MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Infectious Diseases' source. Thu, 26 Jan 2012 18:33:09 +0100 http://www.medworm.com/index.php?rid=5469937&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_9 In 2004, brain tumor stem cells (BTSCs) were isolated from surgical human malignant gliomas. This cancer cell population has been identified as the root for tumor initiation and resistance to therapies. Thus, it is imperative to develop new therapies that can eradicate this subpopulation to improve the prognosis of patients with brain tumors. Our group previously reported the antiglioma effect of the tumor-selective oncolytic adenovirus Delta-24-RGD that is now being tested in a phase I clinical trial for patients with malignant gliomas. We also showed that Delta-24-RGD infects, replicates in, and induces cell death in BTSCs. Interestingly, we observed that adenoviral-infected cells undergo autophagy and that autophagy-related cytoplasmic vacuolization might be part of the lysis process. H... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469937 Mon, 28 Nov 2011 05:00:00 +0100 5469937 http://www.medworm.com/index.php?rid=5469936&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_8 Despite recent advancements in the treatment of cancer, the prognosis for patients with malignant brain tumors remains poor. The success of currently available therapies has been limited in part because of the disseminated nature of these tumors. Furthermore, most of these tumors, when in a high-grade form, are resistant to chemo- and radiotherapy. Taking the above considerations into account, effective treatment of these cancers not only requires the development of new means to target tumor burdens that have dispersed significantly from their site of origin, but also therapeutic approaches which can appropriately discriminate between tumor cell and normal brain. In the past two decades, novel approaches involving the use of oncolytic adenoviruses to target ­malignant brain tumors have... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469936 Mon, 28 Nov 2011 05:00:00 +0100 5469936 http://www.medworm.com/index.php?rid=5469935&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_7 Information regarding the biodistribution and kinetics of spread of oncolytic viruses is crucial for safety considerations in the design of future, more efficient reagents. Although optical imaging can be used to gain this information in rodent models, imaging with radioactive isotopes presents the advantage of being directly applicable to humans, and preclinical imaging data can provide a basis for the design of clinical protocols. In this chapter, we describe the use of the Na/I symporter as a reporter gene, associated with 124I-NaI as a radiotracer, to monitor the anatomical localization, as well as the propagation, of oncolytic viruses using dedicated, preclinical positron emission tomography scanners. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469935 Mon, 28 Nov 2011 05:00:00 +0100 5469935 http://www.medworm.com/index.php?rid=5469934&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_6 Optical imaging of luciferage gene expression has become a powerful tool to track cells and viruses in vivo in small animal models. Luciferase imaging has been used to study the location of infection by replication-defective and replication-competent viruses and to track changes in the distribution of viruses in mouse models. This approach has also been used in oncolytic studies as a noninvasive means to monitor the growth and killing of tumor cells modified with luciferase genes. In this chapter, we describe the techniques used for luciferase imaging as have been applied to track replication-defective and replication-competent adenoviruses in mouse and hamster models of oncolysis and virus pharmacology. Although these methods are simple, the process of obtaining accurate luciferase imagin... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469934 Mon, 28 Nov 2011 05:00:00 +0100 5469934 http://www.medworm.com/index.php?rid=5469933&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_5 Promising results have been reported from numerous studies with replication-selective oncolytic adenoviral mutants as novel treatments for a variety of cancers. Most of these studies were performed in cancer cell lines, dissociated tumor tissue, or animal models, and the predictive utility for efficacy and safety in the clinical setting is unclear. Indeed, the outcome of many clinical trials with viral mutants that demonstrated high efficacy preclinically has so far been disappointing, necessitating better test models. To this end, we developed a methodology using primary human cancer specimens for evaluation of cytotoxicity ex vivo including colorectal liver metastasis, ovarian, breast, colon, and prostate carcinomas. Under optimized culture conditions, primary human tumor tissue remained... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469933 Mon, 28 Nov 2011 05:00:00 +0100 5469933 http://www.medworm.com/index.php?rid=5469932&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_4 Oncolytic (replicating) adenovirus (Ad) vectors are emerging as a promising form of a cancer therapy agent. There has been a need for an appropriate animal model to study oncolytic Ad since human Ad ­replication is usually species specific. We have shown that Syrian (golden) hamsters are an appropriate animal model to study human Ad5-based vectors. Syrian hamsters are immunocompetent, and they allow human Ad5 replication in normal tissues as well as in Syrian hamster cancer cells. The development of the Syrian hamster as a model to study oncolytic Ad vectors has opened avenues to explore the role of host immune response and preexisting immunity in Ad vector efficacy and toxicity/biodistribution following Ad vector administration. Since most of the normal tissues in the Syrian hamster a... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469932 Mon, 28 Nov 2011 05:00:00 +0100 5469932 http://www.medworm.com/index.php?rid=5469931&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_3 Oncolytic (replication-competent) adenoviruses (Ads) represent the most advanced platform for cancer gene therapy. These viral vectors ablate tumors by killing tumor cells in the process of virus replication. As progeny virions are released, they infect remaining cancer cells, generating a bystander effect. Ads engineered for increased cancer specificity produce less damage to normal tissues. First-generation oncolytic Ads have demonstrated acceptable levels of safety while the efficacy was observed only in combination with chemotherapy and/or radiation. Second-generation oncolytic Ads are armed with therapeutic transgenes to increase release, spread, and bystander effect for enhancing the efficacy. Third-generation oncolytic Ads are armed vectors with capsid modifications for transduction... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469931 Mon, 28 Nov 2011 05:00:00 +0100 5469931 http://www.medworm.com/index.php?rid=5469930&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_2 Adenovirus represents a valuable tool for the treatment of cancer, but tumor targeting remains a pending issue. Most common procedures to modify adenovirus genome are time-consuming due to the requirement of multiple cloning steps, and the low efficacy of the recombination process. Here, we present a new method for homologous recombination in yeast to fast construct recombinant adenoviruses. Also, an alternative procedure to purify viral stocks, based on iodixanol gradient is described. Compared to classical methods, iodixanol is nontoxic to cells, which avoids desalting to use in vitro and in vivo. Moreover, viral stocks are more viable and it can be used for large-scale purifications. Finally, a protocol for analyzing blood persistence of modified vector in in vivo biodistribution is pre... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469930 Mon, 28 Nov 2011 05:00:00 +0100 5469930 http://www.medworm.com/index.php?rid=5469929&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_1 Herpes simplex virus-1 (HSV-1) is an enveloped, double-stranded DNA virus that has been used with modification as an oncolytic virus against a number of tumor types. Modifications that make HSV-1 replication-­conditional, i.e., selectively divide in replicating cells make it fulfill a prerequisite criteria for oncolytic viruses. Other appealing features of HSV-1 as an oncolytic virus include its large, modifiable genome; its sensitivity to antiviral agents, such as ganciclovir; and its lack of host cell integration. Here, we review the methods of HSV-1 engineering, through traditional recombination techniques as well as through bacterial artificial chromosome (BAC) technology. We then describe protocols for titering, amplification, and purification of engineered HSV-1-derived oncolytic... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469929 Mon, 28 Nov 2011 05:00:00 +0100 5469929 http://www.medworm.com/index.php?rid=5469928&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_15 Oncolytic viruses, the use of viruses to treat cancer, is emerging as a new option for cancer therapy. Oncolytic viruses, of both DNA and RNA origin, exhibit the ability to preferentially replicate in and kill cancer cells plausibly due to defects in innate immune signaling or translation regulation that are acquired during cellular transformation. Here, we review concepts and assays that describe how to analyze signaling pathways that govern the regulation of Type I IFN production as well as the induction of interferon-stimulated antiviral genes, events that are critical for mounting an effective antiviral response. The following procedures can be used to assess whether innate immune pathways that control antiviral host defense are defective in tumor cells – mechanisms that may help... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469928 Mon, 28 Nov 2011 05:00:00 +0100 5469928 http://www.medworm.com/index.php?rid=5469927&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_14 Oncolytic vaccinia viruses have made some impressive advances over the last 5 years, with a range of ­different backbones displaying significant antitumor responses in preclinical models, and some exciting clinical results being reported against liver cancers. Because the virus is capable of rapid spread within the tumor, has evolved to spread relatively undetected within the blood stream, does not integrate into the host cell chromosome, and can infect almost any cell type, it is well-suited to the requirements for a successful oncolytic. In addition, the extensive clinical use of this virus means that contraindications to its use are known, and approved and experimental antivirals are available. Furthermore, because the virus has a large array of virulence genes whose deletion may ta... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469927 Mon, 28 Nov 2011 05:00:00 +0100 5469927 http://www.medworm.com/index.php?rid=5469926&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_13 We describe an in vitro test called “Tumor Neutralisation Assay” which allows the analysis of bystander antitumor immune effects induced in human peripheral blood mononuclear cells by NDV. There are two variants, one for oncolytic NDV strains and the other one for nonlytic NDV strains. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469926 Mon, 28 Nov 2011 05:00:00 +0100 5469926 http://www.medworm.com/index.php?rid=5469925&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_12 Oncolytic viruses, such as reovirus, offer a promising approach to cancer treatment. Concurrently, oncolytic viruses provide a valuable tool for deciphering unique attributes of cancer cells that support superior virus replication, cell death, or virus dissemination. Through our studies on various cancer cell lines, as well as isogenic cells with and without transformation by oncogenic Ras, we have identified at least four steps of virus replication that can be augmented in transformed cells. Ras transformation can support efficient reovirus uncoating during entry, production of progeny with high infectious capacity, and reovirus-induced apoptosis. Furthermore, Ras transformation also precludes interferon production following reovirus infection, permitting enhanced cell-to-cell virus sprea... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469925 Mon, 28 Nov 2011 05:00:00 +0100 5469925 http://www.medworm.com/index.php?rid=5469924&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_11 Despite significant advances in recent years, treatment of metastatic malignancies remains a significant challenge. There is an urgent need for development of novel therapeutic approaches. Virotherapy approaches have considerable potential, and among them measles virus (MV) vaccine strains have emerged as a promising oncolytic platform. Retargeted MV strains deriving from the Edmonston vaccine lineage (MV-Edm) have shown comparable antitumor efficacy to unmodified strains against receptor expressing tumor cells with improved therapeutic index. Here, we describe the construction, rescue, amplification, and titration of fully retargeted MV-Edm derivatives displaying tumor specific receptor binding ligands on the viral surface in combination with H protein CD46 and SLAM entry ablating mutatio... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469924 Mon, 28 Nov 2011 05:00:00 +0100 5469924 http://www.medworm.com/index.php?rid=5469923&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-340-0_10 Oncolytic viruses are self-amplifying therapeutics that specifically replicate in and kill cancer cells. We have previously shown that vesicular stomatitis virus (VSV) can be used as an oncolytic virus. A strain of VSV harboring a mutation in the M protein (VSVΔ51) was found to exhibit enhanced tumor selectivity over its wild-type counterpart due to its inability to overcome antiviral programs in normal cells and due to the frequent defects in antiviral signaling pathways observed in the majority of tumors. VSVΔ51 can harbor transgenes, is easily propagated and purified to high titers, and shows potent oncolytic activity in several mouse models, including syngeneic CT26-lacZ subcutaneous colon carcinoma models. However, VSV-neutralizing antibodies targeting mainly the VSV-G sur... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5469923 Mon, 28 Nov 2011 05:00:00 +0100 5469923 http://www.medworm.com/index.php?rid=5316519&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_9 Meningococcal mechanisms of adhesion are complex, involving multiple adhesins and their respective target receptors on host cells. Three major surface structures – pili, Opa, and Opc – have been known for some time to mediate meningococcal adhesion to target human cells. More recently, several other relatively minor adhesins have also come to light. The literature on bacterial adhesion mechanisms provides numerous examples of various adhesins acting cooperatively in an apparently hierarchical and sequential manner; in other instances, adhesins may act in concert leading to high avidity interactions, often a prelude to cellular invasion and tissue penetration. Such examples are also present in the case of meningococci, although our knowledge of adhesin cooperation and synergy is... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316519 Sat, 15 Oct 2011 12:59:20 +0100 5316519 http://www.medworm.com/index.php?rid=5316518&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_8 In addition to standard gel-based proteomic approaches, gel-free approaches using isobaric label reagents, such as Tandem Mass Tags (TMT), provide a straightforward method for studying adaptations in microbial proteomes to changing environmental conditions. This approach does not have the known difficulties of 2-D gel electrophoresis with proteins of extreme biochemical properties. The workflow described here was designed to study adaptive responses in bacteria and has been applied to study the response of meningococci to iron limitation. The supplemental use of western blotting allows the confirmation of certain changes in protein abundance identified within the TMT study. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316518 Sat, 15 Oct 2011 12:59:20 +0100 5316518 http://www.medworm.com/index.php?rid=5316517&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_7 Metabolism can be defined as the complete set of chemical reactions that occur in living organisms in order to maintain life. Enzymes are the main players in this process as they are responsible for catalyzing the chemical reactions. The enzyme–reaction relationships can be used for the reconstruction of a network of reactions, which leads to a metabolic model of metabolism. A genome-scale metabolic network of chemical reactions that take place inside a living organism is primarily reconstructed from the information that is present in its genome and the literature and involves steps such as functional annotation of the genome, identification of the associated reactions and determination of their stoichiometry, assignment of localization, determination of the biomass composition, esti... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316517 Sat, 15 Oct 2011 12:59:20 +0100 5316517 http://www.medworm.com/index.php?rid=5316516&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_6 Integral outer membrane proteins (OMPs) play key roles in solute transport, adhesion, and other processes. In Neisseria, they can also function as major protective antigens. Structural, biophysical, and immunological studies of Neisserial OMPs require their isolation in milligram quantities. Purification of any OMP directly from Neisseria would require the growth of large quantities of cell mass, with attendant concerns about safety and convenience. As a result, many investigators have developed methods for expression of OMPs into inclusion bodies in E. coli, followed by refolding of the resolubilized protein. Here we describe such a method, as optimized for the PorA porin but which can be applied, with suitable adaptation, to other OMPs. We also describe an approach to the crystallization... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316516 Sat, 15 Oct 2011 12:59:20 +0100 5316516 http://www.medworm.com/index.php?rid=5316515&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_5 A riboregulated network, in which small RNAs (sRNAs) regulate the stability and thus translation of transcripts (mRNA), has only recently been discovered in prokaryotes. Yet, during the last 5 years, hundreds of sRNAs have been identified in various bacterial species by using a wide variety of both computational and experimental approaches. The majority of the sRNAs interact with the 5′-untranslated region (UTR) of target mRNAs, thereby influencing the stability of target mRNAs, or by either suppressing or upregulating the ribosome entry to the mRNAs influencing translation. Here, we describe experimental approaches successfully used in our laboratory to identify and functionally characterize sRNAs in vivo in our model micro-organism Neisseria meningitidis. (Source: Springer protocol... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316515 Sat, 15 Oct 2011 12:59:20 +0100 5316515 http://www.medworm.com/index.php?rid=5316514&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_4 The human-restricted pathogens Neisseria meningitidis and Neisseria gonorrhoeae are naturally competent for DNA uptake. This trait has been exploited extensively for genetic manipulation of these bacteria in the laboratory. Most transformation protocols were developed for N. gonorrhoeae, but appear to work also for N. meningitidis. In this chapter, we describe a number of protocols for genetic manipulation of N. meningitidis. Specifically, we describe how to (1) obtain knock-out mutants containing antibiotic-resistance markers, (2) generate markerless knock-out mutants, and (3) construct complementation strains. The generation of such mutants provides a valuable resource for studies of bacterial pathogenesis and vaccine development. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316514 Sat, 15 Oct 2011 12:59:20 +0100 5316514 http://www.medworm.com/index.php?rid=5316513&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_3 Rapid clinical and laboratory diagnoses are the foundation for a successful management of serious infections with Neisseria meningitidis. A species-specific multiplex polymerase chain reaction (PCR) coupled with fluidic microarrays using microbeads (the Luminex xMAP™ Technology) can detect pathogens most frequently found in the cerebrospinal fluid of patients. The Luminex suspension array system uniquely combines flow cytometry, microspheres, laser technology, digital signal processing, and traditional chemistry. In this method, the reaction is carried out in one vessel, in which distinctly color-coded bead sets, each conjugated with a different specific nucleic acid reactant, are hybridized with the PCR products, and a reporter molecule is used to quantify the interaction. The flow-... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316513 Sat, 15 Oct 2011 12:59:19 +0100 5316513 http://www.medworm.com/index.php?rid=5316512&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_2 The clinical symptoms induced by Neisseria meningitidis reflect compartmentalized intravascular and intracranial bacterial growth and inflammation. In this chapter, we describe a classification system for meningococcal disease based on the nature of the clinical symptoms. Meningococci invade the subarachnoid space and cause meningitis in as many as 50–70% of patients. The bacteremic phase is moderate in patients with meningitis and mild systemic meningococcemia but graded high in patients with septic shock. Three landmark studies using this classification system and comprising 862 patients showed that 37–49% developed meningitis without shock, 10–18% shock without meningitis, 7–12% shock and meningitis, and 18–33% had mild meningococcemia without shock or meni... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316512 Sat, 15 Oct 2011 12:59:19 +0100 5316512 http://www.medworm.com/index.php?rid=5316511&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_22 DNA-based vaccines are currently being developed for treating a diversity of human diseases including cancers, autoimmune conditions, allergies, and microbial infections. In this chapter, we present a general protocol that can be used as a starting point for developing DNA vaccines to pathogen-derived antigens, using Neisseria meningitidis as an example. In addition, we describe a fusion gene-based vaccine protocol for increasing the potency of DNA vaccines that are based on poorly immunogenic antigens such as short pathogen-derived polypeptides. Finally, we provide a safe and effective protocol for delivery of DNA vaccines, based on intramuscular injection followed by electroporation. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316511 Sat, 15 Oct 2011 12:59:19 +0100 5316511 http://www.medworm.com/index.php?rid=5316510&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_21 Most of the vaccines available today, albeit very effective, have been developed using traditional “old-style” methodologies. Technologies developed in recent years have opened up new perspectives in the field of vaccinology and novel strategies are now being used to design improved or new vaccines against infections for which preventive measures do not exist. The Reverse Vaccinology (RV) approach is one of the most powerful examples of biotechnology applied to the field of vaccinology for identifying new protein-based vaccines. RV combines the availability of genomic data, the analyzing capabilities of new bioinformatic tools, and the application of high throughput expression and purification systems combined with serological screening assays for a coordinated screening proces... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316510 Sat, 15 Oct 2011 12:59:19 +0100 5316510 http://www.medworm.com/index.php?rid=5316509&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_20 The availability of Neisseria genome sequences together with improvements in proteomic technologies provide the opportunity to study at high resolution the immune response to Neisseria meningitidis. In this chapter, we describe a protocol that combines two-dimensional (2D) SDS-PAGE of meningococcal outer membranes with western blotting of human antisera to identify proteins associated with the development of protective antibody responses. This methodology can identify putative vaccine candidates for incorporation in a multi-component serogroup B meningococcal vaccine. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316509 Sat, 15 Oct 2011 12:59:19 +0100 5316509 http://www.medworm.com/index.php?rid=5316508&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_1 Neisseria meningitidis (the meningococcus) causes significant morbidity and mortality in children and young adults worldwide through epidemic or sporadic meningitis and/or septicemia. In this review, we describe the biology, microbiology, and epidemiology of this exclusive human pathogen. N.meningitidis is a fastidious, encapsulated, aerobic gram-negative diplococcus. Colonies are positive by the oxidase test and most strains utilize maltose. The phenotypic classification of meningococci, based on structural differences in capsular polysaccharide, lipooligosaccharide (LOS) and outer membrane proteins, is now complemented by genome sequence typing (ST). The epidemiological profile of N. meningitidis is variable in different populations and over time and virulence of the meningococcus is bas... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316508 Sat, 15 Oct 2011 12:59:19 +0100 5316508 http://www.medworm.com/index.php?rid=5316507&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_19 The process of cellular apoptosis is mediated by a number of microbial pathogens to modulate host defense mechanisms. Inhibition of apoptosis is thought to favor microbial survival, replication or immune evasion, while induction of apoptosis is likely to promote escape of the organisms from host cells. Several studies have reported that infection with Neisseria spp. can inhibit or reduce apoptotic cell death, thus allowing adaptation, intracellular replication, and immune evasion, events that are likely to spread infection. In this chapter, various techniques are described for direct measurement of host cell responses to infection with Neisseria meningitidis and to treatment with pure Neisseria porins, the major proteins found in the outer membrane of the pathogen. (Source: Springer protoc... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316507 Sat, 15 Oct 2011 12:59:19 +0100 5316507 http://www.medworm.com/index.php?rid=5316506&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_18 The pan-Neisseria microarray was the first bacterial microarray to address multiple strains and species, and is a tool specifically developed for the performance of comparative studies within and between species. To achieve this, its design was based upon a detailed comparison of multiple genomes, prior to probe selection, and serial triage to optimize sensitivity and specificity. While this tool can be used for transcriptional comparisons of the same species, such as isogenic mutants, or strains exposed to different environmental conditions, its features are also particularly suited to population and functional studies of unrelated strains. The optimal use of these tools, including the use of single-channel labeling for genomic studies, the biological replication needed to perform robust ... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316506 Sat, 15 Oct 2011 12:59:19 +0100 5316506 http://www.medworm.com/index.php?rid=5316505&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_17 Infectious disease research has been revolutionized by two recent developments in the field of genome biology: (1) the sequencing of the human genome as well as many pathogen genomes and (2) the development of high-throughput technologies including microarray technology, proteomics, and metabolomics. Microarray studies enable a deeper understanding of the genetic evolution of pathogens and investigation of the determinants of pathogenicity on a whole-genome scale. Host studies, in turn, allow for an unprecedented holistic appreciation of the complexities of host cell responses at the molecular level. In combination, host–pathogen studies allow global analysis of gene expression in the infecting bacterium as well as in the infected host cell during pathogenesis, providing a comprehens... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316505 Sat, 15 Oct 2011 12:59:19 +0100 5316505 http://www.medworm.com/index.php?rid=5316504&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_16 The innate immune system utilises a set of receptors, called pattern recognition receptors (PRRs), in order to recognise specific molecular patterns or motifs called pathogen-associated molecular patterns (PAMPs) on invading pathogens. The toll-like receptor (TLR) family of proteins is an integral part of the mammalian innate immune system. We are now beginning to decipher which TLRs are involved in the recognition of particular microbial patterns, but questions remain as to the homo- and heterotypic associations that TLRs form and how these associations affect their activation. Technical advances in fluorescence microscopy has enabled us to investigate the functional associations of TLRs and other PPRs in living cells in response to different pathogens using non-invasive fluorescence imag... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316504 Sat, 15 Oct 2011 12:59:19 +0100 5316504 http://www.medworm.com/index.php?rid=5316503&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_15 A method for exploring protein–protein interactions using hydrogen/deuterium exchange coupled to mass spectrometry is described. The method monitors the exchange of backbone (amide) hydrogens in solutions of deuterated water that primarily occur on portions of the protein exposed to solvent. In the presence of a protein binding partner, regions that experience reduced exchange are either part of the protein–protein interaction interface or undergo conformational changes to reduce accessibility to solvent. This method has the advantage of being used under physiological conditions with unmodified proteins. In this chapter, we describe an approach suitable for probing interactions among relatively large proteins using conventional mass spectrometry systems. The interaction between... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316503 Sat, 15 Oct 2011 12:59:19 +0100 5316503 http://www.medworm.com/index.php?rid=5316502&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_14 Dendritic cells (DC) play a key role in the development of natural immunity to microbes. The DC form a bridge between the innate and adaptive immune system by providing key instructions particularly to antigen naïve T-cells. The interaction of DC with T lymphocytes involves three signals: (1) antigen processing and presentation in context of MHC Class I and/or II, (2) expression of T cell co-stimulatory molecules, and (3) cytokine production. Studying the interactions of DCs with specific pathogens allows for better understanding of how protective immunity is generated, and may be particularly useful for assessing vaccine components. In this chapter, we describe methods to generate human monocyte-derived DCs and assess their maturation, activation, and function, using interaction with... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316502 Sat, 15 Oct 2011 12:59:19 +0100 5316502 http://www.medworm.com/index.php?rid=5316501&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_13 The importance of physical forces in biology is becoming more appreciated. Neisseria gonorrhoeaehas become a paradigm for the study of physical forces in the bacterial world. Cycles of elongations and retractions of Type IV pili enables N. gonorrhoeaebacteria to exert forces on its environment, forces that play major roles in the life cycle of this pathogen. In order to better understand the role of these forces, there is a need to fully characterize them. Here, we present two different techniques, optical tweezers and Polyacrylamide MicroPillars (PoMPs), for measuring pilus retraction forces. Initially designed for N. gonorrhoeae, these assays can be readily modified to study other pilus-bearing bacteria including Neisseria meningitidis. (Source: Springer protocols feed by Infectious Dise... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316501 Sat, 15 Oct 2011 12:59:18 +0100 5316501 http://www.medworm.com/index.php?rid=5316500&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_12 Shear stress levels generated by circulating blood have a strong impact on biological processes taking place in the vasculature. It is therefore important to take them into account when studying infectious agents targeting the endothelium. Here we describe a protocol using disposable laminar-flow chambers and video microcopy to study bacterial infections in an environment that mimics the bloodstream. We initially focused on the interaction of Neisseria meningitidis with human endothelial cells and determined that shear stress is an important factor for the pathogen’s initial adhesion and for the formation of micro-colonies. The experimental set-up can be used to investigate other pathogens that interact with the endothelium as well as with other sites where shear stress is present. (... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316500 Sat, 15 Oct 2011 12:59:18 +0100 5316500 http://www.medworm.com/index.php?rid=5316499&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_11 Neisseria meningitidisis an organism whose environmental niche is limited to the human host. It can frequently colonize the human nasopharynx and has the ability to cause severe systemic infections. These infections can be sporadic, endemic or occur in outbreaks associated with more virulent meningococcal strains. Studies have demonstrated that the meningococcus can form biofilms both in vivo and ex vivo. In this chapter, we discuss methods to establish biofilms in the laboratory for in-depth biochemical, genetic, or microscopic studies. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316499 Sat, 15 Oct 2011 12:59:18 +0100 5316499 http://www.medworm.com/index.php?rid=5316498&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-346-2_10 Neisseria meningitidis is a human specific organism that causes severe sepsis and/or meningitis with high mortality. The disease scenario is rapid and much remains unknown about the disease process and host–pathogen interaction. In this chapter, we describe a protocol for generating a bioluminescently labeled N. meningitidis strain in order to advance our understanding of meningococcal disease progression. We also describe how in vivo bioluminescence imaging (BLI) can be used to observe novel features of the disease dynamics during meningococcal infection. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=5316498 Sat, 15 Oct 2011 12:59:18 +0100 5316498 http://www.medworm.com/index.php?rid=2054010&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_8 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 by flow cytometry methods. The cytometric method we describe here represents a useful tool to investigate to what extent yeasts are able to regulate their glycan surface ... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2054010 Sat, 20 Dec 2008 15:49:40 +0100 2054010 http://www.medworm.com/index.php?rid=2054009&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_7 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 the antigenic, biochemical, and virulence properties of Cryptococcus species. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2054009 Sat, 20 Dec 2008 15:49:39 +0100 2054009 http://www.medworm.com/index.php?rid=2054008&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_6 (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2054008 Sat, 20 Dec 2008 15:49:39 +0100 2054008 http://www.medworm.com/index.php?rid=2054007&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_5 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 bacteria. Accordingly, this methodological approach is ideally suited for the analysis of adhesins expressed in a phase-variable manner and for the study of heterogeneous bacte... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2054007 Sat, 20 Dec 2008 15:49:39 +0100 2054007 http://www.medworm.com/index.php?rid=2054006&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_4 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 the dissection of the complex cross-talk with infectious agents. Accordingly, this chapter describes a plaque assay to identify attenuated pathogens, an infection assay fo... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2054006 Sat, 20 Dec 2008 15:49:39 +0100 2054006 http://www.medworm.com/index.php?rid=2054005&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_3 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 in maintenance of bacterial fitness. Here, we are interested in surface-exposed proteins of Streptococcus pneumoniae, which interact with host proteins including proteins ... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2054005 Sat, 20 Dec 2008 15:49:38 +0100 2054005 http://www.medworm.com/index.php?rid=2054004&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_26 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 the C. albicans proteome targeted by the immune system), but also for evaluating how this pathogen organism interacts with its host to trigger infection. In contrast with... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2054004 Sat, 20 Dec 2008 15:49:37 +0100 2054004 http://www.medworm.com/index.php?rid=2054003&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_25 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) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2054003 Sat, 20 Dec 2008 15:49:37 +0100 2054003 http://www.medworm.com/index.php?rid=2054002&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_24 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) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2054002 Sat, 20 Dec 2008 15:49:37 +0100 2054002 http://www.medworm.com/index.php?rid=2054001&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_23 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, the models provide clear answers regarding protein and gene expression that are not complicated by nonepithelial factors. To study the impact of several host components,... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2054001 Sat, 20 Dec 2008 15:49:36 +0100 2054001 http://www.medworm.com/index.php?rid=2054000&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_22 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) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2054000 Sat, 20 Dec 2008 15:49:33 +0100 2054000 http://www.medworm.com/index.php?rid=2053999&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_21 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, we use Candida albicans for fungal and Shigella flexneri for bacterial pathogens. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053999 Sat, 20 Dec 2008 15:49:32 +0100 2053999 http://www.medworm.com/index.php?rid=2053998&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_20 (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053998 Sat, 20 Dec 2008 15:49:32 +0100 2053998 http://www.medworm.com/index.php?rid=2053997&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_2 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 cell responses at the molecular level. In combination, host-pathogen studies allow global analysis of gene expression in the infecting bacterium as well as in the infect... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053997 Sat, 20 Dec 2008 15:49:31 +0100 2053997 http://www.medworm.com/index.php?rid=2053996&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_19 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 active subunits. Analyses of the proteolysis and the enzymatic activity of caspases therefore represent valuable tools to study apoptotic programs during infection. The ap... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053996 Sat, 20 Dec 2008 15:49:31 +0100 2053996 http://www.medworm.com/index.php?rid=2053995&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_18 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 developing drugs against parasite liver stages. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053995 Sat, 20 Dec 2008 15:49:30 +0100 2053995 http://www.medworm.com/index.php?rid=2053994&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_17 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 cycle, leading from early metacestode vesicles to the production of protoscoleces, can be mimicked under laboratory conditions. The methodology comprises co-cultivation syste... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053994 Sat, 20 Dec 2008 15:49:30 +0100 2053994 http://www.medworm.com/index.php?rid=2053993&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_16 (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053993 Sat, 20 Dec 2008 15:49:30 +0100 2053993 http://www.medworm.com/index.php?rid=2053992&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_15 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 sophisticated search algorithms able to correlate MS information with sequence databases, to name but a few. Here we integrate the typical MS-based strategy for identifying prote... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053992 Sat, 20 Dec 2008 15:49:29 +0100 2053992 http://www.medworm.com/index.php?rid=2053991&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_14 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, in which sera recovered from patients with candidiasis are used to screen a C. albicans genomic DNA expression library. Immunoreactive colonies are detected by reaction w... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053991 Sat, 20 Dec 2008 15:49:28 +0100 2053991 http://www.medworm.com/index.php?rid=2053990&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_13 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 essential to detect virulence gene expression during host invasion. Here, we describe two PCR-based detection methods of Aspergillus gene expression in both plant and mammalian... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053990 Sat, 20 Dec 2008 15:49:28 +0100 2053990 http://www.medworm.com/index.php?rid=2053989&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_12 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) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053989 Sat, 20 Dec 2008 15:49:26 +0100 2053989 http://www.medworm.com/index.php?rid=2053988&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_11 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 plasma membrane or the assembly of the cell wall. For a more specific and efficient treatment and prevention of fungal infection, new therapeutic strategies are needed, inclu... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053988 Sat, 20 Dec 2008 15:49:25 +0100 2053988 http://www.medworm.com/index.php?rid=2053987&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_10 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 observation. For this purpose, different epithelial cell lines are grown on a collagen gel to build up models of intestinal (Caco-2 cell line), vaginal (A431 cell line),... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053987 Sat, 20 Dec 2008 15:49:24 +0100 2053987 http://www.medworm.com/index.php?rid=2053986&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-204-5_1 (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=2053986 Sat, 20 Dec 2008 15:49:24 +0100 2053986 http://www.medworm.com/index.php?rid=4332924&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_9 A general procedure is described for the analysis of gene expression of Candida albicans cultured in a mouse infection model. This technique involves first infecting mice with Candida and subsequently harvesting blood and other tissue at specific time points during infection. The tissues are homogenized and the infecting Candida isolated. Finally, RNA is extracted from recovered Candida cells and subjected to microarray analysis. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332924 Mon, 01 Sep 2008 00:00:00 +0100 4332924 http://www.medworm.com/index.php?rid=4332923&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_8 Animal models are powerful tools to study the pathogenesis of diverse types of candidiasis. Murine models are particularly attractive because of cost, ease of handling, technical feasibility, and experience with their use. In this chapter, we describe methods for two of the most popular murine models of disease caused by Candida albicans. In an intravenously disseminated candidiasis (DC) model, immunocompetent mice are infected by lateral tail vein injections of a C. albicans suspension. Endpoints include mortality, tissue burdens of infection (most importantly in the kidneys, although spleens and livers are sometimes also assessed), and histopathology of infected organs. In a model of oral/esophageal candidiasis, mice are immunosuppressed with cortisone acetate and inoculated in the oral ... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332923 Mon, 01 Sep 2008 00:00:00 +0100 4332923 http://www.medworm.com/index.php?rid=4332922&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_7 Development of Candida spp. biofilms on medical devices such as catheters and voice prosthesis has been recognized as an increasing clinical problem. Simple device removal is often impossible, while in addition, resulting candidal infections are difficult to resolve due to their increased resistance to many antifungal agents. Susceptibility studies of clinical isolates are generally performed according to the CLSI standard, which measures planktonic cell susceptibility, but similar standards have not been designed or applied to testing of cells growing within a biofilm. As consistent biofilms from many strains are more difficult to simultaneously obtain and analyze than are independent planktonic cultures, any standard assay must address these concerns. In the present chapter, optimized co... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332922 Mon, 01 Sep 2008 00:00:00 +0100 4332922 http://www.medworm.com/index.php?rid=4332921&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_6 Biofilm formation is a common complication of the use of prosthetic devices. In clinical settings, biofilms can be comprised of one or more microbial species. In order to investigate the interaction between different species within a biofilm, a reproducible, reliable model system has to be utilized and an appropriate system for species identification applied. The present chapter describes the artificial throat model, a model system for growing mixed species biofilms on shunt prostheses. The model is used in conjugation with fluorescent in situ hybridization (FISH), which facilitates identification and localization of the resident microorganisms within biofilms. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332921 Mon, 01 Sep 2008 00:00:00 +0100 4332921 http://www.medworm.com/index.php?rid=4332920&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_5 A filter disk assay is described, which measures the penetration of antifungal agents through Candida biofilms. The technique involves forming a colony biofilm on a polycarbonate membrane filter, and capping it with a second, smaller membrane filter followed by a wetted paper disk of the type used in zone-of-inhibition assays. The entire assembly is transferred to agar medium containing the antifungal agent of interest. During subsequent incubation, the drug diffuses out of the agar and through the biofilm ‘sandwich’ to the moistened paper disk. The drug concentration in the disk can be determined by measuring the zone of growth inhibition that it produces on medium seeded with an indicator strain of Candida albicans in standard bioassays. Additional procedures are outlined for... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332920 Mon, 01 Sep 2008 00:00:00 +0100 4332920 http://www.medworm.com/index.php?rid=4332919&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_4 Immunoprotection during most forms of candidiasis (oropharyngeal, invasive) is lacking since most candidiasis patients are immunosuppressed either as a result of their allogeneic transplant, cancer chemotherapy, or HIV infection. Consequently, immunization might be considered as an unlikely way to protect patients from such infection. Nonetheless, there are a number of investigations that indicate active immunization or the passive treatment with hyperimmune, specific antibodies can result in protection in models of experimental candidiasis. The former subject, active immunization, is the subject of this chapter. We focus on recent efforts with the Als family of cell wall proteins to serve as a model, and also offer immunization methods in candidiasis models that can be adapted to any anti... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332919 Mon, 01 Sep 2008 00:00:00 +0100 4332919 http://www.medworm.com/index.php?rid=4332918&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_3 Both innate resistance and acquired cell-mediated immunity are involved in an anti-Candida response. Essential components of both the arms of the immune defense against infections by Candida spp. include phagocytic cells, i.e., polymorphonuclear neutrophils (PMNs) and mononuclear phagocytes. A powerful in vitro assay to assess host–pathogen interactions and study pathogenesis is the co-culture of phagocytic cells with a test fungus. The precise contribution of phagocytes to the host defense is usually assessed by determining phagocytosis and killing of Candida spp. blastoconidia. Dissection of the roles of various virulence factors in the infection process will involve the use of both in vitro and ex vivo assays. These assays are very useful as one of the approaches to determine the ... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332918 Mon, 01 Sep 2008 00:00:00 +0100 4332918 http://www.medworm.com/index.php?rid=4332917&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_2 The measurement of antibodies in the external secretions that bathe mucosal surfaces is important in understanding the host response to the opportunistic pathogen, Candida albicans and its determinants of pathogenesis at these sites. The principal immunoglobulin isotype in mucosal secretions is secretory immunoglobulin A (SIgA). Unlike the circulatory system, mucosal surfaces are open systems in which the concentrations of immune factors are affected by diurnal variation, changes in flow rate, complex formation with mucins, and other variables. Thus, it is necessary to control these factors if meaningful data are to be obtained. This chapter outlines methods for the measurement of anti-Candida SIgA antibodies in primary units and shows how to control the factors that influence antibody mea... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332917 Mon, 01 Sep 2008 00:00:00 +0100 4332917 http://www.medworm.com/index.php?rid=4332916&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_18 (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332916 Mon, 01 Sep 2008 00:00:00 +0100 4332916 http://www.medworm.com/index.php?rid=4332915&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_17 We report here a strategy that is useful and successful for large-scale genetic manipulation of C. albicans genes of interest: use of the UAU1 cassette on a Tn7 transposon. Streamlined yet admittedly flawed disruption techniques, such as the one described here, may prove vital to uncovering the genetic basis of fungal virulence. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332915 Mon, 01 Sep 2008 00:00:00 +0100 4332915 http://www.medworm.com/index.php?rid=4332914&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_16 Genetic transformation is the primary method of genetic manipulation of Candida albicans. The lack of a complete sexual cycle prevents application of classical genetic analyses. However, transformation permits introduction into the genome of a wide variety of defined mutations including deletions, insertions, and fusions. Although several methods of transformation are available, the lithium-cation-induced transformation method described here is the most commonly used. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332914 Mon, 01 Sep 2008 00:00:00 +0100 4332914 http://www.medworm.com/index.php?rid=4332913&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_15 Reporter systems are used in Candida albicans in three major experimental areas. These include gene expression, promoter analysis, and protein expression/localization. Heterologous expression in C. albicans is either not effective or inefficient due to the alternative codon usage in Candida, particularly CTG. Consequently, several reporter genes have been constructed by optimizing codons for expression in Candida. The reporter systems include lacZ, luciferase, and GFP. Generally, PCR site directed mutagenesis has been used to construct the modified reporter. Reporter gene vectors are not commercially available for Candida, but they can normally be requested from the laboratories that developed the constructs. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332913 Mon, 01 Sep 2008 00:00:00 +0100 4332913 http://www.medworm.com/index.php?rid=4332912&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_14 Proteomics is the term for the large-scale analysis of proteins. Such studies have contributed greatly to our understanding of gene function in the postgenomic era. Besides identification, the characterization of protein function is also a key objective of proteomic research. One of the major challenges for Candida biologists is how to interpret the large amounts of transcriptional analysis data, and assign function(s) to numerous uncharacterized genes, and the proteins they encode. In this regard, the Candida albicans cell wall and its constituents have been a topic of research for many scientists largely due to its importance as a potential target for antifungal therapy and its role in pathogenesis. Thus, the application of proteomic technology to cell wall analysis is timely, and cell w... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332912 Mon, 01 Sep 2008 00:00:00 +0100 4332912 http://www.medworm.com/index.php?rid=4332911&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_13 Tandem-affinity purification (TAP) tagging systems, developed by the research group of Bertrand Seraphin and others, are a means of isolating physiologically relevant protein and protein–nucleic acid complexes. Where complete (or nearly complete) genome sequence data are available for the organism from which the complexes are isolated, their components can be readily identified using mass spectrometry. The most widely used TAP-tag consists of a proximal calmodulin-binding peptide (CBP) and a distal repeated protein A IgG-binding domain with a cleavage site for the tobacco etch virus (TEV) protease positioned between this and the CBP. This tag is expressed as a co-translational fusion to the protein of interest. Purification is achieved under mild conditions through sequential affinit... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332911 Mon, 01 Sep 2008 00:00:00 +0100 4332911 http://www.medworm.com/index.php?rid=4332910&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_12 It is sometimes necessary to assess the genetic relatedness of isolates to identify the origin of an infection. In addition, evidence is accumulating that drug resistance can be associated with strains from a particular clade and that strains can exhibit anatomical specificity. It may, therefore, be valuable in the near future to screen for strains with a propensity for drug resistance. While a number of methods exist for genetically fingerprinting the infectious fungi, only a few provide the necessary resolution not only for distinguishing whether strains are highly related or unrelated, but also for grouping a strain in a particular clade. Here, we provide the procedures for performing the two methods that have proven most effective in the past 5 years: Southern blot hybridization of res... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332910 Mon, 01 Sep 2008 00:00:00 +0100 4332910 http://www.medworm.com/index.php?rid=4332909&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_11 Intraspecific differentiation of pathogenic microorganisms is a major need in epidemiological studies concerning the source and spread of infections. This requirement is paramount for those etiologic agents of infectious diseases, which are mainly grouped into one species within the genus, such as Candida albicans. Ideally, laboratory methods for biotyping purposes should be sensitive, reproducible, easy, and economical to perform. In addition, the methods should be flexible in their application to taxonomically unrelated pathogens. We have shown that the toxins produced by a selected panel of killer yeasts, each characterized by a wide spectrum of antimicrobial activity, may be used to discriminate strains belonging to the species of the genus Candida and to other species of eukaryotic an... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332909 Mon, 01 Sep 2008 00:00:00 +0100 4332909 http://www.medworm.com/index.php?rid=4332908&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_10 The measurement of virulence using ex vivo and in vitro models is discussed in the context of the human pathogenic yeast, Candida albicans. The models described are of two types. First, reconstituted tissues of various sorts are used that are derived from human carcinomas. The tissues are grown in vitro in complex media, attain a three-dimensional tissue structure, and retain cell-surface antigens typical of the specific tissue. Both adherence and invasion of tissues can be studied following infection with strains of C. albicans (1, 2). Further, one can increase the level of complexity by providing infected tissues with host phagocytes or cytokines such that an immune contribution to protection can be followed (3–5). The second model employs Drosophila melanogaster larvae tha... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332908 Mon, 01 Sep 2008 00:00:00 +0100 4332908 http://www.medworm.com/index.php?rid=4332907&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-151-6_1 A method is described to generate dendritic cells (DCs) from human peripheral blood mononuclear cells (PBMCs). The procedure involves two major steps: (1) preparation of monocytes from human PBMCs and (2) in vitro differentiation of the monocytes into DCs by growth factors and cytokines. Cells obtained in this fashion are screened for the presence or absence of antigenic markers characteristic of DCs by flow cytometry. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4332907 Mon, 01 Sep 2008 00:00:00 +0100 4332907 http://www.medworm.com/index.php?rid=1970076&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_31 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 standard protocols that have proven valuable in the assessment of HCV-specific T-cell responses. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970076 Tue, 01 Jul 2008 04:00:00 +0100 1970076 http://www.medworm.com/index.php?rid=1970075&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_30 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 protocols are described. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970075 Tue, 01 Jul 2008 04:00:00 +0100 1970075 http://www.medworm.com/index.php?rid=1970074&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_3 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) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970074 Tue, 01 Jul 2008 04:00:00 +0100 1970074 http://www.medworm.com/index.php?rid=1970073&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_29 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 reviews development, validation, and early application of a mouse model with a chimeric human liver. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970073 Tue, 01 Jul 2008 04:00:00 +0100 1970073 http://www.medworm.com/index.php?rid=1970072&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_28 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 perfusion and primary hepatocyte isolation that allows us to establish robust and reliable human primary hepatocyte cultures. Moreover, we have demonstrated that these primary... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970072 Tue, 01 Jul 2008 04:00:00 +0100 1970072 http://www.medworm.com/index.php?rid=1970071&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_27 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 JFH1 and all chimeras, except one, can easily be adapted to cultured cells, increasing virus yields by up to three orders of magnitude. Surprisingly, adaptation is achiev... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970071 Tue, 01 Jul 2008 04:00:00 +0100 1970071 http://www.medworm.com/index.php?rid=1970070&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_26 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 from a Japanese patient with fulminant hepatitis) in transfected Huh-7 cells represents a characteristic feature intrinsic to this particular isolate and has very recently m... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970070 Tue, 01 Jul 2008 04:00:00 +0100 1970070 http://www.medworm.com/index.php?rid=1970069&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_25 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 use viral RNA derived from the genotype 2a JFH-1 strain of HCV. Although these systems mark a significant breakthrough for HCV research, the parallel development of a tr... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970069 Tue, 01 Jul 2008 04:00:00 +0100 1970069 http://www.medworm.com/index.php?rid=1970068&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_24 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) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970068 Tue, 01 Jul 2008 04:00:00 +0100 1970068 http://www.medworm.com/index.php?rid=1970067&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_23 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. The HCV infection system contributes to our understanding of HCV virology and may permit development of novel antiviral strategies. (Source: Springer protocols feed by ... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970067 Tue, 01 Jul 2008 04:00:00 +0100 1970067 http://www.medworm.com/index.php?rid=1970066&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_22 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 interactions between viral envelope glycoprotein and cellular receptor or coreceptor or by inhibiting the viral fusion process, would serve as attractive antiviral drugs... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970066 Tue, 01 Jul 2008 04:00:00 +0100 1970066 http://www.medworm.com/index.php?rid=1970065&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_21 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 models have remained limited to chimpanzee (1) and immunodeficient mice carrying engrafted human liver cells (2). HCV pseudoparticles (HCVpp) were the first in vitro infecti... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970065 Tue, 01 Jul 2008 04:00:00 +0100 1970065 http://www.medworm.com/index.php?rid=1970064&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_20 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) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970064 Tue, 01 Jul 2008 04:00:00 +0100 1970064 http://www.medworm.com/index.php?rid=1970063&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_2 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) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970063 Tue, 01 Jul 2008 04:00:00 +0100 1970063 http://www.medworm.com/index.php?rid=1970062&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_19 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) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970062 Tue, 01 Jul 2008 04:00:00 +0100 1970062 http://www.medworm.com/index.php?rid=1970061&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_18 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 well. This duplex measurement greatly increases the efficiency of the HTS while lowering the cost. The usefulness of this approach has been supported by the recent discove... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970061 Tue, 01 Jul 2008 04:00:00 +0100 1970061 http://www.medworm.com/index.php?rid=1970060&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_17 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 subjected to a double selection by G418 and escalating dosages of a compound of choice. Here we describe an alternative screening assay that takes advantage of an HCV replicon t... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970060 Tue, 01 Jul 2008 04:00:00 +0100 1970060 http://www.medworm.com/index.php?rid=1970059&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_16 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 subsequent IFN induction by cleaving critical cellular substrates within the intracellular antiviral signaling pathways upstream of IRF-3 with its major protease, NS3/4A. (Sou... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970059 Tue, 01 Jul 2008 04:00:00 +0100 1970059 http://www.medworm.com/index.php?rid=1970058&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_15 We describe these techniques and highlight selected results. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970058 Tue, 01 Jul 2008 04:00:00 +0100 1970058 http://www.medworm.com/index.php?rid=1970057&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_14 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 peptide/protein database search and immunobiochemistry and functional genomic studies. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970057 Tue, 01 Jul 2008 04:00:00 +0100 1970057 http://www.medworm.com/index.php?rid=1970056&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_13 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 other single stranded. NS5B nucleoside inhibitors block synthesis of both species, whereas nonnucleoside inhibitors inhibit mostly single- tranded RNA synthesis. Our resul... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970056 Tue, 01 Jul 2008 04:00:00 +0100 1970056 http://www.medworm.com/index.php?rid=1970055&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_12 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 provide. More recently, researchers have used similar sequencing methods to identify changes in replicons that represent viral adaptation to engineered mutations, adaptati... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970055 Tue, 01 Jul 2008 04:00:00 +0100 1970055 http://www.medworm.com/index.php?rid=1970054&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_11 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) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970054 Tue, 01 Jul 2008 04:00:00 +0100 1970054 http://www.medworm.com/index.php?rid=1970053&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_10 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) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970053 Tue, 01 Jul 2008 04:00:00 +0100 1970053 http://www.medworm.com/index.php?rid=1970052&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_1 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 divided into two categories by method: those based on signal amplification and those based on target amplification. These two categories are then divided into qualitative, q... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1970052 Tue, 01 Jul 2008 04:00:00 +0100 1970052 http://www.medworm.com/index.php?rid=4461278&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_33 In the last decade, viral kinetic modeling has played an important role in the analysis of HCV RNA decay after the initiation of antiviral therapy. Models have provided a means of evaluating the antiviral effectiveness of therapy and of estimating parameters, such as the rate of virion clearance and the rate of loss of HCV-infected cells, and they have suggested mechanisms of action for both interferon-α and ribavirin. The inclusion of homeostatic proliferation of infected and uninfected hepatocytes in existing viral kinetic models has allowed prediction of most observed HCV RNA profiles under treatment, for example, biphasic and triphasic viral decay and viral rebound to baseline values after the cessation of therapy. In addition, new kinetic models have taken into consideration the... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4461278 Tue, 01 Jul 2008 00:00:00 +0100 4461278 http://www.medworm.com/index.php?rid=4461277&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-394-3_32 Infectious HCV pseudoparticles (HCVpp) can be assembled by display of unmodified and functional HCV glycoproteins on retroviral and lentiviral core particles. HCVpp have been shown to mimic the early infection steps of parental HCV. The presence of a marker gene packaged within these HCV pseudoparticles allows reliable and fast determination of infectivity mediated by the HCV glycoproteins. With this highly flexible system, E1E2 from a broad range of HCV strains can be investigated, including autologous HCV strains from patients’ virus, and it has allowed careful investigation of the humoral response to HCV. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=4461277 Tue, 01 Jul 2008 00:00:00 +0100 4461277 http://www.medworm.com/index.php?rid=1538968&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_5 Serological methods, gene sequencing, and RT-PCR-based methods have all been used for the identification of influenza virus hemagglutinin (HA) subtypes. Compared to serological methods and gene sequencing, RT-PCR is fast, sensitive, and relatively inexpensive. However, since RT-PCR generally lacks the specificity of sequencing or serology, the most practical application of RT-PCR methods for subtype identification is either to target a few of the most important subtypes such as H5 and H7 or to use it in situations where a specific strain is being targeted, such as during an outbreak or with experimental samples. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538968 Fri, 08 Feb 2008 05:00:00 +0100 1538968 http://www.medworm.com/index.php?rid=1538967&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_2 Successful detection of the avian influenza (AI) virus, viral antigen, nucleic acid, or antibody is dependent upon the collection of the appropriate sample type, the quality of the sample, and the proper storage and handling of the sample. The diagnostic tests to be performed should be considered prior to sample collection. Sera are acceptable samples for ELISA or agar gel precipitin tests, but not for real-time RT-PCR. Likewise, swabs and/or tissues are acceptable for real-time RT-PCR and virus isolation. The sample type will also depend on the type of birds that are being tested; oropharyngeal swabs should be collected from poultry, and cloacal swabs should be collected from waterfowl. This chapter will outline the collection of different specimen types and procedures for proper specimen... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538967 Fri, 08 Feb 2008 05:00:00 +0100 1538967 http://www.medworm.com/index.php?rid=1538966&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_8 The hemagglutination-inhibition (HI) assay is a classical laboratory procedure for the classification or subtyping of hemagglutinating viruses. For the avian influenza (AI) virus, the HI assay is used to identify the hemagglutinin (H) subtype of an unknown AI virus isolate or the HA subtype specificity of antibodies to AI virus. Since the HI assay is quantitative, it is frequently applied to evaluate the antigenic relationships between different AI virus isolates of the same subtype. The basis of the HI test is inhibition of hemagglutination with subtype-specific antibodies. The HI assay is a relatively inexpensive procedure utilizing standard laboratory equipment, is less technical than molecular tests, and is easily completed within several hours. However, when working with uncharacteriz... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538966 Fri, 08 Feb 2008 05:00:00 +0100 1538966 http://www.medworm.com/index.php?rid=1538965&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_10 Immunohistochemical methods are commonly used for studying the pathogenesis of the avian influenza (AI) virus by allowing the identification of sites of replication of the virus in infected tissues and the correlation with the histopathological changes observed. In this chapter, the materials and methods for performing immunohistochemical detection of AI virus antigens in tissues are provided. The technique involves the following steps: heat-induced antigen retrieval; binding of a primary antibody to a virus type-specific antigen; antibody-antigen complex binding by a biotinylated secondary antibody; and binding of an enzyme-streptavidin conjugate. The enzyme is then visualized by application of the substrate chromogen solution to produce a colorimetric end product. Demonstration of AI vir... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538965 Fri, 08 Feb 2008 05:00:00 +0100 1538965 http://www.medworm.com/index.php?rid=1538964&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_13 The measurement of avian cellular immunity is critical to understanding the role and regulation of avian lymphocytes following avian influenza (AI) virus infection. Although the ability to measure avian T cell responses has steadily increased over the last few years, few studies have examined the role of cell-mediated immunity in avian species against the AI virus. Because of the structural and functional differences between mammalian and avian immune systems—including MHC architecture, different modes of somatic recombination for antibody production, and the absence of lymph nodes in birds—the extent to which birds and mammals regulate similar immune responses against the AI virus is currently under investigation. The increasing availability of monoclonal antibodies recognizin... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538964 Fri, 08 Feb 2008 05:00:00 +0100 1538964 http://www.medworm.com/index.php?rid=1538963&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_7 The hemagglutination (HA) assay is a tool used to screen cell culture or amnioallantoic fluid harvested from embryonating chicken eggs for hemagglutinating agents, such as type A influenza. The HA assay is not an identification assay, as other agents also have hemagglutinating properties. Live and inactivated viruses are detected by the HA test. Amplification by virus isolation in embryonating chicken eggs or cell culture is typically required before HA activity can be detected from a clinical sample. The test is, to some extent, quantitative [1 hemagglutinating unit (HAU) is equal to approximately 5–6 logs of virus]. It is inexpensive and relatively simple to conduct. Several factors (quality of chicken erythrocytes, laboratory temperature, laboratory equipment, technical expertise ... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538963 Fri, 08 Feb 2008 05:00:00 +0100 1538963 http://www.medworm.com/index.php?rid=1538962&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_1 The avian influenza (AI) virus is type A influenza isolated from and adapted to an avian host. Type A influenza belongs to the orthomyxovirdae virus family, is enveloped, and is pleiomorphic with a size ranging from 80–120 nm (reviewed in [1]). Type A influenza strains are classified by the serological subtypes of the primary viral surface proteins, the hemagglutinin (HA) and neuraminidase (NA). The HA has 16 subtypes (H1–H16) and contains neutralizing epitopes. Antibodies against the NA are not neutralizing, and there are nine neuraminidase or “N” subtypes. The “H” and N subtypes seem to be able to assort into any combination, and many of the 144 possible combinations have been found in natural reservoir species, although some combinations are more comm... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538962 Fri, 08 Feb 2008 05:00:00 +0100 1538962 http://www.medworm.com/index.php?rid=1538961&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_12 Reverse genetics is the creation of a virus from a full-length cDNA copy of the viral genome, referred to as an infectious clone, and is the most powerful genetic tool in modern virology. The generation of influenza A viruses by reverse genetics has lagged mainly due to the inherent technical difficulties of providing a sufficient amount of all eight viral RNAs from cloned cDNA. A breakthrough was made in 1999 by utilizing the cellular enzyme RNA polymerase I for the synthesis of influenza viral RNAs. Although slightly different methods are being used in different laboratories for the rescue of the influenza virus, the basic concept of synthesizing viral RNA using RNA polymerase I remains the same. Coupled with in vitro mutagenesis, reverse genetics can be applied widely to accelerate prog... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538961 Fri, 08 Feb 2008 05:00:00 +0100 1538961 http://www.medworm.com/index.php?rid=1538960&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_4 Real-time RT-PCR (rRT-PCR) is a relatively new technology that has been used for avian influenza (AI) virus detection since the early 2000s for routine surveillance, during outbreaks, and for research. Some of the advantages of rRT-PCR are high sensitivity, high specificity, rapid time-to-result, scalability, cost, and quantitative nature. Furthermore, rRT-PCR can be used with numerous sample types, is less expensive than virus isolation in chicken embryos, and since infectious virus is inactivated early during processing, biosafety and biosecurity are also easier to maintain. This chapter will provide an overview of the USDA-validated rRT-PCR procedure for the detection of type A influenza. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538960 Fri, 08 Feb 2008 05:00:00 +0100 1538960 http://www.medworm.com/index.php?rid=1538959&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_9 The neuraminidase-inhibition (NI) assay is a laboratory procedure for the identification of the neuraminidase (NA) glycoprotein subtype in influenza viruses or the NA subtype specificity of antibodies to influenza virus. A serological procedure for subtyping the NA glycoprotein is critical for the identification and classification of avian influenza (AI) viruses. The macro-procedure was first described in 1961 by D. Aminoff et al. [2] and was later modified to a microtiter plate procedure (micro-NI) by Van Deusen et al. [4]. The micro-NI procedure reduces the quantity of reagents required, permits the antigenic classification of many isolates simultaneously, and eliminates the spectrophotometric interpretation of results. Although the macro-NI has been shown to be more sensitive than the m... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538959 Fri, 08 Feb 2008 05:00:00 +0100 1538959 http://www.medworm.com/index.php?rid=1538958&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_6 The avian influenza (AI) virus is usually isolated and propagated by inoculating either swab or tissue samples from infected birds into the chorioallantoic sac of embryonating chicken eggs. This is the accepted method, but occasionally an isolation may only be successful when inoculated either into the yolk sac or onto the chorioallantoic membrane of embryonating chicken eggs. Chorioallantoic fluid is harvested from eggs with dead or dying embryos and is tested for the presence of hemagglutinating antigen. If hemagglutination-positive, this indicates that the isolate may be the AI virus. The presence of the AI virus may be confirmed by either an agar gel immunodiffusion (AGID) assay, RT-PCR specific for AI virus, or a commercially available immunoassay kit specific for type A influenza. In... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538958 Fri, 08 Feb 2008 05:00:00 +0100 1538958 http://www.medworm.com/index.php?rid=1538957&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_3 The efficient extraction and purification of viral RNA are critical for downstream molecular applications, whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of the avian influenza (AI) virus by molecular methods from experimentally infected birds. Samples can generally be divided into two types: enriched (e.g., virus stocks) and clinical. Clinical type samples, which may be tissues or swab material, are the most difficult to process due to the complex sample composition and possibly low virus titers. In this chapter, two well-established procedures for the isolation of AI virus RNA from common clinical specimen types and enriched virus stocks for further molecular applications will be presented. (Source: Sp... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538957 Fri, 08 Feb 2008 05:00:00 +0100 1538957 http://www.medworm.com/index.php?rid=1538956&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_14 Functional and molecular techniques have both been employed to measure the production of cytokines following influenza infection. Historically, the use of functional or antibody-based techniques was employed in mammalian immunology. In avian immunology, only a few commercial antibodies are available to measure avian cytokines. However, the determination of the genomic sequence of Gallus gallus species has made it possible to measure cytokine induction without monoclonal antibody- or functional-based tests, but rather based on molecular techniques. Although these tests do not measure functionally expressed cytokines, the lack of reagents to identify and quantify avian cytokines makes them critical to extend any measure of cytokine response. Measurement of cytokine induction, based on the de... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538956 Fri, 08 Feb 2008 05:00:00 +0100 1538956 http://www.medworm.com/index.php?rid=1538955&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-279-3_11 Avian influenza (AI) viruses have been isolated from a wide diversity of free-living avian species representing several orders. Isolations are most frequently reported from aquatic birds in the Orders Anseriformes and Charadriiformes, which are believed to be the reservoirs for all AI viruses. Since their first recognition in the late 1800 s, AI viruses have been an important agent of disease in poultry and, occasionally, of nongallinaceous birds and humans. However, the recent highly pathogenic avian influenza (HPAI) H5N1 virus epidemics have increased the awareness of AI viruses and their potential implications among the scientific community, politicians, and the general public. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538955 Fri, 08 Feb 2008 05:00:00 +0100 1538955 http://www.medworm.com/index.php?rid=1538979&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-468-1_13 The microarray has shown tremendous potential for investigating gene expression profiles and expression levels in comparative biology; exploring the regulation mechanisms of gene expression; and evaluating target gene for developing new chemotherapeutic agents, vaccine, and diagnostic methods. In this chapter, we provide a detailed protocol for scientists who wish to investigate gene expression profiles by performing a microarray analysis, including different methods of RNA purification, decontamination, cDNA synthesis, fragmentation, and biotin labeling for hybridization using Affymetrix Staphylococcus aureus chips. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538979 Thu, 27 Sep 2007 04:00:00 +0100 1538979 http://www.medworm.com/index.php?rid=1538978&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-468-1_10 As with many other bacterial species, the most commonly used method to assess staphylococcal biofilm formation in vitro is the microtiter plate assay. This assay is particularly useful for comparison of multiple strains including large-scale screens of mutant libraries. When such screens are applied to the coagulase-negative staphylococci in general, and Staphylococcus epidermidis in particular, they are relatively straightforward by comparison with microtiter plate assays used to assess biofilm formation in other bacterial species. However, in the case of clinical isolates of Staphylococcus aureus, including methicillin-resistant S. aureus, we have found it necessary to employ specific modifications including precoating of the wells of the microtiter plate with plasma proteins and supplem... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538978 Thu, 27 Sep 2007 04:00:00 +0100 1538978 http://www.medworm.com/index.php?rid=1538977&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-468-1_8 In recent years, molecular genetic approaches to the study of the disease pathogenesis of Staphylococcus aureus have resulted in many new biological insights. I describe methods used for targeted disruption of staphylococcal genes leading to loss of gene function, important for studies of staphylococcal proteins and their role in virulence. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538977 Thu, 27 Sep 2007 04:00:00 +0100 1538977 http://www.medworm.com/index.php?rid=1538976&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-468-1_11 Multiple drug resistance to antibiotics is a major public health problem. Many mechanisms may be involved in such resistance. Increasing data have shown that Staphylococcus aureus can invade different types of nonphagocytic cells, which, in turn, may contribute to evasion of the toxicity of certain antibiotics. The fibronectin-binding proteins are required for S. aureus to adhere to and internalize into the host cells. We have shown that a two-component signal transduction system, SaeRS, is essential for bacterial adhesion and invasion of the epithelial cells. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538976 Thu, 27 Sep 2007 04:00:00 +0100 1538976 http://www.medworm.com/index.php?rid=1538975&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-468-1_5 Pulsed-field gel electrophoresis (PFGE) is a genetic typing method that is widely used as a molecular epidemiological tool for studying the genetic diversity of Staphylococcus aureus and numerous other bacterial pathogens. For PFGE, intact bacterial cells are embedded in soft agarose plugs followed by lysis of the cell wall in situ to minimize shearing of the chromosome. The genome, which for S. aureus is approx 2.8 Mb, is then digested with a rare cutting restriction endonuclease and separated by agarose gel electrophoresis. The restriction fragments generated are too large to be resolved by conventional electrophoresis. Therefore, resolution of the bands is achieved in a “contour-clamped homogeneous electrical field“ where electrical current to the gel switches direction betw... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538975 Thu, 27 Sep 2007 04:00:00 +0100 1538975 http://www.medworm.com/index.php?rid=1538974&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-468-1_2 Staphylococcus aureus is a versatile pathogen associated with diverse clinical presentations. Only recently have the genetic factors underlying the virulence of this bacterial species become understood in a significant way. Methicillin-resistant S. aureus (MRSA) strains have been extremely important as nosocomial pathogens in health care facilities for more than three decades. Additionally, infections resulting from community-associated MRSA strains have emerged in the last decade and become a public health problem of global proportions. This changing epidemiology has spurred renewed interest in translating knowledge of the molecular determinants of virulence into rational prevention and control strategies. Four case histories are provided (three involving MRSA and one involving a methicil... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538974 Thu, 27 Sep 2007 04:00:00 +0100 1538974 http://www.medworm.com/index.php?rid=1538973&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-468-1_3 The widespread occurrence of methicillin-resistant Staphylococcus aureus (MRSA) or oxacillin-resistant MRSA is a major cause of concern worldwide. Although mainly located in hospital environments, these microorganisms have been reported to have the capacity to cause infections in the community. Resistance to methicillin implies resistance to all β-lactam antibiotics and, furthermore, MRSA isolates normally harbor resistance to other families of antibiotics such as co-trimoxazole and aminoglycosides. Prompt and accurate detection of MRSA isolates is therefore extremely important in clinical microbiology laboratories. In this chapter, we review the most common methods of susceptibility testing for MRSA. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538973 Thu, 27 Sep 2007 04:00:00 +0100 1538973 http://www.medworm.com/index.php?rid=1538972&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-468-1_17 We review data on the treatment of infections caused by drug-resistant Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA). In this review, we cover findings reported in the English language medical literature up to February 2006. Despite the emergence of resistant and multidrug resistant S. aureus, five effective drugs for which little resistance has been observed are in clinical use: vancomycin, quinupristin-dalfopristin, linezolid, tigecycline, and daptomycin. However, vancomycin is less effective for infections with MRSA isolates that have a high minimum inhibitory concentration in the susceptible range. Linezolid looks promising in the treatment of MRSA pneumonia and skin and soft-tissue infections (SSTIs). Daptomycin displays rapid bactericidal activity in vitr... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538972 Thu, 27 Sep 2007 04:00:00 +0100 1538972 http://www.medworm.com/index.php?rid=1538971&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-468-1_9 Staphylococcal superantigens (SAgs) comprise a large family of exotoxins produced by Staphylococcus aureus strains. These exotoxins are important in a variety of serious human diseases, including menstrual and nonmenstrual toxic shock syndrome (TSS), staphylococcal pneumonias, and a recently described staphylococcal purpura fulminans. In addition, these SAg exotoxins are being increasingly recognized for their possible roles in many other human diseases, such as atopic dermatitis, Kawasaki syndrome, nasal polyposis, and certain autoimmune disorders. To clarify the full spectrum of human diseases caused by staphylococcal SAgs, it is necessary to have assays for them. At present there are 17 well-characterized, serologically distinct SAgs made by S. aureus: TSS toxin-1; staphylococcal entero... Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538971 Thu, 27 Sep 2007 04:00:00 +0100 1538971 http://www.medworm.com/index.php?rid=1538970&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-468-1_6 Multilocus sequence typing (MLST) is a widely accepted method of DNA sequence-based typing that relies on analysis of relatively conserved genes that encode essential proteins. For Staphylococcus aureus, the level of discrimination provided by MLST is sufficient to provide a relatively detailed picture of the global dissemination of the organism. The technique is not restrictive in the precise methodology used to acquire the sequences, but the method of assigning types requires that the data be of high quality. Excellent Web-based tools have been developed and are curated by the groups that launched MLST. These tools have allowed the scheme to be maintained as a coherent global asset and assist users in the analysis of their data. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538970 Thu, 27 Sep 2007 04:00:00 +0100 1538970 http://www.medworm.com/index.php?rid=1538969&cid=s_37125_20_f&fid=37125&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-468-1_14 We describe here proteomic methods to prepare, identify, and analyze protein fractions, allowing the study of S. aureus on the organism level. Coupled with methods analyzing the whole bacterial transcriptome, this approach might contribute to the development of rapid diagnostic tests and to the identification of new drug targets. (Source: Springer protocols feed by Infectious Diseases) Springer protocols feed by Infectious Diseases news http://www.medworm.com/rss/comments.php?id=1538969 Thu, 27 Sep 2007 04:00:00 +0100 1538969