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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%; semi-automated 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.

Background Rivaroxaban (Riva), a direct FXa inhibitor, at 2.5 mg twice-daily (bid) combined with low-dose aspirin (ASA, 100 mg once daily-od) reduced major vascular events vs. ASA alone in subjects with stable coronary artery (CAD) or symptomatic peripheral artery disease (PAD). Whether this benefit is due to the anticoagulant effect or additional FXa-mediated effects through the platelet and endothelial cell thrombin receptors is unknown. Type 2 diabetes mellitus (T2DM) is characterized by high platelet activation and oxidative stress that may contribute to increased cardiovascular risk. Purpose We investigated the effects of Riva (2.5 mg bid) + ASA (100 mg od) vs. ASA (100 mg od) alone on platelet and oxidative biomarkers in subjects with T2DM and stable vascular disease (stable CAD, symptomatic PAD and/or significant carotid stenosis). Methods In this randomized, open-label, cross-over trial, patients were randomized to continue ASA for 4 weeks and then add Riva for 4 weeks, or add Riva in the first 4 weeks and then continue with ASA alone for 4 weeks. Primary endpoints were: in vivo platelet activation and lipid peroxidation, assessed by the urinary excretion of 11-dehydro-TXB2 (TXM) and 8-iso-PGF2alpha (ISOP), respectively. Secondary endpoints included: routine coagulation tests, D-dimer, thrombin generation, serum TXB2, and Riva plasma concentrations. Results Seventy-6 subjects (10 females) were recruited: age 68±7 years (mean±SD); BMI 27.1±3.5 kg/m2; fasting glucose 129±31 mg/dL; HbA1c 6.8±0.9%; serum creatinine 1±0.25 mg/dL; LDL-cholesterol 77±31 mg/dL. Two patients dropped out: one for benign, self-limiting hematuria, one for unwillingness to continue, 8 subjects are completing the study leaving 66 who completed the 8-week randomized treatment and showed no sequence effect. Urinary TXM and ISOP were significantly reduced by Riva+ASA vs. ASA alone: TXM was 260 [195–398] vs. 335 [225–441] pg/mg creatinine and ISOP 722 [601–991] vs. 827 [648–1350] pg/mg creatinine (median [IQR]) on Riva+ASA vs. ASA alone, respectively (p Conclusions In ASA-treated subjects with T2DM and stable vascular disease, the addition of very low-dose Riva restrained incompletely-suppressed lipid peroxidation and platelet activation and modified the kinetics of thrombin formation. These changes may contribute to the beneficial effects of the Riva+ASA combination. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Investigator-initiated study funded by Bayer AG

Background Platelet activation plays a major role in the atherothrombotic complications of diabetes. Thromboxane (TX)A2 is a pro-thrombotic prostanoid, synthesized via cyclooxygenase-1 and released by activated platelets. The metabolism of TXA2 in vivo leads to a major stable end-product, 11-dehydro-TXB2 (TXM), measurable in urine and reflecting the whole-body rate of TXA2 biosynthesis. In two large trials of high-risk, aspirin-treated (mostly, without diabetes) patients, (CHARISMA and HOPE trials), the baseline rate of urinary TXM excretion was an independent predictor of future cardiovascular events. Purpose The aim of the ASCEND (A Study of Cardiovascular Events in Diabetes) TXM sub-study was to investigate the association between baseline urinary TXM and future serious vascular events or revascularization (SVE-R), major bleeds and incident cancer independent of other risk factors and treatment, in people with diabetes and no manifest cardiovascular disease at trial entry. Methods Urinary TXM was measured by a previously GC/MS-validated, immunoassay in 6,487 participants with eligible baseline samples. Analyses excluded 539 participants using NSAIDs. TXM appeared log-normally distributed, so analyses were by quintiles and per SD (=0.622) of continuous loge TXM. The association of loge TXM with outcome was adjusted by basic factors (age, sex, sample volume and randomized treatment allocation) and by the predictors of log TXM (smoking, type 2 diabetes treated with insulin or oral hypoglycaemics, HDL cholesterol, body mass index, urinary albumin/creatinine ratio, eGFR). The association of log TXM with non-vascular, non-cancer MedDRA outcomes was investigated to determine whether TXM had a general effect on outcome. During a mean of 6.6 years follow-up there were 618 SVE-Rs, 206 bleeds and 700 cancers among these patients. Results Log TXM correlated significantly with SVE-R, hazard ratio (HR) per 1 SD of log TXM: 1.13 (1.04–1.23), p=0.003 (Figure 1, panel a), non-significantly with major bleeds [HR 1.15 (1.00–1.32), p=0.055] (Figure 1, panel b), and marginally significantly with cancer [HR 1.09 (1.01–1.17), p=0.03] (Figure 1, panel c). There was no association of log TXM with non-vascular non-cancer MedDRA outcomes (HR per 1 SD, 0.99; 99% CI, 0.94–1.05). Conclusion The rate of urinary TXM excretion, a non-invasive biomarker of TXA2-mediated platelet activation in vivo, is log-linearly associated with serious vascular events independent of other risk factors in people with diabetes. Its potential association with cancer must be viewed as hypothesis-generating and needs confirmation. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): IMI1: Surrogate markers for micro- and macro-vascular hard endpoints for innovative diabetes tools (SUMMIT).

Objective: To perform a clinical and genetic study of two large Italian families (RM-36 and RM-51) showing the cardinal clinical features of Silver syndrome (SS), a rare dominantly inherited form of hereditary spastic paraplegia (HSP) complicated by amyotrophy of the small hand muscles. Methods: Clinical assessment including neurophysiologic, neuropsychological, and neuroimaging evaluations. Genetic studies included linkage and sequence analyses. Results: Using a genome-wide survey in the RM-36 family, a novel locus (SPG38) has been identified and mapped within the 13.1-cM region on chromosome 4p16-p15 between markers D4S432 and D4S1599. The RM-51 family was linked to the SPG4 locus at 2p21-p24 and sequence analysis of SPG4 showed a novel frameshift mutation p.Asp321GlyfsX6. Clinical examination of the affected members carrying the mutation showed high frequency of additional clinical features including decreased vibration sense, pes cavus, temporal lobe epilepsy, and cognitive impairment. Conclusions: This study demonstrates evidence of a novel locus SPG38 for Silver syndrome (SS) and suggests that genetic defects in SPG4 might lead to broad clinical features overlapped with those of SS.

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.

Two mutations (G8363A and A8296G) in the mtDNA (mitochondrial DNA) tRNALys gene have been associated with severe mitochondrial diseases in a number of reports. Their functional significance, however, remains unknown. We have already shown that homoplasmic cybrids harbouring the A8296G mutation display normal oxidative phosphorylation, although the possibility of a subtle change in mitochondrial respiratory capacity remains an open issue. We have now investigated the pathogenic mechanism of another mutation in the tRNALys gene (G8363A) by repopulating an mtDNA-less human osteosarcoma cell line with mitochondria harbouring either this genetic variant alone or an unusual combination of the two mutations (A8296G+G8363A). Cybrids homoplasmic for the single G8363A or the A8296G+G8363A mutations have defective respiratory-chain enzyme activities and low oxygen consumption, indicating a severe impairment of the oxidative phosphorylation system. Generation of G8363A cybrids within a wild-type or the A8296G mtDNA genetic backgrounds resulted in an important alteration in the conformation of the tRNALys, not affecting tRNA steady-state levels. Moreover, mutant cybrids have an important decrease in the proportion of amino-acylated tRNALys and, consequently, mitochondrial protein synthesis is greatly decreased. Our results demonstrate that the pathogenicity of the G8363A mutation is due to a change in the conformation of the tRNA that severely impairs aminoacylation in the absence of changes in tRNA stability. The only effect detected in the A8296G mutation is a moderate decrease in the aminoacylation capacity, which does not affect mitochondrial protein biosynthesis.

We analyzed seven unrelated children with the Smith–Lemli–Opitz syndrome (SLOS) for mutations in the Δ7-sterol reductase gene by using SSCP and direct sequencing. We identified two novel mutations (V330M and R363C) in the DHCR7 gene. Reported mutations found in this study were T93M (3/14 alleles), E448K (2/14), and W151X, G244R, P329L, and R446Q (each found in one allele). The so-called common IVS8-1 G→C was found in three alleles, confirming its relative rarity among Italian SLOS families. By using a scoring system, clinical severity did not seem to correlate with 7DHC levels and type of mutation. Expanding the spectrum of mutations in SLOS, our study does not support direct genotype-phenotype correlation.

We studied nine Italian families with a pure form of autosomal dominant spastic paraplegia (ADHSP) to assess the frequency of mutations in the SPG4 gene. We observed marked intrafamilial variability in both age-at-onset and clinical severity, ranging from severe congenital presentation to mild involvement after age 55 years to healthy carriers of the mutation after age 70. Four of nine probands harboured SPG4 mutations, We identified three new SPG4 mutations, all predicting a loss-of-func-tion with apparently important consequences for spastin function. RT-PCR studies predict loss-of-function as a possible mechanism leading to spastin-related HSP. The current study expands the spectrum of allelic variants in SPG4, confirming their pathological significance in pure AD-HSP and suggesting implications for the presumed function of spastin.

Rifamycin SV, injected intraarticularly at dosages of 500 mg weekly, induced a complete removal of persistent knee effusions in 57 of 60 rheumatoid patients, and in all patients with effusions due to juvenile rheumatoid arthritis (three), ankylosing spondylitis (three), psoriatic arthritis (10), intermittent hydrarthrosis (two) and chondromatosis (two). One rheumatoid patient did not respond to treatment, and two dropped out because of local side effects. The number of infiltrations varied between three and 12, and was correlated with activity grade of local synovitis (P.05). As of this writing, 50 out of 57 rheumatoid patients and 19 out of 20 belonging to the other groups have not had a relapse of effusions after 25 to 36 months and 12 to 20 months respectively. While the changes in the synovial fluid and membrane of the rheumatoid subjects, and the results obtained from experimental arthritis in guinea pigs, confirm the hypothesis that Rifamycin has a local antiinflammatory action in arthritic knee joints, they also suggest that this property alone of the drug is not entirely responsible for its therapeutic action.

The authors studied a family with pure autosomal dominant spastic paraplegia (ADHSP) that showed a marked intrafamilial variability in both age at onset and clinical severity, ranging from severe congenital presentation to mild involvement after age 55. They found a novel mutation in the SPG4 gene, which segregates with the disease in six patients. The mutation affects the consensus donor splice site of SPG4 intron 16, resulting in a premature termination codon at amino acid 578. The data confirm the pathologic significance of SPG4 mutations in pure ADHSP and add to the list of known SPG4 allelic variants.

The mutation of the spatacsin gene is the single most common cause of autosomal recessive hereditary spastic paraplegia with thin corpus callosum. Common clinical, pathological and genetic features between amyotrophic lateral sclerosis and hereditary spastic paraplegia motivated us to investigate 25 families with autosomal recessive juvenile amyotrophic lateral sclerosis and long-term survival for mutations in the spatascin gene. The inclusion criterion was a diagnosis of clinically definite amyotrophic lateral sclerosis according to the revised El Escorial criteria. The exclusion criterion was a diagnosis of hereditary spastic paraplegia with thin corpus callosum in line with an established protocol. Additional pathological and genetic evaluations were also performed. Surprisingly, 12 sequence alterations in the spatacsin gene (one of which is novel, IVS30 + 1 G > A) were identified in 10 unrelated pedigrees with autosomal recessive juvenile amyotrophic lateral sclerosis and long-term survival. The countries of origin of these families were Italy, Brazil, Canada, Japan and Turkey. The variants seemed to be pathogenic since they co-segregated with the disease in all pedigrees, were absent in controls and were associated with amyotrophic lateral sclerosis neuropathology in one member of one of these families for whom central nervous system tissue was available. Our study indicates that mutations in the spatascin gene could cause a much wider spectrum of clinical features than previously recognized, including autosomal recessive juvenile amyotrophic lateral sclerosis.

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.

Aldo Orlacchio, Carlo Massini, Roberto Tiribuzi, Egidia Costanzi, Georgia Makrypidi, Filippo Mattoli, Zaira Esposito, Clarice Patrono, Marialuisa Miele, Mariangela Maiotti, Giuseppe Sancesario, Mauro Zampolini, Antonio Orlacchio, Sabata Martino, Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universita di Perugia, Perugia, Italy; Laboratorio di Neurogenetica, CERC-IRCCS Santa Lucia, Rome, Italy; Dipartimento di Neuroscienze, Universita di Roma ‘Tor Vergata’, Rome, Italy; Struttura Complessa di Neurologia e Neuroriabilitazione, Ospedale Nuovo San Giovanni Battista, ASL3, Foligno (PG), Italy. Contact email: orly@unipg.it

Hereditary spastic paraplegia (HSP) includes a heterogeneous group of neurodegenerative disorders with the characteristics of slowly progressive spasticity and weakness of the lower limbs. It can be inherited in an autosomal-dominant, autosomal-recessive or X-linked fashion.1 Until now, 38 loci have been registered at the HUGO database (http://www.genenames.org/) and 15 causative genes have already been described. The most common form of autosomal-dominant HSP, accounting for approximately 40% of affected individuals, is due to mutations in the SPG4 gene.2 3 The defective SPG4 gene encodes spastin, a 616-amino-acid protein belonging to the AAA family ( A TPases A ssociated with diverse cellular A ctivities). The protein is expressed ubiquitously and has a critical biological role in the regulation of microtubule severing.2 4 The aim of this study was to investigate the SPG4 mutation profile in Romanian patients with spastic paraplegia (SP) and to describe them in the context of the corresponding clinical phenotype. Between 1990 and 2007, a total of 30 unrelated Romanian patients with SP who had emigrated to Italy were included in the study. Twenty-three affected individuals had a family history of autosomal-dominant traits, and 7 patients had no family history or information about the health status of family members was limited. Participants were recruited as Romanian if they were born in, or originated from, Romania. All …

The authors report on a novel frameshift mutation (c.1688insA) in the SPG3A gene resulting in premature translation termination of the gene product atlastin. These data add a new variant to the second disease gene in autosomal dominant hereditary spastic paraplegia (ADHSP) and lend definitive support to its causative role. By combining direct testing of SPAST and SPG3A, at least 50% of ADHSP families can now receive appropriate genetic diagnosis.

Hereditary spastic paraplegia is a clinically and genetically heterogeneous disorder characterized by progressive spasticity of the lower limbs. Seven loci for autosomal dominant pure hereditary spastic paraplegia (ADPHSP) have already been mapped on chromosomes 14q, 2p, 15q, 8p, 12q, 19q, and 2q. We report on an Italian family affected by ADPHSP for which we excluded linkage with the known loci and performed a genome-wide search. Linkage analysis and haplotype construction permitted the identification of a novel ADPHSP locus on the long arm of chromosome 9, designated SPG19. The phenotype was characterized by late onset (range, 36-55 years) and mild disability, with only 1 patient bound to a wheelchair after 31 years of disease. Urinary disturbances (urgency and/or incontinence) were always present, even in young patients with a short disease history.

Hereditary Spastic Paraplegias (HSPs) are heterogeneous neurodegenerative disorders whose etiopathogenesis is still unclear. The identification of pathogenic mutations in a gene (SPG7) encoding a mitochondrial metalloprotease suggested that oxidative phosphorylation (OXPHOS) alterations might underlie HSP in a subgroup of patients. We performed clinical, morphological, biochemical, and molecular genetic studies in six HSP patients and in six sporadic patients to investigate OXPHOS in muscle biopsies. Complicated and pure forms were included in our study. Morphological alterations of the type seen in OXPHOS-related disorders were found in three patients. Five patients showed an isolated defect of complex I activity. No mutations in the SPG7 gene were detected. Our results suggest that OXPHOS defects in HSP patients are more common than previously believed.

Six unrelated Italian children with Wolfram syndrome (WS) were analyzed for mutations in the WFS1. Four novel mutations (1387delCTCT, S443I, 1519del16, and IVS6+16g->a) were identified. In addition, we found two new, probably neutral changes (A684V and R708C). Other previously described variants were a heterozygous I333V in three alleles and the H611R in two. The 1519del16 mutation was carried by two patients whereas the CTCT deletion occurred in three subjects from two apparently unrelated families with WS. The current study expands the spectrum of mutations in WFS1 and represents the first molecular characterization of Italian WS patients. Hum Mutat 17:348-349, 2001. © 2001 Wiley-Liss, Inc.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder characterized by minor facial anomalies, mental retardation, and multiple congenital abnormalities. Biochemically, the disorder is caused by deficient activity of 7-dehydrocholesterol reductase, which catalyzes the reduction of the Δ7 double bond of 7-dehydrocholesterol to produce cholesterol. Recently, mutations in the gene encoding 7-dehydrocholesterol reductase (7DHCR) were found to cause SLOS. We report the first molecular characterization of an Italian SLOS patient. Interestingly, his paternal 7DHCR allele, of Arab origin, harbored a novel P329L mutation which in combination with a maternal splice-site (IVS8-1 G>C) mutation resulted in a relatively milder phenotype. Am. J. Med. Genet. 91:138–140, 2000. © 2000 Wiley-Liss, Inc.

We report on a 5-year-old boy with clinical and neuroradiological evidence of Leigh syndrome and peripheral neuropathy. Skeletal muscle biopsy showed decreased cytochrome c oxidase stain. Ultrastructurally, the nerve biopsy showed a defect of myelination. Biochemical analyses of muscle homogenate showed cytochrome c oxidase deficiency (15% residual activity). SURF1 gene analysis identified a novel homozygous nonsense mutation which predicts a truncated surf1 protein.

We evaluated whether therapeutic blood levels of meloxicam are associated with selective inhibition of monocyte cyclooxygenase (COX)-2 in vitro and ex vivo. Concentration-response curves for the inhibition of monocyte COX-2 and platelet COX-1 were obtained in vitro after the incubation of meloxicam with whole blood samples. Moreover, 11 healthy volunteers received placebo or 7.5 or 15 mg/day meloxicam, each treatment for 7 consecutive days, according to a randomized, double-blind, crossover design. Before dosing and 24 h after the seventh dose of each regimen, heparinized whole blood samples were incubated with lipopolysaccharide (10 microgram/ml) for 24 h at 37 degrees C, and prostaglandin E2 was measured in plasma as an index of monocyte COX-2 activity. The production of thromboxane B2 in whole blood allowed to clot at 37 degrees C for 60 min was assessed as an index of platelet COX-1 activity. The administration of placebo did not significantly affect plasma prostaglandin E2 (21. 3 +/- 7.5 versus 19.1 +/- 4 ng/ml, mean +/- S.D., n = 11) or serum thromboxane B2 (426 +/- 167 versus 425 +/- 150 ng/ml) levels. In contrast, the administration of 7.5 and 15 mg of meloxicam caused dose-dependent reductions in monocyte COX-2 activity by 51% and 70%, respectively, and in platelet COX-1 activity by 25% and 35%, respectively. Although the IC50 value of meloxicam for inhibition of COX-1 was 10-fold higher than the IC50 value of COX-2 in vitro, this biochemical selectivity was inadequate to clearly separate the effects of meloxicam on the two isozymes after oral dosing as a function of the daily dose and interindividual variation in steady-state plasma levels.

More than 50 randomized trials have documented the efficacy and safety of aspirin as an antiplatelet agent and a cardiovascular drug. However, the optimal dose for preventing coronary and cerebral thrombosis has long been a cause of debate. For patients with ischaemic heart disease the range recommended for the prevention of a secondary event, based on strong clinical evidence, is 75-160 mg aspirin/day. For patients with cerebrovascular disease, recommendations range from 30-1300 mg/day. If these patients require a higher dose of aspirin it suggests that a different mechanism of action is involved. This paper considers hypotheses and reports the findings of recent clinical trials. The SALT study compared aspirin with placebo in 1360 patients with TIA or minor ischaemic stroke. It showed an 18% reduction in the risk of stroke or death in patients receiving 75 mg aspirin/day. Five other trials of 55,000 patients with ischaemic cerebrovascular disease compared the protective effect of aspirin (range 30-300 mg/day) with placebo, clopidogrel, or oral anticoagulants. Aspirin was better than placebo, safer than oral anticoagulants, and no different from clopidogrel. The implications of these findings are discussed. Mechanistic studies and randomized clinical trials strongly suggest that the mechanism of action and dose requirement of the antithrombotic effect of aspirin in patients with cerebrovascular disease is the same as that for ischaemic heart disease.

We have evaluated the selectivity in vitro of various conventional nonsteroidal anti-inflammatory drugs (NSAIDs) and new anti-inflammatory compounds (NS-398, L-745,337 and SC58125) in inhibiting the cyclooxygenase activity of platelet prostaglandin endoperoxide synthase (PGHS)-1 and monocyte PGHS-2 in a human whole blood assay. The effects of the compounds towards the cyclooxygenase activity of monocyte PGHS-2 induced in response to lipopolysaccharide (LPS) was evaluated by measuring the levels of PGE2 produced in plasma. The effects of the same inhibitors on platelet PGHS-1 activity were assessed by allowing 1-ml whole blood samples to clot at 37 degrees C for 1 h in the presence of the compounds and measuring immunoreactive TXB2 levels in serum. Under these experimental conditions, most compounds resulted equipotent towards the two isozymes. Differently, meloxicam, nimesulide and diclofenac were approximately 10- to 20-fold more potent in inhibiting the cyclooxygenase activity of monocyte PGHS-2 than platelet PGHS-1. L-745,337, NS-398 and SC58125 achieved selective inhibition of monocyte PGHS-2 (IC50, PGHS-1/IC50, PGHS-2:100) and may provide adequate tools to test the contribution of this novel pathway of arachidonate metabolism to human inflammatory disease and to verify the hypothesis that the common side-effects of NSAIDs are due primarily to their ability to affect the activity of PGHS-1.

The clinical development of low-dose aspirin as an antiplatelet agent provides an interesting paradigm of selective inhibition of a COX isozyme. During this process, we have learned important lessons in the use of analytical tools for studying the dose-dependence and biochemical selectivity of cyclooxygenase inhibition in man as well as observing the clinical correlates of such selectivity. Relatively selective inhibition of platelet COX-1 can be achieved by virtue of the irreversible inactivation of the enzyme and the very short half-life of aspirin in the human circulation (15–20 minutes). A 24-hour dosing interval allows full recovery of COX-1 activity in nucleated cells, but causes cumulative inhibition of enzyme activity in anucleated platelets upon repeated daily dosing with very low doses of the drug1–3. The unique mechanism of permanent inactivation of COX-1 by aspirin involves acetylation of a single amino acid residue (Ser-529) within the polypeptide chain of the enzyme. The strategic position of Ser-529 in the so-called cyclooxygenase channel blocks access of arachidonic acid to the active site of the enzyme, as a consequence of its acetylation by aspirin. Because of the irreversible nature of this modification, cyclooxygenase activity can be restored only by new protein synthesis (occurring within a few hours in nucleated cells) or platelet turn-over (occurring over days).

F2-isoprostanes are prostaglandin (PG) F2-like compounds that are formed in vivo directly by free radical-catalyzed lipid peroxidation. One of the compounds that can be produced in abundance by such mechanism is 8-epi-PGF2 alpha, a potent vasoconstrictor. We have developed an enzyme immunoassay and a radioimmunoassay for measuring urinary concentrations of 8-epi-PGF2 alpha by raising antibodies against this compound. The antisera presented high titers (1/300,000) and provided highly sensitive assays (IC50, 8 and 24 pg/ml, for EIA and RIA, respectively); cross-reactivity with other PG was negligible. The interassay reproducibility of EIA was assessed by measuring the same urine stored frozen in aliquots after solid phase extraction and thin-layer chromatography (17%, n = 13). Measurements of urinary 8-epi-PGF2 alpha by immunoassays were validated using different antisera and by comparison with gas chromatography/mass spectrometry. Healthy volunteers excreted 25 +/- 12 ng of 8-epi-PGF2 alpha/mmol creatinine (n = 19), with no circadian variation over three consecutive 8-hr collection periods (n = 10); preliminary results showed that excretion increased as a function of age. Urinary excretion of 8-epi-PGF2 alpha was unchanged by treatment with two nonsteroidal antiinflammatory drugs, Ibuprofen at 1.2 g/day for 4 days (n = 4) or aspirin as a single administration of 1 g (n = 6). In contrast, the urinary excretion of 11-dehydro-thromboxane B2, a platelet cyclooxygenase-derived metabolite was reduced by more than 80% after aspirin administration.(ABSTRACT TRUNCATED AT 250 WORDS)

The co-administration of nonsteroidal anti-inflammatory drugs (NSAIDs) and antihypertensive agents often, but not always, results in blunting of the effect of antihypertensive therapy. Although NSAIDs have no detectable pressor effects in normal subjects or untreated hypertensive people, they seem to antagonize the action of the majority of antihypertensive agents, making it necessary to increase their dosage, and often preventing proper control of blood pressure, particularly in black and elderly patients. The mechanism of this pharmacodynamic interaction is not completely understood, but may involve inhibition of vascular and renal prostaglandin (PG) synthesis, with resulting vasoconstriction and impaired renal excretion of Na+ and water. Also, suppression of a possible intermediary action of PG in the antihypertensive action of agents such as angiotensin converting enzyme inhibitors has been proposed. Certain antihypertensive drugs, such as Ca(2+)-channel blockers, centrally acting alpha agonists, and diuretics seem less sensitive to antagonism by NSAIDs. Aspirin and sulindac seem to be devoid of pressor effects, and thus provide a safe alternative in patients at risk for this interaction. These compounds may, in selected circumstances, even potentiate the effects of antihypertensive medications. Studies are underway to evaluate the hemodynamic effects of more selective blockers of arachidonate metabolism, such as thromboxane synthase inhibitors and selective inhibitors of PGH synthase-2.

The combination of thromboxane (TX) synthase inhibition and prostaglandin (PG) H2/TXA2 receptor antagonism yields enhanced antithrombotic effects as compared with either intervention alone. However, it is not known whether the enhancing effect of TX synthase inhibition is expressed also in the presence of complete blockade of PGH2/TXA2 receptors. Thus we evaluated the antithrombotic effects of increasing doses of the PGH2/TXA2 receptor antagonist L 670596 alone and in combination with a dose of the TX synthase inhibitor FCE 22178 causing95% inhibition of platelet TXB2 production. In the dog model of electrically induced coronary thrombosis, occlusion time in control animals (n = 14) averaged 72 +/- 29 min. L 670596 alone dose-dependently antagonized platelet PGH2/TXA2 receptors and prolonged occlusion time. The addition of FCE 22178 displaced the dose-occlusion time relation of L 670596 in a parallel fashion without modifying receptor occupancy. In the rabbit model of copper coil-induced carotid artery thrombosis, occlusion was very rapid (14 +/- 4 min) in control animals (n = 17) and was not modified by either aspirin or FCE 22178. L 670596 caused a dose-related receptor blockade and prolongation of occlusion time. The association with FCE 22178 enhanced significantly the antithrombotic effect of L 670596 at all doses. We conclude that the full therapeutic potential of PGH2/TXA2 receptor antagonism is expressed at90% platelet receptor occupancy. The additive effect of TX synthase inhibition suggests that conversion of PGH2 to platelet-inhibitor and vasodilator prostaglandins might be of therapeutic importance, irrespective of the extent of PGH2/TXA2 receptor blockade.

Increased thromboxane (TX) production and modified aspirin sensitivity has been detected in vitro in platelets isolated from patients with polycythemia vera. To verify the relevance of these capacity-related measurements to the actual rate of TXA2 biosynthesis in vivo and its suppression by oral aspirin, we have investigated the urinary excretion of major enzymatic metabolites of TXB2 in 17 patients with polycythemia vera and 23 gender- and age-matched controls. Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were measured by previously validated radioimmunoassays. In addition, urinary immunoreactive leukotriene (LT) E4 was measured to explore the 5-lipoxygenase pathway of arachidonate metabolism. Polycythemic patients had significantly (P.001) higher excretion rates of both 11-dehydro-TXB2 (1,033 +/- 1,050 v 117 +/- 45 pmol/mmol creatinine; mean +/- SD) and 2,3-dinor-TXB2 (725 +/- 676 v 82 +/- 43 pmol/mmol creatinine) than controls. In contrast, urinary LTE4 was not significantly different. Enhanced metabolite excretion did not correlate with the platelet count or with the hematocrit value, and was not related to the current treatment or to a clinical history of thrombotic complications. Platelet TX receptor studies did not show any significant changes in the binding characteristics of two different ligands. A platelet-selective regimen of aspirin therapy (50 mg/d for 7 to 14 days) was associated with greater than 80% suppression in metabolite excretion in nine patients. These results are consistent with abnormal stimuli operating in polycythemia vera to induce a selective enhancement in the platelet biosynthesis of TXA2 without changes in receptor binding. This in vivo abnormality in platelet biochemistry can be largely suppressed by low doses of aspirin.

BACKGROUND Increased platelet thromboxane (TX)A2 production has been described in type IIa hypercholesterolemia. To verify the relevance of these capacity-related measurements to the actual rate of TXA2 biosynthesis in vivo, we studied the urinary excretion of its major enzymatic metabolites in 46 patients with type IIa hypercholesterolemia and 20 age-matched controls. METHODS AND RESULTS Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were measured by previously validated radioimmunoassays. The excretion rate of 11-dehydro-TXB2 was significantly (p less than 0.001) higher in patients (68.7 +/- 35.1 ng/hr, mean +/- SD) than in controls (22.4 +/- 9.4 ng/hr), with metabolite excretion greater than 2 SD of the normal mean in 74% of the patients. Urinary 11-dehydro-TXB2 was significantly (p less than 0.01) correlated with the threshold aggregating concentration of collagen (r = -0.641) and arachidonate (r = -0.734) and with agonist-induced platelet TXB2 production in vitro (r = 0.647 and 0.748, respectively). Moreover, a statistically significant correlation (r = 0.673, p less than 0.001, n = 66) was found between 11-dehydro-TXB2 excretion and total plasma cholesterol. The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin (20 mg/day for 6 months) significantly reduced cholesterol levels by 22-28% and urinary 11-dehydro-TXB2 excretion by 32-42% in 10 patients. However, the reduction in the latter did not correlate with the reduction in the former and may have resulted from a nonspecific effect of simvastatin. Moreover, selective inhibition of platelet cyclooxygenase activity by low-dose aspirin (50 mg/day for 7 days) was associated with cumulative inhibition of 11-dehydro-TXB2 excretion by approximately 70% in six patients. CONCLUSIONS TXA2 biosynthesis is enhanced in the majority of patients with type IIa hypercholesterolemia; this is, at least in part, a consequence of abnormal cholesterol levels, as suggested by the correlation between the two. Low-dose aspirin can largely suppress increased metabolite excretion, thus suggesting that it reflects TXA2-dependent platelet activation in vivo.

The development of RIA techniques for the measurement of urinary eicosanoid metabolites has allowed characterization of their biosynthesis and metabolism in health and disease. PGI2, TXA2, and LTC4 share a very low rate of secretion into the human circulation and rapidly disappear from circulation because of extensive enzymatic degradation. Long-term changes as well as episodic increases in their biosynthesis can be detected in association with various disease states, as reflected by increased excretion of major enzymatic metabolites such as 2,3-dinor-6-keto-PGF1 alpha, 11-dehydro-TXB2, and LTE4. Application of the same investigative approach to other recently identified products of arachidonate metabolism (2) may provide further insight into the pathophysiologic significance of this complex mediator system.

We measured the excretion of a major urinary metabolite of leukotriene (LT) C4, i.e., LTE4, during the infusion of exogenous LTC4 to enable estimation of the rate of entry of endogenous LTC4 into the bloodstream. Four healthy volunteers received 2-h intravenous infusions of vehicle alone and LTC4 at 0.063, 0.32, 1.6, and 2.9 pmol.kg-1.min-1 in random order. Urinary LTE4 was measured before, during, and up to 24 h after the infusions. The fractional elimination of LTE4 was independent of the rate of LTC4 infusion and averaged 4.3 +/- 0.9%. Calculation of the mean rate of entry of LTC4 into the circulation was found to be 0.06 pmol.kg-1.min-1. In addition, we characterized further metabolism of [14C]LTC4. The two major urinary metabolites were the omega- and beta-oxidation products (16-COOH-LTE4 and 14-COOH-LTE3), which accounted for 6-8% of the total infused amount of [14C]LTC4. We conclude that 1) LTC4 is produced at a low rate under physiological circumstances and is rapidly converted in the vasculature to LTE4, 2) changes in the urinary excretion of the latter may reliably reflect short-term changes in the rate of secretion of LTC4, and 3) measurement of the omega- and beta-oxidation products may reflect chronic changes in cysteinyl leukotriene biosynthesis.

Sudden fissuring of an atherosclerotic plaque has been suggested as the primary trigger of transient spontaneous ischemia in both the coronary and cerebral circulation. Measurements of urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2, as well as results of Aspirin trials, have suggested that episodic platelet activation at the site of this acute vascular lesion is mediated, at least partly, by enhanced thromboxane (TX) A2 biosynthesis. Thus, episodic increases in metabolite excretion have been detected in unstable angina. Aspirin (75-325 mg/day) prevents about one third of all fatal and nonfatal thrombotic events in this setting. That a similar "dynamic" thrombotic process occurs during the early phase of acute myocardial infarction is suggested by thromboxane metabolite measurements and by the results of the ISIS-2 trial showing a similar impact of short-term Aspirin therapy to that seen in unstable angina. Percutaneous transluminal coronary angioplasty is associated with transiently enhanced TXA2 biosynthesis and Aspirin-suppressable periprocedural thrombotic complications. On the other hand, both non-insulin-dependent diabetes mellitus and type IIa hypercholesterolemia are associated with a relatively reproducible and persisting abnormality of TXA2-dependent platelet function. This association is likely to reflect a systemic rather than localized stimulus to platelet activation and a continuous rather than episodic alteration. Low-dose (50 mg/day) Aspirin can largely suppress thromboxane metabolite excretion in both diseases. Thus, low-dose Aspirin and/or selective prostaglandin H2/TXA2-receptor antagonists may be important tools to test the hypothesis that TXA2-dependent platelet activation represents an important transducer of the enhanced thrombotic risk associated with these metabolic abnormalities.

Episodic platelet activation has been shown to occur in unstable angina, and aspirin should have an important therapeutic role in the management of these patients. The response to aspirin alone or to aspirin in combination with vasodilators such as heparin and beta-blockers has been assessed in 41 patients with unstable angina. Therapy was added sequentially in the event of recurrence of transient myocardial ischemia. Patients were randomly assigned to two groups. Group 1 (21 patients) received an intravenous infusion of isosorbide dinitrate and oral diltiazem, and group 2 (20 patients) received intravenous aspirin (60 mg the first day and 20 mg on successive days). This dose of aspirin reduced serum thromboxane B2 from 160 +/- 88 ng/ml (mean +/- SD) to undetectable values (less than 6 ng/ml, p less than 0.01). If episodes of ischemic ST segment shift continued, the therapy of group 1 was added to that of group 2 or vice versa; if further ST segment changes were documented, intravenous heparin and oral beta-blockers were added; if episodes of myocardial ischemia persisted, urgent coronary arteriography and myocardial revascularization were performed. Nine patients in group 1 and six in group 2 (p = 0.8) had no further episodes of myocardial ischemia on their initial therapy; 12 additional patients had no further episodes when taking combined therapy of aspirin and vasodilators. Thus, the administration of aspirin alone was not superior to coronary dilators; 30% of all patients continued to have episodes of myocardial ischemia or had a myocardial infarction develop when heparin and beta-blockers were added. Myocardial infarction occurred in one patient on vasodilator therapy alone, in two on combined therapy, and in two on full therapy. These results suggest that in some patients, the stimulus to coronary thrombosis and vasoconstriction occasionally becomes so strong that it cannot be inhibited by certain antagonist drugs. The unstable tendency to continuation of ischemia or evolution to myocardial infarction is not related to the severity of the persisting stenosis. Those patients not promptly responding to combined therapy immediately from admission should have early coronary angiography and aggressive treatment.

The use of antiplatelet agents in chronic glomerular disease is reviewed, with emphasis on drugs affecting thromboxane A2 synthesis or action. The results of short-term and long-term studies in human as well as animal models suggest a role for enhanced intrarenal synthesis of thromboxane A2, though not clearly defining the potential platelet vs glomerular targets of drug action. The availability of novel inhibitors and antagonists may provide new therapeutic strategies against thromboxane-A2-dependent loss of renal function.