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1.  The microstructure of collagen type I gel cross-linked with gold nanoparticles 
Scanning electron microscopy, transmission electron micrsocopy, rheomerty, and electrochemistry were used to provide insight into the microstructure of collagen type I gel (1% w/v) modified with the tiopronin-protected (N-(2-mercaptopropionyl)glycine) gold nanoparticles (TPAu), a multivalent crosslinker. The cross-linking reaction, performed via EDC (1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide) coupling, results in compliant, mechanically stable and continuous gels. The gels contain unusual interconnected collagen-TPAu particles. Electrochemical measurements of 4-hydroxy-(2,2,6,6-tetramethylpiperidine-1-oxyl) (4HT) diffusion within the gel reveal that the gel hindrance is nearly independent of the TPAu concentration. The properties of the collagen-TPAu gel make it suitable for potential biomedical applications, such as delivery of small molecule drugs.
PMCID: PMC3508363  PMID: 22796781
2.  Environmental Xenobiotics and the Antihormones Cyproterone Acetate and Spironolactone Use the Nuclear Hormone Pregnenolone X Receptor to Activate the CYP3A23 Hormone Response Element 
Molecular pharmacology  1998;54(6):1113-1117.
The pregnenolone X receptor (PXR), a new member of the nuclear hormone receptor superfamily, was recently demonstrated to mediate glucocorticoid agonist and antagonist activation of a hormone response element spaced by three nucleotides (DR-3) within the rat CYP3A23 promoter. Because many other steroids and xenobiotics can up-regulate CYP3A23 expression, we determined whether some of these other regulators used PXR to activate the CYP3A23 DR-3. Transient cotransfection of LLC-PK1 cells with (CYP3A23)2-tk-CAT and mouse PXR demonstrated that the organochlorine pesticides transnonachlor and chlordane and the nonplanar polychlorinated biphenyls (PCBs) each induced the CYP3A23 DR-3 element, and this activation required PXR. Additionally, this study found that PXR is activated to induce (CYP3A23)2-tk-CAT by antihormones of several steroid classes including the antimineralocorticoid spironolactone and the antiandrogen cyproterone acetate. These studies reveal that PXR is involved in the induction of CYP3A23 by pharmacologically and structurally distinct steroids and xenobiotics. Moreover, PXR-mediated PCB activation of the (CYP3A23)2-tk-CAT may serve as a rapid assay for effects of nonplanar PCBs.
PMCID: PMC3662300  PMID: 9855641
3.  Transporter-Mediated Protection Against Thiopurine-Induced Hematopoietic Toxicity 
Cancer Research  2008;68(13):4983-4989.
Thiopurines are effective immunosuppressants and anticancer agents, but intracellular accumulation of their active metabolites (6-thioguanine nucleotides, 6-TGNs) causes dose-limiting hematopoietic toxicity. Thiopurine S-methyltransferase (TPMT) deficiency is known to exacerbate thiopurine toxicity. However, many patients are highly sensitive to thiopurines for unknown reasons. We show that Mrp4 is abundant in myeloid progenitors and tested the role of the multidrug-resistance protein 4 (Mrp4), an ATP binding cassette (ATP) transporter of monophosphorylated nucleosides, in this unexplained thiopurine sensitivity. Mrp4-deficient mice experienced Mrp4 gene dosage–dependent toxicity caused by accumulation of 6-TGNs in their myelopoietic cells. Therefore, Mrp4 protects against thiopurine-induced hematopoietic toxicity by actively exporting thiopurine nucleotides. We then identified a single-nucleotide polymorphism (SNP) in human MRP4 (rs3765534) that dramatically reduces MRP4 function by impairing its cell membrane localization. This SNP is common (>18%) in the Japanese population and indicates that the increased sensitivity of some Japanese patients to thiopurines may reflect the greater frequency of this MRP4 SNP.
PMCID: PMC3323115  PMID: 18593894
5.  Cyclic Nucleotide Compartmentalization: Contributions of Phosphodiesterases and ATP-Binding Cassette Transporters 
Cyclic nucleotides [e.g., cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)] are ubiquitous second messengers that affect multiple cell functions from maturation of the egg to cell division, growth, differentiation, and death. The concentration of cAMP can be regulated by processes within membrane domains (local regulation) as well as throughout a cell (global regulation). The phosphodiesterases (PDEs) that degrade cAMP have well-known roles in both these processes. It has recently been discovered that ATP-binding cassette (ABC) transporters contribute to both local and global regulation of cAMP. This regulation may require the formation of macromolecular complexes. Some of these transporters are ubiquitously expressed, whereas others are more tissue restricted. Because some PDE inhibitors are also ABC transporter inhibitors, it is conceivable that the therapeutic benefits of their use result from the combined inhibition of both PDEs and ABC transporters. Deciphering the individual contributions of PDEs and ABC transporters to such drug effects may lead to improved therapeutic benefits.
PMCID: PMC4303346  PMID: 23072381
CFTR; cAMP; efflux; export; MRP4
6.  Initiation of ART during Early Acute HIV Infection Preserves Mucosal Th17 Function and Reverses HIV-Related Immune Activation 
PLoS Pathogens  2014;10(12):e1004543.
Mucosal Th17 cells play an important role in maintaining gut epithelium integrity and thus prevent microbial translocation. Chronic HIV infection is characterized by mucosal Th17 cell depletion, microbial translocation and subsequent immune-activation, which remain elevated despite antiretroviral therapy (ART) correlating with increased mortality. However, when Th17 depletion occurs following HIV infection is unknown. We analyzed mucosal Th17 cells in 42 acute HIV infection (AHI) subjects (Fiebig (F) stage I-V) with a median duration of infection of 16 days and the short-term impact of early initiation of ART. Th17 cells were defined as IL-17+ CD4+ T cells and their function was assessed by the co-expression of IL-22, IL-2 and IFNγ. While intact during FI/II, depletion of mucosal Th17 cell numbers and function was observed during FIII correlating with local and systemic markers of immune-activation. ART initiated at FI/II prevented loss of Th17 cell numbers and function, while initiation at FIII restored Th17 cell numbers but not their polyfunctionality. Furthermore, early initiation of ART in FI/II fully reversed the initially observed mucosal and systemic immune-activation. In contrast, patients treated later during AHI maintained elevated mucosal and systemic CD8+ T-cell activation post initiation of ART. These data support a loss of Th17 cells at early stages of acute HIV infection, and highlight that studies of ART initiation during early AHI should be further explored to assess the underlying mechanism of mucosal Th17 function preservation.
Author Summary
Persistent systemic immune activation is a hallmark of chronic HIV infection and an independent predictor of disease progression. The underlying mechanism is not yet completely understood but thought to be associated with the loss of Th17 cells leading to the disruption of the mucosal barrier and subsequent microbial translocation. However, it remains unclear when these events take place in HIV infection, as the only data available to date are from SIV models. We evaluated the kinetics of Th17 depletion, microbial translocation and subsequent immune activation in early acute HIV infection and the effect of early initiated ART on these events. We discovered that a collapse of Th17 cell number and function, accompanied by local and systemic immune activation, occurs already during acute HIV infection. However, early initiation of ART preserved Th17 number and function and fully reversed any initial HIV-related immune activation. These findings argue for the importance of early events during HIV infection setting the stage for chronic immune activation and for early and aggressive treatment during acute HIV infection.
PMCID: PMC4263756  PMID: 25503054
7.  Pre-analytic factors and initial biomarker levels in community-acquired pneumonia patients 
BMC Anesthesiology  2014;14(1):102.
Blood biomarkers are increasingly used to diagnose, guide therapy in, and risk-stratify community-acquired pneumonia (CAP) patients in emergency departments (EDs). How pre-analytic factors affect these markers’ initial levels in this population is unknown.
In this secondary analysis of consecutive ED patients with CAP from a large multicentre antibiotic stewardship trial, we used adjusted multivariate regression models to determine the magnitude and statistical significance of differences in mean baseline concentrations of five biomarkers (procalcitonin [PCT], C-reactive protein [CRP], white blood cells count [WBC], proadrenomedullin [ProADM], copeptin) associated with six pre-analytic factors (antibiotic or corticosteroid pretreatment, age, gender, chronic renal failure or chronic liver insufficiency).
Of 925 CAP patients (median age 73 years, 58.8% male), 25.5% had antibiotic pretreatment, 2.4%, corticosteroid pretreatment, 22.3%, chronic renal failure, 2.4% chronic liver insufficiency. Differences associated with pre-analytic factors averaged 6.1% ±4.6%; the three largest statistically significant changes (95% confidence interval) were: PCT, +14.2% (+2.1% to +26.4%, p = 0.02) with liver insufficiency; ProADM, +13.2% (+10.2% to +16.1%, p < 0.01) with age above median; CRP, -12.8% (-25.4% to -0.2%, p = 0.05) with steroid pretreatment. In post hoc sensitivity analyses, reclassification statistics showed that these factors did not result in significant changes of biomarker levels across clinically used cut-off ranges.
Despite statistically significant associations of some pre-analytic factors and biomarker levels, a clinically relevant influence seems unlikely. Our observations reinforce the concept of using biomarkers in algorithms with widely-separated cut-offs and overruling criteria considering the entire clinical picture.
Trial registration
Identifier ISRCTN95122877.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2253-14-102) contains supplementary material, which is available to authorized users.
PMCID: PMC4240803  PMID: 25419180
Community-acquired pneumonia; Blood biomarkers; Procalcitonin; C-reactive protein; White blood cells count; Proadrenomedullin; Copeptin; Pre-analytic factors; Pretreatment
8.  Regulation of Coagulation Factor XI Expression by MicroRNAs in the Human Liver 
PLoS ONE  2014;9(11):e111713.
High levels of factor XI (FXI) increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs) in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR-23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK) demonstrated a direct interaction between miR-181a-5p and 3′untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI.
PMCID: PMC4224396  PMID: 25379760
10.  The Arachidonic Acid Metabolome Serves as a Conserved Regulator of Cholesterol Metabolism 
Cell Metabolism  2014;20(5):787-798.
Cholesterol metabolism is closely interrelated with cardiovascular disease in humans. Dietary supplementation with omega-6 polyunsaturated fatty acids including arachidonic acid (AA) was shown to favorably affect plasma LDL-C and HDL-C. However, the underlying mechanisms are poorly understood. By combining data from a GWAS screening in >100,000 individuals of European ancestry, mediator lipidomics, and functional validation studies in mice, we identify the AA metabolome as an important regulator of cholesterol homeostasis. Pharmacological modulation of AA metabolism by aspirin induced hepatic generation of leukotrienes (LTs) and lipoxins (LXs), thereby increasing hepatic expression of the bile salt export pump Abcb11. Induction of Abcb11 translated in enhanced reverse cholesterol transport, one key function of HDL. Further characterization of the bioactive AA-derivatives identified LX mimetics to lower plasma LDL-C. Our results define the AA metabolome as conserved regulator of cholesterol metabolism, and identify AA derivatives as promising therapeutics to treat cardiovascular disease in humans.
Graphical Abstract
•GWAS identifies ALOX5 to associate with plasma cholesterol and HDL-C in humans•Aspirin promotes reverse cholesterol transport (RCT) via Abcb11•Lipoxins and leukotrienes regulate expression of Abcb11•Lipoxin mimetics increase hepatic LDLr thereby lowering LDL-C
Omega-6 polyunsaturated fatty acids, including arachidonic acid (AA), have beneficial cardiovascular effects. Demetz et al. show that Alox5, a key enzyme of the AA pathway, regulates cholesterol in humans. Modulation of the AA pathways genetically or pharmacologically, with aspirin or bioactive AA-mimetics influences cholesterol metabolism including reverse cholesterol transport.
PMCID: PMC4232508  PMID: 25444678
11.  Heterogeneous clinical presentation in ICF syndrome: correlation with underlying gene defects 
European Journal of Human Genetics  2013;21(11):1219-1225.
Immunodeficiency with centromeric instability and facial anomalies (ICF) syndrome is a primary immunodeficiency, predominantly characterized by agammaglobulinemia or hypoimmunoglobulinemia, centromere instability and facial anomalies. Mutations in two genes have been discovered to cause ICF syndrome: DNMT3B and ZBTB24. To characterize the clinical features of this syndrome, as well as genotype–phenotype correlations, we compared clinical and genetic data of 44 ICF patients. Of them, 23 had mutations in DNMT3B (ICF1), 13 patients had mutations in ZBTB24 (ICF2), whereas for 8 patients, the gene defect has not yet been identified (ICFX). While at first sight these patients share the same immunological, morphological and epigenetic hallmarks of the disease, systematic evaluation of all reported informative cases shows that: (1) the humoral immunodeficiency is generally more pronounced in ICF1 patients, (2) B- and T-cell compartments are both involved in ICF1 and ICF2, (3) ICF2 patients have a significantly higher incidence of intellectual disability and (4) congenital malformations can be observed in some ICF1 and ICF2 cases. It is expected that these observations on prevalence and clinical presentation will facilitate mutation-screening strategies and help in diagnostic counseling.
PMCID: PMC3798845  PMID: 23486536
ICF syndrome; DNMT3B; ZBTB24; genotype–phenotype
12.  Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1 
Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotics detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet-drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that cautions should be taken for PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies.
PMCID: PMC3775940  PMID: 23707768
Piperine; pregnane X receptor; cytochrome P450 3A4; multidrug resistance protein 1; drug-drug interaction
13.  Human rhinovirus infections of the lower respiratory tract in hematopoietic stem cell transplant recipients 
Human rhinoviruses (HRVs) are a common cause of upper respiratory infection (URI) in hematopoietic stem cell transplant (HSCT) recipients; yet, their role in lower respiratory illness is not well understood.
We performed a retrospective chart review of HSCT recipients with HRV infection from the time molecular detection methods were implemented at our institution in 2008. Factors associated with proven or possible HRV pneumonia at the first HRV detection were evaluated by univariate and multivariate analysis. We then characterized all episodes of proven and possible HRV pneumonia from the initial HRV infection through a 1-year follow-up period.
Between 2008 and 2011, 63 HSCT recipients had ≥ 1 documented HRV infections. At first HRV detection, 36 (57%) patients had HRV URI and 27 (43%) had proven or possible HRV pneumonia; in multivariate analysis, hypoalbuminemia (odds ratio [OR] 9.5, 95% confidence interval [CI] 1.3–71.7; P=0.03) and isolation of respiratory co-pathogen(s) (OR 24.2, 95%CI 2.0–288.4; P=0.01) were independently associated with pneumonia. During the study period, 22 patients had 25 episodes of proven HRV pneumonia. Fever (60%), cough (92%), sputum production (61%), and dyspnea (60%) were common symptoms. Fifteen (60%) episodes demonstrated bacterial (n=n=7), fungal (n=n=5), or viral (n=n=3) co-infection. Among the remaining 10 (40%) cases of HRV monoinfection, patients’ oxygen saturations ranged from 80% to 97% on ambient air, and computed tomography scans showed peribronchiolar, patchy, ground glass infiltrates.
HRV pneumonia is relatively common after HSCT and frequently accompanied by bacterial co-infection. As use of molecular assays for respiratory viral diagnosis becomes widespread, HRV will be increasingly recognized as a significant cause of pneumonia in immunocompromised hosts.
PMCID: PMC3962254  PMID: 23890179
human rhinovirus; viral pneumonia; hematopoietic stem cell transplantation; HSCT; lower respiratory infections
15.  Etoposide pathway 
Pharmacogenetics and genomics  2009;19(7):552-553.
PMCID: PMC4164627  PMID: 19512958
etoposide; pathway; pharmacogenetics; pharmacogenomics; pharmGKB
16.  Contribution of Abcc4-Mediated Gastric Transport to the Absorption and Efficacy of Dasatinib 
Several oral multikinase inhibitors are known to interact in vitro with the human ATP-binding cassette transporter ABCC4 (MRP4), but the in vivo relevance of this interaction remains poorly understood. We hypothesized that host ABCC4 activity may influence the pharmacokinetic profile of dasatinib and subsequently affect its antitumor properties.
Experimental Design
Transport of dasatinib was studied in cells transfected with human ABCC4 or the ortholog mouse transporter, Abcc4. Pharmacokinetic studies were done in wildtype and Abcc4-null mice. The influence of Abcc4-deficiency on dasatinib efficacy was evaluated in a model of Ph+ acute lymphoblastic leukemia (ALL) by injection of luciferase-positive, p185(BCR-ABL)-expressing Arf(−/−) pre-B cells.
Dasatinib accumulation was significantly changed in cells over-expressing ABCC4 or Abcc4 compared to control cells (P<0.001). Deficiency of Abcc4 in vivo was associated with a 1.75-fold decrease in systemic exposure to oral dasatinib, but had no influence on the pharmacokinetics of i.v. dasatinib. Abcc4 was found to be highly expressed in the stomach, and dasatinib efflux from isolated mouse stomachs ex vivo was impaired by Abcc4-deficiency (P<0.01), without any detectable changes in gastric pH. Abcc4-null mice receiving dasatinib had an increase in leukemic burden, based on bioluminescence imaging, and decreased overall survival compared to wildtype mice (P=0.048).
This study suggests that Abcc4 in the stomach facilitates the oral absorption of dasatinib, and it possibly plays a similar role for other orally-administered substrates, such as acetylsalicylic acid. This phenomenon also provides a mechanistic explanation for the malabsorption of certain drugs following gastric resection.
PMCID: PMC3752901  PMID: 23794731
Abcc4 (Mrp4); oral bioavailability; gastric absorption; dasatinib
17.  Pre-Transplant IgG Reactivity to Apoptotic Cells Correlates with Late kidney Allograft Loss 
Pre-existing serum antibodies have long been associated with graft loss in transplant candidates. While most studies have focused on HLA-specific antibodies, the contribution of non-HLA-reactive antibodies has been largely overlooked. We have recently characterized monoclonal antibodies secreted by B cell clones derived from kidney allograft recipients with rejection that selectively bind to apoptotic cells. Here, we assessed the presence of such antibodies in pre-transplant serum from 300 kidney transplant recipients and examined their contribution to the graft outcomes. Kaplan-Meier survival analysis revealed that patients with high pre-transplant IgG reactivity to apoptotic cells had a significantly increased rate of late graft loss. The effect was only apparent after approximately 1 year post-transplant. Moreover, the association between pre-transplant IgG reactivity to apoptotic cells and graft loss was still significant after excluding patients with high reactivity to HLA. This reactivity was almost exclusively mediated by IgG1 and IgG3 with complement fixing and activating properties. Overall, our findings support the view that IgG reactivity to apoptotic cells contribute to pre-sensitization. Taking these antibodies into consideration alongside anti-HLA antibodies during candidate evaluation would likely improve the transplant risk assessment.
PMCID: PMC4120834  PMID: 24935695
Anti-apoptotic cell antibodies; sensitization; apoptotic cells; kidney transplantation; graft loss; complement
18.  BNP but Not s-cTnln Is Associated with Cardioembolic Aetiology and Predicts Short and Long Term Prognosis after Cerebrovascular Events 
PLoS ONE  2014;9(7):e102704.
We analyzed the prognostic value of b-type natriuretic peptide (BNP) and sensitive cardiac Troponin (s-cTnI) in patients with ischemic stroke or transient ischemic attack (TIA) and their significance in predicting stroke aetiology.
In a prospectively enrolled cohort we measured BNP and s-cTnI levels upon admission. Primary endpoints were mortality, unfavorable functional outcome and stroke recurrence after 90 days and after 12 months. Secondary endpoint was cardioembolic aetiology.
In 441 patients BNP but not s-cTnI remained an independent predictor for death with an adjusted HR of 1.2 (95% CI 1.1–1.4) after 90 days and 1.2 (95% CI 1.0–1.3) after one year. The comparison of the Area under Receiver Operating Characteristic (AUROC) of model A (age, NIHSS) and model B (age, NIHSS, BNP) showed an improvement in the prediction of mortality (0.85 (95% CI 0.79–0.90) vs. 0.86 (95% CI 0.81–0.92), Log Rank p = 0.004). Furthermore the category free net reclassification improvement (cfNRI) when adding BNP to the multivariate model was 57.5%, p<0.0001. For the prediction of functional outcome or stroke recurrence both markers provided no incremental value. Adding BNP to a model including age, atrial fibrillation and heart failure lead to a higher discriminatory accuracy for identification of cardioembolic stroke than the model without BNP (AUC 0.75 (95% CI 0.70–0.80) vs. AUC 0.79, (95% CI 0.75–0.84), p = 0.008).
BNP is an independent prognostic maker for overall mortality in patients with ischemic stroke or TIA and may improve the diagnostic accuracy to identify cardioembolic aetiology.
Trial Registration NCT00390962
PMCID: PMC4114527  PMID: 25072816
19.  Interplay between Vitamin D and the Drug Metabolizing Enzyme CYP3A4 
Cytochrome P450 3A4 (CYP3A4) is a multifunctional enzyme involved in both xenobiotic and endobiotic metabolism. This review focuses on two aspects: regulation of CYP3A4 expression by vitamin D and metabolism of vitamin D by CYP3A4. Enterohepatic circulation of vitamin D metabolites and their conjugates will be also discussed. The interplay between vitamin D and CYP3A4 provides new insights into our understanding of how enzyme induction can contribute to vitamin D deficiency.
PMCID: PMC3549031  PMID: 22985909
Cytochrome P450 3A4; Vitamin D; Drug metabolism; Enterohepatic circulation; Osteomalacia
20.  Endocarditis Caused by Rhodotorula Infection 
Journal of Clinical Microbiology  2014;52(1):374-378.
Rhodotorula is an emerging opportunistic fungal pathogen that is rarely reported to cause endocarditis. We describe a case involving a patient who developed endocarditis due to Rhodotorula mucilaginosa and Staphylococcus epidermidis, proven by culture and histopathology. The case illustrates the unique diagnostic and therapeutic challenges relevant to Rhodotorula spp.
PMCID: PMC3911467  PMID: 24197888
21.  Induction of Oxidative and Nitrosative damage leads to Cerebrovascular Inflammation in Animal Model of Mild Traumatic Brain Injury Induced by Primary Blast 
We investigate the hypothesis that oxidative damage of the cerebral vascular barrier interface (the blood brain barrier, BBB) causes the development of mild traumatic brain injury (mTBI) during primary blast wave spectrum. The underlying biochemical and cellular mechanisms of this vascular layer-structure injury are examined in a novel animal model of shock tube. We first established that low frequency (123 kPa) single or repeated shock wave causes BBB/brain injury through biochemical activation by acute mechanical force that occurs at 6–24 hrs after the exposure. This biochemical damage of the cerebral vasculature is initiated by the induction of free radical generating enzymes NADPH oxidase (NOX1) and inducible nitric oxide synthase (iNOS). Induction of these enzymes by shock wave exposure correlated well with the signatures of oxidative and nitrosative damage (4HNE/3NT) and reduction of the BBB tight junction (TJ) proteins occludin, claudin-5 and zonula occluden 1 (ZO-1) in the brain microvessel. In parallel with TJ protein disruption, the perivascular unit was significantly diminished by single or repeated shock wave exposure coinciding with the kinetic profile. Loosening of the vasculature and perivascular unit was mediated by oxidative stress-induced activation of matrix metalloproteinases and fluid channel aquaporin-4, promoting vascular fluid cavitation/edema, enhanced leakiness of the BBB and progression of neuroinflammation. The BBB leakiness and neuroinflammation were functionally demonstrated in an in vivo model by enhanced permeability of Na-Fl/EB low molecular weight tracers and the infiltration of immune cells across the BBB. The detection of brain cell matters NSE/S100β in the blood samples validated the neuro-astroglial injury in shock wave TBI. Our hypothesis that cerebral vascular injury occurring prior to the development of neurological disorders in mild TBI was further confirmed by the activation of caspase-3 and cell apoptosis mostly around the perivascular region. Thus, induction of oxidative stress and MMPs activation by shock wave underlies the mechanisms of cerebral vascular BBB leakage and neuroinflammation.
PMCID: PMC4007171  PMID: 23466554
Mild Traumatic Brain Injury; Blood-brain barrier; Oxidative stress; Perivascular unit; Neuroinflammation; Primary blast
22.  Corticosteroid treatment for community-acquired pneumonia - the STEP trial: study protocol for a randomized controlled trial 
Trials  2014;15:257.
Community-acquired pneumonia (CAP) is the third-leading infectious cause of death worldwide. The standard treatment of CAP has not changed for the past fifty years and its mortality and morbidity remain high despite adequate antimicrobial treatment. Systemic corticosteroids have anti-inflammatory effects and are therefore discussed as adjunct treatment for CAP. Available studies show controversial results, and the question about benefits and harms of adjunct corticosteroid therapy has not been conclusively resolved, particularly in the non-critical care setting.
This randomized multicenter study compares a treatment with 7 days of prednisone 50 mg with placebo in adult patients hospitalized with CAP independent of severity. Patients are screened and enrolled within the first 36 hours of presentation after written informed consent is obtained. The primary endpoint will be time to clinical stability, which is assessed every 12 hours during hospitalization. Secondary endpoints will be, among others, all-cause mortality within 30 and 180 days, ICU stay, duration of antibiotic treatment, disease activity scores, side effects and complications, value of adrenal function testing and prognostic hormonal and inflammatory biomarkers to predict outcome and treatment response to corticosteroids. Eight hundred included patients will provide an 85% power for the intention-to-treat analysis of the primary endpoint.
This largest to date double-blind placebo-controlled multicenter trial investigates the effect of adjunct glucocorticoids in 800 patients with CAP requiring hospitalization. It aims to give conclusive answers about benefits and risks of corticosteroid treatment in CAP. The inclusion of less severe CAP patients will be expected to lead to a relatively low mortality rate and survival benefit might not be shown. However, our study has adequate power for the clinically relevant endpoint of clinical stability. Due to discontinuing glucocorticoids without tapering after seven days, we limit duration of glucocorticoid exposition, which may reduce possible side effects.
Trial registration
7 September 2009 on NCT00973154.
PMCID: PMC4083867  PMID: 24974155
Corticosteroids; Community-acquired pneumonia; Pulmonary infection; Emergency medicine; Intensive care; Glucocorticoids
23.  Association of Adrenal Function and Disease Severity in Community-Acquired Pneumonia 
PLoS ONE  2014;9(6):e99518.
Rapid and accurate risk stratification in patients with community-acquired pneumonia (CAP) is an unmet clinical need. Cortisol to dehydroepiandrosterone (DHEA) ratio was put forward as a prognostic marker in sepsis. We herein validated the prognostic value of the adrenal hormones DHEA, DHEA-Sulfate (DHEAS), cortisol/DHEA-, cortisol/DHEAS- and DHEA/DHEAS – ratios in patients with CAP.
We assessed severity of illness using the pneumonia severity index (PSI) and measured adrenal hormone concentrations in 179 serum samples of prospectively recruited patients hospitalized with CAP. We calculated spearman rank correlation, logistic regression analysis and Kaplan Meier curves to study associations of adrenal hormones and outcomes.
There was a significant correlation between PSI score and total cortisol (r = 0.24, p = 0.001), DHEAS (r = −0.23, p = 0.002), cortisol/DHEA (r = 0.23, p = 0.003), cortisol/DHEAS (r = 0.32, p = <0.0001) and DHEA/DHEAS (r = 0.20, p = 0.009). In age and gender adjusted logistic regression analysis, cortisol (OR: 2.8, 95% CI: 1.48–5.28) and DHEA (OR: 2.62, 95% CI: 1.28–5.34), but not DHEAS and the different ratios were associated with all-cause mortality. The discriminatory accuracy of cortisol and DHEA in ROC analysis (area under the curve) was 0.74 and 0.61. In Kaplan Meier analysis, patients in the highest deciles of cortisol and DHEA (p = 0.005 and p = 0.015), and to a lesser extent of cortisol/DHEAS ratio (p = 0.081) had a higher risk of death.
Cortisol, DHEAS and their ratios correlate with CAP severity, and cortisol and DHEA predict mortality. Adrenal function in severe pneumonia may be an important factor for CAP outcomes.
PMCID: PMC4049821  PMID: 24910975
24.  Metal artifacts from titanium and steel screws in CT, 1.5T and 3T MR images of the tibial Pilon: a quantitative assessment in 3D 
Radiographs are commonly used to assess articular reduction of the distal tibia (pilon) fractures postoperatively, but may reveal malreductions inaccurately. While magnetic resonance imaging (MRI) and computed tomography (CT) are potential three-dimensional (3D) alternatives they generate metal-related artifacts. This study aims to quantify the artifact size from orthopaedic screws using CT, 1.5T and 3T MRI data. Three screws were inserted into one intact human cadaver ankle specimen proximal to and along the distal articular surface, then CT, 1.5T and 3T MRI scanned. Four types of screws were investigated: titanium alloy (TA), stainless steel (SS) (Ø =3.5 mm), cannulated TA (CTA) and cannulated SS (CSS) (Ø =4.0 mm, Ø empty core =2.6 mm). 3D artifact models were reconstructed using adaptive thresholding. The artifact size was measured by calculating the perpendicular distance from the central screw axis to the boundary of the artifact in four anatomical directions with respect to the distal tibia. The artifact sizes (in the order of TA, SS, CTA and CSS) from CT were 2.0, 2.6, 1.6 and 2.0 mm; from 1.5T MRI they were 3.7, 10.9, 2.9, and 9 mm; and 3T MRI they were 4.4, 15.3, 3.8, and 11.6 mm respectively. Therefore, CT can be used as long as the screws are at a safe distance of about 2 mm from the articular surface. MRI can be used if the screws are at least 3 mm away from the articular surface except for SS and CSS. Artifacts from steel screws were too large thus obstructed the pilon from being visualised in MRI. Significant differences (P<0.05) were found in the size of artifacts between all imaging modalities, screw types and material types, except 1.5T versus 3T MRI for the SS screws (P=0.063). CTA screws near the joint surface can improve postoperative assessment in CT and MRI. MRI presents a favourable non-ionising alternative when using titanium hardware. Since these factors may influence the quality of postoperative assessment, potential improvements in operative techniques should be considered.
PMCID: PMC4032923  PMID: 24914417
Computed tomography (CT); metal artifacts; magnetic resonance imaging (MRI); pilon; tibial plafond
25.  A novel recombinant papillomavirus genome enabling in vivo RNA interference reveals that YB-1, which interacts with the viral regulatory protein E2, is required for CRPV-induced tumor formation in vivo  
YB-1 is considered a negative prognostic marker for different types of cancer. Increased YB-1 protein levels in tumor cells indicate a worse prognosis. In a preceding study comparing the transcripts of CRPV-induced benign papillomas to mRNA levels of malignant epithelial tumors, we identified YB-1 as a gene that is up-regulated in papillomavirus-associated carcinomas and which causes an invasive phenotype in CRPV-positive cells in vitro. Here we demonstrate that YB-1 is a previously unknown factor required for papillomavirus-induced tumor development in the rabbit animal model system. By infecting the animals with a novel recombinant shRNA-expressing CRPV genome, we show that knock-down of YB-1 dramatically reduces papillomavirus-dependent tumor formation in vivo. Consistent with previous reports showing a nuclear distribution of YB-1 proteins as a hallmark of malignancy, we demonstrate a predominantly nuclear localization of YB-1 in CRPV-immortalized cells. Furthermore we give evidence of YB-1 regulating the CRPV URR and thereby viral gene expression and we identified YB-1 as a novel interactor of the CRPV regulatory protein E2. Taken together we hypothesize that YB-1 is essential for papillomavirus-induced tumor formation probably by regulating viral gene expression including expression of the oncogenes E6 and E7.
PMCID: PMC4065403  PMID: 24959377
Papillomavirus; CRPV; rabbit; in vivo; YB-1; E2; viral transcription; protein-protein interaction

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