University Times

Research shows stem cells unnecessary for cloning

In the first demonstration that an animal can be derived directly from a fully differentiated cell, researchers Tao Cheng of Pitt and Xiangzhong (Jerry) Yang of the University of Connecticut report in the journal Nature Genetics they created two mouse pups from a type of blood cell. Their research dismisses the notion that adult stem cells are necessary for successful animal cloning, showing instead that cells that have completely evolved to a specific type not only can be used for cloning, but they may be a better and more efficient starting point.

They say results of their studies provide compelling evidence that Dolly the sheep and other mammals cloned by somatic cell nuclear transfer most likely were derived from fully differentiated cells, not adult stem cells, as most have argued.

Because stem cells have the ability to self-renew and differentiate into any specialized cell type, they have been heralded for their promise for treating a variety of diseases and conditions. Yet, even for cloning of an embryo to the blastocyst stage, from which embryonic stem cells can be generated, adult stem cells have yielded disappointing results, with success rates in the range of 1-5 percent.

In their studies, the researchers compared the efficiency for cloning mice using a fully differentiated blood cell called a granulocyte with its ancestor cells at different stages: hematopoietic stem cells, which are found in bone marrow and give rise to all red and white blood cells, and progenitor cells. Granulocytes are well-characterized white blood cells unique for their segmented nuclei and the numerous granules in the cells’ cytoplasm.

The researchers found the granulocytes were the most efficient donor cells for nuclear transfer among the different lineage cells, with 35-39 percent becoming a blastocyst, an early embryo consisting of about 100-150 cells, compared to 11 percent for the progenitor cells and only 4 percent for the stem cells. Only the granulocytes were able to produce two live cloned pups, although both died within a few hours of birth.

As a control, the researchers performed nuclear transfer using embryonic stem cells; 49 percent developed to the blastocyst stage and 18 cloned pups were born.

“Our results clearly demonstrate that there is no apparent advantage in using either adult stem cells or progenitor cells over fully differentiated cells as nuclear donors,” said Yang, co-corresponding author of the study.

Cheng, associate professor of radiation oncology at the School of Medicine and director of stem cell biology and co-leader of the cancer stem cell program at the University of Pittsburgh Cancer Institute, said, “Even we were surprised to find fully differentiated cells were more efficient for cloning, because granulocytes are not capable of dividing. In fact, we repeated our experiments six times just to be sure. Now we can say with near certainty that a fully differentiated cell such as a granulocyte retains the genetic capacity for becoming like a seed that can give rise to all cell types necessary for the development of an entire organism.”

Since Dolly, animal cloning using adult cells has been accomplished in more than a dozen mammalian species, but the process is highly inefficient. Even if the reconstructed eggs survive to the blastocyst stage, at most only a handful result in live young when implanted into a female.

Many have attributed cloning’s limited success to a theory that clones must be derived from adult stem cells, which reside in a specific area of each tissue and remain quiescent until they are activated by the presence of disease or tissue injury. Yet, if this were true, Yang and Cheng point out, the results of their studies would have found the adult stem cells to be more efficient than the other, more differentiated cells.

“Of the 1,828 nuclear transfers we performed with stem cells, very few could develop to the blastocyst stage and not one clone was produced. With such odds, it’s hard to believe that Dolly and other cloned animals could have possibly been derived from adult stem cells. Much more likely is that these animals were derived from fully differentiated tissue cells,” Yang argues.

The investigators say their current studies may have important implications for regenerative medicine, since the findings suggest the potential of adult stem cells in this arena may be more limited than previously thought. However, of particular interest to Cheng is the relevance of their findings to cancer stem cell research.

“An interesting question to me is whether [stem cell nuclear transfer] can play a role in understanding or even reprogramming the behavior of cancer stem cells. Such studies may potentially reveal a new set of molecular targets that could aid in the treatment of cancer,” said Cheng.

Other Pitt authors of the study were Hongmei Shen of surgery and radiation oncology and Hui Yu and Yifang Song of radiation oncology.

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Colon cancer research funded

Eric Lagasse of the Department of Pathology is among the first 11 recipients of the Jeannik M. Littlefield-American Association for Cancer Research awards for his project, “Metastatic Colon Cancer, Stem Cells and Bioreactors.”

He plans to use an artificial bioreactor that models the environment in liver and colon cancer and to identify and expand stem cells in vitro. Therapy then will be refined and customized pre-clinically before being administered to patients.

The 11 recipients are sharing more than $2.64 million in grants aimed at discovering and developing new ways to treat metastatic colon cancer.

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SSRIs may work by influencing amygdala’s autoreceptor

Although drugs that target the brain’s serotonin system are widely used to treat depression, the basic biological mechanism by which they help to alleviate symptoms is poorly understood. Now, new Pitt research suggests these drugs work by acting on a specific serotonin receptor called the 5-HT1A autoreceptor, which investigators found plays a key role in regulating the response of the amygdala.

The findings, published in the journal Nature Neuroscience, also indicate how changes in 5-HT1A autoreceptors and associated amygdala reactivity may impact a person’s risk for developing depression. Much like a rheostat, these serotonin receptors regulate the brain’s emotional responses and may contribute to one’s vulnerability for depression and other psychiatric disorders.

The amygdala processes clues from the environment about potential threats and generates appropriate behavioral and physiological responses — such as the “fight or flight” response — to these challenges. Research has indicated that depression and other mood disorders, such as anxiety, are associated with emotional brain circuitry problems involving the amygdala.

The 5-HT1A autoreceptor is located on the surface of serotonin neurons, which are responsible for producing the serotonin neurotransmitter and delivering it to several areas of the brain, including the amygdala.

The effect serotonin release has is varied and complex, due to the number of different serotonin receptors found in these brain regions. In contrast, the 5-HT1A autoreceptor’s effect on serotonin neurons in the brain stem is rather uniform: It reduces the neuron’s activity and its subsequent release of serotonin to downstream targets, a process called negative feedback inhibition. As more serotonin is released and becomes available in the brain, the neurotransmitter also acts on these 5-HT1A autoreceptors, which in turn work to reduce serotonin release.

In normal subjects, investigators found those with higher concentrations of the 5-HT1A autoreceptor also had significantly reduced activity in their amygdala. And the concentration of the 5-HT1A autoreceptor accounted for 30-44 percent of the differences in the reactivity of the amygdala among individuals.

“There’s a significant inverse relationship between the density of the 5-HT1A autoreceptor and the amygdala’s reactivity to threatening stimuli, and this mechanism appears to be reflecting the effects of the 5-HT1A on the negative feedback loop that controls the release of serotonin,” said study leader Ahmad R. Hariri, assistant professor of psychiatry and director of the developmental imaging genetics program at the School of Medicine and Western Psychiatric Institute and Clinic.

“This observation could be important in understanding a key molecular pathway that may make some people more vulnerable for developing depression or other psychiatric disorders.

“In our normal subjects, we saw a pattern that suggests the reactivity of the amygdala is regulated by the capacity for negative feedback regulation of serotonin release through the 5-HT1A autoreceptor. Moreover, the pattern suggests that higher levels of serotonin, associated with reduced autoreceptor density and negative feedback, is linked with greater amygdala reactivity.”

In earlier studies, Hariri and colleagues established a brain basis for a gene variant and its relationship to behavior, finding greater amygdala reactivity in people carrying a form of a gene that leads to reduced expression of the serotonin transporter who, because of this variation, are more vulnerable to developing depression in the face of stressful and difficult life circumstances.

Recently, other researchers have noted that this same genetic variation is associated with reduced 5-HT1A density. Moreover, alterations in 5-HT1A autoreceptor density and amygdala reactivity have been documented in depression, and studies suggest that the class of drugs known as selective serotonin reuptake inhibitors (SSRIs) may evoke an antidepressant effect by acting on both the 5-HT1A autoreceptor and amygdala reactivity.

Taken together, Hariri’s latest findings may be important for understanding the molecular mechanisms underlying the effects of the serotonin transporter gene variation on amygdala reactivity.

“Antidepressants, especially the SSRIs, which increase the availability of serotonin, may actually be working by shifting the availability of 5-HT1A autoreceptors and, as a consequence, modulating the reactivity of the amygdala. Such shifts may drive the improvements in depressed mood and affect that result from SSRI treatment,” explained Hariri, who plans to conduct additional studies in both healthy volunteers and patients with depression in order to better understand the specific biological pathways that contribute to risk for the condition as well as predict treatment response.

Other Pitt researchers who contributed to this work were Patrick M. Fisher, Scott K. Ziolko and Julie C. Price of the School of Medicine, and Carolyn C. Meltzer, now at Emory University.

The research was supported by the National Institute of Mental Health of the National Institutes of Health and the National Alliance for Research on Schizophrenia and Depression.

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Oncology diagnostic test research funded

Federico A. Monzon of the Department of Pathology is principal investigator in a $234,000 sponsored-research agreement with Pathwork Informatics LLC for a project to validate the Pathwork Oncology Suite: Tissue of Origin (POS:TOO) test.

POS:TOO, a gene expression microarray-based diagnostic test, is an in-vitro diagnostic designed to evaluate the tissue of origin for metastatic tumors. Its suite of 15 one-against-all tests report independent similarity scores from which a physician may draw a final conclusion as to the likely tissue of origin for the tumor specimen.

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Brief mania may signal bipolar disorder in children

Not all children with bipolar disorder may be getting identified properly because they fall just short of meeting adult-based diagnostic criteria for the disorder, finds a new Pitt study. The findings, from the first study of its kind to delineate the types of symptoms seen in children with bipolar spectrum disorders, were published in the Archives of General Psychiatry.

The researchers found that a significant number of children with symptoms of bipolar disorder were just below the threshold of meeting the two primary classifications of bipolar disorder, mostly because their manic episodes did not last long enough. However, these youth with “subthreshold” mania were similar in most ways to children and adolescents who met the full diagnostic criteria for bipolar disorder.

David Axelson, assistant professor of psychiatry in the School of Medicine and lead author of the study, said, “Some children with bipolar disorder have distinct episodes of manic symptoms that last for many days or weeks at a time, just like it classically presents in adults with bipolar disorder. However, we do not know very much about children who have very clear periods of manic symptoms that do not last for several days. The results from this study suggest that some of these kids likely have bipolar disorder.

“We need more research to figure out which kids go on to become bipolar adults, so it is too early to say that every child with brief periods of manic symptoms is bipolar. However, it is reasonable for clinicians to consider the possibility of bipolar disorder in youth who present with mania that does not reach the duration criteria for adult bipolar disorder.”

The study enrolled 438 participants ages 7-17 at centers at Pitt, Brown University and the University of California at Los Angeles. Bipolar disorder, commonly called manic-depressive illness, is characterized by swings between depression and mania and periods with mixed symptoms.

The researchers hope outcomes from this study, which will follow the participants for five-10 years, will address whether changes should be made to the diagnostic criteria.

The results represent the second in a series of publications from the Course and Outcome of Bipolar Illness in Youths (COBY) study, a multicenter National Institute of Mental Health-funded study led by Boris Birmaher of Pitt’s Department of Psychiatry, Martin Keller of Brown and Michael Strober of UCLA.

The study is the largest to date of pediatric bipolar disorder and the first prospective naturalistic study of children and adolescents with bipolar spectrum disorders. The first published results established the characteristics and short-term outcomes of the disease.

Other Pitt co-authors were Laurel Chiapetta and Satish Iyengar of statistics and Mary Kay Gill, Jeffrey Bridge and Neal Ryan of psychiatry.

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Pitt gets $8.4 million to find best practices against sepsis

The School of Medicine will receive a five-year, $8.4 million, National Institute of General Medical Sciences grant to lead a multi-center investigation on treating sepsis.

Sepsis occurs when the body’s inflammatory response overreacts to an infection, resulting in a cascade of events that begins with septic shock, then compromises vital organs, resulting in organ failure and death.

Sepsis is among the top causes of death in the United States, affecting 750,000 Americans each year, of which 30 percent die. It also is one of the most expensive diseases, with a cost to U.S. hospitals of $17 billion each year.

The Protocolized Care for Early Septic Shock (ProCESS) study will attempt to determine if there is a “golden hour” in the management of sepsis and septic shock when a prompt, rigorous, standardized treatment regimen can be used to improve clinical outcomes and halt the cascade of events that often lead to organ failure and death.

The investigators, led by Derek C. Angus, vice chair of research in the School of Medicine’s Department of Critical Care Medicine, aim to generate comprehensive data on the clinical and biological aspects of standardized treatment for septic shock that may improve care of critically ill patients.

“Sepsis has reached epidemic proportions in the United States, taking as many lives as heart attacks do. It is essential that we conduct this kind of study to identify the ideal way to stop the rampant inflammation before it reaches the point where it becomes so severe and aggressive that it cannot be stopped.”

Up to 2,000 participants who arrive at participating hospital emergency departments will be randomized to receive alternative treatment protocols involving intravenous fluids, drugs that reverse the shock and hemodynamic monitoring during the first six hours of care.

The protocols will be evaluated for clinical effectiveness as evidenced by improved mortality rates and effectiveness in reducing concentrations of biological markers associated with sepsis-related organ dysfunction and cost effectiveness.

“ProCESS will expand our understanding of emergency department sepsis care and could revolutionize that care,” said Donald M. Yealy, vice chair of emergency medicine at the School of Medicine and co-principal investigator of the study.

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Brain trauma research grant renewed

Pitt’s Brain Trauma Research Center has been awarded a $6.3 million grant renewal from the National Institute of Neurological Disorders and Stroke of the National Institutes of Health (NIH) to continue researching the effects of head injury on the brain.

The researchers will look at the factors that often contribute to poor outcomes and investigate new treatments that may lead to better recovery for patients at all levels of brain injury.

The center, housed in the School of Medicine’s Department of Neurological Surgery, is an NIH Center of Excellence originally established at Pitt in 1991. It is one of only three NIH-designated head injury centers in the United States.

Edward Dixon, professor of neurological surgery, anesthesiology, neurobiology, and physical medicine and rehabilitation and director of the center, said, “Since there are so few treatments for brain trauma, our special focus is to translate our research into practical, clinical approaches that can ultimately help the brain-injured patients who are on the road to recovery.”

Current center research projects include a comprehensive study of the links between Alzheimer’s disease and brain trauma; investigations of the mechanisms of nerve cell death and dysfunction; an investigation of learning and memory disruption after injury, which may shed light on post-traumatic amnesia and offer strategies to prevent or treat it, and a two-year neurocognitive follow-up study of severely brain-injured patients who received aggressive treatment for their injuries.

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Pitt research contributes to measurement of extrasolar planet

NASA’s Hubble Space Telescope, in collaboration with Pitt’s Allegheny Observatory and other ground-based observatories, has provided definitive evidence for the existence of the nearest planet outside our solar system. The results were presented Oct. 9 at a meeting of the American Astronomical Society’s Division of Planetary Sciences and will appear in the November issue of the Astronomical Journal.

The planetary system around the star Epsilon Eridani was first detected in 1988 by a Canadian team and confirmed in 2000 by observers at the University of Texas at Austin who interpreted a wobble in the motion of Epsilon Eridani as being caused by the gravitational tug of an unseen planet.

Pitt professor of physics and astronomy George Gatewood strengthened the result that same year. Using data from the Allegheny Observatory, which he directs, he estimated the mass of the planet to be 1.2 times that of Jupiter.

Some astronomers wondered whether the turbulent motion of the young star’s atmosphere was merely mimicking the effects of the star being nudged by a planet’s gravitational pull. The new Hubble observations settle any uncertainty, calculating the planet’s mass as 1.5 times Jupiter’s, within the range calculated by Gatewood.

The planet orbits the Sun-like star Epsilon Eridani, which is 10.5 light-years away (approximately 63 trillion miles) — so close it may be observable by Hubble and large ground-based telescopes in late 2007 when Hubble’s orbit is closest to Epsilon Eridani. Hubble also found that the planet’s orbit is tilted 30 degrees to our line of sight, which is the same inclination as a disk of dust and gas that also encircles Epsilon Eridani.

The research team emphasized that the alignment of the planet’s orbit with the dust disk provides compelling direct evidence that planets indeed are created from disks of gas and dust debris around stars. Although it has long been inferred that planets form from such disks, this is the first time that both have been observed around the same star. (The planets in our solar system share a common alignment, evidence that they were created at the same time in the Sun’s disk, which has long since dissipated.)

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Trial to study stem cell treatment for CMI

The School of Medicine is among the first medical centers in the country participating in a clinical trial to study whether a patient’s own stem cells can treat a form of severe coronary artery disease.

Pitt plans to enroll 10- 20 subjects in the study, known as the Autologous Cellular Therapy CD34-Chronic Myocardial Ischemia (ACT34-CMI) Trial. The trial is the first human phase II adult stem cell therapy study in the nation designed to investigate the efficacy, tolerability and safety of blood-derived selected CD34+ stem cells to improve symptoms and clinical outcomes in patients with chronic myocardial ischemia (CMI), a severe form of coronary artery disease.

Myocardial ischemia is a narrowing of the coronary arteries that results in limited blood flow to the heart, affecting hundreds of thousands of new individuals each year. CMI patients are those who continue to have an insufficient flow of oxygen-rich blood to the heart despite optimum medical intervention.

Joon Sup Lee, associate professor of medicine, is principal investigator of the Pittsburgh clinical trial site and clinical director of the UPMC Cardiovascular Institute.

The study is sponsored by the Cellular Therapies business unit of the Baxter Healthcare Corp. Baxter technology is used to select the patient’s own CD34+ stem cells that are under investigation in this trial.

Participants will have cells containing CD34+ stem cells separated from their blood and processed to select the CD34+ stem cells for use in this investigational therapy.

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Awareness of subliminal ad messages may reduce teen smoking

Many adolescents who start smoking are lured by advertisements and movies that suggest smoking is a glamorous, grown-up activity. However, teens wise to the motives of advertisers may be less inclined to take to cigarettes, a School of Medicine study indicates.

Teens with above-average smoking media literacy (SML) are nearly half as likely to smoke as their less media-literate peers, according to the lead study in the current issue of the Journal of Adolescent Health. The results not only suggest that SML training could be an effective intervention to decrease teen smoking, but they also provide some of the first quantitative evidence linking SML to smoking.

“Many factors that influence a teen’s decision to smoke — like peer influence, parental smoking and risk-seeking tendency — are difficult to change,” said the study’s lead author, Brian Primack, assistant professor in the School of Medicine’s Division of General Internal Medicine. “However, media literacy, which can be taught, may be a valuable tool in efforts to discourage teens from smoking.”

Earlier research by Primack and his colleagues established the reliability and validity of the scale used to measure SML.

In the current study, researchers conducted further analysis, more completely quantifying the relationship between SML and smoking behavior. They found that the median SML score of all of the survey participants was 6.8, and students with scores above the median were half as likely to smoke or to be susceptible to future smoking than those below the median, even after controlling for over a dozen demographic, environmental and intrinsic risk factors for smoking.

The analysis suggests that even minor intervention may be able to influence behavior. According to survey data, a decrease in an SML score of just one point corresponded with a 30 percent increase in a student’s likelihood to smoke or be susceptible to smoking.

Many of the teenage anti-smoking programs tend to rely heavily on negative messages, which often fail to achieve the intended goal. Primack’s study suggests media literacy training could be more useful in decreasing smoking rates.

There could be broader applications to the findings. “Research has linked media exposure to eating behaviors, alcohol abuse, social violence and sexual behavior. Perhaps media literacy will turn out to be valuable in addressing these health-related areas as well,” he said.

Other study authors include Michael Fine and Melanie A. Gold of the School of Medicine and Stephanie R. Land of the Graduate School of Public Health. The study was funded by the Maurice Falk Fund and Tobacco Free Allegheny.