Serum ferritin concentration and recurrence of colorectal adenoma.

Both body iron stores and dietary iron intake have been reported to increase risk of colorectal neoplasms. We assessed whether serum ferritin concentration was associated with recurrence of colorectal adenomas among 733 individuals with baseline determinations of ferritin as part of a multicenter clinical trial of antioxidant supplements for adenoma prevention. All study participants had at least one adenoma removed within 3 months before enrollment, and 269 of them developed one or more adenomas between follow-up colonoscopies conducted 1 and 4 years after enrollment. Baseline serum ferritin concentrations were analyzed both as a log-transformed continuous variable and as a categorical variable, defined as whether iron stores were nonreplete and low (ferritin < or =30 microg/liter), nonreplete and borderline (31-70 microg/liter), replete and adequate (71-160 microg/liter), or replete and high (>160 microg/liter). Analyses were based on multiple logistic regression models, including age, sex, study center, energy, alcohol, fiber, folate, and total fat intake, number of months between colonoscopic examinations, smoking status, and aspirin use. Overall, there was no statistically significant linear association between log ferritin concentration and adenoma recurrence (P = 0.33). Risk of adenoma recurrence was modestly increased among participants with ferritin concentrations >70 microg/liter relative to those with lower ferritin (odds ratio, 1.39; 95% confidence interval, 0.96-2.02). This result seemed more pronounced among women than men. Dietary intake of iron and red meat was inversely associated with adenoma recurrence among participants with replete iron stores but not consistently associated among those with nonreplete stores. Our findings suggest that any role of iron stores and dietary iron in influencing risk of colorectal adenoma recurrence is likely complex.

Imino sugars inhibit the formation and secretion of bovine viral diarrhea virus, a pestivirus model of hepatitis C virus: implications for the development of broad spectrum anti-hepatitis virus agents.

One function of N-linked glycans is to assist in the folding of glycoproteins by mediating interactions of the lectin-like chaperone proteins calnexin and calreticulin with nascent glycoproteins. These interactions can be prevented by inhibitors of the alpha-glucosidases, such as N-butyl-deoxynojirimycin (NB-DNJ) and N-nonyl-DNJ (NN-DNJ), and this causes some proteins to be misfolded and retained within the endoplasmic reticulum (ER). We have shown previously that the NN-DNJ-induced misfolding of one of the hepatitis B virus (HBV) envelope glycoproteins prevents the formation and secretion of virus in vitro and that this inhibitor alters glycosylation and reduces the viral levels in an animal model of chronic HBV infection. This led us to investigate the effect of glucosidase inhibitors on another ER-budding virus, bovine viral diarrhea virus, a tissue culture surrogate of human hepatitis C virus (HCV). Here we show that in MDBK cells alpha-glucosidase inhibitors prevented the formation and secretion of infectious bovine viral diarrhea virus. Data also are presented showing that NN-DNJ, compared with NB-DNJ, exhibits a prolonged retention in liver in vivo. Because viral secretion is selectively hypersensitive to glucosidase inhibition relative to the secretion of cellular proteins, the possibility that glucosidase inhibitors could be used as broad-based antiviral hepatitis agents is discussed. A single drug against HBV, HCV, and, possibly, HDV, which together chronically infect more than 400 million people worldwide, would be of great therapeutic value.

Treatment of chronic hepadnavirus infection in a woodchuck animal model with an inhibitor of protein folding and trafficking.

A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.

Decreased survival of HTLV-I carriers in leprosy patients from the Democratic Republic of the Congo: a historical prospective study.

In this historical prospective study using sera stored for 22 years, we investigated the effect of HTLV-I infection on survival in a population of leprosy patients in the Democratic Republic of the Congo (formerly Zaire). We also determined the distribution of HTLV-I by subpopulation, age, and gender. Stored sera taken from a population of leprosy patients and controls in 1969 were tested for HTLV-I. Follow-up survival data on these patients were obtained in 1991. The sera collected in 1969 from 520 individuals was used to determine the prevalence of HTLV-I. Included in this number were 328 patients resident in the sanatorium. Survival and other data were available for 327 of these. A multivariate survival analysis using a logistic regression model was performed to evaluate the influence of HTLV-I status, age, type of leprosy, gender, duration of hospitalization, and ethnic group on survival. The overall prevalence of HTLV-I among the 520 individuals in the prevalence study was 34%, with 37.4% in the leprosy group and 25.2% in the control group (p < 0.01). Multivariate analysis using logistic regression showed that females of the Mongo and Ngombe ethnic group taken together were significantly more likely to be infected than the other groups (OR = 3.67, 95% CI: 2.14 to 6.30). A comparison of the death rates directly standardized for age and sex showed that the rate was significantly higher for HTLV-I positive (5.5/100 person-years of observation) compared with HTLV-I negative (3.6/100 person-years of observation). A survival analysis using the Cox model showed a risk ratio of 1.4 (CI: 1.04 to 1.89) for those infected with HTLV-I. An increase in the death rate was associated with HTLV-I infection in leprosy inpatients. The decreased survival associated with HTLV-I infection may result from an increased susceptibility to a variety of diseases.

PIP: Preservation of stored sera collected in 1969 from leprosy patients at a sanatorium in the Democratic Republic of the Congo's Equator Province enabled an analysis of the survival of carriers of human T-cell lymphotropic virus type 1 (HTLV-1). The HTLV-1 prevalence in the sera collected from 377 leprosy patients and 143 controls in 1969 was 34% (37.4% and 25.2%, respectively). Multivariate survival analysis was performed to evaluate the impact of HTLV-1 status, age, type of leprosy, gender, duration of hospitalization, and ethnic group on the survival of leprosy patients. Members of the Mongo ethnic group were more likely than those of the Ngombe ethnic group to be infected. After adjustment for age and sex, mortality was significantly higher among HTLV-1-positive cases (5.5 per 100 person-years of observation) than HTLV-1-negative persons (3.6 per 100 person-years of observation). A survival analysis using the Cox model revealed a mortality risk ratio of 1.4 (95% confidence interval, 1.04-1.89) for HTLV-1-positive individuals. The reduced survival observed in HTLV-1-infected leprosy patients presumably resulted from concomitant increased susceptibility to other diseases.

Hepatitis B virus (HBV) envelope glycoproteins vary drastically in their sensitivity to glycan processing: evidence that alteration of a single N-linked glycosylation site can regulate HBV secretion.

The role of N-linked glycosylation and glycan trimming in the function of glycoproteins remains a central question in biology. Hepatitis B virus specifies three glycoproteins (L, M, and S) that are derived from alternate translation of the same ORF. All three glycoproteins contain a common N-glycosylation site in the S domain while M possesses an additional N-glycosylation site at its amino terminus. In the presence of N-butyl-deoxnojirimycin (an inhibitor of alpha-glucosidase) virions and the M protein are surprisingly retained. Preliminary evidence suggests that the retained M protein is hyperglucosylated and localized to lysosomal vesicles. In contrast, the S and L proteins are secreted, and their glycosylation state is unaffected by the presence of the inhibitor. Site-directed mutagenesis provides evidence that virion secretion requires the glycosylation sequon in the pre-S2 domain of M. This highlights the potential role of the M protein oligosaccharide as a therapeutic target.

Aberrant trafficking of hepatitis B virus glycoproteins in cells in which N-glycan processing is inhibited.

The role of N-glycan trimming in glycoprotein fate and function is unclear. We have recently shown that hepatitis B virus (HBV) DNA is not efficiently secreted from cells in which alpha-glucosidase mediated N-glycan trimming is inhibited. Here it is shown that, in cells in glucosidase-inhibited cells, viral DNA, accompanied by envelope and core proteins, most likely accumulate within lysosomal compartments. Pulse-chase experiments show that although the viral glycoproteins (L, M, and S) are dysfunctional, in the sense that they do not mediate virion egress and are not efficiently secreted from the cell, they all still leave the endoplasmic reticulum (ER). Surprisingly, however, the glycoproteins retained within the cell were not rapidly degraded, appearing as aggregates, enriched for L and M, with intracellular half-lives exceeding 20 h. Moreover, by 24 h after synthesis, a substantial fraction of the detained glycoproteins appeared to return to the ER, although a considerable amount was also found in the lysosomes. To our knowledge, this is the first report that shows, as a consequence of inhibiting glycosylation processing, certain glycoproteins (i) become dysfunctional and aggregate, yet still depart from the ER, and (ii) have extended rather than shortened half-lives. Taken together, these data suggest that proper intracellular routing of HBV glycoproteins requires ER glucosidase function. It is hypothesized that failure to process N-glycan causes HBV glycoproteins to aggregate and that impaired protein-protein interactions and trafficking are the result of misfolding.

The dynamics of hepatitis B virus infection.

We consider a cellular model of infection by the hepatitis B virus and describe how it may be used to account for two important features of the disease, namely (i) the wide variety of manifestations of infection and the age dependence thereof, and (ii) the typically long delay before the development of virus-induced liver cancer (primary hepatocellular carcinoma). The model is based on the assumption that the liver is comprised of both immature and mature hepatocytes, with these two subpopulations of cells responding contrastingly upon infection by the virus.

X region deletion variants of hepatitis B virus in surface antigen-negative infections and non-A, non-B hepatitis.

The etiology of non-A, non-B hepatitis (NANBH) in renal dialysis patients was determined. Hepatitis C virus was present in many, but its appearance did not correlate with elevated alanine aminotransaminase. When sera from these patients were tested for antibodies against hepatitis B virus (HBV) X antigen and polymerase, 70% were positive. HBV infection was confirmed by polymerase chain reaction using several HBV-specific primer pairs. However, amplification with X region primers failed to yield products in many patients. Cloning and sequencing of these products demonstrated deletions within the X region. Hence, X-deletion variants of HBV are strongly associated with NANBH in renal dialysis patients.

X region deletion mutants associated with surface antigen-positive hepatitis B virus infections.

BACKGROUND/AIMS: The finding of antibodies against the polymerase of hepatitis B virus in renal dialysis patients before the incubation phase of infection implies underlying virus replication. Hence, the aim of the study was to test for virus during infection. METHODS: Viremia was assayed in virus-infected and control patients using the polymerase chain reaction and Southern blotting. RESULTS: Six months before the appearance of surface antigen, most patients had detectable core region, but few patients were X region positive. Three months after surface antigen appeared, most carriers had detectable core and X products. Three years after surface antigen appeared, 5 of 8 carriers with persistent hepatitis B e antigen and 1 of 8 carriers with corresponding antibody had these products. Cloning and sequencing showed deletions within the X/precore region of viral DNA. CONCLUSIONS: Infection with X region mutants precedes that of wild-type virus, and they reappear after wild-type virus is eliminated in carriers.

Precore and X region mutants in hepatitis B virus infections among renal dialysis patients.

Hepatitis B virus (HBV) variants containing mutations within the X and the precore regions of the viral genome were demonstrated by polymerase chain reaction (PCR) amplification and DNA sequencing in renal dialysis patients with different serological patterns of HBV infection. Among carriers, X region deletion mutants predominated in patients who lost hepatitis B e antigen (HBeAg), or developed anti-HBe, but not in persistently HBeAg-positive patients. The precore region remained wild type in all carriers whether or not they seroconverted from HBeAg to anti-HBe. The frequency of precore and X region mutants was greatest among non-carrier patients with viral antibodies as the only indication of infection and among patients with non-A, non-B hepatitis (NANBH), suggesting an inverse relationship between the presence of wild type HBV markers and the presence of HBV mutants. Furthermore, the detection of one but not the other mutation in many serum samples suggests that these mutations are independently selected for during infection. Finally, the absence of HBV DNA in 21 'uninfected' dialysis patients with normal transaminases and no viral serology, suggests that replication of these mutants is associated with hepatitis. These results have important implications for HBV screening and treatment, as well as for the pathogenesis of chronic infection.

Usage and bioassays in Phyllanthus (Euphorbiaceae). IV. Clustering of antiviral uses and other effects.

A number of species of the genus Phyllanthus (Euphorbiaceae) have been tested for their efficacy as antivirals, partly on the basis of references to traditional usage for the treatment of diseases possibly having a viral origin. There are also many references to indigenous uses and to laboratory assays for other biological activities in this large genus (550+ species). These citations have been arranged by subgenus, section, subsection and species and have been published in three previous papers. This paper summarizes selected clustering of usage and effect by subgeneric taxa. Consideration of the data from ethnobotany, in vitro assays and clinical trials supported the presence of some type of biological activity(s) particularly within the subgenus Phyllanthus. Although the herbaceous species of subgenus Phyllanthus have been extensively used to treat jaundice, and have generally inhibited hepadnavirus DNAp, effects on chronic infection with hepatitis B virus (HBV) or related viruses have generally been negative. Alternative explanations for the wide usage have been little explored. Other medical categories suggested possible leads for research, or possibly, herbal or galenic remedies with bona fide effects. In most cases, the data remain suggestive but not conclusive.

A cellular model to explain the pathogenesis of infection by the hepatitis B virus.

The natural history of infection by the hepatitis B virus (HBV) depends on many factors, including the age and immunological status of the patient, and can range from acute transient infection to subclinical chronic hepatitis. Persistent infection often leads to the development of primary hepatocellular carcinoma. We consider a cellular model of HBV infection based on the hypothesis that the liver contains two populations of cells with contrasting responses to the virus. Our findings show that the model can be used to account for the wide variety of clinical manifestations of infection and can explain the observed age dependence of the main different outcomes of the disease.

Modelling the impact of mass vaccination against hepatitis B. I. Model formulation and parameter estimation.

A new model of the transmission dynamics of hepatitis B virus in countries with high transmission rates is presented. The model is age and sex stratified, and includes details of host demography. Details of hepatitis B natural history, such as the existence of infectious and non-infectious carriers, are included. The biological assumptions of the model are discussed in full and related to the model's equations. Hepatitis B epidemiological data is reviewed with special emphasis on the estimation of model parameter values from field data. A full set of model parameter values are derived. Possible uses of the model in the assessment of strategies of mass vaccination are discussed.

Secretion of human hepatitis B virus is inhibited by the imino sugar N-butyldeoxynojirimycin.

The imino sugar N-butyldeoxynojirimycin (NBDNJ) is a potent inhibitor of the oligosaccharide-trimming enzyme alpha-glucosidase I. Hepatitis B virus (HBV) contains three surface proteins (HBs proteins) of different sizes that are singly or doubly N-glycosylated and are essential for the formation of infectious virus. Therefore, the replication and secretion of HBV in the human hepatoma cell line HepG2 were studied in the presence of NBDNJ. In the stably HBV-transfected HepG 2.2.15 cells and in HBV-infected HepG2 cells, NBDNJ suppressed secretion of HBV particles and caused intracellular retention of HBV DNA. The secretion of subviral particles was less affected. These data suggest that inhibitors of oligosaccharide trimming may be useful for antiviral therapy of hepatitis B and for the study of the intracellular transport of the viral glycoproteins.

Pathogenesis of posttransfusion viral hepatitis in children with beta-thalassemia.

The pathogenesis of posttransfusion hepatitis was determined in 14 children with beta-thalassemia. All had blood samples obtained in 1980 or 1981, were vaccinated against hepatitis B virus in 1983 and had another serum sample collected in 1989. Seven children had detectable antibodies against hepatitis C virus before vaccination, and all were positive in 1989. With specific solid-phase enzyme immunoassays, all children had antibodies against hepatitis B virus, X and polymerase antigens in 1981, and six had one or both antibodies in 1989. Hepatitis B virus infection was confirmed by means of polymerase chain reaction, which demonstrated virus DNA in 13 of the 14 children. The amplification products spanning the X/precore region were smaller than expected, suggesting mutations in this region. Cloning and sequencing of these products revealed deletions spanning part or all of the X gene. The results show that these children were infected with hepatitis B virus even without other markers in serum, that hepatitis B persists years after vaccination and that such infections are associated with the presence of X deletion mutants. Coinfection with hepatitis B and C viruses, the former containing a new class of variants, is common in children with beta-thalassemia.