Methods in molecular medicine最新文献

In the post-Human Genome Project era, awareness of the resources available through the internet is essential to both molecular biologists and clinicians. An overview of the main databases and analytical tools described in this chapter is important to understand the principles upon which hypotheses are generated, experiments are based and conclusions reached. Similarly, an introduction to the terminology of these resources often facilitates their use and adoption into practice. This chapter covers database resources such as NCBI/ Entrez, Ensembl and UCSC as well as analytical tools for sequence alignment, promoter analysis and molecular interactions.

Complex diseases can involve the interaction of multiple genes and environmental factors. Discovering these genes is difficult, and in silico based strategies can significantly improve their detection. Data mining and automated tracking of new knowledge facilitate locus mapping. At the gene search stage, in silico prioritization of candidate genes plays an indispensable role in dealing with linked or associated loci. In silico analysis can also differentiate subtle consequences of coding DNA variants and remains the major method to predict functionality for non-coding DNA variants, particularly those in promoter regions.

Whole-genome association studies of complex diseases hold great promise to identify systematically genetic loci that influence one's risk of developing these diseases. However, the polygenic nature of the complex diseases and genetic interactions among the genes pose significant challenge in both experimental design and data analysis. High-density genotype data make it possible to identify most of the genetic loci that may be involved in the etiology. On the other hand, utilizing large number of statistic tests could lead to false positives if the tests are not adequately adjusted. In this paper, we discuss a two-stage method that sequentially applies a generalized linear model (GLM) and principal components analysis (PCA) to identify genetic loci that jointly determine the likelihood of developing disease. The method was applied to a pilot case-control study of esophageal squamous cell carcinoma (ESCC) that included 50 ESCC patients and 50 neighborhood-matched controls. Genotype data were determined by using the Affymetrix 10K SNP chip. We will discuss some of the special considerations that are important to the proper interpretation of whole genome-wide association studies, which include multiple comparisons, epistatic interaction among multiple genetic loci, and generalization of predictive models.

The rapid increase in the use of microarray studies has generated many questions on how to plan and design experiments that will effectively utilize this technology. Investigators often require answers to questions relating to microarray platforms, RNA samples, options for replication, allocation of samples to arrays, sample sizes, appropriate downstream analysis, and many others. Careful consideration of these issues is critical to ensure the efficiency and reliability of the actual microarray experiments, and will assist in enhancing interpretability of the experimental results.

The activation of mast cells is of pivotal importance in the pathogenesis of allergic conditions. Mast cell activation can provoke rapid increases in microvascular permeability, induce bronchoconstriction after blood flow, stimulate the recruitment and activation of other inflammatory cells, and has come to be associated with the processes of tissue remodeling and fibrosis. Such changes may be mediated by the release of a range of potent mediators of inflammation: preformed in secretory granules, or newly generated, or both. There are major differences in the responsiveness to various stimuli and to pharmacological agents for mast cells from different body compartments. A method is presented here for the purification of mast cells from enzymatically dispersed human tissues. The methods described for the experimental activation of mast cells can be readily adapted to studies with cell lines or mast cells obtained through long-term culture.

CD8 T cells play an important role in the regulation of allergic disease. Human and murine CD8 T cells have been shown to be capable of differentiating into distinct subsets defined by cytokine profiles analogous to the Th1 and Th2 subsets and termed T cytotoxic 1 (Tc1, IFN-gamma producing) and 2 (Tc2, IL-4 producing). Effector cell phenotype can be analyzed in vitro on a single cell basis using intracellular cytokine staining and flow cytometry or analysis of other phenotypic markers. Human PBMC usually contain only very low percentages of effector cells which produce relatively high levels of cytokines required for this kind of analysis. It is therefore necessary to activate the T cells to induce rapid accumulation of cytoplasmic cytokines before analysis. This makes it difficult to analyze the antigen specificity of responding T cells but will indicate the type 1/type 2 bias of the population, reflecting previous exposures to antigen. In this chapter, we provide protocols for the generation of polarized populations of CD8 T effector cells using polyclonal stimulation and for their subsequent analysis by intracellular cytokine staining.

Monoclonal antibodies (mabs) are powerful tools for the quantification, detection, and targeting of specific molecules. Allergen-specific mabs are important for the quantification of major allergens in allergen preparations used for allergen-specific immunotherapy and allergy diagnosis. Indeed, progress in the understanding of the mechanisms of the immunological responses underlying allergic disease would not have been possible without the use of mabs. Quantification assays are also important in the assessment of environmental allergen exposure and monitoring of avoidance procedures.Mabs against human IgE provide the basis for various test systems for the detection of specific and nonspecific IgE. Mabs raised against IgE or defined cytokines or cytokine receptors have potential as neutralizing reagents in vivo for the treatment of allergic diseases.Allergen-specific mabs are also valuable tools for the localization of allergens within their source material and the characterization of allergens derived from natural sources and by recombinant technologies. Furthermore they are often used for the isolation of allergens from complex extracts by affinity chromatography. The procedure described in this chapter has been used successfully to produce mabs against numerous allergens from house dust mites, insect venoms, cat, hens egg white, tree-, grass-, and herb pollens, and fungi, with the ultimate aim of obtaining matched antibody pairs to establish two-site binding assays for the quantification of major allergens. The method has also been used successfully to generate mabs against human IgE.

Proper understanding of the pathogenesis of type I allergy relies on the identification of allergen epitopes. The phage display technique is a relatively new one to define peptide structures that mimic natural epitopes, including conformational B-cell epitopes. Peptides displayed on the phage recognized by an antiallergen antibody mimic the physicochemical properties of the amino acids and are, therefore, called mimotopes. The main advantage of the biopanning technique described in this chapter is that the structure of the antigen/allergen may be completely unknown; the only material needed is an antibody binding to it. The mimotopes generated by this technique display the features of the antigen/allergen but do not crosslink the mast cell-bound IgE-antibodies. Thus mimotopes could be used as a safe alternative to the commonly applied allergen extracts in immunotherapy of allergic patients and direct the immune response toward the desired allergen epitopes. In the selection procedure called biopanning, phages with the mimotopes best recognized by the selecting antibody are amplified. The titers of phages specifically binding to the selection antibody are checked. In this chapter we describe two alternative methods for colony screening: the immunoblot and ELISA.

Aminoacyl-tRNA synthetases (aa-RS) attracted interest as potential targets for new antibacterial compounds. Most organisms express 20 aa-RSs: one for each amino acid. Aa-RSs are essential proteins in all living organisms. When one aa-RS is inhibited, the corresponding tRNA is not charged and is therefore unavailable for translation. This leads to protein synthesis inhibition, which in turn causes cell growth arrest. Consequently, each compound that inhibits any of the aa-RS could be a potential antibacterial agent. Only one aa-RS inhibitor, the Ile-RS inhibitor mupirocin, is currently marketed as an antibacterial agent. We focused on phenylalanyl (Phe)-tRNA synthetase (Phe-RS), but the described methods are not restricted to Phe-RS and might be adapted to other aa-RS.