Adenovirus-mediated gene transfer to liver grafts: An improved method to maximize infectivity

Background. Adenoviral gene therapy in liver transplantation has many potential applications, but current vector delivery methods to grafts lack efficiency and require high titers. In this study, we attempted to improve gene delivery efficacy using three different delivery methods to liver grafts with adenoviral vector encoding the LacZ marker gene (AdLacZ). Methods. AdLacZ was delivered to cold preserved rat liver grafts by: (1) continuous perfusion via the portal vein (portal perfusion), (2) continuous perfusion via both the portal vein and hepatic artery (dual perfusion), and (3) trapping viral perfusate in the liver vasculature by clamping outflow (clamp technique). Results. Using 1x109 plaque-forming units of Ad-LacZ (multiplicity of infection of 0.4), transduction rate in 3-hr preserved liver grafts, determined by 5-bromo-4-chromo-3-indolyl-β-D-galactopyranoside staining and β-galactosidase assay 48 hr after transplantation, was best with clamp technique (21.5±2.7% 5-bromo-4-chromo-3-indolyl-β-D- galactopyranoside-positive cells and 81.1±3.6 U/g β-galactosidase), followed by dual perfusion (18.5±1.8%, 66.6±19.4 U/g) and portal perfusion (8.8±2.5%, 19.7±15.4 U/g). Further studies using clamp technique demonstrated a near-maximal gene transfer rate of 30% at multiplicity of infection of 0.4 with prolonged cold ischemia to 18 hr. Transgene expression was stable for 2 weeks and slowly declined to 7.8±12.1% at day 28. Lack of inflammatory response was confirmed by histopathological examination and liver enzymes. Transduction was selectively induced in hepatocytes with nearly no extrahepatic transgene expression in the lung and spleen. Conclusions. The clamp technique provides a highly efficient viral gene delivery method to cold preserved liver grafts. This method offers maximal infectivity of adenoviral vector with minimal technical manipulation

Are Child and Adolescent Responses to Placebo Higher in Major Depression than in Anxiety Disorders? A Systematic Review of Placebo-Controlled Trials

BACKGROUND: In a previous report, we hypothesized that responses to placebo were high in child and adolescent depression because of specific psychopathological factors associated with youth major depression. The purpose of this study was to compare the placebo response rates in pharmacological trials for major depressive disorder (MDD), obsessive compulsive disorder (OCD) and other anxiety disorders (AD-non-OCD). METHODOLOGY AND PRINCIPAL FINDINGS: We reviewed the literature relevant to the use of psychotropic medication in children and adolescents with internalized disorders, restricting our review to double-blind studies including a placebo arm. Placebo response rates were pooled and compared according to diagnosis (MDD vs. OCD vs. AD-non-OCD), age (adolescent vs. child), and date of publication. From 1972 to 2007, we found 23 trials that evaluated the efficacy of psychotropic medication (mainly non-tricyclic antidepressants) involving youth with MDD, 7 pertaining to youth with OCD, and 10 pertaining to youth with other anxiety disorders (N = 2533 patients in placebo arms). As hypothesized, the placebo response rate was significantly higher in studies on MDD, than in those examining OCD and AD-non-OCD (49.6% [range: 17-90%] vs. 31% [range: 4-41%] vs. 39.6% [range: 9-53], respectively, ANOVA F = 7.1, p = 0.002). Children showed a higher stable placebo response within all three diagnoses than adolescents, though this difference was not significant. Finally, no significant effects were found with respect to the year of publication. CONCLUSION: MDD in children and adolescents appears to be more responsive to placebo than other internalized conditions, which highlights differential psychopathology

The first decade of oligotrophication of Lake Constance

In Lake Constance, after several decades of cutrophication, a decrease in phosphorus loading over the last decade has lead to a partial recovery from eutrophication. Here we analyse the shift in the taxonomic composition of phytoplankton during the first decade of oligotrophication in Lake Constance. During the 1980s, spring total P concentrations decreased from ca. 130 to less than 50 mgr·l–1. This decrease was reflected by an approximately proportional decrease in summer phytoplankton biomass while spring phytoplankton biomass seemed unresponsive. Major taxonomic changes occured during both growth seasons. In spring, the proportion of diatoms, green algae and Chrysophyta increased while the proportion of Cryptophyta decreased. The summer trend was very different: the relative importance of diatoms decreased and Cryptophyta and Chrysophyta increased, while Chlorophyta reached their peak around 1985. These trends are also analysed at the genus level. Comparison with taxonomic trends during the eutrophication period shows the expected reversals in most cases. Comparison with other lakes shows general similarities, with the notable exception that Planktothrix rubescens has never been important in Lake Constance. The increase of diatoms during spring is attributed to their improved competitive performance with increasing Si:P ratios. Their decrease during summer is explained by the increasing silicate removal from the epilimnion by increasing spring populations

Impact of Circulating Cholesterol Levels on Growth and Intratumoral Androgen Concentration of Prostate Tumors

Prostate cancer (PCa) is the second most common cancer in men. Androgen deprivation therapy (ADT) leads to tumor involution and reduction of tumor burden. However, tumors eventually reemerge that have overcome the absence of gonadal androgens, termed castration resistant PCa (CRPC). Theories underlying the development of CRPC include androgen receptor (AR) mutation allowing for promiscuous activation by non-androgens, AR amplification and overexpression leading to hypersensitivity to low androgen levels, and/or tumoral uptake and conversion of adrenally derived androgens. More recently it has been proposed that prostate tumor cells synthesize their own androgens through de novo steroidogenesis, which involves the step-wise synthesis of androgens from cholesterol. Using the in vivo LNCaP PCa xenograft model, previous data from our group demonstrated that a hypercholesterolemia diet potentiates prostatic tumor growth via induction of angiogenesis. Using this same model we now demonstrate that circulating cholesterol levels are significantly associated with tumor size (R = 0.3957, p = 0.0049) and intratumoral levels of testosterone (R = 0.41, p = 0.0023) in LNCaP tumors grown in hormonally intact mice. We demonstrate tumoral expression of cholesterol uptake genes as well as the spectrum of steroidogenic enzymes necessary for androgen biosynthesis from cholesterol. Moreover, we show that circulating cholesterol levels are directly correlated with tumoral expression of CYP17A, the critical enzyme required for de novo synthesis of androgens from cholesterol (R = 0.4073, p = 0.025) Since hypercholesterolemia does not raise circulating androgen levels and the adrenal gland of the mouse synthesizes minimal androgens, this study provides evidence that hypercholesterolemia increases intratumoral de novo steroidogenesis. Our results are consistent with the hypothesis that cholesterol-fueled intratumoral androgen synthesis may accelerate the growth of prostate tumors, and suggest that treatment of CRPC may be optimized by inclusion of cholesterol reduction therapies in conjunction with therapies targeting androgen synthesis and the AR

Quantum Process Tomography of a Universal Entangling Gate Implemented with Josephson Phase Qubits

Quantum logic gates must perform properly when operating on their standard input basis states, as well as when operating on complex superpositions of these states. Experiments using superconducting qubits have validated the truth table for particular implementations of e.g. the controlled-NOT gate [1,2], but have not fully characterized gate operation for arbitrary superpositions of input states. Here we demonstrate the use of quantum process tomography (QPT) [3,4] to fully characterize the performance of a universal entangling gate between two superconducting quantum bits. Process tomography permits complete gate analysis, but requires precise preparation of arbitrary input states, control over the subsequent qubit interaction, and simultaneous single-shot measurement of the output states. We use QPT to measure the fidelity of the entangling gate and to quantify the decoherence mechanisms affecting the gate performance. In addition to demonstrating a promising fidelity, our entangling gate has a on/off ratio of 300, a level of adjustable coupling that will become a requirement for future high-fidelity devices. This is the first solid-state demonstration of QPT in a two-qubit system, as solid-state process tomography has previously only been demonstrated with single qubits [5,6]

Topical NSAIDs for chronic musculoskeletal pain: systematic review and meta-analysis

A previous systematic review reported that topical NSAIDs were effective in relieving pain in chronic conditions like osteoarthritis and tendinitis. More trials, a better understanding of trial quality and bias, and a reclassification of certain drugs necessitate a new review. Studies were identified by searching electronic databases, and writing to manufacturers. We identified randomised, double blind trials comparing topical NSAID with either placebo or another active treatment, in adults with chronic pain. The primary outcome was a reduction in pain of approximately 50% at two weeks, and secondary outcomes were local and systemic adverse events and adverse event-related withdrawals. Relative benefit and number-needed-to-treat (NNT), and relative harm and number-needed-to-harm (NNH) were calculated, and the effects of trial quality, validity and size, outcome reported, and condition treated, were examined by sensitivity analyses. Twelve new trials were added to 13 trials from a previous review. Fourteen double blind placebo-controlled trials had information from almost 1,500 patients. Topical NSAID was significantly better than placebo with relative benefit 1.9 (95% confidence interval 1.7 to 2.2), NNT 4.6 (95% confidence interval 3.8 to 5.9). Results were not affected by trial quality, validity or size, outcome reported, or condition treated. Three trials with 764 patients comparing a topical with an oral NSAID found no difference in efficacy. Local adverse events (6%), systemic adverse events (3%), or the numbers withdrawing due to an adverse event were the same for topical NSAID and placebo. Topical NSAIDs were effective and safe in treating chronic musculoskeletal conditions for two weeks. Larger and longer trials are necessary to fully elucidate the place of topical NSAIDs in clinical practice

Activity and Interactions of Liposomal Antibiotics in Presence of Polyanions and Sputum of Patients with Cystic Fibrosis

BACKGROUND:To compare the effectiveness of liposomal tobramycin or polymyxin B against Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA). METHODOLOGY:Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated. RESULTS:The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations--DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold. CONCLUSIONS:Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections

VITA-D: Cholecalciferol substitution in vitamin D deficient kidney transplant recipients: A randomized, placebo-controlled study to evaluate the post-transplant outcome

<p>Abstract</p> <p>Background</p> <p>Vitamin D does not only regulate calcium homeostasis but also plays an important role as an immune modulator. It influences the immune system through the induction of immune shifts and regulatory cells resulting in immunologic tolerance. As such, vitamin D is thought to exert beneficial effects within the transplant setting, especially in kidney transplant recipients, considering the high prevalence of vitamin D deficiency in kidney transplant recipients.</p> <p>Methods/Design</p> <p>The VITA-D study, a randomized, placebo-controlled, double-blind study with two parallel groups including a total of 200 kidney transplant recipients, is designed to investigate the immunomodulatory and renoprotective effects of cholecalciferol (vitamin D₃) within the transplant setting. Kidney transplant recipients found to have vitamin D deficiency defined as 25-hydroxyvitamin D₃< 50 nmol per liter will be randomly assigned to receive either oral cholecalciferol therapy or placebo and will be followed for one year. Cholecalciferol will be administered at a dose of 6800 International Units daily over a time period of one year.</p> <p>The objective is to evaluate the influence of vitamin D₃substitution in vitamin D deficient kidney transplant recipients on the post-transplant outcome. As a primary endpoint glomerular filtration rate calculated with the MDRD formula (modification of diet in renal disease) one year after kidney transplantation will be evaluated. Incidence of acute rejection episodes, and the number and severity of infections (analyzed by means of C-reactive protein) within the first year after transplantation will be monitored as well. As a secondary endpoint the influence of vitamin D₃on bone mineral density within the first year post-transplant will be assessed. Three DXA analyses will be performed, one within the first four weeks post-transplant, one five months and one twelve months after kidney transplantation.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov NCT00752401</p

The Circadian Deadenylase Nocturnin Is Necessary for Stabilization of the iNOS mRNA in Mice

Nocturnin is a member of the CCR4 deadenylase family, and its expression is under circadian control with peak levels at night. Because it can remove poly(A) tails from mRNAs, it is presumed to play a role in post-transcriptional control of circadian gene expression, but its target mRNAs are not known. Here we demonstrate that Nocturnin expression is acutely induced by the endotoxin lipopolysaccharide (LPS). Mouse embryo fibroblasts (MEFs) lacking Nocturnin exhibit normal patterns of acute induction of TNFα and iNOS mRNAs during the first three hours following LPS treatment, but by 24 hours, while TNFα mRNA levels are indistinguishable from WT cells, iNOS message is significantly reduced 20-fold. Accordingly, analysis of the stability of the mRNAs showed that loss of Nocturnin causes a significant decrease in the half-life of the iNOS mRNA (t1/2 = 3.3 hours in Nocturnin knockout MEFs vs. 12.4 hours in wild type MEFs), while having no effect on the TNFα message. Furthermore, mice lacking Nocturnin lose the normal nighttime peak of hepatic iNOS mRNA, and have improved survival following LPS injection. These data suggest that Nocturnin has a novel stabilizing activity that plays an important role in the circadian response to inflammatory signals