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University of Pittsburgh

January 12, 2012

Research Notes

Stem cells rejuvenate aging mice

Mice bred to age too quickly lived longer than expected after being injected with stem cell-like progenitor cells derived from the muscle of young, healthy animals. Instead of becoming infirm and dying early as untreated mice did, the animals that got the stem/progenitor cells lived two to three times longer than expected, according to School of Medicine research published in the Jan. 3 edition of Nature Communications.

Previous research has revealed stem cell dysfunction in a variety of tissues in old age, but it has been unclear whether that loss of function contributed to the aging process or was a result of it, explained senior investigators Johnny Huard and Laura Niedernhofer. Huard is a faculty member in the departments of orthopaedic surgery and of microbiology and molecular genetics and director of the Stem Cell Research Center at Pitt and Children’s Hospital. Niedernhofer is a faculty member in the Department of Microbiology and Molecular Genetics and the University of Pittsburgh Cancer Institute (UPCI).

“Our experiments showed that mice that have progeria, a disorder of premature aging, were healthier and lived longer after an injection of stem cells from young, healthy animals,” Niedernhofer said. “That tells us that stem cell dysfunction is a cause of the changes we see with aging.”

Their team examined a stem/progenitor cell population derived from the muscle of progeria mice and found that, compared to those from normal rodents, the cells were fewer in number, did not replicate as often, didn’t differentiate as readily into specialized cells and were impaired in their ability to regenerate damaged muscle. The same defects were discovered in the stem/progenitor cells isolated from very old mice.

Huard said, “We wanted to see if we could rescue these rapidly aging animals, so we injected stem/progenitor cells from young, healthy mice into the abdomens of 17-day-old progeria mice. Typically the progeria mice die at around 21-28 days of age, but the treated animals lived far longer — some even lived beyond 66 days. They also were in better general health.”
As the progeria mice age, they lose muscle mass in their hind limbs, hunch over, tremble and move slowly and awkwardly. Affected mice that got a shot of stem cells just before showing the first signs of aging were more like normal mice, and they grew almost as large. Closer examination showed new blood vessel growth in the brain and muscle, even though the stem/progenitor cells weren’t detected in those tissues.

In fact, the cells didn’t migrate to any particular tissue after injection into the abdomen. “This leads us to think that healthy cells secrete factors to create an environment that helps correct the dysfunction present in the native stem cell population and aged tissue,” Niedernhofer said. “In a culture dish experiment, we put young stem cells close to, but not touching, progeria stem cells, and the unhealthy cells functionally improved.”

Pitt co-authors included Mitra Lavasani, Aiping Lu and Minjung Song, all of the Stem Cell Research Center and the Department of Orthopaedics; Andria Robinson of UPCI and the Graduate School of Public Health; Joseph M. Feduska and Bahar Ahani of the Stem Cell Research Center; Jeremy S. Tilstra and Chelsea H. Feldman of the Department of Microbiology and Molecular Genetics, and Paul D. Robbins of the orthopaedic surgery and microbiology and molecular genetics departments and UPCI.

The project was funded by the National Institutes of Health, the Ellison Medical Foundation, the Henry J. Mankin Endowed Chair at the University and the William F. and Jean W. Donaldson endowed chair at Children’s Hospital.

Precision MRI advances

Quantum computing methods are moving researchers closer to the development of nanoscale magnetic resonance imaging (MRI) instruments that could study the properties of single molecules or small groups of molecules in a noninvasive way.

Faculty member Gurudev Dutt of the Department of Physics and Astronomy said, “Our work shows that quantum computing methods reach beyond pure electronic technologies and can solve problems that, earlier, seemed to be fundamental roadblocks to making progress with high-precision measurements.”

He noted, “Traditional MRI techniques don’t work well with such small volumes, so an instrument must be built to accommodate such high-precision work.”

A significant challenge arose for researchers working on the problem of building such an instrument: How does one measure a magnetic field accurately using the resonance of the single electrons within the diamond crystal?

Resonance — an object’s tendency to oscillate with higher energy at a particular frequency — occurs naturally in such examples as musical instruments, children on swings and pendulum clocks. Resonances are particularly powerful because they allow physicists to make sensitive measurements of quantities like force, mass and electric and magnetic fields, Dutt said. “But they also restrict the maximum field that one can measure accurately.”

In magnetic imaging, this means that physicists can detect only a narrow range of fields from molecules near the sensor’s resonant frequency, making the imaging process more difficult.

“It can be done,” said Dutt, “but it requires very sophisticated image processing and other techniques to understand what one is imaging. Essentially, one must use software to fix the limitations of hardware, and the scans take longer and are harder to interpret.”

Dutt, in conjunction with postdoctoral researcher Ummal Momeen and PhD student Naufer Nusran, has used quantum computing methods to circumvent the hardware limitation to view the entire magnetic field.

By extending the field, they have improved the ratio between maximum detectable field strength and field precision by a factor of 10 compared to the standard technique.

Current methods employed for this kind of study inevitably destroy the samples, but the Pitt team’s research, published in the journal Nature Nanotechnology, puts them one step closer to a future nanoscale MRI instrument that could study properties of molecules, materials and cells in a noninvasive way.

“This would have an immediate impact on our understanding of these molecules, materials or living cells and potentially allow us to create better technologies,” said Dutt, adding that he expects further improvements to be made with additional research.

Future vaccines may target cell proteins

Vaccines with broader reach might be made by stimulating specialized immune cells to recognize foreign cell membrane proteins that are shared across bacterial species, say researchers from the School of Medicine and Children’s Hospital in a report published online in Immunity. The approach could be particularly beneficial in preventing infection by multi-drug resistant organisms.

The genetic heritage of organisms such as oysters, frogs and fish indicate that a family of cell-signaling molecules called interleukin-17 (IL-17) arose in evolution before the advent of T cells, one of the main arms of the immune system in humans.

The human IL-17 gene is turned on in a specialized group of immune cells in the T helper-cell lineage, known as Th17 cells, explained senior author Jay K. Kolls, a faculty member in pediatrics and immunology.

“That development led us to think that perhaps Th17 cells confer some immunological advantage for eliminating infectious organisms beyond the antibody strategy that we typically employ when we make vaccines,” explained Kolls, who also is vice chair for translational research in the hospital’s Department of Pediatrics and director of its Richard King Mellon Foundation Institute for Pediatric Research.

The research team exposed mice to Klebsiella pneumoniae bacteria, a common cause of lung infection, and re-exposed them several weeks after they recovered from the first pneumonia. The presence of the germ in both instances led to increased numbers of Th17 cells in the lungs and spleen. But when IL-17 was blocked, the mice still developed immunity to infection.

The antibody response, which is controlled by B cells, did not require IL-17 to become established. Next, the team infected mice bred to lack B cells, which make antibodies. They found that the animals could become immunized against repeat infection as long as IL-17 was unblocked, allowing Th17 cells to develop an immunological memory of the Klebsiella bacteria.

The researchers determined also that while antibodies react to sugar complexes called polysaccharides in the bacterial coat or capsule, Th17 cells respond to protein complexes in the cell membrane. Those proteins, which are integral to the structure of the cell membrane, tend to be similar across bacterial strains, unlike the capsular polysaccharides, which are variable, Kolls said.

“Some current vaccines require generating a response to a number of these capsular sugars for effective immunization,” he said. “An approach that harnesses the stability of the Th17 cell response to common proteins has the potential to simplify vaccination and provide a broader spectrum of coverage. This strategy may be particularly useful against bacteria that have diverse capsular sugars or multi-drug resistant organisms.”

The team included Pitt co-authors John F. Alcorn and Jeremy P. McAleer of pediatrics.

The project was funded by the U.S. Public Health Service.

Modeling better flu shots

Research that focuses on the composition and timing of flu shot design was published in the September-October issue of Operations Research by industrial engineering faculty members Oleg Prokopyev and Andrew Schaefer in conjunction with Mark Roberts, chair of the Department of Health Policy and Management, and Osman Ozaltin, a recent industrial engineering PhD graduate now on the faculty at the University of Waterloo in Ontario.

Each year the Food and Drug Administration (FDA) must make the complicated decision of choosing which strains to include in that season’s vaccine.

Schaefer noted that the strains in the flu shot now are chosen at least six months before the actual flu season. “This leaves a lot of uncertainty because we’re really not sure which strain will emerge. Our models provide insights into a better flu shot.”

Observing which strains are occurring in other parts of the world improves the accuracy of the selection, but the longer the FDA waits to make the decision, the more likely it is that there will be insufficient vaccine available by the start of the flu season.

The Pitt researchers used optimization methods from engineering to examine whether they could improve the yearly decisions made regarding which strains of influenza should be included in the current year’s vaccine.

Their models allow several policy questions to be addressed, Schaefer said. “For example, incorporating more than three strains might increase the societal benefit substantially, particularly under more severe flu seasons.”

The Pitt team’s models allow examination of the effects of many options, such as how many strains to include in the shot, when to make the final decision, how often the FDA should meet to re-examine information about strains in other parts of the world and the potential benefits from improved production methods.

Flu shot production also is limited by the scarcest strain, as the three strains are combined together to compose the shot. Ozaltin noted, “Our model considers all three strains simultaneously, because unanticipated difficulties in growing a strain might result in reductions in the overall flu shot supply.”

More frequent committee meetings could provide additional gains in the benefit, said Prokopyev.

Amino acid variation linked to ulcerative colitis

Pitt researchers are part of a multi-center team that has pinpointed a variant amino acid in a crucial binding site in a genetic variation associated with ulcerative colitis. Their findings were published online in Genes & Immunity.

Variations in genes that regulate immune responses in a region of chromosome 6 long have been linked with susceptibility for many infectious and chronic inflammatory conditions, including ulcerative colitis, said corresponding author Richard H. Duerr, a faculty member in the Department of Medicine and co-director and scientific director of the UPMC Inflammatory Bowel Disease Center.

Ulcerative colitis is characterized by recurrent inflammation of the large intestine that results in diarrhea mixed with blood and abdominal pain.

“We tested more than 10,000 points, called single nucleotide polymorphisms, or SNPs, in the gene sequence in this chromosomal region, and we also tested amino acid variations in human leukocyte antigen (HLA) proteins that were deduced from the SNPs to identify those most important for ulcerative colitis,” Duerr said. “Refining the gene association signals in this region enabled us to better understand the underlying mechanisms of the disease.”

The authors confirmed that an HLA gene called DRB1, which codes for a protein that is involved in the immune response and routinely is tested in tissue matching for organ transplantation, was uniquely related to ulcerative colitis.

Variation, or polymorphism, in that gene altered which amino acid was selected for the 11th position in the DRB1 protein — a key location because it is in a pocket of the so-called binding cleft where other proteins, such as antigens or markers of foreign cells, attach.

“This particular position probably plays a significant role in determining the human immune response to extracellular antigens,” Duerr said. “It ties into theories that ulcerative colitis might result from an abnormal immune response to gastrointestinal bacterial antigens or might be an autoimmune disorder caused by an abnormal immune response to a self-antigen.”

The researchers also looked for a similar relationship between that amino acid position and Crohn’s disease, another chronic inflammatory bowel condition, but did not find a strong association. Still, variants in immune response genes on chromosome 6 likely contribute not only to ulcerative colitis and Crohn’s disease, but also to other immune-mediated diseases such as rheumatoid arthritis and multiple sclerosis, added first author Jean-Paul Achkarof the Cleveland Clinic Digestive Disease Institute.

Pitt co-authors included Bernard Devlin of psychiatry, R. Scott and Miguel Regueiro of medicine and Mohammad I. Kamboh of the Graduate School of Public Health.

The team included researchers from Children’s Hospital, Carnegie Mellon, Harvard Medical School, the Broad Institute of Harvard, the Massachusetts Institute of Technology and the University Medical Center Utrecht in the Netherlands.

The project was funded by the National Institutes of Health, the Crohn’s & Colitis Foundation of America, the Department of Defense, Kenneth and Jennifer Rainin, Gerald and Nancy Goldberg and Victor and Ellen Cohn.

Texting may reduce young adults’ drinking

Text messaging might be an effective way for health care providers to help young adults reduce heavy drinking, according to a study by School of Medicine researchers.

In findings to be published in the March issue of Alcoholism: Clinical and Experimental Research and available online now, emergency medicine faculty member Brian Suffoletto and colleagues found that using text messaging to collect drinking data and to offer immediate feedback and support to young adults discharged from emergency rooms reduced the number of drinks they consumed and the number of binge-drinking episodes.

Suffoletto and his colleagues designed a randomized, 12-week trial of a text messaging-based program involving 45 people ages 18-24 who were discharged from three local emergency departments (EDs).

The researchers found that almost half of the young adults who were screened indicated hazardous drinking behavior. Those who reported previous treatment for alcohol dependence or current treatment for any psychiatric condition were ineligible for the trial.

Over the course of the study, participants in the assessment and intervention groups reported that they drank an average of 1.6 days per week and a maximum of 3.8 drinks per drinking day.

Participants in both groups received a series of standard, automated text-message queries each week about the frequency of their drinking and quantity consumed. In the intervention group, men who reported more than five drinks during any 24-hour period and women who reported more than four received a text message expressing concern about those levels and asking if the participant would be willing to set a goal to reduce drinking for the week. Those who said yes then received messages expressing positive reinforcement and strategies for cutting down.

Those who refused to set goals received a text message encouraging them to reflect on the decision (for example, “It’s OK to have mixed feelings about reducing your alcohol use. Consider making a list of all the reasons you might want to change.”) The program was based on National Institute on Alcohol Abuse and Alcoholism recommendations for alcohol interventions by a primary care clinician.

At three months, participants who were exposed to the text-message intervention had 3.4 fewer heavy drinking days in the preceding month and 2.1 fewer drinks per drinking day when compared to baseline.

The assessment-only group, however, increased drinking over the course of the study, which is inconsistent with prior studies showing a reduction in drinking in patients that undergo assessments. The researchers speculated that the frequent text messaging might have raised the awareness of alcohol use by the participants and improved the accuracy of their responses.

“Each day in the U.S., more than 50,000 adults ages 18-24 visit hospital emergency departments, and more than a third of them report current alcohol abuse or dependence,” said Suffoletto. The emergency department provides a unique opportunity to screen young adults for drinking problems and to intervene, he noted. However, fewer than 15 percent of EDs at trauma centers have formal screening processes, and only 9 percent offer interventions. “That’s often because clinical staff believe they do not have the time or expertise to discuss substance abuse,” Suffoletto added.

“Because we used an automated computer system, our intervention has the ability to provide text messaging-based feedback and support at large scale with minimal cost,” said Suffoletto.

Co-authors included Clifton Callaway and Jeff Kristan of emergency medicine, Kevin Kraemer of medicine and Duncan B. Clark of psychiatry.

The trial was funded by the Emergency Medicine Foundation with support from the Century Council, an organization supported by distillers dedicated to fighting drunk driving and underage drinking.

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The University Times Research Notes column reports on funding awarded to Pitt researchers as well as findings arising from University research.

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