By David N. Leff
Three weeks from now, a thickset, grizzled animal will wake up, emerge from his burrow and look around. If that groundhog can't see his shadow, because the sky is overcast, he'll know that spring is on the way and amble off in search of food. But if the woodchuck does see his shadow, because the sun is shining, he'll know he's in for six more weeks of winter, and will pop back down his to den to hibernate some more.
Groundhog Day, Feb. 2 each year, celebrates this season-predicting prowess of Marmota monax — although in reality he's no smarter than the rest of us.
Virologists and molecular hepatologists have now enlisted M. monax in a different kind of forecasting mission — helping to set up a small-animal model, namely a transgenic mouse, that scopes the progress of hepatitis B infection all the way to liver cancer.
"Woodchucks are good for experimentation," observed virologist Charles Rogler, of Albert Einstein College of Medicine, in New York, "because their hepatitis B virus is not infectious to humans, and also because the pathology of woodchuck hepatitis virus closely parallels that of human hepatitis B infection."
Worldwide, an estimated 350 million men, women and children carry the human hepatitis B virus (HBV), with hot spots of consequent liver cancer throughout Southeast Asia and southern Africa. "The number of carriers, historically," Rogler said, "has been very small in America and focused in certain subpopulations of individuals who have migrated to the U.S. from areas of the world that have very high prevalence."
There are about 125,000 new HBV infections a year in the U.S., according to Julie Lehane, editorial and information director of the American Liver Foundation, in Cedar Grove, N.J. "Some 75 to 80 percent of them recover," she told BioWorld Today, adding that "of those who don't recover, 30 to 40 percent have persistent infection with no symptoms, and many progress to cancer."
Rogler defined an HBV carrier as "a person who has the virus replicating in his or her liver. Carriers who don't know they are carriers," he continued, "may spread the disease to their infants during birth. And the reason that being a carrier has carcinogenic sequellae," he went on, "is because persistent viral infection sets up a continuous pathophysiology in the liver, which leads to cell death and regeneration in the presence of mutation. And wherever you get that process, it's a formula for the selection of precancerous cells and then cancerous cells." (See BioWorld Today, Dec. 29, 1997, p. 1.)
In a recent year, the National Cancer Institute counted some 16,000 cases of primary hepatocellular carcinoma diagnosed in the U.S.
Tolerant Mouse Model Welcomes Woodchuck Cells
Rogler is senior author of a paper in the current Proceedings of the National Academy of Sciences, dated Jan. 6, 1998. Its title: "Liver repopulation with xenogenic hepatocytes in B and T cell-deficient mice leads to chronic hepadnavirus infection and clonal growth of hepatocellular carcinoma."
"These mice," Rogler told BioWorld Today, "are the first small-animal model that transplants hepatocytes [liver cells], which replicate the virus by its natural mechanism." Hepadnaviruses, he explained, "describe the family of viruses of which human hepatitis B virus [HBV] is the prototype, and woodchuck hepatitis B virus [WHV] a family member."
A unique feature of his model, Rogler explained, "is that in it we're transplanting woodchuck cells, which have the natural receptors for the woodchuck virus on them, enabling us to study the infectious process.
"So we can transplant either infected or uninfected hepatocytes from a woodchuck carrier to our transgenic mice," he continued.
Rogler and his team deployed a couple of genetic tricks to combine two types of transgenes into a mouse. First, these doubly transgenic animals are knockouts for a recombinant activation gene (RAG-2), which deprives the rodents of their B cell and T cell immune defenses, rendering them immune-tolerant to foreign antigens — in this case, woodchuck cells.
Secondly, the mice carry a urokinase plasminogen activator transgene, uPA. "This uPA gene," Rogler explained, "compromises the ability of existing liver cells to grow, and provides the permissive microenvironment of a sponge, which can soak up transplanted woodchuck hepatocytes and stimulate them to grow.
"So we now had an animal," he recounted, "that can receive xenogenic hepatocytes, for which its liver acts as a sponge. We injected these woodchuck cells into the spleens of our mice, from which they rapidly got transported into the liver. There, the hepatic microenvironment stimulated them to grow very rapidly, in the absence of rejecting the foreign cells."
One such recipient mouse, transplanted with uninfected woodchuck hepatocytes, developed a persistent viral infection. "Persistent hepatitis B infection," Rogler pointed out, "in real animals and real humans, causes a continuous liver disease called chronic active hepatitis, which leads to liver cancer. The idea that started us on it all," he observed, "was the ability to transplant cells and speed up the carcinogenesis process, while studying the gene-expression changes.
Humanizing System Under Way
"We're working now to refine the model, which," he pointed out, "is an animal model that closely mimics the human disease, but it's not the actual human disease. So if one can adapt the system to transplant human hepatocytes into these mice and get them to grow, then of course many other therapeutic opportunities are open, for many different kinds of viruses, etcetera, that infect the liver."
Meanwhile, Rogler concluded, "The present model is suitable for biotechnology companies that are interested in testing antiviral and anticancer compounds. I'm not totally at liberty to say too much more," he went on, "because there are certain biotech interests involved." *