Hepatitis C virus dynamics and cellular gene expression in uPA-SCID chimeric mice with humanized livers during intravenous silibinin monotherapy.

Legalon SIL (SIL) is a chemically hydrophilized version of silibinin, an extract of milk thistle (Silybum marianum) seeds that has exhibited hepatoprotective and antiviral effectiveness against hepatitis C virus (HCV) in patients leading to viral clearance in combination with ribavirin. To elucidate the incompletely understood mode of action of SIL against HCV, mathematical modelling of HCV kinetics and human hepatocyte gene expression studies were performed in uPA-SCID-chimeric mice with humanized livers. Chronically HCV-infected mice (n = 15) were treated for 14 days with daily intravenous SIL at 469, 265 or 61.5 mg/kg. Serum HCV and human albumin (hAlb) were measured frequently, and liver HCV RNA was analysed at days 3 and 14. Microarray analysis of human hepatocyte gene expression was performed at days 0, 3 and 14 of treatment. While hAlb remained constant, a biphasic viral decline in serum was observed consisting of a rapid 1st phase followed by a second slower phase (or plateau with the two lower SIL dosings). SIL effectiveness in blocking viral production was similar among dosing groups (median ε = 77%). However, the rate of HCV-infected hepatocyte decline, δ, was dose-dependent. Intracellular HCV RNA levels correlated (r = 0.66, P = 0.01) with serum HCV RNA. Pathway analysis revealed increased anti-inflammatory and antiproliferative gene expression in human hepatocytes in SIL-treated mice. The results suggest that SIL could lead to a continuous second-phase viral decline, that is potentially viral clearance, in the absence of adaptive immune response along with increased anti-inflammatory and antiproliferative gene expression in human hepatocytes.

Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells.

The objective of this study was to characterize Huh7 cells' baseline capacity to metabolize drugs and to investigate whether the drug metabolism was enhanced upon treatment with dimethyl sulfoxide (DMSO). The messenger RNA (mRNA) levels of major Phase I and Phase II enzymes were determined by quantitative real-time-polymerase chain reaction (RT-PCR), and activities of major drug-metabolizing enzymes were examined using probe drugs by analysing relevant metabolite production rates. The expression levels of drug-metabolizing enzymes in control Huh7 cells were generally very low, but DMSO treatment dramatically increased the mRNA levels of most drug-metabolizing enzymes as well as other liver-specific proteins. Importantly, functionality assays confirmed concomitant increases in drug-metabolizing enzyme activity. Additionally, treatment of the Huh7 cells with 3-methylcholanthrene induced cytochrome P450 (CYP) 1A1 expression. The results indicate that DMSO treatment of Huh7 cells profoundly enhances their differentiation state, thus improving the usefulness of this common cell line as an in vitro hepatocyte model.

Eating disorders and concurrent psychopathology: a reconceptualisation of clinical need through Rasch analysis.

Therapies for anorexia nervosa (AN), bulimia nervosa (BN), binge eating disorder (BED) and eating disorder not otherwise specified (EDNOS) take as their focus, perhaps of necessity, the eating disorder symptomatology. However, there is increasing evidence of comorbidity of psychopathological mechanisms (e.g. perfectionism, depression) with eating disorders which, left untreated, may diminish any therapeutic effects. Thus identifying the extent of comorbidity in an eating disorder population and assessing the relationship between psychopathological mechanisms and the eating disorder is important. Rasch analysis was applied to the findings from questionnaires (EDI-2; SCL90-R) completed by 105 female patients referred to an eating disorder unit. General psychopathology was found to be more indicative of 'caseness' than eating disorder psychopathology. In particular, interpersonal sensitivity, depression and mild interpersonal aspects of psychoticism emerged as important factors across eating disorders. The comorbidity of psychopathological mechanisms needs to be given consideration in the successful treatment of eating disorders.

The effect of aprotinin on interleukin-8 concentration and leukocyte adhesion molecule expression in an isolated cardiopulmonary bypass system.

We examined the effect of aprotinin on the production of interleukin-8 and the expression of leukocyte adhesion molecules in an isolated cardiopulmonary bypass circuit. Five healthy volunteers each donated 500 ml blood which was divided into equal portions (A) and (B). Portion A was treated with aprotinin 250000 kallikrein inactivator units and added to a circuit primed with a further 250000 kallikrein inactivator units. Portion B was simultaneously treated with 25 ml normal saline and added to an aprotinin-free circuit. Samples of blood were taken at donation, after addition of a drug and at 30-min intervals throughout isolated cardiopulmonary bypass. The interleukin-8 increase was smaller in the aprotinin group but the difference between groups was not statistically significant (p > 0.05). In contrast, aprotinin inhibited the cardiopulmonary bypass-induced increase in monocyte (p < 0.05) and granulocyte (p < 0.01) CD18 and the increase in granulocyte, but not monocyte, CD11b (p < 0.05). Cardiopulmonary bypass had little effect on expression of L-selectin. These results confirm that aprotinin attenuates leukocyte activation during isolated cardiopulmonary bypass.

The role of herpes simplex virus ICP27 in the regulation of UL24 gene expression by differential polyadenylation.

Herpes simplex virus specifies two sets of transcripts from the UL24 gene, short transcripts (e.g., 1.4 kb), processed at the UL24 poly(A) site, and long transcripts (e.g., 5.6 kb), processed at the UL26 poly(A) site. The 1.4- and 5.6-kb transcripts initiate from the same promoter but are expressed with early and late kinetics, respectively. Measurements of transcript levels following actinomycin D treatment of infected cells revealed that the 1.4- and 5.6-kb UL24 transcripts have similar stabilities, consistent with UL24 transcript kinetics being regulated by differential polyadenylation rather than by differential stabilities. Although the UL24 poly(A) site, which gives rise to short transcripts, is encountered first during processing, long transcripts processed at the UL26 site are equally or more abundant; thus, operationally, the UL24 site is weak. Using a series of viral ICP27 mutants, we investigated whether ICP27, which has been suggested to stimulate the usage of weak poly(A) sites, stimulates 1.4-kb transcript accumulation. We found that accumulation of 1.4-kb transcripts did not require ICP27 during viral infection. Rather, ICP27 was required for full expression of 5.6-kb transcripts, and the decrease in 5. 6-kb transcripts relative to 1.4-kb transcripts was not due solely to reduced DNA synthesis. Our results indicate that temporal expression of UL24 transcripts can be regulated by differential polyadenylation and that although ICP27 is not required for processing at the operationally weak UL24 poly(A) site, it does modulate 5.6-kb transcript levels at a step subsequent to transcriptional initiation.

Comparison of the intranuclear distributions of herpes simplex virus proteins involved in various viral functions.

Herpesviral transcription, DNA synthesis, and capsid assembly occur within the infected cell nucleus. To further define the spatial relationship among these processes, we have examined the intranuclear distributions of viral DNA replication, gene regulatory, and capsid proteins using dual label immunofluorescence and confocal microscopy. We observed that several of the viral DNA replication proteins localize preferentially to punctate structures within replication compartments while the major transcriptional activator, ICP4, and the ICP27 regulatory protein show a more diffuse distribution within replication compartments. The viral proteins that show a punctate distribution in replication compartments redistribute from these compartments to prereplicative sites when viral DNA replication is inhibited, whereas viral proteins that show a diffuse distribution remain within replication compartments when viral DNA replication is inhibited. Thus the sites of viral DNA replication and late transcription appear to be distinct but codistribute within the boundaries of replication compartments. The major capsid protein, ICP5, also localizes initially to a diffuse distribution within replication compartments, but during the time of maximal progeny virus assembly, ICP5 becomes localized to punctate structures within replication compartments that are often near the punctate structures occupied by viral DNA replication proteins. Hence the processes of viral DNA replication, late transcription, and capsid assembly show a general overlapping distribution within replication compartments but appear to be located at distinct sites within these regions of the infected cell nucleus.

Assembly of herpes simplex virus replication proteins at two distinct intranuclear sites.

Herpes simplex virus DNA replication proteins amplify the viral genome in large globular replication compartments within infected cell nuclei. In the absence of viral DNA synthesis, the replication proteins accumulate at punctate foci throughout the nucleus referred to as prereplicative sites. To more thoroughly understand the nature of this nuclear assembly process, we have examined the viral and cellular factors involved. First, we demonstrate that six viral replication proteins are sufficient for formation of functional replication compartments in transfected cells in the absence of viral origin-containing DNA. Second, we show that the viral replication proteins form two distinct types of prereplicative sites within infected cells. One type of punctate structure assembles in S-phase cells, colocalizes with cellular DNA synthesis, and contains components of the host-cell replication apparatus as indicated by the presence of Replication Protein A. However, the other class of prereplicative sites is independent of host-cell DNA synthesis as evidenced by their formation in cells arrested in G1 by n-butyrate. These complexes are significantly less abundant and closely correspond with cellular Nuclear Domain 10 structures to which viral DNA has recently been demonstrated to be targeted early in infection (G. G. Maul, A. M. Ishov, and R.D. Everett, 1996, Virology 217, 67-75). Hence, this second type appears to represent the subset of prereplicative sites destined to become replication compartments.

Functional order of assembly of herpes simplex virus DNA replication proteins into prereplicative site structures.

Herpes simplex virus replicates its DNA within nuclear structures called replication compartments. In contrast, in cells in which viral DNA replication is inhibited, viral replication proteins localize to punctate structures called prereplicative sites. We have utilized viruses individually mutated in each of the seven essential replication genes to assess the function of each replication protein in the assembly of these proteins into prereplicative sites. We observed that four replication proteins, UL5, UL8 UL52, and UL9, are necessary for the localization of ICP8 (UL29) to prereplicative sites natural infection conditions. Likewise, four of the seven viral DNA replication proteins, UL5, UL52, UL9, and ICP8, are necessary for the localization of the viral DNA polymerase to prereplicative sites. On the basis of these results, we present a model for prereplicative site formation in infected cells in which the helicase-primase components (UL5, UL8, and UL52), the origin-binding protein (UL9), and the viral single-stranded DNA-binding protein (ICP8) assemble together to initiate the process. This is followed by the recruitment of the viral polymerase into the structures, a step facilitated by the polymerase accessory protein, UL42. Host cell factors can apparently substitute for some of these viral proteins under certain conditions, because the viral protein requirements for prereplicative site formation are reduced in transfected cells and in infected cells treated with drugs that inhibit DNA synthesis.

Herpes simplex ICP27 mutant viruses exhibit reduced expression of specific DNA replication genes.

Herpes simplex virus type 1 mutants with certain lesions in the ICP27 gene show a 5- to 10-fold reduction in viral DNA synthesis. To determine how ICP27 promotes amplification of viral DNA, we examined the synthesis, accumulation, and stability of the essential viral replication proteins and steady-state levels of the replication gene transcripts throughout the course of ICP27 mutant virus infections. These studies reveal that in the absence of ICP27, expression of the UL5, UL8, UL52, UL9, UL42, and UL30 genes is significantly reduced at the level of mRNA accumulation. In contrast to that of these beta genes, ICP8 expression is unaltered in mutant virus-infected cells, indicating that ICP27 selectively stimulates only a subset of herpes simplex virus beta genes. Analysis of multiple ICP27 mutant viruses indicates a quantitative correlation between the ability of these mutants to replicate viral DNA and the level of replication proteins produced by each mutant. Therefore, we conclude that the primary defect responsible for restricted viral DNA synthesis in cells infected with ICP27 mutants is insufficient expression of most of the essential replication genes. Of further interest, this analysis also provides new information about the structure of the UL52 gene transcripts.