Climbing for Silas
23 NAVY DOLPHINS ARE SAFEGUARDING OUR NATIONAL SECURITY — AND ADVANCING HUMAN MEDICINE
38 FOUNDATION FOR BIOMEDICAL RESEARCH AWARDS THREE SCHOLARSHIPS TO OUTSTANDING CAL POLY POMONA PRE-VETERINARY STUDENTS
Climbing for Silas © The Jackson Laboratory
Climbing for Silas © The Jackson Laboratory
Climbing for Silas
The Ethic of Care © Oregon State University
11 A Healthy Bond: By Improving Pain Treatment, Therapy in Dogs, Research Offers Medical Insight for Humans © Kansas State University
12 Fats and Flies
A toddler's devastating disease spurs a Maine boy to use his own legs and lungs to help those who can't.
© National Institutes of Health
16 Tackling Diabetes © Oklahoma State University
18 How Bees Decide What to Be © Johns Hopkins University
20 Tackling a Brain Tumor Deadly to Pups and People © Virginia Tech
23 Bay Watch © Tufts University
11 A Healthy Bond. By improving pain treatment and therapy in dogs, a researcher is offering medical insights for humans.
23 Bay Watch. Navy dolphins are safeguarding our national security, and advancing human medicine at the same time.
26 Body Bacteria Exploring the Skin’s Microbial Metropolis © National Institutes of Health
31 Hopkins Researchers Solve Key Part of Old Mystery in Generating Muscle Mass © Johns Hopkins University
16 Tackling Diabetes. A team of researchers hope that research with animals can lead to help for humans.
32 Cancer Collaboration Could Someday Help Dogs and Their Humans.
32 Cancer Collaboration Could Someday Help Dogs and their Humans © Princeton University
34 Tackling a Fungal Disease © Oklahoma State University
36 Black Death Threat © University of North Carolina
38 Foundation for Biomedical Research Awards Three Scholarships to Outstanding Cal Poly Pomona Pre-Veterinary Students © Foundation for Biomedical Research
40 The Mystery of an Antıcancer Mechanism © University of Rochester
44 For Janice: Legacy of a Short Life © National Institutes of Health
For article submissions & advertising, please email FBR at email@example.com. ResearchSaves is a semi-annual publication. The annual subscription price is $39 and includes shipping and handling. Complimentary issues are available to K-12 teachers, thanks in large measure to the generous sponsorships granted from individual biomedical researchers.
Editor-in-Chief: Michael Stebbins ResearchSaves™ is a registered trademark of the Foundation for Biomedical Research 818 Connecticut Avenue, NW Suite 900 Washington, DC 20006 All articles are reprinted in their entirety with the expressed written consent of the authors and their associated institutions. Each article and photo is protected under U.S. copyright law and remains the sole property of the authors and their respective institutions. © ResearchSaves.org. 2013 All Rights Reserved
The FBR Team
Nahla Al Bassam Administrative Assistant Hometown: Baghdad, Iraq
Welcome to another issue of ResearchSaves. Once again, we have a lot of great stories about kids, animals and families. The common thread that weaves throughout all the stories is how biomedical research is allowing them to live happier, healthier lives. Because of the great response we had to last issue's Liviya's Story, this month's cover story also features an amazing kid with an inspiring story. This time, though, the kid is raising money and awareness for a disease affecting someone else. When Gus La Cosse, a 10 year-old in Maine, heard about kids with SMARD (spinal muscular atrophy with respiratory distress), he knew had to do something to help out. And the rest, well, you'll have to read the story to find out more. It begins on page 4.
Joined FBR: 2008
Liz Hodge Director, Media and Marketing Communications Hometown: Hamilton, MA Joined FBR: 2008
Paul McKellips Executive Vice President Hometown: Neenah, WI
In this issue, we also have interesting profiles of some amazing researchers at the National Institutes of Health, studying everything from fats in flies to germs in mice to cancer in naked mole rats.
Joined FBR: 2007
Have a comment about what you are reading in this issue? Send us a letter-to-the-editor at firstname.lastname@example.org. We'd love to include your thoughts and ideas on how to improve the magazine, as well as hear your comments about this issue's stories.
Cherie Proctor Director of Development
And last but not least, we're printing the winners of our 2012 Animal Research Essay Contest, in partnership with Cal Poly Pomona. The three winners wrote three very different takes on the subject of animal research. I think you'll find each of them interesting in their own right. That's all for this issue. See you in the Fall!
Hometown: Quincy, MA Joined FBR: 2002
Michael Stebbins Director of Research and Editor-in-Chief Hometown: Silver Creek, NY Joined FBR: 1999
Frankie Trull President
The Jackson Laboratory
BY TOM WALSH
Gus La Casse did some math. The Jackson Laboratory
What he concluded was that if each of 25 mountain treks could attract pledges, that money would provide a tremendous boost for SMARD research at The Jackson Laboratory and, he hoped, help find a cure. “My mom works at The Jackson Lab, and one day she came home and told me about SMARD,” 10-year-old Gus La Casse said in July. “It’s pretty rare, but there are quite a few kids in America and kids all over the world who have SMARD. And nobody has a cure.” That insight distressed Gus, who is into climbing and hiking and who realizes that kids with SMARD will never do either. He decided he would spend much of his summer, in effect, doing that for them, while seeking pledges to fund research. It was, Gus says, a “good summer vacation deed.” Most people have never heard of SMARD, an acronym for spinal muscular atrophy with respiratory distress, which Jackson Laboratory Associate Professor Greg Cox, Ph.D., describes as “the rarest of the rare” in terms of geneticbased, neuromuscular degenerative diseases. “Until recently, I had never heard of it, nor have most physicians,” says Cox. “It’s a very debilitating infantile disease. Most of these children don’t live more than two or three years. SMARD is a very early onset motor neuron disease. Motor neurons are the neurons in your spinal cord that extend into every muscle in your body. When these motor neurons get sick—and with SMARD, they actually die—there’s no way to trigger voluntary muscle response, which includes breathing and swallowing. It’s a recessive disease. You have to have two parents who carry the mutation to have a fear of a child developing SMARD.” Regrettably, Cox notes, he can count on the fingers of one hand the number of research laboratories worldwide that are exploring the biomolecular complexities of SMARD. Researchers in those labs make use of a mouse model of SMARD provided through The Jackson Laboratory. It was Greg Cox who discovered the genetic mutation that causes this currently incurable disease that kills infants and toddlers through muscle atrophy and paralysis. While SMARD affects fewer than 1,000 American children at any given time, each case is devastating for the families involved. ➤
for Silas – Photography by FRANÇOISE GERVAIS Spring/Summer 2013
The Jackson Laboratory
Among those families are the parents and grandparents of Silas Werner. As a newborn, Silas baffled a small army of Pittsburgh, Pa., pediatricians before he was finally diagnosed with SMARD, at age three months. After an original diagnosis of botulism and a prognosis of full recovery, agonizing months of hospitalization finally revealed a diagnosis of SMARD. His parents were devastated. When Gus, whose parents Renée and Joe both work at the Laboratory, first heard of Silas and SMARD, he was moved to act. “I do a lot of things that involve movement, like hiking and climbing,” he says. “So I decided I would raise money by doing something for these young kids with SMARD that they will never be able to do, like go hiking. In hiking, you have to breathe a lot, and these SMARD kids will never breathe on their own.” Over his summer break from grade school, Gus trekked up and down 25 mountain trails, most of them within Acadia National Park on Mount Desert Island. His final climb on July 31 was a steep ascent to the summit of Mount Katahdin, Maine’s highest peak at 5,268 feet. It was five miles straight up, and five miles straight down. Among those joining him in the trek was Greg Cox, who joined Gus on many of his climbs. “When people ask me why they should contribute to Climb for Silas, I tell them because kids all over the world have SMARD, and there’s no cure.” “He doesn’t get tired,” Cox says of Gus’ ambition. Apparently, neither does Greg Cox, according to his wife. “The last couple of weekends have been very hot here on Mount Desert Island,” Kathy Cox wrote in late July on the Facebook site Gus created to promote the project. “But Gus, Renée, Greg and team kept hiking for SMARD families. We are so proud of you.”
Gus set an ambitious goal for his fundraising in hopes that Greg Cox can hire a postdoctoral researcher to focus on gaining a better understanding the disease. To spread the word about his project, he was interviewed on a morning talk radio show and wrote an op-ed that he sent to the editorial pages of daily newspapers throughout Maine. “When people ask me why they should contribute to Climb for Silas, I tell them because kids all over the world have SMARD, and there’s no cure,” he wrote. “It’s important that people like Greg Cox find a cure.” Gus has never met Silas, but plans to. He’s been communicating with Lisa and John Werner, Silas’s parents, by e-mail, as well as other SMARD families he’s tracked down online, including a family in England. “There’s another kid, Dakin, who lives in Texas,” Gus says. “He’s four years old, which is quite old for a kid with SMARD. I also heard that there’s a SMARD kid in California who is 18, which is very unusual.” The dust has settled on his summer of climbing, and Gus is back into his usual school routine. But he continues to work toward his fundraising goals to support research and discovery on behalf of a far-flung community of children and parents to whom he is now firmly connected.
Silas Werner is beloved for his sweet disposition. Silas Werner, the boy who inspired Gus La Casse to climb and raise money for SMARD research, is now 2.
The Jackson Laboratory
Despite his condition, his parents say that Silas radiates joy through his happy smile. Speaking haltingly through a ventilator, Silas has now mastered “mama” and “papa,” much to the delight of his parents. Silas’ nurses describe him as “having the sweetest disposition of any baby we’ve ever met.” Nonetheless, Silas requires the around-the-clock care provided by his parents and by visiting nurses. Lisa and John Werner have mastered what Lisa terms “special needs boot camp” to meet Silas’s needs. The Werners are part of the close network of SMARD parents who now have newfound hope for the future. They recently learned that The Jackson Laboratory has a laboratory making strides in SMARD research. They are actively raising funds to support the research and, hopefully, advance the understanding of the disease to the point that doctors can one day help Silas and his peers. Contributions to support the fundraising efforts of Gus, Renée and Joe can be made by visiting their webpage.
When people ask me why they should contribute to Climb for Silas, I tell them because kids all over the world have SMARD, and there’s no cure,” Gus wrote. “It’s important that people like Greg Cox find a cure.” Spring/Summer 2013
BY LEE SHERMAN
Oregon InsertState Submission University
THE ETHIC OF CARE RESPECT FOR ANIMALS GUIDES THEIR TREATMENT IN TEACHING AND RESEARCH “All who care for, use, or produce animals for research, testing or teaching must assume responsibility for their well-being.” Guide for the Care and Use of Laboratory Animals, 2011, The National Research Council.
The three rats snoozing in Cage 57 don’t know it, but they could someday help save thousands of human lives. Snuggled in their EcoFresh bedding, the rodents are digesting a meal that may hold clues to preventing colon cancer, the second-leading cause of cancer deaths in the United States. On their cage, equipped with HEPA air-purification filters and precision temperature controls, hangs a blue index card labeled “Special Diet,” on which a researcher has scrawled “Bruss” in black felt pen. The scrawl is short for Brussels sprouts, those oft-disparaged veggies resembling tiny cabbages that are loaded with promising cancerprevention compounds such as sulphoraphane. To the rats, however, the pale-green pellets in their food tray (Mix AIN93 from Research Diets Inc., with sprouts added) are just dinner. That dichotomy — the rats’ bodily, mental and social needs (rodents are housed with “buddies” for company and “crawl tunnels” for enrichment) versus the precise methods of science — requires researchers to walk a tightrope, always balancing the pressing questions of medicine, for example, against the welfare of animals. The results are key to curing devastating diseases like ALS or Alzheimer’s. Oregon State University, with 600,000 research and teaching animals (mostly fish and other aquatic species) at 30-plus sites across the state, is balancing those interests exceedingly well. That is the judgment of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which in March gave a glowing report after an extensive accreditation study (see “High Grades for Animal Care” – http://oregonstate.edu/terra/2012/10/highgrades-for-animal-care/). Oregon State is the 19th among the nation’s 71 land grant universities to earn full-campus AAALAC accreditation. The snow-white, sprout-eating rats in OSU’s state-ofthe-art rodent facility are just one among 400 vertebrate species that populate the university’s labs, barns, aquariums, ranches and hatcheries. Zebrafish, steelhead, beef cattle, garter snakes, rainbow trout, dairy cows, yellow- and red-legged frogs, copper and canary rockfish, lambs, koi, swine, salmon smolts and llamas are among the half-million-plus warm- and coldblooded creatures that help educate OSU’s students, improve health (both human and animal), protect ecosystems, guide resource management, bolster local economies and engage the public.
– Photo by FRANK MILLER
Every last one of these creatures, from the 2-inch trout fingerling to the 2,000-pound Hereford bull, is the responsibility of Dr. Helen Diggs. If you don’t have an electronic key card, you must knock at a security door to gain admittance to her building on the west end of campus, the base from which Oregon State’s attending veterinarian oversees her vast menagerie. With the welfare of thousands of animals on her mind, she is quick to question, slow to trust (or, as she likes to say, “I trust but verify”). It’s a hyper-vigilance honed over 25
years in the field, some of those years at UC Berkeley where Diggs endured threats from animal-rights activists and had to be escorted to her car by security guards.
Doctor at the Top So when a hamster is lethargic or a horse is lame, she’s on it. But practices haven’t always been so rigorous in the world of animal research. Diggs has been in the field long enough to have seen the transformation. “In the decades before the 1980s, some universities were not caring for their animals as well as they should,” says Diggs, who has overseen research animals since 1985. “The facilities smelled gnarly. There were wooden floors with urine stains, poor temperature control. Regulations weren’t being enforced. No one was watching.” This laxity was not just a problem for the animals. It was also a problem for the science. “Researchers weren’t able to repeat their results. If I’m keeping my rats in a closet and feeding them oatmeal for breakfast, while your rats are getting leftover tacos or pancakes from the student lounge, we can’t validate our findings.” Adds Steve Durkee, another of the university’s leading researchanimal watchdogs: “If rats in one study are getting cereal while those in a second study are getting oranges and M&Ms, you can’t compare the results of the studies. By standardizing and harmonizing how animals are cared for, you create consistency across labs and institutions.” In fact, he notes, prestigious academic journals publish only findings that document the highest standards of animal care. That’s why Diggs’ job has teeth. Sharp ones. “I can shut a program down,” she says. “I’ve never had to do it here. But two times at other universities, I had to actually shut someone down and lock the door. If I have to go in and have a conversation with someone about their animal work, they’ll listen to me. It’s a big deal.” Even though the U.S. Public Health Service mandated in the early ‘70s that all animal research institutions hire an attending vet, the top docs didn’t have any real enforcement power until the mid’80s. That’s when the National Institutes of Health and the U.S. Department of Agriculture cinched up the rules for labs getting federal research dollars. “Attending vets had no real authority back in the early days,” explains Diggs, who reports to Rick Spinrad, vice president of research at Oregon State. “Some didn’t even have keys to the animal facilities. You need someone who’s minding the store, not just a figurehead.”
Oregon State University
Tacos and M&Ms “This is my morning health-status report,” says Diggs, pointing to a spreadsheet on her computer monitor. “Every day, every animalfacility manager checks in with me. Here’s Chad Mueller at the ag experiment station out in Union. Here’s Rob Chitwood at the fish performance lab over by the golf course. Here’s the Linus Pauling building. The Oregon hatchery. The Horse Center. Wherever I am, I can open up this online report and see what’s happening.”
Minding the Store Bob Murray waves his key card in front of a laser-triggered security panel in the $62.5 million, 1-year-old Linus Pauling Science Center, which houses the Department of Chemistry as well as the Linus Pauling Institute. The elevator opens, and he steps inside. One floor down, he flashes his card again, clicking open an electronic steel door into a small anteroom, where he slips on a gauzy yellow “isolation gown” and a pair of puffy blue booties. For a third time, Murray brandishes his key card, unlocking yet another heavy door. He enters the inner sanctum of Oregon State’s gleaming “vivarium” — the small-mammal equivalent of an aquarium or a terrarium — where hundreds of rats and thousands of mice live, as well as a few hamsters. Not one of these furry beings can get a sniffle or a sore toe without Murray knowing about it. “The animals are checked at least twice a day, 24-7,” says Murray, who clocked 35 years in the field, working at the New England Primate Center, Walter Reed Army Medical Center, Letterman Army Medical Center, UC Berkeley and Genentech before coming to OSU last year to manage the lab-animal facilities. “We watch for changes in gait or overall appearance — does the animal’s coat look scruffy? How is the animal’s appetite and hydration? We look for lethargy, weight loss, tumors. Any health problems we report immediately to Dr. Diggs and the researchers.” The vivarium maintains strict controls on temperature and other factors in the animals’ environment.
Murray’s dad worked for the Society for Prevention of Cruelty to Animals in Boston for nearly 30 years. So worrying about animal welfare is practically in his genes. He takes pride in the life-saving research he has observed over the years, like the groundbreaking Herceptin research at Genentech that is being used to treat thousands of women with breast cancer and the malaria vaccine research at Walter Reed. Still, it’s the health and comfort of the whiskered rodents that gets him out of bed every morning at 5 o’clock and keeps him running as he oversees his team of highly trained, certified animal technicians. “I believe strongly in the value of the research we do here, but I’m not a researcher,” Murray says, surveying his domain with the discerning gaze of a seasoned professional. “I’m into animal care.” If Murray were to take you through the 8,000-square-foot facility where researchers investigate the links between nutrients and human health, the first thing you would notice is an obsession with cleanliness. The giant Steris cage washer (which he calls “the heartbeat of the whole facility”) sanitizes racks of cages in two cycles of 180-degree, pressurized water — and that’s after the cages have been blasted with detergent and rinsed in acid. Everywhere you look, technicians and student workers are prepping cages for incoming animals or plying mops on floors that already look immaculate. Viruses and bacteria that could sicken the animals and compromise the research don’t stand a chance. The next thing you would notice is the attention to precision. Automated lighting simulates 12 hours of day, 12 hours of night. Electronic monitors maintain a 68- to 72-degree temperature range. An alarm alerts the staff if temperatures fall outside the range by even 1-degree Fahrenheit. There are ventilation ➤ Spring/Summer 2013
tubes, fume hoods, stainless-steel work stations illuminated with stretchable spotlights. Every last facet of the facility is designed to protect the health and welfare of all its mammalian inhabitants, human as well as rodent. Oregon State University
Not until you reached the bosom of the vivarium would you come upon the rodents. The Brussels sprout-eating residents of the “rat room” were born and raised at an Indiana-based research-animal supply company called Harlan Laboratories, arriving at OSU in ventilated crates via UPS. Firms like Harlan, along with Charles River Labs, Jackson Labs and dozens of others comprise a global mega-industry in the service of science. All must adhere to the same stringent federal requirements that guide OSU’s animal-care personnel. Diets supplemented with cancer-fighting compounds are under investigation in the Linus Pauling Science Center. In the rat room where Rod Dashwood and other researchers in the LPI Cancer Chemoprotection Unit are looking for evidence that cruciferous vegetables like Brussels sprouts and broccoli sprouts can block the formation of colon tumors, dozens of clearplastic cages are stacked, one above another, inside tall metal racks like high-rise condos. When you lean close and peer inside, you’re likely to get a visual jolt. The cold, hard sterility of biomedical science is, you realize, wrapped around hundreds of breathing beings with whiskered snouts and beating hearts. They cuddle together for warmth and companionship. They look out at you with the pinkish eyes characteristic of albino Strain F344, understanding nothing about the scientific enterprise in which they play the leading role.
A Fish Like Me So why do scientists work with animals? What can rats (Rattus norvegicus) or zebrafish (Danio rerio), seemingly so far from Homo sapiens on the tree of life, reveal about human health and disease? Turns out, many basic biological processes such as cell division, organ differentiation, gene mutation and disease formation play out similarly across species. That’s why a rat or a mouse or a fish can act as a stand-in for a human in studies on micronutrients, obesity, aging, ALS, cancer, drug efficacy, infectious disease and any number of other biomedical questions under investigation at Oregon State.
A Whole Lot of Seriousness When researchers use rats, mice or other species to study processes that mimic or parallel human biology, they call it a “model.” One common model is a “knockout mouse.” It works like this: To gauge how certain genes affect certain bodily functions or disease processes, researchers “knock out” or silence the targeted gene and then study what happens when the mice get, for instance, a high-fat diet or a hormonal supplement. Knockout mice are used at OSU to study bone growth, aging, obesity, immunodeficiency and many other intricate areas of human health. But complex animals like mice and rats are used only when there’s no other way to investigate the question at hand, Durkee stresses. Indeed, basic biomedical research begins with cells in a test tube. Only after experiments have shown great promise do scientists advance to animal work. And then, only after the
animal studies achieve high rates of treatment success or cures — along with low risks for harm — do scientists go on to conduct experiments on humans. Steve Durkee’s mother was a subject in one of those experiments, which researchers call “human” or “clinical” trials, when she was battling breast cancer. Durkee likes to direct people to the AAALAC website’s long list of Nobel Prizes in medicine and physiology over the past 110 years. Without the use of lab animals, Frederick Banting and John McLeod wouldn’t have discovered insulin and the mechanism for diabetes, winning the Nobel in 1923. Alexander Fleming, Ernst Chain and Howard Florey wouldn’t have discovered penicillin and its curative powers. Typhoid and yellow fever would still be raging across the land. But Banting and McLeod’s methods with dogs, rabbits and fish probably would fail to pass muster with today’s regulating agencies. It’s not only federal regs that have changed — it’s the moral, philosophical and ethical sensibilities of Americans toward creatures of all kinds. Oregon State biomedical ethicist Courtney Campbell has seen a sea change over the past decade and a half. “Nothing is more important in an animal study than the animal itself,” says Steve Durkee.
“There’s a generational change going on,” says Campbell, who helped lead a series of national ethics workshops for land grant faculty in the 1990s. “The change isn’t limited to animal research at universities — it’s also about food and entertainment and sports. It’s about the treatment of animals at zoos, circuses, aquariums, rodeos. “It’s about our diets — how veganism and vegetarianism were way out in the ‘fringy granola movement’ not that long ago. “We haven’t done a complete cultural 180, but there is definitely a new moral consciousness.”
At the end of the day In the rat room, the “Bruss” eaters live alongside the “brocc” eaters (broccoli sprouts) and the “fat” eaters (high lipids). There’s a control group, too, which eats regular rat chow. That’s so Dashwood can compare the health impacts of an ordinary diet against those of the special diets. At the study’s start, all the animals were injected with the carcinogen found in charred meat — a known cancer-causing compound to which most Americans have been exposed in barbequed burgers or grilled steaks. Once the study is over, the animals will be euthanized, humanely, in strict accordance with the protocols set out by the American Veterinary Medical Association. The researchers will then compare the number and size of colon tumors among the four groups to find out whether eating sprouts made a difference. When they talk about ending the lives of animals used in biomedical research, Diggs, Durkee and Murray all express a resigned sadness. None of them could do their jobs without a total conviction that scientific discovery justifies the animals’ demise — that the death of a rat may someday save the life of a child. Still, it’s unsettling. “Nobody likes it,” muses Murray, his attempt at matter-of-factness not 100 percent convincing. “But it is what it is.”
Kansas State University
A healthy bond: By improving pain treatment, therapy in dogs, research offers medical insight for humans by James Roush
A Kansas State University professor’s research improving postsurgery pain treatment and osteoarthritis therapy in dogs may help develop better ways to treat humans for various medical conditions. From the use of hot and cold packs to new forms of narcotics, James Roush, professor of clinical sciences, is studying ways to lessen pain after surgery and improve care for small animals, particularly dogs. He is working with the clinical patients who come to the College of Veterinary Medicine’s Veterinary Health Center. Because humans and dogs experience some diseases in similar ways, his research may improve how doctors and physicians understand human health, too. “Several of our projects have human applications, particularly one involving intra-articular prolotherapy,” Roush said. Here’s a closer look at three of Roush’s current projects:
A recent project with Ralph Millard, former Veterinary Health Center resident, focuses on ways that hot packing and cold packing affect tissue temperature in beagles and beagle-sized dogs after surgery.
After surgery in both humans and dogs it is common to put a cold pack or hot pack on tissue to prevent and reduce swelling. How long the pack is used and what type of cold or hot pack is used depends on the type of injury and surgery. Roush said that no studies have looked at how deep in the tissue the packs affect temperature and how long the packs must be applied so that the tissue reaches a desired temperature. The researchers studied the temperature and tissue depth that hot and cold packing affected and the time it took to reach that temperature. “We found that you don’t really need to cold pack anything longer than 10 minutes because there is not a great change in temperature after that,” Roush said. When tissue is cold packed, it will stay cold for a while after the ice pack is removed. But when tissue is hot packed and the pack is removed, the tissue temperature will return to normal much more quickly. Leaving the hot or cold pack on the tissue longer than 10 minutes will extend the time that the tissue stays at the same hot or
cold temperature, Roush said. There just will not be a great change in temperature after 10 minutes. The same technique of hot and cold packing after surgery is also used in humans. Although more research in humans is needed, Roush said there is a strong possibility that a similar 10-minute time frame for hot and cold packing may apply to humans as well. The research appears in two upcoming publications in the Journal of Veterinary Research.
For another project, Roush and Matt Sherwood, Veterinary Health Center resident, are using a mat system to study lameness and osteoarthritis in dogs. When dogs step on the mat, it measures the pressure in their step.
The mat system is a useful clinical tool for evaluating and developing treatment of lameness, Roush said. Roush and Sherwood are using the mat for measuring lameness and determining in which leg the lameness is worse. “We’ve designed the study to help improve osteoarthritis treatment,” Roush said. “We will also use it to measure clinical patients when they come in for regular checkups. We can measure their recovery and a variety of other aspects: how they respond to nonsteroidal anti-inflammatories, how they respond to narcotics or how they respond to a surgical procedure that is designed to take that pressure off the joint.”
Roush also is working with Marian Benitez, Veterinary Health Center resident, on an analgesic pharmacology study. Rose McMurphy, professor of clinical sciences, and Butch KuKanich, associate professor of anatomy and physiology, are also involved.
The researchers are studying the effectiveness of a painkiller used to treat dogs and researching potential alternatives to the drug. The same drug also is commonly used to treat pain in humans. “To achieve the drug’s effect, the dosage in dogs is much higher than in people,” Roush said. “It also may not be a very good analgesic in dogs. We want to see if there is an alternative that requires smaller doses and does not have not as much of a discrepancy for patients.” Spring/Summer 2013
National Institutes of Health
THE SOFT BUZZING OF MOSQUITOES FILLS THE AIR while spotted moths with 4inch wingspans flutter past. Bluegreen caterpillars as thick as fingers crawl in a living carpet. Summer picnic nightmare? Nope—this motley collection of insects lives inside plastic cages in Estela Arrese’s biochemistry lab at Oklahoma State University (OSU). Arrese studies these and other insects to learn how they—and we—store food as fat and later break it down for energy. Her discoveries could lead to new ways for farmers to protect their crops from pests, and for health officials to combat mosquito-borne diseases like malaria and West Nile virus. Not only that, but studying these little critters could one day improve our understanding of disorders like diabetes, obesity and heart disease, which relate to how we store and use fat. Saying that we can learn about human biology from mosquitoes and moths “sounds kind of crazy,” Arrese readily admits. But the argument is hardly outrageous. Scientists have been studying insects to better understand human biology for more than 100 years (see “Fruitful Work,” page 6). While we may not look alike, caterpillars, flies and humans use similar methods to regulate fat at the molecular level. “It’s very exciting,” Arrese says of her work. “We are learning new things all the time. It’s a good time to be in this field.”
Fats Are Us All creatures—people, insects, even plants— need fat to survive.
– Photo by SEAN HUBBARD
Most of the fats we eat are called triglycerides. Triglycerides give us more than twice as much energy as carbohydrates or proteins. But before we can use that energy, our bodies must break down the fats. When we digest triglycerides, they get split into their component parts: three fatty acids and a carbohydrate. This splitting is called lipolysis (lipfor “lipid,” or fat, and lysis for “split”), and the enzymes that do the splitting are called lipases.
National Institutes of Health
Fats and Flies
BY STEPHANIE DUTCHEN
Once they’ve been absorbed into our intestines, the fatty acids are recombined into triglycerides and shipped out to our cells through the bloodstream. Some of the fat gets used for energy right away. The rest is stored inside cells in blobs called lipid droplets. Until the 1990s, researchers thought lipid droplets were just beads of oil passively floating around in cells. Then they discovered that the droplets are actually dynamic structures that help regulate when stored fat gets broken down for energy. Now, lipid droplets have taken center stage in fat research.
For instance, insects like silkworms and osquitoes store up lots of fat when they’re young by eating nectar or leaves. They use that fat later when they metamorphose into their adult forms and start flying. They also burn fat when they lay their eggs. If researchers could block the movement of stored fat to the insects’ ovaries, they could interfere with egglaying and stop the bugs from reproducing. That could have an immense impact on pest and disease control.
It’s like an enormous, living puzzle, and Arrese is trying to identify a few protein pieces and figure out how they fit together.
When the body needs extra energy —for instance, when we run a marathon— hormones direct lipases to break down the fats stored in lipid droplets and wash them back into the bloodstream.
“In a cell, there are so many proteins, it’s hard to tell which protein really does the work,” says physicist Donghua Zhou, one of Arrese’s colleagues and collaborators at OSU. These lipid droplets store fat in the cells of the tobacco hornworm, Manduca sexta.
Protein Puzzle Pieces But before any of that can happen, says Arrese, “We need to study a lot and have information at the molecular level.”
Arrese studies insects, including the tobacco hornworm shown here (counterclockwise from top) as a c aterpillar, pupa and adult moth.
She has a lot of questions: Does TGL activate fat mobilization, block it or help another protein? Do other proteins help regulate TGL? What is the function of another lipase, called ATGL? How does the Lsd1 protein help control lipolysis, and is it a critical target for disease control? What does its sister protein, Lsd2, do?
To determine which proteins perform what jobs, biochemists like Arrese have to isolate and purify each one in a test tube before conducting extensive experiments.
She has already discovered the function of a protein called Lsd1 found in lipid droplets.
And purifying proteins is not easy, explains José Luis Soulages, who collaborates with Arrese in the university’s biochemistry department—and who also happens to be her husband.
Arrese also works with the main lipase involved in fat regulation in insects. Fittingly, it’s called triglyceride lipase, or TGL. Now she’s looking into how TGL works.
“After a lifetime, you may say that you’ve found four key proteins,” he says. “And there are probably 100 more. But their functions would never be known without the discovery of those first four.” ➤
These lipid droplets store fat in the cells of the tobacco hornworm, Manduca sexta.
The cycle of making, breaking, storing and mobilizing fats is at the core of how humans—and all animals— regulate their energy. An imbalance in any step can result in disease, including obesity and diabetes. Having too many triglycerides in our bloodstream raises our risk of clogged arteries, leading to heart attack and stroke. Despite their importance, no one yet understands exactly how fat storage and mobilization work. – Photo by ESTELA ARRESE
It’s like an enormous, living puzzle. ‘Shockingly Good’ National Institutes of Health
It’s especially tough to purify Arrese’s proteins because they don’t survive long outside cells and because they’re found in oils. Most biochemistry tests are designed for waterbased molecules—and as you know if you’ve ever shaken a bottle of salad dressing, oil and water don’t get along. That doesn’t deter Arrese. Her lab was the first to purify TGL from fat tissue in insects as well as Lsd1 and Lsd2, and she continues to purify proteins with a characteristic drive for perfection. “When she brings me molecules, they are impeccable,” says Steve Hartson, a biochemist at OSU who collaborates with Arrese. An advantage to using insects like flies and caterpillars is that Arrese can raise hundreds of them in the lab and collect plenty of the proteins she wants—many more than she could from rats or people.
“I think very few people can do that,” he says. “In science, sometimes we are tempted to switch subjects or even fields because things get hard. She just puts in more hours and tries alternate approaches.” And it pays off. Hartson says, “She’s a shockingly good biochemist. She does some incredible things with molecules.” Hartson recalls being “a little intimidated by her at first. She was very focused on doing science. But then she makes a discovery or [gets] key data and breaks into this radiant, beaming person. She’s a real pleasure to work with.”
Getting Her Hands Dirty Arrese acquired her stringent work ethic as a young girl, when she spent summers on her family’s farm in rural Pehuajo, Argentina.
“It puts her on the edge of what’s possible,” explains Hartson. Arrese aims to learn more about the proteins by examining their structures atom by atom.
It’s definitely not a job for people with short attention spans. Luckily, says Soulages, his wife is wellsuited to the work—she has extraordinary concentration, keen powers of observation and a talent for planning efficient experiments. The combination of talent and commitment allows her to pursue a single scientific question for decades without losing focus.
It’s also true at home, where she cultivates a thriving flower garden despite Oklahoma’s unforgiving climate—dry summers, harsh winters, tornadoes and spring hail that drives Arrese outside to throw a protective tarp over her tulips and roses. “Whatever it takes to have flowers, I will do it,” says Arrese. “I don’t care if it’s a challenge. Because I was raised on that farm, I am used to a lot of work.”
Dedication to Science Arrese’s father, a veterinarian, also raised her to love science. She always felt comfortable in the lab he set up in their house, wandering amidst the pipettes and tubes, the microscope and canisters of liquid nitrogen. By the age of 10, she was helping her father and pretending to be a scientist herself. Her favorite task was looking through the microscope. When she was in middle school, she asked her father, “What do I need to study to look through microscopes a lot?”
But she can’t use one of the most common protein imaging techniques, Xray crystallography, because her proteins don’t naturally form the needed crystal structures. Nor can she use the other common technique, called solution nuclear magnetic resonance (NMR), because the proteins are too big. So Arrese collaborates with Zhou, who uses related technology called solidstate NMR. She hopes that soon, she will finally be able to see the structures of her proteins in 3D.
It’s still the case metaphorically in the lab, where she is always ready with a pair of rubber gloves if she needs to dive into the insect cages or help her students with an experiment.
His answer: biochemistry. “And that,” she says, “is what I ended up doing.” The path wasn’t an easy one.
– Photo by AMERICO ARRESE Arrese as a child.
What do I need to study to look through microscopes a lot? “My father always wanted us to be busy doing useful things,” she says. For example, she and her brother were charged with taking care of a big vegetable garden. Rather than resenting the work, Arrese says, “I realized I really liked it. I need that. I need to get my hands dirty.”
She gave birth to her first child while she was still working on her Ph.D. thesis—just before her husband moved to the United States. When she and her daughter joined her husband in Tucson, Arizona, she left behind her extended family, her language and her culture. In Argentina, she had taught herself to read and write English with a dictionary in one hand and scientific journal articles in the other. But she arrived in the U.S. without knowing how to speak the language. She still struggles to speak as fluently—and as much—in English as she does in her native Spanish.
Identify [your] passion and follow it. Despite the communication barrier, Arrese loves English. “It’s more precise,” she says. “I am 50—maybe when I retire, I will study it.”
Along with the lab, Arrese has always felt comfortable—and enjoys experimenting—in the kitchen.
Now Arrese has three graduate students from China, and she’s looking forward to once again expanding her culinary horizons.
When she has to follow a recipe, she says, “It’s a disaster. I want to change it. I want to know what will happen if I add this or that.”
Her husband agrees: “She has to see how people are cooking, and then she works it out on the bench—on the stove.”
When she isn’t grilling her students for cooking tips, Arrese cares for them as though they were her extended family.
Some of her favorite dishes are cod and paella. Among her appreciative consumers are her husband, her two daughters and her students.
“She nurtures her students…in such a way that the excitement of science never wears off and critical thinking is developed,” says Alisha Howard, who did undergraduate research in Arrese’s lab before earning her Ph.D. with Soulages.
“I love cooking because I like good food, because I want to feed my family and also because I need that—to go and create something,” says Arrese. Growing up, she learned how to prepare not only an eclectic array of Argentine dishes, but also Spanish food from an aunt who was a chef and Italian meals from her immigrant grandmother. When she relocated to Tucson, she promptly pestered a neighbor to teach her Mexican cuisine.
National Institutes of Health
Experimenting in the Kitchen
When the family moved again, this time to Oklahoma, a handful of graduate students from India joined her lab and helped her fulfill a decadesold desire to learn to cook Indian food. Whenever these students traveled home, they would bring back suitcases packed with spices for her.
“She seems to be very invested in our future as scientists and as adults,” agrees Zach Hager, a college senior in the lab. Hager particularly appreciates Arrese’s eagerness to work alongside her students at the bench. Colleague Steve Hartson says that Arrese’s “tremendous oneonone mentoring and intellectual and moral support” are an inspiration to her students and ensure that they achieve their potential. “They’re excited by the science they’re doing, and they go on to very successful careers,” he reports. Her students also admire the way she balances the jobs of lab head and mother (her daughters are now 13 and 20). It helps to have her husband nearby. She and Soulages often take turns running long experiments or helping their kids with daily routines. Her ultimate goals, she says, are to raise her children to be healthy and have good lives, and to find out what her handful of proteins do. Soulages describes his wife’s philosophy not as seeking an earthshaking discovery, but as putting in her grains of sand, knowing they will last a long time. He could be talking as easily about her children and students as about her science. For her part, Arrese gives the same advice to all: “Identify [your] passion and follow it. When you really like what you do, it becomes easy. Live and work with passion and quality.”
– Photo by ESTELA ARRESE
Arrese (right, in blue) teaches her students to be efficient and excited about science.
BY DERINDA BLAKENEY
Oklahoma State University
Tackling diabetes TEAM HOPES RESEARCH FROM ANIMALS CAN LEAD TO HELP FOR HUMANS Véronique Lacombe, DVM, Ph.D., brought a long history of comparative medicine research with her when she joined OSU’s veterinary center as an associate professor in physiological sciences. Since she was a veterinary student, Lacombe has had an interest in metabolism, the complex process the body uses to turn food into energy. Specifically, her fields of interest include skeletal and cardiac muscle energetics, glucose transport during diabetes, insulin resistance using small- and large-animal models, as well as cardiovascular complications during diabetes. The main mission of Lacombe and her team is researching mechanisms underlying diabetes, a complex disease for which there is no cure. “Diabetes is an epidemic disease that affects more than 250 million people with almost 10 percent of the population affected in the U.S., and it is expected that the worldwide prevalence will rise to 450 million by 2030,” she says. “As a result, the disease imposes a considerable medical and economic burden on societies. My lab is investigating the regulation of glucose transport in insulin-sensitive tissue. In other words, we are looking at how the glucose (i.e., sugar) in the bloodstream transfers to tissue. This process is the metabolic bottleneck for glucose utilization and fuel production. In addition, this process is altered in people who have diabetes because they have improper production and/or action of insulin, a hormone that is necessary to make that transfer.”
While there is no drug to cure diabetes, human diabetes can be regulated and monitored to avoid complications. “With a diet regimen, exercise and weight loss, diabetics can help manage their disease. When skeletal muscles contract, that process helps transport glucose into cells. Exercise can speed glucose uptake in muscles. However, the process by which contraction enhances glucose transport is unknown, and it is one of the research focuses of my laboratory. Findings from this research could lead to the discovery of a cure for diabetic patients.” Lacombe says glucose is one of the main sources of fuel for the body, and the uptake of glucose from the blood into the cell and its utilization by the cell to produce energy is similar across all species — human and animal. “Because the process is similar, we use both small- and largeanimal models in my lab, spanning from mice to horses. If we find mechanisms responsible for diabetes using these species, it could also have an impact on human health, a concept referred to as one health, one medicine,” she says. “For example, in mice, we can upregulate a protein potentially implicated in the transport of glucose to see if it will help prevent diabetes. As a result, we have now established a line of mice that are resistant to diabetes.
DR. MELODY DE LAAT (LEFT) DEMONSTRATES THE NEW MICRO-ULTRASOUND MACHINE WITH DR. VERONIQUE LACOMBE (CENTER) AND BRITTANY EVANS.
– Photo by PHIL SHOCKLEY / UNIVERSITY MARKETING 16
By transcending species boundaries to include the study of spontaneous and experimental models of human disease, research in comparative medicine can lead to exciting discoveries that will benefit both people and animals.” Lacombe firmly believes that veterinarians’ thorough training puts them in a unique position to improve research and help society. “Veterinarians have such a broad training. We have to know all the different species from fish to elephants. That vast knowledge can be applied in comparative medicine research. It is a career path with many rewards that many veterinary students don’t really think about.” After earning her DVM degree, Lacombe completed her residency in large-animal internal medicine and worked as an equine clinician before focusing on research. “As a clinician first and a scientist second, I am a better researcher and ask questions that are clinically relevant. Clinical veterinary medicine and research are similar processes. In both cases you start with a problem. In clinical veterinary medicine, you have a list of different diagnoses that could be causing the problem. In research, you have different hypotheses you want to prove. One by one, you check them off the list in both areas. In clinical veterinary medicine you have a final diagnosis, and you treat it. In research, you have the correct hypothesis, and you find a cure or you take one step closer to finding a cure. We are responsible to nourish that aspect of the veterinary profession and train the next generation of veterinary scientists.”
Big pictures from little things
BY MATT ELLIOTT
NEW MICRO-ULTRASOUND MACHINE GETS CLEAR PICTURES FROM INSIDE SMALL CRITTERS
Oklahoma State University
Like people, horses can become obese, which can lead to a metabolic disorder such as insulin resistance. Interestingly, cats can get transient diabetes, where the diabetes goes into spontaneous remission. Unlike the rodent models used to study diabetes, which are generated from inbred strains of laboratory mice, these naturally occurring models are genetically diverse and exposed to many of the same environmental factors that humans are, and they are great models of metabolic disorders.
The Center for Veterinary Health Sciences purchased a state-of-the-art ultrasound machine in 2011 that greatly expands researchers’ ability to image small animals. The machine, the VisualSonics Vevo 2100, allows vet school scientists such as its operator, Dr. Véronique Lacombe, to see into the organs and systems of lab animals as small as mice and rabbits. The high-frequency digital imager adds to the vet school’s growing list of imaging technology that already includes MRI and CT scan machines, and a human-size ultrasound machine available at the teaching hospital. Ultrasound uses sound waves to create images of how things work inside bodies by translating how the waves pass through different objects into an image. They are used in everything from imaging fetuses and the hearts of people suffering from cardiovascular disease to cancer. It’s easy to envision their usefulness in veterinary medicine as well, including the booming realm of comparative medicine and preclinical research that focuses on problems afflicting both animals and humans. Naturally, it makes sense to use animals to model human systems in research. The problem is that large ultrasound machines operate at a lower sound frequency to pass through larger patients, such as a human or a horse. It is not as helpful to image a mouse or a rat because the image quality wouldn’t be as good. “With this micro-ultrasound machine, you can’t go very deep, but you’re going to have a spectacular resolution of the specific area that you’re examining,” Lacombe says. “The advantage of this machine is that it has the same features as a human ultrasound machine, but it has been designed to image all the organs of small animals, including of early embryonic and neonatal mice.” The Vevo 2100, believed to be the only one in Oklahoma, is in Lacombe’s Comparative Metabolism Laboratory in the vet school’s physiology department. It has already proven helpful in her research. As diabetes is one of Lacombe’s chief areas of interest, she often works with small animals, such as mice and rats as models of human metabolic diseases. One of the increasingly common condition’s accompanying diabetes is heart disease. Previously, she had to use human ultrasound machines in her animal models that were not as useful in her work. This machine lets her look at everything from the shape of the heart to how effectively it pumps blood, as well as how the heart tissue contracts and relaxes during each heartbeat, letting her “detect subtle heart dysfunction very early on in the process of the disease in our diabetic mice,” Lacombe says. “Also, with this high resolution, you can also inject anything you want into a targeted site, including the brain and the spinal cord. You can also use micro bubble technology,” she says. “You can create small bubbles of air that will go through the heart or blood vessels to mark the passage of what you’re trying to track.” She can also look at blood flow into organs to track vascular diseases. It also lets her deliver gene and stem cells at a targeted site and measure things such as gene therapy effectiveness. And 3-D reconstructions of organs are possible. “It’s a huge improvement since this micro-ultrasound machine greatly expands our understanding of the physiologic and pathophysiological processes in small animal models.” Lacombe says. The Vevo 2100 is available for any researcher in the veterinary college or on OSU’s campus to use, she says. “It can be used by any investigators on campus since it is a common equipment. I think the more people we have using it from different areas of research, the better it would be. I think it could even generate some collaborations between the veterinary school and other colleges on campus.”
Johns Hopkins University
By Vanessa McMains, Ph.D. Johns Hopkins Researchers
Link Reversible “Epigenetic”
Marks to Behavior Patterns
Nurse bees generally remain in the hive ohns Hopkins scientists report what is believed to be the first evidence that complex, reversible behavioral patterns in bees – and presumably other animals – are linked to reversible chemical tags on genes.
The scientists say what is most significant about the new study, described online September 16 in Nature Neuroscience, is that for the first time DNA methylation “tagging” has been linked to something at the behavioral level of a whole organism. On top of that, they say, the behavior in question, and its corresponding molecular changes, are reversible, which has important implications for human health. According to Andy Feinberg, M.D., M.P.H., Gilman scholar, professor of molecular medicine and director of the Center for Epigenetics at Hopkins’ Institute for Basic Biomedical Sciences, the addition of DNA methylation to genes has long been shown to play an important role in regulating gene activity in changing biological systems, like fate determination in stem cells or the creation of cancer cells. Curious about how epigenetics might contribute to behavior, he and his team studied a tried-and-true model of animal behavior: bees. Working with bee expert Gro Amdam, Ph.D., associate professor of life sciences at Arizona State University and the Norwegian University of Life Sciences, Feinberg’s epigenetics team found significant differences in DNA methylation patterns in bees that have identical genetic sequences but vastly different behavioral patterns. Employing a method that allows the researchers to analyze the whole genome at once, dubbed CHARM (comprehensive high-throughput arrays for relative methylation), the team analyzed the location of DNA methylations in the brains of worker bees of two different “professions.” All worker bees are female and, within a given hive, are all genetically identical sisters. However, they don’t all do the same thing; some nurse and some forage.
– Photos by Christofer Bang
Johns Hopkins University
to feed and take care of the queen and her larvae. Forager bees are responsible for gathering pollen and nectar. Nurses are generally younger and remain in the hive to take care of the queen and her larvae. When nurses mature, they become foragers that leave the hive to gather pollen and other supplies for the hive. “Genes themselves weren’t going to tell us what is responsible for the two types of behavior,” Feinberg says. “But epigenetics – and how it controls genes – could.” Feinberg and Amdam started their experiment with new hives populated by bees of the same age. That removed the possibility that any differences they might find could be attributed to differences of age. “When young, age-matched bees enter a new hive, they divvy up their tasks so that the right proportion becomes nurses and foragers,” explains Amdam. It is these two populations that were tested after painstakingly characterizing and marking each bee with its “professional,” or behavioral, category. Analyzing the patterns of DNA methylation in the brains of 21 nurses and 21 foragers, the team found 155 regions of DNA that had different tag patterns in the two types of bees. The genes associated with the methylation differences were mostly regulatory genes known to affect the status of other genes. “Gene sequences without these tags are like roads without stop lights – gridlock,” says Feinberg. Once they knew differences existed, they could take the next step to determine if they were permanent. “When there are too few nurses, the foragers can step in and take their places, reverting to their former practices,” says Amdam. The researchers used this strategy to see whether foraging bees would maintain their foraging genetic tags when forced to start acting like nurses again. So they removed all of the nurses from their hives and waited several weeks for the hive to restore balance. That done, the team again looked for differences in DNA methylation patterns, this time between foragers that remained foragers and those that became nurses. One hundred and seven DNA regions showed different tags between the foragers and the reverted nurses, suggesting that the epigenetic marks were not permanent but reversible and connected to the bees’ behavior and the facts of life in the hive.
Dramatically, Feinberg The researchers say they hope noted, more than half of those regions had their results may begin to shed already been light on complex behavioral issues identified among the in humans, such as learning, 155 regions that change when nurses memory, stress response and mature into foragers. mood disorders, which all involve These 57 regions are likely at the heart of interactions between genetic and the different behaviors epigenetic components similar exhibited by nurses to those in the study. and foragers, says Amdam. “It’s like one of those pictures that portray two different images depending on your angle of view,” she says. “The bee genome contains images of both nurses and foragers. The tags on the DNA give the brain its coordinates so that it knows what kind of behavior to project.” The researchers say they hope their results may begin to shed light on complex behavioral issues in humans, such as learning, memory, stress response and mood disorders, which all involve interactions between genetic and epigenetic components similar to those in the study. A person’s underlying genetic sequence is acted upon by epigenetic tags, which may be affected by external cues to change in ways that create stable – but reversible – behavioral patterns. Authors on the paper include Brian Herb, Kasper Hansen, Martin Aryee, Ben Langmead, Rafael Irizarry and Andrew Feinberg from The Johns Hopkins University, and Florian Wolschin and Gro Amdam of the Norwegian University of Life Sciences and Arizona State University. This work was funded through the NIH Director’s Pioneer Award through the National Institute of Environmental Health Sciences (#DP1ES022579), the Research Council of Norway and the Pew Charitable Trust.
BY SUSAN STEEVES
TACKLING A BRAIN Virginia Tech
PUPS AND PEOPLE
Helen was a typical mom of the 50s and 60s with four kids who all had busy lives. By 1974, one son was married with two youngsters of his own. One daughter was just out of college and the two youngest children were in college. One fall day that year, they each received a call that their mother had been diagnosed with a brain tumor. Seven hours of surgery, months of radiation therapy, and nine months of worry ended in Helen’s death at the age of 55. Immediately after the operation, the surgeon had said that the kind of tumor they found was like “spilled milk.” Today, that’s still most often the story of what happens to people with the deadliest form of brain tumor, glioblastoma, also known as a stage IV glioma or stage IV astrocytoma. Sen. Ted Kennedy lived for 14-1/2 months with one, dying in 2009 even after receiving top-of-the-line medical care. A team of scientists and surgeons at Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences have teamed up to use a new treatment technique on dogs, with the expectation that within a few years they may be performing clinical trials on humans with glial cell tumors. Glioblastomas in canine patients are almost identical to those found in humans and, in fact, occur at least three times more often in dogs. And sadly, mortality patterns for dogs also are almost identical when corrected for normal longevity; dogs survive a few weeks to a few months and humans about nine months with surgery, chemotherapy, and radiation, and 15 months when a drug called temozolomide is added. It’s these fatal facts that give researchers hope that they can learn from our four-legged pals how to beat this deadly demon for both people and pups. “Dogs are the holy grail of a spontaneous model for glioblastomas because the tumors develop just like in people,” says John Rossmeisl, the neurosurgeon in the Virginia-Maryland Regional College of Veterinary Medicine who is using the new procedure on dogs suffering from brain tumors and some other forms of cancer. “We don’t know why these tumors develop. Some of it may be chemicals, cleaning products. Dogs are so similar in makeup to humans that they can be the environmental sentinels for people.” The brain is a mysterious place with an estimated 100 billion neurons, or nerve cells, that fire up to make your thoughts swirl, your senses work, and your body move. Researchers don’t know exactly how the brain functions and they don’t know why glioblastoma multiforme happens. But when the type of glial cells called astrocytes start multiplying uncontrollably inside a person or dog’s skull, it’s difficult to stop. One reason is that these types of tumors weave their tentacles into the surrounding tissue. Traditional radiation and chemotherapy can’t pinpoint tumor cells precisely enough to prevent damage to other tissues.
Trying something new A new treatment, non-thermal irreversible electroporation (N-TIRE), uses two electrodes about one millimeter in size, approximately the same as a needle used for thick thread, placed directly on the tumor cells. Very short pulses of electricity shoot through the device into the cancer cells. The electric pulse is so short that it raises the temperature of the wire by only about one-quarter of a degree. One of the first patients treated with N-TIRE by the Neurology and Neurosurgery Service at the veterinary college in Blacksburg, Va., was a 12-year-old dog sent there because he was having vision problems, seizures, behavioral changes, and was unsteady on his legs. These are some of the same symptoms that humans often exhibit. The tumor takes up space in the inelastic skull that the brain needs to function. So the brain is crowded and some of its normal work is blocked, preventing transmission of signals that control everything from your eyes to your feet. In Helen’s case, she was having some vision, odor perception, and memory problems before the operation, and when the cancer cells not excised during surgery or killed by radiation grew into a tumor again, she lost her ability to speak, again had vision problems, and was unsteady on her feet until finally bedridden. Rossmeisl’s canine patient was diagnosed with a glioblastoma that was too large and rapidly growing to allow for standard surgery. This made the dog a candidate for N-TIRE. Two days after the procedure, a magnetic resonance image (MRI) showed that the dog’s tumor had shrunk to 75 percent its original size. He was put on a regimen of
radiation, and by four months post-procedure, the family pet was in complete remission.
“What we’re really doing is changing the cancer tissue’s properties,” says Rafael Davalos, co-inventor of the procedure. “Whatever area is treated, we can pinpoint it to half a millimeter. Also, it is super-fast, so there’s no heating and no scarring.” The surgeon can see the placement and progress of the electrodes in real time by using an ultrasound or MRI, says Davalos, a biomedical engineer in the Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences, who attends every procedure that Rossmeisl does using N-TIRE. Currently, the standard follow-up protocol of radiation and chemotherapy is used on the canine patients after having a procedure using N-TIRE. Chemotherapy and radiation can be a two-edged sword because they can damage normal tissue and compromise the immune system. But the new treatment may have a two-fold benefit for the immune system. “Irreversible electroporation seems to trigger an immune response, so we may not need any drugs as part of the treatment,” Davalos says. “That means your immune system isn’t weakened any further.” The veterinary college has successfully used N-TIRE on other types of cancers and other animals. Autumn, a chocolate Labrador retriever, was having trouble walking, so owner Sheryl Coutermarsh-Ott took her to the college for orthopedic surgery. They discovered that Autumn had an inoperable tumor on the inside of her thigh and groin. “The tumor was wrapped around her femoral hip joint,” says Coutermarsh-Ott. “The other options were not good. So, after talking to Dr. Rossmeisl, and reading some research papers about N-TIRE, we went ahead. Now she’s doing absolutely fantastic and is clear of cancer.” Then there was the 11-year-old thoroughbred with an ulcerated, growing cancer of the lip. The treatment with N-TIRE almost completely obliterated the tumor and the rest was removed with a carbon dioxide laser. The horse is now back to horsing around.
The risks Despite those successes, the focus of the Virginia Tech-Wake Forest team is on brain tumors, especially glioblastomas, because no really good treatment exists unless the tumors are diagnosed early, which happens rarely. Like humans, dogs that develop glioblastomas tend to be older — seven years and up; for people it’s usually around age 65 or older. More men than women develop this type of tumor.
Non-thermal irreversible electroporation actually makes holes in the cancer cells that cause them to die. But because this treatment does not generate heat and because it can hone in on the tumor so accurately, it doesn’t damage the normal cells, nerves, or normal vascular system around the tumor.
According to the American Cancer Society, about 13,000 Americans will die from brain tumors this year. More than 77 percent of those tumors are glioblastomas, which have a fatality rate of about 50 percent within 15 months and 75 percent in 24 months. That’s actually a much better survival rate than in 1974. This type of brain cancer is called a primary tumor because it develops in the brain. Tumors that grow elsewhere in the body and spread to the brain are called metastatic tumors. Because it’s often too late to save the life of someone with a glioblastoma, Rossmeisl is involved in a related project to investigate how to discover brain cancer early. Currently, by the time symptoms appear, the tumor is often too large and has infiltrated too much of the brain to be treated effectively. “Early detection is something that we really need,” Rossmeisl says. “We need something noninvasive that we can test for in blood or urine samples.”
The human side Dr. Thomas L. Ellis at Wake Forest is working with Rossmeisl, Davalos, and John Robertson to advance research on the use of N-TIRE. Robertson is a veterinary college professor of pathology, the director of the Center for Comparative Oncology in the veterinary college at Virginia Tech, and coordinator of the N-TIRE project. Ellis is a neurosurgeon and professor at the Wake Forest University Brain Tumor Center of Excellence. It’s Ellis who will take N-TIRE to clinical trials for people if given approval by the Wake Forest institutional review board and the FDA. If the procedure advances to human trials, it will first be used for patients who have recurrent glioblastomas. These are the patients who have run out of treatment options. Every month, a couple of patients come to Ellis with a diagnosis of glioblastomas. Although the cases are all different in some ways, they’re also all the same in other ways. “The causes for these tumors aren’t known; there are many factors but no known exposure pattern,” Ellis says. “These tumors are only rarely inherited. They occur sporadically in humans and there are no known exposures to chemicals or other agents that predispose patients to develop them. We don’t know of any brain tumor that is connected with a prior head injury.” Scientists, including Ellis, don’t believe that primary brain tumors are inherited; however, he has had two cases that seem to defy the odds. He had two siblings as patients who had developed mirror-image glioblastomas, and a recent patient’s father also had a glioblastoma. In July 2009, a man from South Carolina who was having trouble with numbers and with spelling his sister’s name came to Ellis. James Rollison thought he was having early onset Alzheimer’s disease at the age of 58. He hadn’t suspected a brain tumor, although his father died of a glioblastoma. But that was what he had. ➤
“The thought never crossed my mind that it was a GBM (glioblastoma multiforme) because my father’s doctor had said there was no way it was hereditary,” Rollison says. But when Rollison started having problems, his family doctor ordered an MRI and then sent Rollison to a neurosurgeon.
Fortunately, Rollison’s tumor was one of the rare ones that was diagnosed early. The retired attorney credits his work and being attuned to his body to sending him to the doctor at the first indication something was wrong. Rollison doesn’t look like, sound like, or act like someone who has had the deadliest brain cancer possible. Rollison was lucky because Ellis is a top-notch surgeon who got the tumor relatively early and was able to remove 100 percent of the visible mass. However, in order to treat the microscopic tumor cells that inevitably remain after surgery, Rollison underwent the standard chemotherapy and radiation regimen. Now he goes back to Wake Forest every three months for a checkup with Dr. Glenn Lesser, of the hematology and oncology department of the university’s Comprehensive Cancer Center, and has a follow-up MRI to make sure the tumor hasn’t returned.
The past, present, and future But not all outcomes are like this and Ellis wants to have more assurance that when he treats a brain tumor patient he’s giving them a chance at life. “In the last 40 years, we’ve made little progress in treating these tumors,” he says. “Using radiation and chemotherapy the (survival) numbers are pretty dismal.” In 1975, when Helen died, chemotherapy wasn’t common because most drugs couldn’t pass through the blood-brain barrier — a layer of closely spaced cells over the brain that stops harmful substances from invading. According to the National Cancer Institute, the rate of brain, spinal cord, and other nervous system cancers then was 5.9 per 100,000 adults. Today the rate is 6.4 per 100,000. Experts say the apparent increase in cases is mainly because of better diagnostic tools. Some of the treatments are also better, such as the MRI that can be used both for diagnostics and treatment. A functional MRI (fMRI) now exists that can monitor neurological blood flow in real time. In addition, more drugs can cross the blood-brain barrier and some better ways to deliver drugs are under development. Still, improved technologies usually buy patients only a few months more than they would have had 37 years ago. “One of the more difficult parts of my job, that never gets any easier, is when a person comes in who was living a normal life the day before and I have to tell them that they have only one to two years to live,” says Ellis. N-TIRE could give Ellis the advantage of reaching deep tumors and giving uniform treatment, he says. So far the procedure on dogs has been “excellent. This is an amazing collaboration with the vet school, Virginia Tech’s biomedical engineering group, and Wake Forest. I truly believe that N-TIRE can become an important new effective tool against glioblastomas.” In the meantime, Rossmeisl and his team continue to use N-TIRE to treat dogs that have no other hope. “So far, the outcomes with dogs have encompassed all the possible clinical outcomes,” he says. He works with Robertson and 22
other collaborators to identify the reasons certain dogs, most notably dogs with snubbed muzzles and broad heads, such as boxers and Boston terriers, are more prone to primary brain tumors. He also wants to make major strides in drug delivery to overcome the blood-brain barrier so the medications go directly where they are needed and stay there. “We believe that glioblastomas have so many ways to evolve and to evade all the treatments we have now that if we can figure out a way to beat those cancers we can cure any tumor,” Rossmeisl says.
Helen’s last Christmas was a joyful event because, though she’d lost her hair from radiation treatment and was wearing wigs and scarves, most of the family thought she was recovering – but, like the majority of these types of tumors, it was discovered too late.
John H. Rossmeisl Jr., associate professor of neurology and neurosurgery in the veterinary college, does research on primary brain neoplasms and traumatic brain injury. He also sometimes works with the college’s patients, such as Autumn, here with her owner, Sheryl CoutermarshOtt. Rossmeisl treated Autumn for an otherwise inoperable tumor in her groin using non-thermal irreversible electroporation. – Photo by Jim Stroup. The electrode for irreversible electroporation for treating cancer was developed by Rafael Davalos, assistant professor in the Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences. – Photo by Jim Stroup.
Neurosurgeon Tom Ellis and his dog, Gandalf. – Photo by Jim Stroup.
Dr. Glenn Lesser of Wake Forest University Medical Center exams Jim Rollison, who has been undergoing brain tumor treatment. – Photo by John McCormick.
Robert E. Neal II, doctoral student in the Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences (SBES); John Robertson, director of the Center for Comparative Oncology in the Virginia-Maryland Regional College of Veterinary Medicine; and Paulo Garcia, SBES postdoctoral associate, position electrodes in a gel to look at contact parameters as they test irreversible electroporation electrodes for potential applications. – Photo by Jim Stroup.
bay watch By Genevieve Rajewski
Sunrise sends streaks of red, violet and orange over the rippling surface of San Diego Bay at Base Point Loma, where 25 undergraduate interns are getting started on the day’s work. They hose down a gently swaying jigsaw of floating docks and pack 10-gallon thermoses with herring, capelin, smelt, mackerel and squid that will fortify their charges—in this case, 60 bottlenose dolphins that intermittently pop out of the water like prairie dogs. This seaside installation is home to the U.S. Navy’s Marine Mammal Program, which in the late 1950s studied how dolphins whip through water, with the goal of improving torpedo, ship and submarine design. Today the Navy trains two species, the bottlenose dolphin and the California sea lion, to help guard ports, personnel and military vessels around the globe. They have served in Vietnam and Iraq. Because dolphins possess the most sophisticated sonar known to man, they are unrivaled in their ability to locate and disable anti-ship mines and booby traps, even in the murkiest waters. Sea lions, with their superb low-light vision and sharp underwater directional hearing (something humans lack), can detect a potential enemy combatant swimmer approaching a ship. Even more importantly, both species can make repeated deep-water dives without suffering the decompression sickness that humans do. What this means is that a single marine mammal, two handlers and a rubber boat can provide the same high level of security as a team of human divers and the naval vessel, crew, physicians and medical equipment needed to keep them from getting “the bends.” So when it comes to providing for these soldiers of the sea, it’s nothing but top-flight. The National Marine Mammal Foundation is the nonprofit charitable organization that provides medical care for the Navy’s marine mammals. Cynthia
Smith, V99, is the executive director and medical director of the foundation, which cares for 120 Navy dolphins and sea lions, ranging in age from newborns to senior citizens. Foundation employees, the U.S. Navy, the U.S. Army, other contractors and consultants staff the $20 million operation at Point Loma, where the animals live and are trained in pens submerged in San Diego Bay. Eleven fulltime veterinarians and 160 trainers tend to every need of this marine security force. The dolphins and sea lions even have personal chefs: Army veterinarians inspect seafood from all over the world to supply the one million pounds of fish they consume each year.
On this day, Smith, who has cared for the Navy mammals for more than a decade, arrives on base promptly at 7:30 a.m. Because the animals get so much exercise swimming in the open ocean, her job is a lot like that of physician for a team of Olympic athletes. It is also somewhat like treating patients from another planet; although scientists have been studying marine mammal physiology for more than a century, little is known about them relative to the landlubber animal kingdom. Take sea lions, which Smith says are like water dogs, so “small animal medicine comes in really handy.” Dolphins, on the other hand, she says, are more of a physiological patchwork of people, pigs and cows. It is those similarities between dolphins and humans, learned by studying and caring for these animals for nearly a halfcentury, that have yielded an unanticipated benefit—a robust mountain of data that is helping to advance human medicine. One tantalizing outcome currently under investigation could lead to a treatment or cure for the 23.6 million Americans who suffer from type 2 diabetes. “Think about it,” says Stephanie VennWatson, V99, the veterinary epidemiologist who heads the clinical research enterprise at the National Marine Mammal Foundation.
Navy dolphins are safeguarding our national security — and advancing human medicine
“How many blood samples do we, as people, give in a lifetime? Well, compare that with these animals, which routinely provide blood samples, have daily health inspections, get annual ultrasound examinations and have access to numerous diagnostic tests over 30 or 40 years,” she says. “The power of the database is endless. We haven’t found the limit to the questions we are able to ask.”
The first fragments of that longitudinal database were incubated at an amusement park in Santa Monica in 1960. Sam Ridgway, an Air Force veterinary officer stationed at nearby Oxnard, would tag along with the local vet, Robert M. Miller, to care for the dolphins and seals that performed at Pacific Ocean Park. They published a few of their cases in professional journals, including the first X-ray of a live dolphin. Ridgway, now regarded as the father of marine mammal medicine, was also assigned to serve on two naval bases, where he first met the military and civilian scientists who were interested in studying the mechanics of how dolphins swim so fast and dive so deep. After completing active duty with the Air Force in October 1962, Ridgway was hired as the animal health officer for the Navy’s first five bottlenose dolphins. Soon after, he landed on a technique that is now the cornerstone of the Navy’s marine mammal care regimen. In 1963, the Washington University physicist C. Scott Johnson trained a dolphin he was using in his auditory research so that Ridgway could do a complete physical examination without removing the animal from the water. Under the leadership of Ridgway, now president of the National Marine Mammal Foundation, the practice of training marine mammals to participate in their medical care has been refined in the decades since. “The goal is to keep the animals comfortable and in their natural environment at all times,” says Cynthia Smith. ➤
Above: Dolphins undergo a physical.
The Navy mammals can contract the same illnesses that affect their relatives in the wild: respiratory infections, diarrhea and other bacterial and viral diseases. The focus on preventive care and rapid detection of illnesses has been so successful, however, that the veterinarians are developing a geriatric medicine program. Many Navy dolphins are now in their 30s and 40s; some are even reaching 50. That’s ancient compared with dolphins in the wild, which typically live into their teens and 20s. Navy sea lions live two or three times longer than their wild counterparts. Dolphins give blood samples, have their temperature taken and provide fecal and urine samples—all while in the water. As semi-land animals, sea lions undergo the same tests while lounging on the f loating docks in San Diego Bay. This morning, Smith pauses on the dock next to Mu, a 33-year-old dolphin. Mu rolls onto one side, bobbing in the water as a veterinarian runs a portable ultrasound over her belly. Four months pregnant, Mu is one of six Navy dolphins due to calf this summer. “It’s an exciting but anxious time,” says Smith, who pauses to borrow a pair of hightech sunglasses from the attending vet, Forrest Gomez. The ultrasound image projects onto the inside of the lenses, allowing Smith to assess the growing fetus despite the harsh sunlight bouncing off the waves.
Prenatal ultrasounds are rare in traditional veterinary practice, so the foundation team looks to human medicine as the standard. “We do ultrasounds at least once a month at this stage [of pregnancy],” says Smith. “We check for a fetal heartbeat and look at fetal development. As the due date approaches, we do more frequent ultrasounds.” Pleased with Mu’s progress, Smith joins the rest of the medical team for a discussion of the day’s cases. After 15 minutes of rounds beside the bay, the group gathers inside the on-site naval veterinary hospital to review two special cases.
human health fields—has been gaining great momentum over the last four or five years,” he notes. “And unlike your average veterinary clinic, we get to regularly work with specialists in the human field, thanks to our access to Navy and Army physicians. Those resources really elevate our wellness program.” For the CT scans, the dolphins traveled to the human naval hospital and underwent the procedure just like human patients do. “Our animals are trained to voluntarily beach and be transported all over the world,” says Jensen. “So they have no problem going into a human hospital—though they do tend to draw a bit of a crowd. We just roll them down the hall [on a gurney], lay them down [on the plastic-covered CT machine] and clean up thoroughly afterwards.” To prevent overheating, the dolphins are continually kept wet with sponges or spray bottles.
In the last month, the veterinarians worked with two interventional radiologists at Naval Medical Center San Diego, the human military hospital, to check the lungs of two dolphins—one treated for a bacterial abscess and the other for a fungal infection. Jenny Meegan, another foundation vet, pulls the dolphins’ CT scans up on a computer monitor and shares the human doctors’ interpretation of the results: both animals are recovering nicely.
in addition to providing direct care to the Navy marine mammals, foundation veterinarians also work on special projects to solve particular clinical problems in marine mammal medicine or to research disease pathology.
“Having that perspective and direction on cases is so helpful,” says Eric Jensen, the Navy Marine Mammal Program’s managing veterinarian. “Comparative veterinary medicine is not new. But the concept of ‘one health’—uniting veterinary medicine and work in the
Smith and Venn-Watson, for example, have enlisted a group of experts in veterinary and human medicine from the University of Texas Southwestern, University of California, San Diego, Dolphin Quest and SeaWorld San Diego to determine why dolphins develop
kidney stones. The project was inspired by Rake, a geriatric male dolphin that went into renal failure five years ago.
The multidisciplinary research team since has discovered that low levels of citrate in the urine may be a risk factor for kidney stones in dolphins—and that appears to be the case in humans, too. They are now working on pinning down the cause of this condition, known as hypocitraturia, with an eye toward developing a treatment for dolphins. The similarities between dolphins and humans mean that many advances in dolphin medicine could influence how physicians understand and treat human diseases. Venn-Watson, a veterinary epidemiologist who holds an MPH from Emory University, said the information contained in the Navy’s marine mammal database significantly surpasses the breadth of any population study she encountered during her two years traveling the world as a project director for the World Health Organization’s Global Foodborne Infections Network. Even a seemingly routine study can yield unimaginable results.
“Amazingly, dolphins are diabeticlike when they need it and non-diabetic when they don’t,” explains Venn-Watson. Dolphins seem to activate insulin resistance, causing temporarily high blood sugar during short overnight fasts, she says. They then revert to a non-insulin-resistant state as soon as they eat a meal. “If we can find and figure out how to flip that switch off and on in humans, that could be a great benefit to people with diabetes,” she says. Venn-Watson believes that dolphins may have evolved this way to stretch the limited stores of glucose found in their extremely high-protein, low-sugar diet, which consists entirely of fish. That notion is supported by research that Ridgway conducted in the 1970s. When dolphins were fed sugar, they had high glucose levels that lasted up to 10 hours. Those studies showed that dolphins’ diabeteslike systems do not have the ability to handle high-sugar meals. The foundation is now working with the nation’s leading research institutes, including the Salk Institute for Biological Studies in La Jolla, Calif., to identify that
This one-health strategy is catching on. Last winter, the National Marine Mammal Foundation welcomed 40 scientists from around the country, more than half of them from human medicine, to brainstorm new research projects. The gathering generated a five-year strategy and a prioritized list of studies in geriatric health, metabolic diseases and infectious diseases.
While creating a treatment plan for Rake, “we talked to other [marine mammal] facilities and people working with wild animals to see if kidney stones had similarly affected any dolphins they’d seen,” says Smith. “As soon as we realized it was a health problem impacting many dolphins, it became a collaborative effort to learn why, and how to prevent and treat it.”
genetic on/off switch in hopes of eventually testing a diabetes cure in mice and then in humans.
Like humans, dolphins have large blood sugar demands because of their large brains. But unlike humans, insulin resistance in dolphins may be advantageous.
“My job is to take that research road map and bring it to life,” says Venn-Watson. Her typical work week involves reaching out to potential collaborators from the human medicine side. The good news is that many are eager to invest their time and expertise in work that could advance both dolphin and human health. For example, foundation veterinarians and human medicine researchers are collaborating to assess whether changes such as mild chronic inf lammation, high cholesterol and decreasing muscle mass seen in aging dolphins—which mimic changes seen in aging humans—are associated with particular health problems and if targeted therapeutics can improve the quality of life in their golden years. “That kind of breakthrough would be a win-win for animals and humans,” says Venn-Watson. “This is one health. By caring for one species, we can care for many.”
Consider this simple exercise: VennWatson compared more than 1,000 fasting and post-feeding blood samples taken from 52 dolphins over seven years. She was surprised to find that the dolphin data did not match similar studies in other animals. Instead, the blood changes in these dolphins mimicked those seen in large-scale studies of people with type 2 diabetes. We humans need plenty of glucose, a sugar transported through the blood, to feed our big brains. The hormone insulin helps our bodies regulate the metabolism of carbohydrates and fats. People with type 2 diabetes either do not produce enough insulin or are immune to insulin’s effects, promoting the buildup of too much glucose in their blood, a condition known as insulin resistance. This blood sugar overload can lead to severe health problems, including heart disease, stroke, blindness, nerve damage and kidney failure.
Cynthia Smith, V99, examines a dolphin’s CT scan; behind her is the veterinary epidemiologist Stephanie Venn-Watson, V99. Mauricio Solano, head of diagnostic imaging at the Cummings School, helped Smith develop CT, ultrasound and X-ray techniques for the Navy marine mammals.
National Institutes of Health
IMAGINE A LANDSCAPE WITH PEAKS AND valleys, folds and niches, cool, dry zones and hot, wet ones. Every inch is swarming with diverse communities, but there are no cities, no buildings, no fields and no forests. You’ve probably thought little about the inhabitants, but you see their environment every day. It’s your largest organ—your skin. “The skin is like our shell. That’s what people see of us first,” says Elizabeth Grice, who just finished a postdoctoral fellowship in genetics at the National Institutes of Health (NIH) in Bethesda, Maryland. “It’s a defining feature, but it’s also an important organ for human health.” Our skin is home to about a trillion microscopic organisms like bacteria and fungi. Together, these creatures and their genetic material—their genomes—up the microbiome of human skin. Grice studies the skin microbiome to learn how and why bacteria colonize particular places on the body. Already, she’s found that the bacterial communities on healthy skin are different from those on diseased skin. She hopes her work will point to ways of treating certain skin diseases, especially chronic wounds. Collage photographs of body parts in four adjacent hexagonsCollage photographs of body parts in four adjacent hexagonsCollage photographs of body parts in four adjacent hexagonsCollage photographs of body parts in four adjacent hexagons “I like to think that I am making discoveries that will impact the way medicine is practiced,” she says.
Entering the Field Growing up in Wisconsin and Iowa, Grice was exposed to biology at a young age—but in a field, not a laboratory.
– Photo by BILL BRANSON, NIH
“My first job was detasseling corn,” she remembers. Pulling the tassel, or pollenproducing flowers, off the tops of corn plants is a way to breed highyield hybrid corn with specific traits. Summer days in the fields were hot and taxing. “That was when I realized I didn’t want to do manual labor,” Grice laughs. When Grice was in middle school, her mother went back to college for a bachelor’s degree in biology. Reading off flashcards to help her mom study sparked Grice’s own interest in science.
Body Bacteria In high school, Grice trained to become a certified nursing assistant and worked in a nursing home. Then she enrolled at Luther College in Decorah, Iowa for a bachelor’s degree in biology, with dreams of being a doctor. When biology professor Marian Kaehler announced a summer research opportunity for seasoned students, Grice—a freshman with no lab experience—knocked on Kaehler’s door 10 minutes later and asked for the job. “She was determined, enthusiastic and confident, and we decided to try it,” Kaehler remembers. “It worked out extraordinarily well.” Grice studied plant genetics in Kaehler’s lab throughout college. She found the environment, with its experiments and challenges, a more comfortable fit than a career focused on seeing patients—or summers breeding corn. Several research internships later, Grice earned a Ph.D. in human genetics and molecular biology from the Johns Hopkins School of Medicine before coming to NIH to tackle bacterial genomics.
BY ALLISON MACLACHLAN
The Good, the Bad and the Acne When you use antibacterial hand soap or take antibiotics, it’s easy to think of bacteria as bad guys. After all, Salmonella and E. coli can give you food poisoning, and Staphylococcus aureus (S. aureus) can cause pneumonia, meningitis or serious wound infections. But bacteria aren’t all bad. Many are harmless, and some are actually very helpful. On the skin, Staphylococcus epidermidis protects us by taking up space that the harmful S. aureus would otherwise colonize.
Your skin was sterile only once in your life—when you were in the womb. Minutes after you were born, bacteria began to colonize it. Your body relies on some of these bacteria as part of its first line of defense.
Like plants, bacteria don’t all fare well in the same environment. Some are better suited to moist, humid folds like the armpit or navel. Others colonize dry expanses like the forearm or oily nooks like the side of the nostril. Grice has surveyed the microbial landscape of human skin like a topographer charts a territory and an anthropologist studies its populations.
Scientists have traditionally studied skin bacteria by smearing a sample of them onto a layer of nutrientrich gel in a Petri dish.
Grace calls this “the great plate count anomaly”—bacteria that grow well in the lab aren’t necessarily major players on the skin. Grice employed a newer technique that uses a gene called 16S rRNA.
It might sound unhealthy or even dangerous to have skin that’s teeming with bacterial colonies. But as Grice points out, it’s completely ordinary.
From a study of 20 different skin sites on a group of healthy people’s bodies, Grice and her colleagues identified three types of environments: moist, dry and sebaceous (oily). Then they investigated which types of bacteria colonize what sites.
But 99 percent of the microbes won’t grow on laboratory plates, because they need to interact with other members of the skin’s bacterial community to survive. It’s also tough to replicate the exact nutrients and environment the skin provides.
The common skin bacterium that causes acne works the same way. “It’s occupying a niche so that other, more potentially harmful bacteria don’t invade,” Grice explains.
Many bacteria on the skin defend themselves by secreting antimicrobial peptides, or small proteins that kill harmful invaders. In protecting themselves, they also protect us.
National Institutes of Health
Exploring the Skin’s Microbial Metropolis
This gene provides the code for part of a bacterial ribosome, the essential machinery needed to make proteins. The 16S rRNA gene is present in every known bacterium, but in each one, it has a slightly different DNA sequence. Scientists can use the sequence of this gene to classify the bacteria. The Petri dish method has uncovered 10 different types of skin bacteria. The method Grice used revealed more than a thousand. Her study was the first to use the technique for such a large survey of human skin. She found that moist areas tend to host similar bacterial communities in all of her volunteers. The same holds for dry and sebaceous areas. Each skin environment determines its bacterial inhabitants just as an outdoor environment determines its plant life—rainforests support leafy trees, while deserts have cacti. Even with these patterns, the skin still has a surprising amount of variation from person to person. ➤
Bacterial diversity is probably a good thing, especially in wounds…
National Institutes of Health
Skin microbiomes are like snowflakes: No two are exactly alike. Your unique pattern depends on things like your age, sex, sun exposure, diet, hygiene and even where you live and work.
Microbes in Medicine By getting a sense of bacteria on healthy skin, Grice hopes to figure out what’s different about the microbes on diseased skin—and maybe even find a way to fix the problem. She’s most excited about applying her work to the chronic wounds that are common in people who have diabetes or spend most of their time in beds or wheelchairs. People with diabetes can lose some of the sensation in their limbs, making it harder for them to feel pain and easier for any of their injuries to fester. On top of that, they may have poor blood flow, which makes healing tough. As Grice explains, your body needs blood to deliver oxygen, immune cells and important proteins to the site of an injury to help cells regenerate.
A Problem Afoot
“It’s such a farreaching problem that it’s clearly an area of need,” says Grice. “That’s what really drives me the most.”
They found that wounds on diabetic mice started to increase in size at the same time as wounds on healthy mice began to heal.
Grice suspects that bacteria make chronic wounds worse because they spur the human immune system to trigger inflammation. Although designed to kill infected cells, inflammation also prevents skin cells from regenerating after an injury.
In about 2 weeks, most healthy mice looked as good as new. But most diabetic mouse wounds had barely healed even after a month.
The immune system acts slightly differently in each of us, thanks to our genetics. Grice’s work takes a microlevel look at interactions among human genes, the immune system and the skin’s bacterial communities.
Defense Mechanisms To investigate what role bacteria play in diabetic wounds, Grice used a group of laboratory mice bred to display common features of diabetes—like wounds that don’t heal well. Grice and her colleagues took skin swabs from both diabetic and healthy mice, and then compared the two. Using the 16S rRNA technique, they found that diabetic mice had about 40 times more bacteria on their skin, but it was concentrated into few species. A more diverse arrabacteria colonized the skin of healthy mice.
Almost 10 percent of the United States population has diabetes, and up to a quarter of these 24 million people will get a painful wound known as a diabetic foot ulcer.
“People with diabetes have high blood sugar, which is known to change the skin’s structure,” says Grice. “These changes likely encourage a specific subset of bacteria to grow.”
These ulcers are very difficult and expensive to treat. And the problem is increasing: As obesity rates rise, diabetes—and diabetic foot ulcers— are becoming more common.
The researchers then gave each mouse a small wound and spent 28 days swabbing the sites to collect bacteria and observing how the skin healed.
Interestingly, bacterial communities in the wounds became more diverse in both groups of mice as they healed—although the wounds on diabetic mice still had less diversity than the ones on healthy mice. “Bacterial diversity is probably a good thing, especially in wounds,” says Grice. “Often, potentially infectious bacteria are found on normal skin and are kept in check by the diversity of bacteria surrounding them.” Then Grice and her colleagues examined differences between healthy and diabetic mice at the genetic level. They focused on the genes that control aspects of the immune system in the skin. They found distinctly different patterns of gene activity between the two groups of mice. As a result, the diabetic mice put out a longerlasting immune response, including inflamed skin. Scientists believe prolonged inflammation might slow the healing process. Grice’s team suspects that one of the main types of bacteria found on diabetic wounds, Staphylococcus, makes one of the inflammationcausing genes more active. Now that they know more about the bacteria that thrive on diabetic wounds, Grice and her colleagues are a step closer to looking at whether they could reorganize these colonies to help the wounds heal.
– Photo by THE JACKSON LABORATOY
Skin isn’t the only place in the body that’s crawling with bacteria. More Than Skin Deep
Grice also spends time studying bacteria that live in the intestines. There too, microbes can be helpful. Certain strains of E. coli in our digestive tracts help keep dangerous bacteria at bay and produce Kand Bcomplex vitamins, which our bodies can’t make enough of on their own. Grice is involved with a study of Hirschsprung disease, a genetic disorder that leaves parts of the digestive tract without enough nerve endings to push wastes out. Some children born with the disease get enterocolitis, a painful inflammation in the gut, and others don’t. Together with geneticist Bill Pavan, who also works at NIH, Grice is looking at gut bacteria to see if their distribution differs between the two groups. If the researchers find a pattern, it might help predict which patients will need surgery to reduce inflammation. Grice and Pavan also think that redistributing some of the bacteria in inflamed intestines might help. Pavan admires Grice’s confidence and dedication to her science, and he also says that working with her is a lot of fun. “She is driven to get highquality research done, but she’s still extremely friendly and interactive on a personal level,” he says. “She has an infectious laugh.”
Highlights included exploring Mayan ruins, relaxing on beaches and snorkeling in the striking coral reefs off the coast. Grice also counts Greece among her favorite destinations because of its architecture and the laidback Mediterranean attitude. “I love Athens and all the old ruins that are just integrated into the city,” she says.
“Most people wouldn’t suspect that I’m very domestic,” says Grice, who lists cooking as one of her hobbies. “You get to a point where you’re comfortable experimenting with recipes and seeing what works.” Grice likes getting creative with her experiments in the kitchen as well as in the lab. “My husband doesn’t really eat vegetables, so it’s always a challenge to work around that,” she laughs.
When she’s home, Grice likes to explore other cultures and civilizations by reading. A selfprofessed bookworm, her favorite genre is historical fiction, including novels about the Tudor period in Britain. Tying her hobbies to her career choice is easy for Grice. “I really like experiencing different cultures, and science is so multicultural—you get to interact with a diverse group of people,” she says.
Taking Exploration Global For Grice, exploring diverse landscapes and populations goes far beyond skin samples. Outside of her work, she enjoys traveling to exotic locations to soak up the culture.
Charting New Ground
She and her husband were married in Belize, a country they chose for its natural beauty and its preserved culture. “It’s one of those places that you feel isn’t overrun by civilization,” she says.
During the preparation of this article, Grice was considering job offers for a faculty position. She decided to join the University of Pennsylvania’s dermatology department and will start working there in January 2012. In her new job, she will continue her research on the wound microbiome and teach graduate and medical students. She hopes that she, like her longtime mentor Marian Kaehler, will inspire and challenge her students.
Grice loves to experience the natural beauty and local culture in countries like Belize, Greece and Costa Rica. – Photo by ELIZABETH GRICE
National Institutes of Health
Skin isn’t the only place in the body that’s crawling with bacteria.
Pavan said Grice is well known for whipping up impressive treats like miniature chocolate mice, which are very popular in the lab. And whenever a labmate has a birthday, Grice brings in a custombaked cake with whatever flavor and frosting the person wants.
“She was just so tough, and I really respected that,” Grice says of Kaehler. “Having a female mentor was also really important to me, because otherwise, how do you picture yourself in that role?” Even now that she’s landed that role, Grice’s ambition isn’t flagging. She aims to sustain a successful research program, improve the way chronic wounds are managed and keep time for personal goals like traveling to new continents. Kaehler, for one, is confident that Grice will succeed. “She has a very strong sense of self, and there’s nothing more important for people making career decisions than knowing where you’re going to find a niche that makes you satisfied and challenged,” she says. Like the bacteria she studies, Grice knows where she thrives.
TEACHI NG THE HIGHEST STANDARDS OF ANIMAL CARE AND USE
We are dedicated to the advancement of COMPASSIONATE CARE
for laboratory animals through EDUCATION, TRAINING and consistent INFORMATION EXCHANGE
for the benefit of human and animal health.
Career Day Kit is a coordinated, nationwide endeavor designed to educate elementary school students about humane and responsible animal research. According to the U.S. Department of Education, there are more than 130,000 elementary and secondary schools in the U.S. If scientists across the country can make one 20-minute presentation in one classroom in every one of these 130,000 schools, we have the potential to reach 2.6 million students in just one year! It is possible to have a HUGE impact by simply taking time out of the day to teach children about animal research. The Career Day Kit empowers you to make effective presentations in elementary school classrooms that will educate children about science careers and animal research. You will help illuminate the importance of animal research and have an impact on their thinking for their entire lives. It is important to reach children early and often with the truth about biomedical research. The future of science and human health is at stake and you can help!
The Foundation for Biomedical Research (FBR) Career Day Kit includes a DVD with lab footage, stickers for the children, a presenter’s “how to” guide, and several useful props. ORDER YOUR CAREER DAY KIT TODAY…ONE FREE KIT PER INSTITUTION. To order, please contact Nahla at 202-457-0654 or send her an email at email@example.com
– Photos by SE-JIN LEE LAB
Johns Hopkins University
Hopkins Researchers Solve Key Part of Old Mystery in Generating
Implications for treating muscular dystrophy and other muscle wasting diseases BY VANESSA MCMAINS, PH.D.
Mice without the gene for myostatin (right) have nearly twice as much muscle mass as normal mice (left). Working with mice, Johns Hopkins researchers have solved a key part of a muscle regeneration mystery plaguing scientists for years, adding strong support to the theory that muscle mass can be built without a complete, fully functional supply of muscle stem cells. “This is good news for those with muscular dystrophy and other muscle wasting disorders that involve diminished stem cell function,” says Se-Jin Lee, M.D., Ph.D., lead author of a report on the research in the August issue of the Proceedings of the National Academy of Sciences and professor of molecular biology and genetics at the Johns Hopkins University School of Medicine. Muscle stem cells, known as satellite cells, reside next to muscle fibers and are usually dormant in adult mammals, including humans. After exercise or injury, they are stimulated to divide and fuse, either with themselves or with nearby muscle fibers, to increase or replace muscle mass. In muscle wasting disorders, like muscular dystrophy, muscle degeneration initially activates satellite cells to regenerate lost tissue, but eventually the renewal cycle is exhausted and the balance tips in favor of degeneration, the researchers explain. Muscle maintenance and growth under healthy, non-injury conditions have been more of a mystery, including the role of myostatin, a protein secreted from muscle cells to stop muscle growth. Blocking myostatin function in normal mice causes them to bulk up by 25 to 50 percent. What is not known is which cells receive and react to the myostatin signal. Current suspects include satellite cells and muscle cells themselves. In this latest study, researchers used three approaches to figure out whether satellite cells are required for myostatin activity. They first looked at specially bred mice with severe defects in either satellite cell function or number. When they used drugs or genetic engineering to block myostatin function in both types of mice, muscle mass still increased significantly compared to that seen in mice with normal satellite cell function, suggesting that myostatin is able to act, at least partially, without full satellite cell function. Second, the researchers guessed that if myostatin directly inhibits the growth of satellite cells, their numbers should increase in the
absence of myostatin. The researchers marked the satellite cells with a permanent dye and then blocked myostatin activity with a drug. Mouse muscle mass increased significantly as expected, but the satellite cells did not increase in number, nor were they found fusing with muscle fibers at a higher rate. According to Lee, these results strongly suggest that myostatin does not suppress satellite cell proliferation. Third, to further confirm their theory that myostatin acts primarily through muscle cells and not satellite cells, the team engineered mice with muscle cells lacking a protein receptor that binds to myostatin. If satellite cells harbor most of the myostatin receptors, removal of receptors in muscle cells should not alter myostatin activity and should result in muscles of normal girth. Instead, what the researchers saw was a moderate, but statistically significant, increase in muscle mass. The evidence once again, they said, suggested that muscle cells are themselves important receivers of myostatin signals.
Everybody loses muscle mass as they age, and the most popular explanation is that this occurs as a result of satellite cell loss....
Lee notes that, since the results give no evidence that satellite cells are of primary importance to the myostatin pathway, even patients with low muscle mass due to compromised satellite cell function may be able to rebuild some of their muscle tone through drug therapy that blocks myostatin activity. “Everybody loses muscle mass as they age, and the most popular explanation is that this occurs as a result of satellite cell loss. If you block the myostatin pathway, can you increase muscle mass, mobility and independence for our aging population?” asks Lee. “Our results in mice suggest that, indeed, this strategy may be a way to get around the satellite cell problem.” Authors on the paper include Se-Jin Lee, Thanh Huynh, Yun-Sil Lee and Suzanne Sebald from The Johns Hopkins University, Sarah WilcoxAdelman of Boston Biomedical Research Institute, Naoki Iwamori and Martin Matzuk of Baylor College of Medicine, and Christoph Lepper and Chen-Ming Fan from the Carnegie Institution for Science.
Insert Submission Princeton University
Cancer collaboration could someday help dogs and their humans
Olga Troyanskaya’s dog Jessy fell ill in early 2006, the vet had painful news. “The doctor told me she had less than a week to live,” Troyanskaya said.
Sorenmo provides samples of tumors that she has extracted from her canine patients during surgery and Troyanskaya analyzes the genes in the tumors.
Troyanskaya sought a second opinion with a dog cancer specialist, with an unexpected result: The appointment launched an innovative research collaboration to learn more about cancer, possibly leading to new treatments for dogs like Jessy and humans as well.
All of Sorenmo’s patients developed cancer through natural processes, just as humans do. She treats them with the same types of therapies humans receive, including radiation, chemotherapy and surgery. She and her colleagues examine each extracted tumor, noting its size and other attributes, but now with the Princeton collaboration, Sorenmo can learn much more about the cancers she treats.
At the appointment, Troyanskaya, a computational biologist at Princeton University, got to talking with Jessy’s canine oncologist, Karin Sorenmo, head of the small animal oncology service and an associate professor of oncology at the Ryan Veterinary Hospital of the University of Pennsylvania. The two researchers discovered that they share a goal to learn about how to understand and treat cancer. Troyanskaya was interested in the genes involved in cancer; Sorenmo wanted to explore the mechanisms of cancer to find new ways to treat her patients, most of them well-loved pets. “A collaboration seemed like a unique way to look at the question of cancer progression,” said Troyanskaya, an associate professor of computer science with a joint appointment in the Lewis-Sigler Institute for Integrative Genomics at Princeton. It is also a way to help dogs like Jessy, she said. For years, Jessy was Troyanskaya’s canine collaborator, accompanying the scientist to the lab where she’d station herself next to her owner’s desk.
Relying on a comparative approach After months of conversations while Jessy was treated with chemotherapy, Troyanskaya and Sorenmo started a collaboration between their two laboratories, which are located about 50 miles apart.
Computational geneticist Olga Troyanskaya of Princeton University (left) and Karin Sorenmo, a veterinary oncologist at the University of Pennsylvania Ryan Veterinary Hospital, pose with Sorenmo’s dog Ruby. The two scientists are collaborating on a study to explore genetic factors contributing to mammary cancer growth in dogs being treated at the hospital, with the goal of enhancing the understanding of cancer in both dogs and humans. – Photo courtesy of OLGA TROYANSKAYA
The study of naturally occurring cancer in animals, and its application to human cancer, is called “comparative oncology.” Sorenmo is a leader in this emerging field. Comparative oncologists recognize that much can be learned about cancer by comparing animals and humans. “Dogs get all the same cancers that humans get,” said Chand Khanna, director of the Comparative Oncology Program at the Center for Cancer Research, which is part of the U.S. National Cancer Institute. “With dogs, we can ask many questions that one cannot ask in mouse preclinical models of cancer and cannot answer in human clinical trials.” Troyanskaya and Sorenmo are looking for answers by studying a type of cancer that poses a high risk for both dogs and humans: mammary cancer. Mammary tumors are the most common kind of tumor in female dogs that have not been spayed. Breast cancer is the most commonly diagnosed cancer in women, striking one in eight women during their lifetime. Yet not all dogs or women succumb to the disease: In some individuals, the tumors are benign and grow slowly over years, while in others the cancerous cells grow rampantly and eventually kill. Troyanskaya and Sorenmo hope to learn more about why this is so.
BY CATHERINE ZANDONELLA
By studying different tumors isolated from a single dog, the investigators can hone in on gene signatures related to tumor development without having to subtract out gene signatures from the rest of the body. “In humans, a lot of times the variability is so large across individuals that it completely masks any signal of progression-related variability between the tumors,” said Troyanskaya. “With dogs, you can look at tumors at different stages of cancer progression in the same genetic background, within each individual.”
Evaluating genetic signatures At Princeton, Troyanskaya’s team has begun evaluating the tumor samples to look for genetic signatures that may be correlated to the progression of mammary cancer in dogs. This type of genomewide search became possible with the sequencing of the canine genome in 2005.
Stray dogs are also the least likely to have been spayed, and the resulting hormone levels put them at much greater risk than spayed animals of developing mammary tumors. To help homeless dogs, Sorenmo started the Penn Vet Shelter Canine Mammary Tumor Program in 2009 to provide cancer treatment to dogs living in shelters. “The shelter program is a way to provide high-quality care for some of the neediest dogs, while helping to further our knowledge of both canine and human breast cancer,” Sorenmo said. Under the program, which is funded by family foundations, shelter dogs are treated for cancer free of charge. Dogs that are later adopted continue to obtain follow-up treatments at no cost to the owner. Lisa Hertzog, a resident of Reading, Pa., has adopted two dogs participating in the program. “It is really a rewarding experience,” she said. “The dogs are really loving, and they give back so much more than what you give them.” Since beginning the collaboration with Troyanskaya, Sorenmo and her colleagues at Penn have operated on more than 60 shelter dogs. Studies of these samples are already starting to reveal factors that may help clinicians predict the progression of the tumors.
Gaining support from dog lovers
To evaluate the genes in the tumors, Troyanskaya’s group, which includes molecular biology graduate student Dmitriy Gorenshteyn, isolates the genetic material in the tumor samples. The investigators look at which genes are turned on, or “expressed,” and which are turned “off.” They try to tie the resulting gene expression pattern to tumor attributes such as rapid growth.
Despite its advantages, comparative oncology — which involves pets rather than laboratory animals — is not yet a mainstream approach to studying human cancer. To fund her initial studies, Troyanskaya received funding from Princeton’s Project X, a fund set up by businesswoman and philanthropist Lynn Shostack to support pioneering and speculative projects.
Several studies of gene expression patterns have been conducted for human breast cancer, but this is possibly the first such study of canine mammary tumors. Nor have comparisons between the gene expression patterns of human and dog tumors been made.
Further help came this January from 2 Million Dogs, a new foundation dedicated to finding cures for canine cancer. Its founder, Luke Robinson, walked 2,000 miles from Austin, Texas, to Boston in 2008 to raise awareness of canine cancer and build a grassroots network of dog lovers and cancer researchers. He met Sorenmo on his walk through Philadelphia, and the foundation awarded its first grant to the collaboration between Sorenmo and Troyanskaya. In January, 2 Million Dogs presented a $50,000 check to the researchers to help them purchase the laboratory supplies for the study of genes involved in canine cancer.
The dog genome provides special challenges because dog breeds have different genetic variations. Using statistical methods, the researchers have already started identifying the canine equivalents of human genes based not only on their sequence, but also on their functions in the body. “The vast majority of dog genes have a corresponding human gene,” Troyanskaya said. An expert in combining computer science with biology, Troyanskaya has developed computer programs that can sort through the data generated by the genome-scale gene expression studies to detect patterns of gene expression that correlate with increased tumor size. The team, which includes quantitative and computational biology graduate student Jonathan Goya, plans to follow up the gene expression studies with a search for other abnormalities such as extra copies of genes, known as copy number aberrations, and changes in protein levels. Eventually the group hopes to link genetic pathways to the progression of a tumor from a harmless overgrowth of cells to a deadly spreading malignancy.
Providing care to shelter dogs At the beginning of the collaboration, Sorenmo collected samples of tumors that she surgically removed from Penn vet clinic patients, typically pet dogs from comfortable homes. But it nagged at her that homeless dogs were not able to get similar care.
Canines are helpful in the study of mammary cancer for another reason: Each female has eight to 10 mammary glands, making it possible to study several tumors — each arising separately from the other and therefore genetically unique — in one individual. Studying separate breast tumors in humans is usually not possible because it is rare for a woman to develop more than one spontaneous tumor in the breast.
“This work is incredibly important to anyone who has had a loved one, whether dog or human, who has had cancer,” said Robinson, whose dog Malcolm died of cancer. Through the work funded by 2 Million Dogs, Troyanskaya and her team hope to find gene expression patterns that govern the transformation of a tumor from a benign to malignant state, contribute to tumor growth and govern metastasis. The investigators anticipate that their studies will be a starting point for developing diagnostic methods that veterinarians and doctors can use to predict whether a newly discovered tumor will grow slowly or rapidly. They also hope to identify novel pathways that could serve as targets of new drugs to treat cancer. Troyanskaya’s faithful laboratory assistant and canine companion Jessy succumbed to cancer six months after beginning her treatment. But Troyanskaya is optimistic that the collaboration that emerged from Jessy’s illness will provide a lasting legacy. “We have the potential to contribute new findings that could lead to better cancer diagnosis and treatments for humans and for our dogs,” she said. Spring/Summer 2013
Oklahoma State University
TACKLING A FUNGAL OSU RESEARCH COULD HELP ‘MANY DOGS AND CATS LIVE A LONG, HEALTHY LIFE’ An OSU educator could have a profound impact on diagnosing and treating a fungal disease, which has become pervasive in Oklahoma. Andrew Hanzlicek, DVM, M.S., diplomate of the American College of Veterinary Internal Medicine in small-animal internal medicine, is an assistant professor of small animal internal medicine at OSU’s Veterinary Hospital. His research on histoplasmosis — a fungal disease endemic in Oklahoma — may change the way the disease is diagnosed and treated. — by DERINDA BLAKENEY
Dr. Andrew Hanzlicek examines Midget, owned by Lois Crain of Ringling, Okla., with Dr. Jennifer Chang, a resident in small-animal internal medicine.
Histoplasma capsulatum, Oklahoma State University
a soil-borne fungus, is found in temperate and subtropical regions throughout the world. In the U.S., histoplasmosis appears in the Ohio, Missouri and Mississippi river valleys as well as in Oklahoma. The disease is a common systemic fungal infection of many dogs and cats in Oklahoma and occurs when microconidia — the mycelial form of Histoplasma sp. found most abundantly in nitrogen-rich soil — is inhaled or sometimes ingested. “I saw this disease when I worked at Texas A&M University and at Kansas State University, but I was surprised at how frequently we see it here at OSU’s Veterinary Hospital,” says Hanzlicek. “Because of its small size, the microconidia can penetrate deeply into the lungs when inhaled. Histoplasmosis is a disease that can affect the respiratory system, the gastrointestinal tract or the skin in cats and dogs. Sometimes it affects the bone marrow, eyes, the brain — it can go virtually anywhere in the body.” Common clinical signs include lethargy, weight loss, anorexia and fever unresponsive to antibiotics. An infected animal — especially a dog — may have diarrhea. Traditionally, the diagnosis of histoplasmosis is made from clinical signs and finding fungal organisms from affected tissue or fluid samples and, in some cases, a fungal culture. “Fungal culture has the disadvantages of lacking sensitivity, requiring specialized laboratories and having a slow turnaround time (weeks).” Currently, Hanzlicek uses a test from MiraVista Diagnostics in Indianapolis. The test can be performed on body fluids of the potentially infected animal. “We can use urine samples, blood samples or fluid from a lung wash,” explains Hanzlicek. “If a protein from the cell wall is in the sample, the dog or cat will test positive for histoplasmosis.” Hanzlicek started a clinical trial in June 2012 to determine how treatment with antifungal therapy affects the antigen test. “From previous information in over 70 dogs and 30 cats, we are convinced this test is accurate, and in some cases, this test has changed the way we diagnose the disease,” he says. “Before this test, we diagnosed histoplasmosis based on clinical signs and finding the fungus through invasive tissue biopsy or needle aspirate procedures. Now it may be as easy as submitting a urine or blood sample. Next, we need to find out if it can also be used to monitor or guide antifungal therapy.
“Right now, the test is used to monitor treatment in humans. We don’t have data on animals, and that’s why we are doing the study,” says Hanzlicek. “When you test positive for histoplasmosis, the test readout gives you a number. For example, it could be 8.2. “If we can diagnose histoplasmosis You treat the person and then retest. The early on and if we treat for the result has to be below a certain number before you stop treatment. If you stop appropriate amount of time, the antifungal therapy too soon, the infection could return. If the test result decreases animals have a pretty good chance with treatment like we expect, it should work the same for animals as it does for of making it. This isn’t an end-of-life humans.” He has applied for grant money to cover the research and hopes to have 20 dogs and 20 cats known to be infected with histoplasmosis in the program. “We will monitor how the antigen test changes during treatment, which will help us decide if we can use the test to monitor treatment. I anticipate it will take 12 to 18 months to gather the data,” he says.
disease. It affects young, otherwise healthy dogs and cats. If we can diagnose it with a simple fluid test and monitor treatment without invasive procedures, we have a better chance of helping many dogs and cats live a long, healthy life.”
University of North Carolina
Black Death Threat UNC’s Bill Goldman battles the next outbreak of the plague before it happens. BY MARK DEREWICZ – photo by Mark Derewicz and Bill Goldman
on the Grand Canyon’s southern rim, a biologist named Eric York found a dead mountain lion with a bloody nose but no other signs of trauma. He took it back to his garage to perform an autopsy, which revealed nothing unusual.
Two days later, York developed a bad cough. He felt weak, achy, tired. His doctor told him he had a flu-like illness and sent him home. Two days after that, York was dead. This time, the autopsy did reveal something. York was stricken with the plague, also known as the Black Death, the same disease that wiped out half of Europe during the fourteenth century. Public-health officials gave antibiotics to everyone who had come in contact with York. No one else died. Disaster averted. But how did York’s doctor miss something as uniquely horrifying as the plague?
Turns out just about every doctor would’ve missed it, according to UNC’s Bill Goldman. “The first symptoms of the plague really are indistinguishable from the flu,” he says. But unlike the flu, the plague is already well on its way to shutting down the lungs by the time a patient begins to feel sick. It’s a sneaky, extremely contagious, and fatal disease, three reasons why governments and researchers think the plague is a bioterrorism threat—a twenty-first-century weapon of mass destruction. In medieval times of war, combatants would catapult infected bodies over city walls. Today, a bioterrorist attack would be stealthier and a lot more dangerous. After the anthrax scare of 2001, the U.S. government pushed for scientists to research various biological warfare threats, such as Yersinia pestis, the bacterium that causes the plague. “I hate to put it this way, but terrorists aren’t going to unload a bunch of rats or fleas into town,” Goldman says. They’ll culture the bacteria in massive amounts. “They’ll try to spread the disease by an aerosol,” he says. Victims wouldn’t smell it or see it. They wouldn’t even feel a thing at first, but the disease would be on a rampage. Thousands of people would get sick but have no idea they had the plague until it was too late to save them.
The plague is such a silent killer because Yersinia pestis doesn’t trigger the same sort of quick immune response that most bacterial infections do. When a person contracts the plague, the bacteria multiply from a few microbes to a billion within 48 hours. But for some reason the lungs— typically very good at getting rid of undesirables—don’t respond. In the case of Eric York, doctors had no way of distinguishing his illness from the flu. Only when symptoms worsen— vomiting, difficulty breathing, coughing up blood—does the plague give itself away. “By then, when it’s recognizable as pneumonic plague, it’s too late to treat it,” Goldman says. The lungs are overrun with bacteria. The pulmonary system is all but shut down. The circulatory system can’t deliver antibiotics into the lungs. Patients suffocate to death. They just can’t breathe anymore. “Here’s the question we wanted to answer,” Goldman says. “Is Y. pestis avoiding detection, or is it actually suppressing the immune responses of the lung?” The answer would give his team clues about how to make the plague less like the Black Death and more like the flu, at least in terms of patient prognosis. Goldman’s samples of Yersinia pestis came from a repository that got its specimens when a Colorado woman died of the plague in 2000. She had been
infected by her cat, which had probably gotten hold of an infected rodent. These specimens are just as deadly now, which is why Goldman’s team was put through stringent security checks before being allowed to work with the organisms. The FBI has active files on each lab member, including Goldman.
One of the reasons Yersinia pestis is such an aggressive killer is because it contains a particularly nasty plasmid—a segment of DNA that is not part of a bacterium’s chromosomes but can replicate and transfer into other living things. Yersinia pestis picked up its deadly plasmid from some other organism thousands of years ago, Goldman says. He wondered how virulent the bacterium would be without that plasmid, so his team took it out and placed a droplet of the specimen on the nose of a single mouse. When the mouse breathed it in, the bacteria didn’t multiply. In fact, they declined in numbers over four days.
“Imagine the worst-case scenarios,” Goldman says. “An aerosol released that exposes a lot of people at once, and no one would have any idea they’ve been exposed. All of a sudden, everyone is sick. Early symptoms are indistinguishable from the flu.” In such cases, a cure would be best. A vaccine would be a close second. The next best thing would be to slow down the disease so treatment has a chance to work. “The plague is susceptible to antibiotics,” Goldman says. “Just not in that last 24 hours.” Bill Goldman is chair of the department of microbiology and immunology in the School of Medicine. He received funding from a National Institutes of Health grant to the Southeast Regional Center of Excellence for Emerging Infections and Biodefense, which is headquartered at UNC-Chapel Hill.
Plague at a glance
The mouse never got sick. This proved that the plasmid is absolutely critical for lung infection to spiral out of control.
The plague was never eradicated; it thrives in the wild.
Then Goldman’s team mixed the nonlethal strain of Yersinia with the deadly strain and documented how they behaved in mouse lungs. The deadly strain multiplied like mad, as Goldman expected, but so did the nonlethal strain.
Few humans are infected anymore.
The organism that causes the plague is now a bioterrorism threat.
In another experiment, his team documented how other, relatively harmless bacteria responded when the deadly Yersinia strain was present in the lungs. “Even the harmless bacteria are able to grow really well when Y. pestis is present,” Goldman says. “They increase from a thousand to between one million and ten million organisms in the lung.” Those once-harmless bacteria wind up aiding Yersinia in blocking the lung’s air passages.
UNC’s Bill Goldman has found a new clue about why the plague is so deadly and how to make it less so.
Although Goldman and his team have indicted that lone plasmid, they’re still trying to pin down the mechanism that allows Yersinia to change the lung into such a permissive playground for pathogens. And if they find that mechanism? “What I’d like to say is, ‘Oh, that will lead us to a drug,’” Goldman says. “But it depends on what the mechanism is.” His team has already identified a Yersinia protein that helps the bacterium multiply inside the lung. “We have a patent on the idea of creating an inhibitor of that protein,” Goldman says, “but we haven’t found an inhibitor yet.” Disabling that lone gene might be less a cure than a shield to keep the disease from progressing so fast, which might give doctors more time to treat patients. “You have to figure out how to defeat the main barriers to treatment,” Goldman says. And in the case of the plague, the main barrier is the speed at which the disease takes hold. A person usually dies within three and a half or four days of contracting pneumonic plague. Goldman says that inactivating the protein his team has identified could keep patients alive longer than usual, and that would give antibiotics more time to work. “If you can change the speed of the infection,” he says, “you’ve solved a major problem.
University of North Carolina
When no one is working in the Goldman lab, sealed and locked doors separate humans from the containers that hold the bacteria. Lab technicians change into protective clothing in a designated chamber between the outer lab and the inner lab where they handle the samples. They attach to their heads a device that continuously pushes air downward to lessen the chance that they’ll breathe in a pathogen. They open specimen containers only under a special hood, into which they reach with gloved hands to conduct experiments.
This approach wouldn’t help everyone infected with the plague. It likely wouldn’t have helped Eric York. But lengthening the time between initial infection and death could be enough to save thousands of lives after a bioterrorism attack.
Bubonic or Pneumonic “The classic bubonic plague is a disease that’s in the wild all the time,” says UNC microbiologist Bill Goldman. “It’s constantly circulating between rodents and the fleas that infect them.” When an infected rodent or flea bites a person, the bacterium Yersinia pestis spreads through the human lymphatic system. Lymph nodes get inflamed and swell into hard nodules, causing incredible pain and eventual death. There is no cure. The inflamed lymph nodes are called buboes—thus the name bubonic plague. But when Yersinia pestis enters the bloodstream, it travels to lungs and patients develop secondary pneumonic plague, which is very contagious. A cough or sneeze can easily transmit the disease, and if someone catches the plague that way, it’s called primary pneumonic plague. “This is the worst form of the disease,” Goldman says. “It’s one reason why the plague is a bioterrorism concern.” Infected patients might not know they have the plague, but they can still spread it.
Foundation for Biomedical Research Awards Three Scholarships
Foundation for Biomedical Research
to Outstanding Cal Poly Pomona Pre-Veterinary Students Scholarships are awarded to three pre-veterinary students who wrote outstanding essays on the need for lab animals in medical and scientific research
Foundation for Biomedical Research (FBR) announced it has awarded three college scholarships to the winners of its 2012 Animal Research Essay Contest, a partnership with Cal Poly Pomona. First place is awarded Catherine Runion (graduating 2013), second place to Megan Ducey-Hardos (2013), and third place to Fiona Lair (2014). The winning essays will be published in the spring 2013 issue of FBR’s semi-annual magazine, ResearchSaves. “It is critical the next generation understand the vital role humane and responsible animal research plays in both human and animal medicine,” said FBR executive vice president Paul McKellips. “Catherine, Megan and Fiona have each demonstrated an excellent grasp of how lab animal research leads to new treatments, therapies and cures for people and animals, and an exceptional ability to communicate that fact.” Catherine Runion is a senior at Cal Poly Pomona and will graduate in December with a degree in animal and veterinary science. Megan Ducey-Hardos is a senior majoring in animal health science student; she hopes to become a registered veterinary technologist upon graduation. Fiona Lair is pursuing her bachelor’s degree in animal science and plans to specialize in veterinary radiology. Students submitted essays on the fundamental role humane and responsible animal research plays in the advancement of human and animal health. Essays were judged by representatives from FBR and Cal Poly Pomona. In addition to college scholarships, FBR has also donated a CurVet™ Rat Training Simulator to Cal Poly Pomona, in order for its pre-veterinary students to learn humane and ethical handling practices of rodents without the use of live animals. For more information about the 2012 Animal Research Essay Contest or the winners, please visit www.fbresearch.org and http://www.csupomona.edu/~agri/ our-college/news.shtml.
And the winners are:
Catherine Pabst Dear Ingrid Newkirk,
ies in the western among the top public universit University Pomona is ranked nic is the second re tech Poly cultu agri State of ia ge forn Cali y. The colle its learn-by-doing philosoph for n al and know anim is the and of s ent State stud United ents, Being a goal for with about four thousand stud rience with animals are, The largest college on campus expe on s hand t rtan impo th, know just how ter goal of improving their heal grea veterinary program myself 1 the with field al anim a career in the ecting animals and giving prot of l most of us is to one day find goa the with life , I know you live your As you are a member of PETA them a voice. ion for helping and I have always had a pass how important animals are to help them if do how learn As a pre-vet student 1know to osed supp I then comes, how am them. However, the question e we have a few first learning a new techniqu or learn from? When we are us. This is a perfect way to hag esop an not have them to practice on and s lung s, with a full set of vein situation. A real life-like dolls that are equipped d representation of real life to do but does not give a goo a catheter into its vein, rt inse you g lettin learn what we are supposed with ok , hand you its paw and be go through more dog is not likely to just sit there e, more animals will have to als to practice our techniqu If we do not have live anim pain in the long run. rtment. With such a Cal Poly is its research depa the agriculture program at swine unit, a beef and p shee a er, Another huge department in cent research enough to have an equine professors all ated educ ly high by huge campus we are lucky ities is run facility. Each one of these facil regulations that arch t stric rese r se unde mou a ed orm and unit is perf , All the research done here field ific the animal’s spec ing their help in to ion ing pass with a the research work ect to the animal first, with most of treatment without a test subj of put the care and welfare of kind any ect perf and create, improve riments but there are students expe e themselves. It is very hard to thes on ing work are there professionals and resumes for see how it works. Not only looks great on applications ul hands on experience that as well. This provides wonderf ation opportunities. future careers or higher educ only see what much more when 1 can not e I have found that I learn so and most memorable rite favo my of In my own personal experienc One . well could touch and feel it as to go down to I am trying to learn, but if 1 student was when I was able in my five years as a pre-vet topics with the hot the of one was experiences that I have had sure I’m tiful cow named plug. Plug, to be done in arch rese led enab that the beef unit and meet a beau in his side the fact that he had a plug d cow. I not love and py hap a pt university and PETA due to exce nothing r that I spent with him I saw see, touch hou to brief able the was I For en, en. rum rum ’s his cow to experience the inside of a got I ugh. With but thro him ing with pass visit was to only got g the food that en. In the bacteria at work processin complex process of the rum and feel the movement and the of ding rstan unde r to get a bette more fear getting a simple show s that one experience I was able dog s; stres of kind ever see Plug in any loved by all and no way shape or form did I s of students. That cow was showed surrounded by a clas be wrong or cruel when d coul this vaccine injection than Plug that ve belie to tion. It is hard for me that make received so much loving atten ed. It is experiences like this benefiting and not being harm and grow in ways I learn me help both sides of the situation arc will that s ersity that offers opportunitie me feel lucky to attend a univ never thought were possible. Sincerely, duate student al veterinary science undergra A proud Cal Poly Pomona anim Catherine Pabst
Megan Ducey-Hardos I am a student at Cal Poly Pomona majoring in animal health science. Like you, throughout my life, I have had a passion for helping animals. My passion has led me to pursue a career in the animal health science field as a veterinary technologist. When I was in high school, I had the same opinion you did about animal research. I thought it was horrible practice that was torturing animals. After visiting our university research facility, I realized my current opinion of this field was very inaccurate. The animals at these facilities were being humanely treated and were given an enriched habitat. During my sophomore year of college my mother was diagnosed with breast cancer. It is hard to see someone you love go through the pain of having a debilitating and often fatal disease. After seeing my mother go through the treatments and therapies, I realized that animal research right now is necessary to find cures, treatments, and therapies for diseases. If it wasn’t for animal research, my mother wouldn’t be here today. I hope for the day that we don’t have to use animals but right now, animal research is needed for the testing of biomedical drugs and studies on certain diseases. When I graduate, I want to pursue a career as a research animal manager to promote the humane treatment of laboratory animals of all species and models. Currently, there are several research projects being conducted at our University that can only be performed in animals. Our IACUC always looks at alternatives and utilized the 3 R’s when reviewing a protocol. If the research can be performed in vitro, they won’t use laboratory animals. Two research projects can only use animals and they could discover the treatments and therapies for Huntington’s disease and GABA transport membrane related diseases. One research project uses Xenopus laevis to study GABA transport membranes. In this research, researchers surgically remove oocytes from female Xenopus laevis frogs. The oocytes are observed under a freeze-fracture and electron microscope. The goal is to see what does or doesn’t affect the GABA transport membrane. Pharmaceutical companies can use the information from this study to develop drugs for illnesses like Huntington’s and Parkinson’s disease. Oocytes of humans can’t be used for this study because they are too small to visualize and study. Frogs have bigger oocytes than any other animal. Frogs are not harmed in the research or tortured. These frog are anesthetized during the procedure and don’t feel any pain from the egg removal. These frogs receive a great degree of enrichment and a wide range of food.
Established in 1981, FBR is the nation’s oldest and largest organization devoted to educating the public about the essential role of biomedical research in the quest for medical advancements, treatments and cures for both humans and animals. For more information, visit fbresearch.org.
The world of animal rese in its early days, anim arch has come a long al research not necess did have cr way since its first util arily in the ization. It is u best intere come a lo true, st of the ani el and unethical pra ng way sinc ctices that mal or hum e the days could be sn wer an particip where dog atched up ants. But w e s, cats, mic off the stre e have e, frogs, o et and exp r any other Thanks to erimented animal various and on at the w numerous him of the environmen interest gro scientist. tal ups, govern animals are policies, animals in ment agen research a even treate cies, and re treated d better th Research a better than an p nim ever. Many need is wha als are provided with ersonal pets that peo ple keep in t is being ex every need th they could amined, a people are t every hou possibly ha eir homes. still able to r of the da ve, unless get a hold and officia y. tha Ev of any num ls coming to make su ber of anim en with adoption pro t re cesses, a ls th a o nd se animals are there are no In research being trea facilities th inspections ted proper roughout th researcher ly. e United St even can g ates, Cal Po The second research project that is performed at our University Research et money to have to go ly Pomona lo th o ro k u a g we t research, h this In ca studies. mountains included, b talogs in ea Facility utilizes mice for Huntington’s disease animal rese of paperw efore a rnest to pu archers are ork and get rchase ani study the huntingtin protein in the cardiac and skeletal muscles of mice. likely deter mouse, let mals, they so m a re a ny d lo b ne a y p p th a are disease ro e Huntington’s m with patients va resemble sh that o R6/2 mice models re complic ls, many w process is ated and le eer amount of work ould-be necessary it takes to ss readily a not only to utilized in this research. At around 12 weeks mice will display the first in the effort get even a cc w ee ep d it te ta out those p d research kes to get stages of the disease which is weight and muscle loss. The researchers been cons eople who a a research idered for are not seri nimal. This plan ap every poss care about the welfare and the pain intensity each mouse experiences. ous enoug during the ible scenari proved, but to ensure h to put co u rs e of the rese o that cou that every When mice display the first stages of the disease, they are humanely ld happen arch. angle has to Re a animal if ny necessary is se euthanasia believes organization o Your a euthanized. f the anima rchers do no ls t want to p They go th ut their ani is experience incurable pain or diseases. We also agree that our rough extr m a ls a in any more ordinary p they work laboratory animals should be humanely euthanized and should not recautions with have and protoco pain then they abso the highest results. If a lutely have experience the later stages of Huntington’s disease. Once euthanized, ls ju quality of lif n animal w to. e they possib st to make sure that er e to fall ill induced th the mouse’s cardiac and skeletal muscles samples are removed and the animals ly can while during the at is, they w st co o ill u u a g ld rs ne into et currents e electrical b inject to tin st used are o e ectrodes he f g accurate obtained.Microel immediate sia and ana ly treated a a study, an illness th lgesia proto to the stud at was not s much as mice muscle fibers. In this research, we found that this disease changes cols to alle y are used th ey vi co a te in uld discomfort ord and discom membrane potential in muscles and in addition reduces chloride and pain th . All appropriate fort are a ne er to make them m at may be ore comfo were not a cessary pa channels. This research can help find treatments and therapies for rtable. Unf necessary rt of the re part of ever ortunately, search pro yday life fo would not pain, illness cess. If pa this debilitating disease. r many peo be necessa , in , ple, and a ry. nimals, ma illness, and discomfo Animal test ny areas o rt f research ing is not a ll about po the sake o king and p f curiosity. rodd Ther sources fo r a problem e has to be a justifia ing around and seei ng how thin ble need a to be solved serves not nd sufficien gs work fo only to help in order fo r t desire fro r research the biomed mechanics m funding proposals ical comm as well. W ev u en ith ni ty, but ana out anima viruses and to b e co ns l research, tomy, physi idered. It bacteria a we would ology, and nd how we or many tr not disease treat and p eatments. revent them know what we know With anim thalidomid als testing, e, or even tragedies lik . We would not have now about Agent Ora vaccinatio e those tha ng e, ca n ns t followed b The sacrifi e avoided. the release ces that ani of mals make that is alive in the nam today, will e of scienc be alive in that at one e are ones the future, point in tim that help ev and e animal te that it is to ery person sting was no people who are no day, but tim w decease es have ch t the highly regarded a anged. Ani re t integral p g u la ted and offi d. It is true mals in rese arts of the the treatm ciated pro arch are no research p ents that th cess ro w respecte ey receive cess, They are experie d are carefu a re ncing the b no t a bused or m and lly reviewed est quality are either istreated. A and docum of life as p nor practic ll ented to en ossible. Fo al or not p body that a sure that th r most are ossible. Ani rtificial mo ey a s m o f research als provide dels simply su an insight can’t. to the work bstitutions ings of the Spring/Summer 2013 ResearchSaves.org 39
University of Rochester
CANCER CURIOSITY: Gorbunova and Seluanov focused on the biology of cancer after noticing that rates of the disease vary widely across a set of 15 species of rodents that included beavers (inset).
University of Rochester
MYSTERY of an Antıcancer Mechanism A genetic twist in a rarely studied animal may have big implications for the fight against cancer in humans. by Jonathan Sherwood ‘04 (MA), ‘09S (MBA)
hone calls at 3 a.m. rarely involve good news. Especially if the caller is a man toting a firearm. But to Vera Gorbunova, an associate professor of biology, the call that woke her was a welcome one. On the phone was a hunter fresh from the swamps of the Montezuma National Wildlife Refuge near Cayuga Lake, east of Rochester.
The hunter knew that the Rochester professor was on the lookout for hard-to-come-by rodents. And beavers fit the bill. Normally protected from hunting and trapping in New York, the mostly nocturnal animals are considered pests in the wildlife refuge because of the damage they can do to the swamp’s white oak and birch trees. Beavers were among the 15 rodents Gorbunova was studying to investigate a hunch she had about cancer. Why, she had asked, can a squirrel live nearly two de-cades—well into the golden years for a relatively small mammal—and not show signs of cancer? Yet mice, if they manage to live past two years old, often succumb to the disease? Is it possible, Gorbunova wondered, that some rodents have ways to protect against cancer that are completely unknown to humans? ➤
University of Rochester
“Selecting the rodents was easy, but getting the tissue samples from each of them was much, much harder. Some of the rodents, like the beaver, are protected species. Some don’t exist in North America. Some weigh more than a hundred pounds. It’s not like you can order them out of a catalog. It took us more than a year of calling and e-mailing all sorts of people to find all the rodents we needed.” “We know that some species of rodents live to an extreme old age and don’t seem to get cancer,” say Gorbunova. “Do some rodents have an undiscovered anticancer mechanism? If so, what are the implications for fighting cancer in humans?” This fall, Gorbunova may have found an intriguing answer in one of the stranger rodents on her list—the east African naked mole rat, mice-like creatures that spend their lives underground in highly social colonies. Naked mole rats can live up to 28 years, the longest lifespan of any rodent, yet they have never been observed to develop cancer. In a paper published in the Proceedings of the National Academy of Sciences, Gorbunova reported that naked mole rats seem to have a genetic ability to stop cells from replicating if too many crowd together. And runaway cellular replication, she notes, is the very definition of cancer. “Gorbunova has put her finger on the mechanism that gives a dramatic cancer resistance to this rodent,” says John Sedivy, a professor of biology and medical science at Brown University. “Her work elegantly demonstrates the value of studying ‘unusual’ animals because this mechanism simply does not appear to exist in mice.”
Searching for such anticancer mechanisms in animals that are naturally long-lived has been the goal of Gorbunova and her husband and long-time collaborator, Andrei Seluanov, an assistant professor of biology, since they arrived at the University in 2004. Attracted by Rochester’s unusually strong combination of both molecular and evolutionary biology, the pair set up a lab in Hutchison Hall, where they now oversee a research team that includes a halfdozen graduate students and a half-dozen undergraduates.
One of only a few research groups across the country to study the seemingly “cancer-proof” naked mole rats, the team focuses on the role of telomerase, an enzyme that plays a key role in cellular replication that has been an important vein of cancer research over the past 25 years. (The team that discovered the enzyme’s role in 1985 received the Nobel Prize in Medicine and Physiology in December.) Something like a molecular housekeeper, telomerase makes sure that the ends of chromosomes—brief sections of DNA called telomeres— stay intact. As each cell divides, its telomeres slowly shorten, eventually resulting in the death of the cell. Without telomerase, the telomeres would shorten much sooner. If telomerase were to act just right, cells conceivably could reproduce forever. For Gorbunova, as with most cancer researchers, studying the disease traditionally has meant focusing on one of two models: mice or humans. A key difference between the two organisms is that in mice, telomerase is very active, allowing cells to reproduce quickly. In humans, telomerase is much less active. On the plus side, that means mice heal from injuries far faster than humans do.But there’s a downside—increased cancer risk as unwanted cells reproduce quickly and indefinitely.
ecause telomerase allows cells to reproduce very quickly, biologists had long assumed that the reason humans suppress the action of telomerase and mice don’t is that mice live on average only about two years. Their risk of getting cancer is low because they’re not likely to live long enough to get the disease. But humans live for 80 years, plenty of time to develop a few cells that will become cancerous. Gorbunova, however, didn’t accept that explanation. In tests of several closely related rodents that varied greatly in lifespan, she explored whether longer-lived animals suppressed their telomerase more than shorter-lived ones.
The list included regular mice, squirrels, otters, and gerbils, as well as more exotic animals such as giant capybara from South America, chinchillas, and the naked mole rat. s Gorbunova studied the tissue samples over the years, she was surprised to find no correlation between how long a rodent lived and the action of its telomerase. Some animals, such as the naked mole rat, lived nearly three decades yet expressed as much telomerase as a regular mouse.
Instead, another correlation came to light—body mass. Larger animals, like humans and capybara, simply have more cells that can become cancerous, and so telomerase is suppressed to reduce the chance that any particular cell will set off a tumorous cascade. But what about the naked mole rat? Despite their long lives and the large numbers of naked mole rats under observation, there has never been a single recorded case of a mole rat contracting cancer, says Gorbunova. Adding to the mystery is the fact that mole rats appear to age very little until the very end of their lives.
That early contact inhibition was so pronounced that when Gorbunova’s team mutated cells to induce a tumor, the growth of cells in the naked mole rats barely changed, whereas mouse cells became fully cancerous. “We think we’ve found the reason these mole rats don’t get cancer, and it’s a bit of a surprise,” says Gorbunova. “It’s very early to speculate about the implications, but if the effect of early contact inhibition can be simulated in humans we might have a way to halt cancer before it starts.”
University of Rochester
“Selecting the rodents was easy, but getting the tissue samples from each of them was much, much harder,” says Gorbunova. “Some of the rodents, like the beaver, are protected species. Some don’t exist in North America. Some weigh more than a hundred pounds. It’s not like you can order them out of a catalog. It took us more than a year of calling and e-mailing all sorts of people to find all the rodents we needed.”
The key, according to Gorbunova, can be found in the action of a gene known as p16, which, in naked mole rats, triggers an early anticancer mechanism that appears to tell the cells to stop replicating. As in many animals, including humans, the mole rats also have a gene called p27 that limits how many cells can crowd together. “In humans and mice, p16 doesn’t play a major role in contact inhibition, but in the naked mole rat p16 gets activated when the cells just begin crowding and arrests cell proliferation,” Gorbunova says. “Cancer cells tend to find ways around p27, but mole rats have a double barrier that a cell must overcome before it can grow uncontrollably. “We believe the additional layer of protection conferred by this twotiered contact inhibition contributes to the remarkable tumor resistance of the naked mole rat,” says Gorbunova. Gorbunova and Seluanov plan to delve deeper into the mole rat’s genetics to see if the animals’ cancer resistance might be applicable to humans.
ANTICANCER KEY? Despite a long lifespan, naked mole rats (this page) have never been observed to have cancer, a disease that occurs in varying rates in squirrels, capybaras, otters (opposite), and other rodents, according to Gorbunova. When Gorbunova and her team began investigating mole rat cells, they were surprised at how difficult it was to grow the cells in the lab. The cells simply refused to replicate once a certain number occupied a space. The mole rats seemed to be able to turn the process of replication off regardless of the action of telomerase.
“The approach is promising as humans also have the p16 gene, but it plays a different role in anticancer protection,” says Gorbunova. “When we learn more about the differences between the gene in humans and naked mole rats we may learn how to activate the earlier protection in human cells.
Other cells, including human cells, also cease replication when their populations become too dense, but the mole rat cells were reaching their limit much earlier than those of other animals. Gorbunova and Seluanov have named the phenomenon “early contact inhibition.”
“It’s also important to study other genes involved in cell-to-cell contact in order to understand how early contact inhibition can serve to cure or even to prevent human cancer.”
“Since cancer is basically runaway cell replication, we realized that whatever was doing this was probably the same thing that prevented cancer from ever getting started in the mole rats,” says Gorbunova.
Jonathan Sherwood ‘04 (MA), ‘09S (MBA) is a senior science writer for University Communications.
National Institutes of Health
AFTER 4 YEARS OF MEDICAL SCHOOL AND 2 YEARS of training to be a brain surgeon, Kevin Tracey had learned to control the emotions he felt for his patients. But everything changed on May 3, 1985, when he met Janice. As part of his training, Tracey was working in the emergency room at Cornell University’s hospital in New York City. At 6:45 p.m., paramedics brought in an 11monthold female with severe burns. Tracey examined her and noted: burns on 75 percent of her body, no broken bones, no other injuries. As he focused on his patient’s needs, Tracey held back tears. Janice, who arrived wrapped in a teddy bear blanket, now writhed and sobbed on a metal gurney. Her oncesoft skin was seared and peeling from her arms, legs and back. She had a 25 percent chance of surviving. Just an hour before, Janice had been giggling on the kitchen floor while her grandma cooked spaghetti. When the grandma turned to drain a 10quart pot of boiling noodles, she tripped and spilled the 212degree water all over the baby. Sweet chuckles turned to inconsolable screams. For the next 3 weeks, Tracey and others watched Janice ride a rollercoaster of recoveries and setbacks. First, she went into a 5hour surgery, where Tracey and his colleagues removed scalded skin and replaced it with thin layers shaved from Janice’s bottom—an area that had been protected by her diaper. Everyone—her family and her medical team—sighed with relief once Janice opened her eyes and smiled. The next night, though, her blood pressure dropped dangerously low, starving her brain, kidneys, lungs and other vital systems of muchneeded oxygen. Her body was in septic shock. Tracey immediately pumped fluids and drugs into her veins to raise her blood pressure and prevent permanent tissue damage.
– Photo by ADAM COOPER
Despite these measures, Janice drifted into a coma. The next day, her organs began working again and she awoke. But then she spiked a fever and her body swelled. Her kidneys—and soon her liver and other organs—stopped working. Janice was experiencing widespread inflammation, a lifethreatening condition called sepsis. As before, Tracey’s infusion brought her back to life.
For Janice: On May 28, Tracey celebrated with her family as Janice turned 1 year old. Instead of machines and monitors, balloons and streamers now filled her room. But the very next day, Janice’s heart stopped. This time, no medical feat could bring her back.
A New Path Stunned by her sudden, unexpected death, Tracey set out to understand what caused it and to prevent the same thing from happening to anyone else.
You can tell when your immune system is working because you get a fever, swollen lymph nodes in your neck, redness around a wound or even a rash or hives. These are the hallmarks of inflammation, an immune response designed to kill foreign invaders. But for Janice, it had a different effect. Her immune system had begun to destroy the very thing it was supposed to protect. Tracey set out to discover why and how.
During the next 2 years, he put his neurosurgery career on hold and focused on medical research.
Tracey’s mentor and others had just identified a type of cytokine abbreviated TNF. Their research in mice suggested that TNF might play a role in infection.
“Most doctors have a patient who affects their lives, who they really wish they could have done something more for,” says Tracey. “Janice is my patient, and she has a lot to do with the path I’ve been on ever since.”
Wondering whether TNF had been involved in Janice’s case, Tracey injected rats with the cytokine. Almost immediately, their blood pressure plummeted and they went into septic shock.
Tracey was particularly curious to figure out why Janice’s blood pressure had dropped so low. He knew her condition stemmed from septic shock, when the body’s immune system reacts violently to a bacterial infection. But he had run all sorts of blood tests to locate an infection and had never found one. If there was no infection, then what caused her immune system to rage out of control? The answer could overturn centuries of thinking about what makes us sick.
Defense, Defense, Defense The immune system is the body’s natural defense against viruses, bacteria and other invaders. Its army is made up of more than 15 different types of white blood cells that produce molecules to defend and protect our bodies. Some white blood cells produce proteins called antibodies that bind to particular invaders and disable their actions. Others make proteins called cytokines that, when released into an infected area, help heal wounds and repair damaged tissue.
National Institutes of Health
Legacy of a Short Life BY EMILY CARLSON
now used to treat inflammationtriggered arthritis in millions of people. Ulf Andersson, a doctor in Sweden, says he had been waiting decades for a medicine like this. He treats kids with inflammatory conditions like juvenile arthritis. When they come to him, many of his patients ache from inflamed joints in their arms and legs, making it difficult for them to move and even grow. The drug can dramatically improve the quality of their lives, Andersson reports. “I have children who have been in wheelchairs for years who now play soccer again.”
Like Janice, the rats had a high white blood cell count, suggesting infection. But again, there was no bacterial infection— just an excess of TNF. These experiments convinced Tracey that too much TNF can cause septic shock in rats. He further reasoned that, since rats and people are biologically similar, TNF probably does the same thing in humans. Committed to finding a better treatment for patients, Tracey and his team created an antibody that could latch onto and immobilize TNF. It worked in laboratory dishes, but could it soak up excess TNF in living organisms? Could it stop septic shock, preventing harm to healthy organs and tissues? To find out, the scientists tested the antibody in baboons whose bloodstreams were filled with live bacteria, a condition known to cause septic shock. Bingo! The antibody protected the animals. While the antiTNF antibody was never developed into a drug, other scientists built on Tracey’s work and soon developed antiTNF medicines that are
White blood cells, part of the immune system, protect us from viruses, bacteria and other invaders. – IMAGE COURTESY JIM EHRMAN, DIGITAL MICROSCOPY FACILITY, MOUNT ALLISON UNIVERSITY
Compassion Cures Andersson describes Tracey not only as a scientific collaborator but also as a close friend. “I admire his human character as much as his genius,” Andersson says. After Andersson’s wife died, Tracey flew from New York to Sweden to cheer up his friend. In the dead of winter, they put on special long skates and glided across parts of the Baltic Sea. Tracey’s comfort and support, says Andersson, brought him back to life. ➤
National Institutes of Health
Her immune system had begun to destroy the very thing it was supposed to protect. Andersson returns the favor twice a year, spending a week with Tracey, his wife and their four daughters.
He also liked caring for people, which steered him toward medicine.
“It is really amazing how he finds the time to perform firstclass science and manage to be the best father and husband I have ever seen,” says Andersson, who admits to copying some of his friend’s family skills with his own children.
Eventually, he decided to become a neurosurgeon—a specialty that allowed him to split his time between the operating room and the research lab. This dual career, he says, made him both a better doctor and a better scientist.
Tracey attends each of his daughters’ soccer games and says he would attend every single practice if he could. He wants them to have fun, follow their passions and gain confidence in all that they do.
“Being a doctor is gratifying in part because of the experience that comes from helping one person at a time,” he says. “But I was always excited about the possibility of discovering something that could help many, many people.”
“Kevin is at his peak performance when he runs along the sidelines providing advice about how to play,” says Andersson. “As an American, his understanding of soccer equals his lack of skill on the skates,” he jokes.
Also, he says, “Doing science is just addictive. It is more like a hobby than a job.”
“We all like being on the water,” Tracey says. “A couple of hours feel like 2 days off.”
The Octopus and the O.R. Tracey decided on a career path when he was just 5 years old, right after his mother died. Sitting on his grandpa’s lap, he asked what had happened to her and why. His grandpa said she had a tumor that, like an octopus, had spread its tentacles throughout her brain, making it impossible to remove. Right then, Tracey said he wanted to be a scientist so other kids didn’t have to suffer like him.
“For every 100 questions, there are another 100 questions. The churning of these questions and the development of new ideas are what drive scientific progress.”
Surprise in the Lab In 2000, Tracey left his medical practice to devote all his energy to the lab. He turned his attention again to creating an antiTNF drug. He and his team developed a chemical called CNI1493. This compound, which could switch off TNF production, had the potential to treat cytokinerelated disorders from sepsis and arthritis to stroke and digestive diseases.
– Photo by ADAM COOPER
For Tracey and coworkers, lively discussions often lead to new questions to explore and answer. 46
For every 100 questions, there are another 100 questions. electrical device to activate the vagus nerve of rats. Within seconds, the animals produced less TNF.
That’s weird, Tracey thought. Why would something designed to target the immune system have an effect in the nervous system?
Since then, he has shown that in animals, stimulating the vagus nerve can block arthritis, sepsis, shock, heart failure and inflammation of the colon and pancreas.
He discovered that CNI1493 tells brain cells to activate a particular nerve, called the vagus nerve. Once activated, the nerve turns down TNF production.
“The groundwork is being laid to try some of these approaches in humans, perhaps within the next year,” he says.
Legacy of Life
We know that the nervous system controls many important functions —from moving muscles to forming thoughts. But until quite recently, scientists believed that the immune system functioned independently.
When Janice died, Tracey didn’t know exactly what happened or why.
Now it’s clear that the brain and other parts of the nervous system —probably a whole lot of nerves, Tracey suspects—help direct our immune responses.Tracey focused on the vagus nerve. This nerve, which means “wandering” in Latin, regulates our heart rate, digestion and other essential functions. It meanders from the brain stem, across the neck and chest, down into the abdomen and ends up in our internal organs, including the spleen. Like the appendix, the spleen is an organ you probably think little about. But it’s actually a major player in the immune system. “Most of our circulating [white blood] cells pass through the spleen every 5 minutes,” says Tracey.
National Institutes of Health
Tracey knew the substance targeted immune cells. What surprised him was that it had an even more powerful effect on brain cells.
“I had no good explanation for what the molecules in her body were doing,” he says. “I know a tremendous amount more now.” He suspects that the baby’s injury impaired her nervous system, which not only led to an overproduction of TNF but also to her heart failure. He also thinks that too much HMGB1— another type of cytokine discovered in his lab—likely triggered her sepsis. More than 25 years later, Janice’s story still influences Tracey every day. “I tell everyone who will listen never to take anything out of the microwave with a baby in their arms,” he says. “Never put hot food on a tablecloth that a child could pull down.” He created an invisible triangle that fills the space between his
Her short life had a big impact. Thebraincommunicateswiththese white blood cellsvia the vagus. Electrical signals fromthebrainzip down the nerveand trigger itsendingsto release amolecule called acetylcholine into the spleen. When acetylcholine binds to special receptors on white blood cells, the cells stop making TNF. Less TNF means less inflammation.
Sketching Out an Idea Since TNF causes septic shock, Tracey wondered how the brain limits production of the cytokine. To work out the answer, he turned to a trick he often uses to capture his thoughts: sketching. When he first examined Janice, he diagrammed her burns and their severity. When he planned to build his daughters a twostory playhouse, he drew a blueprint on the back of a grocery bag. When he talked to middle school students about being a brain surgeon, he went to the blackboard and drew a drill with a spring to help the kids figure out how to bore through the skull without nicking the brain. Now using a whiteboard in his lab, Tracey sketched the brain, the spleen and the vagus nerve running between them.
kitchen sink, stove and refrigerator. From an early age, his girls— now 7 to 15 years old—knew not to step inside it when unsupervised. He even wrote a book called Fatal Sequence: The Killer Within. The title refers to septic shock caused by the immune system. The book describes Janice’s life, how she changed Tracey’s life and what he and others have learned—and applied to help patients—because of her. Tracey’s book describes Although she didn’t Janice’s life and what he live to see it, her and others have learned short life had a big about septic shock. impact, says Tracey. – Photo courtesy of THE “The people who DANA FOUNDATION receive drugs based either directly or indirectly on knowledge that came [because of] her—they’re her legacy.”
The next day, he set up an experiment to see if, by stimulating the vagus nerve, he could dampen TNF production. He used an Spring/Summer 2013
RESEARCH AVES ResearchSaves.org 818 Connecticut Avenue Suite 900 Washington, DC 20006 202-457-0654 www.FBResearch.org