Springer protocols feed by Immunology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Quantifying CD4/CCR5 Usage Efficiency of HIV-1 Env Using the Affinofile System
Entry of HIV-1 into target cells involves the interaction of the HIV envelope (Env) with both a primary receptor (CD4) and a coreceptor (CXCR4 or CCR5). The relative efficiency with which a particular Env uses these receptors is a major component of cellular tropism in the context of entry and is related to a variety of pathological Env phenotypes (Chikere et al. Virology 435:81–91, 2013). The protocols outlined in this chapter describe the use of the Affinofile system, a 293-based dual-inducible cell line that expresses up to 25 distinct combinations of CD4 and CCR5, as well as the associated Viral Entry Receptor Se...
Source: Springer protocols feed by Immunology - December 7, 2015 Category: Allergy & Immunology Source Type: news
Computational Methods to Model Persistence
Bacterial persister cells are dormant cells, tolerant to multiple antibiotics, that are involved in several chronic infections. Toxin–antitoxin modules play a significant role in the generation of such persister cells. Toxin–antitoxin modules are small genetic elements, omnipresent in the genomes of bacteria, which code for an intracellular toxin and its neutralizing antitoxin. In the past decade, mathematical modeling has become an important tool to study the regulation of toxin–antitoxin modules and their relation to the emergence of persister cells. Here, we provide an overview of several numerical met...
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
Population Dynamics Analysis of Ciprofloxacin-Persistent S. Typhimurium Cells in a Mouse Model for Salmonella Diarrhea
In vivo, antibiotics are often surprisingly inefficient at eliminating bacterial pathogens. In the case of ciprofloxacin therapy in a Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium, S. Tm) mouse infection model, this has been traced to tolerant bacterial cells surviving in lymph node monocytes (i.e., classical dendritic cells). To analyze the growth characteristics of these persisters, we have developed a population dynamics approach using mixtures of wild-type isogenic tagged strains (WITS) and a computational model. Here, we are providing a detailed description of the inoculum, the infection experim...
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
Analysis of Macrophage-Induced Salmonella Persisters
A small subpopulation of non-replicating, multidrug-tolerant bacteria is present within clonal populations of many bacterial species. Known as persisters, these bacteria are probably the cause of relapsing infections such as typhoid fever. Formation of non-growing Salmonella persisters is stimulated by macrophage phagocytosis. This chapter outlines methods to identify and study persisters resulting from interactions between bacterial pathogens and their hosts. We use their antibiotic tolerance for isolation and enumeration and developed a method to study the heterogeneity of growth within clonal populations through single-...
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
A Murine Model for Escherichia coli Urinary Tract Infection
Urinary tract infections (UTI) are among the most common bacterial infections of humans. The mouse provides an excellent and tractable model system for cystitis and pyelonephritis caused by Escherichia coli and other uropathogens. Using a well-established model of experimental cystitis in which the bladders of female mice are infected via transurethral catheterization, the molecular details of the pathogenesis of bacterial cystitis have been substantially illuminated in the last decade. Uropathogenic E. coli attach to bladder epithelium (both in human and mouse) via adhesive type 1 pili, establish a replicative niche withi...
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
In Vitro Models for the Study of the Intracellular Activity of Antibiotics
Intracellular bacteria are poorly responsive to antibiotic treatment. Pharmacological studies are thus needed to determine which antibiotics are most potent or effective against intracellular bacteria as well as to explore the reasons for poor bacterial responsiveness. An in vitro pharmacodynamic model is described, consisting of (1) phagocytosis of pre-opsonized bacteria by eukaryotic cells; (2) elimination of non-internalized bacteria with gentamicin; (3) incubation of infected cells with antibiotics; and (4) determination of surviving bacteria by viable cell counting and normalization of the counts based on sample prote...
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
Experimental Evolution of Escherichia coli Persister Levels Using Cyclic Antibiotic Treatments
Persister cells are difficult to study owing to their transient nature and their usually small number in bacterial populations. In the past, numerous attempts have been made to elucidate persistence mechanisms. However, because of the challenges involved in studying persisters and the clear redundancy in mechanisms underlying their generation, our knowledge of molecular pathways to persistence remains incomplete. Here, we describe how to use experimental evolution with cyclic antibiotic treatments to generate mutants with an increased persister level in stationary phase, ranging from the initial ancestral level up to 100&n...
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
Functional Analysis of the Role of Toxin–Antitoxin (TA) Loci in Bacterial Persistence
We have developed a method to analyze the functionality of putative TA loci by expressing them in Escherichia coli. Here, we describe the procedure for cloning recombinant TA genes into inducible plasmids and expressing these in E. coli. Following expression, toxicity, resuscitation of growth, and changes in persister cell formation are assayed. This can confirm whether predicted TA loci are active in E. coli and whether expression can affect persister cell formation. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
A Whole-Cell-Based High-Throughput Screening Method to Identify Molecules Targeting Pseudomonas aeruginosa Persister Cells
Despite its clinical relevance and the fact that the phenomenon of persistence was discovered in the 1940s, little is known about the mechanisms behind persister cell formation. Research in this field has mainly focused on the model organism Escherichia coli and few genetic determinants of persistence have been described in other bacterial species, impairing the development of target-based strategies to combat these antibiotic-tolerant cells. In this chapter we describe a top-down large-scale screening method capable of specifically identifying small molecule compounds that, in combination with conventional antibiotics, si...
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
Single-Cell Detection and Collection of Persister Bacteria in a Directly Accessible Femtoliter Droplet Array
A directly accessible femtoliter droplet array as a platform for single-cell detection and collection of persister bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single cells, long-term culture and observation of single cells in droplets, and collection of identified persisters from single droplets are described in detail. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
Analyzing Persister Physiology with Fluorescence-Activated Cell Sorting
Bacterial persisters are phenotypic variants that exhibit an impressive ability to tolerate antibiotics. Persisters are hypothesized to cause relapse infections, and therefore, understanding their physiology may lead to novel therapeutics to treat recalcitrant infections. However, persisters have yet to be isolated due to their low abundance, transient nature, and similarity to the more highly abundant viable but non-culturable cells (VBNCs), resulting in limited knowledge of their phenotypic state. This technical hurdle has been addressed through the use of fluorescence-activated cell sorting (FACS) and quantification of ...
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
Quantitative Measurements of Type I and Type II Persisters Using ScanLag
The present method quantifies the number of slow-growing bacteria leading to antibiotic persistence in a clonal population. First, it enables discriminating between slow growers that are generated by exposure to a stress signal (Type I persisters) and slow growers that are continuously generated during exponential growth (Type II persisters). Second, the method enables determining the amount of slow growers in a culture. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
Protocol for Determination of the Persister Subpopulation in Candida Albicans Biofilms
In contrast to planktonic cultures of the human fungal pathogen Candida albicans, C. albicans biofilms can contain a persister subpopulation that is tolerant to high concentrations of currently used antifungals. In this chapter, the method to determine the persister fraction in a C. albicans biofilm treated with an antifungal compound is described. To this end, a mature biofilm is developed and subsequently treated with a concentration series of the antifungal compound of interest. Upon incubation, the fraction of surviving biofilm cells is determined by plating and plotted versus the used concentrations of the antifungal ...
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
A Microplate-Based System as In Vitro Model of Biofilm Growth and Quantification
We describe a 96-well microtiter plate-based system as an in vitro model for biofilm formation and quantification. Although in vitro assays are artificial systems and thus significantly differ from in vivo conditions, they represent an important tool to evaluate biofilm formation and the effect of compounds on biofilms. Stainings to evaluate the amount of biomass (crystal violet staining) and the number of metabolically active cells (resazurin assay) are discussed and specific attention is paid to the use of this model to quantify persisters in sessile populations. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
Optimized Method for Measuring Persistence in Escherichia coli with Improved Reproducibility
Monitoring persister cells can be extremely difficult due to their transient and stochastic nature, their low abundance, and their resemblance to Viable But Non-Culturable Cells (VBNCs). To date, the predominant method consists of determining the survival rate of a bacterial population after antibiotic treatment as a function of time or antibiotic concentration. Unfortunately, this method is limited, as it shows high levels of dispersion of the data around the mean, making interpretation difficult. Furthermore, additional reproducibility problems arise from the lack of a standard method, different research groups using dif...
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
