Suppression of hepatitis C virus by the flavonoid quercetin is mediated by inhibition of NS3 protease activity.

Phytochemicals exert antiviral activity and may play a potential therapeutic role in hepatitis C virus (HCV) infection. In this work, we aimed to isolate NS3 inhibitors from traditional Indian medicinal plants that were found, in our earlier study, to inhibit HCV NS3 protease activity and to evaluate their potential to inhibit HCV replication. A potent inhibitory effect of NS3 catalytic activity was obtained with Embelia ribes plant extracts. Quercetin, a ubiquitous plant flavonoid, was identified as the active substance in the fractioned extract. It was found to inhibit NS3 activity in a specific dose-dependent manner in an in vitro catalysis assay. Quercetin inhibited HCV RNA replication as analysed in the subgenomic HCV RNA replicon system. It also inhibited HCV infectious virus production in the HCV infectious cell culture system (HCVcc), as analysed by the focus-forming unit reduction assay and HCV RNA real-time PCR. The inhibitory effect of quercetin was also obtained when using a model system in which NS3 engineered substrates were introduced in NS3-expressing cells, providing evidence that inhibition in vivo could be directed to the NS3 and do not involve other HCV proteins. Our work demonstrates that quercetin has a direct inhibitory effect on the HCV NS3 protease. These results point to the potential of quercetin as a natural nontoxic anti-HCV agent reducing viral production by inhibiting both NS3 and heat shock proteins essential for HCV replication.

Immunohistochemical evaluation of hepatic oval cell activation and differentiation toward pancreatic beta-cell phenotype in streptozotocin-induced diabetic mice.

Oval cell (OvCs) involvement in regeneration is a well known phenomenon in models of liver injury, however, the activation of these cells following streptozotocin (STZ)-induced diabetes has not been studied yet. Differentiation of liver cells toward insulin-producing cells in diabetes has been reported, but the cell phenotype is still unclear. The aim of the present study was to confirm by immunohistochemical analysis, the activation of OvCs and their ability to express pancreatic beta-cell phenotype in STZ-induced diabetic mice. Using specific anti-A6 antibodies for mouse OvCs, we found a three-fold increase in periportal number and two-fold higher density of OvCs in diabetic livers, when compared to controls. Unlike non-diabetic controls, double staining technique showed co-localization of A6 and proinsulin in the cytoplasm of OvCs of diabetic animals, but no insulin staining was detected, probably reflecting the premature character of OvCs differentiation toward beta-cell-like phenotype. These data add valuable information concerning the nature and the stage of functional maturity of liver cells undergoing differentiation toward beta-cell phenotype in STZ-induced diabetic animals.

Population coding with motion energy filters: the impact of correlations.

The codes obtained from the responses of large populations of neurons are known as population codes. Several studies have shown that the amount of information conveyed by such codes, and the format of this information, is highly dependent on the pattern of correlations. However, very little is known about the impact of response correlations (as found in actual cortical circuits) on neural coding. To address this problem, we investigated the properties of population codes obtained from motion energy filters, which provide one of the best models for motion selectivity in early visual areas. It is therefore likely that the correlations that arise among energy filters also arise among motion-selective neurons. We adopted an ideal observer approach to analyze filter responses to three sets of images: noisy sine gratings, random dots kinematograms, and images of natural scenes. We report that in our model, the structure of the population code varies with the type of image. We also show that for all sets of images, correlations convey a large fraction of the information: 40% to 90% of the total information. Moreover, ignoring those correlations when decoding leads to considerable information loss-from 50% to 93%, depending on the image type. Finally we show that it is important to consider a large population of motion energy filters in order to see the impact of correlations. Study of pairs of neurons, as is often done experimentally, can underestimate the effect of correlations.

Localist attractor networks.

Attractor networks, which map an input space to a discrete output space, are useful for pattern completion--cleaning up noisy or missing input features. However, designing a net to have a given set of attractors is notoriously tricky; training procedures are CPU intensive and often produce spurious attractors and ill-conditioned attractor basins. These difficulties occur because each connection in the network participates in the encoding of multiple attractors. We describe an alternative formulation of attractor networks in which the encoding of knowledge is local, not distributed. Although localist attractor networks have similar dynamics to their distributed counterparts, they are much easier to work with and interpret. We propose a statistical formulation of localist attractor net dynamics, which yields a convergence proof and a mathematical interpretation of model parameters. We present simulation experiments that explore the behavior of localist attractor networks, showing that they yield few spurious attractors, and they readily exhibit two desirable properties of psychological and neurobiological models: priming (faster convergence to an attractor if the attractor has been recently visited) and gang effects (in which the presence of an attractor enhances the attractor basins of neighboring attractors).

Cell transformation induced by hepatitis C virus NS3 serine protease.

Persistent infection with hepatitis C virus (HCV) may lead to hepatocellular carcinoma (HCC). It has been suggested that HCV-encoded proteins are directly involved in the tumorigenic process. The HCV nonstructural protein NS3 has been identified as a virus-encoded serine protease. To study whether HCV NS3 has oncogenic activity, nontumorigenic rat fibroblast (RF) cells were stably transfected with an expression vector containing cDNA for the NS3 serine protease (nucleotides 3356-4080). The NS3 serine protease activity was determined in the transfected cells. The transfected cells grew rapidly and proliferated serum independently, lost contact inhibition, grew anchorage independently in soft agar and induced significant tumour formation in nude mice. Cells transfected with an expression vector containing a mutated NS3 serine protease (serine 139 to alanine at the catalytic site) showed no transforming abilities; their growth was dependent on serum and they did not grow anchorage independently in soft agar. Moreover, cells transfected with the NS3 serine protease and treated with the chymotrypsin inhibitors TPCK and PMSF (a serine protease inhibitor) lost their transforming feature. These results suggest that the NS3 serine protease of HCV is involved in cell transformation and that the ability to transform requires an active enzyme.

The addition of mycophenolate mofetil for suppressing hepatitis B virus replication in liver recipients who developed lamivudine resistance--no beneficial effect.

BACKGROUND: Mycophenolate mofetil is used as an immunosuppressive agent in liver transplant recipients. Its active compound, mycophenolic acid, also inhibits the replication of Epstein-Barr virus and human immunodeficiency virus. Based on a study indicating the effectiveness of mycophenolate mofetil on hepatitis B virus (HBV) replication in infected human hepatocyte cells in culture, we examined the efficacy of mycophenolate mofetil in suppressing HBV replication in lamivudine-resistant liver allograft recipients with recurrent HBV infection. METHOD: The study population included four liver allograft recipients (three males, one female), median age 51 years (range 41-57 years), with recurrent HBV infection who proved to be resistant to lamivudine. All received standard maintenance immunosuppression therapy. Median pretreatment serum alanine aminotransferase level was 75 mu/L (range 39-182 mu/L) and HBV DNA level (quantitative dot blot), 70 pg/ml (range: 10-5,000 pg/ml). Mycophenolate mofetil, 1.0 g p.o. twice daily, was administered for 8 weeks, concomitant with a reduction in the maintenance corticosteroid and cyclosporine doses. RESULTS: After mycophenolate mofetil was administered, the serum alanine aminotransferase level increased in two patients, did not change in one, and decreased in one. Serum HBV DNA levels increased in three patients and decreased (nonsignficantly) in only one patient. Two patients complained of abdominal pain and nausea. CONCLUSIONS: Mycophenolate mofetil at the dosage used is not effective in suppressing HBV replication after liver transplantation.

Occlusion, symmetry, and object-based attention: reply to Saiki (2000).

J. Saiki (2000) argued that, because the stimuli used by M. Behrmann, R. S. Zemel, and M. C. Mozer (1998) were confounded by symmetry, conclusions about whether amodally completed objects can benefit from object-based attention are unwarranted. Here, the authors examine J. Saiki's claim further and expand on their view of the mechanisms underlying object-based attention, suggesting that perceptual organization is the process whereby features from a single object are selectively attended. In light of this, they claim that heuristics such as symmetry and collinearity play an important role in the facilitation of features from a single object. In support of this claim, they present data from a further experiment using displays that exploit common fate, another grouping heuristic, and show that, under these conditions, the hallmark of object-based attention, a single-object advantage, is obtained for the occluded (amodally completed) shapes.

Fulminant non--A-G viral hepatitis leading to liver transplantation.

BACKGROUND: All hepatotropic viruses are known to cause fulminant hepatic failure (FHF). However, in 30% to 40% of patients with FHF, the precise cause remains unknown. We aimed to better define this subgroup. METHODS: We evaluated the clinical course and outcome of 7 patients admitted during a 22-month period with fulminant viral hepatitis leading to liver transplantation; none had serologic or molecular evidence of hepatitis A, B, C, D, E, or G viral infection, thus the term non-A-G viral hepatitis. All known etiologies of FHF were excluded. RESULTS: All patients had prodromal symptoms suggestive of viral causes. Mean age was 30 years. The interval between onset of jaundice and appearance of encephalopathy was 23 days (range, 4-50 days). Five patients had grade III/IV encephalopathy. Serum alanine aminotransferase levels showed a single peak of activity. The duration between first symptoms and liver transplantation was 28 days (range, 12-71 days). Results of histological study of the explanted liver showed submassive (4 patients) or massive (3 patients) hepatocyte necrosis. In all patients, results of polymerase chain reaction analysis did not detect hepatitis B virus DNA, hepatitis C virus RNA, or hepatitis G virus RNA in the explanted liver. After transplantation, 2 patients showed (6 months later) increased liver enzyme levels of undetermined cause, and results of a liver biopsy showed mild lobular hepatitis; 1 patient had lymphoproliferative disorder (Epstein-Barr virus-originated); and 1 patient, aplastic anemia, which is known to be associated with seronegative viral hepatitis. The latter patient died, whereas the other 6 patients are alive (survival rate, 86%). CONCLUSIONS: Our patients with non-A-G viral hepatitis had a severe acute onset with progressive FHF requiring liver transplantation. There is some suggestion of recurrent viral disease after transplantation implicating other unknown viruses in the etiology.

Mutations at vicinity of catalytic sites of hepatitis C virus NS3 serine protease gene isolated from hepatocellular carcinoma tissue.

The mechanism of hepatitis C virus (HCV) -induced hepatotocellular carcinoma (HCC) is still unknown, but in vitro studies clearly suggest that HCV proteins exert a direct effect on liver carcinogenesis. HCV NS3 serine protease is known to play a key role in the life cycle of the virus and may interact with the host cellular regulatory proteins. The aim of the present study was to conduct a genetic analysis of the HCV NS3 gene coding for the serine protease isolated from serum, tumor, and nontumor tissue of HCC patients. RNA was extracted and HCV cDNA was amplified by nested reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence comparison yielded unique changes at the vicinity of the catalytic sites of the NS3 clones isolated only from HCC tissue. These changes included the insertion of a "large" and charged amino acid, substitution of a polar with a hydrophobic amino acid, and substitution of a charged with a polar amino acid. Those changes affect the electrostatic charge around the active site, and thus the activity and substrate specificity of the serine protease. This is the first study to define significant amino acid changes at the catalytic domain of the NS3 serine protease gene isolated from HCC tissue.

Information processing with population codes.

Information is encoded in the brain by populations or clusters of cells, rather than by single cells. This encoding strategy is known as population coding. Here we review the standard use of population codes for encoding and decoding information, and consider how population codes can be used to support neural computations such as noise removal and nonlinear mapping. More radical ideas about how population codes may directly represent information about stimulus uncertainty are also discussed.

Streptozotocin and alloxan-based selection improves toxin resistance of insulin-producing RINm cells.

The aim of our study was to develop a method for selection of subpopulations of insulin producing RINm cells with higher resistance to beta cell toxins. Cells, resistant to streptozotocin (RINmS) and alloxan (RINmA), were obtained by repeated exposure of parental RINm cells to these two toxins, while the defense capacity was estimated by the MTT colorimetric method, and [3H]-thymidine incorporation assay. We found that RINmS and RINmA displayed higher resistance to both streptozotocin (STZ) and alloxan (AL) when compared to the parental RINm cells. In contrast, no differences in sensitivity to hydrogen peroxide were found between toxin selected and parental cells. Partial protection from the toxic effect of STZ and AL was obtained only in the parental RINm cells after preincubation of cells with the unmetabolizable 3-O-methyl-glucose. The possibility that GLUT-2 is involved in cell sensitivity to toxins was confirmed by Western blot analysis, which showed higher expression of GLUT-2 in parental RINm compared to RINmS and RINmA cells. In addition to the higher cell defense property evidenced in the selected cells, we also found higher insulin content and insulin secretion in both RINmS and RINmA cells when compared to the parental RINm cells. In conclusion, STZ and AL treatment can be used for selection of cell sub-populations with higher cell defense properties and hormone production. The different GLUT-2 expression in parental and resistant cells suggest involvement of GLUT-2 in mechanisms of cell response to different toxins.

Nitric oxide metabolites in decompensated liver cirrhosis.

High levels of nitric oxide are thought to be the cause of some of the complications associated with decompensated end-stage liver disease. To assess nitric oxide metabolism in cirrhotic patients, we measured the levels of nitric oxide metabolites (nitrosohemoglobin, methemoglobin, nitrate, and nitrite) in normal subjects, in patients with decompensated cirrhosis, in patients with renal failure (model for impaired NO metabolites excretion), and in patients with mononitrates-treated anginal syndrome (model for exogenous nitric oxide). When compared to controls, patients with decompensated cirrhosis exhibited elevated levels of nitrate only. A significant increase of nitrate was also noted in patients receiving exogenous nitrates, whereas patients with impaired excretion had significantly elevated levels of both nitrite and nitrate. In conclusion, nitric oxide metabolism in patients with decompensated cirrhosis is similar to that of patients receiving nitric oxide from an exogenous source. Renal impairment, whether alone or associated with cirrhosis, causes a change in nitric oxide metabolism. These findings may have clinical implications for nitrates treatment in patients with decompensated cirrhosis.

Association of lamivudine resistance in recurrent hepatitis B after liver transplantation with advanced hepatic fibrosis.

BACKGROUND: Orthotopic liver transplantation (OLT) in patients with hepatitis B virus (HBV) infection is known to be associated with a high recurrence rate and poor prognosis. Lamivudine, a nucleoside analogue, is a potent inhibitor of HBV replication, but it is associated with a 14-39% rate of resistance. METHODS: We report on four patients who underwent OLT for HBV infection. In all cases, the HBV infection recurred in the grafted liver and was treated with lamivudine (100 mg daily) on a compassionate-use basis. The patients were monitored closely for serum liver enzymes, hepatitis B surface antigen and HBV DNA (by hybridization). Liver biopsy was performed before and after lamivudine therapy. HBV DNA was amplified from serum for each patient and sequenced through a conserved polymerase domain, the tyrosine-methionine-aspartate-aspartate (YMDD) locus. RESULTS: All four patients exhibited lamivudine resistance 9-20 months after initiation of the drug. In all patients with a clinically mild disease, liver histology findings (12-24 months after lamivudine therapy) showed progressive fibrosis as compared to biopsies performed before lamivudine therapy, with a significant increase (> or =2 points) in the Knodell score in three patients. Moreover, two patients exhibited worsening of the necroinflammatory process. A mutation at the YMDD motif of the HBV polymerase gene was detected in all cases. CONCLUSIONS: Lamivudine resistance frequently occurs in patients with recurrent HBV infection after OLT and is associated with advanced hepatic fibrosis and necroinflammatory process. A combination of antiviral therapies may be necessary.

Efficacy of lamivudine in patients with hepatitis B virus precore mutant infection before and after liver transplantation.

OBJECTIVE: Hepatitis B virus (HBV) precore mutant infection is associated with a more severe liver disease and a poorer response to interferon. We evaluated the efficacy and tolerance of lamivudine to induce complete and sustained suppression of viral replication in seven patients infected with HBV precore mutant (HBeAg-/HBeAb+/HBV DNA+) (in three patients mutation at codon 1896 was detected by direct sequencing). METHODS: Of the seven patients, five had decompensated HBV cirrhosis in a replicative phase and were liver transplant candidates (Group A) and two patients underwent orthotopic liver transplantation (OLT) for HBV liver cirrhosis and developed recurrent HBV infection in the grafted liver (Group B). Lamivudine 100 mg daily was administered orally for a period of 6-75 wk. RESULTS: After 6-8 wk lamivudine therapy was well tolerated and successfully suppressed HBV replication to an undetectable serum level of HBV DNA by polymerase chain reaction in six patients. In Group A, two patients underwent successful OLT with no evidence of HBV reinfection 2-14 months later. Lamivudine was continued after OLT with no episodes of rejection. Three patients died before a suitable liver could be found (one remained serum HBV DNA+ after 6 wk of lamivudine therapy). In Group B, 9-14 months after lamivudine therapy both patients developed lamivudine resistance (increased liver enzymes, reappearance of serum HBsAg and HBV DNA [by hybridization]). In both patients liver histology had progressed and in both, mutation at codon 552 of the HBV polymerase gene was detected. CONCLUSIONS: Lamivudine is well tolerated in patients with decompensated liver cirrhosis due to HBV precore mutant infection who are liver transplant candidates. In four patients (80%) potent suppression of viral replication was detected, allowing OLT to be performed. However, post-OLT, a resistant mutant developed under lamivudine therapy. Combination therapy with other antiviral agents should be evaluated to discourage the emergence of lamivudine-resistant mutants.

Object-based attention and occlusion: evidence from normal participants and a computational model.

One way of perceptually organizing a complex visual scene is to attend selectively to information in a particular physical location. Another way of reducing the complexity in the input is to attend selectively to an individual object in the scene and to process its elements preferentially. This latter, object-based attention process was examined, and the predicted superiority for reporting features from 1 relative to 2 objects was replicated in a series of experiments. This object-based process was robust even under conditions of occlusion, although there were some boundary conditions on its operation. Finally, an account of the data is provided via simulations of the findings in a computational model. The claim is that object-based attention arises from a mechanisms that groups together those features based on internal representations developed over perceptual experience and then preferentially gates these features for later, selective processing.

Reliable disparity estimation through selective integration.

A network model of disparity estimation was developed based on disparity-selective neurons, such as those found in the early stages of processing in the visual cortex. The model accurately estimated multiple disparities in regions, which may be caused by transparency or occlusion. The selective integration of reliable local estimates enabled the network to generate accurate disparity estimates on normal and transparent random-dot stereograms. The model was consistent with human psychophysical results on the effects of spatial-frequency filtering on disparity sensitivity. The responses of neurons in macaque area V2 to random-dot stereograms are consistent with the prediction of the model that a subset of neurons responsible for disparity selection should be sensitive to disparity gradients.

Viremia, genetic heterogeneity, and immunity to hepatitis G/GB-C virus in multiply transfused patients with thalassemia.

BACKGROUND: Thalassemia patients are at high risk for posttransfusion hepatitis. Hepatitis G virus (HGV) has been suspected of being responsible for acute and chronic hepatitis. STUDY DESIGN AND METHODS: The prevalence of HGV infection, its possible association to liver disease, the genetic heterogeneity among the various HGV isolates, and immunity to HGV were studied in 36 thalassemia patients with reverse transcriptase-polymerase chain reaction assay and sequence analysis. RESULTS: HGV RNA was detected in seven patients (19.4%), only two of whom had evidence of hepatitis C virus infection as well. Sequence analysis of the NS3 gene from isolates of the five patients infected with HGV alone revealed 84.7 to 90.9 percent homology at the nucleotide level. Prolonged HGV viremia was not associated with significant liver enzyme elevation. All five patients were chronically infected with the same viral strain. E2 antibodies were detected in 57 percent of the HGV-nonviremic patients and in only 1 of 7 viremic patients. CONCLUSION: HGV is associated with persistent viremia but not with significant biochemical evidence of liver damage. There is some genetic heterogeneity among HGV isolates from thalassemia patients in Israel.

Treatment of colorectal liver metastases by cryotherapy.

One hundred fifty-eight procedures were performed on 136 patients with unresectable hepatic metastases using hepatic cryotherapy to ablate the tumors. The median age was 62 years. Patients included 90 males and 46 females. Fifty-eight patients had synchronous metastases, 55 had bilobar lesions, and 90 had precryo chemotherapy. Median preoperative carcinoembryonic antigen (CEA) level was 14.4 ng/dl. The numbers of lesions treated, frozen, and resected were two and one. Median survival of all patients was 30 months. Survival for 39 patients was 37 months. Patients with a CEA level > 100 ng/dl had a statistically worse survival rate than those with a level < 100 ng/dl (P < .001). Twenty patients underwent recryotherapy with median survival of 34 months. Recurrent disease developed in 78% of patients--82% of the patients developed liver recurrence. Complication rates were comparable to liver resection. Operative mortality was 3.7%. Hepatic cryotherapy is effective and safe in treating colorectal hepatic metastases under ultrasound guidance.

Probabilistic interpretation of population codes.

We present a general encoding-decoding framework for interpreting the activity of a population of units. A standard population code interpretation method, the Poisson model, starts from a description as to how a single value of an underlying quantity can generate the activities of each unit in the population. In casting it in the encoding-decoding framework, we find that this model is too restrictive to describe fully the activities of units in population codes in higher processing areas, such as the medial temporal area. Under a more powerful model, the population activity can convey information not only about a single value of some quantity but also about its whole distribution, including its variance, and perhaps even the certainty the system has in the actual presence in the world of the entity generating this quantity. We propose a novel method for forming such probabilistic interpretations of population codes and compare it to the existing method.