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Photographs by Ric Evans

Two fly-fishing buddies have much more in common than their love for casting lines. They are research partners who have made a discovery that could be the best tool yet for battling one of the greatest healthcare concerns of the 21st century.

Fishing for Answers

Cindy Gill

Bill Klunk and Chet Mathis

In late fall, the streams of Lake Erie along Pennsylvania’s northwest shoreline fill with steelhead trout. The waters churn with these fish, which are driven upstream by an ancient urge to spawn. Every spawning season, too, the shoreline and stream banks around Erie, Pa., fill with people who are eager to catch steelhead that can weigh up to 20 pounds. These muscular trout, with their flashy chrome scales, can strike hooks hard and drag lines down fast. Sometimes, they break the water’s surface, surging up in a twisting leap as the tackle line whizzes and strains. For fishermen like Bill Klunk and Chet Mathis, catching a mighty steelhead is one of the sport’s great thrills.

It’s snowing lightly as the two drive toward one of their favorite fly-fishing spots. The sun has not yet poked above the nearest hillside. Before stepping out of Mathis’s 4Runner, Klunk glances at the thermometer on the dashboard. It’s 20 degrees. They are prepared for the cold, covered with fleece-lined vests, thick neoprene chest waders, and every other trick of insulation they’ve learned in their years of fishing together. Their metal cleats, strapped to their boots, clatter on the frozen ground as they make their way through the woods and along the icy bank.

Once they’re streamside and their gear is settled, Mathis wades through the shore ice into the shifting waters. He’s fit, with the ruddy face of an outdoorsman, even though Mathis, a professor of radiology in the School of Medicine, spends most days in a cramped office on the ninth floor of Pittsburgh’s UPMC Presbyterian Hospital. He casts his line and squints, watching his homemade, handcrafted fly drift in the current. He keeps the line tight so he can quickly set the hook if a “chromer” strikes.

Just upstream stands Klunk, also patiently watching for a steelhead to take his hook. As a boy, he remembers tramping through the woods of south-central Pennsylvania with his father or school chums, heading for secret fishing holes. These days, though, Klunk, an associate professor of psychiatry in the School of Medicine, spends much of his time in his lab along a narrow corridor near the top of Thomas Detre Hall. He is wiry, with a still-boyish face and just-graying hair. His fishing pack contains handcrafted flies, but also salmon eggs, maggots, and other steelhead cuisine. From time to time, unlike Mathis, he tips his fly with some of this bait, as insurance to get the trouts’ attention. “The way we fish is pretty much how we do research,” says Klunk. “Chet’s the purist, and I do whatever works.”

Bill Klunk with a salmon test subject.

The two don’t get distracted when they’re in a stream. They don’t think about upcoming meetings, or grant deadlines, or their joint research. They just think about what they’re doing, standing knee-deep in water, tending their lines. “We have a rule,” says Klunk. “We can talk about fishing at work but not about work while fishing.” It doesn’t take long before Mathis yells from downstream, “Fish on!” His reel whizzes, and a silver flash rockets out of the water in front of Klunk, who smiles.

In the water and in the lab, their most important shared virtues are patience and persistence. Those are the qualities that have kept them going—for at least a decade—on research that Discover Magazine last year ranked among the top science stories.

Klunk came to Pitt first. He arrived in 1984 for a psychiatry residency, newly minted from an MD-PhD training program in neuroscience at Washington University in St. Louis. During those years of training, he was drawn to geriatric psychiatry, especially intrigued by the aging brain. As a medical student, Klunk saw the effects of brain chemistry gone awry. He found he was most interested in working on research that directly connects bench science in the laboratory with behavioral science in the clinic.

In the clinic, he sees in patients what motivates him to go to the lab. He sees people who are confused about the day of the week, where they live, or the names of their children. People who barely communicate, or communicate nonsense, or both. People who have discarded time, who are displaced within their own homes. People whose bright personalities have been swallowed whole into some inner darkness. What he is seeing is Alzheimer’s disease (AD), which occurs most commonly in those beyond the age of 65.

AD is everyone’s concern. Right now, about 14 million baby boomers are likely to develop the disease because the prevalence of AD increases as we age; and, in general, we’re living longer. “If we don’t find answers soon, it will be devastating on multiple fronts,” says Sheldon Goldberg, president and CEO of the national Alzheimer’s Association. “Left unchecked, it is no exaggeration to say that Alzheimer’s disease will destroy the healthcare system and bankrupt Medicare and Medicaid.”

One of the hallmarks of AD is an accumulation of abnormal protein plaques between the brain’s neurons, a feature first described by the German physician Alois Alzheimer in 1906. In the 1980s, a research team at the University of California-San Diego discovered that the renegade plaques contain an abundance of a peptide—or protein fragment—called beta amyloid. This enables researchers to identify beta amyloid in brain tissue.

The protein plaques are smaller than the tip of a pin. To “see” this errant biology, researchers typically use dyes that attach specifically to beta amyloid in thin slices of brain tissue obtained from an autopsy. Microscopic examination shows that, when applied to brain slices, the dye sticks to and stains beta amyloid deposits, confirming the presence of AD.

At Pitt, Klunk embraced an opportunity to work on a beta amyloid research project. “My clinical training made it obvious to me that we needed a window into the brain of Alzheimer’s disease, some way to measure the pathology in a living person,” he says. If the disease’s presence could finally be seen in patients, then researchers would have a real, tangible target against which to evaluate the effects of new treatments. But there’s a huge difference between targeting beta amlyoid on a microscope slide and safely detecting it in a functioning brain.

In 1987, as a fellow in geriatric neuropsychiatry at Pitt, Klunk considered three different dyes as potential objects of research: Congo red, Thioflavin T, and Thioflavin S. Any one of these dyes can be used in the lab to confirm AD in brain-slice tissue. In his preliminary tests, Congo red seemed to have the best ability to bind with beta amyloid (the abnormal protein fragment) in postmortem brain tissue. It also had the most potential for a variety of chemical manipulations, which would increase the possibility of finding a variation that could be safely injected into humans.

The left two columns are of a brain with AD. The right two columns are of a brain without AD. The color gradient reflects levels of beta amyloid density in the brain.

He and a lab assistant began evaluating the dye. Each time, they started with the compound’s basic chemical backbone and added or changed atoms or properties based on self-generated ideas about how to get Congo red safely into a living brain.

This was only one of Klunk’s many projects, so often it had to be put on hold. But Klunk, who is also director of psychiatry at Pitt’s Alzheimer’s Disease Research Center, always returned to it because he relished the process of playing with chemical structures, moving atoms here and there, to see what might happen. AD’s heartbreaking effects on patients, which he routinely saw in the clinic, added to his resolve.

Meanwhile, Mathis joined the Pitt faculty in 1992, eight years after Klunk, and worked as a senior chemist for the PET Facility at UPMC. PET, or positron emission tomography, is a scanning technology that enables medical specialists to see, on a computer screen, full-color pictures that show how chemical substances are handled and processed within the body. PET tracks the body’s biological metabolism and functions. Other scanning techniques, like CTs and MRIs, show primarily the outlines and densities of the body’s physical structures.

Mathis had been working as a research chemist at California’s Lawrence Berkeley National Laboratory. Like Klunk, he studied chemistry as an undergraduate and never stopped. While earning his PhD at the University of California at Davis, he became intrigued with both medicinal chemistry and nuclear medicine, which led him to his expertise in PET research.

In 1994, Klunk and Mathis met when a colleague thought the beta amyloid project might benefit from Mathis’ PET scanning know-how. Mathis’ first impression of Klunk remains true: “He was very enthusiastic about things, even a little bubbly, which is nice.”

At Berkeley, in collaboration with some AD researchers, Mathis had also turned his attention to Congo red. He had injected the dye (with a radiolabel, required for tracking) into mice to see if it would reach the brain, but it didn’t. With a stack of more pressing projects and collaborations accumulating on his office desk, he put the project aside as a dead end.

In working with Congo red, he and Klunk independently encountered the same problem: a phenomenon called the blood-brain barrier. Formed within the vessels that carry blood to the brain, the barrier is a tight web of cells packed closely together so that potentially harmful substances can’t squeeze through. “It’s difficult to fool,” says Mathis.

In the years before meeting Mathis, Klunk made some headway in understanding the limits and potential of Congo red as a possible biomarker for those living with AD. From time to time, he even thought about what that might mean: a long awaited breakthrough in AD, giving physicians the ability to monitor the disease’s severity and gauge the effects of treatment. But he only had access to standard laboratory equipment and conventional scanning methods, such as MRI, not the window into tracking Congo red derivatives that PET technology offered.

Mathis, on the other hand, managed a team of chemists, including technicians, undergraduate students, postdoctoral fellows, and junior faculty members. As the site’s codirector, he had access to the sophisticated chemical-synthesis capabilities of a world-class PET scanning facility. By collaborating with Mathis, Klunk suddenly had massive scientific firepower. He now had access to a team of about a dozen trained chemists who, by sheer numbers, could synthesize and test new molecules much more efficiently. And, with a PET scanner, the team could track the journey of chemical derivatives into the brain.

Together, Klunk and Mathis began the quest in earnest: how to get Congo-red-like-compounds past the blood-brain barrier without destroying the basic chemical properties that bind the dye to beta amyloid. More importantly, they wanted to find a way to do this in people, not just in a lab.

Mathis, Klunk, and their research team would gather regularly in the PET facility’s conference room and start by drawing on a board the chemical structures of possible new Congo red derivatives. Routinely, they revisited the basic chemical structure of the dye and pushed to find even more derivatives. Yet, after testing 100 or so derivatives, the group still hadn’t managed to get past the brain barrier enough—that is, with a sufficient amount to be useful, to be trackable as a PET imaging agent.

The process was slow. Each synthesis took several days or, sometimes, several weeks, depending on its complexity and the vagaries of the workweek. If the new compound showed promise in the test tube, they’d modify it for PET scanning and evaluate it in animal studies. “We just kept varying things,” says Mathis, “to see what effect the variations would have on the basic properties.” As the two continued to synthesize new derivatives, they did develop a system for more readily determining good candidates. In five or six steps, they could eliminate ones that didn’t breach the brain barrier sufficiently, didn’t bind to beta amyloid efficiently, or didn’t leave the brain, which is crucial for ensuring an effective scanning agent.

All this time, they continued to sandwich the Congo red project among all of their other projects and day-to-day responsibilities. With each small victory, the duo was heartened, but there were long stretches with little success. “That’s the nature of research,” says Mathis.

Perhaps it was their love of fishing that kept them from being frustrated. Most days, it’s simply about being in the stream, enjoying the process, anticipating the hit when the big fish grabs the hook and makes the line scream. Dozens of casts and several hours may pass without a hit. In the lab, too, days, weeks, even months go by without a hit. Still, they fish.

By 1999, they had created more than 200 variations on the theme of Congo red’s chemical essence, but they still weren’t getting enough of the compound into the brain for scanning. They talked about what they should do. Their plan was simple. Keep going. Meanwhile, Yanming Wang, a junior faculty member on Mathis’ research team, was looking for a synthesis project. The duo put him to work. But instead of instructing him to search for more Congo red derivatives, they suggested Wang try Thioflavin T, one of the three dyes Klunk first considered 12 years ago.

“That’s a whole different kind of compound from Congo red,” says Klunk, “but we had learned a lot over the years. We decided to get rid of the positive charge on the dye’s chemical structure.” In so doing, they hoped to sneak the dye past the brain barrier’s electrical radar. “We figured that taking the charge away might give it a better chance of getting into the brain, but we didn’t know if that would ruin its binding to beta amyloid.”

Wang quickly synthesized a few Thioflavin T derivatives without a positive charge. Curious, as always, to see the test results, Mathis went to the lab late one night to find out whether one of the new derivatives breached the brain barrier and bound to beta amyloid.

“All of a sudden, this number came up, and it was much better than I expected,” Mathis recalls. “I thought, ‘This can’t be right.’” The calculations were repeated several times, with the same result. “All I could say was, ‘Wow!’” The derivative was about fivefold better than any other compound they had created. “Lo and behold, just one after another, the properties met our criteria. BAM, BAM, BAM, BAM, BAM.” Mathis called Klunk with the incredibly good news. Fish on!

There was no big celebration for this success. Klunk does admit, though, that when Mathis shared the data with the team, “we were all grins and giggles.” But as soon as the meeting ended, everybody went back to work. “We’re not a too-high or too-low kind of group,” he says.

A Swedish colleague, who was aware of the duo’s research, was eager to see if their discovery could, indeed, detect beta amyloid in AD patients. Klunk and Mathis agreed to send it to him. When the researchers in Uppsala, Sweden, received the refined derivative, they promptly named it PIB for Pittsburgh Compound B.

Several months later, on Valentine’s Day 2002, Klunk and Mathis were waiting for a phone call from Sweden. They were anxious to hear the results from the first attempt to use PIB as a PET scanning agent in patients. When the call came, their Swedish colleague’s first words were: “I think we have something to celebrate.”

Initial tests on 16 Swedish patients confirmed PIB’s ability to reveal beta amyloid deposits. Since then, PIB has been injected for scanning into more than 40 people in Uppsala and at UPMC. These ongoing trials continue to verify that PIB binds to beta amyloid protein, doesn’t interfere with other brain tissue, and clears from the brain without any apparent side effects. In other words, it appears PIB works and is safe.

William Thies, vice president for medical and scientific affairs at the national Alzheimer’s Association, says: “The study clearly demonstrates that we now have a tool to detect one of the hallmarks of Alzheimer’s disease in the brains of living patients. This is a significant advance for Alzheimer's research, and we look forward to the discovery of many possible uses for it.”

Steven DeKosky, director of the University of Pittsburgh’s Alzheimer’s Disease Research Center (ADRC), is also enthusiastic about the possibilities. “The major application of this, at least as we conceive of it right now, would be to speed progress in developing medications to fight the disease,” says DeKosky, who is professor and chair of neurology in the School of Medicine.

And Bradley Hyman—professor of neurology and director of an AD research lab in Harvard University’s Center for Aging, Genetics, and Neurodegeneration—concurs that PIB shows promise in helping to develop drugs that “might someday stop or reverse amyloid deposition,” as well as assist in the clinical diagnosis of patients.

Their optimism is understandable. Until now, physicians had no way to pinpoint when the brains of people with AD begin to change. Prior research suggests that the disease’s abnormal plaques may form before any symptoms are apparent. PIB is an important breakthrough that should help researchers answer long-sought questions about how the disease begins and grows and how to hinder its progress. “If you can pick up the pathology before the clinical symptoms begin, then you’re talking very early diagnosis,” says Klunk. “If you can push the diagnosis earlier, you can get a head start in treatment.”

Essentially, Klunk and Mathis have laid the foundation for a new era in AD research. “This is a tool to answer new questions and ask new questions … not just do what we do better, but do new things,” says Klunk. A compound that enables researchers to “see” the damage in an AD brain will also enable researchers to track the disease’s course, monitor its progress or diminishment, gauge the effects of beta-amyloid-targeted drugs, and generate data that can lead to new treatment, theories, and approaches.

In recent months, Klunk and Mathis, along with DeKosky, have been featured nationally in a PBS television special on AD called The Forgetting. Time magazine, CBS News, The Wall Street Journal, Discover Magazine, USA Today, and other media have also reported on their research. The two of them and Wang are named as co-inventors on the patent for this new technology, held by the University of Pittsburgh, and Pitt has recently signed a licensing agreement with a firm called Amersham Health, headquartered just outside London, to develop PIB and other related derivatives as commercial imaging agents for AD.

PIB remains an object of research. Klunk and Mathis continue to explore the compound’s properties, safety, and effectiveness. Within a year or so, PIB may be used, in a research setting, to evaluate the effectiveness of new drug therapies. If all goes well, it could take several more years before PIB becomes commercially available for diagnostic PET scanning. But for a disease that has no curative treatments and can’t be diagnosed with absolute certainty until an autopsy is performed, PIB offers real hope for a better future for those with AD and their caregivers.

Cindy Gill is a senior editor of Pitt Magazine.

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