Viral evolution of hepatitis C in injection drug users.

Injection drug users represent the largest cohort of patients with established hepatitis C virus (HCV) infection as well as the group that is at highest risk for new infections. Most published studies have focused on the clinical consequences of established HCV infection and have not examined the consequences of new infection. The aim of the current study was to measure the virological consequences of HCV in patients with ongoing injection drug use that might pose a risk for new and/or for superinfection with additional strains of HCV. We examined the following groups: (a) those with resolved HCV infection with ongoing injection drug use, (b) those with chronic infection who continued to inject and (c) those with chronic infection who no longer injected. Our study demonstrated a spectrum of responses. The majority of patients appeared to be 'protected' from new infection. None of six patients with resolved infection had detectable HCV RNA by quantitative or qualitative PCR when followed for 1 year. Similarly, despite ongoing injection drug use, no patient with persistent infection had a 'switch' in HCV genotype indicative of possible superinfection. Virological analysis of HCV quasispecies to detect possible infection with new variants of HCV in patients with apparently 'stable' infection, indicated divergence of virus over time, divergence that was unrelated to injection drug behaviour. Thus, patients with ongoing or prior HCV infection appear to develop immunity that protects against further infection with HCV despite repeated exposure.

Evolution of viral quasispecies in interferon-treated patients with chronic hepatitis C virus infection.

BACKGROUND/AIMS: To evaluate whether interferon treatment failure/relapse is related to changes in hepatitis C virus quasispecies complexity (number of variants) or diversity (genetic relatedness of variants). METHODS: We analyzed hypervariable region heterogeneity in hepatitis C virus-infected patients by heteroduplex mobility assay and by phylogenetic analysis of sequenced clones. Sera from 11 patients were tested. Response was defined biochemically and virologically. Patients were treated with 3 or 6 MIU interferon for 6 months and followed up for 6 months. Four patients were non-responders, four were transient responders and three untreated patients served as controls. Three time points were studied for the non-responders (pre-interferon, end of interferon, end of 6 months of follow-up), two for the transient responders (pre-interferon and post follow-up) and two for the controls (1 year apart). A total of 260 clones were examined by heteroduplex mobility assay and 144 clones were sequenced. RESULTS: A linear correlation between heteroduplex mobility and nucleotide substitutions was observed, validating this method for assessment of quasispecies diversity. Although complexity at each time point was similar in all groups, diversity increased significantly with interferon treatment. The percentage of new variants in follow up was significantly higher in non-responders than in controls. These new variants exhibited a greater change in heteroduplex mobility, a higher percentage of changes in amino acids in non-responders compared to controls and were found to cluster separately from pretreatment variants when analyzed phylogenetically. These changes were less marked in transient responders. CONCLUSIONS: These mutations may allow hepatitis C virus to escape antiviral effects of interferon therapy.

Evolution of hepatitis C virus quasispecies in patients with severe cholestatic hepatitis after liver transplantation.

Evolution of hepatitis C quasispecies may be one mechanism by which fibrosing cholestatic hepatitis develops after liver transplantation. In this study, we compared changes in quasispecies complexity and/or divergence in (1) hepatitis C-infected immunosuppressed transplant recipients and in immunocompetent controls; (2) transplant recipients with mild recurrence, and in those with the most severe form of posttransplantation recurrence. Quasispecies were measured in 12 hepatitis C-infected patients pretransplantation and posttransplantation (6 with mild and 6 with severe recurrence), and in 5 immunocompetent patients with similar follow-up, and characterized by heteroduplex mobility and sequence analysis of the hypervariable region. Although the number of variants (complexity) did not change with time in either group, there was a qualitative change in the variants with time (divergence) in immunocompromised, but not in immunocompetent patients. These changes were most marked with severe recurrence, and preceded the development of severe disease. Phylogenetic analysis confirmed that most posttransplantation variants were unrelated to those detected pretransplantation. These observations suggest that in the absence of immune suppression, there is minor evolution of quasispecies. With immune suppression, divergence of quasispecies is enhanced, resulting in selection/emergence of many new variants, particularly in those with fibrosing cholestatic hepatitis. Thus, quasispecies may influence disease progression in immune suppressed populations.

The synergistic action of ethanol and nerve growth factor in the induction of neuronal nitric oxide synthase.

Ethanol alone had no effect on neuronal nitric oxide synthase (nNOS) expression in PC12 cells. However, in the presence of nerve growth factor (NGF), nNOS expression was amplified (threefold, P < 0.05), compared to NGF alone. This increase was eliminated with pretreatment of PC12 cells with staurosporine, suggesting that the effects of ethanol on nNOS expression are mediated by a protein kinase C-dependent pathway.

Chimeric forms of neuronal nitric oxide synthase identify different regions of the reductase domain that are essential for dimerization and activity.

Nitric oxide synthase (NOS) is the enzyme responsible for the conversion of L-arginine to L-citrulline and nitric oxide. Dimerization of the enzyme is an absolute requirement for catalytic activity. Each NOS monomer contains an N-terminal heme-binding domain and a C-terminal reductase domain. It is unclear how the reductase domain is involved in controlling dimerization and whether dimer formation alone controls enzyme activity. Our initial studies demonstrated that no dimerization or activity could be detected when the reductase domain of rat neuronal NOS (nNOS) was expressed either separately or in combination with the heme domain. To further evaluate the reductase domain, a set of expression plasmids was created by replacing the reductase domain of nNOS with other electron-transport proteins, thereby creating nNOS chimeric fusion proteins. The rat nNOS heme domain was linked with either cytochrome P450 reductase, adrenodoxin reductase, or the reductase domain from Bacillus megaterium cytochrome P450, BM-3. All the chimeric enzymes retained the ability to dimerize but were unable to metabolize L-arginine (<8% of wildtype activity levels), indicating that dimerization alone is insufficient to produce an active enzyme. Because the greatest regions of homology between electron-transport proteins are in the flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide phosphate (NADPH) binding regions, we produced truncation mutants within the nNOS reductase domain to investigate the role of these sequences in the ability of nNOS to dimerize and to metabolize L-arginine. The results demonstrated that the deletion of the final 56 amino acids or the NADPH-binding region had no effect on dimerization but produced an inactive enzyme. However, when the FAD-binding site (located between amino acids 920 and 1161) was deleted, both activity and dimerization were abolished. These results implicate sequences within the FAD-binding site as essential for nNOS dimerization but sequences within amino acids 1373 to 1429 as essential for activity.

Both neuronal NO synthase and nitric oxide are required for PC12 cell differentiation: a cGMP independent pathway.

PC12 cells are used as a model system to study neuronal differentiation. Nerve growth factor (NGF) triggers a differentiation pathway in PC12 cells. Neurite outgrowth (a morphological marker of differentiation) in PC12 cells is significantly reduced in the presence of the NOS inhibitor l-NAME, but not d-NAME, implicating NOS in the differentiation process. Previously we have shown that the neuronal NO synthase (nNOS) isoform is induced in PC12 cells in the presence of NGF. Thus, we wished to further evaluate the role of nNOS and NO in PC12 cell differentiation. When a dominant negative mutant nNOS expression vector was transiently transfected into NGF-treated PC12 cells, it significantly reduced PC12 cell neurite outgrowth. Thus, we concluded that the NO required for PC12 cell differentiation, in response to NGF, is produced by nNOS. NO alone was insufficient to induce differentiation as cells treated with the NO donor, sodium nitroprusside did not produce neurites. Treatment of PC12 cells with oxyhemoglobin (an NO scavenger) was also found to significantly reduce the number of neurites produced by PC12 cells treated with NGF. Thus, NO appears to be necessary, but not sufficient, to induce differentiation, and its mode of action appears to be extracellular. A well documented action of NO is to activate soluble guanylate cyclase. Thus, we determined the role of soluble guanylate cyclase activation as a means by which NO induces PC12 cell differentiation. However, in the presence of NGF (to prime PC12 cells for differentiation) and l-NAME (to specifically remove the NO component), 8Br-cGMP (a cGMP analog) failed to induce PC12 cell differentiation. In addition, blockade of sGC activity with specific inhibitors failed to block NGF-induced PC12 cell differentiation. We conclude that the NO required for PC12 cell differentiation is produced by nNOS and that the NO exerts its effects on surrounding PC12 cells in a sGC/cGMP independent manner.

Use of chimeric forms of neuronal nitric-oxide synthase as dominant negative mutants.

Because the functional form of neuronal nitric-oxide synthase (nNOS) is a homodimer, we investigated whether we could disrupt dimer formation with inactive nNOS chimeras acting as dominant negative mutants. To test this hypothesis, we either expressed the heme and reductase regions of rat nNOS as single domains or produced fusion proteins between the rat nNOS heme domain and various other electron-shuttling proteins. A dominant negative potential of these constructs was demonstrated by their ability to reduce NOS activity when transfected into a cell line stably expressing rat nNOS. In the presence of these nNOS mutant proteins, cellular levels of inactive nNOS monomers were significantly increased, indicating that their mechanism of action is through the disruption of nNOS dimer formation. These dominant negative mutants should prove valuable in analyzing the role of nNOS in biological systems.

Feeding practices and dental caries in an urban Canadian population of Vietnamese preschool children.

The aim of this project was to determine the severity of nursing caries, and to examine contributing behavioral factors, in a group of Vietnamese families in British Columbia, Canada. The data collected became the basis for a community-based oral health promotion program. Information on feeding, dental health practices, and dental caries were collected for 60 mother/child pairs. For children > or = 18 mos, prevalence of nursing caries was 64 percent. Sixty-five percent of all children had a naptime bottle, and 85 percent > or = 18 mos had a "comfort" bottle that was carried around, and drunk from during the day. Milk was the most common beverage. A "comfort" bottle was significantly related to the presence of nursing caries, P = 0.02; a naptime bottle had a less significant association, P = 0.07. Dental knowledge questions revealed that all mothers knew that a child who had a "comfort" bottle could get tooth decay, but 63 percent thought that cavities were not a problem in baby teeth.

Growth factor induction of nitric oxide synthase in rat pheochromocytoma cells.

Previous work has suggested that nerve growth factor treatment of PC12 cells induces neuronal nitric oxide synthase, and possibly also endothelial nitric oxide synthase (NOS) and inducible NOS. To further analyze this process we exposed rat pheochromocytoma (PC12) cells to increasing concentrations of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), nerve growth factor (NGF), and vascular endothelial cell growth factor (VEGF). Changes in NOS expression were then analyzed by Western blotting, using antisera generated against the three isoforms of NOS. Our results demonstrate that neuronal NOS was induced by growth factors that promote both differentiation (bFGF, NGF) and proliferation (EGF). nNOS levels were unaffected by VEGF treatment. In contrast, the levels of endothelial and inducible NOS were undetectable in these same cells, suggesting that different clonal lines of PC12 cells have different isoform complements.

Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor.

Since the discovery that epidermal growth factor (EGF) can accelerate opening of the eyelids, the EGF receptor (EGF-R) has been extensively studied and is now considered to be a prototype tyrosine kinase receptor. Binding of EGF or of transforming growth factor-alpha (TGF-alpha) or other related factors activates the receptor and induces cell proliferation and differentiation. Although it is not found on haematopoietic cells, the EGF-R is widely expressed in mammals and has been implicated in various stages of embryonic development. Here we investigate the developmental and physiological roles of this receptor and its ligands by inactivating the gene encoding EGF-R. We find that EGF-R-/- mice survive for up to 8 days after birth and suffer from impaired epithelial development in several organs, including skin, lung and gastrointestinal tract.