University Times

Gene therapy approach reverses diabetic neuropathy in animal model

Researchers from Pitt’s School of Medicine have demonstrated for the first time that gene therapy can reverse diabetic neuropathy. While their studies have so far only involved mice, the results are significant because they provide the earliest evidence that such an approach might some day help people with diabetes, in whom neuropathy is a common complication that causes irreversible nerve damage. Details of the research were presented at the 34th Annual Meeting of the Society for Neuroscience, held this week in San Diego.

More than two thirds of patients with diabetes develop neuropathy, which generally affects the sensory neurons within the peripheral nervous system and is characterized by such symptoms as numbness, tingling, pins and needles or loss of sensation, most often to the legs and feet. As a result, patients may be unaware of the presence of ulcers or infections or those they’ve experienced injury. No treatments exist that can stop progression of neuropathy, let alone reverse any damage to nerves.

Five weeks after a one-time inoculation, diabetic animals receiving gene therapy had complete reversal of established peripheral neuropathy and restoration of lost nerve endings to their feet, reported James R. Goss, research assistant professor of molecular genetics and biochemistry at Pitt’s School of Medicine. He and colleagues had previously found the same gene therapy approach could prevent the development of neuropathy in diabetic animals.

While the exact cause of diabetic neuropathy is unknown, there is evidence to suggest it is associated with a deficiency or dysfunction of certain neurotrophic factors, which are proteins essential for the survival and proper function of neurons.

In addition to Goss, other authors included on the abstract are William F. Goins, Darren Wolfe and Joseph Glorioso III, all from the department of molecular genetics and biochemistry at Pitt’s School of Medicine, and Marina Mata and David Fink, formerly of Pitt but now with the University of Michigan.

Development of working memory that allows voluntary control of behavior

A person’s ability to have voluntary control over behavior improves with age because with development, additional brain processes are used, according to scientists at Pitt’s School of Medicine.

The research, presented recently at the Annual Meeting of the Society for Neuroscience, helps to resolve questions about how working memory – a function that allows people to perform tasks as diverse as making toast to solving complex math problems – develops and changes from childhood to adulthood.

“This study gives us a good picture of how our ability to have voluntary control over our behavior using working memory changes and improves with maturity,” said Beatriz Luna, associate professor of psychiatry at Pitt’s School of Medicine.

“Anyone with kids or teenagers knows that they can make irrational decisions when they are under stress,” Luna said. “That is not just because they are trying to be difficult – kids simply are not yet able to access the brain regions that allow adults to react in a more controlled way. What this may mean is that adolescents may be able to act like adults under normal conditions, but under stress they may go back to a more instinctual, less thought-out response.”

Working memory is where the brain stores information used to make immediate calculations, similar to the random access memory (RAM) in a computer. Like RAM, the information stored in working memory is dumped when it is no longer needed. Working memory allows the brain to take in information and create planned responses using abstract thought.

Without it, human behavior would consist mostly of reflexive actions, and humans would not have been able to develop higher mental abilities.

Luna added: “Understanding working memory will inform us about how thinking occurs and how it is linked to other brain processes – and because working memory also is one of the major brain systems impaired in many psychiatric illnesses, understanding these links could inform the development of new treatments.”

Pitt researchers devise algorithm to pinpoint menopause status

A simple algorithm based on hormone levels and other physiological measures may help women’s health researchers pinpoint a patient’s status in her transition to menopause during a single visit to a medical practitioner or study nurse, according to a multi-center study published in the October issue of the Journal of Women’s Health. Additional studies will be required to verify the algorithm before it can be recommended for use as a predictive measure in the general patient population.

Hormone levels fluctuate wildly during the menopausal transition, and knowing a patient’s status can be a valuable tool for determining overall risk for heart disease, stroke, bone loss and diabetes – risks that increase after menopause, according to B. Delia Johnson, a faculty research associate at Pitt’s Graduate School of Public Health and the study’s lead author.

“For all patients, even those who had hysterectomies, our simplified hormonal algorithm produced a 96 percent accuracy overall compared with other methods currently used to determine menopausal status,” Johnson said.

Researchers devised the algorithm using information from 515 women ages 21 to 86 who are participating in the National Heart, Lung and Blood Institute-sponsored four-center Women’s Ischemia Syndrome Evaluation (WISE) study, which is designed to improve the diagnostic reliability of cardiovascular testing in the evaluation of women with heart disease. One of the major objectives of the WISE study is to evaluate the influence of cyclical hormones, menopausal status and blood reproductive hormone levels on cardiovascular physiology, symptoms and diagnostic testing results.

“Although our one-time measure of blood hormones necessarily provides only a snapshot measure, the WISE hormonal algorithm is an estimate that appears to be reasonably accurate and practical,” Dr. Johnson said.

Additional Pitt researchers involved with the project includes Gretchen Gierach, Sheryl Kelsey and Steven Reis.

Pitt researchers report on new DNA repair enzyme

A newly discovered enzyme described by Pitt researchers in a study published online recently, is believed to play a key role in maintaining the integrity of a cell’s genetic information by allowing its DNA to be replicated despite discovery of a mishap on the sequence that it corrects with a new mistake. The research could have implications for treating some cancers.

In the paper posted on the website of The EMBO Journal, an official journal of the European Molecular Biology Organization, the researchers describe how DNA polymerase Q, or POL-Q, has the exceptional ability to bypass damaged spots in the DNA sequence that are caused by a cell’s normal wear and tear or other abuses. In addition, it is the only known enzyme that orchestrates not only one, but also two steps involved in bypassing common types of DNA damage.

POL-Q is one of 15 different DNA polymerases in human cells. These specialized enzymes carry out the duplication, proofreading and repair of DNA. The researchers found POL-Q’s role is to detect late-stage mishaps in the replication process, specifically those that are found at a juncture called the replication fork, just before separation of the copied parent and daughter strand takes place. Rather than stop the process altogether, which would result in the cell not surviving, POL-Q comes to the rescue by performing its two-step handiwork.

“POL-Q’s two-step actions of insertion and extension, essentially the work that would be performed by two enzymes, are the most efficient of any known DNA polymerase. While a mispaired base may in turn result in a mutation after replication, it seems to be a small price to pay for the cell’s survival,” explained Richard D. Wood, professor of pharmacology and the Richard Cyert chair of Molecular Oncology at Pitt’s School of Medicine and leader of the Molecular and Cellular Oncology Program at the University’s Cancer Institute (UPCI).

“Based on what we have learned, our impression of POL-Q is that it does what is necessary when emergency measures are required. One analogy would be duct tape, which in a pinch can be used to mend a torn piece of luggage, for example. POL-Q does what must be done when it encounters a lesion at the DNA replication fork. It’s an efficient strategy in a crisis,” he added.

Other authors of the EMBO Journal paper are Lee Wei Yang, and Ivet Bahar from Pitt’s Center for Computational Biology and Bioinformatics and department of Molecular Genetics and Biochemistry; Anthony Schuffert, of the University’s Cancer Institute, and Chikahide Masutani and Shigenori Iwai, both from Osaka University, Japan.

The research was supported by grants from the National Cancer Institute of the National Institutes of Health and by the U.S.-Japan Cooperative Cancer Research Program of the Japan Society for the Promotion of Science.

Percentage of sex-related web searches down, business searches up

In the last seven years, the percentage of web searches on sex has declined, while that of business-related searches has gone up, according to a new book on web searching coauthored by Amanda Spink, associate professor of information sciences at Pitt.

Spink and her coauthor Bernard J. Jansen of Pennsylvania State University based their book “Web Search: Public Searching of the Web” on studies conducted between 1997 and 2004.

“It’s very difficult to find information on how people are searching the web because web companies are very cagey about giving any information,” Spink said. “Because we were fortunate enough to get data from different web Companies, ours is about the only book that says what people are really doing.”

The research team found that the information people are searching for on the web has changed significantly, moving away from searches on sex-which have declined by almost 50 percent since 1997. Searches on business and e-commerce have increased by 86 percent.

Within this decline in sex-related searches, however, one type of search stood out, said Spink: When people are looking for multimedia (images, audio, or video) on the Web-which generally means pornography-the Number of queries they search for, and their complexity, increases.

“When people are searching for multimedia, they tend to do more queries, particularly if it’s sexually related,” said Spink. “And when people are searching for multimedia, they seem more agreeable to doing more advanced Search features.”

However, when sex isn’t involved in a search, people don’t seem to want to work as much at searching. Since 1997, searches have remained short and simple, with an average of two words per query and two queries per search session. Also, most people won’t look past the first page of results, so companies vie to land a spot for their pages at the top of the list with sponsored links.

People expect search engines to be simple to use and companies market them as such. However, search engines aren’t actually designed that way. “The fundamentals Of most of the retrieval systems used today were developed back in the1950s and 1960s, and a lot of them haven’t changed,” said Spink. “If you look at web search engines, most of them don’t have as extensive Functionality as the electronic library catalogs.

Also, no search engine covers more than 20 percent of the web, even though people think they’re searching the entire Internet, said Spink. Even “meta” search engines like Dogpile, which get results from many search engines at Once, don’t cover the whole Web. As a result, people may not be searching as effectively as they could be.

Exercising limbs protects brain cells affected by Parkinson’s

In an animal model of Parkinson’s, exercise prevents degeneration of nerve cells that are normally impaired or destroyed by the disease, according to Pitt researchers. Based on their work, which was presented recently at the Annual Meeting of the Society for Neuroscience in San Diego, a small pilot study has been initiated in patients with Parkinson’s to determine if regular exercise has an impact on the progression of their disease.

In Parkinson’s, cells in the brain that contain dopamine, a neurotransmitter essential for purposeful and facile muscle control, progressively die until only a small percentage remains. Dopamine carries signals from the nerve cells, or neurons, located deep inside the brain in an area called the substantia nigra along nerve fibers that end in the brain’s striatum, an area involved in control of movement. In the absence of dopamine, neurons can’t send the appropriate messages for smooth motor control, resulting in the telltale symptoms of Parkinson’s: uncontrollable tremors, rigidity of limbs, slow movements and stooped posture.

In one of the studies presented by Annie D. Cohen, a doctoral student in the department of neurology and Center for Neuroscience at Pitt’s School of Medicine, the researchers examined the brains of rats that had been forced to exercise for seven days before receiving a toxin that normally induces Parkinson’s disease. They found that, compared to animals that had not been exercised, significantly fewer dopamine-containing neurons died.

“Whereas a number of explanations could be offered as to why the exercised animals do so well, we have evidence that indicates it’s because exercise stimulates production of key proteins that are important for survival of neurons,” said the study’s senior author, Michael J. Zigmond, professor of neurology, neurobiology and psychiatry, and co-director of the Parkinson’s Disease Center of Excellence at Pitt’s School of Medicine.

Called neurotrophic factors, these proteins protect neurons and promote their survival. According to the researchers’ studies, one particular neurotrophic factor, glial cell line-derived neurotropic factor, or GDNF, is increased with exercise by 40 percent. “GDNF, and probably other factors as well, may help offset the cell’s vulnerability to the effects of oxidative stress from free radical molecules that are produced by the toxin we use in our rat model,” Zigmond explained.

“Whether exercise can reduce the risk of Parkinson’s disease or can slow down its progression are intriguing questions. We are certainly encouraged that in our experimental models we can demonstrate that this sort of forced exercise improves motor function and protects the neurons affected by the disease,” Zigmond added.

As an extension to the animal research, Zigmond has enlisted Anthony DeLitto and colleagues from Pitt’s School of Health and Rehabilitation Sciences to begin a study whereby patients with Parkinson’s disease are enrolled in a 60-minute exercise program that meets three times a week. The study plans to enroll 20 patients in its initial phase.

Other authors of the abstract include Amina El Ayadi and Amanda Smith from the department of neurology at Pitt’s School of Medicine. Related studies also were presented by Niklas Lindgren and Eva Lin, both from Pitt’s department of neurology and Jane E. Cavanaugh of the Pitt’s department of pharmacology. Their research was supported by grants from the National Institute of Neurological Disorders and Stroke, the United States Army, and the Michael J. Fox Foundation.

Researchers develop neural prosthesis that allows monkey to feed itself using only its brain

Pitt researchers have demonstrated that a monkey can feed itself with a robotic arm simply by using signals from its brain, an advance that could enhance prosthetics for people, especially those with spinal cord injuries.

The robotic arm, or neural prosthesis, is about the size of a child’s arm and moves much like a natural arm, with a fully mobile shoulder, elbow and a simple gripper that allows the monkey to grasp and hold food while its own arms are restrained.

The arm is wired into the monkey’s brain and intercepts signals through electrodes attached to tiny probes that tap into neuronal pathways in the motor cortex, a region of the brain responsible for voluntary movement. The neurons’ collective activity is fed through an algorithm developed at the University and then sent to the arm to tell it what direction to go.

“This is a breakthrough in the development of neural prosthetic devices that will someday lead to devices that could help people who are paralyzed or who have lost limbs,” said Andrew Schwartz, professor of neurobiology at Pitt’s School of Medicine and senior researcher on the project.

The research was detailed at the 2004 Annual Meeting of the Society for Neuroscience in San Diego.

According to Schwartz, a part of the brain that controls movement, called the primary motor cortex, contains neurons that fire like a Geiger counter in different directions. The direction to which a neuron fires fastest is called its “preferred direction.” Many motor cortical cells change their firing rate for each movement, and this activity from the many neurons is routed through the spinal cord to different muscle groups to generate movement.

It takes thousands of neurons firing in concert to allow even the most simple of movements, and it would be impossible to tap into all of them, so the Pitt team developed an algorithm to fill in the missing neuron signals, allowing them to get a useable signal from a manageable number of electrodes. The algorithm they developed to decode the cortical signals acts like a voting machine by using each cell’s preferred direction as a label and taking a continuous tally of the population throughout the intended movement.

Monkeys were trained to reach for targets, and once the electrodes were in place, the algorithm was adjusted while the arms were restrained to assume the animal was intending to reach for targets.

“Each cell is movement-sensitive and has a preferred direction, and each cell’s preferred direction is like a vote,” said Chance Spalding, a bioengineering graduate student in Schwartz’s lab who presented the findings. “When all of the votes are added up it gives us the population vector.” These population vectors accurately predict the velocity of normal arm movement, and in the case of this prosthetic, serve as the control signal to convey the monkey’s intention to the prosthetic arm.

“The next step with this device is to add realistic hand and finger movement,” said Meel Velliste, a postdoctoral fellow in the Schwartz lab. “This presents quite a challenge because there are hundreds of different subtle movements we make with our hands and we will need to interpret all of them.”

The arm was developed by the Pitt researchers and custom-built by Keshen Prosthetics in Shanghai, China. The software that controls the arm was developed at Pitt and Arizona State University. Modifications to the original arm were made at the Robotics Institute at Carnegie Mellon University.

In addition to Schwartz and Velliste and Spalding, other authors include Beada Jarosiewicz and Gordon Kirkwood, both from the University.

When early-life stress occurs determines its impact later

Significant stress early in life can have varying lifelong impacts depending on the timing of the stress exposure, according to a report from scientists at Pitt, Oregon Health & Science University (OHSU), Oregon National Primate Research Center and Emory University. The impact can become even more profound when coupled with stress in adulthood, the research also demonstrates.

In a related but separate study, Pitt and OHSU researchers found the impact of early life stress is counteracted if therapies are initiated at specific times during development. Both studies were presented recently at the annual meeting of the Society for Neuroscience in San Diego.

“Past research conducted in both humans and animals has already established that significant stresses experienced early in life can cause problems in the development of social skills and behavioral problems that can last throughout childhood and into adulthood. This research shows that is not always the case and is at least partially dependent on the timing of the early life stress,” said Judy Cameron, associate professor of psychiatry at the Pitt’s School of Medicine and a scientist in the divisions of Reproductive Sciences and Neuroscience at the OHSU Oregon National Primate Research Center.

“In humans, behavioral problems can manifest themselves in a number of ways including increased anxiety, antisocial behavior, depression, drug and alcohol abuse and suicide. However, to date, little information has been obtained as to whether the timing of early life stress exposure can be linked to differing outcomes. In addition, few studies have been conducted to determine the best methods for preventing or counteracting the lifelong impacts of such early life stress exposure.”

The first several months of life represent a dramatic period for brain development, especially in the neocortical regions of the brain, which are important in developing social skills. The development of neurons in this region takes place at an extremely accelerated rate during the first few months of life. In addition, important changes in brain chemistry are taking place during this period and rapid changes in brain structure and function make the person or animal more susceptible to environmental impacts.

Both studies presented in San Diego were conducted using non-human primates. Past research has shown that monkeys are excellent models for studying the impacts of early childhood stress. Infant monkey behavioral changes caused by stress are very similar to those witnessed in human infants. In addition, studies using monkeys allow researchers to control factors such as the timing and duration of stress exposure, something not possible in human studies. Scientists also have a greater ability to observe the test subjects. In addition, non-human primate studies allow researchers to eliminate uncontrolled environmental factors that can skew the results of human studies.

Experts use technology, teamwork to save dolphin’s dorsal fin

An expert team of marine mammal veterinarians, medical researchers, cosmetic surgeons and dolphin trainers recently joined forces to apply the latest advances in human regenerative medicine in an attempt to restore a bottlenose dolphin’s damaged dorsal fin.

The procedure on Liko, a three-year-old male dolphin at Dolphin Quest on Hawaii’s Big Island, took place on July 30 and marked the first-ever marine mammal application of extracellular matrix tissue repair. Liko (pronounced Lee-ko) continues to undergo pioneering veterinary light emitting diode (LED) therapy to stimulate tissue growth and regeneration in his injured fin.

Liko sustained a tear at the base of his dorsal (top) fin, likely in a game of “chase” with his dolphin cohorts. While wild dolphins have been observed with similar and more severe lacerations that can result in eventual loss of the dorsal fin, Dolphin Quest veterinarians organized the ground-breaking procedure in an effort to keep as much of Liko’s dorsal fin intact as possible. A dolphin’s dorsal fin consists of soft, cartilage-like tissue.

“Liko’s progress has been fantastic and he’s well on his way to healing completely,” said Stephen Badylak, research professor in the department of surgery at Pitt’s School of Medicine and director of the Center for Pre-Clinical Tissue Engineering at the University’s McGowan Institute for Regenerative Medicine.

Badylak developed the use of extracellular matrix for the repair of soft tissues. Once in place, the matrix, a 3-dimensional scaffold void of cells but with structural and functional proteins still intact, serves to recruit the appropriate cells for tissue remodeling without producing scarring.

The extracellular matrix used in Liko’s procedure was derived from pig urinary bladder and provided by ACell(tm) Inc., which Badylak and his team at Pitt custom-designed for Liko in consultation Dolphin Quest co-presidents.

Updates to Liko’s treatment regimen and his progress in recovery are periodically posted on the Dolphin Quest web site at www.dolphinquest.org.