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1.
Arthritis Care Res (Hoboken) ; 65(5): 712-7, 2013 May.
Article in English | MEDLINE | ID: mdl-23589412

ABSTRACT

OBJECTIVE: An annual assessment of cardiovascular (CV) risk factors in rheumatoid arthritis (RA) is recommended, but its practical modalities have not been determined. The objective was to assess the feasibility and usefulness of a standardized CV risk assessment in RA, performed by rheumatologists during outpatient clinics. METHODS: We used a cross-sectional design within a network of rheumatologists. Each rheumatologist included 5 consecutive unselected patients with definite RA. Data collection included standardized assessment of CV risk factors: blood pressure, interpretation of glycemia and of lipid levels, and calculation of the Framingham CV risk score. Outcome criteria included feasibility (missing data and time taken to assess the patients) and usefulness (the CV risk assessment was considered useful if at least 1 modifiable and previously unknown CV risk factor was evidenced). RESULTS: Twenty-two rheumatologists (77% in office-based practice) assessed 110 RA patients. The mean ± SD age was 57 ± 10 years, and the mean ± SD RA duration was 11 ± 9 years; 50 patients (45%) were treated with biologic agents, and 76% were women. Regarding feasibility, missing data were most frequent for glycemia (27% of patients) and cholesterolemia (14% of patients). The mean ± SD duration of the CV risk assessment was 15 ± 5 minutes. The CV risk assessment was considered useful in 33 patients (30%), evidencing dyslipidemia (15% of patients) or high blood pressure (9% of patients) as the most frequently previously unknown CV risk factor. CONCLUSION: The assessment of CV risk factors is feasible, but labor intensive, during an outpatient rheumatology clinic. This assessment identified modifiable CV risk factors in 30% of the patients. These results suggest that RA patients are not sufficiently assessed and treated for CV risk factors.


Subject(s)
Ambulatory Care/methods , Arthritis, Rheumatoid/diagnosis , Arthritis, Rheumatoid/epidemiology , Cardiovascular Diseases/diagnosis , Cardiovascular Diseases/epidemiology , Aged , Arthritis, Rheumatoid/therapy , Cardiovascular Diseases/prevention & control , Cross-Sectional Studies , Feasibility Studies , Female , Humans , Male , Middle Aged , Pilot Projects , Prospective Studies , Rheumatology/methods , Risk Assessment
3.
Eur J Pharmacol ; 400(2-3): 185-94, 2000 Jul 21.
Article in English | MEDLINE | ID: mdl-10988332

ABSTRACT

1-[2-(4-(2-Chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid (SR 27897) is an effective CCK(1) receptor antagonist, while the structurally related molecule 2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid (SR 146131) is a highly potent and specific agonist for the same receptor. To discover how the two molecules interact with the human cholecystokinin (CCK) CCK(1) receptor, we have carried out binding and activity studies with 33-point mutated receptors. Only six mutants showed altered [3H]SR 27897 binding properties, Lys(115), Lys(187), Phe(198), Trp(209), Leu(214) and Asn(333). In contrast, numerous mutations throughout the receptor either reduced SR 146131 agonist potency, Phe(97), Gly(122), Phe(198), Trp(209), Ile(229), Asn(333), Arg(336) and Leu(356) or increased it, Tyr(48), Cys(94), Asn(98), Leu(217) and Ser(359). Only mutations of Phe(198), Trp(209) and Asn(333) affected both SR 27897 and SR 146131 binding or activity. The collated information was used to construct molecular models of SR 27897 and SR 146131 bound to the human CCK(1) receptor. The clear difference in the binding sites of SR 27897 and SR 146131 offers a molecular explanation for their contrasting pharmacological characteristics.


Subject(s)
Indoleacetic Acids/metabolism , Indoles/metabolism , Receptors, Cholecystokinin/metabolism , Thiazoles/metabolism , Amino Acid Sequence , Animals , Binding Sites , Binding, Competitive , COS Cells/metabolism , Hormone Antagonists/metabolism , Hormone Antagonists/pharmacology , Humans , Indoleacetic Acids/pharmacology , Indoles/pharmacology , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Point Mutation , Protein Conformation , Protein Structure, Tertiary , Receptors, Cholecystokinin/agonists , Receptors, Cholecystokinin/antagonists & inhibitors , Receptors, Cholecystokinin/genetics , Thiazoles/pharmacology
5.
Eur J Pharmacol ; 401(1): 17-25, 2000 Jul 28.
Article in English | MEDLINE | ID: mdl-10915832

ABSTRACT

We have investigated the binding site of the subtype specific antagonist SR 144528, (N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2. 1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methoxybenzyl)- pyrazo le-3-carboxamide) on the human cannabinoid CB(2) receptor based on functional studies with mutated receptors. Two serine residues in the fourth transmembrane region, Ser(161) and Ser(165), were singly mutated to the cognate cannabinoid CB(1) receptor residue, alanine, and each gave receptors with wild-type properties for the cannabinoid agonists CP 55,940 (1R,3R,4R)-3-[2-hydroxy-4-(1, 1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol) and WIN 55212-2 (R)-(+)[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl](1-naphthalenyl) methanone, which SR 144528 completely failed to antagonise. Molecular modelling studies show that SR 144528 interacts with residues in transmembrane domains 3, 4, and 5 of the cannabinoid CB(2) receptor through a combination of hydrogen bonds and aromatic and hydrophobic interactions. In addition, the replacement by serine of a nearby cannabinoid CB(2) receptor-specific residue, Cys(175) resulted in wild-type receptor properties with CP 55,940, loss of SR 144528 binding and eight-fold reduced binding and activity of WIN 55212-2, a result compatible with a recently-proposed binding site model for WIN 55212-2.


Subject(s)
Camphanes/metabolism , Pyrazoles/metabolism , Receptors, Drug/metabolism , Amino Acid Sequence , Animals , Benzoxazines , Binding Sites/genetics , Binding, Competitive/drug effects , COS Cells , Camphanes/chemistry , Camphanes/pharmacology , Cyclohexanols/pharmacology , Humans , Luciferases/genetics , Luciferases/metabolism , Models, Molecular , Molecular Sequence Data , Morpholines/pharmacology , Mutagenesis, Site-Directed , Mutation , Naphthalenes/pharmacology , Protein Structure, Tertiary , Pyrazoles/chemistry , Pyrazoles/pharmacology , Receptors, Cannabinoid , Receptors, Drug/antagonists & inhibitors , Receptors, Drug/genetics , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
6.
Eur J Pharmacol ; 389(2-3): 115-24, 2000 Feb 18.
Article in English | MEDLINE | ID: mdl-10688974

ABSTRACT

We hypothesized that charge-charge interactions may be important for the binding of the human cholecystokinin type 1 (CCK(1)) receptor-specific non-peptide full agonist SR 146131, (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid), the competitive antagonist SR 27897, (1-[2-(4-(2-chlorophenyl)thiazol-2-yl) aminocarbonyl indoyl] acetic acid) and the natural octapeptide CCK-8S to the CCK(1) receptor. Alanine replacement studies of positively charged residues in the extracellular domains of the receptor showed that only the R336A mutation affected SR 146131 potency of mutated receptors transiently expressed in monkey kidney epithelial COS-7 cells. Two residues, Lys(115) and Lys(187), were implicated in SR 27897 binding. Only the replacement of Lys(115), Arg(197) and Arg(336) significantly affected CCK-8S binding or activity. These results clearly indicated the importance of certain charged residues, but not others, in SR 146131, SR 27897 and CCK-8S binding. Furthermore, although these molecules probably occupy different binding sites on the CCK(1) receptor, we show that a small non-peptide agonist, SR 146131, can stimulate the dual signaling pathways mediated by the CCK(1) receptor.


Subject(s)
Indoleacetic Acids/metabolism , Indoles/metabolism , Receptors, Cholecystokinin/metabolism , Sincalide/analogs & derivatives , Thiazoles/metabolism , Amino Acid Sequence , Animals , COS Cells , Dose-Response Relationship, Drug , Humans , Inositol Phosphates/metabolism , Molecular Sequence Data , Mutation , Receptor, Cholecystokinin A , Receptors, Cholecystokinin/chemistry , Sincalide/metabolism , Structure-Activity Relationship
7.
Eur J Pharmacol ; 383(3): 339-46, 1999 Nov 03.
Article in English | MEDLINE | ID: mdl-10594328

ABSTRACT

A new highly specific, potent non-peptide agonist for the cholecystokinin subtype 1 receptor (CCK(1)), SR 146131 (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid) was recently described [Bignon, E., Bachy, A., Boigegrain, R., Brodin, R., Cottineau, M., Gully, D., Herbert, J.-M., Keane, P., Labie, C., Molimard, J.-C., Olliero, D., Oury-Donat, F., Petereau, C., Prabonneaud, V., Rockstroh, M.-P., Schaeffer, P., Servant, O.Thurneyssen, O., Soubrié, P., Pascal, M., Maffrand, J.-P., Le Fur, G., 1999. SR 146131: a new, potent, orally active and selective non-peptide cholecystokinin subtype I receptor agonist: I. In vitro studies. J. Pharmacol. Exp. Ther. 289, 742-751]. From binding and activity assays with chimeric constructs of human CCK(1) and the cholecystokinin subtype 2 receptor (CCK(2)) and receptors carrying point mutations, we show that Leu(356), situated in transmembrane domain seven in the CCK(1) receptor, is a putative contact point for SR 146131. In contrast, Leu(356) is probably not in contact with the CCK(1) receptor specific antagonist SR 27897 (1-[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl indoyl]acetic acid), a compound structurally related to SR 146131, since its replacement by alanine, histidine or asparagine gave receptors having wild-type CCK(1) receptor SR 27897 binding affinity. Previous mutational analysis of His(381), the cognate position in the rat CCK(2) receptor, had implicated it as being involved in subtype specificity for SR 27897, results which we confirm with corresponding mutations in the human CCK(2) receptor. Moreover, binding and activity assays with the natural CCK receptor agonist, CCK-8S, show that CCK-8S is more susceptible to the mutations in that position in the CCK(1) receptor than in the CCK(2) receptor. The results suggest different binding modes for SR 27897, SR 146131 and CCK-8S in each CCK receptor subtype.


Subject(s)
Hormone Antagonists/metabolism , Indoleacetic Acids/metabolism , Indoles/metabolism , Leucine/metabolism , Receptors, Cholecystokinin/metabolism , Sincalide/analogs & derivatives , Thiazoles/metabolism , Animals , Binding Sites , COS Cells/metabolism , Humans , Point Mutation , Receptors, Cholecystokinin/agonists , Receptors, Cholecystokinin/genetics , Sincalide/metabolism
8.
Brain Res Mol Brain Res ; 38(1): 54-62, 1996 May.
Article in English | MEDLINE | ID: mdl-8737667

ABSTRACT

Serotonin is a widely distributed neurotransmitter which elicits a range of central activities. We examined the effect of serotonin on cytokine mRNA expression by rat hippocampal astrocytes in primary cultures. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis shows that interleukin-6 (IL6) mRNA is expressed after 10(-12) M serotonin stimulation whereas transforming growth factor-beta (TGF beta) and tumor necrosis factor (TNF alpha) are induced by 10(-10) M serotonin. These inductions appeared after 1 h stimulation for IL6 and TNF alpha, whereas that of TGF beta appeared after 4 h. The present results provide the first evidence that serotonin can influence astrocyte cytokine production, and thus this neurotransmitter may be considered a potential neuroimmunomodulator.


Subject(s)
Astrocytes/drug effects , Cytokines/genetics , Hippocampus/drug effects , RNA, Messenger/biosynthesis , Serotonin/pharmacology , Animals , Astrocytes/metabolism , Base Sequence , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Hippocampus/cytology , Hippocampus/metabolism , Molecular Sequence Data , Polymerase Chain Reaction/methods , Rats , Rats, Sprague-Dawley , Transcription, Genetic
9.
Eur Cytokine Netw ; 3(6): 553-63, 1992.
Article in English | MEDLINE | ID: mdl-1296803

ABSTRACT

The measurement of cytokine mRNA levels is of fundamental importance in the understanding of diverse pathological states. We present a simplification of a polymerase chain reaction-based technique which permits the simultaneous measurement of up to 20 cytokine mRNAs, together with those of several other cellular products, including beta 2-microglobulin and beta-actin. The technique makes use of internal standards bearing multiple PCR primer sites which are identical to those on the mRNAs to be assayed. Known quantities of the standards are added to the cellular RNA and the mixture is co-reverse transcribed and co-amplified. The simplifications described here are based on the fact that each pair of amplicons accumulates in a constant ratio even in the plateau phase of amplification. As a result, no preliminary experiments to determine the limits of the exponential phase of amplification are necessary; the same number of cycles may be chosen for all the mRNAs to be measured, whatever their level in the mixture might be; pipetting errors are avoided since all calculations are based upon the relative quantities of co-amplified material. Here we illustrate the method through a quantitative study of the expression of cytokine mRNAs in U373 human astrocytoma cells before and after stimulation with IL-1 beta. Quantitation was carried out either by incorporating radioactivity in the amplicons or by fluorescence measurements after propidium iodide staining. Only very low numbers of transcripts for IL-6, IL-8, CSF-1, MCP-1 and either Gro alpha or Gro beta were detectable in unstimulated cells. The levels of these cytokine mRNAs increased dramatically following IL-1 beta stimulation and, in addition, transcription of IL-1 beta, TNF alpha, GM-CSF, G-CSF, Gro gamma and MCP-1, some of which have not previously been detected in U373, was initiated in the stimulated cells. At the same time we found that transcripts for IL-2, IL-3, IL-4, IL-5, IFN gamma, huMlP1 alpha and huMlP1 beta were totally absent in this cell line. These results suggest a potentially important role for astrocytes in the local amplification of inflammatory responses in the brain.


Subject(s)
Astrocytes/immunology , Cytokines/genetics , Polymerase Chain Reaction/methods , RNA, Messenger/genetics , Astrocytes/metabolism , Base Sequence , Cell Line , DNA/genetics , DNA Probes , Humans , Interleukin-1/pharmacology , Molecular Sequence Data , RNA, Messenger/analysis , RNA, Messenger/standards , Reference Standards
10.
J Biol Chem ; 267(30): 21830-8, 1992 Oct 25.
Article in English | MEDLINE | ID: mdl-1400493

ABSTRACT

We describe a simplified and reliable polymerase chain reaction-based method for assaying RNAs of low abundancy. The technique involves the co-amplification of cellular RNA-derived cDNA with a multispecific cDNA of synthetic origin added as an internal standard, using primer pairs common to both templates. We show that the co-amplified templates accumulate in a parallel manner throughout both the exponential and nonexponential phases of amplification, even when the starting amounts of the templates differ by up to 2 orders of magnitude. This finding means that preliminary experiments designed to determine either the late exponential region or the amplification efficiency for each pair of primers are unnecessary. This has enabled us to develop a greatly simplified quantitation protocol. We illustrate our approach by quantifying the effect of the immunosuppressor cyclosporin A on the accumulation of interleukin-4, interferon-gamma, and interleukin-2 receptor mRNAs in phytohemagglutinin-stimulated human peripheral blood mononuclear cells.


Subject(s)
Polymerase Chain Reaction/methods , RNA, Messenger/analysis , Base Sequence , Blotting, Southern , Cells, Cultured , Cyclosporine/pharmacology , DNA, Single-Stranded , Humans , Interferon-gamma/genetics , Interleukin-4/genetics , Kinetics , Molecular Sequence Data , Plasmids , Receptors, Interleukin-2/genetics , Titrimetry , Tumor Necrosis Factor-alpha/genetics , beta 2-Microglobulin/genetics
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