Gene expression studies in cultured dendritic cells: new indicators for the discrimination of skin sensitizers and irritants in vitro.

BACKGROUND: The replacement of animal tests for the detection of the sensitizing potential of chemicals is of great importance due to current legislation. One promising approach for the development of an in vitro assay is the exposure of immature dendritic cells (iDCs) to contact sensitizers and irritants, followed by an analysis of the maturation status of the cells. OBJECTIVE: The aim of this study was to further investigate the performance of our previously developed targeted microarray, the immune toxicity chip. In addition, we aimed to identify new marker genes for the discrimination of allergens and irritants using whole-genome microarrays. METHODS: Monocyte-derived iDCs were exposed to contact sensitizers and irritants in concentrations resulting in 10-20% cytotoxicity, as determined by dose-response curves. Changes in gene expression were analysed using the immune toxicity chip and a commercially available whole-genome microarray. RESULTS: Using the immune toxicity chip, we could identify a panel of marker genes suitable to discriminate strong allergens and irritants. Analysis with the whole-genome array revealed additional genes that are differentially expressed after allergen exposure, but not after irritant exposure. Hierarchical clustering of these genes showed distinct groups representing the different chemicals. CONCLUSION: Here we show that our test system based on an immune-specific microarray is suitable for the discrimination of strong allergens and irritants. Genes detected as differentially expressed with the whole-genome array and previously not connected to the maturation process of DCs might be suitable candidate genes for the identification of weaker sensitizers.

MethCancerDB--aberrant DNA methylation in human cancer.

Early detection, classification and prognosis of human cancers by analysis of CpG methylation carry huge diagnostic potential. MethCancerDB collects and annotates genes and sequences from the abundance of published methylation studies and interlinks them to all methylation-relevant bioinformatical resources. MethCancerDB starts with 4720 entries from 348 sources and is freely accessible at http://www.methcancerdb.net.

Oral feeding with glutamine prevents lymphocyte and glutathione depletion of Peyer's patches in endotoxemic mice.

OBJECTIVE: To determine the effect of oral glutamine feeding on lymphocyte subpopulations and glutathione metabolism in Peyer's patches (PPs) of healthy and endotoxemic mice. SUMMARY BACKGROUND DATA: Recent data indicate that nutrients both maintain nitrogen and energy balances and modulate cell and organ function. In particular, glutamine has an impact on gut and immune function. This is of special importance in the perioperative phase. METHODS: Female Balb/c mice were fed a glutamine-enriched diet or a control diet for 10 days. On day 7 25 microg lipopolysaccharide (LPS) or saline was injected. On day 3 after the challenge, mice were killed, total cell yield was determined, and lymphocyte subpopulations (total T cells, CD4+, CD8+ cells, and B cells) were analyzed by flow cytometry. One experimental group was treated with buthionine sulfoximine, a specific inhibitor of glutathione synthesis. The glutathione content in PPs was measured by high-performance liquid chromatography. RESULTS: Glutamine administration led to a significant increase in total cell yield, including T and B cells, in PPs. The LPS-induced reduction of T cells (-45%) and of B cells (-30%) was significantly lower in glutamine-treated mice. Endotoxemia caused a 42% decrease of glutathione in control animals, but not in glutamine-treated animals. As with LPS, buthionine sulfoximine also lowered lymphocyte numbers and glutathione content of the PPs. CONCLUSIONS: Administration of glutamine prevents LPS-stimulated lymphocyte atrophy in PPs, possibly by increasing the glutathione content in the PPs. Therefore, oral glutamine supply seems to be a suitable approach for improving intestinal immunity in immunocompromised patients.

Lipopolysaccharide causes atrophy of Peyer's patches and an increased expression of CD28 and B7 costimulatory ligands.

Intestinal mucosal dysfunction appears to contribute to infectious complications in critically ill patients. The current study was undertaken to investigate whether endotoxin affects lymphocyte subpopulations and the expression of costimulatory signals in Peyer's patches (PP). Female Balb/c mice were given an intraperitoneal injection of 25 microg LPS and sacrified 24 h or 72 h later to determine total cell yield, lymphocyte subpopulations (B-cells, total T-cells, CD4+- and CD8+-cells), the costimulatory molecules CD28, B7.1 (CD80) and B7.2 (CD86) and the percentage of apoptotic cells in PP and in the spleen as well as small intestinal IgA concentration. Lipopolysaccharide (LPS) challenge caused a significant decrease of total cell yield in PP at both time-points (-50+/-28% and -43+/-25%, respectively; P < 0.001). This decrease was significant for all measured lymphocyte subpopulations. In contrast, total cell yield was increased (P < 0.001) in the spleen 24 h (+52+/-13%) and 72 h (+130+/-22%) after LPS. The decrease of lymphocyte numbers in the PP was accompanied by an increased percentage of lymphocytes expressing costimulatory molecules. In this respect, an increased percentage of CD40+CD80+, CD40+CD86+, and of CD4+CD28+ could be demonstrated after LPS administration. In the spleen, the percentage of CD4+CD28+ was also elevated after LPS bolus, however, the percentage of CD40+CD80+ was reduced, and that of CD40+CD86+ was unaltered. The influence of LPS on apoptosis of lymphocytes was time-dependent. The percentage of apoptotic cells 24 h after LPS was increased in PP (P < 0.01), but was unchanged in the spleen. Seventy-two hours after LPS injection, the percentage of apoptotic cells returned to normal in PP. Luminal IgA levels remained unchanged after LPS challenge. In conclusion, our data show that LPS causes atrophy of PP which seems to be counterregulated by an enhanced expression of costimulatory molecules.

Influence of enteral diets supplemented with key nutrients on lymphocyte subpopulations in Peyer's patches of endotoxin-boostered mice.

BACKGROUND AND AIMS: This study was undertaken to compare the effect of different key nutrients on lymphocyte subsets of Peyer's patches (PP) and spleen in endotoxemic mice. METHODS: Female Balb/c mice were fed over a period of 10 days either with an isocaloric and isonitrogenous control diet (Control), a glutamine enriched diet (Diet I) or a diet containing glutamine, arginine, glycine, and n-3 fatty acids (Diet II). On day 7 the mice were challenged intraperitoneally with 25 microg LPS. The lymphocyte subpopulations (B cells, T cells, CD4+ and CD8+) of PP and spleen were analysed by flow cytometry. Glutathione content of small intestinal mucosa and spleen was determined by HPLC and luminal small intestinal IgA by ELISA. RESULTS: Both experimental diets increased the number of B and T cells in the PP and that of T cells in the spleen (P<0.01). Glutathione content in PP and spleen was higher under administration of key nutrients (P<0.05). Diet II reduced luminal small intestinal IgA content in comparison to the two other groups. CONCLUSION: The addition of arginine, glycine and n-3 fatty acids to a glutamine supplemented diet does not enhance lymphocyte numbers in PP and spleen, but reduces intestinal IgA content.

Influence of short-term protein malnutrition of mice on the phenotype and costimulatory signals of lymphocytes from spleen and Peyer's patches.

The objective of this study was to investigate the impact of short-term protein malnutrition (PM) on immunoglobulin A (IgA) production and on the number and phenotype of lymphocytes in Peyer's patches (PP) and in the spleen. Balb/c mice were fed for 4, 7, or 10 d with a protein-deficient diet (0.1% protein). We determined B lymphocytes (CD40(+)), T lymphocytes (CD3(+)), T-helper (CD4(+)), and T-suppressor (CD8(+)) cells and the expression of costimulatory signals B7.1 (CD80) and B7.2 (CD86) on B cells and their counter receptors CD28 and CTLA-4 on T cells by fluorescence-activated cell-sorting analysis. Luminal IgA concentration in the small intestine was determined by an enzyme-linked immunosorbent assay. Four days of PM caused a significant reduction in the number of mononuclear cells in the spleen (5.6 x 10(7) +/- 1 x 10(7) versus 2. 4 x 10(7) +/- 0.5 x 10(7), P < 0.001) and the PP (13 x 10(6) +/- 3 x 10(6) versus 8.6 x 10(6) +/- 2 x 10(6), P < 0.01). There was a relative increase of T cells in the spleen and a relative increase of B cells in the PP. Luminal IgA content of small intestine was significantly reduced after 4 d of PM (242 +/- 55 microg versus 173 +/- 39 microg, P < 0.05) and remained at about this level until day 10 of PM. Four days after PM, the costimulatory signals B7.1 and B7. 2 on B cells were upregulated in the PP but markedly downregulated in the spleen, which was inversely related to the expression of the counter receptor CD28 on T-helper cells. We conclude that short-term PM increases the activation of B cells in the PP but reduces the relative number and activation state of splenic B cells. Only 4 d of PM caused a systemic and intestinal immunodepression, as indicated by a markedly decreased content of mononuclear cells in the PP and the spleen.