Genome-free hepatitis B virion levels in patient sera as a potential marker to monitor response to antiviral therapy.

Complete virions of hepatitis B virus (HBV) contain a DNA genome that is enclosed in a capsid composed of the HBV core antigen (HBcAg), which is in turn surrounded by a lipid envelope studded with viral surface antigens (HBsAg). In addition, HBV-infected cells release subviral particles composed of HBsAg only (HBsAg 'spheres' and 'filaments') or HBsAg enveloping HBcAg but devoid of viral DNA ('empty virions'). The hepatitis B e antigen (HBeAg), a soluble antigen related to HBcAg, is also secreted in some HBV-infected patients. The goals of this study were to explore the levels of empty virions in HBV-infected patients before and during therapy with the nucleotide analog tenofovir disoproxil fumarate (TDF) that inhibits HBV DNA synthesis and the relationships of empty virions to complete virions, HBsAg and HBeAg. HBV DNA, HBcAg and HBsAg levels were determined in serum samples from 21 patients chronically infected with HBV and enrolled in clinical TDF studies. Serum levels of empty virions were found to exceed levels of DNA-containing virions, often by ≥ 100-fold. Levels of both empty and complete virions varied and were related to the HBeAg status. When HBV DNA replication was suppressed by TDF, empty virion levels remained unchanged in most but were decreased (to the limit of detection) in some patients who also experienced significant decrease or loss of serum HBsAg. In conclusion, empty virions are present in the serum of chronic hepatitis B patients at high levels and may be useful in monitoring response to antiviral therapy.

Activity of adefovir dipivoxil against all patterns of lamivudine-resistant hepatitis B viruses in patients.

One hundred and thirty-one post-liver transplantation patients with chronic hepatitis B and failing lamivudine therapy with detectable serum hepatitis B virus (HBV) deoxyribonucleic acid by hybridization assays or > or =1 x 10(6) copies/mL by polymerase chain reaction, and elevated alanine transaminase levels despite continuous lamivudine, were enrolled in an open-label study of adefovir dipivoxil. The B and C domains of HBV polymerase were sequenced for baseline samples to determine the presence of lamivudine resistance mutations. The results showed that 98% of the samples had tyrosine-methionine-aspartate-aspartate (YMDD) mutations, indicating a strong correlation between the above clinical definition of lamivudine treatment failure and the presence of YMDD mutations. In addition to the rtM204V/I and the rtL180M mutations, the mutation rtV173L was identified in 19% of patients. Four major patterns of lamivudine-resistant HBV were identified: rtL180M + rtM204V (60%), rtV173L + rtL180M + rtM204V (19%), rtM204I (9%) and rtL180M + rtM204I (9%). Treatment with adefovir dipivoxil showed similar antiviral efficacy in patients with lamivudine-resistant virus from all four patterns.

Transient disruption of intercellular junctions enables baculovirus entry into nondividing hepatocytes.

Baculovirus infection has extended the capabilities for transfection of exogenous genes into a variety of mammalian cell types. Because rat hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented chemically defined medium are an excellent model system for studying liver function in vitro, we investigated the ability of baculoviruses to infect and deliver exogenous genes to cells in this culture system. Efficient delivery to hepatocytes in short-term culture becomes restricted to peripheral cells, or "edge" cells, as the hepatocytes acquire intercellular junctions and form islands with time in culture. This barrier to baculovirus entry can be overcome, and the percentage of internal cells within the hepatocyte islands that are infected with the baculovirus can be increased more than 100-fold, when cells are subjected to transient calcium depletion before and during infection. These findings suggest that at least in some cell types, such as hepatocytes, baculovirus entry may require contact with the basolateral surface. We conclude from this study that recombinant baculovirus infection following transient depletion of extracellular calcium results in delivery of exogenous genes to at least 75% of hepatocytes in long-term DMSO culture, thereby making it possible for the first time to carry out gain-of-function and loss-of-function studies in this cell system.

Resistance of hepatitis B virus to antiviral drugs: current aspects and directions for future investigation.

Despite the existence of vaccines, chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Interferon therapy successfully controls infection in only a small percentage of chronically infected individuals. The recent approval of the nucleoside analogue lamivudine for the treatment of chronic HBV infection has ushered in a new era of antiviral therapy. While lamivudine is highly effective at controlling viral infection short-term, prolonged therapy has been associated with an increasing incidence of viral resistance. Thus, it appears that lamivudine alone will not be sufficient to control chronic viral infection in the majority of individuals. In addition to lamivudine, several new nucleoside and nucleotide analogues that show promising antihepadnaviral activity are in various stages of development. Lamivudine resistance has been found to confer cross-resistance to some of these compounds and it is likely that resistance to newer antivirals may also develop during prolonged use. Drug resistance therefore poses a major threat to nucleoside analogue-based therapies for chronic HBV infection. Fortunately, combination chemotherapy (antiviral therapy with two or more agents) can minimize the chance that resistance will develop and can be expected to achieve sustained reductions in viral load, provided that suitable combinations of agents are chosen. Here we review the basis of drug resistance in HBV, with emphasis on aspects that are likely to affect drug choice in future.

Cross-resistance testing of antihepadnaviral compounds using novel recombinant baculoviruses which encode drug-resistant strains of hepatitis B virus.

Long-term nucleoside analog therapy for hepatitis B virus (HBV)-related disease frequently results in the selection of mutant HBV strains that are resistant to therapy. Molecular studies of such drug-resistant variants are clearly warranted but have been difficult to do because of the lack of convenient and reliable in vitro culture systems for HBV. We previously developed a novel in vitro system for studying HBV replication that relies on the use of recombinant baculoviruses to deliver greater than unit length copies of the HBV genome to HepG2 cells. High levels of HBV replication can be achieved in this system, which has recently been used to assess the effects of lamivudine on HBV replication and covalently closed circular DNA accumulation. The further development of this novel system and its application to determine the cross-resistance profiles of drug-resistant HBV strains are described here. For these studies, novel recombinant HBV baculoviruses which encoded the L526M, M550I, and L526M M550V drug resistance mutations were generated and used to examine the effects of these substitutions on viral sensitivity to lamivudine, penciclovir (the active form of famciclovir), and adefovir, three compounds of clinical importance. The following observations were made: (i) the L526M mutation confers resistance to penciclovir and partial resistance to lamivudine, (ii) the YMDD mutations M550I and L526M M550V confer high levels of resistance to lamivudine and penciclovir, and (iii) adefovir is active against each of these mutants. These findings are supported by the limited amount of clinical data currently available and confirm the utility of the HBV-baculovirus system as an in vitro tool for the molecular characterization of clinically significant HBV strains.

Use of the hepatitis B virus recombinant baculovirus-HepG2 system to study the effects of (-)-beta-2',3'-dideoxy-3'-thiacytidine on replication of hepatitis B virus and accumulation of covalently closed circular DNA.

(-)-Beta-2',3'-Dideoxy-3'-thiacytidine (lamivudine [3TC]) is a nucleoside analog which effectively interferes with the replication of hepatitis B virus (HBV) DNA in vitro and in vivo. We have investigated the antiviral properties of 3TC in vitro in HepG2 cells infected with recombinant HBV baculovirus. Different types of information can be obtained with the HBV baculovirus-HepG2 system because (i) experiments can be carried out at various levels of HBV replication including levels significantly higher than those that can be obtained from conventional HBV-expressing cell lines, (ii) cultures can be manipulated and/or treated prior to or during the initiation of HBV expression, and (iii) high levels of HBV replication allow the rapid detection of HBV products including covalently closed circular (CCC) HBV DNA from low numbers of HepG2 cells. The treatment of HBV baculovirus-infected HepG2 cells with 3TC resulted in an inhibition of HBV replication, evidenced by reductions in the levels of both extracellular HBV DNA and intracellular replicative intermediates. The effect of 3TC on HBV replication was both dose and time dependent, and the reductions in extracellular HBV DNA that we observed agreed well with the previously reported efficacy of 3TC in vitro. As expected, levels of HBV transcripts and extracellular hepatitis B surface antigen and e antigen were not affected by 3TC. Importantly, the HBV baculovirus-HepG2 system made it possible to observe for the first time that CCC HBV DNA levels are lower in cells treated with 3TC than in control cells. We also observed that the treatment of HepG2 cells prior to HBV baculovirus infection resulted in a slight increase in the efficacy of 3TC compared to treatments starting 24 h postinfection. The treatment of HepG2 cells with the highest concentration of 3TC tested in this study (2 microM) prior to the initiation of HBV replication markedly inhibited the accumulation of CCC DNA, whereas treatment with the same concentration of 3TC at a time when CCC HBV DNA pools were established within the cells was considerably less effective. In addition, our results suggest that in HepG2 cells, non-protein-associated relaxed circular HBV DNA and particularly CCC HBV DNA are considerably more resistant to 3TC treatment than other forms of HBV DNA, including replicative intermediates and extracellular DNA. We conclude from these studies that the HBV baculovirus-HepG2 system has specific advantages for drug studies and can be used to complement other in vitro model systems currently used for testing antiviral compounds.

Fabrication of high-spatial-frequency gratings through computer-generated near-field holography.

We demonstrate an innovative method for fabrication of high-spatial-frequency grating structures. This technique makes use of the near-field diffraction patterns from computer-generated phase holograms for lithographic fabrication of grating structures with periods that are one half that of the phase hologram mask. Linear, rectilinear, and circular gratings were fabricated with this technique. Experimental results from gratings with periods to 0.5 mum and feature sizes to ~0.2 mum are presented.

Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus.

A novel transient mechanism for studying hepatitis B virus (HBV) gene expression and replication using recombinant HBV baculovirus to deliver the HBV genome to HepG2 cells was generated. In HBV baculovirus infected HepG2 cells, HBV transcripts, and intracellular and secreted HBV antigens are produced; replication occurs as evidenced by the presence of high levels of intracellular replicative intermediates and protected HBV DNA in the medium. Density-gradient analysis of extracellular HBV DNA indicated that the DNA was contained predominantly in enveloped HBV virions. Covalently closed circular (CCC) DNA is present indicating that, in this system, HBV core particles are capable of delivering newly synthesized HBV genomes back into the nuclei of infected cells. HBV gene expression is driven exclusively from endogenous promoters. Levels of HBV gene expression and replication can be achieved in HBV baculovirus-infected HepG2 cells which far exceed levels found in HepG2 2.2.15 cells. HBV baculovirus infection of HepG2 cells lends itself readily to experimental manipulation as follows: 1) HBV expression can be initiated any time relative to seeding of HepG2 cells; 2) levels of HBV replication can be regulated over a wide range simply by changing the baculovirus multiplicity of infection; 3) HBV replication is readily detectable by one day post infection with HBV baculovirus and persists at least through day eleven post infection; and (4) the transient nature of the infection can be extended and/or enhanced by superinfecting the cultures. We conclude that infection of HepG2 cells by HBV recombinant baculovirus represents a simple to use and highly flexible system for studying the effects of antivirals and/or cytokines on HBV production and for understanding HBV replication and pathogenesis at the molecular level.

Predicting likelihood of seeking help through the employee assistance program among salaried and union hourly employees.

AIMS: This research investigated belief, social support and background predictors of employee likelihood to use an Employee Assistance Program (EAP) for a drinking problem. DESIGN: An anonymous cross-sectional survey was administered in the home. Bivariate analyses and simultaneous equations path analysis were used to explore a model of EAP use. SETTING: Survey and ethnographic research were conducted in a unionized heavy machinery manufacturing plant in the central states of the United States. PARTICIPANTS: A random sample of 852 hourly and salaried employees was selected. MEASUREMENTS: In addition to background variables, measures included: likelihood of going to an EAP for a drinking problem, belief the EAP can help, social support for the EAP from co-workers/others, belief that EAP use will harm employment, and supervisor encourages the EAP for potential drinking problems. FINDINGS: Belief in EAP efficacy directly increased the likelihood of going to an EAP. Greater perceived social support and supervisor encouragement increased the likelihood of going to an EAP both directly and indirectly through perceived EAP efficacy. Black and union hourly employees were more likely to say they would use an EAP. Males and those who reported drinking during working hours were less likely to say they would use an EAP for a drinking problem. CONCLUSIONS: EAP beliefs and social support have significant effects on likelihood to go to an EAP for a drinking problem. EAPs may wish to focus their efforts on creating an environment where there is social support from coworkers and encouragement from supervisors for using EAP services. Union networks and team members have an important role to play in addition to conventional supervisor intervention.

Oligonucleotides with novel, cationic backbone substituents: aminoethylphosphonates.

Oligonucleotide (2-aminoethyl)phosphonates in which the backbone consisted of isomerically pure, alternating (2-aminoethyl)-phosphonate and phosphodiester linkages have been prepared and characterized. One of these single isomer oligonucleotides (Rp) formed a more stable duplex with DNA or RNA than its corresponding natural counterpart. Hybrid stability was more pH-dependent, but less salt-dependent than a natural duplex. The specificity of hybridization was examined by hybridization of an oligonucleotide containing one (2-aminoethyl)phosphonate to oligonucleotides possessing mismatches in the region opposite to the aminoethyl group. In contrast to oligonucleotides containing (aminomethyl)-phosphonate linkages, oligonucleotide (2-aminoethyl)phosphonates were completely stable to hydrolysis in aqueous solution. These oligonucleotides were resistant to nuclease activity but did not induce RNase H mediated cleavage of a complementary RNA strand. Incubation in a serum-containing medium resulted in minimal degradation over 24 hours. Studies of cell uptake by flow cytometry and confocal microscopy demonstrated temperature dependent uptake and intracellular localization. (2-Aminoethyl)phosphonates represent a novel approach to the introduction of positive charges into the backbone of oligonucleotides.

Follow-up study of young attenders at an alcohol unit.

A follow-up study of all under 21 year olds satisfying DSM-III-R criteria for alcohol dependence treated as in-patients at an alcohol treatment unit over a 10-year period was conducted. A total of 52 individuals were identified and of these 80% (44) were traced. The mean period of follow-up was just over 4 years. A poor outcome (no abstinence or controlled drinking for the past 6 months) was adjudged in 61.4% (27) of the cases. This was associated with indicators of early personality difficulties and unsatisfactory schooling experiences. There was considerable concurrent illicit drug use associated with continuing problem drinking. Both good and poor outcome groups initially had striking forensic histories and significant differences developed over the follow-up period with the poor outcome group continuing to have marked legal problems. In addition the poor outcome group continued to make heavy demands on the medical services over the period. One person died from an alcohol related cause. Greater attention to the specific needs of this group is required and specialized treatment units for young problem drinkers is suggested.

(Aminomethyl)phosphonate derivatives of oligonucleotides.

Oligothymidylate (aminomethyl)phosphonates have been prepared, and their enzymatic and physicochemical properties have been studied. The individual isomers of the protected dimers have been separated, characterized, and incorporated into oligonucleotides in which the backbone consists of alternating (aminomethyl)phosphonate and phosphodiester linkages. One of these net neutral, single isomer oligonucleotides forms a duplex with its complementary sequence which is more stable than the corresponding natural counterpart, whereas the other isomer is considerably less stable. Specificity of hybridization is maintained, as determined by the reduction in melting temperature observed upon the introduction of mismatches into the complementary strand of the duplex. The (aminomethyl)-phosphonate linkage is stable toward enzymatic degradation but can be hydrolyzed in aqueous solution at elevated temperature.

Regression of thyrotoxic ophthalmopathy following lithium withdrawal.

The case of a bipolar patient who developed thyrotoxicosis with severe exophthalmos while on lithium therapy is described. The patient had required two surgical decompressions of the right orbit to relieve pressure, which occurred secondary to progression of the exophthalmos, and was scheduled for further surgery. Lithium therapy was discontinued because of poor compliance to the medication and intolerable polyuria. The exophthalmos improved dramatically within 72 hours of the withdrawal of lithium. A severe form of exophthalmos resulting from lithium therapy has not been described in the literature. The case described here adds to the body of information about the possible causes of thyrotoxic ophthalmopathy.

Shop steward handling of alcohol-related problems.

Using direct observations and extensive field interviewing over a 4-year period, this paper examines practices and beliefs of shop stewards in their effects on drinking patterns and consequences. It also includes response frequencies from a random sample survey questionnaire (n = 984) that are consistent with the qualitative analysis of steward behavior. Several themes are extracted which position steward handling of alcohol-related cases as intervening between disciplinary measures of supervisors and consequences of work-related drinking of union employees.

Integration and exchange in multidisciplinary alcohol research.

How do anthropologists effectively enter into cross-disciplinary discourse and influence research direction in a multi-disciplinary field like alcohol studies? We answer: by aiming for an improved qualitative product, and by establishing and participating in exchange structures that cross over disciplinary lines. Using examples from our current study of work environment and alcohol practices, we explain how such an exchange structure was developed around the three areas of: (1) ethnography and local theory, (2) numerical archival data, and (3) survey research. We describe the nature of the exchange and the interplay among the three research segments. In contrast to single-method analysis, whether quantitative or qualitative, we maintain that team efforts can produce knowledge products that are conceptually dense and have higher validity and generalizability.

Ocular vascular disease: in-office primary care diagnosis.

Central retinal artery occlusion occurs most commonly between the ages of 50 and 70 years, and nearly one-half (45%) of patients also have carotid artery disease. Other causes of vision-threatening vascular disease include atherosclerosis, embolism, hypertension, diabetes mellitus, and valvular disease. Symptoms vary, depending on the ocular structures involved. The patient's symptoms are an important clue to the diagnosis of peripheral or posterior retinal vascular occlusion, macular blood vessel disease, intravitreal hemorrhage, optic nerve ischemia, and ocular ischemic syndrome. The patient's ocular symptoms should lead to investigation for clinical signs of ocular vascular disease (eg, hemorrhage, "hard" or "soft" exudates, neovascularization, retinal edema, pallor, emboli, vessel narrowing, or atriovenous crossing changes).

Obstacles to effective alcohol policy in the workplace: a case study.

Based on a case study of an assembly plant in a large US corporation, this paper reports on cultural and environmental influences on the form and process of workplace alcohol policy. Utilizing both ethnographic and survey methods, research findings indicate that although alcohol policies are in place, management and union leaders are unclear about their source and content, and the general employee population perceives alcohol availability and on-job drinking as poorly controlled. In grounded theory fashion, two conceptual themes are employed for organizing empirical explanations of the weakened policy and under-controlled drinking. These are: (1) the dual alcohol policy dilemma, and (2) the union-management debate over authority to discipline. The ambivalent nature of alcohol policy and organizational mechanisms involved in its implementation become risk factors for developing workplace-related alcohol problems.

Minimization of workplace alcohol problems: the supervisor's role.

Based on findings from an ethnographic analysis of an assembly plant in a large manufacturing industry, this paper delineates a conceptual framework for "minimization" that proposes to explain the interactional processes leading to systemized weakening of alcohol problem awareness and action in the unionized workplace. In the course of 50 semi-structured interviews and observations with union representatives and management, the role of the first-line supervisor emerged as pivotal to minimization. Individual case studies from the ethnography and responses from a survey sample of 1000 employees are used to illustrate how goals and processes of the workplace culture impede forthright action and clear lines of decision making on alcohol issues.

Aligning lithography on opposite surfaces of a substrate.

Equipment has been developed for aligning lithographic features between opposite surfaces of substrates to within 1 microm. It will work with opaque substrates and allows registration to existing features on the other surface.