Methods in molecular medicine最新文献

Allergen-specific T-cell lines established from allergic patients provide the opportunity of investigating T-cell functions at the poly- or oligoclonal level. T-cell lines are useful in determining the presence or absence of antigen-specific T-cell reactivity. However, to obtain detailed knowledge of the action of T cells with clearly defined features, for example epitope specificity or phenotype, T-cell clones are necessary.The frequency of allergen-specific T cells in peripheral blood mononuclear cells (PBMC) tends to be low and so stimulation of PBMC with single allergens often results in low allergen-specific reactivity or requires high doses of the allergen. In contrast, the stimulation of PBMC with whole allergen extract results in stronger reactivity because a greater spectrum of T-cell specificities is addressed. Therefore, for the investigation of polyclonal reactivity toward single allergens it is useful to establish T-cell lines, which represent an allergen-specific enrichment of T cells from the respective individual. These T cells are poly- or oligoclonal and might possess different epitope specificities. The method described here is based on experiences with human T-cell lines and clones specific for several allergens from grass pollens and tree pollens.

The complete genome sequences of about 300 bacteria (mostly pathogenic) have been determined, and many more such projects are currently in progress. The detection of bacterial genes that are non-homologous to human genes and are essential for the survival of the pathogen represent a promising means of identifying novel drug targets. We present a subtractive genomics approach for the identification of putative drug targets in microbial genomes and demonstrate its execution using Pseudomonas aeruginosa as an example. The resultant analyses are in good agreement with the results of systematic gene deletion experiments. This strategy enables rapid potential drug target identification, thereby greatly facilitating the search for new antibiotics. It should be recognized that there are limitations to this computational approach for drug target identification. Distant gene relationships may be missed since the alignment scores are likely to have low statistical significance. In conclusion, the results of such a strategy underscore the utility of large genomic databases for in silico systematic drug target identification in the post-genomic era.

This chapter describes reliable flow cytometric methods for assessment of two important physiologic characteristics of bacteria, membrane potential and membrane permeability, which can provide indications of the effects of antimicrobial agents on microorganisms.

A problem frequently encountered by the biological scientist is the identification of a previously unknown gene or protein sequence, where there are few or no clues as to the biochemical function, ligand specificity, gene regulation, protein-protein interactions, tissue specificity, cellular localization, developmental phase of activity, or biological role. Through the process of bioinformatics there are now many approaches for predicting answers to at least some of these questions, often then allowing the design of more insightful experiments to characterize more definitively the new protein.

Complex diseases and traits are influenced by a combination of genetic and environmental risk factors, some of which may be known, and many of which are unknown. It is possible to estimate the relative importance of the influence of genes and environment on a trait by studying correlations in the trait in related individuals. Known risk factors can be measured and included in the statistical models to understand disease etiology better. The joint effect of specific genes and environmental exposures can be estimated by measuring these in individuals, not necessarily related, with and without the disease of interest or with a range of trait values. These methods are illustrated by considering two example analyses in detail. The first is an analysis of a study of adolescent twins, quantifying the effect of genes and environment, including measured sun exposure, on the density of nevi. The second is an analysis of a case-control study, examining the joint effect of the GSTT1 gene and vegetable intake on risk of colorectal cancer.

Human cytomegalovirus (HCMV) is one of the largest known DNA viruses. It is ubiquitous, and following resolution of primary productive infection, it persists in the human host by establishing a lifelong latent infection in myeloid lineage cells such as monocytes and their progenitors. Most adults with HCMV infection are healthy but it can cause neurologic deficits in infants, and remains an important cause of morbidity and mortality in the immunosuppressed patient. Microarray-based studies of HCMV have provided useful information about genes that are transcriptionally active during both productive and latent phases of infection. This chapter describes how to study genes in HCMV using microarrays and two cell types (productively infected human foreskin fibroblasts, and latently infected primary human myeloid progenitor cells).

Allergens are molecules with the capacity to elicit IgE responses in humans. When stimulated with allergens, most allergic patients respond with production of IgE specific for several proteins/allergens in the source material. The standardization of allergen extracts is essential in order to control variability and to achieve consistency and reproducibility in a clinical setting. Because the IgE binding capacity of an allergen extract is related to the content of one or a few major allergens, it is important that the standardization procedure ensures consistency, not only in the overall IgE binding potency, but also in the content and ratio of individual major allergens. Owing to the complexity of allergen extracts, a key element in standardization of allergen extracts is the use of standards. This chapter describes the principles for standardization of allergen extracts to be used by research laboratories. Other chapters in this volume describe methods in detail.

Specific allergen immunotherapy is an effective treatment for IgE-mediated allergic disease and involves T- and B-cell mediated events. IgE receptors on the surface of antigen-presenting cells facilitate the presentation of allergens in the presence of specific IgE antibody resulting in T-cell activation. Interference with these IgE-dependent mechanisms by 'blocking' IgG antibodies may downregulate T-cell responses and manifest as a reduction in allergic responses in vivo. The vigor of proliferative responses by T-cell clones is representative of the binding of allergen-IgE complexes to B cells. Therefore, a simplified assay can be employed that measures the binding of allergen-IgE complexes to B cells instead of a more complex assay involving proliferative assays using antigen-specific T-cell clones. Allergen-IgE complexes can be easily detected by flow cytometry and this simplified technique is called the IgE-facilitated allergen binding (IgE-FAB) assay which is described in this chapter.

The study of monoclonal human T-cell populations has had a fundamental impact on our current knowledge of the function, specificity, and mechanisms of activation of these cells. The frequency of antigen-specific T cells in peripheral blood is low, necessitating several enrichment steps prior to the isolation of individual clones. Two different methods of limiting dilution cloning are described in this chapter, the choice of which depends on the availability of starting materials. After isolation, T-cell clones are expanded and then tested for specificity and cryopreserved, both of which are described.Recently, time-saving variations to the above methods have emerged. However, they require the use of sophisticated equipment and reagents, making them less economical than the established techniques.

Allergens are characterized by their ability to be bound by gE. The Swiss-Prot protein database currently lists a partial or complete amino acid sequence of in excess of about 350 allergens. It is not clear how allergens participate in the process of allergic sensitization, the generation of specific T-helper type 2 (Th2) lymphocytes, which play a crucial role in stimulating B lymphocytes to produce allergen-specific IgE.T-helper (Th) cells play a key role in the regulation of immune responses. The recognition of antigen by T cells is complex and it can trigger qualitatively differential signaling. Therefore, it is conceivable that epitopes or antigenic determinants recognized by Th cells may influence the quality of immune response. The aim of this chapter is to describe the way in which T-cell epitopes can be identified (mapped). This is particularly important because knowledge of the precise T-cell epitopes of allergens can give important information on the pathogenesis of allergy and can help to develop better preparations for the diagnostics and/or immunotherapy of allergy.