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Objectives— Apolipoprotein E (apoE) exerts antiatherogenic effects but precise mechanisms remain unclear. We here investigated the effect of apoE on intracellular signaling by interleukin-1β (IL-1β), a proinflammatory cytokine present in atherosclerotic lesions. Methods and Results— IL-1β-induced expression and activation of inducible nitric oxide synthase and cyclooxygenase-2 were inhibited by apoE in vascular smooth muscle cells (VSMCs). These inhibitory effects were linked to the suppression of both NF-κB and activating protein-1 (AP-1) transactivation, suggesting that the interruption of IL-1β signaling occurs upstream of transcription factors. Studies in VSMCs overexpressing IL-1β signaling intermediates revealed that NF-κB transactivation was inhibited by apoE in MyD88- and IRAK1- but not in TRAF6-transfected cells. Furthermore, apoE prevented IRAK1 phosphorylation and IRAK1-TRAF6 but not MyD88-IRAK1 complex formation. Inhibitory effects of apoE on IL-1β signaling were abolished after silencing LDL receptor-related protein-1 (LRP1) expression with siRNA. In addition, inhibitors of adenylyl cyclase and protein kinase A (PKA) restored IL-1β signaling in apoE-treated VSMCs, whereas apoE stimulated PKA activity. ApoE inhibited VSMC activation in response to IL-18 but not to tumor necrosis factor-α or polyinosinic:polycytidylic acid. Conclusion— ApoE targets IRAK-1 activation and thereby interrupts IL-1β and IL-18 signaling in VSMCs. This antiinflammatory effect represents a novel antiatherogenic activity of apoE.

Objective The SAH gene locus has recently been proposed to be involved in obesity-related hypertension in Japanese individuals. Methods To replicate independently the initial findings in another ethnic group, we scanned the entire SAH gene in 190 Caucasian chromosomes. A total of 651 patients with essential hypertension and 776 controls (PEGASE Study) were genotyped for all identified variants using allele-specific oligonucleotides, and single nucleotide polymorphism as well as haplotype analyses were carried out. We also performed transient transfection experiments, northern and western blots, immunoprecipitation, and acyl-coenzyme A synthetase activity assays. Results We identified five polymorphisms in the promoter region (C−1808T, G−1606A, −962ins/del, G−451A, T−67C), two in introns 5 and 7 (T+9/In5C, A+20/In7T), and one missense variant (K359N). Carriage of the −1606A allele was significantly associated with hypertension [odds ratio (OR) 1.28, P = 0.049] as was 359N (OR 1.35, P = 0.048) compared with non-carriers. Conversely, for −962del, the OR for hypertension was 0.80 (P = 0.042). The SAH alleles −1606A and 359N, but not −962ins/del, displayed a raising effect on body mass index (BMI; P = 0.004 and P = 0.030, respectively) in hypertensive as well as in control individuals. After adjustment for BMI in hypertensive individuals, only the OR associated with −962ins/del remained significant (OR 0.77, P = 0.028). Functional analyses in BHK did not reveal differences for SAH 359N or 359K-containing constructs, formally excluding K359N as the functional variant. Conclusion We confirm recent evidence that the SAH locus is associated with obesity-related hypertension, in which pathophysiological context SAH variants affecting blood pressure remain, however, to be shown.

Because of the shortage of agalsidase-β supply between 2009 and 2012, patients with Fabry disease either were treated with reduced doses or were switched to agalsidase-α. In this observational study, we assessed end organ damage and clinical symptoms with special focus on renal outcome after 2 years of dose-reduction and/or switch to agalsidase-α. A total of 89 adult patients with Fabry disease who had received agalsidase-β (1.0 mg/kg body wt) for >1 year were nonrandomly assigned to continue this treatment regimen (regular-dose group, n=24), to receive a reduced dose of 0.3-0.5 mg/kg and a subsequent switch to 0.2 mg/kg agalsidase-α (dose-reduction-switch group, n=28), or to directly switch to 0.2 mg/kg agalsidase-α (switch group, n=37) and were followed-up for 2 years. We assessed clinical events (death, myocardial infarction, severe arrhythmia, stroke, progression to ESRD), changes in cardiac and renal function, Fabry-related symptoms (pain, hypohidrosis, diarrhea), and disease severity scores. Determination of renal function by creatinine and cystatin C-based eGFR revealed decreasing eGFRs in the dose-reduction-switch group and the switch group. The Mainz Severity Score Index increased significantly in these two groups (P=0.02 and P

Fabry disease (FD) is a progressive multisystemic disorder, treatable with recombinant enzyme replacement therapy (agalsidase). However, recent studies suggest an endogenous inhibition of agalsidase in patients with FD, as reported for other lysosomal storage diseases. To assess the clinical consequences of serum-mediated agalsidase inhibition in affected patients, we determined the agalsidase inhibition status of 168 patients (68 male) with FD and compared outcomes of inhibition-positive patients with those of inhibition-negative patients. The assessment included clinical events during time on agalsidase, determination of renal and cardiac function, and evaluation of FD-related symptoms. The frequency of serum-mediated agalsidase inhibition was 40% in agalsidase-treated males. Inhibition did not depend on the compound initially used (agalsidase-α or -β). Agalsidase inhibition was associated with higher lyso-globotriaosylceramide levels and worse disease severity scores in patients. Compared with agalsidase inhibition-negative men, agalsidase inhibition-positive men showed greater left ventricular mass (P=0.02) and substantially lower renal function (difference in eGFR of about -30 ml/min per 1.73 m(2); P=0.04), which was confirmed by a longitudinal 5-year retrospective analysis. Additionally, affected patients presented more often with FD-typical symptoms, such as diarrhea, fatigue, and neuropathic pain, among others. Therefore, patients with poor clinical outcome on agalsidase should be tested for agalsidase inhibition. Future studies are warranted to determine if affected patients with FD benefit from acute reduction of anti-agalsidase antibodies or long-term immune modulation therapies to suppress agalsidase inhibition and to identify mechanisms that minimize antibody generation against agalsidase.

Hypertension is a chronic age-related disorder, affecting nearly 20% of all adult Europeans. This disease entails debilitating cardiovascular complications and is the leading cause for drug prescriptions in Europeans older then 50 years. Intensive research over the past two decades failed so far to identify common genetic polymorphisms with major impact on blood pressure or associated cardiovascular phenotypes, suggesting that multiple genes each with a minor impact, along with gene-gene and gene-environment interactions play a role. The European Project on Genes in Hypertension (EPOGH) is a large-scale family-based study, in which participants from 7 different populations were phenotyped and genotyped according to standardised procedures. This review article summarizes the initial 5-year findings and puts these observations into perspective against other published studies. EPOGH demonstrated that phenotype-genotype relations strongly depend on host factors, such as gender and lifestyle, in particular salt intake as reflected by the 24-hour urinary excretion of sodium. EPOGH therefore highlights the concept that phenotype-genotype relations can only be studied within a defined ecogenetic context. © 2006 Bentham Science Publishers Ltd. ispartof: Current Hypertension Reviews vol:2 issue:4 pages:275-281 status: published

Background and Purpose— Carotid intima-media thickness (IMT) reflects generalized atherosclerosis and is predictive of future vascular events. Evidence exists that carotid IMT is heritable, and genetic studies can provide clues in the pathogenesis of atherosclerosis. Methods— We recruited 470 white ischemic stroke patients, measured common carotid artery (CCA) IMT, and analyzed 54 polymorphisms with suspected roles in atherosclerosis. Results— Among the polymorphisms tested, the angiotensin-converting enzyme insertion/deletion, osteopontin (OPN) T-443C, monocyte chemoattractant protein-1 (MCP-1) G-927C, and MCP-1 A-2578G polymorphisms were associated with CCA–IMT in age-gender–adjusted analysis. In multivariate analysis, the association between the OPN and MCP-1 polymorphisms remained significant. The OPN-443C allele was associated with increased IMT in the dominant model (0.053 mm for the TC and CC genotypes; P =0.001). The MCP-1-927C allele was associated with increased IMT in the additive model (0.040 mm for each C allele; P =0.001), and the MCP-1-2578 G allele was associated with decreased IMT in the recessive model (0.088 mm for the GG genotype; P =0.002). Conclusions— The OPN and MCP-1 genes, coding for 2 cytokines with known roles in atherosclerosis, may contribute to increased carotid IMT and warrant further study.

Background The polymorphic angiotensinogen (AGT) gene is one of the most promising candidates for blood pressure (BP) regulation and essential hypertension. Objectives To investigate whether AGT haplotype analysis adds significant information compared to single polymorphism analysis with respect to different BP phenotypes in an untreated hypertensive sample. Methods Two hundred and twelve untreated hypertensive subjects of Caucasian origin were genotyped for the AGT polymorphisms C-532T, A-20C, C-18T, and G-6A. Results In single variant analyses, untreated hypertensives, carrying the AGT -532T or -6A alleles had significantly higher systolic blood pressure (SBP) and diastolic blood pressure (DBP), as well as ambulatory BP values compared to respective non-carriers. In haplotype-based analyses, combining all four AGT promoter variants, we demonstrate that AGT haplotypes containing different allele combinations at positions -532 and -6 were significantly associated with different BP values: (1) -532T and -6A with higher, (2) -532C and -6G with lower, (3) -532C and -6A with intermediate BP values. Since the result for the -532C/-20A/-18C/-6G haplotype was due to differences between non-carriers and carriers of this haplotype on both chromosomes, a recessive inheritance model for BP effects could be assumed. Conclusions Our results designate the C-532T and G-6A as the best candidates for functional studies on the AGT gene. Haplotype-based analyses should greatly aid in the dissection of the genetic basis of complex traits, such as BP regulation and hypertension.

Peripheral nerve stimulation limits the use of whole-body gradient systems capable of slew rates > 80 T/m/s and gradient strengths > 25 mT/m. The stimulation threshold depends mainly on the amplitude of the induced electric field in the patient's body, and thus can be influenced by changing the total magnetic flux of the gradient coil. A gradient system was built which allows continuous variation of the field characteristics in order to permit the use of full gradient performance without stimulation (slew rate 190–210 T/m/s, Gmax 32–40 mT/m). The system consists of a modular six-channel gradient coil designed with a modified target field method, two three-channel amplifiers, and a six-channel gradient controller. It is demonstrated that two coils on one gradient axis can be driven by two amplifiers in parallel, without significant changes in image quality. Scaling of the field properties and stimulation threshold according to the current polarity and ratio of both coil sets was verified in both phantom and volunteer studies. Magn Reson Med 47:800–808, 2002. © 2002 Wiley-Liss, Inc.

Objectives: Whether a human immunodeficiency virus (HIV)-associated vasculitis in-part accounts for occlusive large artery disease remains uncertain. We aimed to identify the histopathological features that characterize large vessel changes in HIV sero-positive as compared to sero-negative patients with critical lower limb ischemia (CLI). Materials and Methods: Femoral arteries obtained from 10 HIV positive and 10 HIV negative black African male patients admitted to a single vascular unit with CLI requiring above knee amputation were subjected to histopathological assessment. None of the HIV positive patients were receiving antiretroviral therapy. Results: As compared to HIV negative patients with CLI, HIV positive patients were younger (p,0.01) and had a lower prevalence of hypertension (10 vs 90%, p,0.005) and diabetes mellitus (0 vs 50%, p,0.05), but a similar proportion of patients previously or currently smoked (80 vs 60%). 90% of HIV positive patients, but no HIV negative patient had evidence of adventitial leukocytoclastic vasculitis of the vasa vasorum (p,0.0001). In addition, 70% of HIV positive, but no HIV negative patient had evidence of adventitial slit-like vessels. Whilst T-lymphocytes were noted in the adventitia in 80% of HIV positive patients, T-lymphocytes were noted only in the intima in HIV negative patients. The presence of femoral artery calcified multilayered fibro-atheroma was noted in 40% of HIV positive and 90% of HIV negative patients with CLI. Conclusions: An adventitial vasculitis which characterizes large artery changes in CLI in HIV-infected as compared to noninfected patients, may contribute toward HIV-associated occlusive large artery disease.

The Ca 2+ - and bicarbonate-activated soluble adenylyl cyclase (sAC) has been identified recently as an important mediator of aldosterone signaling in the kidney. Nuclear sAC has been reported to stimulate cAMP response element–binding protein 1 phosphorylation via protein kinase A, suggesting an alternative cAMP pathway in the nucleus. In this study, we analyzed the sAC as a potential modulator of endothelial stiffness in the vascular endothelium. We determined the contribution of sAC to cAMP response element–mediated transcriptional activation in vascular endothelial cells and kidney collecting duct cells. Inhibition of sAC by the specific inhibitor KH7 significantly reduced cAMP response element–mediated promoter activity and affected cAMP response element–binding protein 1 phosphorylation. Furthermore, KH7 and anti-sAC small interfering RNA significantly decreased mRNA and protein levels of epithelial sodium channel-α and Na + /K + -ATPase-α. Using atomic force microscopy, a nano-technique that measures stiffness and deformability of living cells, we detected significant endothelial cell softening after sAC inhibition. Our results suggest that the sAC is a regulator of gene expression involved in aldosterone signaling and an important regulator of endothelial stiffness. Additional studies are warranted to investigate the protective action of sAC inhibitors in humans for potential clinical use.

Background. Primary healthcare is the foundation of a country’s healthcare system. Without an efficient and cost-effective programme, the level of healthcare offered across all levels of health management is adversely affected. Objective. To analyse the effectiveness of the management of hypertension and diabetes mellitus (DM) among two distinct patient populations, one with significant cardiovascular risk factors and the other without. Method. We performed a case control study of a high-risk group of patients presenting with chronic critical limb ischaemia (CLI) to the Divisions of Vascular Surgery at Charlotte Maxeke Johannesburg Academic Hospital and Chris Hani Baragwanath Academic Hospital, and a randomly selected group of ‘healthy’ community participants from Johannesburg’s South Western Townships (Soweto). Results. We assessed 217 patients with CLI and 1 030 participants from the community. We assessed the number of patients who were not achieving their therapeuatic targets, among those known to be hypertensive (CLI: 44.7%; community: 59.9%) and diabetic (CLI: 83.5%; community: 66%). Undiagnosed diabetes affected 10.8% of patients with CLI and 11% of the community sample. Conclusion. Traditional vascular risk factors are managed poorly at both primary healthcare and at tertiary care levels. There is a need to identify factors that will address this issue.

Synapse function requires the cell-adhesion molecules neurexins (Nrxn) and neuroligins (Nlgn). Although these molecules are essential for neurotransmission and prefer distinct isoform combinations for interaction, little is known about their transcriptional regulation. Here, we started to explore this important aspect because expression of Nrxn1-3 and Nlgn1-3 genes is altered in mice lacking the transcriptional regulator methyl-CpG-binding protein2 (MeCP2). Since MeCP2 can bind to methylated CpG-dinucleotides and Nrxn/Nlgn contain CpG-islands, we tested genomic sequences for transcriptional activity in reporter gene assays. We found that their influence on transcription are differentially activating or inhibiting. As we observed an activity difference between heterologous and neuronal cell lines for distinct Nrxn1 and Nlgn2 sequences, we dissected their putative promoter regions. In both genes, we identify regions in exon1 that can induce transcription, in addition to the alternative transcriptional start points in exon2. While the 5′-regions of Nrxn1 and Nlgn2 contain two CpG-rich elements that show distinct methylation frequency and binding to MeCP2, other regions may act independently of this transcriptional regulator. These data provide first insights into regulatory sequences of Nrxn and Nlgn genes that may represent an important aspect of their function at synapses in health and disease.

White matter lesions (WML) are clinically relevant since they are associated with strokes, cognitive decline, depression, or epilepsy, but the underlying etiology in young adults without classical risk factors still remains elusive. Our aim was to elucidate the possible clinical diagnosis and mechanisms leading to WML in patients carrying the D313Y mutation in the α-galactosidase A (GLA) gene, a mutation that was formerly described as nonpathogenic. Pathogenic GLA mutations cause Fabry disease, a vascular endothelial glycosphingolipid storage disease typically presenting with a symptom complex of renal, cardiac, and cerebrovascular manifestations. We performed in-depths clinical, biochemical and genetic examinations as well as advanced magnetic resonance imaging analyses in a pedigree with the genetically determined GLA mutation D313Y. We detected exclusive neurologic manifestations of the central nervous system of the "pseudo"-deficient D313Y mutation leading to manifest WML in 7 affected adult family members. Furthermore, two family members that do not carry the mutation showed no WML. The D313Y mutation resulted in a normal GLA enzyme activity in leukocytes and severely decreased activities in plasma. In conclusion, our results provide evidence that GLA D313Y is potentially involved in neural damage with significant WML, demonstrating the necessity of evaluating patients carrying D313Y more thoroughly. D313Y might broaden the spectrum of hereditary small artery diseases of the brain, which preferably occur in young adults without classical risk factors. In view of the existing causal therapy regime, D313Y should be more specifically taken into account in these patients.

Objectives A systematic review of the role of cytokines in clinical medicine as diagnostic, prognostic, or predictive biomarkers in pancreatic ductal adenocarcinoma was undertaken. Materials and Methods A systematic review was conducted according to the 2009 PRISMA guidelines. PubMed database was searched for all original articles on the topic of interest published until June 2015, and this was supplemented with references cited in relevant articles. Studies were evaluated for risk of bias using the Quality in Prognosis Studies tools. Results Forty one cytokines were investigated with relation to pancreatic ductal adenocarcinoma (PDAC) in 65 studies, ten of which were analyzed by more than three studies. Six cytokines (interleukin[IL]-1β, -6, -8, -10, vascular endothelial growth factor, and transforming growth factor) were consistently reported to be increased in PDAC by more than four studies; irrespective of sample type; method of measurement; or statistical analysis model used. When evaluated as part of distinct panels that included CA19-9, IL-1β, -6 and -8 improved the performance of CA19-9 alone in differentiating PDAC from healthy controls. For example, a panel comprising IL-1β, IL-8, and CA 19–9 had a sensitivity of 94.1% vs 85.9%, specificity of 100% vs 96.3%, and area under the curve of 0.984 vs 0.925. The above-mentioned cytokines were associated with the severity of PDAC. IL-2, -6, -10, VEGF, and TGF levels were reported to be altered after patients received therapy or surgery. However, studies did not show any evidence of their ability to predict treatment response. Conclusion Our review demonstrates that there is insufficient evidence to support the role of individual cytokines as diagnostic, predictive or prognostic biomarkers for PDAC. However, emerging evidence indicates that a panel of cytokines may be a better tool for discriminating PDAC from other non-malignant pancreatic diseases or healthy individuals.

Background. A common problem in clinical practice is predicting whether a patient will be continent after treatment of a severe perineal injury. Several tests have been described. Anal manometry is unreliable; continence can be normal with low pressures, and poor with high or normal pressures. Endo-anal ultrasound only illustrates anatomical sphincter integrity. The saline continence test involves the quite unphysiological instilling of saline into the rectum, and assessing seepage. What is needed in the prediction of continence is a normal stool simulator. Method. We propose the use of powdered instant mashed potato reconstituted with water to the consistency of faeces. About 100 - 150 ml is introduced into the rectum using a catheter-tipped syringe. The patient is instructed to walk around for half an hour. On return the underwear is examined for any soiling. If there is no leakage the colostomy may be reversed. Results. Over the past 15 years, 53 patients have undergone this test. In 47 patients there was no leakage, all had their stomas reversed, and none was incontinent during follow-up. Conclusion. The dynamic continence challenge is an accurate physiological test that allows clinicians to simulate the effects of colostomy reversal and assess a patient’s continence before actually proceeding to the reversal.

Although an increased aortic pulse wave velocity (PWV) may be employed to predict cardiovascular risk, the extent to which this may be limited by decreases in PWV in peripheral arterial disease (PAD) is uncertain. Applanation tonometry (SphygmoCor software) and vascular B-mode ultrasound were employed to assess carotid-femoral PWV and carotid intima-media thickness (IMT) respectively in 1030 randomly selected healthy adults, and in 217 patients with chronic critical lower limb ischemia (CLI). Patients with CLI had a marked attenuation of age-related increases in aortic PWV (slope of the multivariate adjusted age-log PWV relationship: CLI group=0.23±0.07, Controls=0.54±0.03, p

Patients with coronary heart disease (CHD) have higher overall and cardiovascular mortality than the general population (1). They can decrease the risk of suffering a further CHD event by Lowering their blood pressure (2) Controlling their cholesterol level (3, e1) Giving up smoking (4, e2) Changing their eating habits (5) Increasing their physical activity (6) The regularly updated guidelines of the Joint European Societies promulgate evidence-based recommendations for the prevention of cardiovascular diseases in clinical practice (7, e3– e5). In 1995/96 and 1999/2000 the European Society of Cardiology (ESC) carried out the surveys EUROASPIRE I and II (EUROpean Action on Secondary Prevention through Intervention to Reduce Events) to examine the implementation of these recommendations in CHD patients. EUROASPIRE is a multicenter study to evaluate secondary prevention in CHD patients in Europe. To this end, cross-sectional surveys of hospital patients with CHD were conducted in nine (EUROASPIRE I) and 15 (EUROASPIRE II) regions across Europe. EUROASPIRE I and II showed inadequate secondary prevention of CHD in Europe (8, 9) and found no essential changes in risk and lifestyle factors (10). These overall findings also applied to the area around Munster, the German study region in EUROASPIRE I and II (11, 12). Given the importance of cardiovascular diseases for the population, these results prompted the ESC to carry out a third EUROASPIRE survey of CHD patients, extended to 22 European regions (13, 14). The aim of the study described here was to investigate the temporal trends of cardiovascular risk factors in CHD patients in the Munster region, using the data from all three EUROASPIRE surveys.

Target values for cardiovascular risk factors in patients with coronary heart disease (CHD) are stated in guidelines for the prevention of cardiovascular disease. We studied secular trends in risk factors over a 12-year period among CHD patients in the region of Münster, Germany.The cross-sectional EUROASPIRE I, II and III surveys were performed in multiple centers across Europe. For all three, the Münster region was the participating German region. In the three periods 1995/96, 1999/2000, and 2006/07, the surveys included (respectively) 392, 402 and 457 ≤ 70-year-old patients with CHD in Münster who had sustained a coronary event at least 6 months earlier.The prevalence of smoking remained unchanged, with 16.8% in EUROASPIRE I and II and 18.4% in EUROASPIRE III (p=0.898). On the other hand, high blood pressure and high cholesterol both became less common across the three EUROASPIRE studies (60.7% to 69.4% to 55.3%, and 94.3% to 83.4% to 48.1%, respectively; p0.001 for both). Obesity became more common (23.0% to 30.6% to 43.1%, p0.001), as did treatment with antihypertensive and lipid-lowering drugs (80.4% to 88.6% to 94.3%, and 35.0% to 67.4% to 87.0%, respectively; p0.001 for both).The observed trends in cardiovascular risk factors under-score the vital need for better preventive strategies in patients with CHD.

Reverse remodeling (RR) after cardiac resynchronization therapy (CRT) is associated with favorable clinical outcomes in heart failure (HF). The renin-angiotensin-aldosterone system (RAAS) is involved in the remodeling process.We assessed the association between RR and 8 common RAAS gene variants, which were determined by TaqMan assays, in 156 outpatients with chronic HF. RR was defined as a15% decrease in left ventricular end systolic volume (LVESV) at 9 (interquartile range 7-12) months after CRT. We matched 76 patients who did not show RR (RR-) to 80 RR+ control subjects by age, sex, HF etiology, New York Heart Association (NYHA) functional class and left ventricular ejection fraction (LVEF). The frequency of the minor allele of the NR3C2 gene (rs5522 C/T), encoding the mineralocorticoid receptor, was higher in RR- than in RR (24/126 vs 10/150; P value after false discovery rate correction:.0193). Conversely, LVESV decreased significantly less after CRT in carriers of the NR3C2 minor C allele (P = .02). After adjustment for age, sex, NYHA functional class, previous myocardial infarction, atrial fibrillation, and LVEF, RR- remained independently associated with NR3C2 C allele carriage (odds ratio 3.093, 95% confidence interval 1.253-7.632).The association of RR- after CRT with a common polymorphism in the mineralocorticoid receptor gene involved in aldosterone signaling suggests a possible role for variants in RAAS genes in progressive LV function decline, despite apparently effective CRT.

Background: Tyrosine kinase inhibitors (TKIs) are effective in treating malignant disorders and were lately suggested to have an impact on non-malignant diseases. However, in some inflammatory conditions like rheumatoid arthritis (RA) the in vivo effect seemed to be moderate. As most TKIs are taken up actively into cells by cell membrane transporters, this study aimed to evaluate the role of such transporters for the accumulation of the TKI Imatinib mesylates in RA synovial fibroblasts as well as their regulation under inflammatory conditions. Methodology/Principal Findings: The transport and accumulation of Imatinib was investigated in transporter-transfected HEK293 cells and human RA synovial fibroblasts (hRASF). Transporter expression was quantified by qRT-PCR. In transfection experiments, hMATE1 showed the highest apparent affinity for Imatinib among all known Imatinib transporters. Experiments quantifying the Imatinib uptake in the presence of specific transporter inhibitors and after siRNA knockdown of hMATE1 indeed identified hMATE1 to mediate Imatinib transport in hRASF. The anti-proliferative effect of Imatinib on PDGF stimulated hRASF was quantified by cell counting and directly correlated with the uptake activity of hMATE1. Expression of hMATE1 was investigated by Western blot and immuno-fluorescence. Imatinib transport under disease-relevant conditions, such as an altered pH and following stimulation with different cytokines, was also investigated by HPLC. The uptake was significantly reduced by an acidic extracellular pH as well as by the cytokines TNFa, IL-1b and IL-6, which all decreased the expression of hMATE1-mRNA and protein. Conclusion/Significance: The regulation of Imatinib uptake via hMATE1 in hRASF and resulting effects on their proliferation may explain moderate in vivo effects on RA. Moreover, our results suggest that investigating transporter mediated drug processing under normal and pathological conditions is important for developing intracellular acting drugs used in inflammatory diseases.

I read with interest and would like to commend Lin et al. on their article [1]. They give an excellent description of how to perform a diagnostic laparoscopy in trauma, including therapeutic maneuvers and tips on how not miss injuries, specifically occult bowel perforations. I have a few questions regarding the management algorithm of the two patient groups. Omental evisceration in a clinically benign abdomen is not an immediate indication for laparotomy [2, 3]. Peritonitis itself should have been included as a criterion leading to laparotomy in both groups. Why did bowel evisceration go to laparotomy in group B and not to laparoscopy? Were the surgeons involved able to repair bowel perforations? The argument that non-therapeutic laparoscopy is less invasive than non-therapeutic laparotomy, though true, is not a reason to perform laparoscopy on all patients with penetrating trauma. I also question the very high rate of non-therapeutic laparotomy in group A (22/38). If they had no signs of peritonitis or bowel evisceration should they not have had radiological investigation and/or serial abdominal examinations in the ward? Selective nonoperative management has been shown to be safe and effective [4]. Depending on the site of injury, radiological imaging may have been the more appropriate diagnostic tool. An example is the right upper quadrant. Most stable liver injuries can be managed conservatively [5]. Is it possible that laparoscopy may have been avoided in the 17 patients with liver injuries? I think an interesting study from your institution would examine the use of therapeutic laparoscopy in hemodynamically stable patients following penetrating trauma that have peritonitis or CT scan evidence of significant injuries that in the past would have mandated a laparotomy.

The prevalence of heart failure is 2–3% of the general population and affects millions of people. In recent years, considerable progress has been made decoding the pathophysiology of this multi-factorial trait. Still the search for new variables with significant impact on the development of heart failure is an ongoing process. As part of the VPH2 project, a data mining study was conducted aiming specifically at extracting new knowledge from a population suffering from heart failure In particular, the population consists of patients suffering from post-mitral infarction development of myocardial remodelling. The aim of the study was to apply data mining methodologies in order to classify the patients in those who developed late onset heart failure against those that did not develop the trait. Data derived from a multiple genetic variant analysis added predictive value to this study. The methodology followed, the results and the clinically important findings are presented in this work.

The use of prophylactic antibiotics before endoscopic retrograde cholangiopancreatography (ERCP) is recommended by all major international gastroenterological societies, especially in the presence of an obstructed biliary system. Their use is intended to decrease or eliminate the incidence of complications following the procedure, namely cholangitis, cholecystitis, septicaemia, and pancreatitis.To assess the benefits and harms of antibiotics before elective ERCP in patients without evidence of acute or chronic cholecystitis, or acute or chronic cholangitis, or severe acute pancreatitis.We searched The Cochrane Hepato-Biliary Group Controlled Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, MEDLINE, EMBASE, Science Citation Index Expanded, and LILACS until March 2010. Relevant medical and surgical international conference proceedings were also searched.Only randomised clinical trials were included in the analyses, irrespective of blinding, language, or publication status. Participants were patients that underwent elective ERCP that were not on antibiotics, without evidence of acute or chronic cholecystitis, cholangitis, or severe acute pancreatitis before the procedure. We compared patients that received prophylactic antibiotics before the procedure with patients that were given placebo or no intervention before the procedure.The review was conducted according to the recommendations of The Cochrane Collaboration as well as the Cochrane Hepato-Biliary Group. Review Manager 5 was used employing fixed-effect and random-effects models meta-analyses.Nine randomised clinical trials (1573 patients) were included in the analyses. The majority of the trials had risks of bias. When all patients providing data for a certain outcome were included, the fixed-effect meta-analyses significantly favoured the use of prophylactic antibiotics in preventing cholangitis (relative risk (RR) 0.54, 95% CI 0.33 to 0.91), septicaemia (RR 0.35, 95% CI 0.11 to 1.11), bacteriaemia (RR 0.50, 95% CI 0.33 to 0.78), and pancreatitis (RR 0.54, 95% CI 0.29 to 1.00). In random-effects meta-analyses, only the effect on bacteriaemia remained significant. Overall mortality was not reduced (RR 1.33, 95% CI 0.32 to 5.44). If one selects patients in whom the ERCP resolved the biliary obstruction at the first procedure, there seem to be no significant benefit in using prophylactic antibiotics to prevent cholangitis (RR 0.98, 95% CI 0.35 to 2.69, only three trials).Prophylactic antibiotics reduce bacteriaemia and seem to prevent cholangitis and septicaemia in patients undergoing elective ERCP. In the subgroup of patients with uncomplicated ERCP, the effect of antibiotics may be less evident. Further research is required to determine whether antibiotics can be given during or after an ERCP if it becomes apparent that biliary obstruction cannot be relieved during that procedure.

International audience; To identify genetic susceptibility factors conferring increased risk of venous thrombosis (VT), we conducted a multistage study, following results of a previously published GWAS that failed to detect loci for developing VT. Using a collection of 5862 cases with VT and 7112 healthy controls, we identified the HIVEP1 locus on chromosome 6p24.1 as a susceptibility locus for VT. Indeed, the HIVEP1 rs169713C allele was associated with an increased risk for VT, with an odds ratio of 1.20 (95% confidence interval 1.13-1.27, p = 2.86 x 10(-9)). HIVEP1 codes for a protein that participates in the transcriptional regulation of inflammatory target genes by binding specific DNA sequences in their promoter and enhancer regions. The current results provide the identification of a locus involved in VT susceptibility that lies outside the traditional coagulation/fibrinolysis pathway.

Objective— Osteoprotegerin (OPG) has been reported to be involved in the development of atherosclerotic disease, and OPG gene variation has been associated with plasma OPG levels and different cardiovascular disease phenotypes. However, the genetic architecture of the OPG promoter and its transcriptional regulation are poorly characterized. Methods and Results— We identified 1008 bp of the OPG 5′-flanking region to be sufficiently transcriptionally active in osteosarcoma cell lines and generated serial promoter deletion constructs. Individual subcloning revealed the existence of 3 molecular haplotypes (MolHaps): [T −960 -A −946 -G −900 -T −864 ; MolHap1, wild type], [T −960 -G −946 -G −900 -T −864 ; MolHap2], [C −960 -G −946 -A −900 -G −864 ; MolHap4]. Compared to MolHap1, transcriptional activities of MolHaps 2 and 4 were significantly reduced ( P =0.0018). Whereas introduction of the −159C allele reduced transcriptional activities of the full-length constructs ( P =0.0014), it significantly increased activities of the deletion constructs ( P =0.0005). Electrophoretic mobility shift, competition, and chromatin immunoprecipitation assays revealed specific DNA:protein interactions for the MolHaps with Sp1 and NF-1, and identified Egr1 interacting exclusively with the −159T allele. Conclusions— We propose new structural and transcriptional features within the OPG promoter region and identified MolHaps being differentially transcriptionally active and allele-dependently interacting with a proximal polymorphic site.

Antibiotic prophylaxis for endoscopic retrograde cholangiopancreatography (ERCP) is controversial. We set out to assess the current antibiotic prescribing practice among South African endoscopists who perform ERCPs, and then reviewed international guidelines and relevant studies.Our audit of South African endoscopists who perform ERCPs took the form of a questionnaire. For the literature review a Pubmed search was performed from 1978 to March 2008, and these findings were compared with the current practice in South Africa.No specific protocols were being implemented widely in South Africa, and there was a marked difference in the practice between surgical and medical gastroenterologists, with surgeons using antibiotics more often. There was also a wide spectrum of antibiotic types that were being used. The Pubmed search revealed only 7 randomised controlled trials, with little consensus between them as to the absolute indications for prophylactic antibiotics in ERCP.Guidelines on antibiotic prophylaxis for ERCP are based on poor evidence. Varied opinions on its indications in South Africa may reflect the situation in other countries as well.

The CXC chemokine receptor 3 (CXCR3) has been linked to autoimmune and inflammatory disease, allograft rejection, and ischemic nephropathy. CXCR3 is expressed on endothelial and smooth muscle cells. Although a recent study posited that antagonizing of CXCR3 function may reduce atherosclerosis, the role of CXCR3 in controlling physiological vascular functions remains unclear. This study demonstrates that disruption of CXCR3 leads to elevated mean arterial pressures in anesthetized and conscious mice, respectively. Stimulation of isolated resistance vessels with various vasoconstrictors showed increased contractibility in CXCR3−/− mice in response to angiotensin II (ANG II) and a decreased vasodilatation in response to acetylcholine (ACh). The increased contractibility was related to higher ANG II type 1 receptor (AT1R) expression, whereas the decreased vasodilatation was related to lower M3-ACh receptor expression in the mesenteric arteries of CXCR3−/− mice compared with wild-type mice. The vasodilatatory response to ACh could be antagonized by the nonselective ACh receptor antagonist atropine and the selective M3 receptor antagonist 4-DAMP, but not by M1, M2, and M4 receptor antagonists. Additionally, EMSA studies revealed that transcription factors SP-1 and EGR-1 interact as a complex with the murine AT1R promoter region. Furthermore, we could show increased expression of SP-1 in CXCR3−/− mice indicating an imbalanced SP-1 and EGR-1 complex formation which causes increased AT1R expression and hypertension. The data indicate that CXCR3 receptor is important in vascular contractility and hypertension, possibly through upregulated AT1R expression.

Earlier studies have demonstrated the interaction between ADD1 and ACE in relation to arterial properties. We investigated whether arterial characteristics might also be related to interactions of ADD1 with other renin-angiotensin system genes. Using a family-based sampling frame, we randomly recruited 1064 Flemish subjects (mean age, 43.6 years; 50.4% women). By means of a wall-tracking ultrasound system, we measured the properties of the carotid, femoral and brachial arteries. In multivariate-adjusted analyses, we assessed the multiple gene effects of ADD1 (Gly460Trp), AGT (C-532T and G-6A) and AT1R (A1166C). In ADD1 Trp allele carriers, but not in ADD1 GlyGly homozygotes (P-value for interaction < or =0.014), femoral cross-sectional compliance was significantly higher (0.74 vs 0.65 mm(2) kPa(-1); P=0.020) in carriers of the AT1R C allele than in AT1R AA homozygotes, with a similar trend for femoral distensibility (11.3 vs 10.2 x 10(-3) kPa(-1); P=0.055). These associations were independent of potential confounding factors, including age. Family-based analyses confirmed these results. Brachial diameter (4.35 vs 4.18 mm) and plasma renin activity (PRA) (0.23 vs 0.14 ng ml(-1) h(-1)) were increased (P< or =0.005) in AGT CG haplotype homozygotes compared with non-carriers, whereas the opposite was true for brachial distensibility (12.4 vs 14.4 x 10(-3) kPa(-1); P=0.011). There was no interaction between AGT and any other gene in relation to the measured phenotypes. ADD1 and AT1R interactively determine the elastic properties of the femoral artery. There is a single-gene effect of the AGT promoter haplotypes on brachial properties and PRA. ispartof: Journal of Human Hypertension vol:23 issue:1 pages:55-64 ispartof: location:England status: published

OBJECTIVE— Genetic and environmental factors modulate the susceptibility to diabetic nephropathy, as initiating and/or progression factors. The objective of the European Rational Approach for the Genetics of Diabetic Complications (EURAGEDIC) study is to identify nephropathy susceptibility genes. We report molecular genetic studies for 127 candidate genes for nephropathy. RESEARCH DESIGN AND METHODS— Polymorphisms were identified through sequencing of promoter, exon, and flanking intron gene regions and a database search. A total of 344 nonredundant SNPs and nonsynonymous variants were tested for association with diabetic nephropathy (persistent albuminuria ≥300 mg/24 h) in a large type 1 diabetes case/control (1,176/1,323) study from three European populations. RESULTS— Only one SNP, rs2281999, located in the UNC13B gene, was significantly associated with nephropathy after correction for multiple testing. Analyses of 21 additional markers fully characterizing the haplotypic variability of the UNC13B gene showed consistent association of SNP rs13293564 (G/T) located in intron 1 of the gene with nephropathy in the three populations. The odds ratio (OR) for nephropathy associated with the TT genotype was 1.68 (95% CI 1.29–2.19) (P = 1.0 × 10−4). This association was replicated in an independent population of 412 case subjects and 614 control subjects (combined OR of 1.63 [95% CI 1.30–2.05], P = 2.3 × 10−5). CONCLUSIONS— We identified a polymorphism in the UNC13B gene associated with nephropathy. UNC13B mediates apopotosis in glomerular cells in the presence of hyperglycemia, an event occurring early in the development of nephropathy. We propose that this polymorphism could be a marker for the initiation of nephropathy. However, further studies are needed to clarify the role of UNC13B in nephropathy.

In genome-wide studies, the intercellular adhesion molecule-1 (ICAM-1) locus has been associated with cardiovascular and inflammatory bowel diseases. To determine the functional relevance of five missense ICAM-1 variants (G241R; I316V; P352L; K469E; R478W), we generated wild-type and variant proteins [M2(241R); M3(469E); M4(352L); M5(478W); M6(316V); M7(352L/469E)] and transiently transfected CV1 cells. Reverse transcription PCR, western blot, and ELISA did not reveal any differences in mRNA and protein expression levels for any construct. Conversely, in pulse-chase experiments, compared with wild-type (90-120 min), M3 and M5 possessed a prolonged half-life of approximately 150 min, whereas M2, M4, and M7 displayed a decreased half-life of approximately 60-75 min, implying differences in protein degradation. Our results do not indicate a major impact of missense variants on ICAM-1 biological function, even if G241R and K469E were functional in pulse-chase experiments. Whether these differences in protein stability exert measurable functional consequences needs to be elucidated further.

Human thrombopoietin (TPO) is involved in cardiovascular disease as it regulates megakaryocyte development and enhances platelet adhesion/aggregation. The THPO promoter structure is still controversial. By reverse transcription-PCR, we confirm that THPO transcription is cell line-dependently initiated at two alternative promoters, which we newly designated P1a and P1. We subsequently electrophoretically scanned and resequenced these portions in 95 and 57 patients with cardiovascular disease, respectively, and identified seven variants (-1450/del58bp, C-920T [rs2855306], A-622G, C-413T [rs885838], C+5A, G+115A, and C+135T). After subcloning of 1032 bp of THPO P1 in pGL3-basic vector, five molecular haplotypes (MolHaps1-5) were observed: [A(-622)-C(-413)-C(+5)-G(+115); wild type (wt)], [A(-622)-T(-413)-C(+5)-G(+115)], [G(-622)-T(-413)-C(+5)-G(+115)], [A(-622)-C(-413)-A(+5)-G(+115)], [A(-622)-C(-413)-C(+5)-A(+115)], and analyzed in reporter gene assays in HEK293T and HepG2 cells. MolHaps 2, 4, and 5 were significantly more active than wt (all p valuesor =0.01) in HEK293T cells, MolHap3 exerted a substantial loss of promoter activity (p0.0001 in HEK293T and p0.01 in HepG2, compared with wt). Electrophoretic mobility shift assays revealed that A-622G and C-413T individually differed from MolHaps in their DNA-protein interaction patterns. Supershift and chromatin immunoprecipitation assays identified CCAAT/enhancer-binding protein delta as the binding protein exclusively for the -622A allelic portion.