Molecular mechanisms for hyperinsulinaemia induced by overproduction of selenium-dependent glutathione peroxidase-1 in mice.

AIMS/HYPOTHESIS: We previously observed hyperglycaemia, hyperinsulinaemia, insulin resistance and obesity in Gpx1-overexpressing mice (OE). Here we determined whether these phenotypes were eliminated by diet restriction, subsequently testing whether hyperinsulinaemia was a primary effect of Gpx1 overexpression and caused by dysregulation of pancreatic duodenal homeobox 1 (PDX1) and uncoupling protein-2 (UCP2) in islets. METHODS: First, 24 male OE and wild-type (WT) mice (2 months old) were given 3 g (diet-restricted) or 5 g (full-fed) feed per day for 4 months to compare their glucose metabolism. Thereafter, several mechanistic experiments were conducted with pancreas and islets of the two genotypes (2 or 6 months old) to assay for beta cell mass, reactive oxygen species (ROS) levels, mitochondrial membrane potential (Deltapsi(m)) and expression profiles of regulatory proteins. A functional assay of islets was also performed. RESULTS: Diet restriction eliminated obesity but not hyperinsulinaemia in OE mice. These mice had greater pancreatic beta cell mass (more than twofold) and pancreatic insulin content (40%) than the WT, along with an enhanced Deltapsi(m) and glucose-stimulated insulin secretion in islets. With diminished ROS production, the OE islets displayed hyperacetylation of H3 and H4 histone in the Pdx1 promoter, elevated PDX1 and decreased UCP2. CONCLUSIONS/INTERPRETATION: Overproduction of the major antioxidant enzyme, glutathione peroxidase 1, caused seemingly beneficial changes in pancreatic PDX1 and UCP2, but eventually led to chronic hyperinsulinaemia by dysregulating islet insulin production and secretion.

Lipopolysaccharide and interferon-gamma-induced nitric oxide production and protein oxidation in mouse peritoneal macrophages are affected by glutathione peroxidase-1 gene knockout.

This study investigated the role of glutathione peroxidase-1 (GPX1) in protein oxidation in peritoneal macrophages. Macrophages isolated from both wild-type (WT) and GPX1 knockout (KO) mice were activated by lipopolysaccharide (LPS, 1 microg/ml) and interferon-gamma (IFN, 10 U/ml for 24 or 48 h in the presence or absence of 1 microM diquat (DQ), 250 microM aminoguanidine (AG, an inhibitor of inducible nitric oxide synthase), and (or) 100 microM diethyldithiocarbamate (DETC, an inhibitor of Cu,Zn-SOD). In the KO macrophages, there was no protein band detected by Western blot with anti-GPX1 antibody and 98% reduction in total GPX activity compared with WT cells. Nitric oxide (NO) synthesis was greatly enhanced after 24 h by GPX1 knockout and DQ, but inhibited by AG or DETC. Protein carbonyl formation in total cell extract was clearly associated with NO synthesis as higher levels of protein carbonyl were detected in activated KO than WT macrophages, and DQ enhanced slightly while AG or DETC virtually blocked its formation. A similarly marginal effect of GPX1 KO was observed on protein nitration. The LPS/IFN/DQ-induced DNA fragmentation was blocked by AG, but not by DETC. Cell viability at 48 h was decreased by the LPS/IFN activation and further reduced by the addition of DQ, but restored by AG. In conclusion, GPX1 affects the NO production in activated peritoneal macrophages and protects these cells against NO-associated protein oxidation.

Temporal pathogenesis of experimental neonatal woodchuck hepatitis virus infection: increased initial viral load and decreased severity of acute hepatitis during the development of chronic viral infection.

Acute hepatitis B virus (HBV) infections either resolve or progress to chronicity. Identification of early deviations in host-virus responses associated with these outcomes can further differentiate cause-effect mechanisms that initiate and maintain chronicity. Neonatal woodchucks were infected experimentally with the woodchuck hepatitis virus (WHV) at 3 days of age. At 8 or 14 weeks of age (i.e. , the early- or mid-acute stage of infection), whole blood and large surgical biopsies of the liver were obtained from infected animals and uninfected controls. These were stored for later correlating histopathologic responses and viral load with the subsequently determined outcome of infection. As of 1 year postinfection, half of the surgically treated infected woodchucks had developed self-limited infections, while the other half developed chronic infections. The self-limited outcome was characterized by decreased viral load in acute-phase liver and plasma and a generally robust acute hepatic inflammatory response. Comparisons at the same early time points revealed that the chronic outcome was characterized by increasing initial viral load in liver and plasma, and a detectable, but diminished, acute hepatic inflammation. These cotemporal comparisons indicate that there is an early host-response deviation during the acute phase of a developing chronic infection. Continued analysis of the tissues banked from this study will facilitate further temporal characterization of acute-phase mechanisms that determine resolution versus chronicity in WHV infection. Understanding such mechanisms may be useful in the rational design of therapy for established chronic HBV infection.

Hepatic expression of the woodchuck hepatitis virus X-antigen during acute and chronic infection and detection of a woodchuck hepatitis virus X-antigen antibody response.

The expression and localization of the woodchuck hepatitis virus X-antigen (WHxAg) was examined and compared with other markers of a woodchuck hepatitis virus (WHV) infection using rabbit antisera generated against recombinant WHxAg produced in bacteria. Cellular fractionation studies showed that WHxAg was localized to the soluble and cytoskeletal fractions of the cell when assayed by immunoprecipitation of [35S]-met-cys labeled extracts derived from primary cultures of acute WHV-infected hepatocytes. Immunohistochemical examination of liver from chronic WHV-infected animals showed WHV core antigen (WHcAg) and WHxAg expression in non-neoplastic tissue. The WHxAg was found localized to the cytoplasm of infected cells, similar to WHcAg. WHxAg expression was diminished in the foci of altered hepatocytes and in hepatocellular adenomas but was found in only 1 of 11 hepatocellular carcinomas (HCC). Hepatic biopsies from woodchucks experimentally inoculated with WHV were examined during the acute phase of infection and during convalescence for WHcAg and WHxAg expression by immunohistochemistry. Concurrent expression of WHcAg and WHxAg was observed during the viremic phase of infection. The two antigens exhibited similar localization to the cell cytoplasm, similar distribution within the liver lobule, and similar patterns of clearance during convalescence. An immune response to WHxAg was documented in some woodchucks following acute WHV infection. These studies further define the woodchuck model of HBV infection and should allow for the investigation of the role of hepadnaviral X-antigen expression in the pathogenesis of chronic hepatitis and HCC.

Characterization and immortalization of woodchuck hepatocytes isolated from normal and hepadnavirus-infected woodchucks (Marmota monax).

Primary woodchuck (Marmota monax) hepatocytes from normal woodchucks and woodchucks with chronic woodchuck hepatitis virus (WHV) infection were cultured in either a conventional serum-containing medium or a serum-free medium. The de novo synthesis of the plasma proteins albumin, transferrin, fibrinogen, and complement C3 were identical under both conditions. However, expression of the WHV and the synthesis of nitric oxide were diminished under serum-free conditions. Primary woodchuck hepatocytes cultured in conventional, serum-containing medium were immortalized utilizing the simian virus 40 T antigen oncogene. Immortalized hepatic cell lines retained differentiated functions of nitric oxide synthesis and expression of complement C3. The woodchuck hepatocyte culture model will supplement current experimental methods, allowing investigation of hepadnaviral pathogenesis, including hepatocarcinogenesis in vitro.

Enhanced neutralization of feline immunodeficiency virus by complement viral lysis.

The ability of complement to inactivate feline immunodeficiency virus (FIV) was examined. Treatment of virus with complement plus sub-neutralizing titers of antiserum resulted in a significant reduction in the virus titer compared with treatment of the virus with complement or antibody alone. One of the mechanisms by which cat complement inactivates FIV was shown to be by viral lysis as determined by a reverse transcriptase release assay. Kinetic studies revealed that viral lysis is initiated soon after the addition of complement to a mixture of virus and antiserum. Treatment of FIV with normal non-complement-inactivated human serum resulted in virus inactivation and release of viral RT in the absence of specific antiserum. It appears that FIV activates complement directly through the classical pathway and that integrity of the membrane attack components is a requirement for FIV lysis by human serum. The vulnerability of two distinct isolates of FIV to complement lysis was compared using complement from different species. Oradell isolate was more sensitive to complement lysis than the Petaluma isolate as assessed by reverse transcriptase release. It appears that factors intrinsic to the virus isolate may influence the amplitude of complement-dependent viral lysis.

New enzyme immunoassays for the serologic detection of woodchuck hepatitis virus infection.

The woodchuck and the woodchuck hepatitis virus (WHV) have been used as a model of hepatitis B virus infection and its disease sequelas. Serologic responses to WHV infection have been described in previous reports from this laboratory by using virus-specific radioimmunoassays (RIAs) for WHV surface antigen, antibody to WHV core antigen, and antibody to WHsAg. In this study, we developed and evaluated new enzyme immunoassays (EIAs) for these WHV serologic markers. Relative to the established RIAs, the EIAs were either improved or comparable in their sensitivity and specificity, and in their utility for monitoring experimental WHV infection and classifying woodchucks into serological diagnostic categories. These EIA systems are amenable to the quantitative titration of antibodies and quantitation of WHV antigens in serum, and ultimately should allow improved resolution of virologic and humoral immune responses of woodchucks to WHV infection.

Characterization of two monoclonal antibodies against feline immunodeficiency virus gag gene products and their application in an assay to evaluate neutralizing antibody activity.

Monoclonal antibodies (MAbs) 3B7 and 1C11 were produced against the gag gene products of feline immunodeficiency virus (FIV). These MAbs reacted strongly with FIV p24 in Western blots (immunoblots) and recognized p50 with a lower intensity. They specifically bound antigens in the cytoplasm of FIV-infected cells as determined by indirect immunofluorescence and immunocytochemistry. Although neither MAb inhibited viral replication in vitro, they were useful in a simple assay for the detection and quantification of infectious virus and neutralizing antibody activity. The assay utilizes Crandell feline kidney cells and requires 4 days for completion. Neutralizing antibodies in cats were detected 3 to 4 weeks after experimental infection with FIV. Antibody titres progressively increased during the first year of infection reaching high titres which were maintained 2.5 years post-infection. The MAbs produced should be valuable reagents for the monitoring of viral replication in cells or tissues from FIV-infected cats and for other in vitro applications.

Anti-idiotypic antibodies to feline leukemia virus: an approach for retroviral immunization strategies.

The present study describes an approach to the development and use of anti-idiotypic antibodies as a possible immunization strategy to prevent retroviral infection. The rationale for using anti-idiotypes (anti-Ids) to try to elicit an antigenic-specific immune response is examined, and the production and characterization of polyclonal and monoclonal anti-Ids are described. Several techniques were used to determine antigenic mimicry and anti-Id subtypes. The potential use of anti-Ids in feline leukemia virus (FeLV) receptor studies and vaccine trials in vivo were investigated. Results from these studies suggest that the anti-Id strategy is feasible for the FeLV model. Polyclonal Ab2 reagents were developed that blocked virus-receptor binding and thus inhibited viral infection in vitro and induced humoral immune responses in 6- to 8-week old kittens characterized by production of Ab3 with the ability to bind the original FeLV envelope protein gp70 as assessed by Western blot analysis.

Antibody response to reverse transcriptase in cats infected with feline immunodeficiency virus.

The antibody response in cats to feline immunodeficiency virus (FIV) reverse transcriptase (RT) was followed for 3 years. Eight of the nine cats used in this study produced reverse transcriptase-inhibiting (RTI) antibodies. Relative inhibitory means of 2.9%, 18.4%, 33%, and 47% were found 6, 12, 24, and 36 months, respectively, after infection with FIV. The enzyme activity was suppressed by greater than or equal to 78% with the use of 100 micrograms of FIV-associated IgG. The RTI antibodies were FIV-specific, as they did not inhibit other mammalian retroviral polymerases, including feline leukemia virus RT. An RT-inhibition assay with sera in the presence of protein A and immunoblot analysis showed that antibody binding to FIV RT protein p62 is independent of antibody ability to block enzyme activity. Viral RT released by detergent-treated virus was stable for more than 6 weeks at 4 degrees C, whereas its activity was reduced by 50% after 2 weeks at 37 degrees C. Because significant concentrations of RTI antibodies are detected only at 1 to 2 years after infection, they can be used to determine the approximate time of virus infection and as a marker for disease progression.

Testing of nucleoside analogues in cats infected with feline leukemia virus: a model.

In the present communication we evaluate the feline leukemia virus (FeLV) infection of cats as a model for antiretroviral chemotherapy studies. Additionally, we report the results of testing the antiviral effect of the compounds, 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxycytidine, 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine against FeLV replication in vitro. Cumulative data from experiments in which FeLV-infected cats were treated with AZT at different stages of experimental infection are also evaluated.

3'-Azido-3'-deoxythymidine in feline leukemia virus-infected cats: a model for therapy and prophylaxis of AIDS.

Due to similarities between human immunodeficiency virus and feline leukemia virus, the etiological agents of acquired immunodeficiency syndromes in humans and cats, the feline system was used as a model to conduct preliminary investigations as to the efficacy of the thymidine analogue 3'-azido-3'-deoxythymidine (AZT) as a therapeutic and preventive agent against retroviruses. In vitro evaluations of AZT cytotoxicity and its antiviral effects were conducted. Subsequently, 50 6-week-old specific pathogen free kittens were inoculated with a highly immunosuppressive strain of Richard-Feline Leukemia Virus. These cats were randomly subdivided into smaller groups with initiation of AZT treatment at variable times postinfection. All animals were periodically monitored for circulating infectious virus particles and virus-neutralizing antibodies. Their clinical condition was closely followed throughout the 6-week AZT treatment phase and for several months thereafter. The results indicate that AZT prevents retrovirus infection if administered immediately following virus exposure, and may also reduce retrovirus replication if administered to previously infected animals. Some of the treated cats developed neutralizing antibodies against the virus and became resistant to subsequent viral challenge. Future trials with this drug, both for the prevention and treatment of retroviral diseases in humans and animals, are warranted.