우리는 Takaji Waki​​ta 각각 JFH1 및 J6CF의 분리를위한 젠스 Bukh에 감사를드립니다. 아울러 우리는 찰스 허-7.5 세포에 대한 쌀과 9E10 항체, E2 특정 항체 CBH-23에 대한 스티븐 였어, 그리고 실험 바이러스학, 유용한 제안 및 토론을위한 Twincore학과의 모든 구성원 감사드립니다.

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+replication+of+hepatitis+C+virus+in+cell+culture.">Complete replication of hepatitis C virus in cell culture.</a> Science. 309, 623-626 (2005).</li><li>Wakita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+infectious+hepatitis+C+virus+in+tissue+culture+from+a+cloned+viral+genome.">Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.</a> Nat. Med. 11, 791-796 (2005).</li><li>Witteveldt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD81+is+dispensable+for+hepatitis+C+virus+cell-to-cell+transmission+in+hepatoma+cells.">CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells.</a> J. Gen. Virol. 90, 48-58 (2009).</li><li>Bitzegeio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+hepatitis+C+virus+to+mouse+CD81+permits+infection+of+mouse+cells+in+the+absence+of+human+entry+factors.">Adaptation of hepatitis C virus to mouse CD81 permits infection of mouse cells in the absence of human entry factors.</a> PLoS Pathog. 6, e1000978 (2010).</li><li>Lindemann, D., Goepfert, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+foamy+virus+envelope+glycoproteins.">The foamy virus envelope glycoproteins.</a> Curr Top Microbiol Immunol. 277, 111-129 (2003).</li><li>Brohm, C. <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+determinants+important+for+hepatitis+C+virus+p7+function+in+morphogenesis+by+using+trans-complementation.">Characterization of determinants important for hepatitis C virus p7 function in morphogenesis by using trans-complementation.</a> J. Virol. 83, 11682-11693 (2009).</li><li>Steinmann, E., Brohm, C., Kallis, S., Bartenschlager, R., Pietschmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+trans-encapsidation+of+hepatitis+C+virus+RNAs+into+infectious+virus-like+particles.">Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles.</a> J. Virol. 82, 7034-7046 (2008).</li><li>Koutsoudakis, G. <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+the+early+steps+of+hepatitis+C+virus+infection+by+using+luciferase+reporter+viruses.">Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses.</a> J. Virol. 80, 5308-5320 (2006).</li><li>Frentzen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Completion+of+hepatitis+C+virus+replication+cycle+in+heterokaryons+excludes+dominant+restrictions+in+human+non-liver+and+mouse+liver+cell+lines.">Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.</a> PLoS Pathog. 7, e1002029 (2011).</li><li>Lindemann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+particle-associated+glycoprotein+signal+peptide+essential+for+virus+maturation+and+infectivity.">A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity.</a> J. Virol. 75, 5762-5771 (2001).</li><li>Blight, K. J., McKeating, J. A., Rice, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+permissive+cell+lines+for+subgenomic+and+genomic+hepatitis+C+virus+RNA+replication.">Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication.</a> J. Virol. 76, 13001-13014 (2002).</li></ol>

TP는 Biotest에서 컨설팅 수수료를 받았다.

우리는 HCV 복제를 배제 지배적인 부정적인 제한 사항 분석을 위해 교양 세포 heterokaryon 형성을 유발하는 두 가지 방법을 제시. 이 절차를 사용하여 우리는 다양한 인간 이외의 간장 및 murine 간 세포 라인의 지배 constitutively 표현이나 바이러스 유발 요인의 존재를 제외. 제한 요인 전염성 자손의 HCV 조립 및 릴리스를 방지하는 경우 첫 번째 분석은 주로 분석합니다. 이 경우에는 포장 세포가 HCV replico...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> DMEM </td><td> Invitrogen, 카를 스루에, 독일 </td><td> 41965-039 </td></tr><tr><td> L-글루타민 </td><td> Invitrogen, 카를 스루에, 독일 </td><td> 25030-024 </td></tr><tr><td> 비 필수 아미노산 </td><td> Invitrogen, 카를 스루에, 독일 </td><td> 11140-035 </td></tr><tr><td> 페니실린 / 스트렙토 마이신 </td><td> Invitrogen, 카를 스루에, 독일 </td><td> 15140-122 </td></tr><tr><td> 태아 송아지 혈청 </td><td> PAA, Cölbe, 독일 </td><td> A15151 </td></tr><tr><td> α-E2 (CBH23) </td><td> 친절 스티븐 였어 10 제공 </td><td></td></tr><tr><td> ATP </td><td> 시그마, Steinheim, 독일 </td><td> A2833-106 </td></tr><tr><td> 글루타티온 </td><td> 시그마, Steinheim, 독일 </td><td&gt; G4251-1G </td></tr><tr><td> Blasticidin </td><td> Invivo 세대, 샌디에고, 미국 </td><td> 개미-BL-1 </td></tr><tr><td> G418 (geneticin) </td><td> Invitrogen, 카를 스루에, 독일 </td><td> 11811-064 </td></tr><tr><td> 폴리에틸렌 - 글리콜-1500 </td><td> 로슈, 만하임, 독일 </td><td> 10783641001 </td></tr><tr><td> Paraformaldehyde </td><td> 로스, 카를 스루에, 독일 </td><td> 0335.3 </td></tr><tr><td> 트리톤 X-100 </td><td> 로스, 카를 스루에, 독일 </td><td> 3051.2 </td></tr><tr><td> 염소 혈청 </td><td> 시그마, Steinheim, 독일 </td><td> G9023-5mL </td></tr><tr><td> α-NS5A (9E10) </td><td> 친절 찰스 쌀 7에 의해 제공 </td><td></td></tr><tr><td> DAPI (4 &#39;, 6&#39;-diamidino-2-phenylindole dihydrochloride) </td><td> Invitrogen </td><td> D1306 </td></tr><tr><td> 알렉사 - 형석 546 - 염소 안티 - 인간 IgG </td><td> Invitrogen, 카를 스루에, 독일 </td><td> A21089 </tD&gt; </tr><tr><td> 알렉사 - 형석 488 - 염소 안티 - 마우스 IgG </td><td> Invitrogen, 카를 스루에, 독일 </td><td> A10680 </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen, 카를 스루에, 독일 </td><td> 11668-019 </td></tr><tr><td> CellTracker CMTMR </td><td> Invitrogen, 카를 스루에, 독일 </td><td> C2927 </td></tr><tr><td> CellTracker CMFDA </td><td> Invitrogen, 카를 스루에, 독일 </td><td> C2925 </td></tr><tr><td> Fluoromount </td><td> 시그마, Steinheim, 독일 </td><td> F4680-25ML </td></tr><tr><td> 기타 모든 화학 물질 </td><td> 로스, 카를 스루에, 독일 </td><td></td></tr><tr><td> 세포 배양 자료 </td><td> Sarstedt, Nümbrecht, 독일 </td><td></td></tr></tbody></table>

two methods of heterokaryon formation to discover hcv restriction factors

C 형 간염 바이러스 (HCV)는 인간과 침팬지에 제한된 호스트 범위 hepatotropic 바이러스입니다. HCV RNA의 복제 인간이 아닌 간장과 murine 세포 라인에서 관찰되었지만, 효율이 매우 낮은이었고, HCV replicon을 사용하여 필요한 장기적인 선정 절차가 지배적인 항생제가 선택할 수있는 마커 1-5 구성하고 표현. 시험 관내 연구에서 HCV 따라서 바이러스 생활주기의 바이러스 항목과 완성을위한 허용 인간 간암 세포 라인으로 제한됩니다. 좁은 종의 tropism을 HCVs으로 인해 전체 HCV 복제주기에게 6-8 격려하고 사용할 수있는 면역 적격 작은 동물 모델이 없습니다. 마우스 원산지 예 비 인간의 세포에서 HCV의 비효율적인 복제 가능성 필수 호스트 종속 요소 및 / 또는 제한 요인의 표현의 유전자 호환성 부족으로 인한 것입니다. 우리는 HCV 전파가 지배적인 제한 FA에 의해 억압 받고 있는지 여부를 조사비 간장 조직이나 마우스 간 세포 라인에서 파생된 인간의 세포 라인 중의 ctors. 이를 위해, 우리는 체세포 융합에 의존하는 두 개의 독립적인 조건부 횡단 complementation 방법을 개발했습니다. 두 경우 모두, 바이러스 복제 사이클의 완성은 heterokaryons에서만 가능합니다. 따라서 전염성 바이러스 자손의 드 노보 생산을 측정하여 결정됩니다 성공 횡단 complementation은, 지배적인 규제의 부재를 나타냅니다. 특히, 루시페라제 transgene를 들고 subgenomic HCV replicons가 적극 허용 인간 간암 세포 (어 - 7.5 세포)로 transfected되었다. 그 후,이 세포들은 공동 배양해와 HCV 구조 단백질 코어, 봉투 1과 2 (E1, E2) 및 액세서리 단백질 P7과 NS2를 표현하는 다양한 인간과 murine 세포에 융합되었다. 세포 융합은 폴리에틸렌 글리콜 - (PEG)와 치료에 의해 시작되었다고 제공, 문화가 전염성 바이러스 PA를 출시수용체에 의존 방식으로 순진 세포를 감염 rticles. 셀 입력, RNA 번역, 복제 및 바이러스 조립을 포함한 완전한 바이러스 생활주기에 지배적인 제한의 영향을 평가하기 위해, CD81의 내생적인 표현이 부족 인간 간 세포주 (허-7 Lunet N 전지 9)을 이용했다 HCV의 필수 항목 계수. ectopically 표현 CD81의 부재에서 이러한 세포는 HCV 감염 10 ~ 근본적 내화물입니다. 중요한 것은 공동 배양해과 인간 CD81하지만 부족 적어도 또 다른 중요한 세포 엔트리 계수 (예 : SR-BI, CLDN1, OCLN)를 표현하는 세포와 융합 경우에만 결과 heterokaryons 감염에 대한 필요한 HCV 항목 요소의 전체 집합을 표시합니다. 따라서 지배적인 제한 요인 HCV 복제주기의 완성을 억제하는 경우에는 분석, 우리는 위에서 언급한 crit를 충족 인간 및 마우스 유래의 다양한 세포로 Lunet N 개의 세포를 융합eria. 공동 교양 세포 프로토 타입 거품 바이러스의 돌연변이 높은 fusogenic 바이러스성 봉투 단백질 (PFV 11)로 transfected하고 이후 감염 HCV 입자 (HCVcc)와 도전을 때, 전염성 바이러스의 드 노보 제작이 관찰되었다. 이것은 HCV가 성공적 따라서 이들 세포 라인에있는 지배적인 제한 요인의 표현을 배제 heterokaryons 년에 복제주기를 완료했음을 나타냅니다. 이 소설 조건부 횡단 complementation 방법 HCV 특정 지배적인 제한 요인의 표현을위한 셀 라인 및 기본 세포의 대형 패널을 구분할 도움이 될 것입니다.

Watch this Scientific Journal Video about Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors at JoVE.com

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

우리는 조건부를위한 두 가지 방법을 설명<em&gt; 트랜스</emC 형 간염 바이러스 (HCV) 조립 및 heterokaryon 형성에 의존하는 완전한 바이러스 생활주기의 완성&gt;-complementation. 이 기술은 감염 HCV 자손의 생산을 배제 지배적인 제한 요소를 표현하는 세포 라인을위한 화면으로 적합합니다.

Heterokaryon 형성의 두 가지 방법 HCV 제한 요소를 발견하기

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

two-methods-heterokaryon-formation-to-discover-hcv-restriction

Research

JoVE Journal

Immunology and Infection

11.5K 조회수.  Twincore, Centre for Experimental and Clinical Infection Research. 우리는 조건부를위한 두 가지 방법을 설명 트랜스-complementation. 이 기술은 감염 HCV 자손의 생산을 배제 지배적인 제한 요소를 표현하는 세포 라인을위한 화면으로 적합합니다.

비디오: Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

세포 독성 T Lymphocytes에 의해 베타 세포 죽음의 중재를 평가하는 방법

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

연구

면역학 및 감염병학

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

제한 효소 기반의 복제 방법을 평가하기 위해<em&gt; 시험관</em&gt; HIV-1 서브 타입 C 개그 - MJ4 키메라 바이러스의 복제 능력

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

설립과 연구에 높은 처리량 설정 최적화<em&gt; 표피 포도 구균</em&gt;와<em&gt; Mycobacterium의 박테</em&gt; 감염 신약 개발의 모델로

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-&#947;, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-&#947;, TNF-&#945;, and MIP-1&#945; were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

방법은 가연성 담배 제품 준비의 세포 독성 및 면역 억제를 평가하기

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

CXCR6.Gfp 기자 마우스를 사용하여 건강하고 병에 걸린 간에서 면역 세포의 장기 생체 내에 다중 광자 현미경 이미징

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

방법은 말라리아에 단일 염기 다형성 유전자형 녹는 고해상도의 감도를 증가

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

유도 발현 시스템을 적용하면 세포 내 신호와 세균 독성 요인의 간섭을 연구하기 위해

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

방법은 세균 Pyomelanin 생산을 억제하고 산화 스트레스에 감도에 대응 증가를 확인하는 방법

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

영상 병인과의 Cording를 해독하고<em&gt; 미코 농양</em&gt; Zebrafish의 배아에서

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

방법은 작은 RNA를과의 리보솜 점유 규제 역할을 조사하기 위해<em&gt; 말라리아 원충</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...