Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons.

Diacylglycerol lipase (DAGL)-α and -ß are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently lacking. Here, we describe the identification of a highly selective DAGL inhibitor using structure-guided and a chemoproteomics strategy to characterize the selectivity of the inhibitor in complex proteomes. Key to the success of this approach is the use of comparative and competitive activity-based proteome profiling (ABPP), in which broad-spectrum and tailor-made activity-based probes are combined to report on the inhibition of a protein family in its native environment. Competitive ABPP with broad-spectrum fluorophosphonate-based probes and specific ß-lactone-based probes led to the discovery of α-ketoheterocycle LEI105 as a potent, highly selective, and reversible dual DAGL-α/DAGL-ß inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metabolism including abhydrolase domain-containing protein 6, abhydrolase domain-containing protein 12, monoacylglycerol lipase, and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concentration-dependently reduced 2-AG levels, but not anandamide levels, in Neuro2A cells. We show that cannabinoid CB1-receptor-mediated short-term synaptic plasticity in a mouse hippocampal slice model can be reduced by LEI105. Thus, we have developed a highly selective DAGL inhibitor and provide new pharmacological evidence to support the hypothesis that "on demand biosynthesis" of 2-AG is responsible for retrograde signaling.

A study on the optics of copper indium gallium (di)selenide (CIGS) solar cells with ultra-thin absorber layers.

We present a systematic study of the effect of variation of the zinc oxide (ZnO) and copper indium gallium (di)selenide (CIGS) layer thickness on the absorption characteristics of CIGS solar cells using a simulation program based on finite element method (FEM). We show that the absorption in the CIGS layer does not decrease monotonically with its layer thickness due to interference effects. Ergo, high precision is required in the CIGS production process, especially when using ultra-thin absorber layers, to accurately realize the required thickness of the ZnO, cadmium sulfide (CdS) and CIGS layer. We show that patterning the ZnO window layer can strongly suppress these interference effects allowing a higher tolerance in the production process.

Inclined fluid-film flow with bottom filtration.

We investigate the interfacial instability of the steady uniform flow of a fluid-film layer down a permeable incline. A theoretical model is obtained by coupling the clear fluid flow equations governing the fluid film with Darcy's law for the filtration of fluid through the porous medium. A linear stability analysis is conducted and the effect of various parameters on the state of neutral stability is investigated. A simplified model is developed by reducing the dimensionality of the problem, which is then employed in order to determine the nonlinear effects on the stability of the equilibrium flow.

Reliability of spike and burst firing in thalamocortical relay cells.

The reliability and precision of the timing of spikes in a spike train is an important aspect of neuronal coding. We investigated reliability in thalamocortical relay (TCR) cells in the acute slice and also in a Morris-Lecar model with several extensions. A frozen Gaussian noise current, superimposed on a DC current, was injected into the TCR cell soma. The neuron responded with spike trains that showed trial-to-trial variability, due to amongst others slow changes in its internal state and the experimental setup. The DC current allowed to bring the neuron in different states, characterized by a well defined membrane voltage (between -80 and -50 mV) and by a specific firing regime that on depolarization gradually shifted from a predominantly bursting regime to a tonic spiking regime. The filtered frozen white noise generated a spike pattern output with a broad spike interval distribution. The coincidence factor and the Hunter and Milton measure were used as reliability measures of the output spike train. In the experimental TCR cell as well as the Morris-Lecar model cell the reliability depends on the shape (steepness) of the current input versus spike frequency output curve. The model also allowed to study the contribution of three relevant ionic membrane currents to reliability: a T-type calcium current, a cation selective h-current and a calcium dependent potassium current in order to allow bursting, investigate the consequences of a more complex current-frequency relation and produce realistic firing rates. The reliability of the output of the TCR cell increases with depolarization. In hyperpolarized states bursts are more reliable than single spikes. The analytically derived relations were capable to predict several of the experimentally recorded spike features.

Transjugular intrahepatic portosystemic shunts in the treatment of refractory ascites: results in 48 consecutive patients.

PURPOSE: To assess the efficacy, morbidity, and mortality involved in the creation of transjugular intrahepatic portosystemic shunts (TIPS) in the treatment of patients with refractory ascites in Child-Pugh classes B and C. MATERIALS AND METHODS: Forty-eight consecutive patients with refractory ascites were treated with TIPS creation in a tertiary care institution. They were followed for a median of 337 days (range, 3-1376 d). RESULTS: TIPS significantly decreased the portohepatic pressure gradient (20.7 +/- 5.9 mm Hg vs. 6.8 +/- 4.1 mm Hg; P < .0001). Seventy-three percent of patients had complete or partial response. One year after TIPS creation, survival was 73% in Child class B patients and 56% in Child class C patients. Thirteen patients experienced procedural complications (portal vein thrombosis, peritoneal bleeding, acute renal failure, pneumothorax, hemoptysis, spontaneous bacterial peritonitis, and heart failure) and TIPS creation was considered the cause of death in five patients (10.4%). Primary patency was 65% at 3 months and 23% at 1 year, but shunt obstruction was accessible for a second intervention. Ten patients (21%) had de novo encephalopathy after TIPS creation. CONCLUSIONS: This series suggests that TIPS is an effective treatment for refractory ascites; however, it is a challenging procedure and serious complications--usually renal and heart failure--can be seen. A careful selection of patients is mandatory.

Genetic heterogeneity of hypervariable region 1 of the hepatitis C virus (HCV) genome and sensitivity of HCV to alpha interferon therapy.

Hepatitis C virus (HCV) populations persist in vivo as a mixture of heterogeneous viruses called quasispecies. The relationship between the genetic heterogeneity of these variants and their responses to antiviral treatment remains to be elucidated. We have studied 26 virus strains to determine the influence of hypervariable region 1 (HVR-1) of the HCV genome on the effectiveness of alpha interferon (IFN-alpha) therapy. Following PCR amplification, we cloned and sequenced HVR-1. Pretreatment serum samples from 13 individuals with chronic hepatitis C whose virus was subsequently eradicated by treatment were compared with samples from 13 nonresponders matched according to the major factors known to influence the response, i.e., sex, genotype, and pretreatment serum HCV RNA concentration. The degree of virus variation was assessed by analyzing 20 clones per sample and by calculating nucleotide sequence entropy (complexity) and genetic distances (diversity). Types of mutational changes were also determined by calculating nonsynonymous substitutions per nonsynonymous site (K(a)) and synonymous substitutions per synonymous site (K(s)). The paired-comparison analysis of the nucleotide sequence entropy and genetic distance showed no statistical differences between responders and nonresponders. By contrast, nonsynonymous substitutions were more frequent than synonymous substitutions (P

Prostate-specific antigen in acute hepatitis and hepatocellular carcinoma.

BACKGROUND: Prostate-specific antigen (PSA) is the most important tumor marker in prostate cancer diagnosis and follow-up. Its catabolism by the liver has not influenced its use as a prostate marker until the recent report of a significant increase in a man and a woman with acute hepatitis. In addition, PSA was detected in liver tumor extracts, which warranted its evaluation in liver cytolysis and hepatocellular carcinoma. In this study, PSA was evaluated in a cohort of both sexes presenting either acute hepatitis or hepatocellular carcinoima. METHODS: Forty-two patients with acute hepatitis (21 male patients, 21 female patients) and 54 patients with hepatocellular carcinoma (31 male patients, 23 female patients) were tested for PSA by equimolar immunoassay (Abbott AxSYM Total PSA, Abbott Diagnostics, Rungis, France) and for relevant liver biological parameters (alpha-fetoprotein, alanine aminotransferase, aspartate aminotransferase, total bilirubin, and prothrombin rate). RESULTS: PSA was undetectable in all the female patients and was consistent with age in the males (PSA median and range in acute hepatitis, 0.36 microg/l (range, 0.05-1.3); in hepatocellular carcinoma, 0.36 microg/l (range, 0.02-3.9)). It did not correlate with alpha-fetoprotein and aminotransferases. CONCLUSIONS: Our results confirm the well-established reliability of PSA, and show that PSA remains a valid prostate marker in patients with acute hepatitis and hepatocellular carcinoma.

Transjugular intrahepatic portosystemic shunt in the treatment of refractory bleeding from ruptured gastric varices.

The optimal management of ruptured gastric varices in patients with cirrhosis has not been codified yet. The present study reports the use of transjugular intrahepatic portosystemic shunt (TIPS) in patients with refractory gastric variceal bleeding. Thirty-two consecutive patients were included. All had been unresponsive to vasoactive agents infusion, sclerotherapy, and/or tamponade and were considered poor surgical candidates. They were followed-up until death, transplantation, or at least 1 year (median: 509 days; range 4 to 2,230). Hemostasis was achieved in 18 out of 20 patients actively bleeding at the time of the procedure. In the whole sample of 32 patients, rebleeding rates were 14%, 26%, and 31%, respectively at 1 month, 6 months, and 1 year. De novo encephalopathy was observed in 5 (16%) patients. Seven patients experienced complications and consequently 4 of these patients died. TIPS primary patency rates were 84%, 74%, and 51%, respectively, at 1 month, 6 months, and 1 year. For the same periods of time, survival rates were 75%, 62%, and 59%. These results suggest that TIPS can be used in cirrhotic patients with refractory gastric variceal bleeding and are effective in achieving hemostasis as well as in preventing rebleeding.

Lack of effect of propranolol in the prevention of large oesophageal varices in patients with cirrhosis: a randomized trial. French-Speaking Club for the Study of Portal Hypertension.

OBJECTIVE: Beta-blockers have been shown to reduce portal pressure in patients with cirrhosis and limit the development of portosystemic shunts in portal hypertensive animals. Thus, a randomized double-blind trial was conducted to evaluate propranolol in the prevention of the development of large oesophageal varices in patients with cirrhosis without varices or with small varices. METHODS: One hundred and two patients received long-acting propranolol (160 mg/day) and 104 patients received a placebo. At inclusion, there was no significant difference between the two groups in terms of clinical characteristics or biochemical tests. At 2 years, the size of varices was estimated on video recordings. RESULTS: One-third of the patients were lost to follow-up, and 95%/97% of the remaining patients were compliant in the propranolol and placebo groups, respectively. At 2 years, the proportion of patients with large varices was 31% in the propranolol group and 14% in the placebo group (P< 0.05). Three and four patients bled in the propranolol and placebo groups, respectively, and nine and ten died, respectively. CONCLUSION: This trial suggests that propranolol administration cannot be recommended for the prevention of the development of large oesophageal varices in patients with cirrhosis; thus other studies are needed in selected subgroups of patients.

Pharmacokinetics of alphaIFN-2b in chronic hepatitis C virus patients undergoing chronic hemodialysis or with normal renal function: clinical implications.

In this prospective controlled study, the pharmacokinetic profiles of alpha-interferon 2b (alphaIFN-2b) were determined by the enzyme-linked immunosorbent assay method in hepatitis C virus-positive (HCV+) dialysis and nonuremic patients, after a single subcutaneous injection of 3 million units. Ten HCV+/RNA+ patients (group A) with a normal renal function (mean serum creatinine: 1.03 +/- 0.26 [SD] mg/dl) and 10 HCV+/RNA+ patients undergoing chronic hemodialysis (group B) were included. The pharmacokinetic profiles of alphaIFN were determined after the very first subcutaneous injection of the drug. Plasma alphaIFN concentrations were determined before the injection and then 1, 2, 3, 4, 6, 8, 12, 16, 20, 24, 28, 32, and 36 h after the injection. They were assessed by means of an enzyme-linked immunosorbent assay test. Patients from both groups had a similar body surface area. It was found that in group B: (1) the mean maximum (SD) serum alphaIFN concentration (Cmax) was significantly higher (52 +/- 12 pg/ml) than in group A (39 +/- 12 pg/ml; P = 0.03); (2) the time at which Cmax occurred (Tmax) was significantly higher (10 +/- 3 h) than in group A (7.5 +/- 2 h; P = 0.05); (3) the observed area under the plasma alphaIFN concentration-time curve was about twice as much, i.e., 936 +/- 212 pg x h/ml, as that for group A (485 +/- 184 pg x h/ml; P < 0.0001); and (4) the alphaIFN half-life was significantly longer (9.6 +/- 2.9 h) than in group A (5.3 +/- 1.3 h). As early as 24 h after the alphaIFN injection was given, the drug was no longer detectable in nonuremic patients' sera, whereas it could be detected up to the next injection in all of the dialysis patients' sera. When trough levels of alphaIFN were measured just before the 10th injection, they were always below the threshold level in the 10 patients from group A, i.e., 4.1 pg/ml, whereas in group B they were measurable for four of nine patients (P = 0.05) and ranged between 5.8 and 36.1 pg/ml. Severe neurologic side effects were observed only in group B, i.e., in three patients. Hemoglobin levels did significantly decrease but only in group B patients, and this was significantly correlated with the Cmax (r = 0.67; P = 0.03). This is the first controlled study to demonstrate that the clearance of alphaIFN is about twice as low in dialysis patients as in nonuremic patients. These results might be of relevance when deciding the optimal alphaIFN therapy scheme for HCV+ patients, either with normal renal function or undergoing chronic hemodialysis.

Better efficacy of a 12-month interferon alfa-2b retreatment in patients with chronic hepatitis C relapsing after a 6-month treatment: a multicenter, controlled, randomized trial. Le Groupe D'étude et De Traitement du Virus De L'hépatite C (Get.Vhc).

We studied the efficacy of three interferon alfa-2b (IFN-2b) regimens for the retreatment of patients with chronic hepatitis C (CHC) with prior complete response followed by relapse. Consecutive patients with CHC who had a complete biochemical response but relapse after a first course of 6 months of IFN with 3 million units (MU) given subcutaneously three times per week were enrolled in the study. Six to 24 months after the end of the first treatment, the patients were randomly assigned to receive IFN with either the same regimen (group 1), a regimen of 12 months with 3 MU (group 2), or a regimen of 6 months with 10 MU (group 3). Sustained biochemical response was defined as normal serum alanine transaminase (ALT) values during the follow-up and sustained virological response as a clearance of hepatitis C virus (HCV) RNA from the serum at the end of follow-up (6 months' posttreatment). Histological improvement was defined as a decrease of 1 point in Metavir score between the first liver biopsy and a biopsy performed at 6 months' postretreatment. Two hundred forty-seven patients were randomized: 75 to group 1, 91 to group 2, and 81 to group 3. In an intent-to-treat analysis, 12%, 36.3%, and 18.5% of patients had a sustained biochemical response after retreatment in groups 1, 2, and 3, respectively (P <.001); 13. 8%, 32.4%, and 17.2% of patients had a sustained virological response after retreatment in groups 1, 2, and 3, respectively (P <. 05). A low viral load and patients in group 2 were independently associated with a sustained biochemical response. A low Knodell score index before treatment, patients with a high level of ALT before retreatment, genotype 3, low viral load, and patients in group 2 were independently associated with sustained virological response. Younger age, a high level of ALT, a low level of gamma-glutamyl transferase before retreatment, low viral load, and patients in group 2 were independently associated with sustained biochemical and virological response. Among the 80 patients with repeated liver biopsies, 47.6% had improved histological activity scores; this improvement was associated with a sustained biochemical and virological response. In patients with CHC initially treated with 3 MU of IFN given subcutaneously three times per week over a 6-month period, and who subsequently developed a relapse after a biochemical response, retreatment with a regimen of 3 MU of IFN given three times per week for 12 months produced better biochemical and virological sustained response rates than regimens involving a higher dose or a shorter duration of retreatment. The biochemical and virological sustained response was associated with histological improvement.

Baseline level and early suppression of serum HCV RNA for predicting sustained complete response to alpha-interferon therapy.

The relationship between serum hepatitis C virus (HCV) RNA and the outcome of alpha-interferon (alpha-IFN) therapy in patients with chronic hepatitis C has important implications for therapeutic research and clinical care. Serum HCV RNA was tested for HCV genotype and quantified by a standardized reverse transcriptase-polymerase chain reaction assay as a measure of viral load in a cohort of 130 patients with chronic hepatitis C treated with alpha-IFN at a standard dose of 3 million units three times a week scheduled for 6 (n = 50) or 12 months (n = 76). Twenty-one of 126 evaluable patients (16.7%) developed a sustained complete response to alpha-IFN according to biochemical and virological criteria. The 3 pretreatment independent factors associated with a sustained complete response were a low baseline serum HCV RNA concentration, non-1 HCV genotype, and female sex. A multivariate logistic regression model, with pretreatment and month 1 variables, showed that a lower baseline serum HCV RNA concentration, female sex, and a greater suppression of RNA were the significant predictors of sustained complete response. The lowest baseline serum HCV RNA concentration was observed in patients with genotype 2 infection and the greatest decrease in HCV RNA from baseline to month 1 in those with genotype 3. The findings suggest that measuring HCV RNA in serum before and soon after beginning treatment can be helpful for selecting patients who are most likely to have a sustained complete response to standard schedule of alpha-IFN and for identifying patients in whom alternative strategies should be examined.

Genes of the major histocompatibility complex class II influence the outcome of hepatitis C virus infection.

BACKGROUND & AIMS: The host's immune response may influence the course of hepatitis C virus (HCV) infection. The aim of this study was to investigate the distribution of HLA class II alleles in white subjects who spontaneously recovered from HCV infection compared with that in patients with persistent infection. METHODS: HLA-DRB1 and -DQB1 typing were performed in 103 consecutive patients with persistent HCV infection (HCV antibody positive, HCV RNA positive) and in 25 subjects with transient HCV infection (HCV antibody positive, persistently negative HCV RNA). RESULTS: No significant differences between subjects with transient or persistent infection were observed for age, sex, source of infection, or HCV serotype. The frequency of DQB1*0301 and DRB1*1101 alleles was higher in patients with transient infection than in those with persistent infection (84% vs. 30.8%, 40% vs. 9.8%; P < 0.01 and P < 0.02, respectively [Bonferroni correction]). DRB1 and DQB1 alleles did not influence viral load as an independent factor. Mean Knodell's scores were lower in patients with DQB1*0301 allele (6.12 +/- 0.4) than in those negative for DQB1*0301 (7.37 +/- 0.3; P < 0.05). CONCLUSIONS: Our results suggest that host- rather than virus-related factors are probably involved in the spontaneous clearance of HCV.

Histopathologic analysis of transjugular intrahepatic portosystemic shunts.

This prospective study was undertaken in 17 patients treated with 22 transjugular intrahepatic portosystemic shunt (TIPS) procedures and aimed to evaluate the nature and causes of short- and long-term shunt abnormalities. Specimens were collected after autopsy in 6 patients and after liver transplantation in 11 patients; the time from shunting ranging from 4 to 385 days. During the first 2 weeks the shunt surface was covered by poorly organized fibrin platelet clots that were mixed with inflammatory and red blood cells. Thereafter, a pseudointima developed, initially made of loose granulation tissue that was formed by edema, myofibroblasts, neo-capillaries, collagen fibers, and inflammatory cells. This pseudo-intima extended with time as the myofibroblasts increased in number, as the collagen fibers thickened, and as the inflammatory cells grew more scarce. Its thickness ranged from 50 to 3,500 microm, the maximal values being observed in the middle of the shunt. The shunt was obstructed by a clot in 4 patients (18%). In 7 shunts (31.8%) the pseudo-intima was thicker than 1,000 microm and was referred to as hyperplastic pseudo-intima. It showed more dense inflammatory infiltrate and anarchic deposition of more scarce collagen fibers. Pseudo-intima hyperplasia was associated with previously diagnosed thrombosis, which had been treated by dilatation in 2 cases and by biliary deposits in 3 cases, while it remained unexplained in 2 cases. Accordingly, this study confirms the following: 1) that early TIPS obstruction results from thrombosis; 2) that 2 weeks after insertion, the stent is covered by a smooth pseudo-intima; 3) that thereafter, pseudo-intimal hyperplasia is the main cause of TIPS stenosis or occlusion; and 4) that biliary secretions and previous thrombosis could be important triggering mechanisms for this pseudo-intima overgrowth.