Expression of the chemokine receptor CCR6 in the Lewis lung carcinoma (LLC) cell line reduces its metastatic potential in vivo.

Chemokines and their receptors play important roles in various aspects of tumoral processes, and evidence was provided for their critical involvement in determining the metastatic destination of tumor cells. Here, we analyzed in vitro and in vivo, how CCR6 expression could alter the behavior of Lewis lung carcinoma (LLC) cells, which were shown to express low levels of the CCR6 ligand, CCL20 (LARC), both in vitro and in vivo. The expression of CCR6 significantly decreased the number of metastases in immunocompetent C57BL/6 mice, without affecting the tumor-forming ability of LLC cells. This was correlated with a decrease in clonogenicity in soft and hard agar, and with increased adhesion to type-IV collagen. These two observations made in basal conditions were enhanced when CCL20 was added to the assay medium. Thus, expression of CCR6 in tumor cells, associated with the local production of CCL20, decreased the metastatic potential of the LLC line. We propose a model, in which the expression of a chemokine receptor in tumor cells can act as a metastasis-suppressor, or a metastasis-promoting factor, according to the expression, or the absence of expression of the cognate ligand(s) in the tumor.

Deorphanization of G-protein-coupled receptors.

G-protein-coupled receptors constitute one of the major families of drug targets. Orphan receptors, for which the ligands and function are still unknown, are an attractive set of future targets for presently unmet medical needs. Screening strategies have been developed over the years in order to identify the natural ligands of these receptors. Natural or chimeric G-proteins that can redirect the natural coupling of receptors toward intracellular calcium release are frequently used. Potential problems include poor expression or trafficking to the cell surface, constitutive activity of the receptors, or the presence of endogenous receptors in the cell types used for functional expression, leading to nonspecific responses. Many orphan receptors characterized over the last 10 years have been associated with previously known bioactive molecules. However, new and unpredicted biological mediators have also been purified from complex biological sources. A few old and recent examples, including nociceptin, chemerin, and the F2L peptide are illustrated. Future challenges for the functional characterization of the remaining orphan receptors include the potential requirement of specific proteins necessary for quality control, trafficking or coupling of specific receptors, the possible formation of obligate heterodimers, and the possibility that some constitutively active receptors may lack ligands or respond only to inverse agonists. Adapted expression and screening strategies will be needed to deal with these issues.

[Pain management in bone metastasis of pulmonary origin: new interventional and metabolic techniques]. / Nouvelles techniques interventionnelles et métaboliques dans la prise en charge des métastases osseuses.

Invasion of bone by a metastatic lesion is the most common cause of pain in cancer patients. Pain management in these patients is an important and difficult task. The pain is not always properly controlled by high doses of specific medication, radiation therapy or chemotherapy. When these therapies do not provide adequate pain relief, percutaneous vertebroplasty, cementoplasty, radiofrequency ablation and internal radiotherapy appear to be elegant and efficient complementary alternative pain control methods.

[Smoking and pregnancy: survey among women enrolled in an independent worker insurance program]. / Tabac et grossesse. Etude de l'Assurance maladie des professions indépendantes en Ile-de-France.

OBJECTIVES: In order to further improve its prevention program entitled "Maternity childhood follow-up", the Paris region health insurance program for independent workers carried out a survey among female participants. The survey was designed to assess women's smoking behavior and evaluate reception of information concerning smoking-related risks and support available to stop smoking. The women's suggestions were also collected. MATERIALS AND METHODS: A questionnaire was sent by the physicians in charge of the Ile de France region to the 3525 women who had reported a pregnancy. RESULTS: 1099 answers were received (31%) The mean age of responders was 34.3 4.7 years. Their educational level was high (76% had a university degree). Before pregnancy, 37% were smokers. At the first trimester of pregnancy, 17% were smokers, 15% at the second trimester and 14% at the third trimester. Two years after childbirth, 20.5% were smokers. The proportion of women who stopped smoking was higher for those with a low Fagerström score. 29% of women said they did not receive any information about the harmful consequences of smoking. To cease smoking during pregnancy, 70% are helped by family and friends, 20% by health professionals; 3.5% took nicotine substitutes. CONCLUSION: This survey emphasises the need for improving information to pregnant women for alerting them to the dangers of smoking and for training the relevant health professionals.

[Characterization of new chemoattractant agents in leukocytes]. / Caractérisation de nouveaux récepteurs d'agents chimioattractants pour les leucocytes.

G protein-coupled receptors constitute one of the largest gene families in mammals. About a hundred orphan receptors still exist, for which the ligands and functions are unknown. We have recently identified the natural ligands of two orphan receptors expressed in dendritic cells and monocytes/macrophages. Chemerin, product of the gene Tig-2, was characterized as the ligand of the chemR23 receptor. The protein is synthesized as an inactive precursor, prochemerin, which requires the proteolytic removal of the last 6 or 7 amino acids, in order to generate a high affinity ligand of chemR23. Two neutrophil proteases, elastase and cathepsin G, are able to mediate this conversion. Besides, a peptide derived from the intracellular protein heme-binding protein (HBP) has been characterized as the first specific ligand of the FPRL2 receptor. The role of these two new systems in the control of physiological and pathological inflammatory reactions is presently being studied.

Adenosine A2A receptor gene disruption provokes marked changes in melanocortin content and pro-opiomelanocortin gene expression.

A2A receptor knockout (A2AR-/-) mice are more anxious and aggressive, and exhibit reduced exploratory activity than their wild-type littermates (A2AR+/+). Because alpha-melanocyte-stimulating hormone (alpha-MSH) influences anxiety, aggressiveness and motor activity, we investigated the effect of A2AR gene disruption on alpha-MSH content in discrete brain regions and pro-opiomelanocortin (POMC) expression in the hypothalamus and pituitary. No modification in alpha-MSH content was observed in the hypothalamus and medulla oblongata where POMC-expressing perikarya are located. In the arcuate nucleus of the hypothalamus, POMC mRNA levels were not affected by A2AR disruption. Conversely, in A2AR-/- mice, a significant increase in alpha-MSH content was observed in the amygdala and cerebral cortex, two regions that are innervated by POMC terminals. In the pars intermedia of the pituitary, A2AR disruption provoked a significant reduction of POMC mRNA expression associated with a decrease in alpha-MSH content. By contrast, in the anterior lobe of the pituitary, a substantial increase in POMC mRNA and adrenocorticotropin hormone concentrations was observed, and plasma corticosterone concentration was significantly higher in A2AR-/- mice, revealing hyperactivity of their pituitary-adrenocortical axis. Together, these results suggest that adenosine, acting through A2A receptors, may modulate the release of alpha-MSH in the cerebral cortex and amygdala. The data also indicate that A2A receptors are involved in the control of POMC gene expression and biosynthesis of POMC-derived peptides in pituitary melanotrophs and corticotrophs.

Caffeine reduces hypnotic effects of alcohol through adenosine A2A receptor blockade.

The role of the adenosine A(2A) receptor in the hypnotic effects of ethanol was assessed in mice. The duration of the loss of righting reflex following acute ethanol administration was shorter for A(2A) receptor-deficient mice (A(2A)R KO) than for wild-type mice (A(2A)R WT), whereas the fall in body temperature was not different between the two phenotypes. In contrast, the duration of the loss of righting reflex was increased in A(2A)R KO mice versus controls after administration of pentobarbital. Dipyridamole, an inhibitor of adenosine uptake, increased the sleep time observed following administration of ethanol in CD1 mice and in A(2A)R WT but not in A(2A)R KO mice. SCH 58261, a selective A(2A) receptor antagonist, unlike DPCPX, a selective A(1) receptor antagonist, shortened the duration of the loss of righting reflex induced by ethanol, thus mimicking the lack of receptor in deficient mice. Finally, the non-selective adenosine receptor antagonist caffeine (25 mg/kg) reduced ethanol-induced hypnotic effects. These results indicate that the activation of A(2A) receptors that follows an increase in extracellular adenosine levels caused by the administration of high doses of ethanol plays a role in its hypnotic effects. Thus, A(2A) receptor antagonists may be useful therapeutic agents for alleviating ethylic coma.

Immunodetection of proteins from grapes and yeast in a white wine.

The objective of this study was to analyze the origin of proteins of a Chardonnay wine. Three various polyclonal antibodies raised against must, yeast, and bacteria proteins were produced. For microorganisms, only the secreted macromolecules were used. To this end, yeast and bacteria were cultured in a model medium under conditions close to those of winemaking. Results obtained using these specific antibodies indicate that most of the wine proteins came from grapes and many of them were glycoproteins. Some proteins of this Chardonnay wine came from the yeast; they were released during the alcoholic fermentation and consisted of high molecular weight mannoproteins. In contrast, no bacteria proteins were detected in this Chardonnay wine.

Lack of CB1 cannabinoid receptors modifies nicotine behavioural responses, but not nicotine abstinence.

Cannabis is the most widely consumed illicit drug and its consumption is currently associated with tobacco, which contains another psychoactive compound, namely nicotine. Interactions between cannabinoids and other drugs of abuse, such as opioids, have been previously reported. The aim of the present study was to evaluate the possible role of CB1 cannabinoid receptor in responses induced by acute and repeated nicotine administration by using knockout mice lacking the CB1 cannabinoid receptor and their wild-type littermates. Acute nicotine (0.5, 1, 3 and 6 mg/kg, sc) administration decreased locomotor activity and induced antinociceptive responses in the tail-immersion and the hot-plate test, in wild-type animals. The antinociceptive effects in the tail-immersion test were significantly enhanced in CB1 knockout mice. In wild-type mice nicotine (0.5 mg/kg, sc) produced a significant rewarding effect, as measured by a conditioned place preference paradigm. This response was absent in CB1 knockout mice. Finally, a model of mecamylamine-induced abstinence in chronic nicotine-treated mice (10 mg/kg/day, sc) was developed. Mecamylamine (1 and 2 mg/kg, sc) precipitated several somatic signs of nicotine withdrawal in wild-type dependent mice. However, no difference in the severity of nicotine withdrawal was observed in CB1 knockout mice. These results demonstrate that some acute effects and motivational responses elicited by nicotine can be modulated by the endogenous cannabinoid system and support the existence of a physiological interaction between these two systems.

[Secondary prevention of myocardial infarction in the Ile de France]. / Prévention secondaire de l'infarctus du myocarde en Ile-de-France.

This enquiry was carried out to evaluate the measures of secondary prevention at 6 months and over of myocardial infarction in the ile de France region with respect to the recommendations of scientific societies and results of large scale therapeutic trials. A questionnaire was completed for the 1,215 patients selected from data obtained from the hospital discharge summary, interrogation and examination of the patient, and a telephone conversation with the attending physician. The data covered cardiovascular risk factors, the main clinical parameters, the results of biological tests and investigations carried out for risk stratification, plus different elements of therapeutic management. Compared with previous studies of the same type, this enquiry showed a favourable tendency towards the prescription of antithrombotic drugs and betablockers (98.3% and 82.4% of patients, respectively), and to patients with reputedly normal blood pressure values (84.7%). A positive result concerning the reduction in the number of smokers (17.4%) and the increase in lipid lowering prescriptions should be tempered by the fact that advice about stopping smoking was rarely given and that the quantitative target of LDL cholesterol was often ignored. Finally, the prescriptions of ACE inhibitors, physical exercise and cardiac rehabilitation remained well below the recommendations or recent scientific data.

Amino-terminal truncation of CXCR3 agonists impairs receptor signaling and lymphocyte chemotaxis, while preserving antiangiogenic properties.

The interferon (IFN)-inducible chemokines, specifically, IFN-gamma-inducible protein-10 (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), share a unique CXC chemokine receptor (CXCR3). Recently, the highly specific membrane-bound protease and lymphocyte surface marker CD26/dipeptidyl peptidase IV (DPP IV) was found to be responsible for posttranslational processing of chemokines. Removal of NH(2)-terminal dipeptides by CD26/DPP IV alters chemokine receptor binding and signaling, and hence inflammatory and anti-human immunodeficiency virus (HIV) activities. CD26/DPP IV and CXCR3 are both markers for Th1 lymphocytes and, moreover, CD26/DPP IV is present in a soluble, active form in human plasma. This study reports that at physiologic enzyme concentrations CD26/DPP IV cleaved 50% of I-TAC within 2 minutes, whereas for IP-10 and Mig the kinetics were 3- and 10-fold slower, respectively. Processing of IP-10 and I-TAC by CD26/DPP IV resulted in reduced CXCR3-binding properties, loss of calcium-signaling capacity through CXCR3, and more than 10-fold reduced chemotactic potency. Moreover, IP-10 and I-TAC cleaved by CD26/DPP IV acted as chemotaxis antagonists and CD26/DPP IV-truncated IP-10 and Mig retained their ability to inhibit the angiogenic activity of interleukin-8 in the rabbit cornea micropocket model. These data demonstrate a negative feedback regulation by CD26/DPP IV in CXCR3-mediated chemotaxis without affecting the angiostatic potential of the CXCR3 ligands IP-10 and Mig.

In vivo labelling of the adenosine A2A receptor in mouse brain using the selective antagonist [3H]SCH 58261.

The selective A2A receptor antagonist [3H]SCH 58261 was injected intravenously in mice and the radioactivity accumulating in various brain regions was determined by tissue sampling. Radioactivity levels in regions of interest such as the striatum were highest 15 min after injection and quickly declined thereafter (30 min and 1 h postinjection) in a time-dependent manner. The amount of labelling was ranked as follows: striatum (4.6 +/- 0.3 fmol/mg protein) >> cortex > hippocampus > pons = hypothalamus > cerebellum (0.5 +/- 0.05 fmol/mg protein). Specific labelling of the A2A receptor occurred in striatum and cortex because significantly less radioactivity accumulated in these areas from adenosine A2A receptor knockout mice as compared to wild-type littermates. In control outbred CD1 mice, a striatum-to-cerebellum ratio of 7.6 +/- 0.6 was found. At 30 min postinjection, the nonselective adenosine receptor antagonist caffeine reduced the radioactivity due to [3H]SCH 58261 in the striatum by 32% at 1 mg/kg i.p. and by 66% at the stimulant dose of 6.25 mg/kg i.p. Radioactivity in the striatum was lowered, respectively, by 66 and 86% 30 min after injection of 3 or 10 mg/kg i.p. doses of unlabelled SCH 58261. The present results indicate that [3H]SCH 58261 directly labels striatal A2A receptors in vivo. Thus [3H]SCH 58261 is an excellent tool for studying brain distribution and A2A receptor occupancy of various compounds ranging from xanthines, such as caffeine, to other A2A antagonists.

Distribution of an orphan G-protein coupled receptor (JP05) mRNA in the human brain.

JP05, also called GPR72 or GIR, is an orphan G-protein-coupled receptor, GPCR, showing significant structural similarity to the tachykinin receptors. The anatomical distribution of JP05 mRNA was first described in the central nervous system of the mouse, and recently the human JP05 orphan receptor gene has been cloned. In the present study the distribution of JP05 mRNA was examined in the human forebrain using in situ hybridization analysis. The results revealed a wide but discrete distribution of the transcript with strongly JP05 mRNA expressing cells, presumably neurons, present in the cerebral cortex (layer II), hippocampus (pyramidal CA3 neurons and granule cells), amygdala (basal and periamygdaloid cortical nuclei), in the endopiriform nucleus, diagonal band of Broca, thalamus (nucleus reuniens, parafascicular nucleus) and hypothalamus (posterior, dorsal, and around the medial mammillary). Weaker signals were detected in the deeper cortical layers and throughout the striatum. A few positive cells were evident in the raphe but not in the substantia nigra or pontine nuclei. The results indicate significant similarities between human and mouse brain with regard to JP05 mRNA expression. The distribution patterns of JP05 mRNA in the human brain suggest involvement in control of emotions and of neuroendocrine, cognitive and motor functions.

Acute and chronic caffeine administration differentially alters striatal gene expression in wild-type and adenosine A(2A) receptor-deficient mice.

In order to assess for the respective involvement of adenosine A(1) and A(2A) receptors (A(2A)-R) in the consequences of short- and long-term caffeine exposure on gene expression, the effects of acute caffeine administration on striatal, cortical, and hippocampal expression of immediate early genes (IEG), zif-268 and arc, and the effects of long-term caffeine or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) exposure (once daily for 15 days) on striatal gene expression of substance P, enkephalin, and glutamic acid decarboxylase isoforms, GAD65 and GAD67, were evaluated in wild-type and A(2A)-R-deficient (A(2A)-R(-/-)) mice. In situ hybridization histochemistry was performed using oligonucleotides followed by quantitative image analysis. Our results demonstrated that a biphasic response of IEG expression to acute caffeine observed in the wild-type striatum was resumed in a monophasic response in the mutant striatum. In the cerebral cortex and hippocampus, the effect of caffeine was weak in wild-type, whereas in mutant mice it induced a 2-3-fold increase in the IEG expression to restore a level similar to the wild-type basal expression. Chronic caffeine and DPCPX-mediated regulation in neuropeptide and GADs striatal gene expression typically showed the mimicking of alterations resulting from the A(2A)-R genetic deficiency in 25 mg/kg caffeine-treated wild-type mice as well as the dose-dependent normalization of substance P and enkephalin expression in A(2A)-R(-/-) mice. These results indicate that, depending on the dose, the blockade of A(2A)-R or A(1) receptors by caffeine is preferentially revealed leading to highly differential alterations in striatal gene expression and they also suggested the central role of these two receptors on the control of dopaminergic functions.

Distribution of metabotropic glutamate receptor DmGlu-A in Drosophila melanogaster central nervous system.

L-glutamate is the excitatory neurotransmitter at neuromuscular junctions in insects. It may also be involved in neurotransmission within the central nervous system (CNS), but its function therein remains elusive. The roles of glutamatergic synapses in the Drosophila melanogaster CNS were investigated, with focus on the study of DmGluRA, a G-protein-coupled glutamate receptor. In a first attempt to determine the function of this receptor, we describe its distribution in the larval and adult Drosophila CNS, using a polyclonal antibody raised against the C-terminal sequence of the protein. DmGluRA is expressed in a reproducible pattern both in the larva and in the adult. In particular, DmGluRA can be found in the antennal lobes, the optic lobes, the central complex, and the median bundle in the adult CNS. However, DmGluRA-containing neurons represented only a small fraction of all CNS neurons. DmGluRA immunoreactivity was not detected at the larval neuromuscular junction nor in the body wall muscles. The correlations between DmGluRA distribution and previously described glutamate-like immunoreactivity patterns, as well as the implications of these observations concerning the possible functions of DmGluRA in the Drosophila CNS, are discussed.

Urokinase plasminogen activator and plasmin efficiently convert hemofiltrate CC chemokine 1 into its active.

We have previously isolated from human hemofiltrate an N-terminally truncated form of the hemofiltrate CC chemokine 1 (HCC-1), and characterized HCC-1[9-74] as a strong agonist of CCR1, CCR5, and to a lower extent CCR3. In this study, we show that conditioned media from human tumor cell lines PC-3 and 143B contain proteolytic activities that convert HCC-1 into the [9-74] form. This activity was fully inhibited by inhibitors of urokinase-type plasminogen activator (uPA), including PA inhibitor-1, an anti-uPA mAb, and amiloride. Pure preparations of uPA processed HCC-1 with high efficiency, without further degrading HCC-1[9-74]. Plasmin could also generate HCC-1[9-74], but degraded the active product as well. The kinetics of HCC-1 cleavage by uPA and plasmin (Michaelis constant, K(m), of 0.76 +/- 0.4 microM for uPA, and 0.096 +/- 0.05 microM for plasmin; catalytic rate constant, k(cat): 3.36 +/- 0.96 s(-1) for uPA and 6 +/- 3.6 s(-1) for plasmin) are fully compatible with a role in vivo. The activation of an abundant inactive precursor into a broad-spectrum chemokine by uPA and plasmin directly links the production of uPA by numerous tumors and their ability to recruit mononuclear leukocytes, without the need for the transcriptional activation of chemokine genes.

Identification of a novel human ADP receptor coupled to G(i).

We have cloned and expressed a novel human G-protein-coupled receptor closely related to the human P2Y(12) receptor. It corresponds to the orphan receptor called GPR86. GPR86 proved to be a G(i)-coupled receptor displaying a high affinity for ADP, similar to the P2Y(12) receptor and can therefore be tentatively called P2Y(13). In 1321N1 cells, the P2Y(13) receptor coupled to the phosphoinositide pathway only when coexpressed with Galpha(16). Inositol trisphosphate formation was stimulated equipotently by nanomolar concentrations of ADP and 2MeSADP, whereas 2MeSATP and ATP were inactive. In CHO-K1 cells expressing the P2Y(13) receptor, ADP and 2MeSADP had a biphasic effect on the forskolin-stimulated accumulation of cAMP: inhibition at nanomolar concentrations and potentiation at micromolar levels. In the same cells, ADP and 2MeSADP also stimulated the phosphorylation of Erk1 and Erk2, in a pertussis toxin-sensitive way. The tissue distribution of P2Y(13) was investigated by reverse transcriptase-polymerase chain reaction, and the predominant signals were obtained in spleen and brain. Although these can be discriminated by tissue distribution and some pharmacological features, the P2Y(12) and P2Y(13) receptors form a subgroup of related P2Y subtypes that is structurally different from the other P2Y subtypes but share coupling to G(i) and a high affinity for ADP.

The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila.

Frizzled (Fz) signaling regulates cell polarity in both vertebrates and invertebrates. In Drosophila, Fz orients the asymmetric division of the sensory organ precursor cell (pI) along the antero-posterior axis of the notum. Planar polarization involves a remodeling of the apical-basal polarity of the pI cell. The Discs-large (Dlg) and Partner of Inscuteable (Pins) proteins accumulate at the anterior cortex, while Bazooka (Baz) relocalizes to the posterior cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell.