Detection and localization of bile duct leaks after cholecystectomy using Gd-EOB-DTPA-enhanced MR cholangiography: retrospective study of 16 patients.

OBJECTIVE: To determine if gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance (MR) cholangiography can detect and localize bile duct leaks in postcholecystectomy patients. MATERIAL AND METHODS: Four blinded independent radiologists performed a retrospective review of 16 consecutive patients who underwent MR cholangiography with intravenous Gd-EOB-DTPA for the evaluation of possible biliary leak. Image quality, ductal opacification, and presence and location of any bile leak were evaluated. An independent observer determined the criterion standard using a consensus of all chart, clinical, and imaging findings. RESULTS: All 6 bile leaks confirmed at endoscopic retrograde cholangiopancreatography were diagnosed by all reviewers (sensitivity, 100%). Of the 10 patients with no leak, only one reader incorrectly diagnosed a bile leak in a single case (specificity, 98%). The accuracy for detection of the site of leak with Gd-EOB-DTPA-enhanced MR cholangiography was 80%. CONCLUSION: Gadolinium-EOB-DTPA-enhanced MR can detect bile leaks with a high sensitivity and specificity.

Predicting risk of mortality in dialysis patients: a retrospective cohort study evaluating the prognostic value of a simple chest X-ray.

BACKGROUND: Clinical outcomes of dialysis patients are variable, and improved knowledge of prognosis would inform decisions regarding patient management. We assessed the value of simple, chest X-ray derived measures of cardiac size (cardiothoracic ratio (CTR)) and vascular calcification (Aortic Arch Calcification (AAC)), in predicting death and improving multivariable prognostic models in a prevalent cohort of hemodialysis patients. METHODS: Eight hundred and twenty-four dialysis patients with one or more postero-anterior (PA) chest X-ray were included in the study. Using a validated calcification score, the AAC was graded from 0 to 3. Cox proportional hazards models were used to assess the association between AAC score, CTR, and mortality. AAC was treated as a categorical variable with 4 levels (0,1,2, or 3). Age, race, diabetes, and heart failure were adjusted for in the multivariable analysis. The criterion for statistical significance was p<0.05. RESULTS: The median CTR of the sample was 0.53 [IQR=0.48,0.58] with calcification scores as follows: 0 (54%), 1 (24%), 2 (17%), and 3 (5%). Of 824 patients, 152 (18%) died during follow-up. Age, sex, race, duration of dialysis, diabetes, heart failure, ischemic heart disease and baseline serum creatinine and phosphate were included in a base Cox model. Both CTR (HR 1.78[1.40,2.27] per 0.1 unit change), area under the curve (AUC)=0.60[0.55,0.65], and AAC (AAC 3 vs 0 HR 4.35[2.38,7.66], AAC 2 vs 0 HR 2.22[1.41,3.49], AAC 1 vs 0 HR 2.43[1.64,3.61]), AUC=0.63[0.58,0.68]) were associated with death in univariate Cox analysis. CTR remained significant after adjustment for base model variables (adjusted HR 1.46[1.11,1.92]), but did not increase the AUC of the base model (0.71[0.66,0.76] vs. 0.71[0.66,0.76]) and did not improve net reclassification performance (NRI=0). AAC also remained significant on multivariable analysis, but did not improve net reclassification (NRI=0). All ranges were based on 95% confidence intervals. CONCLUSIONS: Neither CTR nor AAC assessed on chest x-ray improved prediction of mortality in this prevalent cohort of dialysis patients. Our data do not support the clinical utility of X-ray measures of cardiac size and vascular calcification for the purpose of mortality prediction in prevalent hemodialysis patients. More advanced imaging techniques may be needed to improve prognostication in this population.

Dynamics of heat shock protein 60 in endothelial cells exposed to cigarette smoke extract.

Heat shock protein 60 (HSP60), expressed on the surface of endothelial cells (ECs) stressed by e.g. oxidized LDL or mechanical shear, was shown to function as an auto-antigen and thus as a pro-atherosclerotic molecule. The aim of this study was to determine whether cigarette smoke chemicals can lead to the activation of the "HSP60 pathway." It was also our aim to elucidate the dynamics of HSP60 from gene expression to endothelial surface expression and secretion. Here we show for the first time that the exposure of human umbilical vein endothelial cells (HUVECs) to cigarette smoke extract (CSE) results in an up-regulation of HSP60 mRNA. Live cell imaging analysis of a HSP60-EYFP fusion protein construct transfected into ECs revealed that mitochondrial structures collapse in response to CSE exposure. As a result, HSP60 is released from the mitochondria, transported to the cell surface, and released into the cell culture supernatant. Analysis of HSP60 in the sera of healthy young individuals exposed to secondhand smoke revealed significantly elevated levels of HSP60. Cigarette smoking is one of the most relevant risk factors for atherosclerosis. Herein, we provide evidence that cigarette smoke may initiate atherosclerosis in the sense of the "auto-immune hypothesis of atherosclerosis."

Cadmium is a novel and independent risk factor for early atherosclerosis mechanisms and in vivo relevance.

OBJECTIVE: Although cadmium (Cd) is an important and common environmental pollutant and has been linked to cardiovascular diseases, little is known about its effects in initial stages of atherosclerosis. METHODS AND RESULTS: In the 195 young healthy women of the Atherosclerosis Risk Factors in Female Youngsters (ARFY) study, cadmium (Cd) level was independently associated with early atherosclerotic vessel wall thickening (intima-media thickness exceeding the 90th percentile of the distribution; multivariable OR 1.6[1.1.-2.3], P=0.016). In line, Cd-fed ApoE knockout mice yielded a significantly increased aortic plaque surface compared to controls (9.5 versus 26.0 mm(2), P<0.004). In vitro results indicate that physiological doses of Cd increase vascular endothelial permeability up to 6-fold by (1) inhibition of endothelial cell proliferation, and (2) induction of a caspase-independent but Bcl-xL-inhibitable form of cell death more than 72 hours after Cd addition. Both phenomena are preceded by Cd-induced DNA strand breaks and a cellular DNA damage response. Zinc showed a potent protective effect against deleterious effects of Cd both in the in vitro and human studies. CONCLUSIONS: Our research suggests Cd has promoting effects on early human and murine atherosclerosis, which were partly offset by high Zn concentrations.

T-cells from advanced atherosclerotic lesions recognize hHSP60 and have a restricted T-cell receptor repertoire.

Atherosclerosis is a multifactorial, chronic-inflammatory disease for which the underlying cause remains unknown. It is also well documented that T-cells are among the first cells to migrate into the arterial intimal vessel layer, but their function there is still unexplained. Clinical and experimental data have provided evidence that atherosclerosis starts as an autoimmune reaction based on humoral and cellular immunity against a phylogenetically highly conserved stress protein, heat shock protein 60 (HSP60). In the present study, we phenotypically characterized T-cells from endarterectomized specimens of the carotid artery, and tested their reactivity to human HSP60. In addition, the T-cell receptor repertoire of the T-cell lines was defined by immunoscope analysis. We found a mixed population of CD4(+) and CD8(+) intralesional T-cells, with a slight predominance of CD8(+) cells. IFN-gamma production prevailed over IL-4 production. The T-cell reaction against human HSP60 was significantly increased in intralesional cells compared to peripheral T-cells. The lesion-derived T-cells showed an oligoclonally-restricted repertoire, in contrast to the polyclonal pattern of PBMC. These results clearly show that HSP60 is a major antigenic candidate, and that an oligoclonal T-cell expansion takes place in advanced human atherosclerotic lesions.

Increased serum cadmium and strontium levels in young smokers: effects on arterial endothelial cell gene transcription.

OBJECTIVE: Metal constituents of tobacco have long been suspected to contribute to cardiovascular diseases. In this study, we determined the serum concentrations of aluminum, cadmium (Cd), cobalt, copper, iron, manganese, nickel, lead, strontium (Sr), and zinc of young nonsmokers, passive smokers, and smokers. METHODS AND RESULTS: Cd and Sr were found to be significantly increased in smokers compared with nonsmokers. The effects of these metals on primary arterial endothelial cells were then assessed using microarray technology and real-time polymerase chain reaction (RT-PCR). The data showed that Sr does not interfere with endothelial cell transcription. In contrast, the effects of Cd in amounts delivered to the human body by smoking were dramatic. CONCLUSIONS: Arterial endothelial cells responded to Cd exposure by massively upregulating metal and oxidant defense genes (metallothioneins) and by downregulating a number of transcription factors. In addition, the mRNA of the intermediate filament protein vimentin, crucial for the maintenance of cellular shape, was reduced. Surprisingly, a number of pro-inflammatory genes were downregulated in response to Cd. The present data suggest that by delivering Cd to the human body, smoking deregulates transcription, exerts stress, and damages the structure of the vascular endothelium; furthermore, in contrast to the effects of cigarette smoke as a whole, Cd seems to possess anti-inflammatory properties.

Gene expression profiling of human endothelial cells exposed to 50-Hz magnetic fields fails to produce regulated candidate genes.

To address the question of a possible effect of magnetic fields (MF) at 50 Hz on living systems, gene expression analyses were performed on human primary vascular endothelial cells exposed to MF of various intensities compared to control cells. Exposure protocols included continuous exposure at a single intensity (10 and 700 microT), intermittent exposure at a single intensity (700 microT), and continuous exposure to a variable-intensity field (10-30 microT). The transcriptional response of the cells was investigated using oligonucleotide microarrays containing up to 30 000 unique features. Although in individual experiments genes were identified where the expression appeared to be affected by exposure to MF, none of these genes were regulated in the same manner in subsequent repetition experiments. This is the first report of a transcriptome-wide analysis of the effects of MF exposure on human cells. The lack of a reproducible effect of MF on the expression of any genes in our investigation adds further weight to the evidence that 50-Hz MF are not capable of interacting with biological systems and thus do not represent an endothelial stress factor.

Cigarette smoke metal-catalyzed protein oxidation leads to vascular endothelial cell contraction by depolymerization of microtubules.

Smoking is a significant risk factor for development of atherosclerosis. However, the pathophysiology of smoking-mediated vessel wall damage is not understood. With tools ranging from analytical chemistry to cell biology, we show that cigarette smoke contains metals that catalyze the direct oxidation of cellular proteins by smoke oxidants. Oxidation of cellular proteins causes a loss of microtubule function, culminating in microtubule depolymerization and proteasome-dependent degradation of alpha-tubulin. As a consequence of the microtubule collapse, cytoskeletal structures as well as intermediate filaments break down, leading finally to a contraction of vascular endothelial cells. We observed a smoke extract-induced, calpain-dependent degradation of the intracellular form of platelet-endothelial cell adhesion molecule 1/CD31, as well as a release of P-selectin/CD62P, IL-6, and IL-8 from endothelial cells into the supernatant. Increased levels of soluble CD62P and IL-6 are well known to be associated with smoking in humans. Increased permeability of the vascular endothelium is a crucial event in atherogenesis. This work highlights the compounds and mechanisms by which cigarette smoke induces leakiness of the vascular endothelium.

Progression of arteriovenous bypass restenosis in mice exposed to a 50 Hz magnetic field.

The controversy over whether magnetic fields (MF) produced by electrical wiring and appliances contribute to diseases such as cancer has been debated in the literature for more than 2 decades. These extremely low frequency fields at 50 or 60 Hz are omnipresent in the industrialized world and have been linked to various forms of cancer by epidemiological studies. Little has been published investigating any possible role of MF and cardiovascular disease, and this is the first study looking specifically at the effect of exposure to high-intensity MF on the development and progression of restenosis. A mouse arteriovenous bypass model was used, and mice were exposed to MF for periods of 1, 2, or 3 weeks. Neointima formation, infiltration of mononuclear cells, and heat shock protein 60 expression were all studied at the conclusion of the exposure regimen. Animals exposed to the MF for 1 week showed significantly smaller neointima formation compared with control mice exposed to a null field, although this difference was not observed in mice exposed for 2 or 3 weeks. No difference was found between mice exposed to MF and controls in any of the other parameters investigated.

Development and evaluation of an in vitro model for the analysis of cigarette smoke effects on cultured cells and tissues.

INTRODUCTION: Smokers have an increased risk for a variety of diseases. Among the most prominent is atherosclerosis, the leading cause of death in the Western world. Although this conjunction is accepted knowledge, the basic biological mechanisms and the identities of the active tobacco smoke constituents surprisingly are still unknown. One reason for this is the lack of accurate in vitro models. METHODS: Cell culture experiments, including cell morphology and cell death analyses, high-performance liquid chromatography, and liquid chromatography coupled to mass spectrometry via an electrospray ionization interface allowing collision-induced dissociation analyses, were applied. RESULTS AND DISCUSSION: In this study, we present and validate an in vitro model that has proven to be useful for standardized studies of cellular and histological effects of cigarette smoke. The system consists of a cigarette smoke sampling device in which water-soluble cigarette smoke constituents pass over from the gas phase into the aqueous phase resulting in nicotine concentrations identical to the in vivo concentrations, suggesting in vivo similar conditions for gas-to-liquid compound exchange.

Cigarette smoke is an endothelial stressor and leads to cell cycle arrest.

The molecular mechanisms underlying the atherogenic activity of cigarette smoke have yet to be fully elucidated. In the present study, genome-wide microarray analysis was performed on endothelial cells exposed to an aqueous cigarette smoke extract (CSE) for 3, 7, and 24 h, to obtain a better insight into how smoking may lead to endothelial damage. Microarray analysis showed the transcriptional response to CSE was dominated by heat shock, stress responsive, and inflammatory genes, along with genes encoding for anti-oxidant and metal detoxification proteins. The heat shock response was shown to be a result of short lived reactive species of CSE, with the abrogation of the effect by the addition of old CSE, the anti-oxidant N-acetyl cysteine, or the removal of metals from CSE implying that reactive oxygen species are the main culprit. This was further supported by a strong decline in the level of intracellular protein oxidation levels seen under these conditions compared to freshly prepared CSE. Mitochondrial integrity was also found to be significantly compromised after CSE treatment, resulting in a threefold increase in depolarised mitochondria after 6 h. Finally, cell cycle analysis showed the induction of G1 cell cycle arrest. An increased stress and inflammation response indicates that endothelial damage from smoking could contribute to immune cell infiltration, while decreased growth rates reduce endothelial layer repair, promoting atherogenesis.

A cost-effective web-based teaching file system.

Teaching files are common in radiology. Although there is an increasing role of digital technology in radiology departments, today's teaching files have not yet seen the application of this new technology. This may have been due in part to poor or incomplete implementation in many commercial software packages. We have demonstrated that by utilizing free software from the Internet, a web-based teaching file system, which is easy to use, low cost, and secure, can be created.

Optimization of an Escherichia coli system for cell-free synthesis of selectively N-labelled proteins for rapid analysis by NMR spectroscopy.

Cell-free protein synthesis offers rapid access to proteins that are selectively labelled with [15N]amino acids and suitable for analysis by NMR spectroscopy without chromatographic purification. A system based on an Escherichia coli cell extract was optimized with regard to protein yield and minimal usage of 15N-labelled amino acid, and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of up to 1.8 mg of human cyclophilin A per mL of reaction medium were obtained by expression of a synthetic gene. Equivalent yields were obtained using transcription directed by either T7 or tandem phage lambdapR and pL promoters, when the reactions were supplemented with purified phage T7 or E. coli RNA polymerase. Nineteen samples, each selectively labelled with a different 15N-enriched amino acid, were produced and analysed directly by NMR spectroscopy after ultracentrifugation. Cross-peaks from metabolic by-products were evident in the 15N-HSQC spectra of 13 of the samples. All metabolites were found to be small molecules that could be separated readily from the labelled proteins by dialysis. No significant transamination activity was observed except for [15N]Asp, where an enzyme in the cell extract efficiently converted Asp-->Asn. This activity was suppressed by replacing the normally high levels of potassium glutamate in the reaction mixture with ammonium or potassium acetate. In addition, the activity of peptide deformylase appeared to be generally reduced in the cell-free expression system.

Validation of fentanyl pharmacokinetics in patients undergoing coronary artery bypass grafting.

PURPOSE: The current emphasis on more rapid recovery and earlier tracheal extubation after cardiac surgery requires greater precision in administering opioids to reap their benefits while minimizing the duration of postoperative respiratory depression. Therefore, we aimed to define a pharmacokinetic model that accurately predicts fentanyl concentrations before, during, and after cardiopulmonary bypass (CPB) in patients undergoing coronary artery bypass grafting (CABG). METHODS: Parameters for two-compartment and three-compartment models were estimated by applying population pharmacokinetic modelling to fentanyl concentration vs time data measured in 29 patients undergoing elective, primary CABG. The ability of these models to predict fentanyl concentrations in a second series of ten patients undergoing CABG was then assessed. RESULTS: A simple, three-compartment model had excellent predictive ability, with a median prediction error (PE = ([Fentanyl]meas - [Fentanyl]pred)/[Fentanyl]pred x 100%) of -0.5%, and a median absolute PE (APE = /PE/) of 14.0%. In comparison to the two-compartment models, linear regression of measured:predicted concentration ratios indicated that the three-compartment model was free of systematic and time-related changes in bias (P < 0.05). The parameters of this three-compartment model are: V1 15.0 l, V2 20.0 l, V3 86.1 l, Cl1 1.08 L x min(-1), Cl2 4.90 L x min(-1), and Cl3 2.60 L x min(-1). CONCLUSIONS: Our pharmacokinetic model provides a rational foundation for designing fentanyl dose regimens for patients undergoing CABG. When combined with previously published information regarding intraoperative fentanyl pharmacodynamics, dose regimens that reliably achieve and maintain desired fentanyl concentrations throughout the intraoperative period can be designed to achieve specific therapeutic goals.