实验动物技术

Rapid growth in the field of antibody engineering occurred after it was shown that functional antibody fragments could be secreted into the periplasmic space and even into the medium of Escherichia coli by fusing a bacterial signal peptide to the antibody’s N-terminus (1,2). These findings a ...

For cloning and expressing the antigen-binding variable (Fv) portion of an antibody in Escherzchia coli, vectors have been constructed that combine the two variable regions (VH and VL) with a peptide linker (1–3). The genetic information for VH and VL is generally amplified from hybridoma cel ...

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. Ce ...

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 mononuc ...

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 muc ...

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, the ...

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 wi ...

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 tiss ...

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 mur ...

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 v ...

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 incr ...

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 u ...

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 ...

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 con ...

Candida albicans is a major fungal systemic pathogen in humans. Genetic manipulation of C. albicans is unwieldy. 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. Streamlin ...

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 i ...

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 us ...

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 ...

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 ava ...

Upon uptake into a host cell, the intracellular bacterium Legionella pneumophila is not degraded on the lysosomal pathway but efficiently establishes a highly specialized replicative vacuole in which it readily multiplies. As many Icm/Dot type 4 secretion translocated bacterial ...