Adenosine stress cardiac magnetic resonance imaging for the assessment of ischemic heart disease

Aims  This prospective study was designed to determine the diagnostic value of adenosine stress cardiac magnetic resonance imaging (CMRI) in patients referred to elective coronary angiography. Methods and results  Myocardial perfusion measurements at rest and adenosine stress were performed in 141 patients (105 men, 36 women, mean age 63.4 years) at 1.5 T with a Turbo Flash sequence. Stress-induced perfusion deficits were correlated to angiographic stenoses ≥75%. The overall sensitivity for CMRI depicting coronary artery disease (CAD) with relevant stenoses was 90.4%, the specificity was 77.4%, the positive predictive value was 85.9%, the negative predictive value was 84.2% and the accuracy 85.2%. Subgroup analysis was performed for 3-vessel disease (n = 44, sensitivity 92.3%, specificity 75.0%), 2-vessel disease (n = 43, sensitivity 92.6%, specificity 92.9%), 1-vessel disease (n = 27, sensitivity 93.1%, specificity 71.4%) and patients without CAD (n = 27, specificity 70.4%) as well as for patients with prior myocardial infarction (n = 44, sensitivity 92.9%, specificity 86.7%), prior coronary artery bypass surgery (n = 21, sensitivity 88.2%, specificity 66.7%), prior coronary interventions (n = 88, sensitivity 91.9%, specificity 75.0%), or diabetics (n = 27, sensitivity 90.5%, specificity 83.3%). Conclusion  Our study shows that stress perfusion CMRI can accurately predict relevant CAD and contributes to the identification of hemodynamic relevant stenoses in patients scheduled for coronary angiography. C. Doesch and A. Seeger have equally contributed to this publication.     

The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein

The hepatitis C virus (HCV) non-structural (NS) 5A protein plays an essential role in the replication of the viral RNA by the membrane-associated replication complex (RC). Recently, a putative NS5A inhibitor, BMS-790052, exhibited the highest potency of any known anti-HCV compound in inhibiting HCV replication in vitro and showed a promising clinical effect in HCV-infected patients. The precise mechanism of action for this new class of potential anti-HCV therapeutics, however, is still unclear. In order to gain further insight into its mode of action, we sought to test the hypothesis that the antiviral effect of BMS-790052 might be mediated by interfering with the functional assembly of the HCV RC. We observed that BMS-790052 indeed altered the subcellular localization and biochemical fractionation of NS5A. Taken together, our data suggest that NS5A inhibitors such as BMS-790052 can suppress viral genome replication by altering the proper localization of NS5A into functional RCs.     

Uropathogenic Escherichia coli induces chronic pelvic pain

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a debilitating syndrome of unknown etiology often postulated, but not proven, to be associated with microbial infection of the prostate gland. We hypothesized that infection of the prostate by clinically relevant uropathogenic Escherichia coli (UPEC) can initiate and establish chronic pain. We utilized an E. coli strain newly isolated from a patient with CP/CPPS (strain CP1) and examined its molecular pathogenesis in cell culture and in a murine model of bacterial prostatitis. We found that CP1 is an atypical isolate distinct from most UPEC in its phylotype and virulence factor profile. CP1 adhered to, invaded, and proliferated within prostate epithelia and colonized the prostate and bladder of NOD and C57BL/6J mice. Using behavioral measures of pelvic pain, we showed that CP1 induced and sustained chronic pelvic pain in NOD mice, an attribute not exhibited by a clinical cystitis strain. Furthermore, pain was observed to persist even after bacterial clearance from genitourinary tissues. CP1 induced pelvic pain behavior exclusively in NOD mice and not in C57BL/6J mice, despite comparable levels of colonization and inflammation. Microbial infections can thus serve as initiating agents for chronic pelvic pain through mechanisms that are dependent on both the virulence of the bacterial strain and the genetic background of the host.     

Assessment of myocardial viability in a reperfused porcine model: evaluation of different MSCT contrast protocols in acute and subacute infarct stages in comparison with MRI

OBJECTIVE: To assess myocardial viability in acute and subacute infarcts using different multislice spiral computed tomography contrast protocols with magnetic resonance imaging (MRI) correlation. METHODS: Seven pigs were studied with 64-multislice spiral computed tomography and MRI (1.5 T) at a median of 1 and 21 days after temporary occlusion of the second diagonal branch. Computed tomography was performed at 3, 5, 10, and 15 minutes after injection of contrast medium. Contrast agent was applied either as a bolus (protocol 1; n = 7 for the first; n = 5 for the second scan) or as a bolus plus 30 mL of subsequent 0.1 mL/s low-flow (protocol 2; n = 7 for the first; n = 6 for the second scan). Finally, histological sections were obtained. Volumes of infarcted myocardium were assessed as the percentage of the left ventricle. Computed tomography attenuation values were obtained, and image quality was assessed. RESULTS: When compared with protocol 1, protocol 2 provided greater Hounsfield unit attenuation difference between viable and nonviable myocardium at 5, 10, and 15 minutes (P = 0.19; 0.003; 0.0006) and an additional significant contrast between nonviable myocardium and ventricular blood at 3 and 5 minutes (P < 0.001). Image quality was rated significantly higher with the use of protocol 2 at 5, 10, and 15 minutes (P < or = 0.027) and for all time points use of protocol 2 resulted in improved correlation of acute and subacute infarct size with MRI. CONCLUSIONS: Good correlation of infarct zones with MRI was achieved for both acute and subacute infarcts. With the use of a bolus/low-flow protocol, image quality was substantially improved by means of a higher tissue contrast.     

Sixty-four-slice CT in the assessment of global and regional left ventricular function: Comparison with MRI in a porcine model of acute and subacute myocardial infarction

The purpose was to assess 64-slice CT in the analysis of global and regional ventricular function, using a model of acute and subacute myocardial infarction in comparison with cine-MRI. Seven pigs underwent standard MSCT and MRI examination a median 1 and 21 days following creation of reperfused myocardial infarction. Endocardial and epicardial contours were manually defined and ventricular volumes calculated according to Simpson’s method. Results were compared by Pearson’s correlation coefficient and Blant-Altman analysis. Wall motion was assessed on cine-images and evaluated by kappa statistics. MSCT revealed a strong correlation with cine-MRI regarding quantification of end-diastolic volume (EDV; r = 0.97), end-systolic volume (ESV; r = 0.97), stroke volume (SV; r = 0.94), ejection fraction (EF; r = 0.95) or myocardial mass (MM; r =0.94 ). Minor overestimation was observed for EDV and ESV (bias −1.7 ml; −1.5 ml; P=0.095; 0.025), whilst the mean difference for EF was found to be negligible (bias 0.9%; P = 0.18). Both modalities showed a 96.2% segmental agreement in regional wall motion (weighted-kappa 0.91 for 238 segments). This was true for both acute and subacute infarct phase and MSCT, and thereby enabled accurate intraindividual follow-up of segmental dysfunction. Sixty-four-slice CT allows for reliable analysis of global cardiac function and, moreover, provides accurate evaluation of wall motion in acute and subacute myocardial infarct. Scheule and Kopp both contributed equally. This study was funded by an institutional “Fortune Grant” (project no. 1500-0-0). There is no conflict of interest.     

Urothelial Cultures Support Intracellular Bacterial Community Formation by Uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) causes most community-acquired and nosocomial urinary tract infections (UTI). In a mouse model of UTI, UPEC invades superficial bladder cells and proliferates rapidly, forming biofilm-like structures called intracellular bacterial communities (IBCs). Using a gentamicin protection assay and fluorescence microscopy, we developed an in vitro model for studying UPEC proliferation within immortalized human urothelial cells. By pharmacologic manipulation of urothelial cells with the cholesterol-sequestering drug filipin, numbers of intracellular UPEC CFU increased 8 h and 24 h postinfection relative to untreated cultures. Enhanced UPEC intracellular proliferation required that the urothelial cells, but not the bacteria, be filipin treated prior to infection. However, neither UPEC frequency of invasion nor early intracellular trafficking events to a Lamp1-positive compartment were modulated by filipin. Upon inspection by fluorescence microscopy, cultures with enhanced UPEC intracellular proliferation exhibited large, dense bacterial aggregates within cells that resembled IBCs but were contained with Lamp1-positive vacuoles. While an isogenic fimH mutant was capable of forming these IBC-like structures, the mutant formed significantly fewer than wild-type UPEC. Similar to IBCs, expression of E. coli iron acquisition systems was upregulated by intracellular UPEC. Expression of other putative virulence factors, including hlyA, cnf1, fliC, kpsD, and the biofilm adhesin yfaL also increased, while expression of fimA decreased and that of flu did not change. These results indicate that UPEC differentially regulates virulence factors in the intracellular environment. Thus, immortalized urothelial cultures that recapitulate IBC formation in vitro represent a novel system for the molecular and biochemical characterization of the UPEC intracellular life cycle.Urinary tract infections (UTI) are the second most common infectious disease, in which uropathogenic Escherichia coli (UPEC) causes approximately 80% of community-acquired infections and 40% of nosocomial infections (51, 62, 63). UTI result in seven million clinic visits per year and cost $3.5 billion in treatment, representing a significant burden on the health care system (37). Half of all women will suffer a UTI during their lifetime, with a 25% recurrence rate within 6 months (10, 15). In 50% of these recurrent infections, the same UPEC strain causes both the primary and relapsing UTI (16, 25).Several host-pathogen interactions between urothelial cells and UPEC have been characterized, including UPEC induction of apoptosis, suppression of cytokine secretion, and invasion of urothelial cells (3, 4, 9, 11, 30, 31, 38, 40, 61). UPEC invasion can be mediated by the Dr adhesin, which binds type IV collagen and decay-accelerating factor, or by type 1 pili, which binds mannose residues by the FimH adhesin. Invasion by either mechanism can result in a persistent infection (18, 19, 41, 43, 44, 56, 57). When type 1 pilus-expressing UPEC is internalized, UPEC proliferates and differentiates into intracellular bacterial communities (IBCs), compact aggregates of intracellular bacteria with biofilm-like properties that have been characterized in a murine UTI model (1, 26, 71). IBCs are primarily identified visually by their morphology and location. IBCs are globular in shape, are tightly packed with coccoid bacteria, and typically occupy most of the host cell cytoplasm, causing urothelial cell distortion. Formation occurs 6 to 24 h postinfection, and IBCs express antigen 43 and type 1 pili and secrete a polysaccharide matrix (1, 26). Bacterial genes leuX, fimH, and surA are required for IBC formation, and several iron acquisition systems are upregulated in IBCs (22, 26, 48, 71). Similar to the murine model, IBC-like structures have been identified during human UTIs. Exfoliated cells exhibiting features resembling IBCs were found in patient urine samples, and UPEC isolates originating from asymptomic bacteriura, pyelonephritis, and cystitis patients formed IBCs in the murine UTI model (17, 52).Replicating the conditions required for IBC formation in vitro has proven to be difficult. One report described formation of IBC-like structures in a bladder carcinoma cell line after host cell permeabilization with a detergent (12). The absence of in vitro models is potentially due to the lack of cell lines that sufficiently recapitulate urothelial characteristics. Here we establish an in vitro model of IBC formation by pharmacologic manipulation of immortalized human urothelial cells. Our immortalized cell lines retain numerous characteristics of urothelial cells, including differentiation, inflammatory response, and apoptosis (3, 4, 30, 31, 39, 49, 64, 65). The IBC-like structures reported here are visually and morphologically similar to IBCs formed in the murine UTI model, occur with comparable kinetics, and similarly upregulate iron acquisition systems. Additionally, IBC formation is attenuated in a UPEC mutant lacking fimH. Thus, we have developed a model for studying UPEC IBC formation in vitro.     

Evaluation of micro-parallel liquid chromatography as a method for HTS-coupled actives verification

The identification of biologically active compounds from high-throughput screening (HTS) can involve considerable postscreening analysis to verify the nature of the sample activity. In this study we evaluated the performance of micro-parallel liquid chromatography (microPLC) as a separation-based enzyme assay platform for follow-up of compound activities found in quantitative HTS of two different targets, a hydrolase and an oxidoreductase. In an effort to couple secondary analysis to primary screening we explored the application of microPLC immediately after a primary screen. In microPLC, up to 24 samples can be loaded and analyzed simultaneously via high-performance liquid chromatography within a specially designed cartridge. In a proof-of-concept experiment for screen-coupled actives verification, we identified, selected, and consolidated the contents of "active" wells from a 1,536-well format HTS experiment into a 384-well plate and subsequently analyzed these samples by a 24-channel microPLC system. The method utilized 0.6% of the original 6-microl 1,536-well assay for the analysis. The analysis revealed several non-biological-based "positive" samples. The main examples included "false" enzyme activators resulting from an increase in well fluorescence due to fluorescent compound or impurity. The microPLC analysis also provided a verification of the activity of two activators of glucocerebrosidase. We discuss the benefits of microPLC and its limitations from the standpoint of ease of use and integration into a seamless postscreen workflow.     

Granulocyte colony-stimulating factor crosses the placenta and stimulates fetal rat granulopoiesis

We studied the effect of recombinant human granulocyte colony- stimulating factor (rhG-CSF) administration to pregnant rats upon fetal and neonatal myelopoiesis. Pregnant rats were treated with rhG-CSF twice daily for 2, 4, and 6 days before parturition. rhG-CSF crossed the placenta and reached peak fetal serum concentrations 4 hours after administration. Peak fetal serum levels were 1,000-fold lower than levels detected in the dam. Hematopoietic effects of rhG-CSF were assessed by cytologic analysis of the newborn blood, spleen, bone marrow, thymus, and liver. White blood cell counts were increased twofold to fourfold in newborns. This increase was due to circulating numbers of polymorphonuclear cells (PMN). rhG-CSF induced a myeloid hyperplasia in the newborn marrow consisting of immature and mature myeloid cells in the day-2 and day-4 treated pups. Bone marrow of pups treated for 6 days contained mostly hyper-segmented PMN with little or no increase in myeloid precursors. An increase in the number of postmitotic (PMN, bands, and metamyelocytes) and mitotic (promyeloblasts, myeloblasts, and metamyeloblasts) myeloid cells in the spleen of neonates was observed. No change was detected in splenic lymphocytes or monocytes. No effect of rhG-CSF was noted in the newborn liver or thymus. These results demonstrate that maternally administered rhG-CSF crosses the placenta and specifically induces bone marrow and spleen myelopoiesis in the fetus and neonate. The significant myelopoietic effects of rhG-CSF at low concentrations in the fetus suggest an exquisite degree of developmental sensitivity to this cytokine and may provide enhanced defense mechanisms to the neonate.     

白花前胡中前胡香豆素D和前胡香豆素E的分离和鉴定

从中药白花前胡(Peucedanum Praeruptorum Dunn)根中分到5个化合物,经理化常数、波谱数据及化学反应分别鉴定为前胡香豆素D(I),Pd-Ib(II),前胡香豆素E(III),nodakenetin(IV)和scopoletin(V)。其中化合物I和III为两个新化合物,与巳知化合物凯林内酯的化学沟通确定了其绝对构型,其化学结构分别为3'(S),4'(S)-二乙酰氧基-3',4'-二氢邪蒿内酯(I)和3'(R)-惕各酰氧基-4'-酮基-3',4'-二氢邪蒿内酯(III)。化合物IV和V为首次从该植物中分离得到。通过DEPT,¹H-¹HCOSY和¹³C-¹HCOSY等实验归属了II的碳氢信号。