Genetic variants in PPARGC1B and CNTN4 are associated with thromboxane A2 formation and with cardiovascular event free survival in the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT).

BACKGROUND AND AIMS: Elevated urinary 11-dehydro thromboxane B2 (TxB2), a measure of thromboxane A2 formation in vivo, predicts future atherothrombotic events. To further understand this relationship, the genetic determinants of 11-dehydro TxB2 and their associations with cardiovascular morbidity were investigated in this study. METHODS: Genome-wide and targeted genetic association studies of urinary 11-dehydro TxB2 were conducted in 806 Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) participants. RESULTS: The strongest associations were in PPARGC1B (rs4235745, rs32582, rs10515638) and CNTN4 (rs10510230, rs4684343), these 5 single nucleotide polymorphisms (SNPs) were independently associated with 11-dehydro TxB2 formation. Haplotypes of 11-dehydro TxB2 increasing alleles for both PPARGC1B and CNTN4 were significantly associated with 11-dehydro TxB2, explaining 5.2% and 4.5% of the variation in the whole cohort, and 8.8% and 7.9% in participants not taking aspirin, respectively. In a second ASCOT population (n = 6199), addition of these 5 SNPs significantly improved the covariate-only Cox proportional hazards model for cardiovascular events (chisq = 14.7, p=0.01). Two of the risk alleles associated with increased urinary 11-dehydro TxB2 were individually associated with greater incidences of cardiovascular events - rs10515638 (HR = 1.31, p=0.01) and rs10510230 (HR = 1.25, p=0.007); effect sizes were larger in those not taking aspirin. CONCLUSIONS: PPARGC1B and CNTN4 genotypes are associated with elevated thromboxane A2 formation and with an excess of cardiovascular events. Aspirin appears to blunt these associations. If specific protection of PPARGC1B and CNTN4 variant carriers by aspirin is confirmed by additional studies, PPARGC1B and CNTN4 genotyping could potentially assist in clinical decision making regarding the use of aspirin in primary prevention

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Simultaneous double 1,3-dipolar cycloaddition reactions involving bisnitrones or bisdipolarophiles. 1H NMR investigation of the conformational preferences of N-methyl- and N-phenylisoxazolidines

Bisnitrones 1a and 1b reacted with N-methylmaleimide giving bisisoxazolidines. Diastereospecific reaction of the phenyl substituted dipole 1a gave 3a whilst the N-methyl dipole 1b furnished diastereomeric adducts 3b, 4 and 7 classified as trans,trans, 3b, cis,cis, 4 and cis,trans adducts 7 (major) according to the relative orientation of the 3-H and 4-H protons on each isoxazolidine ring. Similar behaviour was observed in reaction of mono dipoles N-benzylideneaniline N-oxide and N-benzylidenemethylamine N-oxide with phenylenedimaleimide 2. The N-phenyl dipole reacted highly selectively furnishing the trans,trans adduct 8a whilst the N-methyl dipole again gave trans,trans8b, cis,cis10 and cis,trans adducts 9 (major). Some of the N-methyl substituted isoxazolidines (3b, 7, 8b, 9b) displayed a number of very broad signals in their rt 1H NMR spectra which sharpened (and duplicated) on cooling. By analogy to the corresponding 1H NMR data of the “hemi-adducts” 5 and 6, and with reference to crystal structure data for 5c [Fig. 1], it was shown that for this group of adducts the 3-H and 4-H protons are trans orientated. The isoxazolidine ring in these adducts equilibrates between the o- and i-conformations [Fig. 2] and at −40 °C each conformer can be clearly identified in the 1H NMR spectrum. No line broadening was observed in the 1H NMR spectra of any of the N-phenyl substituted adducts. The conformational freedom of the adducts is thus dictated by the size of the N-isoxazolidine substituent and the relative orientation of the 3-H and 4-H protons. All new cycloadducts reported, 3–10, are prepared as racemic mixtures and stereochemical information portrayed in the drawings implies relative and not absolute relations

Simultaneous double 1,3-dipolar cycloaddition reactions involving bisnitrones or bisdipolarophiles. 1h nmr investigation of the conformational preferences of n-methyl- and n-phenyl-isoxazolidines

Binitrones 1a and 1b reacted with N-methylmaleimide giving bisisoxazolidines. Diastereospecific reaction of the phenyl substituted dipole 1a gave 3a whilst the N-methyl dipole 1b furnished diastereomeric adducts 3b, 4 and 7 classified as trans,trans, 3b, cis,cis, 4 and cis,trans adducts 7 (major) according to the relative orientation of the 3-H and 4-H protons on each isoxazolidine ring. Similar behaviour was observed in reaction of mono dipoles N-benzylideneaniline N-oxide and N-benzylidenemethylamine N-oxide with phenylenedimaleimide 2. The N-phenyl dipole reacted highly selectively furnishing the trans,trans adduct 8a whilst the N-methyl dipole again gave trans,trans 8b, cis,cis 10 and cis,trans adducts 9 (major). Some of the N-methyl substituted isoxazolidines (3b, 7, 8b, 9b) displayed a number of very broad signals in their rt H-1 NMR spectra which sharpened (and duplicated) on cooling. By analogy to the corresponding H-1 NMR data of the "hemi-adducts" 5 and 6, and with reference to crystal structure data for 5c [Fig. 1], it was shown that for this group of adducts the 3-H and 4-H protons are trans orientated. The isoxazolidine ring in these adducts equilibrates between the o- and i-conformations [Fig. 2] and at -40 degreesC each conformer can be clearly identified in the H-1 NMR spectrum. No line broadening was observed in the H-1 NMR spectra of any of the N-phenyl substituted adducts. The conformational freedom of the adducts is thus dictated by the size of the N-isoxazolidine substituent and the relative orientation of the 3-H and 4-H protons. All new cycloadducts reported, 3-10, are prepared as racemic mixtures and stereochemical information portrayed in the drawings implies relative and not absolute relations

Building Capability and Capacity: The Establishment of an Oncology Unit in the Solomon Islands

PURPOSETo describe the establishment of an oncology unit at the National Referral Hospital (NRH) in the Solomon Islands, a low-income nation in the South Pacific.METHODSA scoping visit was carried out in 2016 to assist in the development of coordinated cancer services and to establish a medical oncology unit at the NRH at the request of the Medical Superintendent. This was followed by an observership visit to Canberra by an NRH doctor training in oncology in 2017. After a request from the Solomon Islands Ministry of Health, the Australian Government Department of Foreign Affairs and Trade (DFAT) arranged an in-country multidisciplinary mission under the Royal Australasian College of Surgeons/Royal Australasian College of Physicians Pacific Islands Program to help in the commissioning of the NRH Medical Oncology Unit in September 2018. Staff training and education sessions were held. The team, with the assistance of an Australian Volunteers International Pharmacist, has helped the NRH staff to develop localized Solomon Islands Oncology Guidelines. Donated equipment and supplies have helped with the initial establishment of the service. A second DFAT Oncology mission visit was made in 2019 followed by two NRH oncology nurses visiting Canberra on observership later that year and support of the Solomon's doctor to pursue postgraduate education in cancer sciences. Ongoing mentorship and support has been maintained.RESULTSThe island nation now has a sustainable oncology unit delivering chemotherapy treatments and management of patients with cancer.CONCLUSIONA collaborative multidisciplinary team approach by professionals from the high-income country working with colleagues from the low-income nation with coordination of different stakeholders was the key to this successful initiative in improving cancer care

Site selectivity in the addition of ketoximes to activated allenes and alkynes; N- versus O-alkylation

Reaction of ketoximes with methyl propiolate afforded geometrical isomers of the methyl 3-(hydroxyimino)propanoates 4 and of the O-vinyl oximes 5 as well as the 2-isoxazoline 6. With dimethyl penta-2,3-diendioate 8c reaction progressed via an O-alkylation to give the O-oxime ethers 9, only in the case of cyclopentanone oxime was the spirocyclic dihydroazepinol 11 also obtained, its identity has been confirmed by an X-ray structure determination