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A Method for Examining Glycans Surface Expression of Yeasts by Flow CytometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Recognition of pathogenic yeasts by host cells is based on components of the yeast cell wall, which are considered part of its virulence attributes. Cell wall glycans play an important role in the continuous interchange that regulates the balance between saprophytism and parasitism and between resistance and infection. Flow cytometry is a useful method for probing surface yeast glycans in order to compare their expression depending on strains and growth conditions. By using different monoclonal or polyclonal antibodies, levels of β- and α-linked mannosides as well as β-glucans can be successfully evaluated ...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Screening of Virulence Traits in Legionella pneumophila and Analysis of the Host Susceptibility to Infection by Using the Dictyostelium Host Model SystemEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The social soil amoeba Dictyostelium discoideum has been established as a host model for several human pathogens including Legionella pneumophila. The complete genome sequence, the genetic tractability, and the phagocytic characteristics of Dictyostelium generate many opportunities for the study of host-pathogen interactions. Important applications of this haploid model organism are (i) the use of Dictyostelium cells as a screening system for bacterial virulence, (ii) the use of Dictyostelium mutant cells to identify genetic host determinants of susceptibility and resistance to infection, and (iii) experiments that allow t...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Characterizing Host Receptor Recognition by Individual Bacterial PathogensEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A critical determinant of host range and specificity relies on the ability of pathogenic bacteria to recognize eukaryotic cell surface molecules via specialized adhesins. The specific adhesin-receptor interaction allows pathogens to tightly bind to their target cells, thereby facilitating the colonization of host tissues. Therefore, the identification and characterization of bacterial adhesins is a major topic in infection biology. This chapter focuses on a rapid and simple method for the analysis of adhesin-receptor interactions that permits the characterization of receptor binding properties at the level of single bacter...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Introduction: Fungal PathogensEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Isolation and Purification of Antigenic Components of CryptococcusEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The encapsulated fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are significant agents of life-threatening infections, particularly in persons with suppressed cell-mediated immunity. This chapter provides detailed methodology for the purification of two of the major antigen fractions of C. neoformans: glucuronoxylomannan (GXM) and mannoprotein (MP). GXM is the primary component of the polysaccharide capsule, which is the major cryptococcal virulence factor. In contrast, MPs have been identified as key antigens that stimulate T-cell responses. Purification of GXM and MP should assist investigators studying...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Surface-Exposed Adherence Molecules of Streptococcus pneumoniaeEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Surface-exposed proteins of pathogenic bacteria are considered as potential virulence factors through their direct contribution to host-pathogen interactions. The specific interaction of bacterial proteins with host proteins often subverts the physiologic function of host-derived proteins, and therefore the bacterial proteins are considered as key players in the infectious process. The direct binding of host proteins is exploited by the pathogens for colonization, host tissue invasion, or immune evasion. Strikingly, surface proteins such as ABC transporters are also implicated in bacterial pathogenesis through their role i...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Phagocytosis of Candida albicans by RNAi-Treated Drosophila S2 CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Phagocytosis is a highly conserved aspect of innate immunity. Drosophila melanogaster has an innate immune system with many similarities to that of mammals and has been used to successfully model many aspects of innate immunity. The recent availability of Ribo Nucleic Acid interference (RNAi) libraries for Drosophila has made it possible to efficiently screen for genes important in aspects of innate immunity. We have screened an RNAi library representing 7216 fly genes conserved among metazoans to identify proteins required for the phagocytosis of the human fungal pathogen Candida albicans.
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Oral Mucosal Cell Response to Candida albicans in Transgenic Mice Expressing HIV-1Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Controlled studies on the immunopathogenesis of mucosal candidiasis in HIV infection have been hampered by the lack of a relevant animal model. We have previously reported that oral Candida infection in CD4C/HIV transgenic mice expressing gene products of HIV-1 in immune cells and developing an AIDS-like disease closely mimics oropharyngeal candidiasis in human HIV infection. The role of defective dendritic cells and CD4+ T cells in impaired induction of protective immunity and in the phenotype of chronic oral carriage of C. albicans can now be investigated under controlled conditions in these transgenic mice.
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Proteomic Profiling of Serologic Response to Candida albicans During Host-Commensal and Host-Pathogen InteractionsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe the typical proteomic strategy to globally and integratively profile these host antibody responses and determine serum antibody signatures. This approach is based on the combination of classic immunoproteomics or serologic proteome analysis (two-dimensional electrophoresis followed by quantitative Western blotting and mass spectrometry) with data mining procedures. This global proteomic stratagem is a useful tool not only for obtaining an overview of different anti-Candida antibodies that are being elicited during the host-fungus interaction and, consequently, of the complex C. albicans immunome (the subset of ...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Models of Oral and Vaginal Candidiasis Based on In Vitro Reconstituted Human Epithelia for the Study of Host-Pathogen InteractionsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This protocol describes the setup, maintenance, and characteristics of models of oral and vaginal candidiasis based on well-established three-dimensional organotypic tissues of human oral and vaginal mucosa. Infection experiments are highly reproducible and can be used for the direct analysis of pathogen/epithelial cell interactions. Using the models, the several stages of infection by wild-type Candida albicans strains, the consequence of gene disruption of putative virulence factors in mutant cells, and the evaluation of the host immune response can be evaluated by histologic, biochemical, and molecular methods. As such,...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Endothelial Cell Stimulation by Candida albicansEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe the use of real-time PCR and enzyme immunoassays to measure the effects of C. albicans on the endothelial cell production of E-selectin and tumor necrosis factor α in vitro.
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Introduction: Host ResponsesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Fungal and Bacterial Killing by NeutrophilsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Neutrophils are professional phagocytes of the innate immune system that are essential to control bacterial and fungal infections. These cells engulf and kill invading microbes. Additionally, activated neutrophils are able to release neutrophil extracellular traps (NETs). These fibers consist of chromatin decorated with antimicrobial proteins to trap and kill microbes. Appropriate quantitative methods are required to understand the nature of interactions of neutrophils with pathogens. Here we present assays to measure killing mediated by phagocytosis, by NETs, by a combination of both, and by granular extract. As examples,...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Modulation of Caspase Activation by Toxoplasma gondiiEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Apoptosis plays crucial roles for the outcome of infection with various infectious agents. The host’s apoptotic program may be modulated after infection in order to combat the pathogen or to restrict the immune response. In addition, distinct microorganisms alter the apoptotic program of the host in order to meet the requirements for their further distribution. The activation of caspases (i.e., cysteine proteases with specificity for aspartic acid residues) preludes the disassembly of the cell in response to apoptosis-inducing stimuli. This depends on the proteolytic cleavage of inactive proforms into catalytically a...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Transcriptome Analyses in the Interaction of Neisseria meningitidis with Mammalian Host CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
As in many other areas of basic and applied biology, research in infectious diseases has been revolutionized by two recent developments in the field of genome biology: first, the sequencing of the human genome as well as that of many pathogen genomes; and second, the development of high-throughput technologies such as microarray technology, proteomics, and metabolomics. Microarray studies enable a deeper understanding of genetic evolution of pathogens and investigation of determinants of pathogenicity on a whole-genome scale. Host studies in turn permit an unprecedented holistic appreciation of the complexities of the host...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Introduction: ParasitesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Axenic In Vitro Cultivation of Echinococcus multilocularis Metacestode Vesicles and the Generationof Primary Cell CulturesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Parasitic helminths are a major cause of disease worldwide, yet the molecular mechanisms of host-helminth interaction and parasite development are only rudimentarily studied. A main reasons for this lack of knowledge are the tremendous experimental difficulties in cultivating parasitic helminths under defined laboratory conditions and obtaining sufficient amounts of parasite material for molecular analyses. For one member of this neglected group of pathogens, the fox-tapeworm Echinococcus multilocularis, we have established and optimized in vitro cultivation systems by which the major part of the parasite’s life cycl...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Culture of Exoerythrocytic Stages of the Malaria Parasites Plasmodium falciparum and Plasmodium vivaxEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The two most prevalent human malaria parasites, Plasmodium falciparum and Plasmodium vivax, cause the majority of malaria-related morbidity and mortality. Compared with our knowledge about the erythrocytic stages, we understand little about the liver exoerythrocytic (EE) stages of the human malaria parasites. Our recent development of a hepatocyte line from normal human liver tissue is crucial for successful culturing of the liver stages of both P. falciparum and P. vivax. This technical advancement should be an important tool for directly studying developmental biology of the EE stages of the human malaria parasites and d...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Identification of the Candida albicans Immunome During Systemic Infection by Mass SpectrometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Over the past two decades, mass spectrometry (MS) has ceased to be a fairly exotic technique banished from the protein scientists’ mind to become a seminal tool for deciphering the information encoded in the genomes of many biological species. Clues to this shift in the modus operandi for characterizing their proteomes stem from the progressive availability of full genome sequences and well-annotated protein databases of many model (micro)organisms, the development both of soft ionization methods for large biomolecules (peptides and proteins) and of innovative instrumentation designs, and the introduction of sophisti...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Real-Time and Semiquantitative RT-PCR Methods to Analyze Gene Expression Patterns During Aspergillus-Host InteractionsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Aspergillus species are infamous for causing several plant and animal diseases that directly (e.g., invasive aspergillosis) or indirectly (e.g., consumption of toxic food supplies) can lead to high rates of morbidity in humans and animals worldwide. Despite progress in molecular information and manipulation of Aspergillus spp., including genome sequence availability and suitable transformation methodologies, efforts to control Aspergillus diseases are still far from satisfactory, due in part to lack of knowledge of fungal virulence attributes. In order to obtain meaningful insights on the disease mechanism(s), it is essent...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Antibody-Based Strategy to Identify Candida albicans Genes Expressed During InfectionsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Investigators have long used antibody-based screening strategies to identify Candida albicans immunogenic proteins and the genes that encode them during infections. With the recent availability of the C. albicans genome sequence and the development of genomic and proteomic technologies, it is now possible to efficiently conduct large-scale screening in standard research labs. C. albicans proteins and genes identified with a variety of screening methods have been implicated as important determinants of candidal virulence and exploited as vaccine and therapeutic targets. In this chapter, we describe methods used in our lab, ...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Experimental Infection of Rodent Mammals for Fungal Virulence TestingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Invasive fungal infections comprise a group of serious and life-threatening diseases affecting immunocompromised patients. Molecular analysis of fungal virulence involves the deletion of genes that are suspected for contributing to fungal pathogenesis. Phenotypic analysis of the generated mutants includes in vivo infection experiments in order to assign a function during fungal disease to a gene of interest.
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

In Vitro Systems for Studying the Interaction of Fungal Pathogens with Primary Cells from the Mammalian Innate Immune SystemEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The incidence of invasive fungal diseases has increased over the past decades, particularly in relation with the increase of immunocompromised patient cohorts (e.g., HIV-infected patients, transplant recipients, immunosuppressed patients with cancer). Opportunistic fungal pathogens such as Candida spp. are most often associated with serious systemic infections. Currently available antifungal drugs are rather unspecific, often with severe side effects. In some cases, their prophylactic use has favored emergence of resistant fungal strains. Major antifungal drugs target the biosynthesis of lipid components of the fungal plas...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Introduction: Bacterial PathogensEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Human Epithelial Model Systems for the Study of Candida infections In Vitro: Part II. Histologic Methods for Studying Fungal InvasionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Although the role of invasion in the virulence of Candida albicans has been demonstrated, the mechanism that governs fungal invasion is not fully understood. Among the tools that exist to fill these gaps in knowledge, in vitro tissue models based on reconstituted human epithelia (RHE) have already been developed. Such models are designed to study more reproducably the fungus-host relationship, as they eliminate the complexity and variability found in vivo. Herein we describe the preparation of these RHE and their application in study of the invasion properties of C. albicans by further histologic processing and microscopic...
Source: Springer protocols feed by Infectious Diseases - December 20, 2008 Category: Infectious Diseases Source Type: info

Detection and Quantification of Serum or Plasma HCV RNA: Mini Review of Commercially Available AssaysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The treatment schedule (combination of compounds, doses, and duration) and the virological follow-up for management of antiviral treatment in patients chronically infected by HCV is now well standardized, but to ensure good monitoring of the treated patients, physicians need rapid, reproducible, and sensitive molecular virological tools with a wide range of detection and quantification of HCV RNA in blood samples. Several assays for detection and/or quantification of HCV RNA are currently commercially available. Here, all these assays are detailed, and a brief description of each step of the assay is provided. They are div...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Structural and Functional Analysis of the HCV p7 ProteinEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present here in vitro assays designed to purify synthetic p7 by RP-HPLC, to investigate its ion-channel properties by means of planar lipid-bilayer assays and patch-clamp recordings after reconstitution into liposomes, and to analyze its structural features by circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD).
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

HCV Replicons: Overview and Basic ProtocolsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Subgenomic replicons have been the first efficient cell-culture system for HCV and still are a valuable tool for studying different aspects of RNA replication. A variety of replicons based on different viral isolates and vector designs have been established. Here, I give a brief overview of viral isolates, applicable host-cell lines, replicon structures, and general considerations regarding replicon experiments, supplemented by basic protocols for in vitro transcription, electroporation, selection of replicon cells, transient replication assays, and northern hybridization.
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Reverse Transcription PCR-Based Sequence Analysis of Hepatitis C Virus Replicon RNAEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Since the advent of efficient cell-culture methods for HCV replication and, more recently, infection, there has been a need to efficiently sequence the viral RNA in these systems. This need is especially urgent in light of the error-prone nature of HCV RNA replication, which leads to a variety of interesting mutations. The adaptation of hepatitis C replicons to cell culture, which greatly increased their replication capacity, and the subsequent identification of viral point mutations responsible for this adaptation are prime examples of the type of phenotype–genotype connection that viral RNA sequencing methods can p...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Studying HCV RNA Synthesis In Vitro with Replication ComplexesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
HCV replication complexes are well-organized protein and lipid structures responsible for HCV RNA replication. The nonstructural protein NS5B, an RNA-dependent RNA polymerase, is the catalytic subunit of these replication complexes. After being isolated from HCV replicon-containing cells as a crude membrane fraction, these replication complexes have been shown to remain active in synthesis of viral RNA under proper assay conditions. Under the improved assay conditions presented here, we recently showed that isolated replication complexes are able to synthesize two species of nascent viral RNA, one double stranded and the o...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Proteomics Study of the Hepatitis C Virus Replication ComplexEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
RNA replication of HCV occurs in the multiprotein complexes associated with the endoplasmic reticular (ER) membranes. The HCV NS3 to NS5B proteins are necessary and sufficient for HCV RNA replication in the cell, but cellular proteins in the HCV replication complex (RC) have not been determined. Several methods have been used to isolate the HCV RC, including crude cell extract preparation, subcellular fractionation, and affinity purification. The components of the HCV RC can be separated by two-dimensional electrophoresis and then determined by proteolytical digestion and mass spectrometry analysis in conjunction with pept...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Investigation of the Hepatitis C Virus Replication ComplexEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe these techniques and highlight selected results.
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Regulation of Interferon Regulatory Factor 3-Dependent Innate Immunity by the HCV NS3/4A ProteaseEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Interferon regulatory factor 3 (IRF-3) is a ubiquitously expressed latent cellular transcription factor that plays a pivotal role in control of innate, type I interferon (IFN) antiviral responses. After viral infections, IRF-3 is activated by specific C-terminal phosphorylation, which induces its dimerization and nuclear translocation, whereupon IRF-3 activates the transcription of type I IFNs and a number of other antiviral effector genes. Many viruses have evolved strategies that antagonize signaling mechanisms leading to IRF-3 activation. Recent studies have shown that hepatitis C virus blocks IRF-3 activation and subse...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Selection and Characterization of Drug-Resistant HCV Replicons In Vitro with a Flow Cytometry-Based AssayEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Because HCV RNA-dependent RNA polymerase is error-prone and the viral RNA has a high turnover rate, the genetic diversity of HCV is very high both in vitro and in vivo. The mutation rate in long-term replicon cultures approaches 3.0 × 10-3 base substitutions/site/year in this in vitro replication model. A direct consequence of the high mutation rate is the rapid emergence of drug-resistant variants, both in cell culture and in patients. Selectable replicons have been used extensively to isolate and characterize drug-resistant HCV genomes in vitro. Typically, replicon cells are plated at a low density and then subject...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

High-Throughput Screening of HCV RNA Replication Inhibitors by Means of a Reporter Replicon SystemEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Efforts to find effective treatment for hepatitis C virus (HCV) have been hampered by the lack of a robust in vitro infectious tissue-culture system for this virus. A subgenomic replicon system was first developed in 1999 and has since been extensively optimized to accommodate the need for conveniently measuring HCV replication in vitro and widely adopted in HCV drug-discovery efforts. Here we describe the adaptation of a modified replicon system for a high-throughput screening (HTS) in anti-HCV drug discovery. In this system, the antiviral activity and cytotoxicity of any experimental compound are measured from a single w...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Inhibition of HCV Replication by Small Interfering RNAEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) have been reported to suppress gene expression significantly. HCV seems a suitable candidate for targets of siRNAs, as HCV is a positive single-strand RNA virus and replicates in the cytoplasm. Efficient inhibition by siRNAs requires access to target RNAs, which usually possess secondary structure. We have shown that shRNAs suppressing the HCV internal ribosomal entry site (IRES) can inhibit different HCV genotypes grown in cell culture and replicon replication, suggesting the potential of siRNA as an additional therapeutic option against HCV infection.
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Simultaneous Detection of Anti-HCV Antibody and HCV Core AntigenEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
HCV infection is usually diagnosed by means of an enzyme immune assay for the detection of antibody against HCV. The window period between infection and seroconversion remains a dramatic problem in the transfusional and diagnostic setting. In this chapter, we report (i) procedures for assays using two different approaches designed to reduce the window period and (ii) performance in terms of specificity and sensitivity in the detection of both antibody and antigen, and we compare their efficacy with that of commercial assays.
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Cellular Receptors and HCV EntryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
After attachment to specific receptors on the surfaces of target cells, hepatitis C virus (HCV) particles are thought to be internalized to endosomes, where low pH induces fusion between the viral and cellular membranes, delivering the HCV genome into the cytoplasm. Here, we describe methods to study the early events in HCV infection; the interactions with cellular receptors and the mechanism of entry.
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Studying HCV Cell Entry with HCV Pseudoparticles (HCVpp)Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
HCV infection leads in 50 to 80% of cases to chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma. Interferons and the nucleoside analog ribavirin form the basis for treatment but are not sufficiently effective and have numerous side effects. Although about 300 million people worldwide are estimated to be infected, the characterization of HCV biology and associated pathologies and development of new therapeutics have been slow. Systems that support HCV replication and particle formation in vitro have emerged only over the last few years, over 15 years after the discovery of the virus. The available infection mod...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Screening of Small-Molecule Compounds as Inhibitors of HCV EntryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The hepatitis C virus (HCV) has infected some 170 million people worldwide, and is expected to pose a significant medical problem for the foreseeable future. No vaccine is presently available, and the current antiviral therapies (pegylated interferon-α and ribavirin) are characterized by limited efficacy, high costs, and substantial side effects. Initiation of infection requires attachment of the HCV virus to the cell surface followed by viral entry and represents a critical determinant of tissue tropism and pathogenesis. Small molecules that inhibit the virus at the stage of viral entry, for example, by blocking the...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Isolation of JFH-1 Strain and Development of an HCV Infection SystemEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Detailed analysis of hepatitis C virus (HCV) has been hampered by the lack of an appropriate viral culture system and small animal models of infection. My group and others have recently reported the production of infectious virus after full-length HCV RNA transfection into Huh-7 cells. This system depends primarily on isolation of a JFH-1 strain from a patient with fulminant hepatitis. The JFH-1 strain belongs to genotype 2a and has high colony-formation efficiency when tested with a subgenomic replicon system. Here, I describe various protocols for isolation of the JFH-1 strain and construction of the HCV infection system...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Measuring HCV Infectivity Produced in Cell Culture and In VivoEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Recently described systems for efficiently growing HCV in cell culture provide powerful new tools with which to dissect the life cycle of this important human pathogen. This chapter describes methods for measuring the infectivity of HCV produced in cell culture or recovered from animals experimentally infected with the cell culture–produced virus.
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Genotype 1a HCV (H77S) Infection SystemEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
HCV is a small RNA virus belonging to the genus Hepacivirus within the virus family Flaviviridae. Infection with HCV often leads to chronic liver diseases including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Current therapy, based on the use of interferon-α (IFN-α) in combination with ribavirin, results in limited success, especially in patients infected with the most prevalent genotype 1 viruses. Better therapies are needed, but the inability to propagate HCV in cell culture hampers antiviral drug-discovery efforts. Recently, fully permissive cell-culture systems have been developed that...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Full-Length Infectious HCV ChimerasEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
One hallmark of HCV is its pronounced genetic plasticity, caused by error-prone RNA replication, which probably contributes to its remarkable ability to establish chronic infections. On the basis of phylogenetic analyses, HCV variants are classified into six genotypes (GTs), each comprising a variable number of subtypes. Presumably, these genetic differences, which range from 33 to 35% at the nucleotide level among genotypes and from 22 to 25% between subtypes, are reflected by divergent biological properties of the respective isolates. The unprecedented replication efficiency of the JFH1 isolate (a GT2a strain derived fro...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Adaptation of the Hepatitis C Virus to Cell CultureEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A major breakthrough in the field of HCV research was the development of a system that supports the production of infectious virus particles. The key to this achievement was the molecular cloning of a genotype 2a genome, designated JFH1, which replicates to exceptionally high levels and at the same time supports virus particle assembly and release. A major drawback of this system was, however, the rather low yield of infectious particles obtained with the JFH1 genome as well as with most JFH1-derived virus chimeras. One approach to overcoming this hurdle is adaptation of the HCV genomes to cell culture. We found that both ...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Primary Human Hepatocyte Culture for HCV StudyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Studies of HCV pathogenesis and antiviral research have been hampered by the lack of adequate cell-culture and small-animal models. The culturing of human primary hepatocytes would greatly facilitate the model development in HCV research. The availability of robust infectious virus, JFH1 (i.e., genotype 2) strain, will further increase the interest in using primary hepatocyte cultures. This cell model system will significantly enhance research in the areas of antiviral research and host–virus interaction, but obtaining pure and viable human primary hepatocytes is not trivial. We have optimized a method of liver perfu...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

In Vivo Study of HCV in Mice with Chimeric Human LiversEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Estimates of hepatitis C virus infection include 170 million people worldwide, who face increased risk of development of cirrhosis, liver failure, and hepatocellular carcinoma. Standard of care therapy with pegylated interferon and ribavirin is effective in just half of patients, is challenged by substantial treatment-related morbidity, and is prohibitively expensive in most parts of the world. New therapeutics for treatment and prevention are clearly needed. Development of effective therapies has been significantly hampered by difficulties in establishing in vitro and in vivo models of viral replication. This chapter revi...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Immunohistochemical Detection of HCV Proteins in Liver TissueEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe here a study using a novel immunohistochemical method effective on fixed, archived specimens, including liver biopsies and surgical resection samples. The initial protocol uses a biotin-detection system but can also be used in a polymer-detection system. This protocol offers easy, precise, and strong staining resolution with distinct patterns consistent with the liver pathology, irrespective of the viral HCV genotype examined. This approach provides applications for diagnosis as well as for exploratory pathological studies.
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Determination of HCV-Specific T-Cell ActivityEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The magnitude and breadth of T-cell responses against HCV are associated with the outcome of HCV infection. Parameters of HCV-specific T-cell responses that are frequently assessed in clinical immunological studies include proliferation of T cells in response to HCV antigens, frequency of HCV-specific T cells secreting cytokines, and changes in antigen specificity during the course of HCV infection. Common techniques for assessing these parameters such as 3H-thymidine incorporation assay, cytokine ELISpot assay, and strategies for epitope mapping and identification of minimal optimal epitopes are outlined, and detailed pro...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info

Analysis of HCV-Specific T Cells by Flow CytometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Flow cytometry has become an essential research tool because of the increase in the number of its {applications.} The development of an increasing number of monoclonal antibodies (mAbs) and fluorochromes, and of instruments capable of multicolor detection, allows the acquisition of a large amount of phenotypic and functional information in a single assay. In addition, flow-cytometry techniques have overcome critical problems of conventional assays, such as the use of radioactive reagents to assess proliferation and cytotoxicity of virus-specific T cells. Here, we provide both an overview of available techniques as well as ...
Source: Springer protocols feed by Infectious Diseases - July 1, 2008 Category: Infectious Diseases Source Type: info