Enzo Ragazzoni

Aspirin-insensitive thromboxane biosynthesis in essential thrombocythemia is explained by accelerated renewal of the drug target

Essential thrombocythemia (ET) is characterized by enhanced platelet generation and thrombotic complications. Once-daily low-dose aspirin incompletely inhibits platelet thromboxane A(2) (TXA(2)) in the majority of ET patients. In the present study, we investigated the determinants of aspirin-insensitive platelet TXA(2) biosynthesis and whether it could be further suppressed by changing the aspirin dose, formulation, or dosing interval. In 41 aspirin-treated ET patients, the immature platelet count predicted serum TXB(2) independently of platelet count, age, JAK-2 V617F mutation, or cytoreduction (β = 3.53, P = .001). Twenty-one aspirin-treated patients with serum TXB(2) ≥ 4 ng/mL at 24 hours after dosing were randomized to the following 7-day regimens in a crossover design: enteric-coated aspirin 100 mg twice daily, enteric-coated aspirin 200 mg once daily, or plain aspirin 100 mg once daily. A twice-daily regimen caused a further 88% median (IQR, 78%-92%, P < .001) TXB(2) reduction and normalized the functional platelet response to aspirin, as assessed by urinary 11-dehydro-TXB(2) excretion and the VerifyNow Aspirin assay. Doubling the aspirin dose reduced serum TXB(2) only partially by 39% median (IQR, 29%-54%, P < .05). We conclude that the abnormal megakaryopoiesis characterizing ET accounts for a shorter-lasting antiplatelet effect of low-dose aspirin through faster renewal of platelet cyclooxygenase-1, and impaired platelet inhibition can be rescued by modulating the aspirin dosing interval rather than the dose.

Interleukin-1β and Interleukin-6 Specifically Increase the Release of Prostaglandin E2 from Rat Hypothalamic Explants in vitro

It has previously been shown that the cytokines interleukin-1 beta and interleukin-6 (IL-1 beta and IL-6) stimulate directly the release of corticotrophin-releasing-hormone-41 from the rat hypothalamus in vitro, while IL-1 beta can also stimulate the release of somatostatin. These effects can be antagonized by drugs which block prostaglandin (PG) synthesis. PGs are also involved in the control of hypothalamic neuropeptides by other neurotransmitters. In the present study, we have characterized the production of PGs from the rat hypothalamus in vitro, and investigated the effects of IL-1 beta and IL-6, as well as the neurotransmitters norepinephrine, acetylcholine and 5-hydroxytryptamine, on the acute release of PGs, using a well-validated acute hypothalamic incubation system. The rate of release of PGs [PGE2, PGF2 alpha, 6-keto-PGF1 alpha (6KPGF1 alpha) and thromboxane B2 (TXB2) in the medium was found to stabilize after 60 min of preincubation and thereafter remain constant, with TXB2 being the predominant species. Twenty-minute incubation in the presence of human recombinant IL-1 beta or IL-6, in the dose range 1-100 U/ml, had no effect on the release of PGF2 alpha, 6KPGF1 alpha or TXB2; however, the release of PGE2 was significantly increased by both IL-1 beta and IL-6. The effect of IL-1 beta was antagonized by both indomethacin and dexamethasone. None of the other neurotransmitters tested had any effect on the release of any of the PGs.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of small intestinal bacterial overgrowth in Parkinson's disease

Parkinsons disease is associated with gastrointestinal motility abnormalities favoring the occurrence of local infections. The aim of this study was to investigate whether small intestinal bacterial overgrowth contributes to the pathophysiology of motor fluctuations. Thirty‐three patients and 30 controls underwent glucose, lactulose, and urea breath tests to detect small intestinal bacterial overgrowth and Helicobacter pylori infection. Patients also underwent ultrasonography to evaluate gastric emptying. The clinical status and plasma concentration of levodopa were assessed after an acute drug challenge with a standard dose of levodopa, and motor complications were assessed by Unified Parkinsons Disease Rating Scale–IV and by 1‐week diaries of motor conditions. Patients with small intestinal bacterial overgrowth were treated with rifaximin and were clinically and instrumentally reevaluated 1 and 6 months later. The prevalence of small intestinal bacterial overgrowth was significantly higher in patients than in controls (54.5% vs. 20.0%; P = .01), whereas the prevalence of Helicobacter pylori infection was not (33.3% vs. 26.7%). Compared with patients without any infection, the prevalence of unpredictable fluctuations was significantly higher in patients with both infections (8.3% vs. 87.5%; P = .008). Gastric half‐emptying time was significantly longer in patients than in healthy controls but did not differ in patients based on their infective status. Compared with patients without isolated small intestinal bacterial overgrowth, patients with isolated small intestinal bacterial overgrowth had longer off time daily and more episodes of delayed‐on and no‐on. The eradication of small intestinal bacterial overgrowth resulted in improvement in motor fluctuations without affecting the pharmacokinetics of levodopa. The relapse rate of small intestinal bacterial overgrowth at 6 months was 43%.

Validation of leukotriene B4 measurements in exhaled breath condensate.

Abstract:Objective: To qualitatively validate an enzyme immunoassay to measure leukotriene B4 in exhaled breath condensate. Exhaled breath condensate is a new non-invasive method to monitor airway inflammation.¶Subjects: Twenty-two subjects with different lung diseases attended the outpatient clinic on one occasion for exhaled breath condensate collection.¶Methods: Samples were pooled together and purified by reverse-phase high-performance liquid chromatography. The fractions eluted were assayed for leukotriene B4 by enzyme immunoassay.¶Results: A single peak of leukotriene B4-like immunoreactivity co-eluting with leukotriene B4 standard (retention time: 24 min) was identified by enzyme immunoassay. Reverse phase-high performance liquid chromatography peak of leukotriene B4 was clearly separated from those of 6-trans- leukotriene B4 (retention time: 14 min) and leukotriene B5 (retention time: 18 min) for which the antiserum used in the enzyme immunoassay had the highest cross-reactivity. Leukotriene B4 recovery was 64%.¶Conclusions: This study provides evidence for the presence of leukotriene B4 in the exhaled breath condensate and the specificity of the enzyme immunoassay used.

Pharmacokinetic variability of antiretroviral drugs and correlation with virological outcome: 2 years of experience in routine clinical practice

OBJECTIVES To assess the inter-individual and intra-individual plasma concentration variabilities of non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) in routine clinical practice and to investigate their relationships with virological failure. METHODS We retrospectively enrolled HIV-infected patients undergoing therapeutic drug monitoring (TDM) of NNRTIs and PIs during routine outpatient visits. Plasma drug concentrations were measured by HPLC-UV and were considered therapeutic if above the proposed minimum efficacy trough concentration. Inter-individual and intra-individual variabilities were evaluated through the coefficient of variation (CV). RESULTS A total of 457 PI and 172 NNRTI plasma concentrations were measured from 363 patients (HIV-RNA <50 copies/mL in 70.8%, median CD4 count 434 cells/mm(3)). NNRTIs showed less inter-individual (CV(inter) 54.8% versus 84.3%) and intra-individual (CV(intra) 19.0% versus 38.1%) pharmacokinetic variabilities than PIs. Intra-individual variability was constantly lower than inter-individual variability for each drug. Subtherapeutic drug concentrations were observed in 106 samples (16.9%). Older age (P = 0.020) and higher viral load (P = 0.013) were associated with subtherapeutic levels. Patients with therapeutic levels had a viral load of <50 copies/mL more frequently than those with subtherapeutic levels (74.8% versus 63.2%, P = 0.020). The estimated proportion with virological failure at 24 weeks was 0.21 in patients with suboptimal baseline drug levels and 0.08 in those with optimal levels (P < 0.001). In the multivariate analysis, therapeutic drug levels showed an independent negative association with virological failure (P = 0.004). CONCLUSIONS A wide inter-individual and limited intra-individual pharmacokinetic variabilities, together with the demonstration of a concentration-response relationship, suggest that TDM is a useful tool for the clinical management of patients treated with NNRTIs or PIs.

Validation of 8-isoprostane and prostaglandin E2 measurements in exhaled breath condensate

AbstractObjective:To qualitatively validate radioimmunoassays for 8-isoprostane and prostaglandin (PG) E2 in exhaled breath condensate. Subjects:Twenty-two subjects with different lung diseases attended the outpatient clinic on one occasion for exhaled breath condensate collection. Methods:Samples were pooled together and purified by reverse phase high performance liquid chromatography (RP-HPLC). The eluted fractions were assayed for 8-isoprostane-like immunoreactivity and PGE2-like immunoreactivity by radioimmunoassays. In addition, simultaneous measurements of exhaled breath condensate unextracted samples with two anti-8-isoprostane and anti-PGE2 sera with different cross-reactivity were performed. Results:A single peak of 8-isoprostane-like immunoreactivity and PGE2-like immunoreactivity co-eluting with 8-isoprostane (retention time: 13 min) and PGE2 (retention time: 21 min) standards, respectively, was identified by radioimmunoassays. Testing with two different antisera showed similar results for both 8-isoprostane-like immunoreactivity (limits of agreement = 4.5 pg/ml and – 4.1 pg/ml, n = 12) and PGE2-like immunoreactivity (limits of agreement = 6.1 pg/ ml and – 6.1 pg/ml, n = 12). Conclusion: This study provides evidence for the specificity of the radioimmunoassays for 8-isoprostane and PGE2 in exhaled breath condensate. This is critical for proposing these markers as a non-invasive way for monitoring airway inflammation.

Involvement of nitric oxide in basal and interleukin-1β-induced CRH and ACTH release in vitro

NG-Nitro-L-arginine, an inhibitor of nitric oxide synthase, does not influence CRH basal release but is able to modify ACTH basal secretion and to block interleukin-1 beta-induced CRH and ACTH release from rat hypothalamic and anterior pituitary cell cultures in vitro. The data suggest a different involvement of NO at hypothalamic and pituitary level.

Anaphylaxis increases 8-iso-prostaglandin F2α release from guinea-pig lung in vitro

8-Iso-prostaglandin F2alpha, release from isolated and perfused guinea-pig lung was measured by radioimmunoassay. 8-Iso-prostaglandin F2alpha release was detectable under basal conditions and increased 10-fold during antigen-induced bronchoconstriction, concomitant with the increase of thromboxane B2 and prostaglandin E2. The anti-8-iso-prostaglandin F2alpha serum used in the radioimmunoassay seems to be quite specific for this compound. Pretreatment of lungs with indomethacin (a non-selective inhibitor of cyclooxygenase) reduced 8-iso-prostaglandin F2alpha release under basal conditions and completely abolished the increase observed during lung anaphylaxis. Pretreatment of lungs with NS 398 (N-[2-cyclohexyl]-4-nitrophenyl methanesulphonamide), a selective inhibitor of cyclooxygenase-2, did not change basal or antigen-induced 8-iso-prostaglandin F2alpha release at all. We conclude that under basal conditions guinea-pig lung perfusates contain low levels of 8-iso-prostaglandin F2alpha-like immunoreactivity, which increase 10-fold during antigen-induced bronchoconstriction. This isoprostane seems to be derived from the cyclooxygenation of arachidonic acid via the constitutive form of cyclooxygenase.

Inhibition of heme oxygenase in the central nervous system potentiates endotoxin-induced vasopressin release in the rat

Previous in vitro studies have shown that increases in endogenous carbon monoxide (CO) generation via activation of the enzyme heme oxygenase (HO) within the rat hypothalamus are associated with the reduced release of the neuropeptides, vasopressin (AVP) and oxytocin, while evidence concerning corticotrophin-releasing hormone (CRH) is controversial. The present study investigated whether there is also a functional relationship between the HO-CO pathway and AVP and corticosterone (Cort) in vivo. Male Wistar rats were challenged with bacterial lipopolysaccharide (LPS) at doses producing significant activation of the hypothalamo-pituitary-adrenal (HPA) axis. LPS was given alone or after pretreatment with the HO inhibitor Sn-protoporphyrin-9 (SnPP9). The latter was injected either intraperitoneally (i.p.) or by intracerebroventricular (i.c.v.) route. SnPP9 given i.p. failed to modify either basal or LPS-stimulated levels of AVP and Cort. On the contrary, i.c.v. SnPP9 strongly potentiated LPS-induced AVP release and significantly enhanced basal serum Cort levels, although it failed to potentiate stimulation by LPS. The LPS + i.c.v. SnPP9 also significantly reduced the hypothalamic stores of AVP compared to controls, correlating with increased circulating levels of AVP. Taken collectively, these data are in concordance with previous in vitro observations showing that the HO-CO pathway acts centrally to attenuate endotoxin-stimulated AVP release, while having less effects on the pituitary-adrenal axis.

Release of vasoactive intestinal polypeptide from the rat gastric fundus.

1 Auxotonic responses and release of vasoactive intestinal polypeptide‐like immunoreactivity (VIP‐LI) induced by electrical field stimulation (EFS) were studied in longitudinal muscle strips from the gastric fundus of reserpinized rats suspended between parallel platinum electrodes in Krebs solution containing atropine (1 μm), 5‐hydroxytryptamine (3 μm) and bovine serum albumin (50 mg l−1). 2 EFS (supramaximal voltage, 1 ms, 0.25–32.0 Hz, trains of 2 min) induced frequency‐dependent relaxations. 3 EFS at frequencies ≥ 8 Hz also produced significant increases in VIP‐LI release. 4 VIP‐LI release induced by EFS at 16 Hz no longer occurred in the presence of tetrodotoxin (1 μm) or a Ca2+‐free medium. 5 Detection of VIP‐LI upon activation of inhibitory non‐adrenergic, non‐cholinergic neurones indicates that VIP meets the ‘detectable release’ criterion for an inhibitory neurotransmitter in the rat gastric fundus.