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University of Pittsburgh

November 8, 2012

Research Notes

Male fertility restored after treatment for cancer

An injection of banked sperm-producing stem cells can restore fertility to male primates who become sterile due to cancer drug side effects, according to researchers at the School of Medicine and Magee-Womens Research Institute (MWRI). In the animal study, which was published in Cell Stem Cell, previously frozen stem cells restored production of sperm that successfully fertilized eggs to produce early embryos.

Some cancer drugs work by destroying rapidly dividing cells. As it is not possible to discriminate between cancer cells and other rapidly dividing cells in the body, the precursor cells involved in making sperm can be wiped out inadvertently, leaving the patient infertile, said senior investigator Kyle Orwig, faculty member in the  Department of Obstetrics, Gynecology and Reproductive Sciences and an MWRI investigator.

“Men can bank sperm before they have cancer treatment if they hope to have biological children later in their lives,” he said. “But that is not an option for young boys who haven’t gone through puberty.”

Even very young boys, though, have spermatogonial stem cells in their testicular tissue that are poised to begin producing sperm during puberty. To see whether it was possible to restore fertility using these cells, Orwig and his team biopsied the testes of prepubertal and adult male macaque monkeys and cryopreserved the cells from the small samples. The monkeys then were treated with chemotherapy agents known to impair fertility.

A few months after chemotherapy treatment, the team re-introduced each monkey’s own spermatogonial stem cells back in to his testes using an ultrasound-guided technique. Sperm production was established from transplanted cells in nine out of 12 adult animals and three out of five prepubertal animals after they reached maturity.

In another test, spermatogonial stem cells from other unrelated monkeys were transplanted into infertile animals, which created sperm with the DNA fingerprint of the donor to allow easy tracking of their origin. In lab tests, sperm from transplant recipients successfully fertilized 81 eggs, leading to embryos that developed to the morula and blastocyst stages, which are the stages that normally precede implantation in the mother’s uterus. Donor parentage was confirmed in seven of the embryos.

“This study demonstrates that spermatogonial stem cells from higher primates can be frozen and thawed without losing their activity, and that they can be transplanted to produce functional sperm that are able to fertilize eggs and give rise to early embryos,” Orwig said.

The findings are encouraging because several centers in the United States and abroad are banking testicular tissue for boys in anticipation that new stem cell-based therapies will be available in the future to help them have their own biological children.

Orwig directs the fertility preservation program, a collaboration between MWRI, Magee-Womens Hospital, Children’s Hospital and the University of Pittsburgh Cancer Institute that offers education and treatment options for children and adults who are at risk of becoming infertile due to medical problems including cancer.

“Many questions remain to be answered,” Orwig noted. “Should we re-introduce the spermatogonial cells as soon as treatment is over, or wait until the patient is considered cured of his disease, or when he is ready to start a family? How do we eliminate the risk of cancer recurrence if we give back untreated cells that might include cancer cells?”

Co-authors of the paper included lead author Brian P. Hermann, David K. Cooper, Angus W. Thomson, Gerald P. Schatten and others from the School of Medicine, as well as collaborators from the Oregon National Primate Research Center, University of California-Davis, ITxM Diagnostics and CaridianBCT.

The project was funded by Magee-Womens Research Institute and Foundation, The Richard King Mellon Foundation and the National Institutes of Health (NIH) Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD).

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Grant funds placental research

NICHD has awarded a $5 million grant to a Pitt project examining the molecular and cellular controls of placental metabolism.

Because the placenta plays a central role in supporting fetal development, metabolic dysfunction of the placenta may hinder fetal growth, and subsequently render the growth-restricted newborn susceptible to a host of childhood and adult diseases, researchers say.

By harnessing the power of new, rapidly evolving mouse genetic and epigenetic technologies, as well as high throughput genomics and lipidomics analyses, the study will strive to answer fundamental systems-based questions in placental biology, and offer a novel view on metabolic pathways that underlie a clinical conundrum.

Yoel Sadovsky, faculty member in obstetrics, gynecology reproductive sciences, microbiology and molecular genetics and the Clinical and Translational Science Institute (CTSI), as well as director of the Magee-Womens Research Institute, is program and project principal investigator. Other PIs are Yaacov Barak, faculty member in of obstetrics, gynecology reproductive sciences, and J. Richard Chaillet, faculty member in microbiology and molecular genetics.

Other Pitt collaborators/co-investigators are Tianjiao Chu, obstetrics, gynecology and reproductive sciences; Valerian Kagan, vice chair, environmental and occupational health (EOH); W. Tony Parks, pathology, and Vladimir Tyurin, EOH.

Also participating are researchers from  Ohio State University, the University of Calgary, McGill University and Mt. Sinai School of Medicine.

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Genomic areas associated with IBD identified

An international team that includes researchers from the School of Medicine has uncovered 71 genomic regions associated with inflammatory bowel disease (IBD), which includes Crohn’s disease and ulcerative colitis. The results, published in the Nov. 1 issue of Nature, increase the number of known IBD-associated genomic regions to 163 and suggest that genes involved in defense against infection also play a key role in IBD.

Crohn’s disease and ulcerative colitis affect as many as 1.4 million people in the United States alone, noted Richard Duerr, faculty member in medicine, co-director and scientific director of the UPMC Inflammatory Bowel Disease Center and one of 12 co-investigators. Both conditions are characterized by chronic inflammation of the intestine, typically leading to diarrhea and abdominal pain, and sometimes rectal bleeding.

“This research was conducted to fill gaps in our understanding of the genetic predisposition and biological pathways leading to Crohn’s disease and ulcerative colitis,” Duerr said. More than 75,000 IBD and control study subjects from 15 countries, as well as 100 scientists and physicians, contributed to the study.

The research team analyzed data from 15 studies on the genetics of IBD, identifying more than 25,000 single nucleotide polymorphisms (SNPs) or genetic variations with at least suggestive evidence for association with either Crohn’s disease, ulcerative colitis or both. That information was followed up by data from an additional set of more than 41,000 IBD and control samples at 11 centers around the world, including the University, to verify that 163 genomic regions, including 71 newly identified ones, are associated with IBD.

Seventy percent of the IBD-associated genomic regions also are home to genetic variants that are associated with other immune-mediated, chronic inflammatory diseases, particularly psoriasis and ankylosing spondylitis, the researchers noted. Also, the IBD-associated genomic regions are enriched with genes linked to immune deficiencies that increase susceptibility to certain infections. The study uncovered pathways shared between responses to mycobacterial infections, such as tuberculosis and leprosy, and those predisposing people to IBD.

“It’s possible that the biological mechanisms intended to protect us from infection go awry and overreact, triggering inflammation that characterizes IBD,” Duerr noted.

The project was funded in part by the National Institute of Diabetes and Digestive and Kidney Diseases, the Crohn’s & Colitis Foundation of America and the International IBD Genetics Consortium.

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Off-label antipsychotic use common in VA nursing homes

More than one in four older veterans residing in U.S. Department of Veterans Affairs (VA) community living centers received antipsychotic medications, and more than 40 percent of those veterans had no documented evidence-based reason for such medications, according to research from Pitt and the VA Pittsburgh Medical Center.

The study found similar rates of antipsychotic use as studies in non-VA nursing homes. The findings will be published in the November issue of the journal Medical Care and are available online.

“Our study adds to the growing evidence base that antipsychotics have been overused in nursing homes, and the VA is not immune to this problem,” said lead author Walid Gellad of the School of Medicine and the Graduate School of Public Health’s Department of Health Policy and Management. “Behavioral symptoms in dementia patients are difficult to treat and, in most cases, nursing home staff are doing what they can to keep patients comfortable and safe. We have to find better ways to do this, though.”

Antipsychotics have limited efficacy in alleviating behavioral problems in dementia patients, and several studies associate their use with an increased risk of mortality.

“The VA already is undertaking several initiatives to address the use of antipsychotics in VA nursing homes, including increasing the availability and integration of psychologist services and piloting behavioral modification programs,,” said Gellad, also a core faculty member with the VA Center for Health Equity Research and Promotion and a primary care physician in the VA Pittsburgh Healthcare System.

Gellad and his colleagues collected data on veterans age 65 and older who were admitted for 90 or more days to one of the 133 VA community living centers between January 2004 and June 2005, the most recent years for which data could be collected.

Of the 3,692 veterans, 948 — or 25.7 percent — received an antipsychotic. Among that group, 59.3 percent had an evidenced-based reason for use. The U.S. Food and Drug Administration (FDA) has approved antipsychotics for use in treating psychiatric illness, such as schizophrenia, bipolar disorder and Tourette syndrome.

Veterans residing in Alzheimer’s or dementia special care units had 66 percent greater odds of receiving an antipsychotic, and residents with aggressive behavior had nearly three times greater odds of receiving an antipsychotic medication. Among those residents with dementia, veterans with no evidence of psychosis for which an antipsychotic would be appropriate were just as likely to receive an antipsychotic as those with documented psychosis.

“We couldn’t determine if clinicians are using antipsychotics in residents with dementia without considering the risks, or whether they are considering the risks but have determined that the behavioral symptoms are sufficiently problematic that the potential benefits outweigh the risks of therapy,” Gellad said. “The VA is supporting efforts for better documentation in patients’ medical records of the risk vs. benefit discussions regarding the use of these antipsychotics.”

Residents receiving multiple medications of any type, particularly those receiving antidepressants, were more likely to receive antipsychotics.

The FDA issued a warning in 2005 for the atypical antipsychotics, emphasizing their association with increased mortality when used for behavioral disorders in elderly residents with dementia. The warning was extended to all antipsychotics in 2008.

“Our data was collected prior to these warnings, so we cannot draw conclusions about whether they make a difference in current practices,” said Gellad. “However, despite the lack of an FDA warning at the time that the data for our study was collected, reports had already appeared in print about the risks of these drugs.”

Co-authors included Sherrie L. Aspinall, pharmacy and therapeutics; Steven M. Handler, biomedical informatics; Roslyn A. Stone, biostatistics; Nicholas Castle, health policy and management; Todd P. Semla; Chester B. Good, Michael J. Fine and Joseph T. Hanlon, all of the Department of Medicine, and Maurice Dysken.

The researchers were supported by a VA Career Development Award and grants from the National Institute of Aging, the National Institute of Mental Health, the National Institute of Nursing Research and the Agency for Healthcare Research and Quality.

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Kids help researchers fight flu

Hundreds of school children on Election Day — a scheduled day off from school — wore proximity sensors to help Pitt researchers learn more about the spread of influenza and the impact of school closures on slowing epidemics.

Dubbed the “Social Mixing and Respiratory Transmission in Schools,” or SMART Schools, study, the deployment is part of a U.S. Centers for Disease Control and Prevention (CDC) effort to create a national policy on school response to flu and pandemics.

Charles Vukotich Jr., senior project manager at the Graduate School of Public Health, said: “This unprecedented study will contribute greatly to our knowledge on influenza outbreaks and what we can do to disrupt their spread.”

On Nov. 5, a proximity sensor — called a “mote” — was sent home with each participating child at Borland Manor elementary and North Strabane intermediate schools in the Canon-McMillan School District. The children wore the motes, which weigh three ounces and are about the size of a beeper, on lanyards all day at school Monday, all day while home on Tuesday, and all day at school Wednesday.

The battery-powered motes send out a weak signal to detect other motes and record when they detect one. The researchers will use the data collected by the motes to determine how often children come in contact with each other.

“We know that children can drive influenza outbreaks, but we don’t know how or why,” said Shanta Zimmer, faculty member in the School of Medicine. “Knowing their interaction and contact patterns will give us much needed real-world data that can be used to conduct research, test hypotheses and run computer simulations.”

The data will allow researchers to investigate whether limiting movement between classes during the school day, increasing vaccination campaigns, instituting educational programs, changing sick leave policies or instituting programs that encourage hand sanitizer use are better interventions for controlling the spread of flu.

This is the second year of the SMART Schools study, and the first time the motes have been sent home with the children on a scheduled day off from school. Last year children in eight schools in the Canon-McMillan School District in Washington County and Propel charter schools in Allegheny County wore the motes during the school day. Elementary, middle and high schools were included.

Preliminary results from last year’s mote deployments showed that each child over all grades interacted with an average of 109 other children during the school day. High school students interacted more than middle school students. The students interacted the most at mid day.

The children also are given diaries to record who they come in contact with during a 24-hour period. This helps the researchers incorporate information about people who might not be wearing the sensors.

In the diary, relationships are identified with generic terms, such as mother, friend or teacher, to maintain confidentiality.

“When the students took the motes home overnight last year, we found that they would still cluster together after school and well into the evening,” Vukotich said. “This provides us some evidence that simply closing schools for a few days will not stop children from interacting with each other.”

Other Pitt researchers participating in the SMART Schools study are Hasan Guclu, Shawn T. Brown and John Grefenstette, all of public health.

Researchers from Johns Hopkins, the University of Liverpool and the CDC also are participating.

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Grant funds research to aid oil extraction

With a $1.3 million grant from the U.S. Department of Energy, Pitt researchers are developing an economical CO2 thickener that could improve crude oil extraction significantly and expand accessible domestic oil reserves.

Current oil-extraction methods in the United States involve oil being “pushed” from underground layers of porous sandstone or limestone reservoirs using a first-water-then-CO2 method. CO2 — which is obtained from natural CO2 reservoirs and piped to oil reservoirs — pushes and dissolves oil from underground layers of porous rock, but its viscosity is too low to extract oil efficiently.

As such, it tends to “finger” through the oil rather than sweep oil forward toward the production well. This process, “viscous fingering,” results in oil production companies recovering only a small fraction of the oil that is in a field.

During the late 1990s, Pitt researchers demonstrated that it was possible to design additives that could greatly enhance CO2’s viscosity, but the compounds were costly and environmentally problematic.

Principal coinvestigator Eric Beckman, George M. Bevier Professor of Engineering in the Swanson School of Engineering, said: “In this proposal, we’re looking at designing candidates that can do the job at a reasonable cost.”

Beckman and Robert Enick, principal co-investigator and Bayer Professor and vice chair for research in the Department of Chemical and Petroleum Engineering, intend to build upon earlier Pitt models of CO2 thickeners, but with a more affordable design.

“An affordable CO2 thickener would represent a transformational advance in enhanced oil recovery,” said Enick. “If a thickener could be identified that could increase the viscosity of the CO2 to a value comparable to that of the oil in the underground layers of rock, then the fingering would be inhibited, the need to inject water would be eliminated and more oil would be recovered more quickly using less CO2.”

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Omega-3 aids working memory in young adults

Pitt researchers have determined that healthy persons ages 18-25 can improve their working memory even further by increasing their omega-3 fatty acid intake. Their findings have been published online in PLOS One.

Bita Moghaddam, project investigator and faculty member in neuroscience, said: “Before seeing this data, I would have said it was impossible to move young healthy individuals above their cognitive best. We found that members of this population can enhance their working memory performance even further, despite their already being at the top of their cognitive game.”

Led by Rajesh Narendarn, project principal investigator and faculty member in radiology, the research team sought healthy young men and women from all ethnicities to boost their omega-3 intake with supplements for six months. They were monitored through phone calls and outpatient procedures.

Before they began taking the supplements, all participants underwent positron emission tomography (PET) imaging and their blood samples were analyzed. They then were asked to perform a working memory test in which they were shown a series of letters and numbers. The young adults had to keep track of what appeared one, two and three times prior, known as a simple “n-back test.”

“What was particularly interesting about the presupplementation n-back test was that it correlated positively with plasma omega-3,” said Moghaddam. “This means that the omega-3s they were getting from their diet already positively correlated with their working memory.”

After six months of taking Lovaza — an omega-3 supplement approved by the Food and Drug Administration — the participants were asked to complete this series of outpatient procedures again. It was during this last stage, during the working memory test and blood sampling, that the improved working memory of this population was revealed.

“So many of the previous studies have been done with the elderly or people with medical conditions, leaving this unique population of young adults unaddressed,” said Matthew Muldoon, project co-investigator and faculty member in medicine. “But what about our highest-functioning periods? Can we help the brain achieve its full potential by adapting our healthy behaviors in our young adult life? We found that we absolutely can.”

Although the effects of omega-3s on young people were a focus, the Pitt team also was hoping to determine the brain mechanism associated with omega-3 regulation. Previous rodent studies suggested that removing omega-3 from the diet might reduce dopamine storage (the neurotransmitter associated with mood as well as working memory) and decrease density in the striatal vesicular monoamine transporter type 2 (commonly referred to as VMAT2, a protein associated with decision making). Therefore, the Pitt researchers posited that increasing VMAT2 protein was the mechanism of action that boosted cognitive performance. But PET imaging revealed this was not the case.

“It is really interesting that diets enriched with omega-3 fatty acid can enhance cognition in highly functional young individuals,” said Narendarn. “Nevertheless, it was a bit disappointing that our imaging studies were unable to clarify the mechanisms by which it enhances working memory.”

Ongoing animal modeling studies in the Moghaddam lab indicate that brain mechanisms that are affected by omega-3s may be influenced differently in adolescents and young adults than they are in older adults. With this in mind, the Pitt team will continue to evaluate the effect of omega-3 fatty acids in this younger population to find the mechanism that improves cognition.

Other Pitt researchers involved in the project included William G. Frankle, psychiatry, and Neal S. Mason, radiology.

The research was supported by grants from the National Institute on Drug Abuse and the American Reinvestment and Recovery Act of 2009.

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Mathematical modeling used to study neuron behavior in brain

While scientists know that neurons in the brain anatomically organize themselves into network camps, or clusters, the implications of such groupings on neural dynamics have remained unclear until now.

Using mathematical modeling, two Pitt researchers have found that neurons team up to sway particular outcomes in the brain and take over the nervous system in the name of their preferred action or behavior.

The findings will be published in the November print issue of Nature Neuroscience.

“Through complex mathematical equations, we organized neurons into clustered networks and immediately saw that our model produced a rich dynamic wherein neurons in the same groups were active together,” said Brent Doiron, faculty member in mathematics.

Together with Ashok Litwin-Kumar, a doctoral student in neural computation in Pitt and Carnegie Mellon’s Center for the Neural Basis of Cognition, Doiron found that, like a political race, the brain’s neurons divide into a collection of candidates (clusters) with various preferences, each with its own network of supporters (neurons) interacting on a competitive playing field (the cortex).

The mathematical models show that few neural teams can be highly active and “in the lead” at any given time, advocating for their stimulus or response preference and suppressing the preferences of the other teams.

However, when Doiron and Litwin-Kumar introduced a stimulus to select only a few groups in the network, the competition quickly became unbalanced. Similar to selective funding during a campaign, the parties with “more campaign money” (neurons influenced by their preferred stimulus) had a higher probability of “winning the race” (or taking over the nervous system).

“We found that stimulation actually reduces the firing rate variability among neurons, an observation that is consistent with recent cortical readings,” said Doiron. “Our results show that even weak stimuli can substantially change our balanced network dynamics, making brain dynamics much more predictable.”

When there was no stimulus, Doiron and Litwin-Kumar saw that the landscape of winners and losers was very random in time, with clusters randomly transitioning from the lead position to secondary positions and vice versa.

These significant fluctuations over long periods of time mimicked recorded activity in the spontaneously active brain. While past models of unclustered brains could not capture this key dynamic, this new model gives a plausible explanation for how spontaneous neural activity arises and is maintained.

These results explore how the wiring of the nervous system can influence stochastic (or unpredictable) brain dynamics, especially when the brain is spontaneously active. Doiron said there has been significant research on how the brain responds to input — such as during tasks like remembering a phone number or grasping a cup. However, the neuroscience community has just begun to think about what is happening in the brain when there are no inputs and the brain seemingly is idle.

“Unlike the quiet states of your computer between processing jobs, the brain has a highly variable and random political fight going on when there are no immediate tasks,” said Doiron. “This fight likely consolidates factions and keeps specific networks well linked, a feature that can be crucial for proper brain functioning in driven states.”

The research was supported by the National Science Foundation, the Sloan Foundation and the U.S. Department of Defense.

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Carotid artery change in menopause may mean more cardiovascular disease risk

Substantial changes in the diameter and thickness of a section of carotid artery in perimenopausal women may indicate a higher risk of developing cardiovascular disease, the leading cause of death in women, according to Pitt researchers.

Epidemiologists studied 249 women aged 42-52 from the Pittsburgh site of the Study of Women’s Health Across the Nation (SWAN) observational study. Each participant was given up to five ultrasound scans during transitional phases of menopause to measure the thickness and diameter of a section of the carotid artery. Researchers noted significant increases in the average thickness (0.017 mm per year) and diameter (0.024 mm per year) of the carotid artery during the late perimenopausal stage, the period of time when menstruation ceases for more than three consecutive months.

These increases were significantly higher than those found in the premenopausal stage.

Samar R. El Khoudary, lead author of the study and a public health faculty member, said: “These data highlight late perimenopause as a stage of vascular remodeling during which arteries become more vulnerable, regardless of a woman’s age and ethnicity.”

The study is available online and will be printed in the January 2013 issue of Menopause: The Journal of the North American Menopause Society.

The findings also suggest that the changes in the diameter of the arterial wall may occur first in response to lower levels of estrogen during perimenopause.

The thickening of the arterial wall likely follows as the body adjusts to the increased stress from the dilated artery, said El Khoudary. Late perimenopause also is the time during which women gain weight and face changes in lipid profiles and body fat distribution.

Those risk factors in combination with the vascular changes may place older women at risk for developing atherosclerosis, said El Khoudary.

“Our current study highlights late perimenopause as a time when early intervention strategies targeting cardiovascular disease might yield the greatest benefit,” she added.

Pitt contributing authors included Joyce T. Bromberger and Kim Sutton-Tyrrell, both of public health, and Karen Matthews and Rebecca C. Thurston, both of the School of Medicine.

SWAN received support from NIH, the Department of Health and Human Services through the National Institute on Aging, the National Institute of Nursing Research and the NIH Office of Research on Women’s Health.

The Study of Women’s Health Across the Nation Heart was supported by the National Heart, Lung and Blood Institute.

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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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