Somos gratos a Takaji Wakita e Bukh Jens para JFH1 e isolados J6CF, respectivamente. Além disso, agradecemos Charles Rice para Hein-7.5 células e do anticorpo 9E10, Steven Foung para o anticorpo específico CBH-E2-23, e todos os membros do Departamento de Virologia Experimental, Twincore para sugestões e discussões.

<ol>
	<li>Zhu, Q., Guo, J. T., Seeger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+hepatitis+C+virus+subgenomes+in+nonhepatic+epithelial+and+mouse+hepatoma+cells.">Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells.</a> J. Virol. 77, 9204-9210 (2003).</li><li>Kato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonhepatic+cell+lines+HeLa+and+293+support+efficient+replication+of+the+hepatitis+C+virus+genotype+2a+subgenomic+replicon.">Nonhepatic cell lines HeLa and 293 support efficient replication of the hepatitis C virus genotype 2a subgenomic replicon.</a> J. Virol. 79, 592-596 (2005).</li><li>Ali, S., Pellerin, C., Lamarre, D., Kukolj, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+subgenomic+replicons+in+the+human+embryonic+kidney+293+cell+line.">Hepatitis C virus subgenomic replicons in the human embryonic kidney 293 cell line.</a> J. Virol. 78, 491-501 (2004).</li><li>Date, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+2a+hepatitis+C+virus+subgenomic+replicon+can+replicate+in+HepG2+and+IMY-N9+cells.">Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells.</a> J. Biol. Chem. 279, 22371-22376 (2004).</li><li>Chang, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+hepatitis+C+virus+(HCV)+RNA+in+mouse+embryonic+fibroblasts:+protein+kinase+R+(PKR)-dependent+and+PKR-independent+mechanisms+for+controlling+HCV+RNA+replication+and+mediating+interferon+activities.">Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities.</a> J. Virol. 80, 7364-7374 (2006).</li><li>Zhong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+hepatitis+C+virus+infection+in+vitro.">Robust hepatitis C virus infection in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 9294-9299 (2005).</li><li>Lindenbach, B. 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>

TP recebeu honorários de consultoria da Biotest.

Apresentamos dois métodos para induzir a formação heterocário em células em cultura para a análise de dominantes negativos restrições que impedem a replicação do HCV. Utilizando estes procedimentos que excluída a presença de um factor dominante expressa constitutivamente ou induzida por vírus em vários figado humano não-e em linhas celulares de murídeo do fígado. O primeiro ensaio analisa principalmente se os fatores de restrição de impedir a montagem HCV e liberação de progênie infecciosa. Uma vez...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> DMEM </td><td> Invitrogen, Karlsruhe, Alemanha </td><td> 41965-039 </td></tr><tr><td> L-glutamina </td><td> Invitrogen, Karlsruhe, Alemanha </td><td> 25030-024 </td></tr><tr><td> Non-aminoácidos essenciais </td><td> Invitrogen, Karlsruhe, Alemanha </td><td> 11140-035 </td></tr><tr><td> A penicilina / estreptomicina </td><td> Invitrogen, Karlsruhe, Alemanha </td><td> 15140-122 </td></tr><tr><td> Soro fetal de vitelo </td><td> PAA, Cölbe, Alemanha </td><td> A15151 </td></tr><tr><td> α-E2 (CBH23) </td><td> gentilmente cedido por Steven Foung 10 </td><td></td></tr><tr><td> ATP </td><td> Sigma, Steinheim, Alemanha </td><td> A2833-106 </td></tr><tr><td> Glutationa </td><td> Sigma, Steinheim, Alemanha </td><td&gt; G4251-1G </td></tr><tr><td> Blasticidina </td><td> Invivo Gen, San Diego, EUA </td><td> Ant-bl-1 </td></tr><tr><td> G418 (geneticina) </td><td> Invitrogen, Karlsruhe, Alemanha </td><td> 11811-064 </td></tr><tr><td> Polietileno-glicol-1500 </td><td> Roche, Mannheim, Alemanha </td><td> 10783641001 </td></tr><tr><td> Paraformaldeído </td><td> Roth, Karlsruhe, Alemanha </td><td> 0.335,3 </td></tr><tr><td> Triton X-100 </td><td> Roth, Karlsruhe, Alemanha </td><td> 3051,2 </td></tr><tr><td> Soro de cabra </td><td> Sigma, Steinheim, Alemanha </td><td> G9023-5mL </td></tr><tr><td> α-NS5A (9E10) </td><td> Gentilmente cedido por Charles Rice 7 </td><td></td></tr><tr><td> DAPI (4 &#39;, 6&#39;-diamidino-2-fenilindol dicloridrato) </td><td> Invitrogen </td><td> D1306 </td></tr><tr><td> Alexa-Fluor 546 - cabra anti-IgG humana </td><td> Invitrogen, Karlsruhe, Alemanha </td><td> A21089 </td&gt; </tr><tr><td> Alexa-Fluor 488 - cabra anti-IgG de rato </td><td> Invitrogen, Karlsruhe, Alemanha </td><td> A10680 </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen, Karlsruhe, Alemanha </td><td> 11668-019 </td></tr><tr><td> CellTracker CMTMR </td><td> Invitrogen, Karlsruhe, Alemanha </td><td> C2927 </td></tr><tr><td> CellTracker CMFDA </td><td> Invitrogen, Karlsruhe, Alemanha </td><td> C2925 </td></tr><tr><td> Fluoromount </td><td> Sigma, Steinheim, Alemanha </td><td> F4680-25ML </td></tr><tr><td> Todos os outros produtos químicos </td><td> Roth, Karlsruhe, Alemanha </td><td></td></tr><tr><td> De cultura de células materiais </td><td> Sarstedt, Nümbrecht, Alemanha </td><td></td></tr></tbody></table>

two methods of heterokaryon formation to discover hcv restriction factors

Vírus da hepatite C (HCV) é um vírus hepatotrópico com uma gama de hospedeiros restrita aos seres humanos e chimpanzés. Embora HCV replicação de RNA tem sido observada em linhas de células humanas não-hepáticas e de murídeo, a eficiência foi muito baixa e necessários procedimentos de longo prazo de selecção utilizando HCV replicão constrói expressando dominantes antibiótico-seleccionáveis ​​marcadores 1-5. HCV in vitro de pesquisa é, portanto, limitados a linhas celulares de hepatoma humano permissivas para a entrada do vírus e conclusão do ciclo de vida do vírus. Devido a HCVs tropismo espécies estreito, não existe nenhum modelo animal imunocompetente pequena disponível que sustenta o ciclo de replicação do HCV completa 6-8. Replicação ineficiente do HCV em células não-humanos, por exemplo de origem do rato é provavelmente devido à falta de incompatibilidade genética de factores do hospedeiro essenciais de dependência e / ou expressão de factores de restrição. Investigamos se a propagação HCV é suprimida por fa restrição dominantectors em qualquer linhas de células humanas derivadas de tecidos não-hepáticas, ou em linhas de células do fígado de rato. Para este fim, desenvolvemos dois independentes condicionais de complementação trans métodos que dependem somática fusão celular. Em ambos os casos, a conclusão do ciclo de replicação viral só é possível em os heterocariontes. Por conseguinte, a complementação trans-sucesso, o que é determinado por medição da produção de novo de descendência virai infecciosa, indica ausência de restrições dominantes. Especificamente, subgenómico replicões de HCV que transportam um transgene de luciferase foram transfectados para altamente permissivas células de hepatoma humano (Huh-7.5 células). Subsequentemente, estas células foram co-cultivados e fundidos a várias células humanas e de murino expressando HCV núcleo proteínas estruturais, envelope 1 e 2 (E1, E2) e acessório proteínas p7 e NS2. Desde que a fusão celular foi iniciada por meio de tratamento com polietileno-glicol (PEG), a cultura libertado infecciosa virai aaARTIGOS que as células infectadas sem tratamento prévio de uma forma do receptor-dependente. Para avaliar a influência das restrições dominantes sobre o ciclo de vida viral, incluindo entrada de célula, tradução do RNA, replicação e montagem do vírus, que se aproveitou de uma linha celular humana do fígado (Huh-7 células Lunet N 9) que não tem expressão endógena de CD81, um factor de entrada essencial do HCV. Na ausência de CD81 ectopicamente expressa, estas células são essencialmente refractário para infecção por VHC 10. Importante, quando co-cultivadas e fundidos com células que expressam CD81 humano, mas falta pelo menos um outro fator fundamental a entrada de células (ou seja, SR-BI, CLDN1, OCLN), apenas os resultantes heterocários exibir o conjunto completo de fatores de entrada HCV necessárias para a infecção. Portanto, para analisar se factores de restrição dominantes suprimir a conclusão do ciclo de replicação do HCV, que fundiram células Lunet N com várias células de origem humana e do rato que preenchem a crit acima mencionadoeria. Quando a co-cultura de células foram transfectadas com uma proteína do envelope viral altamente fusogénicos mutante do vírus sincicial protótipo (PFV 11) e subsequentemente desafiados com partículas infecciosas HCV (HCVcc), de novo de produção de vírus infeccioso foi observada. Isso indica que o HCV concluído com êxito o seu ciclo de replicação em heterocárions descartando assim a expressão de fatores de restrição dominantes nessas linhas celulares. Estas novas condicionais de complementação-trans métodos serão úteis para pesquisar uma grande painel de linhas celulares e células primárias para a expressão de factores específicos de HCV de restrição dominantes.

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

Descrevemos dois métodos para condicional<em&gt; Trans</em&gt; Complementação de vírus da hepatite C (HCV) de montagem e da realização do ciclo de vida completo viral, que se baseiam na formação de heterocarionte. Estas técnicas são adequados para triagem de linhagens de células que expressam fatores de restrição dominantes, que impedem a produção de progênie HCV infeccioso.

Dois métodos de formação de heterocários Descobrir Fatores de restrição HCV

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 visualizações.  Twincore, Centre for Experimental and Clinical Infection Research. Descrevemos dois métodos para condicional Trans Complementação de vírus da hepatite C (HCV) de montagem e da realização do ciclo de vida completo viral, que se baseiam na formação de heterocarionte. Estas técnicas são adequados para triagem de linhagens de células que expressam fatores de restrição dominantes, que impedem a produção de progênie HCV infeccioso.

Vídeo: 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.

Métodos para avaliar Mediated Beta Morte celular por linfócitos T citotóxicos

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

Imunologia e Infecção

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.

A Enzima de Restrição método baseado Clonagem para avaliar a<em&gt; In vitro</em&gt; Replicação Capacidade de HIV-1 do subtipo C gag-MJ4 quiméricos Vírus

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.

Criação e Otimização de um Setup de Alto Rendimento para o Estudo<em&gt; Staphylococcus epidermidis</em&gt; E<em&gt; Mycobacterium marinum</em&gt; Infecção como um modelo para a Descoberta de Drogas

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.

Métodos para avaliar a citotoxicidade e Imunossupressão de combustível tabaco Preparações Produto

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.

Long Term Intravital Multiphoton Microscopia de imagem de células imunes no saudáveis ​​e doentes do fígado por meio 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, ...

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.

Métodos para aumentar a sensibilidade de alta resolução de derretimento Single Nucleotide Polymorphism Genotipagem em Malária

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.

Aplicando um sistema de expressão induzível para estudar interferência de fatores de virulência bacteriana com Sinalização Intracelular

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).

Métodos para inibir bacteriana Pyomelanin Produção e determinar o correspondente aumento de sensibilidade ao estresse oxidativo

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.

Decifrar e Imagem Patogênese e Cording de<em&gt; Mycobacterium abscessus</em&gt; Em embriões Zebrafish

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.

Métodos para investigar o papel de Regulamentação dos Pequenos RNAs e Ribosomal Ocupação de<em&gt; Plasmodium falciparum</em

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