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In order to define the extent of T cell heterogeneity and clonality, unique DNA sequences in the junctional region in rearranged T cell receptor (TcR) genes can be studied. For this purpose we have adapted a non-denaturing nucleic acid gel electrophoresis procedure to detect TcR junctional diversity. Detection of junctional diversity is based upon electrophoretic separation of single stranded (ss) and double stranded (ds) DNA molecules via mobility shifts due to nucleotide sequence polymorphism. To examine the capacity of this nucleic acid gel electrophoresis procedure to detect nucleotide sequence polymorphism in the CDR 3 region within TcR Vβ gene family sequences polymerase chain reaction (PCR) amplified TcR Vβ 5.1/5.4 and Vβ14 cDNA sequences were analyzed. The results of this study showed that (1) the single strand conformation polymorphism (SSCP) procedure has a low capacity to discriminate between diverse TcR Vβ cDNA sequences due to comigration of the ssDNA molecules, which results in an underestimation of the heterogeneity in a given T cell population; (2) comigrating ssDNA and/or dsDNA (homoduplex) molecules can be separated by the formation of heteroduplex molecules; these heteroduplex molecules provide essential additional information on the degree of nucleotide sequence polymorphism in the CDR 3 region within the TcR Vβ cDNA sequences; (3) the double strand conformation polymorphism (DSCP) procedure provides a fast and reliable procedure to detect junctional diversity within the sequences tested. Using DSCP a more detailed assessment of amplified TcR Vβ cDNA sequences can be obtained as compared with SSCP analysis only. Data obtained by gel analysis were very similar to those obtained by conventional bacterial cloning and DNA sequencing procedures on the corresponding cDNA clones. In conclusion, this new gel electrophoresis procedure allows a direct assessment of the extent of T cell heterogeneity and clonality by screening junctional diversity in TcR chain encoding sequences in clinical conditions with (oligo)clonal expansion of T lymphocytes. Molecular Sequence Numbers: GENBANK: S82223; Chemicals/CAS: Receptors, Antigen, T-Cell, alpha-beta

Background: Application of transcriptomics technology in human nutrition intervention studies would allow for genome-wide screening of the effects of specific diets or nutrients and result in biomarker profiles. Objective: The aim was to evaluate the potential of gene expression profiling in blood cells collected in a human intervention study that investigated the effect of a high-carbohydrate (HC) or a high-protein (HP) breakfast on satiety. Design: Blood samples were taken from 8 healthy men before and 2 h after consumption of an HP or an HC breakfast. Both breakfasts contained acetaminophen for measuring the gastric emptying rate. Analysis of the transcriptome data focused on the effects of the HP or HC breakfast and of acetaminophen on blood leukocyte gene expression profiles. Results: Breakfast consumption resulted in differentially expressed genes, 317 for the HC breakfast and 919 for the HP breakfast. Immune response and signal transduction, specifically T cell receptor signaling and nuclear transcription factor κB signaling, were the overrepresented functional groups in the set of 141 genes that were differentially expressed in response to both breakfasts. Consumption of the HC breakfast resulted in differential expression of glycogen metabolism genes, and consumption of the HP breakfast resulted in differential expression of genes involved in protein biosynthesis. Conclusions: Gene expression changes in blood leukocytes corresponded with and may be related to the difference in macronutrient content of the breakfast, meal consumption as such, and acetaminophen exposure. This study illustrates the potential of gene expression profiling in blood to study the effects of dietary exposure in human intervention studies. © 2006 American Society for Nutrition.

In this study, we investigated the development of T cell responses in mice after administration of a mannosylated ovalbumin peptide (M-OVA323-339). Immunization with M-OVA323-339 in complete adjuvant resulted in enhanced antigen presentation in draining lymph nodes. Monitoring the fate of CFSE-labeled ovalbumin peptide-specific TCR transgenic CD4+ T cells revealed that immunization with M-OVA323-339 induced normal clonal expansion, recirculation and CD62L expression of antigen-specific T cells in vivo. However, these T cells developed only poor effector functions, reflected by minimal IFN-γ production, low IgG2a levels in serum and poor peptide-specific delayed-type hypersensitivity (DTH) responses. This diminished inflammatory response was associated with decreased infiltration of T cell blasts and macrophages. Importantly, also mice with functional effector T cells did not mount a robust DTH response after a challenge with M-OVA323-339 in the ear, although their T cells responded normally to M-OVA323-339 in vitro. In conclusion, mannosylated peptide induces proliferation of T cells with impaired Th1 cell effector functions and additionally abrogates the activity of pre-existing effector T cells. © The Japanese Society for Immunology. 2007. All rights reserved.