Lysophosphatidic acid (LPA), via interaction with its G-protein coupled receptors, is involved in various pathological conditions. Extracellular LPA is mainly produced by the enzyme autotaxin (ATX). Using fibroblast-like synoviocytes (FLS) isolated from synovial tissues of patients with rheumatoid arthritis (RA), we studied the expression profile of LPA receptors, LPAinduced cell migration, and interleukin (IL)-8 and IL-6 production. We report that FLS express LPA receptors LPA 1-3 . Moreover, exogenously applied LPA induces FLS migration and secretion of IL-8/IL-6, whereas the LPA 3 agonist L-sn-1-Ooleoyl-2-methyl-glyceryl-3-phosphothionate (2S-OMPT) stimulates cytokine synthesis but not cell motility. The LPA-induced FLS motility and cytokine production are suppressed by LPA 1/3 receptor antagonists diacylglycerol pyrophosphate and (S)-phosphoric acid mono-(2-octadec-9-enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl]-propyl) ester (VPC32183). Signal transduction through p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and Rho kinase is involved in LPA-mediated cytokine secretion, whereas LPA-induced cell motility requires p38 MAPK and Rho kinase but not p42/44 MAPK. Treatment of FLS with tumor necrosis factor-␣ (TNF-␣) increases LPA 3 mRNA expression and correlates with enhanced LPA-or OMPT-induced cytokine production. LPA-mediated superproduction of cytokines by TNF-␣-primed FLS is abolished by LPA 1/3 receptor antagonists. We also report the presence of ATX in synovial fluid of patients with RA. LPA 1/3 receptor antagonists and ATX inhibitors reduce the synovial fluid-induced cell motility. Together the data suggest that LPA 1 and LPA 3 may contribute to the pathogenesis of RA through the modulation of FLS migration and cytokine production. The above results provide novel insights into the relevance of LPA receptors in FLS biology and as potential therapeutic targets for the treatment of RA.

Sphingosine-1-phosphate (S1P), via interaction with its G protein-coupled receptors, regulates various physiological and pathological responses. The present study investigated the role of S1P/S1P receptor signaling in several functional responses of human fibroblast-like synoviocytes (FLSs) that may contribute to the pathogenesis of rheumatoid arthritis (RA). We report that FLSs express the S1P 1 , S1P 2 , and S1P 3 receptors. Moreover, exogenously applied S1P induces FLS 1) migration, 2) secretion of inflammatory cytokines/chemokines, and 3) protection from apoptosis. Using specific S1P receptor agonists/antagonists, we determined that S1P stimulates FLS migration through S1P 1 and S1P 3 , induces cytokine/chemokine secretion through S1P 2 and S1P 3 , and protects from cell apoptosis via S1P 1 . The S1P-mediated cell motility and cytokine/chemokine secretion seem to be regulated by the p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, and Rho kinase signal transduction pathways. Interestingly, treatment of FLSs with tumor necrosis factor-a increases S1P 3 expression and correlates with the enhancement of S1P-induced cytokine/ chemokine production. Our data suggest that S1P 1 , S1P 2 , and S1P 3 play essential roles in the pathogenesis of RA by modulating FLS migration, cytokine/chemokine production, and cell survival. Moreover, the cytokine-rich environment of the inflamed synovium may synergize with S1P signaling to exacerbate the clinical manifestations of this autoimmune disease.-Zhao, C., M. J. Fernandes, M. Turgeon, S. Tancrède, J. Di Battista, P. E. Poubelle, and S. G. Bourgoin.

The properties and regulation of the mammalian polyamine transport system are still poorly understood. In estrogen-responsive ZR-75-1 human breast cancer cells, which display low polyamine biosynthetic activity, putrescine and spermidine were internalized with high affinity (Km = 3.7 and 0.5 microM, respectively) via a single class of saturable transporter shared by both substrate types, or via distinct but closely similar carriers. The Vmax, but not the Km of polyamine transport was rapidly and synergistically up-regulated by estrogens and insulin. The steady decay in transport activity observed in hormone-deprived cells was accelerated by retinoic acid. The enhancement of uptake activity resulting from polyamine depletion was amplified 3-fold by estrogens and insulin despite profound growth inhibition, indicating that the cooperative hormonal induction of polyamine transport is dissociated from cell growth status. Polyamine uptake was under feedback inhibition by at least three distinct mechanisms in these cells, namely (i) the induction of a short-lived protein not actively synthesized without ongoing uptake or upon polyamine deletion; (ii) a more latent, protein synthesis-independent "trans-inhibition" mechanism; and (iii) a post-carrier, cycloheximide-sensitive mechanism limiting substrate accumulation. The complexity of these multiple levels of feedback transport inhibition is in keeping with the cytotoxicity of excessive polyamine content.

The mechanism of polyamine uptake in mammalian cells is still poorly understood. The role of inorganic cations in polyamine transport was investigated in ZR-75-1 human breast cancer cells. Although strongly temperature dependent, neither putrescine nor spermidine uptake was mediated by a Na+ cotransport mechanism. In fact, Na+ and cholinium competitively inhibited putrescine uptake relative to that measured in a sucrose-based medium. On the other hand, ouabain, H+, Na+, and Ca2+ ionophores, as well as dissipation of the K+ diffusion potential, strongly inhibited polyamine uptake in keeping with a major role of membrane potential in that process. Polyamine transport was inversely dependent on ambient osmolality at near physiological values. Putrescine transport was inhibited by 70% by decreasing extracellular pH from 7.2 to 6.2, whereas spermidine uptake had a more acidic optimum. Deletion of extracellular Ca2+ inhibited putrescine uptake more strongly than chelation of intracellular Ca2+. In fact, bound divalent cations were absolutely required for polyamine transport, as shown after brief chelation of the cell monolayers with EDTA. Either Mn2+, Ca2+, or Mg2+ sustained putrescine uptake activity with high potency (Km = 50-300 microM). Mn2+ was a much stronger activator of spermidine than putrescine uptake, suggesting a specific role for this metal in polyamine transport. Other transition metals (Co2+, Ni2+, Cu2+, and Zn2+) were mixed activators/antagonists of carrier activity, while Sr2+ and Ba2+ were very weak agonists, while not interfering with Ca2+/Mg(2+)-dependent transport. Thus, polyamine uptake in human breast tumor cells is negatively affected by ionic strength and osmolality, and is driven, at least in part, by the membrane potential, but not by the Na+ electrochemical gradient. Moreover, the polyamine carrier, or a tightly coupled accessory component, appears to have a high-affinity binding site for divalent cations, which is essential for the uptake mechanism.

Live-vector human immunodeficiency virus (HIV) vaccines are an integral part of a number of HIV vaccine regimens currently under evaluation that have yielded promising results in pre-clinical testing. In this report, a non-pathogenic protozoan parasitic vector, Leishmania tarentolae, which shares common target cells with HIV-1, was used to express full-length HIV-1 Gag protein. Immunization of BALB/c mice with recombinant L. tarentolae led to the expansion of HIV-1 Gag-specific T cells and stimulated CD8 + T cells to produce gamma interferon in response to specific viral Gag epitopes. A booster immunization with recombinant L. tarentolae elicited effector memory HIV-1 Gag-specific CD4 + T lymphocytes and increased antibody titres against HIV-1 Gag. Most importantly, immunization of human tonsillar tissue cultured ex vivo with Gag-expressing L. tarentolae vaccine vector elicited a 75 % decrease in virus replication following exposure of the immunized tonsils to HIV-1 infection. These results demonstrated that recombinant L. tarentolae is capable of eliciting effective immune responses in mice and human systems, respectively, and suggest that this novel non-pathogenic recombinant vaccine vector shows excellent promise as a vaccination strategy against HIV-1.