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Microarray technology has provided the ability to analyse the expression profiles for thousands of genes in parallel. The need for highly specialised equipment to use certain types of microarrays has restricted the application of this technology to a small number of dedicated laboratories. High-density filter-based cDNA microarrays provide a low-cost option for performing high-throughput gene expression analysis. We have used a model system in which filter-based cDNA microarrays representing over 4000 known human genes were used to monitor the kinetics of gene expression in human peripheral blood mononuclear cells (PBMCs) stimulated with phytohaemagluttinin (PHA). Using software-based cluster analysis, we identified 104 genes that altered in expression levels in response to PHA stimulation of PBMCs and showed that there was a considerable overlap between genes with similar temporal expression profiles and similar functional roles. Comparison of microarray quantitation with quantitative PCR showed almost identical expression profiles for a number of genes. Coupled with the fact that our findings are in agreement with a large number of independent observations, we conclude that the use of filter-based cDNA microarrays is a valid and accurate method for high-throughput gene expression profiling.

Many of the vascular endothelial changes associated with inflammation can be induced in vitro by the cytokines tumour necrosis factor, IL-1, and IFN gamma. On the other hand, although IL-4 is a powerful mediator of leucocyte function, its influence on endothelial cells has not yet been fully determined. In this study the effect of IL-4 on human umbilical vein endothelial cells has been investigated. It is shown that IL-4 stimulates DNA synthesis over the first 24 h in culture, followed by dramatic alterations in endothelial cell morphology in which the cobblestone appearance of cells grown to confluence in medium changes to a monolayer composed of islands of tightly packed polygonal cells. The morphological changes induced by IL-4 were accompanied by a reorganization of the intracellular vimentin matrix from a diffuse pattern to a perinuclear concentration observed by staining with vimentin antibodies. Similar patterns of vimentin staining were also observed with an antibody to the low affinity IgE receptor (CD23) normally associated with IL-4 activation of B lymphocytes. Our results suggest that this represents cross reactivity between CD23 and vimentin rather than the appearance of CD23 in endothelial cells. Amino acid sequence comparison between CD23 and vimentin indicated significant homology between these two molecules, including a leucine zipper-like motif. Our results suggest that IL-4 may be an important regulator of endothelial cell morphology and function in inflammation.

MyD-1 (CD172) is a member of the family of signal regulatory phosphatase (SIRP) binding proteins, which is expressed on human CD14+ monocytes and dendritic cells. We now show a novel role for MyD-1 in the regulation of the innate immune system by pathogen products such as lipopolysaccharide (LPS), purified protein derivative (PPD), and Zymosan. Specifically, we demonstrate that ligation of MyD-1 on peripheral blood mononuclear cells (PBMCs) inhibits tumor necrosis factor alpha (TNFα) secretion but has no effect on other cytokines induced in response to each of these products. In an attempt to understand the molecular mechanisms underlying this surprisingly selective effect we investigated signal transduction pathways coupled to MyD-1. Ligation of the SIRP was found to recruit the tyrosine phosphatase SHP-2 and promote sequential activation of phosphatidylinositol (PI) 3-kinase, phospholipase D, and sphingosine kinase. Inhibition of LPS-induced TNFα secretion by MyD-1 appears to be mediated by this pathway, as the PI 3-kinase inhibitor wortmannin restores normal LPS-driven TNFα secretion. MyD-1-coupling to this PI 3-kinase-dependent signaling pathway may therefore present a novel target for the development of therapeutic strategies for combating TNFα production and consequent inflammatory disease. (Blood. 2003;102:2532-2540)

Recent findings about pathogens and innate immune system interactions have opened new opportunities for adjuvants designs. We have elaborated a new approach, in which gangliosides are incorporated into the outer membrane complex of Neisseria meningitidis (Nm) to form very small size proteoliposomes (VSSP). VSSP, used as monotherapy, demonstrated a unique ability to render immunogenic highly tolerated gangliosides. These results drove our attention to the immunopotentiating properties of VSSP. Here, we examined the VSSP adjuvant effect on the humoral and cellular responses, dendritic cell (DC) activation, and differentiation of Th cells. Also, the role of LPS in VSSP effect was dissected. This study reveals that VSSP is a potent adjuvant for dendritic cells activation and Th1 differentiation.

Recent findings in the interactions between pathogens and the innate immune system, particularly with dendritic cells (DC), have opened new opportunities for adjuvants designs. We have elaborated a new approach, in which gangliosides are incorporated into the outer membrane complex of Neisseria meningitidis to form very small size proteoliposomes (VSSP). VSSP demonstrated a unique ability to render highly tolerated gangliosides immunogenic. These results drove our attention to the immunopotentiating properties of VSSP. Here we examined the VSSP adjuvant effect on dendritic cell activation. Also the role of LPS on this effect was dissected. This study reveals that VSSP is a potent adjuvant for DC activation.

We report from three independent centers that, in human tonsillar B lymphocytes, human IL4 switches on a series of second messenger changes, the precise sequence of which constitutes a novel signal transduction cascade. It involves an immediate and transient elevation of inositol 1,4,5-trisphosphate and Ca2+ levels. This is followed several minutes later by a sustained rise in cellular cyclic adenosine monophosphate concentration, the triggering of which involves both the Ca2+ rise and an additional, as yet unidentified, IL4-generated signal. Both the products of the initial inositol lipid hydrolysis and the delayed cyclic adenosine monophosphate accumulation are essential for the later induction of CD23 expression, a major phenotypic change promoted in these cells by IL4. The striking contrast between these findings and those that have been observed for the IL4 triggering of murine B cells is discussed.