Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

OBJECTIVES: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). METHODS: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). RESULTS: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. CONCLUSIONS: These recommendations are an important step to achieve high quality ANA testing.

Inhibitors of phosphodiesterases PDE2, PDE3, and PDE4 do not increase the sinoatrial tachycardia of noradrenaline and prostaglandin PGE1 in mice.

Phosphodiesterases PDE2, PDE3, and PDE4 are expressed in murine sinoatrial cells. PDE3 and/or PDE4 reduce heart rate but apparently do not influence the tachycardia mediated through sinoatrial ß1- and ß2-adrenoceptors despite the high content of sinoatrial cAMP. The function of PDE2 is, however, uncertain. Prostaglandin PGE1 elicits sinoatrial tachycardia through EP receptors, but the control by phosphodiesterases is unknown. We investigated on spontaneously beating right atria of mice the effects of the PDE2 inhibitors Bay 60-7550 and EHNA on basal beating and the tachycardia produced by noradrenaline (3 nM) and PGE1 (1 µM). Bay 60-7550 (1 µM), but not EHNA (10 µM), increased basal sinoatrial beating. EHNA also failed to produce tachycardia in the presence of the adenosine deaminase inhibitor 2'-deoxycoformycin (10 µM), remaining inconclusive whether PDE2 reduces basal sinoatrial beating. Rolipram (10 µM) and cilostamide (300 nM) caused moderate tachycardia. The tachycardia evoked by Bay 60-7550 was similar in the absence and presence of rolipram. Noradrenaline elicited stable tachycardia that was not increased by Bay 60-7550. A stable tachycardia caused by PGE1 was not increased by the inhibitors of PDE2, PDE3, and PDE4. Unlike PDE3 and PDE4 which reduce murine basal sinoatrial beating, a possible effect of PDE2 needs further research. The stable tachycardia produced by noradrenaline and PGE1, together with the lack potentiation by the inhibitors of PDE2, PDE3, and PDE4, suggests that cAMP generated at the receptor compartments is hardly hydrolyzed by these phophodiesterases. Evidence from human volunteers is consistent with this proposal.

Function of cardiac beta1- and beta2-adrenoceptors of newborn piglets: role of phosphodiesterases PDE3 and PDE4.

The structures of porcine and human beta(2)-adrenoceptors differ but the repercussions for porcine cardiac function are unknown. We investigated the function of porcine beta(2)-adrenoceptors in 3 cardiac regions, sinoatrial node, left atrium and right ventricle of newborn piglets. Both (-)-noradrenaline and (-)-adrenaline caused sinoatrial tachycardia: 60+/-10% and 62+/-7% of the maximum response (E(max)) to (-)-noradrenaline (-logEC(50)=9.0) and (-)-adrenaline (-logEC(50)=7.5) respectively, were resistant to antagonism by the beta(1)-selective CGP20712A (2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide) (300 nM) but antagonized by beta(2)-selective ICI118551 (erythro(+/-)-[1-(2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol) (50 nM), consistent with mediation through beta(2)-adrenoceptors. The phosphodiesterase3-selective inhibitor cilostamide and phosphodiesterase4-selective inhibitor rolipram did not affect catecholamine chronotropic potencies. Only small CGP20712A-resistant positive inotropic effects of (-)-adrenaline were detected in the left atria (13+/-2% of E(max)) and ventricular trabeculae (14+/-5% of E(max)). The atrial inotropic responses to (-)-noradrenaline and (-)-adrenaline faded; fades were prevented by rolipram but not cilostamide or concurrent cilostamide+rolipram respectively. (-)-Noradrenaline (ICI118551 present) increased left atrial cAMP levels through beta(1)-adrenoceptors that were markedly enhanced by rolipram but unaffected by cilostamide. Concurrent cilostamide+rolipram uncovered inotropic and cAMP responses to (-)-adrenaline (CGP20712A present). We conclude that sinoatrial beta(2)-adrenoceptors are more important than beta(1)-adrenoceptors in the mediation of tachycardia caused by both (-)-noradrenaline and (-)-adrenaline in the newborn piglet. beta(2)-adrenoceptors have only a minor role in the mediation of left atrial and ventricular inotropic effects of (-)-adrenaline. Catecholamine-evoked tachycardia is not controlled by PDE3 or PDE4. PDE4, but not PDE3, controls the atrial inotropic and cAMP beta(1)-adrenoceptor-mediated responses to (-)-noradrenaline. Both PDE3 and PDE4 blunt left atrial inotropic and cAMP responses to (-)-adrenaline through beta(2)-adrenoceptors.

Phosphodiesterases do not limit beta1-adrenoceptor-mediated sinoatrial tachycardia: evidence with PDE3 and PDE4 in rabbits and PDE1-5 in rats.

The mammalian heart expresses at least five phosphodiesterases (PDE1-5). Catecholamines produce surges of inotropically relevant cAMP through beta(1)-adrenoceptor stimulation. cAMP is mainly hydrolysed by PDE3 and/or PDE4 thereby blunting contractility. Basal sinoatrial beating rate in mouse, rat, piglet and rabbit sinoatrial cells is reduced by PDE3 and/or PDE4 through hydrolysis of cAMP. However, in rodents, the tachycardia elicited by catecholamines through production of cAMP by beta-adrenoceptor activation is not controlled by PDE3 and PDE4, despite a blunting effect of PDE3 or/and PDE4 on basal sinoatrial beating, but it is unknown whether PDE3 limits catecholamine-evoked tachycardia in the rabbit. Since rabbit sinoatrial cells are an important model for pacemaker research, we investigated whether the positive chronotropic effects of (-)-noradrenaline on spontaneously beating right atria of the rabbit are potentiated by inhibition of PDE3 with cilostamide (300 nM). We also studied the sinoatrial effects of the PDE4 inhibitor rolipram (10 microM) and its influence on the responses to (-)-noradrenaline. For comparison, we investigated the influence of cilostamide and rolipram on the positive inotropic responses to (-)-noradrenaline on rabbit left atria and right ventricular papillary muscles. Cilostamide and concurrent cilostamide + rolipram, but not rolipram alone, increased sinoatrial rate by 15% and 31% of the effect of (-)-isoprenaline (200 microM) but the PDE inhibitors did not significantly change the chronotropic potency of (-)-noradrenaline. In contrast in papillary muscle, the positive inotropic effects of (-)-noradrenaline were potentiated 2.4-, 2.6- and 44-fold by cilostamide, rolipram and concurrent cilostamide + rolipram, respectively. In left atrium, the positive inotropic effects of (-)-noradrenaline were marginally potentiated by cilostamide, as well as potentiated 2.7- and 32-fold by rolipram and by concurrent cilostamide and rolipram respectively. To compare the influence of PDE1-5 on basal sinoatrial rate and (-)-noradrenaline-evoked tachycardia, we investigated on rat right atria the effects of selective inhibitors. The PDE4 inhibitor rolipram and non-selective inhibitor isobutyl-methylxanthine caused tachycardia with -logEC(50)s of 7.2 and 5.0 and E(max) of 18% and 102% of (-)-isoprenaline, respectively. Rolipram did not change the chronotropic potency of (-)-noradrenaline. At high concentrations (10-30 microM), the PDE1, PDE3 and PDE5 inhibitors 8-methoxymethyl-3-isobutyl-1-methylxanthine, cilostamide and sildenafil, respectively, caused marginal tachycardia but did not significantly change the chronotropic potency of (-)-noradrenaline. The PDE2-selective inhibitor erythro-9-[2-hydroxy-3-nonyl]adenine caused marginal bradycardia at 30 microM and tended to reduce the chronotropic potency of (-)-noradrenaline. Rabbit PDE3 reduces basal sinoatrial rate. Although PDE4 only marginally reduces rate, under conditions of PDE3 inhibition, it further reduces sinoatrial rate. Both PDE3 and PDE4 control atrial and ventricular positive inotropic effects of (-)-noradrenaline. In contrast, neither PDE3 nor PDE4 limit the sinoatrial tachycardia induced by (-)-noradrenaline. In the rat, only PDE4, but not PDE1, PDE2, PDE3 and PDE5, reduces basal sinoatrial rate. None of the five rat PDEs limits the (-)-noradrenaline-evoked tachycardia. Taken together, these results confirm and expand evidence for our proposal that the cAMP-compartment modulating basal sinoatrial rate, controlled by PDE3 and/or PDE4, is different from the PDE-resistant cAMP compartment involved in beta(1)-adrenoceptor-mediated sinoatrial tachycardia.

Ontogenic changes of the control by phosphodiesterase-3 and -4 of 5-HT responses in porcine heart and relevance to human atrial 5-HT(4) receptors.

BACKGROUND AND PURPOSE: Atrial inotropic responses to 5-HT mediated through 5-HT(4) receptors fade, presumably through phosphodiesterase (PDE) activity. We investigated the influence of a selective inhibitor of PDE3 (cilostamide) or of PDE4 (rolipram) on the fade of 5-HT responses in atrial muscle. EXPERIMENTAL APPROACH: 5-HT responses were compared, ex vivo, on sinoatrial beating rate of newborn piglets, porcine atrial and ventricular force, and human atrial force. cAMP levels were assessed in piglet atrium. KEY RESULTS: 5-HT-evoked sinoatrial tachycardia did not fade and was not potentiated by cilostamide (300 nmol.L(-1)) or rolipram (1 micromol.L(-1)). Inotropic responses to 5-HT faded in atria from piglets, adolescent pigs and humans. Cilostamide reduced atrial fade of 5-HT responses in adolescent pigs and humans but not in newborn piglets. Cilostamide disclosed 5-HT ventricular responses in newborn, but not adolescent pigs. Rolipram reduced fade of atrial 5-HT responses in newborn and adolescent pigs but not in humans. Concurrent cilostamide + rolipram abolished fade of 5-HT responses in porcine left atria and facilitated ventricular 5-HT responses, but did not reduce residual fade in human atrium in the presence of cilostamide. 5-HT-evoked increases in cAMP faded; fade was abolished by concurrent cilostamide + rolipram. CONCLUSIONS AND IMPLICATIONS: PDE3-induced control of porcine 5-HT responses differed in atrium and ventricle and changed with age. PDE3 and PDE4 jointly prevented fade of inotropic and cAMP responses to 5-HT in porcine atrium. Unlike porcine atria, only PDE3 induced fade of 5-HT responses in human atria.

Diazepam enhances inotropic responses to dopamine in rat ventricular myocardium.

Diazepam inhibits phosphodiesterase type 4 and enhances the effect of some 3',5'-cyclic adenosine monophosphate (cAMP)-dependent positive inotropic drugs. We sought to determine whether diazepam and the selective phosphodiesterase type 4 inhibitor rolipram enhances the contractile response and cAMP levels induced by dopamine in rat myocardium. Dopamine (3-100 microM) produced concentration-dependent positive inotropic effects (-log EC50 = 5.21 +/- 0.2, n = 5), which were augmented in the presence of 10 microM diazepam (-log EC50 = 5.40 +/- 0.08, n = 6, P < 0.05) or 1 microM rolipram (-log EC50 = 5.41 +/- 0.1, n = 6, P < 0.05). The effect of diazepam was not mimicked by 100 microM gamma-aminobutyric acid nor it was antagonized by a 5 microM concentration of the blockers of central and peripheral type benzodiazepine receptors, flumazenil and PK 11195. cAMP levels (pmol/g) produced by dopamine (744.4 +/- 111.8, n = 5) in this tissue were enhanced by the presence of diazepam (1073 +/- 97.7, n = 6, P < 0.05) or rolipram (1034.0 +/- 245.2, n = 5, P < 0.05). Therefore, diazepam, like rolipram, augments the inotropic and biochemical effects of dopamine in rat myocardium. This effect is not mediated by benzodiazepine receptors but is probably the consequence of the phosphodiesterase type 4 inhibitory activity of diazepam.

Phosphodiesterase PDE3 blunts the positive inotropic and cyclic AMP enhancing effects of CGP12177 but not of noradrenaline in rat ventricle.

1.--The cardiostimulant effects of CGP12177, mediated through a beta(1)-adrenoceptor site with low affinity for (-)-propranolol, are potentiated by the nonselective PDE inhibitor IBMX but the role of PDE isoenzymes is unknown. We studied the effects of the PDE3-selective inhibitor cilostamide (300 nM) and PDE4-selective inhibitor rolipram (1 microM) on the positive inotropic and cyclic AMP-enhancing effects of CGP12177 and noradrenaline in right ventricular strips of rat. 2.--CGP12177 (under (-)-propranolol 200 nM) only increased contractile force in the presence of either cilostamide or rolipram with -logEC(50)M 6.7 (E(max)=23% over basal) and 7.1 (E(max)=50%) respectively. The combination of cilostamide and rolipram caused CGP12177 to enhance contractile force with -logEC(50)M=7.7 and E(max)=178%. 3.--The positive inotropic effects of noradrenaline (-logEC(50)M=6.9) were potentiated by rolipram (-logEC(50)M=7.4) but not by cilostamide (-logEC(50)M=7.0). 4.--In the presence of rolipram and (-)-propranolol, noradrenaline (2 microM) and CGP12177 (10 microM) produced matching inotropic effects but failed to increase cyclic AMP levels. 20 microM (-)-noradrenaline increased cyclic AMP levels, a response further enhanced by rolipram. 5.--Both PDE3 and PDE4 of rat ventricle appear to hydrolyse cyclic AMP generated through the low-affinity beta(1)-adrenoceptor site, thereby preventing inotropic responses of CGP12177. When (-)-noradrenaline interacts with the beta(1)-adrenoceptor, the generated cyclic AMP is hydrolysed only by PDE4, thereby reducing cardiostimulation.

The phosphodiesterase 3 inhibitor cilostamide enhances inotropic responses to glucagon but not to dobutamine in rat ventricular myocardium.

The effects of phosphodiesterase (PDE) inhibitors (1-3) on tissue cAMP concentrations and the inotropic responses to dobutamine and glucagon were investigated in electrically driven right ventricular strips of the rat heart. Dobutamine (0.3-100 microM) produced a concentration-dependent positive inotropic effect which was not affected by 50 nM (+/-)-1-(2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy)-3-((1-methylethyl)amino)-2-butanol hydrochloride (ICI 118551), a beta2-receptor antagonist, but was virtually abolished by 0.3 microM (+/-)-2-hydroxy-5-(2-((2-hydroxy-3-(4-(1-methyl-4-(trifluoromethyl)-1H-imidazol-2-l)phenoxy)propyl)amino)ethoxy)-benzamide methanesulfonate (CGP 20712A), a beta1-receptor antagonist. Glucagon (0.01-1 microM) also enhanced the contractility of the preparation in a concentration-dependent way. Selective inhibitors of PDE 1 8-methoxymethyl-3-isobutyl-1-methylxantine (MIMX, 1 muM), PDE 2 erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA, 1 microM) and PDE 3 cilostamide (0.1 microM) did not affect basal contractility. Cilostamide increased the positive inotropic effects of glucagon but not those of dobutamine. MIMX and EHNA did not alter the effects of either dobutamine or glucagon. Dobutamine (3 microM), but not glucagon (0.1 microM), increased tissue levels of cAMP. 1 microM of MIMX or EHNA were devoid of effects and failed to alter the effects of dobutamine and glucagon on cAMP. Cilostamide (0.1 microM) did not increase the effects of dobutamine but caused glucagon to enhance cAMP. The pharmacological and biochemical data presented in this study can be explained quantitatively by a cell compartment model in which PDE 3 appears to be colocalized with the contractile machinery responsible for the effects of glucagon but not those of dobutamine. Neither PDE 1 nor PDE 2 appears to regulate the inotropic effects of dobutamine and glucagon in rat ventricular myocardium.