University Times

Combination stem cell-gene therapy approach seen as potential CF treatment

Patients with cystic fibrosis (CF) could potentially be treated using their own stem cells that have been manipulated by gene therapy, suggests a study reported recently in the Proceedings of the National Academy of Sciences (PNAS). The authors, who represent five institutions, including Pitt’s School of Medicine and Children’s Hospital of Pittsburgh, demonstrate for the first time that human bone marrow-derived adult stem cells can be coaxed to differentiate into airway epithelial cells and that encoding these cells with the gene that is defective in CF restores an important cellular function essential for keeping the airways clear of mucus and air-borne irritants.

“Our results provide proof of principle that a cell-based therapy using marrow stromal stem cells is both a feasible and promising clinical approach. We plan to further investigate its potential and are hopeful that we can perform a small clinical trial within the next two to three years,” noted senior author Jay K. Kolls, professor of pediatrics, immunology and molecular genetics and biochemistry at the University’s School of Medicine and chief, Division of Pediatric Pulmonology and Laboratory of Lung Immunolgy and Host Defense at Children’s Hospital of Pittsburgh.

CF is the most common, fatal genetic disorder affecting Caucasians and is characterized by a mutation of a gene that sits on the surface of epithelial cells, including those that line the airways in the lungs. The gene, transmembrane conductance regulator (CFTR), is responsible for channeling chloride out of cells, an essential function that influences the level of airway surface liquid. This fluid sits atop the airway cells and provides support for the hair-like projections called cilia that sweep mucus and dirt away from the cell. When the level of liquid is too low, as is the case in CF, mucus builds up and the area becomes an attractive environment for bacteria to colonize and cause infection.

Finding ways to correct the gene defect responsible for CF has been the focus of much research and some clinical studies are even looking at gene therapy approaches. To date, no studies have evaluated the potential of adult stem cells or the use of stem cells in combination with ex vivo gene therapy, whereby the CFTR gene is delivered to the cells in a laboratory setting before their introduction into the patient.

While some studies have suggested the potential for bone marrow derived stem cells to differentiate into airway epithelial cells, Kolls and his co-authors have provided significant proof. Using a method they developed and report in PNAS, stem cells taken from normal subjects and placed in a culture with mature epithelial cells for 14 days differentiated into the specialized cells, taking on their shape and displaying their characteristic protein markers. Importantly, stem cells aspirated from the bone marrow of three CF patients and co-cultured in the same manner also produced airway epithelial cells, but, as expected, were deficient for the CFTR gene. To correct the genetic flaw, the researchers used the Maloney murine leukemia virus as a vector to shuttle the CFTR gene into the stem cells.

A technique called polymerase chain reaction, or PCR, confirmed the gene was expressed in the newly differentiated airway epithelial cells, reported Guoshun Wang of Louisiana State University (LSU), where Kolls previously had worked, and Bruce A. Bunnell of Tulane University, the study’s first authors. Moreover, the gene therapy-treated cells from CF patients had the same developmental potential as bone marrow-derived stem cells from healthy people.

To determine whether the gene therapy actually corrected the CFTR defect, the researchers exposed the cells to a medium containing labeled chloride and a molecule that regulates the intake and secretion of salts and water necessary for cellular function. If the CFTR gene were not functioning properly, the chloride taken in by the cell would not be able to be secreted. Yet, as the authors reported, the CFTR-corrected stem cells secreted significantly more chloride than stem cells from CF patients without the inserted gene.

“Being able to demonstrate that the gene permitted enhanced chloride channeling was critical to determining the potential of the stem cell-based therapy in the clinical setting,” said Kolls.

Although he acknowledges that more work needs to be conducted, including developing a way to identify the specific subpopulation of bone marrow stem cells that are key to successful engraftment, Kolls is optimistic about the possibility that a patient’s own stem cells – which would not be subject to immune system attack – could be manipulated by gene therapy and used successfully as a treatment for CF. The ex vivo approach to gene therapy may offer several advantages, including the ability to screen the cells before actually delivering them to patients. In addition, the virus vector being studied by Kolls is bigger than others being used in trials and has the capacity for both the gene and its accessory apparatus, which may help ensure long-term function.

To further study the potential of the stem cell approach, including in CF patients, Kolls and colleagues from Children’s Hospital, in collaboration with the University of Pittsburgh, are seeking funding from the National Institutes of Health (NIH) to establish a center focusing on the development of adult stem cell-based therapies for diseases affecting the lung, heart and blood vessels.

In addition to Kolls, Wang and Bunnell, other authors of the PNAS study include Richard G. Painter, Nicholas A. Lanson, Jr., and Donna Bertucci, from LSU; Blesilda C. Quiniones, Susan Tom, Jeffrey L. Spees, Alexandra Peister, and Darwin J. Prockop from Tulane; Daniel J. Weiss of the University of Vermont College of Medicine and Vincent G. Valentine of the Ochsner Clinic Foundation in New Orleans. The Louisiana Gene Therapy Research Consortium and the National Heart, Lung and Blood Institute of the NIH supported their research.

New protocol for reducing anti-rejection drugs after intestinal transplants show less rejection

Transplant researchers at the University’s Thomas E. Starzl Transplantation Institute have dramatically improved intestinal transplant graft survival, and reduced rejection and infection rates by successfully using a novel immunosuppression minimization protocol, thus improving patients’ overall quality of life and avoiding the use of several anti-rejection drugs, which can cause serious infections and major complications. Because the intestine is especially prone to rejection and infection, results of this innovative clinical protocol presented recently at the Third International Conference on Immunosuppression at the Manchester Grand Hyatt, San Diego, have the potential to advance the field of organ transplantation.

“Transplant recipients do not want to be overwhelmed with a lot of anti-rejection medications, which can often lead to more complications. The fact that we have been able to significantly reduce the amount of anti-rejection drugs in this group of patients has enabled many of them to live full and productive lives,” said Kareem Abu-Elmagd, professor of surgery at the University’s School of Medicine and director of the Intestinal Rehabilitation and Transplant Center at Pitt’s Medical Center’s (UPMC) Thomas E. Starzl Transplantation Institute and lead author of the study.

Of the 123 small bowel patients (65 intestine, 25 liver/intestine and 33 multivisceral) who received transplants under the immunosuppression minimization protocol since July 2001, 109 recipients are alive with 107 having functioning grafts. One-year survival rate is 96 percent. Sixty-nine percent of patients are taking a single dose of immunosuppression, some once a day, while others twice a week.

The protocol involves giving a one-time pre-transplant dose of either the drug thymoglobulin – a drug that kills and depletes T cells – key immune system cells that target the donor organ, or a similar drug called campath, which depletes both T and B cells, immune system cells involved in organ rejection.

Following transplantation, the 103 patients who received thymoglobulin and the 20 patients who received campath received the standard anti-rejection drug tacrolimus and none of the patients received steroids. Tapering of tacrolimus was attempted after 120 days.

While 43 percent of patients experienced some level of rejection before initial weaning, none showed evidence of chronic rejection. Patients under the campath protocol did slightly better than those treated with thymoglobulin.

Of the 123 intestinal transplants, 55 involved children, while the other 68 were adult cases.

The current success of this novel anti-rejection protocol should allow major improvement in both the long-term efficacy and quality of life after intestinal and multivisceral transplants, according to the researchers.

“This study, which is the first of its kind in the small bowel patient population at the University of Pittsburgh, was intentionally done because the intestine is an organ most capable of producing an immune response. With the success of reducing the anti-rejection drugs in these patients, we recommend this treatment regimen for other patients undergoing abdominal organ transplants (i.e., liver, kidney, pancreas) or thoracic organ transplants (i.e., heart or lung). At UPMC, the protocol has already been applied to the other organ transplant recipients, after the encouraging results with the initial intestinal transplant recipients,” added Abu-Elmagd.

UPMC has more clinical experience with intestinal transplants than any other transplant center in the world. Since May 1990, more than 400 patients have undergone various intestinal transplants at UPMC.

Survival rates also continue to improve each year; approximately 90 percent of patients survive after the first year. The overall five-year patient survival rate is 70 percent and 10-year patient survival rate is 42 percent. An adult and child transplanted more than 14 years ago at the University of Pittsburgh are the world’s longest surviving recipients of a liver-intestine transplant.

SHRS sets up 1st of its kind research center

The University’s School of Health and Rehabilitation Sciences (SHRS) has been awarded a five-year, $4.25 million grant from the federal government’s National Institute of Disability and Rehabilitation Research (NIDRR) to establish the Rehabilitation Engineering Research Center (RERC) on Telerehabilitation. The only research center of its kind, the RERC will study and develop methods, systems and technologies for the remote delivery of rehabilitation services via the information superhighway.

“By creating this center of excellence, we have been afforded the incredible opportunity to invent the state-of-the-art in telerehabilitation,” said David Brienza, director of the RERC and associate professor of rehabilitation science and technology. The research team, led by Brienza and co-director Michael McCue, will take input from the disability community to create a consumer-driven design and usable technology to create a model telerehabilitation system that will be used worldwide.

“The top-notch resources in western Pennsylvania will provide a great contribution to the RERC’s projects,” said McCue, who is an associate professor of rehabilitation science and technology at SHRS. “We are fortunate to have a vibrant technology community and an active disability community; partnered together they will be an invaluable tool in developing cutting-edge programs and technology to solve current service-delivery problems.”

The RERC research team will be partnering with National Science Foundation-sponsored e-Design Center in Pitt’s department of industrial engineering; the Medical Robotics Group at Carnegie Mellon University; Anthrotronix, Inc.; AT Science, Inc., and the Center for Telemedicine Law. Infrastructure will be based on existing, award-winning infrastructure currently in use by the University’s medical center. The Three-Rivers Center for Independent Living will help by providing user input. In addition, researchers will draw on the expertise and resources of their colleagues from the departments of rehabilitation science and technology, health information management and communication science and disorders at SHRS.

“The collective work of our faculty continually strives to make our world more accessible to all people with disabilities. This RERC will enable us to reach out to the disability community in innovative ways,” said Clifford Brubaker, dean of SHRS. “This grant further establishes the University as a leading center in rehabilitation, dedicated to developing and delivering the best in rehabilitation services to the people who need them the most.”

Over 54 million Americans have a disability. Many are isolated from rehabilitation services due to geography or physical limitations. Researchers plan to develop and study the necessary infrastructure and architecture and develop and test programs that will provide for the remote delivery of a comprehensive spectrum of rehabilitation services.

“The community of people with a disability is an underserved population. Without a doubt, people with disabilities face a significant lack of accessible services. Many are isolated geographically because they don’t live close to services and some are isolated physically because they are unable to leave their homes or communities,” said Katherine D. Seelman, associate dean for disability programs at SHRS, and communication director of the grant.

“Telerehabilitation can deliver the needed services in an efficient and effective manner.”

Projects in the RERC aim to help people with disabilities by providing an interface with experts at leading rehabilitation programs like those at Pitt. Experts will provide services including communication therapy for children with disabilities, assessment for wheeled-mobility devices, and accessibility assessment of the home and work environment. Researchers also plan to develop an automated job coach that will prepare people with disabilities to enter the workforce. Health care providers in underserved areas will be able to consult with experts as well, helping to alleviate the shortage of rehabilitation professionals.

Additionally, through the RERC, SHRS plans to take a leadership role in establishing telerehabilitation across the United States by influencing public policy and developing a telerehabilitation curriculum.

NASA may need to change sleep-wake scheduling protocols

New University research shows the human body has difficulty adjusting to dramatic time changes such as those experienced by working shifts or traveling across time zones.

The NASA-funded study, detailed in a recent edition of Aviation, Space and Environmental Medicine, was designed to examine the protocols the space agency uses to assign sleep-wake schedules that ensure astronauts are always able to handle their demanding tasks at peak performance. The findings suggest changes should be made in the way NASA schedules sleep periods on missions, but also have meaning for anyone who has had to deal with a significant time change and still function.

“Many of us find that we have to change our sleep schedule, perhaps to accommodate work or school start times, or a change in our commute time,” said Timothy H. Monk, professor of psychiatry at Pitt’s School of Medicine and lead author. “We often wonder if we should make the change all at once, or more gradually over several days or weeks. This research has the eventual aim of helping us make that decision in the best way possible.”

According to Monk, early in the history of manned space flight, NASA realized that it had to have a method for assigning sleep periods to correspond to astronauts’ biological clock rhythms if they were to get enough rest to do their assignments. “If they scheduled sleep for the wrong time, an astronaut could end up having disrupted and unrefreshing sleep, leaving them feeling tired and irritable, and perhaps more apt to make mistakes.”

Getting the right amount of sleep at the right time is more complicated in space than it is on Earth. On Earth, people are used to having time cues tell their bodies when it is time to sleep or to wake up. The strongest of these is the 24-hour day-night cycle, which comes from the fact that we live and have evolved on a planet with a 24-hour rotation. Like most animals we have a biological clock in our head, which is able to keep time, getting us ready for sleep at night and wakefulness during the day using rhythms with a period of about 24 hours – referred to as circadian rhythms. In orbit, the sunrise-sunset cycle lasts for a mere 90 minutes, and after the absence of the natural 24-hour cycle for three months or more, the biological clock starts to weaken. When the biological clock gets thrown off balance, sleep and alertness suffer.

Complicating the issue is the need for astronauts to be awake and alert to undertake sensitive mission goals – say docking with another vessel – at specific times that may fall during a time at which they are normally asleep.

To reconcile an astronaut’s need for sleep with their busy schedules, NASA originally developed guidelines referred to as “Appendix K.” These guidelines specified how much time had to be set aside for sleep and for the transitions to and from it. It also specified by how much an astronaut’s bedtime could change from one day to the next. It favored “trickling in” changes rather gradually, using phase delays to later bedtimes (by up to 2 hours) wherever possible.

The concept is similar to the terrestrial example of the common traveler’s advice to move one’s bedtime ahead or back a little at a time in the week before an overseas trip to help minimize jet lag.

“The thought was that mission schedulers could trickle in a series of two-hour phase delays without incurring any negative consequences as far as sleep quality and alertness,” Monk said. “However, based on the findings from this experiment, that assumption might be quite wrong.”

The researchers observed participants, who volunteered to spend 16 days on a “mission” at the University’s time isolation facilities, and tested them for alertness, mood and core body temperature – the best standard for assessing circadian rhythms. At the same time they recorded their sleep to assess its duration and quality. The experiment involved a series of nine repeated two-hour delays in bedtime.

During the study, Monk and his colleagues found that the circadian pacemaker did adjust itself – but only by about one hour per night rather than the two hours required by NASA’s protocol. Because of that, subjects eventually experienced a massive flattening in the amplitude of their circadian temperature rhythm indicating that the biological clock was not doing its job properly. This led to significant disruptions in sleep and lowered alertness while awake.

More research needs to be done before scientists can advise NASA on how to change its guidelines.

“There is always some cost to performing tasks when we expect to be asleep, but by the end of the series of experiments, of which this is the first part, we shall be able to advise NASA which approach – gradual delays, gradual advances, all at once – will lead to the least disruption of an astronaut’s sleep and alertness,” Monk said.

Co-authors include Daniel J. Buysse, Bart D. Billy and Jean M. DeGrazia. The National Institute on Aging provided additional research support.

First synthesis of anticancer compound disorazole performed

Pitt researchers have accomplished the first synthesis of the rare natural anticancer compound disorazole, according to a paper published in the November issue of the Journal of the American Chemical Society. This synthesis is critical for the continued study and pharmaceutical development of the substance.

“We are very excited about the potential of this compound,” said Peter Wipf, University professor of chemistry and director of the Combinatorial Chemistry Center and the Center for Chemical Methodologies and Library Development at Pitt, who coauthored the paper with Pitt chemistry graduate student Tom Graham. “Disorazole is among the most potent antiproliferative agents known,” Wipf said.

Disorazole was first identified in 1994 in the fermentation broth of the myxobacterium Sorangium cellulosum, but has been difficult to study in its natural form. Prior to Graham and Wipf’s research, no viable method of synthesizing the compound existed.

Pharmaceutical companies have expressed interest in developing disorazole as an anticancer drug. Wipf and Graham are conducting studies of disorazole’s anticancer potential in collaboration with Billy W. Day, associate professor of pharmaceutical sciences in Pitt’s School of Pharmacy.

The paper is available online at http://pubs.acs.org. This research was supported by a grant from the National Institutes of Health.

Pitt researchers find potential new method to slow spread of breast cancer

Pitt researchers announced recently at the annual meeting of the American Society for Cell Biology that they have found a way to suppress migration of breast cancer cells in vitro. Their findings, which they stressed were “exciting, but preliminary,” could have applications for slowing the spread of breast cancer through the body.

Partha Roy, assistant professor of bioengineering at Pitt, and Kenneth Jacobson, professor of cell and developmental biology at the University of North Carolina, Chapel Hill, found that when breast cancer cells were made to express high levels of the protein profilin, they migrated significantly less than did those with normal levels of the protein.

Previous studies had shown that breast cancer cells that were more invasive-that migrated more quickly throughout the body-had lower levels of profilin than other breast cancer cells. “So we thought, ‘Why don’t we overexpress the protein and see whether we can slow down the migration?’ ” said Roy.

Roy will next determine the best method of slowing cell migration by investigating other ways of perturbing profilin, such as inhibiting its expression. “If you take out the profilin, the cell is probably not going to be able to migrate efficiently, either,” said Roy. “There must be an optimum window of profilin concentration that cells require to migrate efficiently.”

Roy estimates those studies will take a few years, after which his group will begin tests using mice to see whether their cancer metastasizes more slowly if they are injected with cells with or without perturbed profilin.

Depending on the results of the mice studies, the researchers’ findings could be used to design therapeutic interventions for breast cancer.

Drug may cut drinking in alcoholics with bipolar disease

An anticonvulsant drug may help cut alcohol use in patients who have one of the most difficult-to-treat combinations of mental illnesses – bipolar disorder and alcoholism – according to a study done at Pitt’s School of Medicine.

Results of the research, the first randomized controlled trial to date that addresses this difficult problem, are detailed in a recent edition of Archives of General Psychiatry, and show the drug valproate is effective at reducing drinking when combined with a patient’s regular treatment program.

Approximately 61 percent of bipolar patients abuse alcohol or other substances, a higher proportion than in any other psychiatric illness, including schizophrenia, major depression and anxiety disorders, and a rate that is ten times higher than that of the general population.

These patients are at greater risk for suicide and have more profound manic and depressive episodes. In addition, they have a difficult time adhering to treatments, which contributes to a Catch-22 of worsening health and cognitive functioning and increasing incidences of hospitalizations and use of costly psychiatric services.

“Patients with bipolar disorder and alcoholism are in a very precarious situation,” said lead author Ihsan Salloum, associate professor of psychiatry at the University’s School of Medicine. “They have high disability rates and ultimately the presence of these two disorders lead to higher death rates. Substance abuse makes it much more difficult for these individuals to stick with medication regimens. Our results show that valproate could be an important element to getting control of this dangerous combination of illnesses.”

The researchers found that patients who were given valproate in addition to their normal treatments drank less often and drank less overall.

Despite the serious nature of bipolar disorder combined with alcoholism, very few interventions to address both illnesses have been looked at. A number of issues have complicated the effort, including ethical problems relating to taking bipolar patients off of their medications to participate in a research study and problems relating to the difficulty in isolating the effects of a particular treatment on one set of symptoms, such as mania or drinking. This situation has created a serious gap in knowledge of how best to treat patients with both bipolar disorder and an addiction.

This study was designed to address both issues. Patients were kept on their normal medication and therapy routine to avoid the danger of triggering mania or depression, and the study medication was added in a way that would allow the researchers to accurately measure its impact on drinking.

“This finding could have a significant impact on the quality of life for these patients,” said Salloum. “Most treatments for these individuals are designed to concentrate on the bipolar disorder with the hope that the alcoholism will ease as well. While common drugs used to treat bipolar disorder, such as lithium, can have a positive effect on regulating mood and preventing episodes of mania and depression, studies have shown it has no meaningful effect on drinking, a treatment rationale that leaves much to be desired in its effect on the associated, but equally significant illness of alcoholism.”

This study showed that valproate appears to decrease heavy drinking independent of measurable effect on mood state. Researchers believe the drug works by affecting the GABAergic system in the brain, affecting mechanisms involved in reducing both alcohol use and withdrawal symptoms.

Other researchers, all from Pitt, included: Jack R. Cornelius, professor of psychiatry; Dennis C. Daley, associate professor of psychiatry; Levent Kirisci, associate professor of pharmaceutical sciences; Jonathan M. Himmelhoch, professor of psychiatry, and Michael E. Thase, professor of psychiatry.

The study was funded by grants from the National Institute of Alcohol Abuse and Alcoholism and the National Institute of Mental Health.