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.

Australia antigen (a hepatitis-associated antigen): purification and physical properties.

Australia antigen [Au(1)], a particle associated with viral hepatitis, was isolated from the plasma of a patient with chronic anicteric hepatitis and leukemia who had received radioactive phosphorus. We have found that the immunoreactivity and appearance of Au(1) in the electron microscope were not altered by treatment with enzymes including trypsin, pronase, lipase, phospholipase C, ribonuclease, deoxyribonuclease, amylase, and neuraminidase. In contrast, other serum constituents were degraded by these enzymes. Therefore, treatment of the patient's plasma with many enzymes was exploited as an initial step for the isolation of Au(1). Subsequently, Au(1) was purified from the enzyme-treated (32)P-labeled plasma by gel filtration through Sephadex G-200 and centrifugation through sucrose and in cesium chloride gradients. There were no detectable human serum components in the purest fractions, as tested by immunoelectrophoresis and immunodiffusion. The density of the purified Au(1) was 1.21 in CsCl. The particle measured about 200 A in diameter, was predominantly spherical in shape and appeared to be composed of subunits. Nucleic acids were not detected by spectrophotometric, radiochemical, and chemical analyses. Immunoreactivity of purified Au(1) was destroyed by heating for 1 hr at 85 degrees C but was stable at 56 degrees C. Treatment with Carnoy's solution (3 parts ethanol:1 part glacial acetic acid) followed by pronase disrupted the particles as seen with the electron microscope. These findings, combined with other published information on Australia antigen and viral hepatitis, suggest that the bulk of Australia antigen in the blood of this patient is an incomplete virus or virus capsid.

The nature of australia antigen and its relation to antigen-antibody complex formation.

There is considerable data to support the hypothesis that Australia antigen is an infectious agent that causes hepatitis in man. (a) Association with acute viral hepatitis. (b) Association with chronic hepatitis. (c) Virus-like appearance under the electron microscope (200-A particles). (d) Transmission of Au(1) from man to man. (e) Transmission and passage of partially purified Au(1) to an animal host (infant African green monkey). (f) Localization [with fluorescent anti-Au(1)] of Au(1) in the nuclei of liver cells of patients with hepatitis and/or Au(1) in their blood. (g) Distribution of Au(1) in institutions, disease groups, and populations is consistent with the distribution of an infectious agent. (h) RNA identified in Au(1) particles isolated from blood. (i) Apparent replication of Australia antigen in tissue cultures of human liver cells. There is also considerable evidence that Australia antigen has many of the characteristics of a serum protein polymorphism. Since neither of these hypotheses has been rejected they can be combined to make a third hypothesis, namely, that Australia antigen is an infectious agent which causes hepatitis in some people infected with it and that it has the characteristics of an (inhertied) serum protein polymorphism. We propose calling agents of this postulated class "Icrons."

The localization of Australia antigen by immunofluorescence.

We have studied the localization of Australia antigen, a particulate substance associated with hepatitis, by means of the fluorescent antibody technique. Preparations were made from 61 liver biopsy specimens taken from patients with infectious hepatitis, serum hepatitis, and a variety of other diseases. When tested with fluorescein-conjugated rabbit anti-Au(1) antisera all 26 patients who had Au(1) in their serum had specific fluorescence in their liver cells. The fluorescence appeared in three forms: as discrete particles within the nucleus, diffuse fluorescence of the entire nucleus, and fluorescence of the nuclear rim. Occasionally there were also fluorescent particles in the cytoplasm. Other specimens were tested with the fluorescent antibody including a variety of human tissues, buffy coat smears, peripheral lymphocyte cultures, and cells obtained from bile and duodenal drainage. Among these specimens, fluorescence was found in the cytoplasm of a few cells in the bone marrow of two patients with hepatitis and Au(1) in their serum, and in the liver, spleen, mesentery, and testis of one patient with leukemia, chronic hepatitis, and Au(1) in his serum. We have shown that the presence of fluorescent particles in the liver cells is strongly associated with the presence of Au(1) in the serum and the diagnosis of viral hepatitis. We believe that this study adds support to the hypothesis that Australia antigen is an antigenic determinant of a virus capable of causing hepatitis.

Albumin Naskapi: a new variant of serum albumin.

An apparently new Variant of human serum albumin, albumin Naskapi, has been found in high frequency in the Naskapi Indians of Quebec and, in lower frequency,in other North American Indians. The family and population data of the albumin are consistent with its inheritance as a simple autosomal trait Controlled by a gene designated Al Naskapi. This gene is allelic with the gene Al(A) which controls the common albumin.Both homozygotes and heterozygotes have been distinguished. This is the first report of a homozygote for an albumin Variant.

Autosomal linkage between the albumin and Gc loci in humans.

Naskapi and Montagnais families segregating for albumin Naskapi give evidence for close linkage of the Gc and albumin loci with a high probability. One possible case of crossover is included in the data.

Serum ferritin as a predictor of host response to hepatitis B virus infection.

With hemodialysis patients, a high serum ferritin before there was serological evidence of hepatitis B virus infection increased the likelihood that the infection would be persistent. This finding suggested that hepatitis B virus is likely to infect and actively replicate in liver cells with the propensity for increased ferritin synthesis. The virus itself could stimulate the synthesis of ferritin in a cyclic positive feedback mechanism that increases intracellular ferritin concentration and, eventually, intracellular iron. Transformed liver cells have low iron content, do not replicate hepatitis B virus, and require iron for growth. Infected, nonmalignant liver cells could supply iron to the transformed cells and nourish their expansion.

Australia antigen: detection and transmission in shellfish.

Australia antigen was found in clams contaminated by drainage of untreated sewage from a coastal hospital. In closed-system aquariums, the antigen was ingested by clams and transmitted to previously uninfected clams. In opensystem aquariums, the titer of Australia antigen decreased with time, suggesting viral concentration rather than replication.

DNA polymerase activity as an index of lymphocyte stimulation: studies in Down's syndrome.

The ability of peripheral blood lymphocytes to respond to phytohemagglutinin (PHA) in vitro was studied in patients with Down's syndrome. The response was measured by the increase in DNA polymerase activity and the rate of incorporation of tritiated thymidine by the cultured lymphocytes. These activities were significantly lower in PHA-stimulated lymphocytes from patients with Down's syndrome compared with age- and sex-matched, mentally retarded patients without Down's syndrome from the same institution and the normal healthy volunteers. The impairment in response to PHA does not seem to be related to the presence of Australia antigen in patients with Down's syndrome or to institutionalization itself. In contrast to DNA polymerase activity and thymidine-(3)H uptake, there was no significant difference in the percentage of blast transformation in the three groups studied. The poor response of the lymphocytes from patients with Down's syndrome to a mitogenic stimulus could reflect an impairment of cellular immune functions in these patients which may be one of the factors contributing to the vulnerability of these patients to repeated or persistent infections.

Iron-binding proteins and risk of cancer in Taiwan.

The relationship of serum ferritin and transferrin levels to risk of cancer was examined in a population of 21,513 Chinese male government workers in Taiwan who have been followed prospectively since 1975. On the basis of a previous study in the Solomon Islands, increased ferritin and decreased transferrin levels were predicted for those men who developed cancer. The results were consistent with the prediction. The mean serum ferritin was higher at the start of the study in 192 men who had died of cancer or who had developed primary hepatocellular carcinoma (PHC) as of July 1983, as compared to their controls. The mean serum transferrin level was lower in men who had died of cancers other than PHC. The estimate of relative risk of cancer death for a man with 200 ng ferritin/ml and 200 mg transferrin/dl, as compared to a man with levels of 20 ng/ml and 400 mg/dl, respectively, is 2.9. These serum iron-binding protein levels are at the extremes of the "normal" range. Men who subsequently died of cancer had lower hemoglobin, lower hematocrit, lower albumin, and higher globulin levels at the start of the study than did the controls. These results are consistent with the hypothesis that increased iron stores increase the risk of cancer. However, direct assessment of iron stores prior to disease was not possible, and the same constellation of findings may be consistent with other explanations.

Forecasting the development of primary hepatocellular carcinoma by the use of risk factors: studies in West Africa.

An association between hepatitis B virus (HBV) and primary hepatocellular carcinoma (PHC) has been found in several studies in Africa, Asia, and elsewhere. In this paper we considered the interrelations between several events related to HBV infection, which include the presence of: 1) hepatitis B surface antigen (HBsAg), 2) antibody to hepatitis B core antigen (anti-HBc), 3) antibody to the surface antigen (anti-HBs), 4) chronic liver disease, 5) elevated alpha-fetoprotein, and 6) PHC. With the use of preliminary epidemiologic data, risk factors related to these events were calculated. We suggested that the interactions between these events and HBV infection in parents be used to estimate the risk of PHC for an individual in this environment.

Studies of parents of children with acute leukemia.

In a case-control study, 70 mothers and 24 fathers of children with acute leukemia (AL) were compared with 70 mothers and 24 fathers of normal children. Three significant differences (p smaller than 0.05) were found when 35 factors were compared among the mother pairs and one difference among the father pairs. Mothers of children with AL, though alike in most respects to their matched controls, had a significantly lower number of monocytes than their controls. This was a new observation. The mothers of the children with AL also had higher levels of gamma-globulin, IgA, and IgG (Philadelphia only), which confirmed previous observations. The fathers and mothers had higher levels of basophils. These findings direct attention to the immune systems, particularly the mononuclear cells, of the parents of children with AL, as a focus for further studies on the etiology and pathogenesis of childhood leukemia.

Iron nutrition and tumor growth: decreased tumor growth in iron-deficient mice.

Groups of 15 mice of three different laboratory strains (BALB/c, C3H/He, DBA/2) were fed on a low iron diet (5 mg iron/kg diet), and three similar groups of 15 mice were maintained on a normal iron diet (312 mg iron/kg diet). When the low iron diet group became iron deficient, tumor cells (5 x 10(5) cells/mouse) of CA07-A (colon adenocarcinoma), HE129 (hepatoma), and M119 (mammary adenocarcinoma) were inoculated s.c. in BALB/c, C3H/He, and DBA/2 mice, respectively. All mice developed tumors, tumors grew more slowly, and the mean tumor sizes were smaller in the low iron diet group at nearly all weekly observations in all three strains of mice. No apparent differences in the behavior, activity (e.g., movement, climbing, running, grooming, etc.), and appearance were observed between low iron diet and normal iron diet mice. The mean body weight of mice at transplantation was less in the low iron than in the normal iron groups for the BALB/c strain but higher in the low iron groups of C3H/He and DBA/2 mice, indicating that food intake of mice on a low iron diet was not impaired. These results suggest that iron nutrition of the host affects tumor growth; tumor cells grow better in an iron-rich environment. This knowledge should be considered when designing treatment for patients with cancer. Iron oversupply in cancer patients might enhance tumor growth and adversely affect cancer therapy.

Susceptibility to persistence of Australia antigen.

To test the hypothesis that liver function is different in populations with inherited susceptibility to persistent Australia antigen (hepatitis B surface antigen [HBsAg]), Sardinians living in Turin, Italy, were compared with their native Turinese neighbors. The study was controlled for age, sex, place of birth, and presence or absence of persistent HBsAg. Slight differences in liver function were found in the Sardinians compared with the Turinese. Their values were in the direction of abnormality. Sardinians also had considerably higher serum gamma-globulin levels than the Turinese. The levels of alpha2-globulin by electrophoresis correlated with the presence of HBsAg.

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.

Feasibility of controlling or eradicating the hepatitis B virus.

A hepatitis B vaccine, prepared from the hepatitis B surface antigen in human blood has been available since the early 1980s. A recombinant vaccine, which can be supplied in unlimited amounts, was recently introduced. Based on current data, these vaccines are highly effective and safe. The most effective procedures for controlling hepatitis B virus is the vaccination of newborn infants. After universal vaccination of newborns has been ensured, vaccination of susceptible persons in older age groups could be instituted. Any attempt at eradicating hepatitis B virus will require international cooperation, and ideally the hepatitis B virus vaccine should be integrated into other childhood vaccination programs.

Terbutaline and ephedrine in asthmatic children.

The effects of terbutaline, ephedrine, and placebo on the cardiovascular and pulmonary systems have been compared in 24 asthmatic children. Ephedrine and terbutaline were both found to be effective bronchodilators, with onset of action within 30 minutes. The bronchodilator effect of ephedrine was maintained for three hours, while terbutaline was active for five hours. Terbutaline caused significantly greater improvement in pulmonary functions than did ephedrine. Both terbutaline and ephedrine were associated with clinically insignificant changes in blood pressure and pulse rate. The only significant side effect observed was hand tremor in children receiving terbutaline and this appeared only early in the course of drug treatment. There was no evidence of tolerance to the bronchodilator effect of ephedrine or terbutaline after eight weeks of therapy.

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.

Viruses similar to hepatitis B virus (Icrons).

In 1971, on the basis of the unusual clinical and epidemiologic characteristics of hepatitis B virus, we postulated the existence of a class of viruses that we termed Icrons. An increased understanding of the molecular biology of hepatitis B virus resulted in the discovery, by Summers and his colleagues, of the woodchuck hepatitis virus. This virus is common in the North American woodchuck (Marmota monax) and is associated with primary cancer of the liver in this animal. Subsequently similar viruses were found in Beechey ground squirrels in California by Marion and her coworkers and domesticated (Pekin) ducks from the United States by Mason and his coworkers. In the latter the virus infects the liver and presumably is associated with disease of this organ. The discovery of additional viruses similar to hepatitis B virus in animals other than man and their association with cancer of the liver encourages the continuing search for other virus-cancer associations for which prevention methods might be effective.