Long-term plasma levels of leptin and adiponectin in Rett syndrome.

OBJECTIVE: Rett syndrome is a progressive neurological disorder affecting almost exclusively females after age 6 months and characterised by acquired microcephaly, psychomotor retardation, growth failure, purposeless hand movements, autistic-like behaviour and wide-based and stiff legged gait. Leptin and adiponectin, peptides secreted by adipose tissue, are involved in the regulation of body weight and energy expenditure. DESIGN AND PATIENTS: We investigated in patients with Rett syndrome the variations of plasma leptin and adiponectin and their relation over a 2-year period. Sixteen female patients, mean age at the basal time 9.4 +/- 4.3 years, with classical Rett syndrome were enrolled. Controls were 16 healthy female subjects, mean age at the basal time 9.9 +/- 3.4 years. MEASUREMENTS: Blood samples were withdrawn in the morning at the baseline, 12 months after and 24 months after; plasma leptin and adiponectin concentrations were detected by ELISA. RESULTS: In patients, leptin concentrations significantly increased, while adiponectin concentrations significantly decreased. Both leptin and adiponectin values were significantly higher than those found in controls at each time. Leptin significantly correlated with adiponectin in patients, while there was not a significant correlation in controls. CONCLUSION: Since all patients were not obese, we might hypothesize that in Rett syndrome leptin and adiponectin might participate to clinical manifestations other than weight balance.

Rett syndrome and plasma leptin levels.

OBJECTIVE: To describe in patients with Rett syndrome (classic and preserved-speech variant) plasma leptin levels and their relationship to BMI (body mass index) and age. STUDY DESIGN: Female patients (n = 48; age range 3-20 years) affected by classic Rett syndrome were enrolled into the study. Eleven female patients, age range 3 to 20 years, with preserved-speech variant Rett syndrome were included in the study. Controls were 24 healthy female subjects, age range 3 to 20 years. Blood samples (3 mL) were withdrawn from an antecubital vein in the morning; plasma leptin concentrations were detected by enzyme-linked immunosorbent assay method. RESULTS: Patients with classic Rett syndrome and preserved-speech variant had leptin values significantly higher than controls. Leptin concentrations did not significantly differ between patients with classic Rett and preserved-speech variant. Leptin values positively correlated with age and BMI. CONCLUSIONS: Because in all patients the increased leptin concentrations were not associated to obesity, we hypothesize that in patients with Rett syndrome leptin might participate to clinical manifestations other than weight balance.

Pharmacokinetics and pharmacodynamics of neutrophil-associated ciprofloxacin in humans.

OBJECTIVE: To study the possibility that the penetration of the antibiotic ciprofloxacin into polymorphonuclear leukocytes (PMN) may be associated with some changes in cell reactivity. DESIGN: Superoxide anion and chemiluminescence generation induced by formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) were studied ex vivo in 12 healthy volunteers (mean age, 53.15 +/- 16.3 years; mean body weight, 71.23 +/- 6.9 kg) at fixed intervals up to 72 hours from the administration of a single oral dose of 250 mg ciprofloxacin. Cytosolic free calcium levels ([Ca2+]i) in resting and stimulated cells were also evaluated. The dynamic parameters of the effects on PMNs were compared with the kinetic profile of the drug in plasma and in PMNs. RESULTS: Superoxide generation induced by the stimulating agents increased significantly, reaching a peak after 12 hours (+116% [p < 0.001] for fMLP and +66% [p < 0.05] for PAF). Similarly, chemiluminescence production showed a threefold increase in the response to the stimulating agents 12 hours after drug administration (p < 0.001). The increase in [Ca2+]i in stimulated PMNs was significantly potentiated (p < 0.001). The mathematic analysis of the effects of ciprofloxacin showed that time to maximal activity was between 10.4 hours (PAF-dependent [Ca2+]i increase), and 15 hours (fMLP-induced superoxide anion and chemiluminescence production). The ratio of PMNs to plasma ciprofloxacin concentration increased progressively, from 0.5 at 30 minutes to 10.4 after 24 hours. In addition, time to maximal activity and half-life differed in PMNs and in plasma (4.66 versus 1.90 hours and 13.03 versus 7.28 hours, respectively). CONCLUSIONS: Ciprofloxacin administration induced a long-lasting enhancement of PMN reactivity to fMLP and PAF. The levels of the drug in the cells were greater and more sustained in the time than those in plasma.

Adenosine system and cell calcium translocation: interference of calcium channel blockers.

Adenosine is able to inhibit in vitro neutrophil functions induced by formyl-methionyl-leucyl-phenylalanine (FMLP) and A23187, but not phorbol 12-myristate 13-acetate (PMA). The inhibiting activity on A23187 is reversed by increasing extracellular Ca2++ concentration. The calcium entry blocker flunarizine shows an activity very similar to that of adenosine. Both adenosine and flunarizine prevent Ca++ influx into activated neutrophils as detected by the fluorescent Ca++ chelator Quin-2. Finally, flunarizine binds to the neutrophil membrane and adenosine competitively inhibits flunarizine binding as assessed by 1H-Nuclear Magnetic Resonance (1H-NMR) technique, thus indicating that the two agents share a common binding site on the cell membrane.

Heparin inhibition of polymorphonuclear leukocyte activation in vitro. A possible pharmacological approach to granulocyte-mediated vascular damage.

The "in vitro" effects of heparin on different functions of human polymorphonuclear leukocytes were studied. Granulocyte aggregation, enzyme release induced by FMLP and zymosan-activated serum and superoxide anion and chemiluminescence generated by FMLP were assessed. Heparin (25-500 micrograms/ml) was able to inhibit in a dose-dependent way cellular aggregation and degranulation induced either by FMLP or by zymosan-activated serum. FMLP-dependent superoxide anion generation and chemiluminescence were specifically inhibited by heparin at the concentration of 25 micrograms/ml. Our results showed that heparin "in vitro" inhibits all the aspects of the functional and metabolic granulocyte activation. A possible protecting effect of the drug on leukocyte-mediated tissue injury and vascular damage is discussed.

Nitroprusside in vitro inhibits platelet aggregation and intracellular calcium translocation. Effect of haemoglobin.

The biologically final active compound of nitrovasodilators is now supposed to be nitric oxide (NO), a labile substance identical to EDRF. The effects of nitroprusside on platelet functions were studied in vitro. Platelet aggregation induced by several stimuli (ADP, collagen, arachidonic acid and PAF) was inhibited by increasing concentrations of the drug (1-50 uM); interestingly, the potency of nitroprusside is higher when PAF is employed as stimulating agent in comparison with the other agonists (ED50 = 2 uM for ADP, 2.5 uM for A.A., 4.5 uM for collagen and 0.3 uM for PAF-induced aggregations). The concomitant addition of haemoglobin is able to reverse the inhibitory effect of nitroprusside, according to the view that haemoglobin possesses a high affinity for NO, thus antagonizing the effect of this compound. Nitroprusside was also able to inhibit intracellular calcium translocation, as studied with the Quin 2 technique, induced by PAF and arachidonic acid. Fron these observations the hypothesis may be suggested that nitroprusside inhibits platelet functions by mimicking the endogenous NO, and that the intracellular calcium metabolism is involved in the inhibitory activity of the drug.

Defibrotide inhibits Ca2+ dependent neutrophil activation: implications for its pharmacological activity in vascular disorders.

Defibrotide (DEF) is a polydeoxyribonucleotide agent provided with profibrinolytic and antithrombotic properties. Moreover, an antiischemic, cardioprotective effect of the drug has recently been demonstrated in experimental animals. Increasing evidence exists of the important role played by neutrophils in the development of tissue damage during chronic and acute ischemia and in the early phases of reperfusion. In order to evaluate whether the overall cytoprotective effect of DEF could be based, at least in part, on a neutrophil-involving mechanism, the authors studied the in vitro effects of the drug on human neutrophil activation triggered by several specific stimuli. The drug dose-dependently (10-100 microM) inhibited enzyme release, superoxide anion generation, and chemiluminescence induced in neutrophils by the chemoattractant oligopeptide fMLP and by the divalent cation ionophores A23187 and ionomycin. The increase of extracellular calcium concentration from 0.5 to 2.0 mM antagonized the inhibitory effect of DEF. The use of the fluorescent probe Fura 2/AM led them to show that DEF is able to reduce the cytosolic free calcium increase following specific stimulation by affecting extracellular calcium entrance. Such a behavior resembles that of calcium-antagonistic drugs, thus suggesting that DEF works, at least in part, similarly to calcium entry blockers. Such an activity on cell calcium translocation could represent the underlying molecular mechanism of cytoprotection. Finally, the inhibitory action on neutrophil functions may play a role in tissue protection during ischemic injury.

Experimental model of short-time exercise-induced preconditioning in POAD patients.

Regular physical exercise improves walking performance in patients affected with peripheral obliterative arterial disease (POAD). The mechanisms underlying the phenomenon are still controversial. In order to verify the hypothesis that physical conditioning of lower limbs on a treadmill and ischemic preconditioning of the heart could share some biological aspects, 14 POAD subjects underwent a training program on the treadmill consisting of five repeated submaximal exercises at five-minute and two-hour intervals preceding the maximal tolerance test. Moreover, a protocol with two daily submaximal walking exercises over one week was also performed. Pain-free and total walking distance were measured before and after they performed the program. Moreover, plasma levels of adenosine and adenosine triphosphate (ATP) were measured and polymorphonuclear (PMN) leukocyte activity was studied together with rheologic parameters. Pain-free distance was prolonged by 15.4% and 14.3%, and total distance was prolonged by 23.1% and 26.9%, in the exercises with five-minute and two-hour intervals, respectively. After one week of daily exercises, the onset of pain and the end of the test were delayed by 24% and 43.7%, respectively. An improvement in blood rheology and a reduced PMN reactivity were also observed with the three protocols, associated with an increase in plasma levels of adenosine and ATP. Similarly to ischemic preconditioning in the heart, the possibility is suggested that an adenosine-mediated mechanism may contribute to the development of physical conditioning in treadmill-trained POAD patients.

Inhibition of neutrophil function in vitro by heparan sulfate.

The profibrinolytic and antithrombotic glycosaminoglycan heparan sulfate (HS) was tested in vitro on some neutrophil functions induced by several stimuli. HS 1-500 micrograms/ml was able to significantly inhibit, in a dose-dependent fashion, superoxide anion generation, lysozyme and beta-glucuronidase release from neutrophils stimulated with the formylated oligopeptide fMLP, the ionophore A23187, and Platelet Activating Factor. Such an effect could represent an additional therapeutical benefit in those pathological conditions in which neutrophil activation contributes to tissue injury and vascular damage.

Heparin inhibits FMLP and Con-A dependent activation of human polymorphonuclear leucocytes in vitro.

The effects in vitro of heparin on different functions of human neutrophilic leucocytes were assessed. Granulocyte aggregation, enzyme release and superoxide anion generation induced by FMLP (10(-6) M), ConA (30 micrograms/ml), and A 23187 (5 microM) were evaluated. Heparin was able to inhibit, in a dose-dependent way, FMLP-mediated and ConA-mediated granulocyte activation. Heparin inhibited cellular aggregation at the final concentration of 100-500 micrograms/ml, while the inhibiting effect on enzyme release and superoxide anion production was present at lower concentrations (10-100 micrograms/ml). No activity was observed on A 23187-induced granulocyte stimulation. The inhibiting effect of heparin on FMLP-induced granulocyte activation proved to be time-dependent. The hypothesis that an interaction of heparin with cells may possibly initiate the inhibitory effect is proposed.

In vitro inhibition of granulocyte function by timegadine, a new anti-inflammatory agent.

Timegadine, a new non-acidic anti-inflammatory agent, inhibits in a concentration-dependent way the FMLP-induced release of beta-glucuronidase and lysozyme from human granulocytes. Superoxide generation and directed migration are also inhibited. As this effect on granulocytes occurs at drug concentrations inhibiting the lipoxygenase pathway of arachidonic acid metabolism, the hypothesis that these two phenomena could be related is discussed.

Defibrotide in vitro inhibits neutrophil activation by a Ca++-involving mechanism.

Defibrotide, a polydeoxyribonucleotide provided with a pro-fibrinolytic and prostacyclin-like activity, was studied as an inhibitor of polymorphonuclear leucocyte activation in vitro. It was found capable of dose-dependently (1-8 X 10(-5) M) inhibiting FMLP-induced activation, as shown by a decrease of enzyme release and free-radical formation (superoxide anion generation and chemiluminescence). A similar inhibiting activity was observed on A23187-induced activation. An increase in extracellular Ca++ concentration significantly prevented the effect of defibrotide on ionophore stimulation. When PMA was employed as stimulating agent, the drug did not show any inhibiting effect. Finally the pre-treatment of cells with theophylline markedly reduced the inhibition by defibrotide of FMLP- and A23187-dependent activation. Since the stimulation of neutrophils by FMLP and A23187 depends on the increase of cytoplasmic free-calcium availability or extracellular calcium entrance respectively, whereas PMA activation is completely independent from any Ca++ change, the inhibiting effect of defibrotide could be attributed to a Ca++-involving mechanism.

Polymorphonuclear leucocytes as a model of Ca++ and Mg++-dependent cellular activation. Effect of calcium entry blockers.

Flunarizine inhibited FMLP- and A23187-induced aggregation, enzyme release and O2- generation from human PMN as a function of its concentration. A23187-dependent PMN aggregation was also studied in media devoid of Ca++ or Mg++. Flunarizine (2.4 X 10(-5)M) significantly affected not only Ca++-supported but also Mg++-sustained PMN aggregation. The inhibiting effect of the drug was reversed by increasing the level of Ca++ (1.2 mM) or Mg++ (2 mM). Nifedipine, another Ca++-entry blocker, was shown to inhibit enzyme release and O2- generation induced by FMLP and A23187 as a function of its concentration, but only slightly affected PMN aggregation at very high concentration (10(-4)M). A role for flunarizine as a specific Mg++-entry blocker is suggested.

Platelet-dependent granulocyte activation in vitro: effect of ticlopidine.

The presence of platelets or platelet release products is known to augment the injury of endothelial cells caused by stimulated neutrophils (PMN leukocytes). In our in vitro studies, platelet-rich or platelet-poor plasma (PRP or PPP) was placed in one compartment and a PMN suspension in the other of a modified Boyden chamber divided by a dialysis membrane. The addition of the aggregating substance (ADP, collagen) to PRP but not to PPP was followed by PMN activation as shown by enzyme release and O-2 generation. The in vivo treatment with ASA completely prevented the platelets from triggering PMN activation. The in vitro addition of a thromboxane synthetase inhibitor (imidazole 10(-3) M) or a lipoxygenase inhibitor (NDGA 10(-6) M) did not show any effect on platelet-dependent PMN activation, thus suggesting that neither TxA2 nor lipoxygenase by-products are involved. Finally, in vitro and in vivo treatment with ticlopidine blunted the stimulating activity of the platelets on PMN. Our data further support the hypothesis that a sequential platelet-PMN interaction may occur, and that the therapeutic effect of some antiplatelet drugs may be partly due to a protective effect against platelet-dependent PMN-mediated vascular damage.

Pharmacological induced depression of E-rosette forming cells (RFC) of normal healthy subjects. Restoration by levamisole and thymopoietin pentapeptide.

The authors describe a pharmacological inhibition of E-Rosette formation by the "in vitro" addition of adenosine and theophylline. In the same experimental conditions TP5 and levamisole are able to reverse this inhibiting activity. The role of the cyclic nucleotides (cAMP and cGMP) in the regulation of the lymphocytes response is discussed.

Adenosine inhibits polymorphonuclear leukocyte in vitro activation: a possible role as an endogenous calcium entry blocker.

Adenosine is able to in vitro inhibit FMLP-dependent activation of polymorphonuclear leukocytes as evaluated by enzyme release, superoxide anion generation and chemiluminescence production. The inhibiting effect is more relevant when A23187 is employed as stimulating agent. In this case the effect is significantly reversed by increasing concentrations of extracellular calcium. Since A23187-dependent activation is strictly dependent on Ca++ influx into the cell, the hypothesis is suggested that adenosine could act by regulatory mechanisms involving membrane calcium transport.

Isradipine inhibits PMN leukocyte function. A possible interference with the adenosine system.

The dihydropyridine derivative isradipine is able to inhibit PMN leukocyte function, such as enzyme release and free radical generation, following the activation with specific stimuli. Moreover, the drug prevents calcium influx into the cells as detected by the specific fluorescent dye FURA 2/acetoxymethylester. The specific adenosine receptor antagonist theophylline is able to partially remove the inhibiting activity, thus suggesting a possible interference of isradipine with the adenosine system. Such a cell-protecting activity adds further rationale to the employment of isradipine in those conditions, such as acute and chronic ischaemia and reperfusion damage, in which PMN leukocyte-dependent tissue injury represents a relevant pathogenetic mechanism.