Hyperglycemia Induces Trained Immunity in Macrophages and Their Precursors and Promotes Atherosclerosis.

BACKGROUND: Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. METHODS: Bone marrow-derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr-/- mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. RESULTS: In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow-derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr-/- mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow-derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. CONCLUSIONS: Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

Differential Gene Expression in Macrophages From Human Atherosclerotic Plaques Shows Convergence on Pathways Implicated by Genome-Wide Association Study Risk Variants.

Objective- Plaque macrophages are intricately involved in atherogenesis and plaque destabilization. We sought to identify functional pathways in human plaque macrophages that are differentially regulated in respect of (1) plaque stability and (2) lipid content. We hypothesized that differentially regulated macrophage gene sets would relate to genome-wide association study variants associated with risk of acute complications of atherosclerosis. Approach and Results- Forty patients underwent carotid magnetic resonance imaging for lipid quantification before endarterectomy. Carotid plaque macrophages were procured by laser capture microdissection from (1) lipid core and (2) cap region, in 12 recently symptomatic and 12 asymptomatic carotid plaques. Applying gene set enrichment analysis, a number of gene sets were found to selectively upregulate in symptomatic plaque macrophages, which corresponded to 7 functional pathways: inflammation, lipid metabolism, hypoxic response, cell proliferation, apoptosis, antigen presentation, and cellular energetics. Predicted upstream regulators included IL-1ß, TNF-α, and NF-κB. In vivo lipid quantification by magnetic resonance imaging correlated most strongly with the upregulation of genes of the IFN/ STAT1 pathways. Cross-interrogation of gene set enrichment analysis and meta-analysis gene set enrichment of variant associations showed lipid metabolism pathways, driven by genes coding for APOE and ABCA1/G1 coincided with known risk-associated SNPs (single nucleotide polymorphisms) from genome-wide association studies. Conclusions- Macrophages from recently symptomatic carotid plaques show differential regulation of functional gene pathways. There were additional quantitative relationships between plaque lipid content and key gene sets. The data show a plausible mechanism by which known genome-wide association study risk variants for atherosclerotic complications could be linked to (1) a relevant cellular process, in (2) the key cell type of atherosclerosis, in (3) a human disease-relevant setting.

Acute myocardial infarction activates distinct inflammation and proliferation pathways in circulating monocytes, prior to recruitment, and identified through conserved transcriptional responses in mice and humans.

AIMS: Monocytes play critical roles in tissue injury and repair following acute myocardial infarction (AMI). Specifically targeting inflammatory monocytes in experimental models leads to reduced infarct size and improved healing. However, data from humans are sparse, and it remains unclear whether monocytes play an equally important role in humans. The aim of this study was to investigate whether the monocyte response following AMI is conserved between humans and mice and interrogate patterns of gene expression to identify regulated functions. METHODS AND RESULTS: Thirty patients (AMI) and 24 control patients (stable coronary atherosclerosis) were enrolled. Female C57BL/6J mice (n = 6/group) underwent AMI by surgical coronary ligation. Myocardial injury was quantified by magnetic resonance imaging (human) and echocardiography (mice). Peripheral monocytes were isolated at presentation and at 48 h. RNA from separated monocytes was hybridized to Illumina beadchips. Acute myocardial infarction resulted in a significant peripheral monocytosis in both species that positively correlated with the extent of myocardial injury. Analysis of the monocyte transcriptome following AMI demonstrated significant conservation and identified inflammation and mitosis as central processes to this response. These findings were validated in both species. CONCLUSIONS: Our findings show that the monocyte transcriptome is conserved between mice and humans following AMI. Patterns of gene expression associated with inflammation and proliferation appear to be switched on prior to their infiltration of injured myocardium suggesting that the specific targeting of inflammatory and proliferative processes in these immune cells in humans are possible therapeutic strategies. Importantly, they could be effective in the hours after AMI.

Black-blood multicontrast imaging of carotid arteries with DANTE-prepared 2D and 3D MR imaging.

PURPOSE: To prospectively compare the black-blood ( BB black blood ) imaging efficiency of a delay alternating with nutation for tailored excitation ( DANTE delay alternating with nutation for tailored excitation ) preparation module with conventional double inversion-recovery ( DIR double inversion recovery ) and motion-sensitive driven equilibrium ( MSDE motion-sensitive driven equilibrium ) preparation modules and to introduce a new three-dimensional ( 3D three-dimensional ) T1-weighted magnetic resonance (MR) imaging sequence. MATERIALS AND METHODS: Carotid artery wall imaging was performed in 10 healthy volunteers and 15 patients in accordance with an institutional review board-approved protocol. Two-dimensional ( 2D two-dimensional ) turbo spin-echo ( TSE turbo spin echo ) and 3D three-dimensional fast low-angle shot ( FLASH fast low-angle shot ) sequences served as readout modules. DANTE delay alternating with nutation for tailored excitation -prepared T1-, T2-, and proton density-weighted 2D two-dimensional TSE turbo spin echo images, as well as T1-weighted 3D three-dimensional DANTE delay alternating with nutation for tailored excitation -prepared FLASH fast low-angle shot (hereafter, 3D three-dimensional DASH DANTE-prepared FLASH ) images, were acquired in the region of the carotid artery bifurcation. For comparison, 2D two-dimensional DIR double inversion recovery -prepared, 2D two-dimensional MSDE motion-sensitive driven equilibrium -prepared multicontrast TSE turbo spin echo , and 3D three-dimensional MSDE motion-sensitive driven equilibrium -prepared FLASH fast low-angle shot (hereafter, 3D three-dimensional MERGE MSDE-prepared FLASH ) MR images were also acquired. The effective contrast-to-noise ratio ( CNReff effective contrast-to-noise ratio ) per unit time was calculated for all sequences. Paired t tests were performed to test within-group differences in vessel wall CNReff effective contrast-to-noise ratio . RESULTS: The CNReff effective contrast-to-noise ratio of DANTE delay alternating with nutation for tailored excitation -prepared T1-, T2-, and proton density-weighted sequences was 27.3, 14.7, and 25.7 mm(-1)min(-1/2), respectively; this represented an improvement of approximately 25%-100% (P < .05) when compared with the CNReff effective contrast-to-noise ratio attained with existing methods. The 3D three-dimensional DASH DANTE-prepared FLASH technique proved to be a fast (<2 seconds per section) and high-spatial-resolution (0.6 mm isotropic) BB black blood technique with higher (75%-100% improvement, P < .001) signal-to-noise ratio efficiency than the 3D three-dimensional MERGE MSDE-prepared FLASH technique. CONCLUSION: The DANTE delay alternating with nutation for tailored excitation -prepared multicontrast 2D two-dimensional BB black blood technique is a promising new tool for MR imaging of carotid artery walls. Additionally, the 3D three-dimensional DASH DANTE-prepared FLASH sequence enables 3D three-dimensional high-spatial-resolution fast T1-weighted imaging of carotid artery walls. ©RSNA, 2014 Online supplemental material is available for this article .

GPR109A and vascular inflammation.

GPR109A has generated expanding interest since its discovery as the receptor for niacin a decade ago, along with deorphanisation as the receptor for endogenous ligand 3-hydroxy-butyrate shortly after. This interest is generated especially because of the continuing exploration of niacin's "pleiotropic" mechanisms of action and its potential in the "cross-talk" between metabolic and inflammatory pathways. As GPR109A's primary pharmacological ligand in clinical use, niacin has been used for over 50 years in the treatment of cardiovascular disease, mainly due to its favourable effects on plasma lipoproteins. However, it has become apparent that niacin also possesses lipoprotein-independent effects that influence inflammatory pathways mediated through GPR109A. In addition to its G-protein-mediated effects, recent evidence has emerged to support alternative GPR109A signalling via adaptive protein ß-arrestins. In this article, we consider the role of GPR109A and its downstream effects in the context of atherosclerosis and vascular inflammation, along with insights into strategy for future drug development.

In-vivo quantitative T2 mapping of carotid arteries in atherosclerotic patients: segmentation and T2 measurement of plaque components.

BACKGROUND: Atherosclerotic plaques in carotid arteries can be characterized in-vivo by multicontrast cardiovascular magnetic resonance (CMR), which has been thoroughly validated with histology. However, the non-quantitative nature of multicontrast CMR and the need for extensive post-acquisition interpretation limit the widespread clinical application of in-vivo CMR plaque characterization. Quantitative T2 mapping is a promising alternative since it can provide absolute physical measurements of plaque components that can be standardized among different CMR systems and widely adopted in multi-centre studies. The purpose of this study was to investigate the use of in-vivo T2 mapping for atherosclerotic plaque characterization by performing American Heart Association (AHA) plaque type classification, segmenting carotid T2 maps and measuring in-vivo T2 values of plaque components. METHODS: The carotid arteries of 15 atherosclerotic patients (11 males, 71 ± 10 years) were imaged at 3 T using the conventional multicontrast protocol and Multiple-Spin-Echo (Multi-SE). T2 maps of carotid arteries were generated by mono-exponential fitting to the series of images acquired by Multi-SE using nonlinear least-squares regression. Two reviewers independently classified carotid plaque types following the CMR-modified AHA scheme, one using multicontrast CMR and the other using T2 maps and time-of-flight (TOF) angiography. A semi-automated method based on Bayes classifiers segmented the T2 maps of carotid arteries into 4 classes: calcification, lipid-rich necrotic core (LRNC), fibrous tissue and recent IPH. Mean ± SD of the T2 values of voxels classified as LRNC, fibrous tissue and recent IPH were calculated. RESULTS: In 37 images of carotid arteries from 15 patients, AHA plaque type classified by multicontrast CMR and by T2 maps (+ TOF) showed good agreement (76% of matching classifications and Cohen's κ = 0.68). The T2 maps of 14 normal arteries were used to measure T2 of tunica intima and media (T2 = 54 ± 13 ms). From 11865 voxels in the T2 maps of 15 arteries with advanced atherosclerosis, 2394 voxels were classified by the segmentation algorithm as LRNC (T2 = 37 ± 5 ms) and 7511 voxels as fibrous tissue (T2 = 56 ± 9 ms); 192 voxels were identified as calcification and one recent IPH (236 voxels, T2 = 107 ± 25 ms) was detected on T2 maps and confirmed by multicontrast CMR. CONCLUSIONS: This carotid CMR study shows the potential of in-vivo T2 mapping for atherosclerotic plaque characterization. Agreement between AHA plaque types classified by T2 maps (+TOF) and by conventional multicontrast CMR was good, and T2 measured in-vivo in LRNC, fibrous tissue and recent IPH demonstrated the ability to discriminate plaque components on T2 maps.

Eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome) masquerading as acute ST-elevation myocardial infarction with complete resolution after immunosuppressive therapy: a case report.

BACKGROUND: Eosinophilic granulomatosis with polyangiitis (EGPA, formerly known as Churg-Strauss syndrome) is a rare autoimmune condition characterized by inflammation of small- and medium-sized blood vessels, which usually presents with systemic vasculitis preceded by airway allergic hypersensitivity. CASE SUMMARY: Here, we report a highly unusual case of acute ST-elevation myocardial infarction in a young and fit man with no cardiovascular risk factors. His emergency coronary angiography revealed disproportionately severe widespread coronary artery disease. We describe the diagnostic challenges with emphasis on meticulous history-taking (deep hyponasal voice, anosmia, and childhood asthma), supported by timely blood markers (peripheral eosinophilia and raised CRP), and multi-modal imaging (severe paranasal sinusitis on cranial magnetic resonance imaging and multiple lung infiltrates with small patches of ground-glass appearance on thoracic computed tomography), to reach a diagnosis of EGPA coronary vasculitis with particular reference to the American College of Rheumatology EGPA classification. Importantly, with prompt immunosuppression, his coronary lesions resolved completely without the need of any surgical or percutaneous revascularisation. He remained well and asymptomatic on maintenance immunosuppressants at 1 year follow-up. DISCUSSION: This case highlighted the rare but recognized involvement of the coronary arteries in systemic EGPA vasculitis, which can sometimes mimic atherosclerotic coronary disease and acute coronary syndrome.

T2 mapping MRI technique quantifies carotid plaque lipid, and its depletion after statin initiation, following acute myocardial infarction.

BACKGROUND & AIMS: A recently-validated, highly-sensitive T2 mapping magnetic resonance (MRI) technique accurately quantifies carotid plaque lipid. The aims of this study were to determine: (i) the extent of carotid plaque lipid in patients with acute coronary syndromes (ACS); (ii) the effects of initiation of high-intensity statin on plaque lipid content and (iii) whether plaque lipid content is related to standard or 'functional' blood lipid measurements. METHODS: Statin naïve subjects presenting with ACS underwent carotid artery MRI at 3 T scanner to quantify plaque lipid. Patients were subsequently commenced on high dose statin as part of clinical care and underwent a second MRI after three months. Plaque composition was measured using objective semi-automated techniques. RESULTS: 23 out of 24 patients had measurable lipid. Three months after statin initiation there was a significant reduction in carotid lipid percentage [from 10.3% (7.2-14.2) to 7.4% (5.4-10.0), p = 0.002] and a significant increase in fibrous percentage [from 83.3% ±â€¯6.6-85.5% ±â€¯4.8, p = 0.039]. None of the studied functional blood biomarkers were related to either baseline carotid plaque lipid content or its propensity to change with statin treatment. CONCLUSIONS: T2-mapping demonstrated depleted carotid plaque lipid following the initiation of high-intensity statin treatment. Standard or 'functional' blood biomarkers were dissociated from plaque lipid content or changes with treatment. These findings further reinforce the importance of disease characterisation over risk factor assessment. Subject to clinical trial findings, quantification of plaque lipid may provide the basis for an approach to identify patients suitable for intensive lipid reduction regimes.

Plaque imaging to refine indications for emerging lipid-lowering drugs.

Statins have been effective in reducing adverse cardiovascular events. Their benefits have been proportional to the level of plasma LDL-cholesterol reduction and seem to extend to patients with 'normal' levels of cholesterol at outset. Statins are also inexpensive and have a favourable side-effect profile. As a result, they are used widely (almost indiscriminately) in patients with atherosclerotic vascular disease, and in those at risk of disease. Next generation lipid-modifying drugs seem unlikely to offer the same simplicity of application. The recent trials of new classes of lipid modifying drugs underline the need for a risk stratification tool which is not based on patients' category of diagnosis (for example, post-myocardial infarction) but based on the characterization of disease in that individual patient. Mechanistic staging, a process that matches the target of the drug action with an identifiable disease characteristic, may offer an opportunity to achieve more precise intervention. The upshots of this targeted approach will be greater efficacy, requiring smaller clinical trials to demonstrate effectiveness; a reduced number needed to treat to yield benefits and more cost-effective prescribing. This will be important, as purchasers require ever more rigorous demonstration of both efficacy and cost-effectiveness. In this context, we will discuss available pharmacological strategies of lipid reduction in anti-atherosclerotic treatment and how plaque imaging techniques may provide an ideal method in stratifying patients for new lipid-modifying drugs.

Inflammatory processes in cardiovascular disease: a route to targeted therapies.

Inflammatory processes are firmly established as central to the development and complications of cardiovascular diseases. Elevated levels of inflammatory markers have been shown to be predictive of future cardiovascular events. The specific targeting of these processes in experimental models has been shown to attenuate myocardial and arterial injury, reduce disease progression, and promote healing. However, the translation of these observations and the demonstration of clear efficacy in clinical practice have been disappointing. A major limitation might be that tools currently used to measure 'inflammation' are insufficiently precise and do not provide information about disease site and activity, or discriminate between functionally important activation pathways. The challenge, therefore, is to make measures of inflammation that are more meaningful, and which can guide specific targeted therapies. In this Review, we consider the roles of inflammatory processes in the related pathologies of atherosclerosis and acute myocardial infarction, by providing an evaluation of the known and emerging inflammatory pathways. We highlight contemporary techniques to characterize and quantify inflammation, and consider how they might be used to guide specific treatments. Finally, we discuss emerging opportunities in the field, including their current limitations and challenges that are the focus of ongoing study.

Quantification of carotid plaque lipid content with magnetic resonance T2 mapping in patients undergoing carotid endarterectomy.

BACKGROUND AND PURPOSE: Techniques to stratify subgroups of patients with asymptomatic carotid artery disease are urgently needed to guide decisions on optimal treatment. Reliance on estimates of % luminal stenosis has not been effective, perhaps because that approach entirely disregards potentially important information on the pathological process in the wall of the artery. METHODS: Since plaque lipid is a key determinant of plaque behaviour we used a newly validated, high-sensitivity T2-mapping MR technique for a systematic survey of the quantity and distribution of plaque lipid in patients undergoing endarterectomy. Lipid percentage was quantified in 50 carotid endarterectomy patients. Lipid distribution was tested, using two imaging indices (contribution of the largest lipid deposit towards total lipid (LLD %) and a newly-developed LAI 'lipid aggregation index'). RESULTS: The bifurcation contained maximal lipid volume. Lipid percentage was higher in symptomatic vs. asymptomatic patients with degree of stenosis (DS ≥ 50%) and in the total cohort (P = 0.013 and P = 0.005, respectively). Both LLD % and LAI was higher in symptomatic patients (P = 0.028 and P = 0.018, respectively), suggesting that for a given plaque lipid volume, coalesced deposits were more likely to be associated with symptomatic events. There was no correlation between plaque volume or lipid content and degree of luminal stenosis measured on ultrasound duplex (r = -0.09, P = 0.53 and r = -0.05, P = 0.75), respectively. However, there was a strong correlation in lipid between left and right carotid arteries (r = 0.5, P <0.0001, respectively). CONCLUSIONS: Plaque lipid content and distribution is associated with symptomatic status of the carotid plaque. Importantly, plaque lipid content was not related to the degree of luminal stenosis assessed by ultrasound. Determination of plaque lipid content may prove useful for stratification of asymptomatic patients, including selection of optimal invasive treatments.

Quantification of Lipid-Rich Core in Carotid Atherosclerosis Using Magnetic Resonance T2 Mapping: Relation to Clinical Presentation.

OBJECTIVES: The aim of this study was to: 1) provide tissue validation of quantitative T2 mapping to measure plaque lipid content; and 2) investigate whether this technique could discern differences in plaque characteristics between symptom-related and non-symptom-related carotid plaques. BACKGROUND: Noninvasive plaque lipid quantification is appealing both for stratification in treatment selection and as a possible predictor of future plaque rupture. However, current cardiovascular magnetic resonance (CMR) methods are insensitive, require a coalesced mass of lipid core, and rely on multicontrast acquisition with contrast media and extensive post-processing. METHODS: Patients scheduled for carotid endarterectomy were recruited for 3-T carotid CMR before surgery. Lipid area was derived from segmented T2 maps and compared directly to plaque lipid defined by histology. RESULTS: Lipid area (%) on T2 mapping and histology showed excellent correlation, both by individual slices (R = 0.85, p < 0.001) and plaque average (R = 0.83, p < 0.001). Lipid area (%) on T2 maps was significantly higher in symptomatic compared with asymptomatic plaques (31.5 ± 3.7% vs. 15.8 ± 3.1%; p = 0.005) despite similar degrees of carotid stenosis and only modest difference in plaque volume (128.0 ± 6.0 mm3 symptomatic vs. 105.6 ± 9.4 mm3 asymptomatic; p = 0.04). Receiver-operating characteristic analysis showed that T2 mapping has a good ability to discriminate between symptomatic and asymptomatic plaques with 67% sensitivity and 91% specificity (area under the curve: 0.79; p = 0.012). CONCLUSIONS: CMR T2 mapping distinguishes different plaque components and accurately quantifies plaque lipid content noninvasively. Compared with asymptomatic plaques, greater lipid content was found in symptomatic plaques despite similar degree of luminal stenosis and only modest difference in plaque volumes. This new technique may find a role in determining optimum treatment (e.g., providing an indication for intensive lipid lowering or by informing decisions of stents vs. surgery).

Loss of heterozygosity and mutations are the major mechanisms of RB1 gene inactivation in Chinese with sporadic retinoblastoma.

We investigated sequence alternation, promoter methylation, and loss of heterozygosity (LOH) of the RB1 gene as possible mechanisms of its inactivation in retinoblastoma. In 42 Chinese patients with sporadic retinoblastoma, the promoter and entire coding region of RB1 were examined for sequence changes. Status of methylation of the CpG-rich island at the 5'end was determined by methylation specific PCR assay. We detected 15 RB1 mutations in 38% (16/42) of the retinoblastoma patients, among them 19% (8/42) were germ-line mutations. A total of nine novel mutations were identified: E54X, S114X, I126S, g73779insG, D718N, IVS2+1G>C, IVS14+1G>C, IVS21+1G>C, and a complex alteration g78177G>T/g78176insTT leading to 543X. Most of them are likely to affect the RB1large pocket domain through the production of truncated gene products. None of the DNA samples showed methylation at the RB1promoter. In 15 cases where both normal and cancerous retinoblastoma tissue specimens were available, allelic loss according to microsatellite markers within or distal to the RB1 locus was analyzed and immunohistological staining for RB1 expression performed. Among them, frequency of LOH at 13q14 was found to be high at 60% (9/15) with no segregation with unilateral tumors. All these nine tumors did not express RB1 protein, showing an association of LOH at the RB1 locus with its loss of expression in retinoblastoma. Our results indicate that the RB1 gene in sporadic retinoblastoma is commonly inactivated because of loss-of-function mutations and loss of heterozygosity but not by the epigenetic phenomenon of promoter hypermethylation.

Cardiometabolic interventions - focus on transcriptional regulators.

Cardiovascular disease (CVD) remains the largest healthcare burden in the Western world; and the increasing prevalence of type II diabetes mellitus, at least partially driven by a trend in lifestyle changes associated with global economic development, is likely to fuel this CVD burden worldwide. Over the past two decades, there has been an increased awareness of the convergence of risk factors contributing to both cardiovascular disease and diabetes leading to the concept of the metabolic syndrome, and the realisation of the opportunity to intervene at this intersection to simultaneously target CVD and metabolic dysfunction. This brings together the fields of cardiovascular medicine, diabetology, and increasingly clinical immunology for a unified and concerted effort to reduce risk for both conditions simultaneously. The discovery of the targeted pathways of drugs already in clinical use such as fibrates and thiazolidinediones (TZD) has led to accelerated basic and clinical research into selective and dual PPAR-α and PPAR-γ agonists, which can theoretically target glucose, lipid and lipoprotein metabolism, as well as potentially exerting inhibitoryeffects in vascular inflammation, all of which might be predicted to reduce atherosclerosis. In this article, we will discuss the basic science as well as recent clinical development in the pursuit of optimal cardiometabolic intervention along with insight into strategies for future drug development.

Nicotinic acid receptor GPR109A is down-regulated in human macrophage-derived foam cells.

Nicotinic acid (NA) regresses atherosclerosis in human imaging studies and reduces atherosclerosis in mice, mediated by myeloid cells, independent of lipoproteins. Since GPR109A is expressed by human monocytes, we hypothesized that NA may drive cholesterol efflux from foam cells. In THP-1 cells NA suppressed LPS-induced mRNA transcription of MCP-1 by 76.6±12.2% (P<0.01) and TNFα by 56.1±11.5% (P<0.01), yet restored LPS-induced suppression of PPARγ transcription by 536.5±46.4% (P<0.001) and its downstream effector CD36 by 116.8±19.8% (P<0.01). Whilst direct PPARγ-agonism promoted cholesterol efflux from THP-1 derived foam cells by 37.7±3.1% (P<0.01) and stimulated transcription of LXRα by 87.9±9.5% (P<0.001) and ABCG1 by 101.2±15.5% (P<0.01), NA showed no effect in foam cells on either cholesterol efflux or key RCT genes transcription. Upon foam cell induction, NA lost its effect on PPARγ and cAMP pathways, since its receptor, GPR109A, was down-regulated by foam cell transformation. This observation was confirmed in explanted human carotid plaques. In conclusion, despite NA's anti-inflammatory effect on human macrophages, it has no effect on foam cells in reverse cholesterol transport; due to GPR109A down-regulation.

Fatal Israeli spotted fever in a UK traveler to South Portugal.

A 63-year-old previously healthy woman developed a severe systemic infection 5 days after returning from a holiday to Southern Portugal. She subsequently died, and polymerase chain reaction of a blood sample was positive for Rickettsia conorii ssp israeliensis. The prevalence of severe forms of this illness in the Mediterranean Basin is discussed.