BY Youlet

Abstract: Objectives: The purpose of this study was to investigate the hypothesis that responses to the ATP test and head-up tilt test (HUT) may be correlated with different purinergic profiles. Design and setting: The ATP and HUT identify distinct subsets of patients with neurally mediated syncope (NMS). Adenosine and its A2A receptors (A2AR) may be implicated in the pathophysiology of NMS in patients with positive HUT. Nothing is known about the purinergic profile of patients with positive ATP. Patients and measures: This prospective study includes a consecutive series of patients with suspected NMS. All patients underwent both HUT and ATP. Before testing, samples were collected for measurement of baseline adenosine plasma level (APL) and expression. Results: A total of 46 patients (25 men and 21 women) with a mean age of 57±18 years were enrolled. The HUT test was positive in 27 patients and the ATP test in 20. Both tests were positive in 9 and negative in 8. High APL was associated with high probability of positive HUT while low APL was associated with high probability of positive ATP. Expression of A2AR was lower in patients with positive ATP than in those with positive HUT. Conclusion: These findings indicate that patients with NMS present different purinergic profiles and that responses to HUT and ATP are correlated with these profiles.

Abstract: We examined the short-course expression of various parameters involved in the adenosinergic signalling of a human T cell line during in vitro decrease of the medium culture oxygen tension mimicking in vivo hypoxia. Fall of 92 mmHg in oxygen tension of culture medium induced in CEM, a CD4+ human T cell line, a continuous production of hypoxia-inducing factor-1α with a plateau value at 9 h, a rapid increase in adenosine production peaking at 3 h and a decrease in adenosine deaminase peaking at 6 h. The adenosine A(2A) receptor (A(2A)R) protein level of CEM cells was enhanced with a peak at 6 h. Intracellular 3',5'-cyclic adenosine monophosphate accumulated in CEM cells with a maximal level at 9 h. These results show that a human-cultured T cells line can upregulate its own adenosine production and A(2A)R expression during exposure to acute hypoxia. Hypoxia-increased stimulation of the adenosinergic signalling of T cells may have immunosuppressive properties and, consequently, A(2A)R agonists may have therapeutic relevance.

Abstract: The fibromyalgia syndrome (FMS) affects 2%–10% of the population. Its diagnosis is only based on a history of widespread pain involving limbs and trunk and mild or greater tenderness to digital palpation of tender points.1 While the role of several neurotransmitters including serotonin, dopamine and SP has been suggested, the influence of endogenous adenosine has never been investigated. Adenosine exerts strong antinociceptive effects mostly via the activation of A1 adenosine receptors2 following its release by endothelial cells and myocytes3 into the extracellular space where it is degraded by the enzyme adenosine deaminase (ADA). Large amounts of ADA are found at the surface of mononuclear cells …

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Abstract: During ischaemia, the extracellular level of adenosine increases, which has cytotoxic effects. In endothelium, cell surface adenosine deaminase (ADA) complexing CD26 is coordinately induced during ischaemia as part of an adaptative response by eliminating adenosine. We examined whether a similar mechanism exists for mononuclear cells. We studied mononuclear cell surface ADA (MCADA) and dipeptidyl-peptidase IV activity (DPPIV) of membrane CD26 during percutaneous transluminal coronary angioplasty (PTCA) as a model of ischaemia–reperfusion. Enzymatic activities were compared with levels of ischaemia-modified albumin (IMA), a marker of ischaemia-reperfusion.

Abstract: Adenosine is a modulator of nociceptive pathways, both at the spinal and supraspinal levels. Adenosine A1 and A2A receptors (A1R, A2AR) are expressed in the basal ganglia where they are the target of caffeine, the most widely use psychoactive drug which acts as an antagonist to both types of receptors. Given the controversial role of A2AR versus A1R in modulating pain in brain areas, mice received intracerebroventricular injection of Adonis, an agonist-like monoclonal antibody with high specificity for the A2AR and were subjected to behavioral tests investigating nociceptive thresholds. We report that Adonis led to a significant dose-dependent increase in hot-plate and tail-flick latencies in mice and that such increase was prevented by caffeine and ZM 241385, a specific A2AR antagonist. The Adonis antinociceptive effects were also inhibited by naloxone, a non selective antagonist for opioid receptors, suggesting that Adonis acts, at least in part, through the stimulation of the endogenous opioid system. These results confirm the A2AR as a target for pain control and Adonis as a potential drug with therapeutic interest.

Abstract: Immunocompetent cells express various G-protein-coupled receptors that transduce extracellular signals across the plasma membrane. Among them, CXCR4 and CCR5 chemokines receptors and adenosine A(2A) receptors (A(2A)R) are involved in inflammatory processes. Considering that A(2A)R activation may have incidence on CXCR4 and CCR5 protein expression through heterologous desensitization process, we tested Adonis, an agonist-like monoclonal antibody to A(2A)R on CD4+ CEM T-cells. We found that Adonis inhibited the CEM cell growth, up-regulated A(2A)R and down-regulated CXCR4 and CCR5 without modifying the CD4 expression. By reducing the expression of CXCR4 and CCR5 chemokines receptors utilized as entry co-receptors by HIV-1 during viral infection of CD4 expressing cells, Adonis stimulation of A(2A)R appears as a valuable means to treat infected cells.

Abstract: The synthesis of new C-6 1,2,3-triazole adenosine derivatives via microwave assisted 1,3-dipolar cycloaddition as key step is described. The binding on membranes of cells that over express A1 adenosine receptors (A1AR) was also evaluated. Among them, four compounds increased cAMP production, in a dose-dependent manner acting as antagonists of the A1AR, while two compounds act as agonists.

Abstract: The second extracellular loop of the A2A receptor (A2AR) of adenosine was used to immunize mice for production of Adonis, an IgM monoclonal antibody. Adonis bound to the immunogen peptide and the native receptor in ELISA with KD values in 6.51–12.35 nM range. It recognized a linear epitope of 7 amino acids (LFEDVVP) at the C-terminal part of the external loop. Adonis revealed a 45-kDa band in lysate of human peripheral blood mononuclear cells in Western blotting in denaturing conditions. This served to monitor the up-regulation of the A2AR expression by caffeine. Adonis stimulated the cAMP production and inhibited the cell proliferation of an A2AR transfected stable cell line. These results confirm the immunogenicity and the functional relevance of the second extracellular loop of the A2AR. They suggest that Adonis may be of clinical use in various pathological situations to measure the regulation of the A2AR expression and to act as A2AR agonist drug.

Abstract: OBJECTIVE: Chronic heart failure (CHF) is accompanied by increased adenosine plasma levels (APLs). It is unknown whether adenosine release occurs at the peripheral level or whether the myocardium itself is the source of adenosine release. To answer this question, we evaluated APLs in the coronary sinus of CHF patients during a resynchronization procedure and compared the values with those at the peripheral level. We also investigated a possible correlation between APLs and ischemia-modified albumin (IMA) levels, a useful marker of tissue ischemia. Patients and measures: 19 men and 7 women were prospectively included. Blood samples for APLs were collected simultaneously from a brachial vein (peripheral) and from the coronary sinus. Blood samples for brain natriutretic peptide (BNP) and IMA were collected from a brachial vein. RESULTS: APLs from the brachial vein were higher than those from the coronary sinus (1.69 vs 0.75microM p < 0.01). IMA levels were correlated with APLs from the brachial vein (r = 0.59, p < 0.01). BNP concentrations were correlated with APLs from the brachial vein (r = 0.73, p < 0.001) but not with APLs from the coronary sinus. (r = 0.38, p > 0.05). BNP concentrations and IMA levels were correlated (r = 0.71, p < 0.001). CONCLUSIONS: In CHF patients, adenosine release occurs at a peripheral level and not at the myocardium level.

Abstract: Aims: High adenosine plasma levels and high expression of adenosine A2A receptors are observed in patients with unexplained syncope and a positive head up tilt test (HUT). This study aimed to evaluate the single nucleotide polymorphism (SNP) c.1364 T>C) which is the most commonly found polymorphism in the A2A receptor gene, in patients with unexplained syncope undergoing HUT. Methods and Results: 105 patients with unexplained syncope who underwent HUT were included. Fifty-two had a positive test. Receptor genotype determinations were performed in patients and in 121 healthy subjects. Genotype (TT, CC, TC) was determined from DNA leucocytes. The distribution of the polymorphism showed significant (p<0.0001) difference when the results of HUT were analyzed. Fifty two per cent of patients with a positive HUT had a CC genotype and 34.6% a TC genotype, whereas 13.2% of the patients with a negative HUT had a CC genotype and 71.7% a TC genotype. Patients with a CC genotype had a higher incidence of spontaneous syncopal episodes. Conclusions: In the patients with unexplained syncope, a significant association between high incidence of syncopal episodes, positive HUT and the presence of the CC variant in the adenosine A2A receptor gene was elicited.

Abstract: The cross talk between different membrane receptors is the source of increasing research. We designed and synthesized a new hetero-bivalent ligand that has antagonist properties on both A(1) adenosine and mu opiate receptors with a K(i) of 0.8+/-0.05 and 0.7+/-0.03muM, respectively. This hybrid molecule increases cAMP production in cells that over express the mu receptor as well as those over expressing the A(1) adenosine receptor and reverses the antalgic effects of mu and A(1) adenosine receptor agonists in animals.

Abstract: OBJECTIVE: Adenosine (ADO) is an endogenous nucleoside, which has been involved in blood pressure failure during severe systemic inflammatory response syndrome (severe SIRS) after cardiac surgery with cardiopulmonary bypass (CPB). Adenosine acts via its receptor subtypes, namely A1, A2A, A2B, or A3. Because A2A receptors are implicated in vascular tone, their expression might contribute to severe SIRS. We compared adenosine plasma levels (APLs) and A2A ADO receptor expression (ie, B, K, and mRNA amount) in patients with or without postoperative SIRS. PATIENTS:: This was a prospective comparative observational study. Forty-four patients who underwent cardiac surgery involving CPB. Ten healthy subjects served as controls. MEASUREMENTS AND RESULTS: Among the patients, 11 presented operative vasoplegia and postoperative SIRS (named complicated patients) and 33 were without vasoplegia or SIRS (named uncomplicated patients). Adenosine plasma levels, K, B, and mRNA amount (mean +/- SD) were measured on peripheral blood mononuclear cells. Adenosine plasma levels, B, and K were significantly higher in complicated patients than in uncomplicated patients (APLs: 2.7 +/- 1.0 vs 1.0 +/- 0.5 micromol l, P < 0.05; B: 210 +/- 43 vs 65 +/- 26 fmol/mg, P < 0.05; K: 35 +/- 10 vs 2 +/- 1 nM, P < 0.05). In uncomplicated patients, APLs remain higher than in controls (1 +/- 0.5 vs 0.6 +/- 0.25 micromol/L; P < 0.05).Mean arterial pressure was inversely correlated to APLs (R = -0.58; P < 0.001) and B (R = -0.64; P < 0.001) leading to an increased requirement of vasoactive drugs during the postoperative period in vasoplegic patients. CONCLUSIONS: High expression of A2A ADO receptor and high APLs may be a predictive factor of postoperative severe SIRS after CPB.

Abstract: Myocardial damage is a frequent complication of cardiac surgery by direct mechanical trauma during the surgical procedure and by myocardial ischemia, which occurs during the cardiopulmonary bypass (CBP). Because the concentrations of biomarkers in the blood collected from the coronary sinus are the best witness of the myocardial damages, we measured the levels of specific cardiac troponin I (c-TnI) and nonspecific (adenosine, myoglobin) markers of left ventricular damages in the coronary sinus of patients during cardiac surgery. Thirty patients who underwent aortic valve replacement for aortic stenosis were included. Blood samples were collected in the coronary sinus and in the radial artery at the beginning (T0), at the end of the CBP (T1), and then 24 hours later (T2). At T0 and T1, adenosine, c-TnI, and myoglobin levels were significantly higher in the coronary sinus than in the radial artery (T0: adenosine: mean +27%; c-TnI: +41%; myoglobin: +28%; T1: adenosine: mean +58%; c-TnI: +58%; myoglobin: +25%; p < .001). These parameters were significantly higher in the coronary sinus at T1 compared with T0 (adenosine: +50%; c-TnI: +229%; myoglobin: +94%; p < .01) and in the radial artery (adenosine: +21%; c-TnI: +194%; myoglobin: +98%; p < .01). At T2, c-TnI further increased. Damages to the myocardium during cardiac surgery are minimal in our surgical conditions but are probably underestimated when using markers measured at the peripheral level. However, most of the damages appear several hours after the surgery.

Abstract: L’adénosine est un nucléoside ubiquitaire, libéré dans le milieu extracellulaire via un système de diffusion facilitée. Il y régule de nombreuses fonctions physiopathologiques par fixation sur ses récepteurs cellulaires transmembranaires couplés aux protéines G : A1, A2A, A2B et A3. L’expression des récepteurs A2A sur diverses cellules est montrée avoir un rôle important dans différentes pathologies. La mesure de ces récepteurs utilise actuellement des ligands radioactifs. L’utilisation d’anticorps monoclonaux pourrait permettre la mise au point d’un dosage non isotopique de ces récepteurs, adapté à la routine clinique. Nous avons présenté ici la production et la caractérisation initiale d’un anticorps monoclonal anti-récepteur A2A de l’adénosine (mAb anti-A2AR). Des souris BALB/c ont été immunisées avec un peptide synthétique de 30 acides aminés correspondant à la seconde boucle extracellulaire du récepteur A2A de l’adénosine. L’anticorps monoclonal est produit, in vitro, par fusion des cellules de rate de souris immunisées et de celles d’un myélome murin, puis, in vivo, par implantation intrapéritonéale de l’hybridome sécréteur aux souris homologues. Le mAb anti-A2AR est caractérisé par Enzyme-Linked Immunosorbent Assay (ELISA), Western blot et épitope mapping (PEPSCAN). Un anticorps monoclonal d’isotype IgM kappa a été obtenu. L’analyse montre que ce mAb anti-A2AR présente une bonne spécificité vis-à-vis du peptide synthétique et du récepteur membranaire natif exprimé à la surface des cellules mononuclées humaines. L’anticorps reconnaît une séquence linéaire du récepteur A2A de l’adénosine en Western blot dans lequel le récepteur est dénaturé par réduction. L’analyse par PEPSCAN précise que le mAb anti-A2AR se lie spécifiquement à la partie C-terminale de la seconde boucle extracellulaire du récepteur A2A, représenté par l’épitope linéaire LFEDVVP. Le peptide synthétique, correspondant à la seconde boucle extracellulaire du récepteur A2A de l’adénosine, a permis d’induire avec succès un anticorps monoclonal anti-récepteur A2A qui reconnaît spécifiquement sa partie C-terminale. Le clone d’hybridome sécréteur est stable et permet d’obtenir de grandes quantités d’anticorps monoclonal qui seront nécessaires pour poursuivre sa caractérisation en vue de ses possibles applications cliniques.

Abstract: La chirurgie cardiaque au cours de la circulation extracorporelle (CEC) s’accompagne des lésions myocardiques qui ont plusieurs origines. Le dosage des marqueurs biologiques d’ischémie dans le sinus coronaire constitue une approche originale permettant de faire la part de la souffrance myocardique en temps réel au cours de cette opération. Le but de notre étude est d’une part, d’évaluer les concentrations dans le sinus coronaire des marqueurs spécifiques (troponine TnIc) ou non spécifiques (adénosine Ado, myoglobine, albumine modifiée IMA), d’autre part, de comparer les valeurs obtenues à partir des prélèvements effectués dans le sinus coronaire avec celles obtenues dans l’artère radiale. Chez 30 patients opérés, en ce qui concerne l’Ado, la myoglobine et la TnIc, leurs concentrations sont plus élevées dans le sinus coronaire par apport à la périphérie. Ceci dès au moment de la mise en fonction de la CEC, ce qui suggère une libération faible mais permanente de ces marqueurs dans le sinus coronaire, en l’absence de processus ischémique évident. A l’arrêt de la CEC il existe toujours une augmentation de ces trois marqueurs à la fois dans le sinus coronaire et à moins degré en périphérie. La libération de ces marqueurs en particulier d’Ado reflète en temps réel la souffrance myocardique inhérente au geste chirurgical lui même. A 24h après l’arrêt de la CEC, l’augmentation en particulier de TnIc est plus importante et est en relation avec le processus ischémique qui accompagne le syndrome inflammatoire postopératoire. En ce qui concerne l’IMA, sa concentration est plus élevée en périphérie que dans le sinus coronaire, ceci pourrait être le témoin d’une atteinte des territoires mésentériques au cours de la CEC.

Abstract: La Biologie Clinique participe à la prise en charge diagnostique et au suivi thérapeutique des patients en réalisant des analyses dont la qualité technique satisfait des contrôles stricts et dont la pertinence clinique est bien établie. La première condition est maintenant facilement remplie grâce au progrès dans l’instrumentation et les systèmes informatiques; la seconde condition réclame l’établissement de « valeurs de référence » permettant de discriminer les patients selon leur statut clinique, pathologique ou non. Les critères de choix (age, sexe, origine, etc…) de la cohorte sont prépondérants quant à la qualité opérationnelle et à la pertinence de ces valeurs de référence. L’exploration de la fonction thyroïdienne chez la femme enceinte est une bonne illustration de ce problème. Quoique des doutes se soient exprimés, l’opinion selon laquelle les hormones thyroïdiennes (HT) maternelles n’avaient aucun rôle dans le développement du fœtus a longtemps prévalu. De ce fait, les valeurs de référence étaient celles de femmes hors grossesse malgré l’observation d’une élévation de la T4 totale circulante, non accompagnée, il est vrai, d’une augmentation de la T4 libre. L’état de grossesse comme la présence du fœtus étaient ignorées. Les données obtenues au cours des vingt dernières années (passage trans-placentaire de la T4, concentration très faible de T4 chez le fœtus, expression précoce de récepteurs des HT, régulation thyroïdienne prise en charge par le fœtus et non plus par la mère, …) ont clairement montré l’importance de la T4 maternelle, mais non de la T3, sur le développement psycho-moteur du fœtus pendant la première partie de la grossesse. Au cours de la deuxième partie de la grossesse, le fœtus devient capable de synthétiser les HT et d’en réguler la production dans la mesure où la mère fournit un apport convenable en iode. La pathologie thyroïdienne du fœtus n’étant plus seulement celle de la mère, de nouvelles valeurs de référence déterminées chez des femmes enceintes, devenaient nécessaires. Le travail est en cours.

Abstract: INTRODUCTION : In collaboration with the National Center of Parasitology, Entomology and Malaria Control, provincial and operational district staff of the Ministry of Health and WHO, in vitro and PCR studies were continued in 2004 by the Laboratory of Molecular Epidemiology at the Pasteur Institute of Cambodia with the aim to: i) Assess the in vitro response of P. falciparum to several antimalarial drugs, ii) Differentiate recrudescence and re-infection among in vivo treatment failure cases by genotyping of P. falciparum , iii) Compare the in vitro data with the in vivo outcomes, iv) Assess the evolution of the P. falciparum drug resistance over the last year. This report summarizes the data collected during year 2004. CONCLUSION : The in vitro data in 2004 showed a worrying decreased susceptibility to almost antiantimalarial drugs tested except chloroquine, of which, the in vitro resistance is likely to be stable. The raise of IC50 values of mefloquine and the correlations between the activity of artesunate and those of quinine, chloroquine particularly mefloquine suggest that the combination of artesunate + mefloquine which is currently used as the first line treatment may become an invalid association for malaria treatment in Cambodia (2). The alternative association need to be further evaluated while the result of responses to piperaquine is satisfying in the north of the country. The apparent decreased of mefloquine susceptibility measured in vitro call for additional in vivo surveys of artesunate efficacy in Cambodia, especially in its western part. Indeed, by this close collaboration, the in vivo study is always necessary to clarifying the situation of antimalarial drug resistant in Cambodia.

Abstract: The second extracellular loop of the A(2A) receptor (A(2A)R) of adenosine was used to immunize mice for production of Adonis, an IgM monoclonal antibody. Adonis bound to the immunogen peptide and the native receptor in ELISA with K(D) values in 6.51-12.35 nM range. It recognized a linear epitope of 7 amino acids (LFEDVVP) at the C-terminal part of the external loop. Adonis revealed a 45-kDa band in lysate of human peripheral blood mononuclear cells in Western blotting in denaturing conditions. This served to monitor the up-regulation of the A(2A)R expression by caffeine. Adonis stimulated the cAMP production and inhibited the cell proliferation of an A(2A)R transfected stable cell line. These results confirm the immunogenicity and the functional relevance of the second extracellular loop of the A(2A)R. They suggest that Adonis may be of clinical use in various pathological situations to measure the regulation of the A(2A)R expression and to act as A(2A)R agonist drug.

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