Seeking Immunity
UMaine scientists use zebrafish to study the building blocks of innate disease resistance that could one day benefit human health

About the Photo:  Carol Kim's interest in applying molecular virology and microbiology to benefit the biomedical field and aquaculture industry brought her to UMaine in 1998, where she set up the state's first and largest zebrafish facility.

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In the room saturated in white light and the sound of rushing water, it's 82 degrees. In rows of shoebox-size tanks stacked five high dart slivers of silver — tiny fish no bigger than grains of rice, adults two inches long and all ages in between.

Even if it's been years since your last childhood trip to the store to bring home a plastic bag bulging with water containing an aquarium fish, the sight of more than 40,000 zebrafish in a laboratory at the University of Maine can still make your heart skip a beat. It all comes flooding back: the excitement and wonder of watching zebras native to India's Ganges River swimming just inches from your face.

Today, what's even more remarkable about this hardy, popular home aquarium fish is the splash it's making in basic science. Zebrafish as model organisms are now comparable in importance to mice and fruit flies in the study of genetics and disease prevention. Evidence of the research potential of the little striped vertebrates is in the prevalence of zebrafish labs at medical schools nationwide and the explosion of the resulting scientific literature being published.

Zebrafish are being studied to better understand such human conditions as congenital disease, cancer and aging. At UMaine, the zebrafish facility of Carol Kim, associate professor of biochemistry, microbiology and molecular biology, is a hub of activity, facilitating the initiatives of as many as five campus scientists conducting research in such areas as microbiology, toxicology, immunology, developmental biology and genetics. In her research on innate immunity and infectious diseases, Kim collaborates with scientists across the country and abroad.

Zebrafish hold the promise of very basic scientific and applied research breakthroughs for Kim, a faculty member in the Department of Biochemistry, Microbiology and Molecular Biology, with affiliations with the Functional Genomics graduate program offered by UMaine, Jackson Laboratory and Maine Medical Center Research Institute, part of the Graduate School of Biomedical Science. In her research, she is studying the biological factors that supplement and prolong the body's immune response to infection. Working on the molecular level, she is studying how cells respond to infection, contributing basic knowledge that could one day lead to disease prevention or more effective vaccines in humans, and in other mammals and fish species.

"We're using the zebrafish as a model for the immune response to infectious disease," Kim says. "The zebrafish is a powerful model system that will allow us to better understand the immune system and implement preventative measures against infection for humans, as well as fish."
 

Zebrafish make ideal model organisms in science for many of the same reasons they are popular in home aquariums. They are easy to care for and breed, and they are resilient, tolerating fluctuations in water temperatures. A female can lay up to 300 eggs each week. Zebras can live for up to five years.

For researchers, two of the most important characteristics of zebrafish are their rapid and viewable development, and their biological traits that mimic those of humans. Zebrafish eggs are transparent. Under a microscope, scientists can watch the embryonic growth that occurs in two to four days following fertilization. Development is so rapid that a single cell multiples to take on a fish shape within 24 hours.

Zebrafish can serve as models for human developmental biology, neurobiology, toxicology and genetic disease. While they are lower vertebrates, their genes, developmental processes, anatomy, physiology and behaviors bear similarities to those of humans, according to ZFIN, the Web-based Zebrafish Information Network of the Zebrafish International Resource Center at the University of Oregon.

"There are some differences in the zebrafish model; namely, that it is not a mammal," says Kim. "In UMaine's partnership with Jackson Lab (the world's largest mammalian genetic research facility, where the mouse is studied as a model for human disease), our zebrafish facility is complementary. I think that the zebrafish model system soon will rival the mouse system as more reagents, antibodies and cell lines are developed. It already rivals the mouse in developmental biology and toxicology."

In recent years, scientists like Kim have turned to zebrafish as models for the study of immunity, and infectious viral and bacterial disease. Key to Kim's work is the identification of the genes and molecular processes involved in innate immunity — the natural ability of multicelled organisms to ward off pathogens. Her studies of infectious disease in zebrafish bridge the biomedical and applied application fields because they have the potential to lead to a better understanding of disease development, resistance, diagnosis and treatment in other vertebrates, including humans and fish.
Kim's interest in applying molecular virology and microbiology to benefit the biomedical field and aquaculture industry brought her to UMaine in 1998, where she set up the state's first and now largest zebrafish facility. Up to that point, much of the genetically based research involving zebrafish focused on developmental biology and neurobiology. Kim was among the first to use the zebrafish to study infectious diseases.

What started as a small laboratory with 250 brood stock has grown to a climate-controlled facility in the new wing of Hitchner Hall, housing more than 40,000 zebrafish at all stages of development.
 

Kim's focus is on the role of toll-like receptor (TLR) signal pathways that are key to innate immunity. First identified in the fruit fly, TLRs are proteins found on the surface of certain cells. The receptors act as defense mechanisms, recognizing and binding with molecules of bacteria or viruses, and signaling the cell nucleus of the invading microbial infection. The result is an innate immune response — the release of infection-fighting molecules, such an cytokines.

Such innate response is a primitive physiological feature still shared among insects and vertebrates like the mouse, zebrafish and human. Unlike adaptive immunity that depends on virus- or bacteria-specific antibodies or vaccines, innate immunity as the body's first line of defense provides an immediate, vigorous, nonspecific inflammatory response to pathogens. Indeed, the stronger the innate immune response in the mouse, zebrafish or human, the more vigorous the adapted immune response.

In their efforts to better understand how the immune system responds to viral infection, Kim and UMaine researchers Stephen Altmann, Mark Mellon and Daniel Distel had a major breakthrough in 2003. The scientists isolated and confirmed the function of a zebrafish gene that produces interferon, an infection-fighting protein known for its ability to inhibit the growth of virus.

The UMaine researchers were the first to document the presence of interferon in any fish species. According to the Web of Science, the report of their discovery in the Journal of Virology is among the top 1 percent of papers cited by other scientists in that field in the last two years.

Since then, Kim has been cloning in zebrafish the immunogene known as Mx, which is activated by interferon. First discovered in mice with an inborn resistance to influenza virus, Mx bears a 50 percent resemblance to antiviral Mx proteins in humans. The important diagnostic tool in assessing interferon activity has been cloned in a variety of mammal, bird and fish species, but not in zebrafish until research was completed by a team of scientists from UMaine, Cornell and Boston's Brigham and Women's and Children's hospitals.

In an effort to understand disease-fighting responses in humans, more immune-related genes in the zebrafish need to be identified. Earlier this year, Kim and another research team focusing on the Mx and interferon proteins were the first to describe how an experimental infection of snakehead rhabdovirus developed and elicited an antiviral response in zebrafish. They focused on the symptoms of disease and the immune response in zebrafish embryos and adults.

Targeted gene disruptions can be used in conjunction with pathogen challenge to alter immunity to infection, according to the research team of scientists from UMaine, the University of Hamburg in Germany and Dalhousie University in Canada, writing in the February 2005 issue of the Journal of Virology. Differences in mortality rates, pathogenesis and gene expression may provide clues about the role of genes linked to immunity.
 

Kim is now collaborating with Nick Trede at the Huntsman Cancer Institute at the University of Utah to establish a transgenic (genetically modified) line of zebrafish that would have fluorescence to indicate activation of the TLR signaling pathways. When viewed using a special microscope, the mutant fish and their embryos have the potential to show scientists how the different proteins along the pathway function when the organism is compromised by disease.

Researchers also hope to identify genes that could improve or exaggerate the response of the TLR pathway.

"To identify what genes are responsible for such changes could mean that one day, we can identify humans with — or who are more susceptible to — disease," Kim says. "Using animal models, we're hoping to mimic the abnormality."

That approach is at the heart of Kim's most recent research project, funded by a more than $405,000 grant from the National Institutes of Health (NIH). She is collaborating with Dartmouth Cystic Fibrosis Research Development Program researchers to develop a zebrafish model for studying cystic fibrosis.

According to NIH's National Human Genome Research Institute, cystic fibrosis is the most common, fatal genetic disease in the United States. About 30,000 people in the U.S. have the disease, which is caused by a single mutated gene — the Cystic Fibrosis Transmembrane Regulator (CFTR).

In normal cells, the CFTR protein serves as a channel, allowing cells to release chloride as part of the immune response system. However, in people with cystic fibrosis, the protein is defective and the cells do not release the chloride, resulting in an improper salt balance and production of thick mucus.

In her lab, Kim will experimentally infect zebrafish with bacterial strains from cystic fibrosis patients in an effort to better understand why they are so pathogenic. The number of bacterial strains and the many CFTR gene mutations (more than 900, according to the National Human Genome Research Institute) make the microbiology portion of the project statistically strong. It is basic science that will be arduous in its compilation and analysis, but valuable. The bacteria preferentially affects cystic fibrosis patients with chronic infection that causes chronic inflammation.

"We're hoping to determine some of the key factors of the innate immune response that contribute to the detrimental inflammatory response seen in cystic fibrosis patients," Kim says. "If we can establish a way to control inflammation, cystic fibrosis patients will have a better outcome."

By Margaret Nagle
September-October, 2005

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