스를 보여줍니다. E.는 대장균 MG1655 bioluminescence는 낮은에서 녹색으로 표시하고, 높은 수준의 보라색입니다.

<ol>
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관심 없음 충돌이 선언 없습니다.

유전자 치료의 맥락에서 환자에게 치료 유전자 전달을위한 생화학 제제의 사용은 큰 약속 3-5 나타났습니다. 바이러스처럼, 박테리아의 본래 생물학적 특성은 특히 암의 맥락에서, 셀이나 조직에 효율적으로 DNA 전달을 허용합니다. 그것은 박테리아가의 자연스럽게 할 수있는 것으로 표시되었습니다 체계적으로 중 외부 로컬 복제 높은 수준의 (비 침습적 종) 또는 종양 세포 (병원균)에 발?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 4T1 세포 라인 </td><td> ATCC </td><td> CRL-2539 </td><td> 자발적으로 발생하는 BALB / C 유방 종양에서 파생 Syngeneic 유방암 모델 </td></tr><tr><td> DMEM </td><td> 시그마 - 알드리치 </td><td> D6429 </td><td> Dulbecco의 수정 된 이글의 매체 </td></tr><tr><td> PBS </td><td> 시그마 - 알드리치 </td><td> D8537 </td><td> 인산염은 식염 버퍼 </td></tr><tr><td> Xenogen IVIS </td><td> 캘리퍼스 생명 과학 </td><td></td><td> 2D 이미지에 대한 IVIS 100, 3D에 대한 IVIS 스펙트럼. </td></tr><tr><td> Luria 국물 밀러 (LB) </td><td> 시그마 - 알드리치 </td><td> L2542 </td><td> E.의 성장 매체 대장균 </td></TR&gt; <tr><td> 에리스로 마이신 </td><td> 시그마 - 알드리치 </td><td> E5389 </td><td> 항생 물질 </td></tr><tr><td> 스트렙토 마이신 </td><td> 시그마 - 알드리치 </td><td> S9137 </td><td> 항생 물질 </td></tr><tr><td> MF1nu/nu 마우스 </td><td> 할렌 (UK) </td><td> 069 (뉴) / 070 (뉴 / +) </td><td> Hsd : Athymic 누드 - Foxn1nu </td></tr><tr><td> Balb / C 마우스 </td><td> 할렌 (UK) </td><td> 066 </td><td> Haplotype : H-2 D </td></tr><tr><td> Gavage 바늘 </td><td> 수의사 기술 솔루션 (UK) </td><td> DE009 </td><td> 22G X 38mm 바로 gavage 바늘 </td></tr><tr><td> IV 주입 주사기 </td><td> BD BioSciences </td><td> 309309-1 ML </td><td> 28 G X ½ 인치 마이크로 미세 IV 바늘과 인슐린 주사기. </td></tr><tr><td> 종양 접종에 대한 주사기 </td><td> 브라운 </td><td> 9161376V </td><td> Omnifix 26 G X &amp;frac12; 인치 바늘 </td></tr></tbody></table>

bioluminescent bacterial imaging in vivo

이 동영상은 암 유전자 및 세포 치료 박테리아의 사용에 중점을두고, 라이브 쥐 세균 매매의 연구 전신 bioluminesce 이미징 (BLI)의 사용에 대해 설명합니다. 박테리아가 조직 관리에 따라 종양 내에 우선적으로 성장하는 자연 능력을 보유하고 암 치료를위한 벡터의 매력적인 클래스를 제시한다. 박테리아는 박테리아와 동시에 종양 사이트의 럭스 유전자 카세트 허가 BLI 검색을 표현하는 설계. 시간이 지남에 따라 종양 내에 위치와 박테리아의 수준은 쉽게 검토 할 수 있습니다, 2, 3 차원 시각화. 방법은 박테리아 종과 종양 이종 이식 유형의 다양한 적용됩니다. 이 문서는 피하 종양 베어링 마우스에서 발광 생물 박테리아의 분석을위한 프로토콜을 설명합니다. BLI하여 위장관 (GIT)의 공생 박테리아의 시각화도 설명되어 있습니다. 이 강력하고 저렴한, 실시간 이미징 전략 represenTS 암 특정 유전자 치료의 연구, 그리고 전염성 질병의 맥락에서 생체 내 박테리아의 연구를위한 이상적인 방법입니다. 이 동영상은 럭스 태그 E.을 공부에 대한 절차를 설명 IVIS 시스템 공간과 측두엽 판독 달성 활용 BLI을 보여주는 라이브 쥐 대장균.

Watch this Scientific Journal Video about Bioluminescent Bacterial Imaging In Vivo at JoVE.com

Bioluminescent Bacterial Imaging In Vivo

이 문서의 관리에 대해 설명합니다<em&gt; 룩스 태그</em&gt; 마우스에 세균 및 이후<em&gt; 생체 내</emIVIS의 bioluminescence 이미징을 사용하여&gt; 분석.

발광 생물 세균 이미징<em&gt; 생체 내</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Research

JoVE Journal

Immunology and Infection

Bioluminescent Bacterial Imaging In Vivo

15.1K 조회수.  University College Cork. 이 문서의 관리에 대해 설명합니다 룩스 태그 마우스에 세균 및 이후 생체 내 분석.

비디오: Bioluminescent Bacterial Imaging In Vivo

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

라이브 호스트에 트렌스 제닉 Leishmania의 기생충의 생체내 이미징에서

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

연구

면역학 및 감염병학

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

백혈구 호밍 및 마이 그 레이션의 비침습 영상 생체내에</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

전체 리프 이미징을 사용하여 박테리아 세포를 라이브 Stomatal 응답을 평가

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

사이 Synergism을 맡은 이후로 발광 생물 이미징을 사용하여 연쇄상 구균 pneumoniae</em유아 마우스에서>와 인플루엔자 바이러스

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

카테고리의 감지 세균성 병원균에 대한 &#39;발광 생물&#39;리포터 파지

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes Scapularis 진드기의 타액, 침샘 및 Hemolymph 컬렉션

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

생체 내 이미징 방법에 급성 및 만성 염증을 구별하는

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

세대와의 멀티 표현형 높은 콘텐츠 상영<em&gt; Coxiella burnetii의</em&gt; 트랜스포존 돌연변이

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

생체 외 및 생체 내 모델에서 혈관 벽에서 흐름 조건에 세균 부착을 연구하기 위해

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.