Vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs: meta-analyses of individual participant data from randomised trials.

BACKGROUND: The vascular and gastrointestinal effects of non-steroidal anti-inflammatory drugs (NSAIDs), including selective COX-2 inhibitors (coxibs) and traditional non-steroidal anti-inflammatory drugs (tNSAIDs), are not well characterised, particularly in patients at increased risk of vascular disease. We aimed to provide such information through meta-analyses of randomised trials. METHODS: We undertook meta-analyses of 280 trials of NSAIDs versus placebo (124,513 participants, 68,342 person-years) and 474 trials of one NSAID versus another NSAID (229,296 participants, 165,456 person-years). The main outcomes were major vascular events (non-fatal myocardial infarction, non-fatal stroke, or vascular death); major coronary events (non-fatal myocardial infarction or coronary death); stroke; mortality; heart failure; and upper gastrointestinal complications (perforation, obstruction, or bleed). FINDINGS: Major vascular events were increased by about a third by a coxib (rate ratio [RR] 1·37, 95% CI 1·14-1·66; p=0·0009) or diclofenac (1·41, 1·12-1·78; p=0·0036), chiefly due to an increase in major coronary events (coxibs 1·76, 1·31-2·37; p=0·0001; diclofenac 1·70, 1·19-2·41; p=0·0032). Ibuprofen also significantly increased major coronary events (2·22, 1·10-4·48; p=0·0253), but not major vascular events (1·44, 0·89-2·33). Compared with placebo, of 1000 patients allocated to a coxib or diclofenac for a year, three more had major vascular events, one of which was fatal. Naproxen did not significantly increase major vascular events (0·93, 0·69-1·27). Vascular death was increased significantly by coxibs (1·58, 99% CI 1·00-2·49; p=0·0103) and diclofenac (1·65, 0·95-2·85, p=0·0187), non-significantly by ibuprofen (1·90, 0·56-6·41; p=0·17), but not by naproxen (1·08, 0·48-2·47, p=0·80). The proportional effects on major vascular events were independent of baseline characteristics, including vascular risk. Heart failure risk was roughly doubled by all NSAIDs. All NSAID regimens increased upper gastrointestinal complications (coxibs 1·81, 1·17-2·81, p=0·0070; diclofenac 1·89, 1·16-3·09, p=0·0106; ibuprofen 3·97, 2·22-7·10, p<0·0001; and naproxen 4·22, 2·71-6·56, p<0·0001). INTERPRETATION: The vascular risks of high-dose diclofenac, and possibly ibuprofen, are comparable to coxibs, whereas high-dose naproxen is associated with less vascular risk than other NSAIDs. Although NSAIDs increase vascular and gastrointestinal risks, the size of these risks can be predicted, which could help guide clinical decision making. FUNDING: UK Medical Research Council and British Heart Foundation.

RENEB Inter-Laboratory Comparison 2021: The Cytokinesis-Block Micronucleus Assay.

The goal of the RENEB inter-laboratory comparison 2021 exercise was to simulate a large-scale radiation accident involving a network of biodosimetry labs. Labs were required to perform their analyses using different biodosimetric assays in triage mode scoring and to rapidly report estimated radiation doses to the organizing institution. This article reports the results obtained with the cytokinesis-block micronucleus assay. Three test samples were exposed to blinded doses of 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 13 mA, ∼75 keV, 1 Gy/min). These doses belong to 3 triage categories of clinical relevance: a low dose category, for no exposure or exposures inferior to 1 Gy, requiring no direct treatment of subjects; a medium dose category, with doses ranging from 1 to 2 Gy, and a high dose category, after exposure to doses higher than 2 Gy, with the two latter requiring increasing medical attention. After irradiation the test samples (no. 1, no. 2 and no. 3) were sent by the organizing laboratory to 14 centers participating in the micronucleus assay exercise. Laboratories were asked to setup micronucleus cultures and to perform the micronucleus assay in triage mode, scoring 500 binucleated cells manually, or 1,000 binucleated cells in automated/semi-automated mode. One laboratory received no blood samples, but scored pictures from another lab. Based on their calibration curves, laboratories had to provide estimates of the administered doses. The accuracy of the reported dose estimates was further analyzed by the micronucleus assay lead. The micronucleus assay allowed classification of samples in the corresponding clinical triage categories (low, medium, high dose category) in 88% of cases (manual scoring, 88%; semi-automated scoring, 100%; automated scoring, 73%). Agreement between scoring laboratories, assessed by calculating the Fleiss' kappa, was excellent (100%) for semi-automated scoring, good (83%) for manual scoring and poor (53%) for fully automated scoring. Correct classification into triage scoring dose intervals (reference dose ±0.5 Gy for doses ≤2.5 Gy, or reference dose ±1 Gy for doses >2.5 Gy), recommended for triage biodosimetry, was obtained in 79% of cases (manual scoring, 73%; semi-automated scoring, 100%; automated scoring, 67%). The percentage of dose estimates whose 95% confidence intervals included the reference dose was 58% (manual scoring, 48%; semiautomated scoring, 72%; automated scoring, 60%). For the irradiated samples no. 2 and no. 3, a systematic shift towards higher dose estimations was observed. This was also noticed with the other cytogenetic assays in this intercomparison exercise. Accuracy of the rapid triage modality could be maintained when the number of manually scored cells was scaled down to 200 binucleated cells. In conclusion, the micronucleus assay, preferably performed in a semi-automated or manual scoring mode, is a reliable technique to perform rapid biodosimetry analysis in large-scale radiation emergencies.

RENEB Inter-Laboratory Comparison 2021: The Dicentric Chromosome Assay.

After large-scale radiation accidents where many individuals are suspected to be exposed to ionizing radiation, biological and physical retrospective dosimetry assays are important tools to aid clinical decision making by categorizing individuals into unexposed/minimally, moderately or highly exposed groups. Quality-controlled inter-laboratory comparisons of simulated accident scenarios are regularly performed in the frame of the European legal association RENEB (Running the European Network of Biological and Physical retrospective Dosimetry) to optimize international networking and emergency readiness in case of large-scale radiation events. In total 33 laboratories from 22 countries around the world participated in the current RENEB inter-laboratory comparison 2021 for the dicentric chromosome assay. Blood was irradiated in vitro with X rays (240 kVp, 13 mA, ∼75 keV, 1 Gy/min) to simulate an acute, homogeneous whole-body exposure. Three blood samples (no. 1: 0 Gy, no. 2: 1.2 Gy, no. 3: 3.5 Gy) were sent to each participant and the task was to culture samples, to prepare slides and to assess radiation doses based on the observed dicentric yields from 50 manually or 150 semi-automatically scored metaphases (triage mode scoring). Approximately two-thirds of the participants applied calibration curves from irradiations with γ rays and about 1/3 from irradiations with X rays with varying energies. The categorization of the samples in clinically relevant groups corresponding to individuals that were unexposed/minimally (0-1 Gy), moderately (1-2 Gy) or highly exposed (>2 Gy) was successfully performed by all participants for sample no. 1 and no. 3 and by ≥74% for sample no. 2. However, while most participants estimated a dose of exactly 0 Gy for the sham-irradiated sample, the precise dose estimates of the samples irradiated with doses >0 Gy were systematically higher than the corresponding reference doses and showed a median deviation of 0.5 Gy (sample no. 2) and 0.95 Gy (sample no. 3) for manual scoring. By converting doses estimated based on γ-ray calibration curves to X-ray doses of a comparable mean photon energy as used in this exercise, the median deviation decreased to 0.27 Gy (sample no. 2) and 0.6 Gy (sample no. 3). The main aim of biological dosimetry in the case of a large-scale event is the categorization of individuals into clinically relevant groups, to aid clinical decision making. This task was successfully performed by all participants for the 0 Gy and 3.5 Gy samples and by 74% (manual scoring) and 80% (semiautomatic scoring) for the 1.2 Gy sample. Due to the accuracy of the dicentric chromosome assay and the high number of participating laboratories, a systematic shift of the dose estimates could be revealed. Differences in radiation quality (X ray vs. γ ray) between the test samples and the applied dose effect curves can partly explain the systematic shift. There might be several additional reasons for the observed bias (e.g., donor effects, transport, experimental conditions or the irradiation setup) and the analysis of these reasons provides great opportunities for future research. The participation of laboratories from countries around the world gave the opportunity to compare the results on an international level.

Autosomal recessive hereditary spastic paraplegia with thin corpus callosum: a novel mutation in the SPG11 gene and further evidence for genetic heterogeneity.

BACKGROUND AND PURPOSE: Autosomal Recessive Hereditary Spastic Paraplegia with Thin Corpus Callosum (AR-HSPTCC) is a clinically and genetically heterogeneous complicated form of spastic paraplegia. Two AR-HSPTCC loci have been assigned to chromosome 15q13-15 (SPG11) and chromosome 8p12-p11.21 respectively. Mutations in the SPG11 gene, encoding the spatacsin protein, have been found in the majority of SPG11 families. In this study, involvement of the SPG11 or 8p12-p11.21 loci was investigated in five Italian families, of which four consanguineous. METHODS: Families were tested for linkage to the SPG11 or 8p12-p11.21 loci and the SPG11 gene was screened in all the affected individuals. RESULTS: Linkage was excluded in the four consanguineous families. In the only SPG11-linked family the same homozygous haplotype 4.2 cM across the SPG11 locus was shared by all the three affected siblings. A novel c.2608A>G mutation predicted to affect the splicing was found in exon 14 of the SPG11 gene. DISCUSSION: This collection of families contributes to highlight the intra and inter locus heterogeneity in AR-HSPTCC, already remarked in previous reports. In particular, it confirms heterogeneity amongst Italian families and reports a new mutation predicted to affect splicing in the spatacsin gene.

A NOVEL BIOLOGICAL DOSIMETRY ASSAY AS A POTENTIAL TOOL FOR TRIAGE DOSE ASSESSMENT IN CASE OF LARGE-SCALE RADIOLOGICAL EMERGENCY.

In case of mass radiological emergencies, new strategies involving biological and clinical endpoints are requested for an efficient triage classification of casualties. For this purpose, we developed a novel protocol combining the two most established cytogenetic methods used in biological dosimetry (dicentric and micronucleus assays) into a single one, in order to have a time-saving, inexpensive and potentially automatable instrument to be used for triage purposes in case of large-scale radiological events. This method could be considered as a 'three in one' assay allowing the simultaneous scoring of chromosome aberrations and micronuclei on a single slide, and also enabling to discriminate between metaphases in first and second cell division without the Fluorescence plus Giemsa staining. This method needs further validation through inter-comparisons involving biological dosimetry laboratories, to verify its reproducibility. Moreover, the possibility to apply the already existing software for automation for dicentric and micronucleus assays could be also verified.

Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects.

Acetylation of platelet cyclooxygenase by oral aspirin is dose dependent and cumulative with repeated administration. However, no single dose of aspirin has been found to be completely selective of platelet thromboxane (TX) synthesis inhibition in man. We determined the dose dependence, cumulative nature and selectivity of aspirin effects on platelet TXB(2) and renal prostaglandin (PG) and prostacyclin (PGI(2)) production. We measured, by radioimmunoassay, serum TXB(2) levels after whole blood clotting and urinary excretion of PGE(2), PGF(2alpha), and 6-keto-PGF(1alpha), before and after single or repeated oral aspirin doses given to 46 healthy subjects. Single doses of 6-100 mg aspirin resulted in a linear (r = 0.92, P < 0.01) inhibition of platelet TXB(2) production, ranging from 12 to 95% after 24 h. A daily dose of 0.45 mg/kg given for 7 d produced a cumulative and virtually complete inhibition of platelet TXB(2) production, without significantly reducing the urinary excretion of PGE(2), PGF(2alpha), and 6-keto-PGF(1alpha) in both healthy men and women. The platelet inhibitory effect of this regimen was maintained unaltered throughout 1 mo of therapy, with no evidence of cumulative inhibition of renal PG-synthesis. Moreover, furosemide-induced renal PGI(2) synthesis and renin release were unaffected by chronic low-dose aspirin. Following cessation of aspirin therapy, platelet TXB(2) production returned toward control values at a similar rate as after a single higher dose. WE CONCLUDE THAT IN HEALTHY SUBJECTS: (a) aspirin causes a dose-dependent inhibition of platelet TXA(2) production, with no obvious sex-related difference; (b) the inhibitory effect of daily low-dose aspirin is cumulative on platelet TXA(2) but not on renal PG-synthesis; (c) during chronic low-dose aspirin therapy, renal PGI(2)-producing cells are readily activable by furosemide at a time of virtually complete suppression of platelet cyclooxygenase activity.

Estimated rate of thromboxane secretion into the circulation of normal humans.

We have measured the excretion of a major urinary metabolite of thromboxane B2 (TxB2), i.e., 2,3-dinor-TxB2, during the infusion of exogenous TxB2 over a 50-fold dose range to enable estimation of the rate entry of endogenous TxB2 into the bloodstream. Four healthy male volunteers received 6-h i.v. infusions of venhicle alone and TxB2 at 0.1, 1.0, and 5.0 ng/kg X min in random order. They were pretreated with aspirin at a dose of 325 mg/d in order to suppress endogenous TxB2 production. Urinary 2,3-dinor-TxB2 was measured before, during, and up to 24 h after the infusions and in aspirin-free periods, by means of radioimmunoassay. The nature of the extracted immunoreactivity was characterized by thin-layer chromatography and confirmed by negative ion-chemical ionization gas chromatography/mass spectrometry. Aspirin treatment suppressed urinary 2,3-dinor-TxB2 excretion by 80%. The fractional elimination of 2,3-dinor-TxB2 was independent of the rate of TxB2 infusion and averaged 5.3 +/- 0.8%. Interpolation of metabolite values obtained in aspirin-free periods onto the linear relationship between the quantities of infused TxB2 and the amount of metabolite excreted in excess of control values (y = 0.0066x, r = 0.975, P less than 0.001) permitted calculation of the mean rate of entry of endogenous TxB2 into the circulation as 0.11 ng/kg X min. The rate of disappearance of immunoreactive TxB2 from the circulation was monoexponential over the first 10 min with an apparent half-life of 7 min. This corresponded to a maximal estimate of the plasma concentration of endogenous TxB2 of 2.0 pg/ml. These results suggest that ex vivo platelet activation and/or analytical problems confound estimates of endogenous thromboxane release based on plasma TxB2 and provide a rationale for seeking longer-lived enzymatic metabolites of TxB2 in plasma.

Distinct roles of prostaglandin H synthases 1 and 2 in T-cell development.

Prostaglandin G and H synthases, or cyclooxygenases (COXs), catalyze the formation of prostaglandins (PGs). Whereas COX-1 is diffusely expressed in lymphoid cells in embryonic day 15.5 thymus, COX-2 expression is sparse, apparently limited to stromal cells. By contrast, COX-2 is predominant in a subset of medullary stromal cells in three- to five-week-old mice. The isozymes also differ in their contributions to lymphocyte development. Thus, experiments with selective COX-1 inhibitors in thymic lobes from normal and recombinase-activating gene-1 knockout mice support a role for this isoform in the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP). Concordant data were obtained in COX-1 knockouts. Pharmacological inhibition and genetic deletion of COX-2, by contrast, support its role during early thymocyte proliferation and differentiation and, later, during maturation of the CD4 helper T-cell lineage. PGE2, but not other PGs, can rescue the effects of inhibition of either isoform, although it acts through distinct EP receptor subtypes. COX-dependent PG generation may represent a mechanism of thymic stromal support for T-cell development.

Functional significance of renal prostacyclin and thromboxane A2 production in patients with systemic lupus erythematosus.

We have examined the urinary excretion of stable immunoreactive eicosanoids in 23 female patients with systemic lupus erythematosus (SLE), 16 patients with chronic glomerular disease (CGD), and 20 healthy women. SLE patients had significantly higher urinary thromboxane B2 (TXB2) and prostaglandin (PG) E2 excretion and significantly lower 6-keto-PGF1 alpha than did healthy women. In contrast, CGD patients only differed from controls for having reduced 6-keto-PGF1 alpha excretion. The group of SLE patients with active renal lesions differed significantly from the group with inactive lesions for having a lower creatinine clearance and urinary 6-keto-PGF1 alpha and higher urinary TXB2. Higher urinary TXB2 excretion was associated with comparable platelet TXB2 production in whole blood, undetectable TXB2 in peripheral venous blood, and unchanged urinary excretion of 2,3-dinor-TXB2. A significant inverse correlation was found between urinary TXB2 and creatinine clearance rate (CCr). In contrast, the urinary excretion of 6-keto-PGF1 alpha showed a significant linear correlation with both CCr and para-aminohippurate clearance rate (CPAH). In four SLE and seven CGD patients, inhibition of renal cyclooxygenase activity by ibuprofen was associated with a significant reduction in urinary 6-keto-PGF1 alpha and TXB2 and in both CCr and CPAH. However, the average decrease in both clearances was 50% lower in SLE patients than in CGD patients, when fractionated by the reduction in urinary 6-keto-PGF1 alpha or PGE2 excretion. We conclude that the intrarenal synthesis of PGI2 and TXA2 is specifically altered in SLE. Such biochemical alterations are associated with changes in glomerular hemodynamics and may play a role in the progression of SLE nephropathy.

Evidence for a direct stimulatory effect of prostacyclin on renin release in man.

THE OBJECTIVES OF THIS INVESTIGATION WERE: (a) to characterize the time and dose dependence of the effects of prostacyclin (PGI(2)) on renin release in healthy men; (b) to define whether PGI(2)-induced renin release is secondary to hemodynamic changes; (c) to determine the plasma and urine concentrations of 6-keto-PGF(1alpha) (the stable breakdown product of PGI(2)) associated with renin release induced by exogenous or pharmacologically enhanced endogenous PGI(2). Intravenous PGI(2) or 6-keto-PGF(1alpha) infusions at nominal rates of 2.5, 5.0, 10.0, and 20.0 ng/kg per min were performed in each of six normal human subjects; in three of them, PGI(2) infusion was repeated after beta-adrenergic blockade and cyclooxygenase inhibition. PGI(2), but not 6-keto-PGF(1alpha), caused a time- and dose-dependent increase of plasma renin activity, which reached statistical significance at 5.0 ng/kg per min and was still significantly elevated 30 min after discontinuing the infusion. Although combined propranolol and indomethacin treatment significantly enhanced the hypotensive effects of infused PGI(2), it did not modify the dose-related pattern of PGI(2)-induced renin release. Plasma 6-keto-PGF(1alpha) levels rose from undetectable levels (<7.5 pg/ml) in a stepwise fashion during increasingly higher infusion rates of PGI(2) or 6-keto-PGF(1alpha). The threshold concentration of plasma 6-keto-PGF(1alpha) associated with a statistically significant stimulation of renin release was approximately 200 pg/ml. Upon discontinuing PGI(2) or 6-keto-PGF(1alpha) infusion, the disappearance of 6-keto-PGF(1alpha) from blood showed an identical biphasic behavior, the initial phase having an apparent t((1/2)) of 3.2 min. The intravenous infusion of furosemide, which is known to stimulate renin release via a cyclooxygenase-dependent mechanism, caused a three-to fourfold increase of urinary 6-keto-PGF(1alpha) excretion rate, concomitant with the elevation of plasma renin activity levels, in six healthy women. 6-Keto-PGF(1alpha) remained undetectable in peripheral venous plasma throughout the study. WE CONCLUDE THAT IN HUMAN SUBJECTS: (a) PGI(2)-induced renin release occurs with a dose and time dependence similar to its reported platelet effects; (b) PGI(2)-induced renin release is not mediated by adrenergic stimuli or cyclooxygenase-dependent mechanisms secondary to hemodynamic changes; (c) furosemide-induced renin release is associated with increased renal PGI(2) formation; and (d) PGI(2) appears to act as a local modulator rather than a circulating hormone in controlling juxtaglomerular function.

Abnormally high thromboxane biosynthesis in homozygous homocystinuria. Evidence for platelet involvement and probucol-sensitive mechanism.

Homocystinuria due to homozygous cystathionine beta-synthase deficiency is an inborn error of metabolism characterized by a high incidence of thrombosis and premature atherosclerosis. We evaluated TXA2 biosynthesis in vivo and several in vitro tests of platelet function in 11 homocystinuric patients and 12 healthy controls. In vitro, patients' platelet aggregation was within control values as were TXB2 formation, fibrinogen binding, and ATP secretion in response to thrombin. In contrast, the urinary excretion of 11-dehydro-TXB2, a major enzymatic derivative of TXA2, was > 2 SD of controls in all patients (1,724 +/- 828 pg/mg creatinine, mean +/- SD, in patients vs. 345 +/- 136 in controls, P < 0.001). The administration to four patients of low-dose aspirin (50 mg/d for 1 wk) reduced metabolite excretion by > 80%. The recovery of 11-dehydro-TXB2 excretion over the 10 d that followed aspirin cessation occurred with a pattern consistent with the entry into the circulation of platelets with intact cyclooxygenase activity. Prolonged partial reduction in the abnormally high excretion of both 11-dehydro-TXB2 and 2,3-dinor-TXB2, was also observed in seven patients who ingested 500 mg daily for 3 wk of the antioxidant drug probucol. These results provide evidence for enhanced thromboxane biosynthesis in homocystinuria and for its partial dependence on probucol-sensitive mechanisms. Furthermore, the elevated TXA2 formation in homocystinuria is likely to reflect, at least in part, in vivo platelet activation.

Reduced platelet thromboxane formation in uremia. Evidence for a functional cyclooxygenase defect.

A qualitative platelet abnormality and a bleeding tendency are frequently associated with renal failure and uremia. We demonstrated previously that uremic patients display an abnormal platelet aggregation to arachidonic acid and reduced malondialdehyde production in response to thrombin and arachidonic acid. The objectives of this investigation were: (a) to compare platelet prostaglandin (PG) and thromboxane (TX) production in whole blood and in platelet-rich plasma (PRP) of 21 uremic patients and 22 healthy subjects; (b) to evaluate the concentration and activity of platelet PG- and TX-forming enzymes; (c) to assess the functional responsiveness of the platelet TXA(2)/PGH(2) receptor; (d) to explore the hemostatic consequences of partially reduced TXA(2) production.Platelet immunoreactive TXB(2) production during whole blood clotting was significantly reduced, by approximately 60%, in uremic patients as compared to age- and sex-matched controls. Exogenous thrombin (5-30 IU/ml) failed to restore normal TXB(2) production in uremic platelets. Uremic PRP produced comparable or slightly higher amounts of TXB(2) than normal PRP at arachidonate concentrations 0.25-1 mM. However, when exposed to substrate concentrations >2 mM, uremic PRP produced significantly less TXB(2) than normal PRP. To discriminate between reduced arachidonic acid oxygenation and altered endoperoxide metabolism, the time course of immunoreactive TXB(2) and PGE(2) production was measured during whole blood clotting. The synthesis and release of both cyclooxygenase-derived products was slower and significantly reduced, at all time intervals considered. Furthermore, PGI(2) production in whole blood, as reflected by serum immunoreactive 6-keto-PGF(1alpha) concentrations, was significantly reduced in uremic patients as compared with healthy subjects. PGH synthase levels, as determined by an immunoradiometric assay, were not significantly different in platelets from uremic patients as compared to control platelets. A single 40-mg dose of aspirin given to five healthy volunteers reduced their serum TXB(2) to levels found in uremic patients. This was associated with a significant increase of threshold aggregating concentrations of ADP and arachidonic acid and prolongation of bleeding time. Substantially similar threshold concentrations of U46619, a TXA(2) agonist, induced aggregation of normal and uremic platelets. Prostacyclin induced a significant elevation of uremic platelet cyclic AMP, which was suppressed by U46619, further suggesting normal responsiveness of the TXA(2)/PGH(2) receptor. WE CONCLUDE THAT: (a) an abnormality of platelet arachidonic acid metabolism exists in uremia, leading to a reduced TXA(2) production; (b) the characteristics of this abnormality are consistent with a functional cyclooxygenase defect; (c) reduced TXA(2) production may partially explain the previously described abnormality of platelet function in uremia.

NOS evaluations in human dental pulp-capping with MTA and calcium-hydroxide.

The aim of this study is to compare mineral trioxide aggregate (MTA) with calcium hydroxide when used as pulp-capping material in human teeth. 40 teeth were divided into groups based on clinical diagnosis: healthy and hyperaemic. The teeth were pulp capped with MTA and calcium hydroxide. We localized the eNOS and iNOS by immunohistochemistry, tested their mRNA expression by RT-PCR and protein levels by western blots. The evaluation of the samples was based on the cell inflammatory response and on the pulp tissue organization. In particular, evaluation of eNOS and iNOS differences between the various groups and the cellular evolution after the first 7 days from the treatment, and at a distance of 28 days. Our results suggest that there are differences in localization and expression between eNOS and iNOS in dental pulp. Our study has helped us to better understand the effects that calcium hydroxide and MTA have on pulp tissue.

Coxibs, Traditional NSAIDs, and Cardiovascular Safety Post-PRECISION: What We Thought We Knew Then and What We Think We Know Now.

The aim of the present review is to analyze how thinking about the cardiovascular safety of nonsteroidal antiinflammatory drugs has evolved during the past two decades, and discuss to what extent the additional information from the Prospective Randomized Evaluation of Celecoxib Integrated Safety Versus Ibuprofen or Naproxen study may alter our current mechanistic understanding and/or clinical practice.

In Silico Modeling of the Antiplatelet Pharmacodynamics of Low-dose Aspirin in Health and Disease.

The influence of platelet turnover on cyclooxygenase (COX-1) inhibition by low-dose aspirin remains largely uncharacterized due to limited feasibility of studying aspirin pharmacodynamics in bone marrow precursors. We developed an in silico compartmental model describing the aspirin effects on COX-1 activity in a population of megakaryocytes (MK) and in peripheral platelets. Model parameters were inferred from the literature and calibrated using measurements of serum thromboxane B2 (sTXB2 ), as proxy of COX-1 activity in peripheral platelets, in 17 healthy subjects and 24 patients with essential thrombocythemia (ET). The model reproduced well the average time-course of sTXB2 inhibition in healthy (accuracy = 10.4%), the reduced inhibition of sTXB2 observed in ET, and the effect of different dosing regimens. In conclusion, the in silico model accurately describes COX-1 inactivation by low-dose aspirin in MK and platelets in different clinical settings, and might help personalize aspirin regimens in conditions of altered megakaryopoiesis.

Low-Dose Aspirin Acetylates Cyclooxygenase-1 in Human Colorectal Mucosa: Implications for the Chemoprevention of Colorectal Cancer.

The mechanism of action of low-dose aspirin in the prevention of colorectal cancer (CRC) remains largely hypothetical. We aimed to compare the effects of low-dose aspirin (100 mg/day for 7 days) given to 40 individuals undergoing CRC screening on the extent of cyclooxygenase (COX)-1 acetylation at serine-529 (AceCOX-1), in blood platelets vs. colorectal mucosa, at 7 (group 1) and 24 h (group 2) after dosing. A significantly (P < 0.01) lower %AceCOX-1 was detected in colonic and rectal mucosa (average 64%) vs. platelets (average 75%) in both groups. This effect was associated with an average 46% (P < 0.01) and 35% (P < 0.05) reduction in prostaglandin (PG) E2 levels and phosphorylated S6 (p-S6) levels, respectively. Rectal mucosal levels of p-S6/S6 significantly (P < 0.01) correlated with PGE2 . These findings demonstrate that low-dose aspirin produces long-lasting acetylation of COX-1 and downregulation of p-S6 in human colorectal mucosa, an effect that may interfere with early colorectal carcinogenesis.

On-pump Cardiac Surgery Enhances Platelet Renewal and Impairs Aspirin Pharmacodynamics: Effects of Improved Dosing Regimens.

On-pump cardiac surgery may trigger inflammation and accelerate platelet cyclooxygenase-1 renewal, thereby modifying low-dose aspirin pharmacodynamics. Thirty-seven patients on standard aspirin 100 mg once-daily were studied before surgery and randomized within 36 hours postsurgery to 100 mg once-daily, 100 mg twice-daily, or 200 mg once-daily for 90 days. On day 7 postsurgery, immature and mature platelets, platelet mass, thrombopoietin, glycocalicin, leukocytes, C-reactive protein, and interleukin-6 significantly increased. Interleukin-6 significantly correlated with immature platelets. At day 7, patients randomized to 100 mg once-daily showed a significant increase in serum thromboxane (TX)B2 within the 24-hour dosing interval and urinary TXA2 metabolite (TXM) excretion. Aspirin 100 mg twice-daily lowered serum TXB2 and prevented postsurgery TXM increase (P < 0.01), without affecting prostacyclin metabolite excretion. After cardiac surgery, shortening the dosing interval, but not doubling the once-daily dose, rescues the impaired antiplatelet effect of low-dose aspirin and prevents platelet activation associated with acute inflammation and enhanced platelet turnover.

Radioimmunoassay of 11-dehydrothromboxane B2 in human plasma and urine.

Because of the discrepancy between the capacity of platelets to synthesize thromboxane B2 ex vivo and the actual synthetic rate in vivo, measurement of thromboxane B2 in plasma is highly influenced by sampling-related artifacts. We have developed and validated a radioimmunoassay for a major enzymatic derivative of thromboxane B2 with an extended plasma half-life, i.e., 11-dehydrothromboxane B2. The binding of the tracer is displaced by as low as 1 pg/ml of the homologous ligand, with a high degree of specificity for the open ring structure as well as for the omega side-chain. This method can detect changes in the plasma concentration and urinary excretion of 11-dehydrothromboxane B2 associated with stimulated short-term increases of thromboxane B2 secretion in the human circulation.

Fractional conversion of thromboxane B2 to urinary 11-dehydrothromboxane B2 in man.

Thromboxane (TX) B2, the chemically stable hydration product of pro-aggregatory TXA2, undergoes two major pathways of metabolism in man, resulting in the formation of 2.3-dinor-TXB2 and 11-dehydro-TXB2, respectively. We have measured the excretion of the latter during the infusion of exogenous TXB2 over a 50-fold dose range in order to examine the fractional conversion of TXB2 to urinary 11-dehydro-TXB2 and to re-assess the rate of entry of endogenous TXB2 into the circulation. Four healthy male volunteers received 6-h intravenous infusions of the vehicle alone and TXB2 at 0.1, 1.0 and 5.0 ng.kg-1.min-1 in random order. They were pretreated with aspirin 325 mg/d in order to suppress endogenous TXB2 production. Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were measured before, during and up to 24 h after the infusions and in aspirin-free periods, by means of NICI-GC/MS-validated radioimmunoassays. Aspirin treatment suppressed urinary 11-dehydro-TXB2 by 91%. The fractional elimination of 11-dehydro-TXB2 was independent of the rate of TXB2 infusion and averaged 6.8 +/- 0.7%, as compared to 6.4 +/- 0.9% for 2,3-dinor-TXB2. Interpolation of 11-dehydro-TXB2 values obtained in aspirin-free periods onto the linear relationship between the quantities of infused TXB2 and the amount of metabolite excreted in excess of control values (y = 0.0058x, r = 0.94, P less than 0.001) permitted calculation of the mean rate of entry of endogenous TXB2 into the circulation as 0.12 ng.kg-1.min-1. We conclude that: (a) urinary 11-dehydro-TXB2 is at least as abundant a conversion product of exogenously infused TXB2 as 2,3-dinor-TXB2; (b) its excretion increases linearly as a function of the rate of entry of TXB2 into the circulation up to approx. 40-fold the calculated rate of secretion of endogenous TXB2; (c) the latter is consistent with previous estimates based on monitoring of the beta-oxidation pathway of TXB2 metabolism.

Fractional conversion of thromboxane A2 and B2 to urinary 2,3-dinor-thromboxane B2 and 11-dehydrothromboxane B2 in the cynomolgus monkey.

Following the intravenous administration of thromboxane (TX) B2, the stable hydration product of TXA2, to human and nonhuman primates the most abundant urinary metabolites are 2,3-dinor-TXB2 and 11-dehydro-TXB2. However, it is not known whether fractional conversion of TXB2 to its enzymatic metabolites is an accurate representation of TXA2 metabolism. Thus, we have compared the metabolic disposition of synthetic TXA2 and TXB2 via the beta-oxidation and 11-OH-dehydrogenase pathways in vivo in the monkey. TXA2 or TXB2 (20 ng/kg) was intravenously administered to four cynomolgus monkeys pretreated with aspirin in order to suppress endogenous TXA2 production. Urinary TXB2, 2,3-dinor-TXB2 and 11-dehydro-TXB2 were measured before, during and up to 24 h after thromboxane administration by means of reversed-phase high-performance liquid chromatography radioimmunoassay. Aspirin treatment suppressed urinary 2,3-dinor-TXB2 and 11-dehydro-TXB2 by approx. 75%. A similar fractional conversion of TXA2 and TXB2 into 2,3-dinor-TXB2 and 11-dehydro-TXB2 was found. These results suggest that TXA2 is hydrolyzed to TXB2 prior to enzymatic degradation and that metabolites of the latter represent reliable indices of TXA2 biosynthesis. Due to the variability in the conversion of thromboxanes into 2,3-dinor-TXB2 and 11-dehydro-TXB2, the measurement of both metabolites seems to represent a more reliable index of acute changes in TXA2 production.