Selective expression of the V beta 14 T cell receptor on Leishmania guyanensis--specific CD8+ T cells during human infection.

Peripheral blood mononuclear cells from subjects never exposed to Leishmania were stimulated with Leishmania guyanensis. We demonstrated that L. guyanensis-stimulated CD8(+) T cells produced interferon (IFN)- gamma and preferentially expressed the V beta 14 T cell receptor (TCR) gene family. In addition, these cells expressed cutaneous lymphocyte antigen and CCR4 surface molecules, suggesting that they could migrate to the skin. Results obtained from the lesions of patients with localized cutaneous leishmaniaisis (LCL) showed that V beta 14 TCR expression was increased in most lesions (63.5%) and that expression of only a small number of V beta gene families (V beta 1, V beta 6, V beta 9, V beta 14, and V beta 24) was increased. The presence of V beta 14 T cells in tissue confirmed the migration of these cells to the lesion site. Thus, we propose the following sequence of events during infection with L. guyanensis. After initial exposure to L. guyanensis, CD8(+) T cells preferentially expressing the V beta 14 TCR and secreting IFN- gamma develop and circulate in the periphery. During the infection, these cells migrate to the skin at the site of the parasitic infection. The role of these V beta 14 CD8(+) T cells in resistance to infection remains to be determined conclusively.

A limited role for beta-selection during gamma delta T cell development.

T cells belong to two distinct lineages expressing either alpha beta or gamma delta TCR. During alpha beta T cell development, it is clearly established that productive rearrangement at the TCR beta locus in immature precursor cells leads to the expression of a pre-TCR complex. Signaling through the pre-TCR results in the selective proliferation and maturation of TCR beta+ cells, a process that is known as beta-selection. However, the potential role of beta-selection during gamma delta T cell development is controversial. Whereas PCR-RFLP and sequencing techniques have provided evidence for a bias toward in-frame VDJ beta rearrangements in gamma delta cells (consistent with beta-selection), gamma delta cells apparently develop normally in mice that are unable to assemble a pre-TCR complex due to a deficiency in TCR beta or pT alpha genes. In this report, we have directly addressed the physiologic significance of beta-selection during gamma delta cell development in normal mice by quantitating intracellular TCR beta protein in gamma delta cells and correlating its presence with cell cycle status. Our results indicate that beta-selection plays a significant (although limited) role in gamma delta cell development by selectively amplifying a minor subset of gamma delta precursor cells with productively rearranged TCR beta genes.

Expression of T cell receptor genes in the thymus: localization of transcripts in situ and comparison of mature and immature subsets.

We have analyzed the expression of T cell receptor (TcR) genes in the thymus using in situ RNA hybridizations with probes to the constant regions of the TcR alpha, beta, gamma and delta chains. Localization of transcripts revealed low TcR alpha mRNA levels in the thymus cortex and very low levels in the subcapsular region. In contrast, TcR beta message was very abundant in the cortex. TcR gamma or delta mRNA+ thymocytes showed a scattered, predominantly cortical localization. In contrast to gamma, TcR delta transcripts were abundant in the subcapsular region. Control experiments with sorted TcR alpha/beta or gamma/delta cells revealed a detection efficiency of 75%-85% for the respective TcR mRNA and data on TcR gene expression in mature, CD3+ thymocytes were consistent with previous reports. The analysis of immature, CD3- thymocyte subsets, however, revealed a virtual absence of TcR alpha transcripts and an unexpectedly high proportion of cells (14%-29%) expressing the gene for the TcR delta chain. The data are discussed in view of current models of lineage relationships in the thymus.

DNA binding properties of peroxisome proliferator-activated receptor subtypes on various natural peroxisome proliferator response elements. Importance of the 5'-flanking region.

The three subtypes of the peroxisome proliferator-activated receptors (PPARalpha, beta/delta, and gamma) form heterodimers with the 9-cis-retinoic acid receptor (RXR) and bind to a common consensus response element, which consists of a direct repeat of two hexanucleotides spaced by one nucleotide (DR1). As a first step toward understanding the molecular mechanisms determining PPAR subtype specificity, we evaluated by electrophoretic mobility shift assays the binding properties of the three PPAR subtypes, in association with either RXRalpha or RXRgamma, on 16 natural PPAR response elements (PPREs). The main results are as follows. (i) PPARgamma in combination with either RXRalpha or RXRgamma binds more strongly than PPARalpha or PPARbeta to all natural PPREs tested. (ii) The binding of PPAR to strong elements is reinforced if the heterodimerization partner is RXRgamma. In contrast, weak elements favor RXRalpha as heterodimerization partner. (iii) The ordering of the 16 natural PPREs from strong to weak elements does not depend on the core DR1 sequence, which has a relatively uniform degree of conservation, but correlates with the number of identities of the 5'-flanking nucleotides with respect to a consensus element. This 5'-flanking sequence is essential for PPARalpha binding and thus contributes to subtype specificity. As a demonstration of this, the PPARgamma-specific element ARE6 PPRE is able to bind PPARalpha only if its 5'-flanking region is exchanged with that of the more promiscuous HMG PPRE.

Fat poetry: a kingdom for PPAR gamma.

Adipose tissue is not an inert cell mass contributing only to the storage of fat, but a sophisticated ensemble of cellular components with highly specialized and complex functions. In addition to managing the most important energy reserve of the body, it secretes a multitude of soluble proteins called adipokines, which have beneficial or, alternatively, deleterious effects on the homeostasis of the whole body. The expression of these adipokines is an integrated response to various signals received from many organs, which depends heavily on the integrity and physiological status of the adipose tissue. One of the main regulators of gene expression in fat is the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma), which is a fatty acid- and eicosanoid-dependent nuclear receptor that plays key roles in the development and maintenance of the adipose tissue. Furthermore, synthetic PPARgamma agonists are therapeutic agents used in the treatment of type 2 diabetes.This review discusses recent knowledge on the link between fat physiology and metabolic diseases, and the roles of PPARgamma in this interplay via the regulation of lipid and glucose metabolism. Finally, we assess the putative benefits of targeting this nuclear receptor with still-to-be-identified highly selective PPARgamma modulators.

Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors controlling the expression of genes involved in lipid homeostasis. PPARs activate gene transcription in response to a variety of compounds including hypolipidemic drugs as well as natural fatty acids. From the plethora of PPAR activators, Scatchard analysis of receptor-ligand interactions has thus far identified only four ligands. These are the chemotactic agent leukotriene B4 and the hypolipidemic drug Wy 14,643 for the alpha-subtype and a prostaglandin J2 metabolite and synthetic antidiabetic thiazolidinediones for the gamma-subtype. Based on the hypothesis that ligand binding to PPAR would induce interactions of the receptor with transcriptional coactivators, we have developed a novel ligand sensor assay, termed coactivator-dependent receptor ligand assay (CARLA). With CARLA we have screened several natural and synthetic candidate ligands and have identified naturally occurring fatty acids and metabolites as well as hypolipidemic drugs as bona fide ligands of the three PPAR subtypes from Xenopus laevis. Our results suggest that PPARs, by their ability to interact with a number of structurally diverse compounds, have acquired unique ligand-binding properties among the superfamily of nuclear receptors that are compatible with their biological activity.

The peroxisome proliferator-activated receptor alpha is a phosphoprotein: regulation by insulin.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily implicated in adipocyte differentiation. The observations that PPAR alpha is a regulator of hepatic lipid metabolism and that the insulin-sensitizing thiazolidinediones are ligands for PPAR gamma suggest that cross-talk might exist between insulin signaling and PPAR activity, possibly through insulin-induced PPAR phosphorylation. Immunoprecipitation of endogenous PPAR alpha from primary rat adipocytes prelabeled with [32P]-orthophosphate and pretreated for 2 h with vanadate and okadaic acid demonstrated for the first time that PPAR alpha is a phosphoprotein in vivo. Treatment with insulin induced a time-dependent increase in PPAR phosphorylation showing a 3-fold increase after 30 min. Insulin also increased the phosphorylation of human PPAR alpha expressed in CV-1 cells. These changes in phosphorylation were paralleled by enhanced transcriptional activity of PPAR alpha and gamma. Transfection studies in CV-1 cells and HepG2 cells revealed a nearly 2-fold increase of PPAR activity in the presence of insulin. In contrast, insulin had no effect on the transcriptional activity of transfected thyroid hormone receptor in CV-1 cells, suggesting a PPAR-specific effect. Thus, insulin stimulates PPAR alpha phosphorylation and enhances the transcriptional activity of PPAR, suggesting that the transcriptional activity of this nuclear hormone receptor might be modulated by insulin-mediated phosphorylation.

FLIP prevents apoptosis induced by death receptors but not by perforin/granzyme B, chemotherapeutic drugs, and gamma irradiation.

FLICE-inhibitory protein, FLIP (Casper/I-FLICE/FLAME-1/CASH/CLARP/MRIT), which contains two death effector domains and an inactive caspase domain, binds to FADD and caspase-8, and thereby inhibits death receptor-mediated apoptosis. Here, we characterize the inhibitory effect of FLIP on a variety of apoptotic pathways. Human Jurkat T cells undergoing Fas ligand-mediated apoptosis in response to CD3 activation were completely resistant when transfected with FLIP. In contrast, the presence of FLIP did not affect apoptosis induced by granzyme B in combination with adenovirus or perforin. Moreover, the Fas ligand, but not the perforin/granzyme B-dependent lytic pathway of CTL, was inhibited by FLIP. Apoptosis mediated by chemotherapeutic drugs (i.e., doxorubicin, etoposide, and vincristine) and gamma irradiation was not affected by FLIP or the absence of Fas, indicating that these treatments can induce cell death in a Fas-independent and FLIP-insensitive manner.

MRI monitoring of focal cerebral ischemia in peroxisome proliferator-activated receptor (PPAR)-deficient mice.

Peroxisome proliferator-activated receptors (PPARs) are a potential target for neuroprotection in focal ischemic stroke. These nuclear receptors have major effects in lipid metabolism, but they are also involved in inflammatory processes. Three PPAR isotypes have been identified: alpha, beta (or delta) and gamma. The development of PPAR transgenic mice offers a promising tool for prospective therapeutic studies. This study used MRI to assess the role of PPARalpha and PPARbeta in the development of stroke. Permanent middle cerebral artery occlusion induced focal ischemia in wild-type, PPARalpha-null mice and PPARbeta-null mice. T(2)-weighted MRI was performed with a 7 T MRI scan on day 0, 1, 3, 7 and 14 to monitor lesion growth in the various genotypes. General Linear Model statistical analysis found a significant difference in lesion volume between wild-type and PPAR-null mice for both alpha and beta isotypes. These data validate high-resolution MRI for monitoring cerebral ischemic lesions, and confirm the neuroprotective role of PPARalpha and PPARbeta in the brain.

Xenopus peroxisome proliferator activated receptors: genomic organization, response element recognition, heterodimer formation with retinoid X receptor and activation by fatty acids.

Peroxisome proliferator activated receptors are ligand activated transcription factors belonging to the nuclear hormone receptor superfamily. Three cDNAs encoding such receptors have been isolated from Xenopus laevis (xPPAR alpha, beta, and gamma). Furthermore, the gene coding for xPPAR beta has been cloned, thus being the first member of this subfamily whose genomic organization has been solved. Functionally, xPPAR alpha as well as its mouse and rat homologs are thought to play an important role in lipid metabolism due to their ability to activate transcription of a reporter gene through the promoter of the acyl-CoA oxidase (ACO) gene. ACO catalyzes the rate limiting step in the peroxisomal beta-oxidation of fatty acids. Activation is achieved by the binding of xPPAR alpha on a regulatory element (DR1) found in the promoter region of this gene, xPPAR beta and gamma are also able to recognize the same type of element and are, as PPAR alpha, able to form heterodimers with retinoid X receptor. All three xPPARs appear to be activated by synthetic peroxisome proliferators as well as by naturally occurring fatty acids, suggesting that a common mode of action exists for all the members of this subfamily of nuclear hormone receptors.

Interleukins (IL)-1 and IL-2 control IL-2 receptor alpha and beta expression in immature thymocytes.

Functional high-affinity interleukin-2 receptors (IL-2R) contain three transmembrane proteins, IL-2R alpha, beta and gamma. We have investigated the expression of IL-2R alpha and beta genes in immature mouse thymocytes. Previous work has shown that during differentiation these cells transiently express IL-2R alpha on their surface. Stimulation of IL-2R alpha+ and IL-2R alpha- immature thymocytes with phorbol 12-myristate 13-acetate and calcium ionophore induces synthesis of IL-2R alpha and IL-2R beta mRNA. Most of this response depends on autocrine stimulation by IL-2. IL-1 synergizes with IL-2 to induce a 120-fold increase in IL-2R alpha mRNA and a 14-fold increase in IL-2R beta mRNA levels. A large proportion of the stimulated cells contains both transcripts. These interleukins do not induce any differentiation to more mature phenotypes. Collectively, these results show that IL-2 plays a major role in the regulation of IL-2R expression in normal immature thymocyte. We suggest that this response to interleukins may be part of a homeostatic mechanism to increase the production of immature thymocytes during stress.

Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers.

The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, beta, and gamma) regulate the peroxisomal beta-oxidation of fatty acids by induction of the acyl-CoA oxidase gene that encodes the rate-limiting enzyme of the pathway. Gel retardation and cotransfection assays revealed that PPAR alpha heterodimerizes with retinoid X receptor beta (RXR beta; RXR is the receptor for 9-cis-retinoic acid) and that the two receptors cooperate for the activation of the acyl-CoA oxidase gene promoter. The strongest stimulation of this promoter was obtained when both receptors were exposed simultaneously to their cognate activators. Furthermore, we show that natural fatty acids, and especially polyunsaturated fatty acids, activate PPARs as potently as does the hypolipidemic drug Wy 14,643, the most effective activator known so far. Moreover, we discovered that the synthetic arachidonic acid analogue 5,8,11,14-eicosatetraynoic acid is 100 times more effective than Wy 14,643 in the activation of PPAR alpha. In conclusion, our data demonstrate a convergence of the PPAR and RXR signaling pathways in the regulation of the peroxisomal beta-oxidation of fatty acids by fatty acids and retinoids.

Critical roles of the nuclear receptor PPARbeta (peroxisome-proliferator-activated receptor beta) in skin wound healing.

The PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma belong to the nuclear hormone receptor superfamily. While all three receptors are undetectable in adult mouse interfollicular epidermis, PPARbeta expression and activity is strongly re-activated by inflammatory stimuli during epidermal injury. The pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) stimulates transcription of the PPARbeta gene via an activator protein-1 site in its promoter and it also triggers the production of PPARbeta ligands in keratinocytes. This increase of PPARbeta activity in these cells up-regulates the expression of integrin-linked kinase and 3-phosphoinositide-dependent kinase-1, which phosphorylates protein kinase B-alpha (Akt1). The resulting increase in Akt1 activity suppresses apoptosis and ensures the presence of a sufficient number of viable keratinocytes at the wound margin for re-epithelialization. Together, these observations reveal that PPARbeta takes on multiple roles and contributes favourably to the process of wound closure.

The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta 2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12.

Within 1 day of infection with Leishmania major, susceptible BALB/c mice produce a burst of IL-4 in their draining lymph nodes, resulting in a state of unresponsiveness to IL-12 in parasite-specific CD4+ T cells within 48 h. In this report we examined the molecular mechanism underlying this IL-12 unresponsiveness. Extinction of IL-12 signaling in BALB/c mice is due to a rapid down-regulation of IL-12R beta2-chain mRNA expression in CD4+ T cells. In contrast, IL-12R beta2-chain mRNA expression was maintained on CD4+ T cells from resistant C57BL/6 mice. The down-regulation of the IL-12R beta2-chain mRNA expression in BALB/c CD4+ T cells is a consequence of the early IL-4 production. In this murine model of infection, a strict correlation is shown in vivo between expression of the IL-12R beta2-chain in CD4+ T cells and the development of a Th1 response and down-regulation of the mRNA beta2-chain expression and the maturation of a Th2 response. Treatment of BALB/c mice with IFN-gamma, even when IL-4 has been produced for 48 h, resulted in maintenance of IL-12R beta2-chain mRNA expression and IL-12 responsiveness. The data presented here support the hypothesis that the genetically determined susceptibility of BALB/c mice to infection with L. major is primarily based on an up-regulation of IL-4 production, which secondarily induces extinction of IL-12 signaling.

The effects of gamma-hydroxybutyrate, baclofen and GABAB receptors on brain activity and sleep in mice and humans

Currently, there is an increased interest in γ-hydroxybutyric acid (GHB) and its effects onsleep. This compound, sometimes referred to as 'rape drug', was recently approved as atreatment for the sleep disorder narcolepsy. Although several studies suggest that GHBinduces slow-wave sleep duration and improves sleep quality by increasing EEG slow-waveactivity, others question its ability to induce physiological sleep. GHB's mechanism of actionis still unclear, although in vivo and in vitro it seems to act at high doses as a low-affinityagonist of GABAB receptors. Furthermore, the role GABAB receptors play in sleep and theelectroencephalogram (EEG) is largely unknown.The aim of this project was therefore to investigate the effects of GHB on sleep and EEG, theinvolvement of GABAB receptors in mediating these effects, as well as the intrinsic role ofeach GABAB receptor subunit in the regulation of sleep. Thus, we administered GHB andbaclofen (BAC, a high-affinity agonist at GABAB receptor) to mice lacking the different GABABreceptor subunits and to healthy human volunteers.Our results, both in mice and humans, showed that GHB produced slow waves exclusivelythrough the stimulation of GABAB receptors, but did not induce physiological sleepnecessary to reduce sleep need and to increase cognitive performance. Unlike GHB, BACaffected the homeostatic regulation of sleep (sleep need) and induced a delayedhypersomnia. Finally, GABAB receptor and its subunits seem to play an important role insleep and in particular its circadian distribution.