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Biomedical subjects

B Tennant

Publications and source records attributed to B Tennant.

At least 19 recordsLinked to original sources
PubMed2001

Large nontransplanted hepatocellular carcinoma in woodchucks: treatment with adenovirus-mediated delivery of interleukin 12/B7.1 genes.

BACKGROUND: Cytokine-based gene therapy strategies efficiently stimulate immune responses against many established transplanted tumors, leading to rejection of the tumor. In this study, we investigated the therapeutic potential of cancer immunotherapy in a clinically more relevant model, woodchucks with primary hepatocellular carcinomas induced by woodchuck hepatitis virus. METHODS: Large (2-5 cm), established intrahepatic tumors were given an injection once with 1 x 10(9) plaque-forming units of AdIL-12/B7.1, an adenovirus vector carrying genes for murine interleukin 12 and B7.1, or of AdEGFP, the control virus, and regression of the tumors was then monitored. Five animals were used in total. RESULTS: In four tumor-bearing animals, the antitumor response was assessed by autopsy and histologic analysis within 1-2 weeks after treatment. In all animals treated with AdIL-12/B7.1 therapy versus AdEGFP therapy, we observed substantial tumor regression (P =.006; two-sided unpaired Student's t test) accompanied by a massive infiltration of T lymphocytes. These tumors also contained increased levels of CD4(+) and CD8(+) T cells and interferon gamma (IFN gamma). In continuously growing tumor nodules given an injection of the control virus or in nontumoral liver, no such effects (i.e., tumor regression and increased levels of CD4(+) and CD8(+) T cells and IFN gamma) were detected. In the fifth animal, monitored for long-term antitumor efficacy by magnetic resonance imaging (MRI) after intratumoral vector administration by MRI guidance, the tumor was almost completely eliminated (> or = 95%) 7 weeks after treatment. CONCLUSION: Adenovirus vector-based immunotherapy appears to be an effective treatment of large nontransplanted (orthotopic) tumors that acquire malignant characteristics in a stepwise process, reflecting the real-world scenario of hepatocellular carcinoma in humans.

Adenoviridae↗
PubMed2001

Antiviral L-nucleosides specific for hepatitis B virus infection.

A unique series of simple "unnatural" nucleosides has been discovered to inhibit hepatitis B virus (HBV) replication. Through structure-activity analysis it was found that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides beta-L-2'-deoxycytidine, beta-L-thymidine, and beta-L-2'-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (alpha, beta, and gamma) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 10(8) genome equivalents/ml of serum and there was no drug-related toxicity. In addition, the decline in woodchuck hepatitis virus surface antigen paralleled the decrease in viral load. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

Animals↗
PubMed2001

Seasonal changes in serum leptin, food intake, and body weight in photoentrained woodchucks.

Male woodchucks (Marmota monax) were maintained in northern vs. southern hemisphere photoperiods, provided feed and water ad libitum, and evaluated every 2 wk for 23 mo for body weight, absolute and relative food intake, body temperature, serum testosterone, and serum concentrations of leptin measured using an anti-mouse leptin enzyme-linked immunoassay. During late spring and summer, body weight increased 56 +/- 4% above winter nadirs, and during the autumn and early winter weights decreased 27 to 43% below midsummer maxima. Serum leptin initially increased during increases in body weight, in the late spring, reached peak values (490 +/- 32 pg/ml) in summer during the initial decline in body weight, and later decreased along with body weight to reach basal values (20 +/- 5 pg/ml) in late winter. Spontaneous declines in food intakes in summer began 2-6 wk before resulting declines in body weight and occurred during increases in leptin >100 pg/ml. The rate of decline in food intakes was greatest when serum leptin was at or near peak values. Food intake increased in late winter when leptin was low and 7-10 wk before resulting increases in body weight. Testis recrudescence occurred when leptin was declining to near basal levels. The results suggest that leptin is involved in the hormonal regulation of the circannual cycle in the drive for voluntary food intake in this species.

Animals↗
PubMed2001

Antiviral beta-L-nucleosides specific for hepatitis B virus infection.

Three simple, related nucleosides, beta-L-2'-deoxycytidine (LdC), beta-Lthymidine (LdT), and beta-L-2'-deoxyadenosine (LdA), have been discovered to be potent, specific and selective inhibitors of the replication hepatitis B virus (HBV), as well as the closely related duck and woodchuck hepatitis viruses (WHV). Structure-activity relationship analysis indicates that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific anti-hepadnavirus activity. The simple nucleosides had no effect on the replication of 15 other RNA and DNA viruses, and did not inhibit human DNA polymerases (alpha, beta and gamma) or compromise mitochondrial function. The nucleosides are efficiently converted intracellularly into active triphosphate metabolites that have a long half-life. Once-daily oral administration of these compounds in the woodchuck efficacy model of chronic HBV infection reduced viral load by as much as 10(8) genome equivalents/ml serum and there was no drug-related toxicity. In addition, a decline in WHV surface antigen (WHsAg) paralleled the decrease in viral load. This class of nucleosides displays an excellent overall safety profile. The first compound, LdT, has already entered clinical trials and LdC, currently being developed as a prodrug, is expected to enter the clinic in the near future. These compounds have the potential for use in combination therapy with the goal of achieving superior viral suppression and diminishing the onset of resistance.

Animals↗
PubMed2000

Transduction efficacy, antitumoral effect, and toxicity of adenovirus-mediated herpes simplex virus thymidine kinase/ ganciclovir therapy of hepatocellular carcinoma: the woodchuck animal model.

Gene therapy for hepatocellular carcinoma (HCC) has shown some promise, but its evaluation requires relevant experimental models. With this aim, we present an evaluation of the interest of using the woodchuck model of HCC to assess in vivo gene transfer efficiency. We tested the transduction efficacy of the adenoviral vectors directing lacZ gene product expression under the control of the cytomegalovirus and alpha-fetoprotein (AFP) regulatory sequences. We have also investigated whether an adenoviral cytomegalovirus-thymidine kinase (Tk) vector might induce an antitumoral effect in this model. Our results demonstrate that with direct intratumoral and intrahepatic arterial injections, transduction of a significant proportion of tumor cells occurred even in large HCC nodules. Furthermore, due to intra-arterial anastomoses, direct intratumoral injection led to transduction of some noninjected HCC nodules. Moreover, direct intratumoral injection of a herpes simplex virus-1 Tk-encoding vector induced, on ganciclovir administration, a significant antitumoral effect in the two animals evaluated. However, in one animal, massive hepatic failure occurred due to Tk expression in nontumor cells. These results emphasize the need to target the expression of the Tk gene to tumor cells using a hepatoma-specific promoter such as AFP promoter. However, we showed that, in vivo, lacZ expression as driven by the AFP promoter was extremely low, thus emphasizing some potential pitfalls when using this approach. Altogether, our data stress the need to test gene therapy-based strategies in such in vivo animal models of HCC and evaluate gene transduction, expression, and biological activity, as well as its potential toxicity.

Adenoviridae↗
PubMed1998

Aberrant expression of a cytokeratin in a subset of hepatocytes during chronic WHV infection.

Chronic infection of woodchucks with woodchuck hepatitis virus (WHV) invariably leads, within 2-4 years, to the appearance of hepatocellular carcinoma (HCC). HCC is preceded by an extended period of chronic liver damage, probably resulting from the immune response to viral antigens. It may be that infection itself also induces changes in the hepatocyte population. To begin to identify some of the changes in the liver prior to the appearance of HCC, monoclonal antibodies (MAbs) were generated from mice immunized with hepatocytes from a woodchuck chronically infected with WHV or with a tumor lysate. Immunofluorescence microscopy was used to select MAbs that reacted with host markers whose patterns of expression would distinguish chronically infected from uninfected liver or from liver tumors. One of these MAbs (2F2) reacted strongly with a subset of hepatocytes in chronically infected liver; a similar staining pattern was not detected in uninfected or transiently infected liver. Evidence is presented that this strong staining reaction reflects the overexpression or accumulation of the hepatocyte-specific intermediate filament protein, cytokeratin K18, a protein previously implicated in cryptogenic cirrhosis of the liver in humans (Ku, N. O. , Wright, T. L., Terrault, N. A., Gish, R., and Omary, M. B. J. Clin. Invest. 99: 19-23, 1997). Double immunofluorescent staining with antibodies to K18 and M-envelope protein of WHV suggested that strong reactivity to K18 was limited to cells expressing high levels of one or both of the large viral-envelope proteins, M and L; however, high expression of these viral proteins was not always associated with a strong K18 staining reaction.

Animals↗
PubMed1998

Lamivudine therapy of WHV-infected woodchucks.

Hepatitis B viruses establish a chronic, productive, and noncytopathic infection of hepatocytes. Viral products are produced by transcription from multiple copies (5-50) of covalently closed circular (ccc) viral DNA. This cccDNA does not replicate, but can be replaced by DNA precursors that are synthesized in the cytoplasm. The present study was carried out to determine if long-term treatment with an inhibitor of viral DNA synthesis would lead to loss of virus products, including cccDNA, from the liver of woodchucks chronically infected with woodchuck hepatitis virus. Viral DNA synthesis was inhibited with the nucleoside analog, lamivudine (2'-deoxy-3'-thiacytidine). Lamivudine treatment produced a slow but progressive decline in viral titers in serum, to about 0.3% or less of the initial level. However, even after maintenance of drug therapy for 3-12 months, > 95% of the hepatocytes in most animals were still infected. Significant declines in the percentage of infected hepatocytes and of intrahepatic cccDNA levels were observed in only three woodchucks, two in the group receiving lamivudine and one in the placebo control group. Moreover, virus titers eventually rose in woodchucks receiving lamivudine, suggesting that drug-resistant viruses began to spread through the liver starting at least as early as 9-12 months of treatment. Three types of mutation that may be associated with drug resistance were found at this time, in a region upstream of the YMDD motif in the active site of the viral reverse transcriptase. The YMDD motif itself remained unchanged. Not unexpectedly, the lamivudine therapy did not have a impact on development of liver cancer.

Amino Acid Sequence↗
PubMed1998

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.

1-Deoxynojirimycin↗
PubMed1998

In vivo effects of mutations in woodchuck hepatitis virus enhancer II.

Woodchuck hepatitis virus (WHV) enhancer II (EnII) is located upstream of the major pregenomic RNA promoter and is thought to play an important role in the insertional activation of the N-myc2 gene during WHV hepatocarcinogenesis. WHV EnII is recognized by at least three host transcription factors: HNF-1, HNF-4, and Oct-1. Here, the roles of these EnII-binding factors in viral transcription and replication have been further examined. In HepG2 cells transiently transfected with a chloramphenicol acetyltransferase (CAT) gene whose expression is dependent upon EnII, mutations in either the HNF-1 or the HNF-4 site strongly reduced CAT activity, while ablation of the Oct-1 site decreased CAT expression only twofold. Mutations in more than one site completely abolished reporter expression. These same mutations were also tested in an overlength WHV genome for their impact on viral replication and gene expression. In transfected HepG2 cells, lesions in the HNF-1 site inactivated pregenomic RNA expression and viral reverse transcription, with only minimal effects on the expression of other viral mRNAs. By contrast, Oct-1 site lesions had no effect on either viral RNA synthesis or DNA replication, and HNF-4 site lesions produced a modest reduction of pregenomic RNA but had no impact on viral DNA synthesis. Testing of the mutants in susceptible woodchucks revealed that, as expected, viruses with lesions in the HNF-1 site were nearly noninfectious, while mutants with lesions at the Oct-1 site were fully replication competent. HNF-4 site mutants were replication competent but may display reduced levels of replication in the intact animal host. We conclude that (i) EnII is primarily devoted to the regulation of pregenomic RNA in WHV, (ii) HNF-1 is essential for EnII function in vivo, and (iii) HNF-4 plays a demonstrable but adjunctive role in EnII function.

Animals↗
PubMed1997

Long-term entrainment of circannual reproductive and metabolic cycles by Northern and Southern Hemisphere photoperiods in woodchucks (Marmota monax).

Woodchucks were exposed to simulated Northern Hemisphere (boreal) or Southern Hemisphere (austral) natural photoperiods in groups of 17 males and 17-18 females at 20-23 degrees C for 69 mo and examined monthly. Photoentrainment of endogenous cycles was evaluated based on dates of peak body weight, peak testis volume, increased serum testosterone in males, and increased serum progesterone in females. Boreal photoperiods entrained and synchronized annual cycles in 15 of 17 males and in all 17 females; 2 males never entrained and free-ran at 9- to 11-mo intervals. Austral photoperiods phase-advanced cycles by approximately 6 mo after 2.5 yr in 34 of 35 animals. Four entrained males became refractory after 4 yr, free-running at 6- to 10-mo intervals. Photoentrained boreal animals became phase-advanced by 1 mo during the first 2 yr, and then had 12-mo cycles for 4 yr. In Year 5, on average, boreal cycles included initial testosterone elevations in mid-January (vs. mid-July in australs), parturition in early March (vs. early September in australs), and peak body weight in mid-July (vs. late January in australs). The results confirm that endogenous circannual cycles of woodchucks can be entrained and synchronized for 6 yr by daily changes in photoperiod similar to those of midnorthern latitudes, and can be re-entrained and phase-advanced 6 mo by photoperiods of midsouthern latitudes.

Animals↗
PubMed1997

Estrus, fertility, early embryo development, and autologous embryo transfer in laboratory woodchucks (Marmota monax).

Reproductive parameters were studied in female laboratory woodchucks over a 6-year period. The pregnancy rate in adult females after ad libitum exposure to a male (n = 643) was 72%; that after limited (4 to 8 h) exposure with video observations (n = 31) was only 37%. However, limited exposure resulted in a 75% ovulation rate, suggesting a problem with fertilization and not ovulation. The ovulation rate was 0% in the absence of mating (n = 10), suggesting that spontaneous ovulation is not the usual situation in this species. With ad libitum mating, fertility was greater (P < 0.05) for 2- to 4-year-old females (73%) than for 1-year-old (56%) or > or = 5-year-old (58%) females. Mean (+/-SEM) litter size at birth was greater (P < 0.05) for 2- to 5-year-old animals (4.0 +/- 0.1) than for 1-year-old animals (2.9 +/- 0.2). However, in 1-year-old females the neonatal mortality was low, and the litter size at weaning (2.4 +/- 0.3 pups) did not differ from that of older females (2.7 +/- 0.1 pups). The measurement of vulval diameter every 3 to 4 days in 31 adult females suggested that enlargement to > or = 7 mm was indicative of proestrus or estrus and was a good indicator of willingness to accept mounting by males. Vulval swelling > or = 7 mm (n = 31) lasted 5 to 47 days. Fertile mating occurred from 3 to 30 days after vulval diameter reached > or = 7 mm, near the time of peak enlargement (10.1 +/- 0.2 mm), and was followed in several days by a decrease in vulval size. Fifty-three females were allowed only limited (6 to 10 h) video-observed exposure to males, with (n = 26) or without (n = 27) another 12 to 24 h of unobserved exposure, and were examined surgically for ovulation and pregnancy at known times after mating. There were no consistent differences among nonovulating, ovulating nonfertile, and fertile females in the longest duration of mating (7.2 +/- 0.4 min) or in the number of matings lasting > or = 1 min (5.7 +/- 0.8). Compared with nonfertile females, fertile females experienced more matings > or = 3 min in duration (3.3 +/- 0.4 versus 1.8 +/- 0.2) and > or = 5 min in duration (2.9 +/- 0.4 versus 1.1 +/- 0.1). At postmating days 1, 2, 3, 4, 5, 6, and 7 the embryos were observed to be 1-, 4-, 4- to 32-cell morulae, 16- to 60-cell morulae, free-floating blastocysts, anti-measometrial peri-implantation blastocysts, and attached blastocysts respectively. Autologous transfer of day-5 uterine embryos of normal appearance into the noncontiguous contralateral uterine horn was performed in five females that had been subjected to a hemi-ovariectomy. Transfers resulted in pregnancy and litters in four of the five females. The results suggest that this large, circannual sciurid rodent can be successfully and predictably bred as a laboratory animal model, has vulval changes indicative of proestrus, is principally an induced ovulator, has an early embryo development rate similar to that of rats and mice, and may be amenable to reproductive technologies developed for more common laboratory rodents.

Animals↗