Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. We have utilized two independent approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14؉ blood monocytes and their derived DCs. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays complementary to 6300 human genes showed that ϳ40% of the genes were expressed in DCs. A total of 255 genes (4%) were found to be regulated during DC differentiation or maturation. Most of these genes were not previously associated with DCs and included genes encoding secreted proteins as well as genes involved in cell adhesion, signaling, and lipid metabolism. Protein analysis of the same cell populations was done using two-dimensional gel electrophoresis. A total of 900 distinct protein spots were included, and 4% of them exhibited quantitative changes during DC differentiation and maturation. Differentially expressed proteins were identified by mass spectrometry and found to represent proteins with Ca 2؉ binding, fatty acid binding, or chaperone activities as well as proteins involved in cell motility. In addition, proteomic analysis provided an assessment of post-translational modifications. The chaperone protein, calreticulin, was found to undergo cleavage, yielding a novel form. The combined oligonucleotide microarray and proteomic approaches have uncovered novel genes associated with DC differentiation and maturation and has allowed analysis of post-translational modifications of specific proteins as part of these processes.

Rapamycin has been shown to affect translation. We have utilized two complementary approaches to identify genes that are predominantly affected by rapamycin in Jurkat T cells. One was to compare levels of polysomebound and total RNA using oligonucleotide microarrays complementary to 6,300 human genes. Another was to determine protein synthesis levels using two-dimensional PAGE. Analysis of expression changes at the polysome-bound RNA levels showed that translation of most of the expressed genes was partially reduced following rapamycin treatment. However, translation of 136 genes (6% of the expressed genes) was totally inhibited. This group included genes encoding RNA-binding proteins and several proteasome subunit members. Translation of a set of 159 genes (7%) was largely unaffected by rapamycin treatment. These genes included transcription factors, kinases, phosphatases, and members of the RAS superfamily. Analysis of [35 S]methionine-labeled proteins from the same cell populations using two-dimensional PAGE showed that the integrated intensity of 111 of 830 protein spots changed in rapamycin-treated cells by at least 3-fold (70 increased, 41 decreased). We identified 22 affected protein spots representing protein products of 16 genes. The combined microarray and proteomic approach has uncovered novel genes affected by rapamycin that may be involved in its immunosuppressive effect and other genes that are not affected at the level of translation in a context of general inhibition of cap-dependent translation.Rapamycin is a macrolide antibiotic originally isolated from Streptomyces hygroscopicus (1). It is a potent immunosuppressant with therapeutic applications in the prevention of organ allograft rejection and in the treatment of autoimmune disease (2-6). The importance of rapamycin as an immunosuppressant drug has focused attention on its mechanism of action. Rapamycin has a similar biochemical structure to cyclosporin A and FK506. However, unlike cyclosporin A and FK506, rapamycin is not a calcineurin inhibitor (7). The primary mode of immunosuppressive action of rapamycin is an antiproliferative action reflecting the ability of the drug to disrupt signaling by T cell growth-promoting lymphokines such as IL-2 1 and IL-4 (8). The growth-inhibitory effects of rapamycin are not limited to T cells, since this drug inhibits the proliferation of many mammalian cell types as well as that of yeast cells (9).Rapamycin blocks progression of the cell cycle at the G 1 phase by binding to FKBP12 (FK506-binding protein) (10, 11). The rapamycin-FKBP12 complex inhibits mTOR (mammalian target of rapamycin), also referred to as FRAP (FKBP-rapamycin-associated protein) (9). Targets of mTOR include 4E-BP1 and the 40 S ribosomal protein S6 kinase, p70 s6k (12-16). Rapamycin-induced inhibition of p70 s6k activity and subsequent dephosphorylation of the ribosomal S6 protein lead to a selective translational repression of mRNA containing a polypyrimidine-rich tract (TOP) motif at their 5Ј terminus (17). 4E-BP1 is a small heat-and...

The specific and sensitive definitions of disease bracket the true values; the relatively small range indicates that the current study provides good estimations of incidence and prevalence of Parkinson's disease for recent years in France.

4E-BP1 plays a major role in translation by inhibiting cap-dependent translation initiation. Several reports have investigated the regulation of 4E-BP1 phosphorylation, which varies along with cell differentiation and upon various stimulations, but very little is known about the regulation of its expression. In a first part, we show that the expression of 4E-BP1 protein and transcript decreases in hematopoietic cell lines cultivated in the presence of phorbol 12-myristate 13-acetate (PMA). This decrease depends on the activation of the ERK/ mitogen-activated protein kinases. 4E-BP1 expression also decreases when the p38/mitogen-activated protein kinase pathway is activated by granulocyte/macrophage colony-stimulating factor but to a lesser extent than with PMA. In a second part, we examine how 4e-bp1 promoter activity is regulated. PMA and granulocyte/ macrophage colony-stimulating factor induce Egr-1 expression through ERK and p38 activation, respectively. Using a dominant negative mutant of Egr, ZnEgr, we show that this transcription factor is responsible for the inhibition of 4e-bp1 promoter activity. In a third part we show that histidine decarboxylase, whose activity and expression are inversely correlated with 4E-BP1 expression, is a potential target for the translational machinery. These data (i) are the first evidence of a new role of ERK and p38 on the translational machinery and (ii) demonstrate that 4E-BP1 is a new target for Egr-1.

The efficient Ag presenting and immunostimulatory capacity of dendritic cells (DCs) has led to the use of tumor Ag-pulsed DCs in treatment regimens for cancer. Although vaccine studies involving tumor lysate-pulsed DCs have been performed, little, if any, information is available on the effects of phagocytic uptake of tumor lysate on DC biology and function. We have investigated gene expression pattern differences between unpulsed DCs and tumor lysate-pulsed-DCs, using Affymetrix MG-U74Av2 oligonucleotide arrays, which contain ∼12,000 genes and expressed sequence tags. Upon 24 h tumor lysate pulsing, the levels of 87 transcripts increased at least 3-fold while the levels of 121 transcripts were reduced by one-third or more, with accompanying p values <0.01. Most of these genes encoded proteins important for DC effector functions including cytokines, chemokines, and receptors, such as IL-12p40, macrophage inflammatory protein-2, and IL-6; Ag presentation, such as carboxypeptidase D and H2-DM; cell adhesion (e.g., EGF-like module containing, mucin-like, hormone receptor-like sequence 1, rhoB); and T cell activation. Interestingly, we observed a high level of expression of a novel member of the class A scavenger receptor family, macrophage receptor with collagenous structure (Marco). Marco is thought to play an important role in the immune response by mediating binding and phagocytosis, but also in the formation of lamellipodia-like structures and of dendritic processes. Therefore, we have identified a repertoire of genes that likely play a role in DC function.

No abstract

We recently reported evidence for differential regulation of the translation machinery during human myeloid differentiation, specific to the monocytic/macrophage pathway or to the granulocytic pathway. A decrease in translation rates and concomitant regulation of two repressors of translation initiation, 4E-BP1 and 4E-BP2 (elF4E-binding proteins 1 and 2), occur in cells induced to differentiate along the monocytic/macrophage pathway or along the granulocytic pathway. Induction of HL-60 and U-937 cell differentiation into monocytes/macrophages results in a dephosphorylation and consequent activation of 4E-BP1. In contrast, following treatment of HL-60 cells with retinoic acid (RA) which results in a granulocytic differentiation of these cells, 4E-BP1 protein expression is decreased whereas 4E-BP2 protein expression is strongly increased. In this study, we further investigated the regulation of 4E-BP1 and 4E-BP2 in the RA-induced differentiation process using the NB4 promyelocytic cell line and the RA maturation-resistant NB4 subclones, NB4-R1 and NB4-R2. RA treatment resulted in a decrease in 4E-BP1 protein and mRNA expression and concomitant increase in 4E-BP2 protein expression, in NB4 cells, but not in NB4-R1 and NB4-R2 cells. The increase in 4E-BP2 protein expression was not correlated to an increase in 4E-BP2 mRNA level suggesting a post-transcriptional regulation of 4E-BP2 expression. In RA-primed cells, cAMP induce maturation of NB4-R1, but not NB4-R2 cells. cAMP treatment resulted in a down-regulation of 4E-BP1 protein and mRNA expression in RA-primed NB4-R1, but not NB4-R2 cells. However, 4E-BP2 expression was not modified in both cell types following cAMP treatment. This indicates that 4E-BP1 down-regulation is associated with granulocytic maturation, whereas post-transcriptional regulation of 4E-BP2 expression is associated with the early action of RA. Leukemia (2000Leukemia ( ) 14, 1909Leukemia ( -1914

Prescription of the psychotropic drugs plays an important role in the choice of the drugs ingested for the IDO. It might make potentially "dangerous" drugs available for the patient. Physicians have always to balance the benefit of the treatment against the risk of drug overdose.