University Times

Efforts to clone primates move forward

Using newer cloning techniques, including the “gentle squeeze” method described by South Korean researchers who earlier this year reported creating the first cloned human embryonic stem cell line, Pitt scientists have taken a significant step toward successful therapeutic cloning of nonhuman primate embryos.

It is the first time researchers have applied methods developed in the Seoul laboratory to nonhuman primate eggs. Resulting cloned embryos progressed to the blastocyst stage, a developmental step in which the embryo resembles a hollow, fluid-filled cavity surrounded by a single layer of cells. Called the inner cell mass, this layer contains embryonic stem cells. Growth of a cloned nonhuman primate egg to the blastocyst stage is farther along the developmental spectrum than ever achieved before, according to Gerald Schatten, director of the Pittsburgh Development Center at Magee-Womens Research Institute and his colleagues.

Calvin Simerly, associate professor of obstetrics, gynecology and reproductive sciences at the University’s School of Medicine and the study’s first author, recently presented the findings at Cell Biology 2004, the 44th annual meeting of the American Society for Cell Biology in Washington, D.C.

“We’ve made improvements by adapting some of the Korean methods and have been able to overcome some of the hurdles we were seeing before,” said Schatten, senior author of the study and professor of obstetrics, gynecology and reproductive sciences and cell biology and physiology in Pitt’s School of Medicine. “This is a significant step forward and gives us hope for eventually being able to derive embryonic stem cells through therapeutic cloning.”

In therapeutic cloning, limited cell division is induced in an unfertilized egg cell to produce embryonic stem cells. In reproductive cloning, an egg cell with a donor nucleus is transferred into a living surrogate female in an attempt to make a successful pregnancy.

Stem cells are believed to be a key ingredient in the body’s self-repair system – blank slates that can develop into multiple cell types such as nerve, blood, bone or muscle. Stem cell-based approaches may hold promise for treating or curing diabetes, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), heart disease, stroke, spinal cord injury and genetic diseases. Scientists believe that embryonic stem cells may have the most versatility in potential cell-based treatments, but intensive research continues on both embryonic and adult-derived stem cells.

Schatten and his colleagues are focusing much of their research on strategies to derive embryonic stem cells from nonhuman primates. Such cells could be used as a template for human embryonic stem cell study, and answer many questions about how embryonic stem cells work and whether they can be used safely and effectively against disease or injury.

The Pittsburgh team also is attempting to clone nonhuman primates as a way to generate better research models for human disease so that studies can obtain more accurate results with fewer animals.

While the current study represents significant progress, many barriers to cloning nonhuman primates remain.

Reporting in the journal Science in April 2003, Schatten, Simerly and their colleagues described fundamental flaws they observed in nonhuman primate embryonic development despite using the techniques of nuclear transfer that had resulted in successful cloning of Dolly the sheep, mice and other domestic animals. In the 2003 study, researchers found basic molecular obstacles that blocked normal cell development, such as absent or deficient proteins, chaotic mitotic spindle structures and misaligned chromosomes. While cell division superficially seemed normal, chromosomal problems existed within each individual cell.

The most recent study appears to have broken that impasse. “We’ve had better development to the blastocyst stage in laboratory culture, which may help us to achieve cloned primate embryonic stem cells,” said Simerly. “There are primate embryonic stem cells now, but no cloned primate embryonic stem cells.”

Developing cloned primate stem cells is vitally important to evaluate the preclinical safety and immune-tolerance of stem cell transplantation. The primary medical reason for performing therapeutic cloning is to make stem cells that are genetic – and hopefully, therefore, immune matches of the patient’s own cells – so that they are not rejected. Being able to generate primate stem cells by nuclear transfer may soon permit transplant investigations to learn whether stem cells created in this way are truly immune-matched.

In addition to replicating the South Korean method of gently squeezing out the egg’s nucleus rather than the traditional practice of removing genetic material with a vacuum needle, Pittsburgh researchers performed nuclear transfer with eggs that had not yet achieved full maturity. Cells prepare to divide by moving through various phases of meiosis before actual division takes place at mitosis.

Eggs were enucleated at late meiosis stage I, when the number of chromosomes begins to be reduced by half to make the resulting egg cell ready for fertilization by a sperm cell, which would provide a similar complement of chromosomes. In other animal cloning work, eggs had been enucleated at metaphase II, part of the second stage of meiosis.

Once the maternal genetic material was removed, it was replaced by donor nuclei from rhesus cumulus and fibroblast cells. Cumulus cells surround the developing egg. Fibroblast cells make up connective tissues.

Reproductive cloning remains elusive, however, and the Pittsburgh team’s experience thus far indicates the possibility for successful cloning of primates (and perhaps humans) is even more remote than previously believed. Schatten’s group made 135 cloned monkey embryos and transferred them into 25 surrogate female rhesus macaques. No pregnancies were established.

Pitt researchers’ success in achieving cloned primate embryo development to the blastocyst stage is a significant advancement over first-generation nuclear transfer techniques that had been used to produce cloned primate embryos. In the past, such cloned embryos generally stopped growing at the 8- to 16-cell stage. Even so, cellular development continues to be somewhat flawed, indicating improper nuclear reprogramming and/or other incompatibilities. Spindle abnormalities, motor deficiencies and other chromosomal anomalies were observed. Embryos created through nuclear transfer appear to be inferior to fertilized ones.

“What this shows is that the Korean method for efficient human somatic cell nuclear transfer is equally effective for nonhuman primates, allowing the further progress toward development of an animal model which parallels human biology,” said Schatten. “This approach does not violate federal or state laws, and allows for preclinical investigations that would not be ethically feasible in humans. Our hope is to help advance the preclinical and fundamental knowledge accurately and swiftly so that perhaps clinical trials on stem cell donations might be responsibly considered within this decade.”

The study also further illuminates the science of cloning.

“While it would be very important to be able to develop genetically identical primates for disease research, which is one of our aims, we also are investigating the feasibility of therapeutic cloning of stem cells,” said Simerly. “If we can test these techniques in rhesus monkeys, it would go a long way toward discovering whether it is possible to create immune-matched stem cells.”

A paper describing these findings in more detail is being published will appear in the Dec. 11 issue of the journal Developmental Biology.

Shampoo ingredient affects neurons of rats

An antimicrobial agent found in many shampoos and hand lotions and widely used in industrial settings inhibits the development of particular neuron structures that are essential for transmitting signals between cells, according to a Pitt study presented at Cell Biology 2004, the 44th annual meeting of the American Society for Cell Biology, held at the Washington Convention Center.

Prolonged exposure to low levels of methylisothiazolinone (MIT) restricted growth of axons and dendrites of immature rat nerve cells in culture, apparently by disengaging the machinery of a key enzyme that is activated in response to cell-to-cell contact, and may have potentially damaging consequences to a developing nervous system, according to the report.

“While more research is needed to determine what effect MIT would have in rodent models, both at the cellular level and to a developing nervous system, our results thus far suggest there is potential that everyday exposure to the chemical could also be harmful to humans. I would be particularly concerned about occupational exposure in pregnant women and the possibility of risk to the fetus,” said senior author Elias Aizenman, professor of neurobiology at Pitt’s School of Medicine.

Aizenman became interested in MIT as an offshoot to his primary area of research on the mechanisms of neuronal cell death. The first he heard of the chemical was when its name came up in a literature search for compounds with specific chemical properties that he thought would incite a particular cell death pathway he recently had identified. As it turned out, MIT activated a different, novel pathway, but Aizenman said he remained intrigued, in large part because of the considerable lack of scientific data about a compound that he came to realize was listed on numerous consumer product labels and was widely used in industry.

As an antimicrobial agent, or biocide, MIT and related compounds kill harmful bacteria that like to grow near moisture or water. As such, they often are found in personal care products, as well as in water-cooling systems and at factories that require water for manufacturing. Since learning about MIT, Aizenman has not found any published neurotoxicity reports, or concrete data in any public documents filed with the Environmental Protection Agency.

The first set of studies he and his team published in 2002 in The Journal of Neuroscience involved acute exposure to mature rat neurons. They reported that 10-minute exposure at a high concentration – roughly 100 times the dose used in their current study – was lethal to these cells.

To understand what effect chronic exposure would have on immature, developing neurons, the researchers kept cells in a media solution containing low concentrations of MIT for 18 hours. In a standard culture, an immature neuron will in such time develop an axon, the extension from the cell body used for sending signals to other cells, and several dendrites, elaborate projections that receive incoming information. But after exposure to MIT, the cells had few, if any, axons and dendrites, with the inhibition of their growth being dose-dependent, reported Kai He, a postdoctoral fellow working with Aizenman.

Additional studies revealed that MIT significantly hindered tyrosine phosphorylation, a process that initiates molecular events during cell-to-cell contact, and that a particular protein enzyme was its target. This enzyme, focal adhesion kinase (FAK), is known to be important for outgrowth of axons and dendrites as well as necessary for cell signaling. But to kick into action, FAK must undergo tyrosine phosphorylation, whereby collections of molecules called phosphate groups are added to FAK’s sequence of amino acids. Like all proteins, FAK is comprised of a unique sequence of some 20 amino acids, including tyrosine, so any change, such as through phosphorylation, essentially changes its function. To pinpoint the exact site along FAK’s sequence where tyrosine phosphorylation was being inhibited, the researchers had to determine which of its tyrosines were targeted by MIT. Digging further, they found that one tyrosine in particular (amino acid 576 in the protein sequence) was more substantially affected.

“Since we know FAK to be highly expressed in the nerve tissue during brain development, this study suggests that phosphorylation of tyrosine 576 may be critical for axon and dendrite outgrowth,” Dr. He reported.

The authors, who also included Carl F. Lagenaur, associate professor of neurobiology at Pitt’s School of Medicine, plan additional research to further understand the molecular mechanisms underlying MIT’s neurotoxic effect on cells, as well as studies involving whole animals. They are hopeful that their work will stimulate additional research by other groups as well as bring heightened awareness about the potential risks from human exposure.

“This chemical is being used more and more extensively, yet there have been no neurotoxicity studies in humans to indicate what kind and at what level exposure is safe. I realize it’s a big leap to suggest there may be a parallel between environmental exposure and the noticeably higher rates of diagnosed childhood developmental disabilities, but I would caution that based on our data, there very well could be neurodevelopmental consequences from MIT. Clearly, more study is needed, with both scientists and government regulators equally engaged,” Aizenman added.

Almost one in five people in SW PA have a disability

Almost one-fifth of people over the age of 5 in Southwestern Pennsylvania-nearly half a million people-report having a disability, according to a new study by Pitt researchers.

“The size of the population with disabilities is rather surprising,” said Ralph Bangs, codirector of the Urban and Regional Research Program in Pitt’s Center for Social and Urban Research (UCSUR), who coauthored the study with José René Argueta, a Pitt Ph.D. candidate in political science. “It particularly affects working-age and elderly people, and to a lesser extent, children. So the need for services and the need for attention to this group is big.” “Disability” was defined in the 1990 Americans with Disabilities Act as a “physical or mental impairment that substantially limits one or more of the major life activities.” The Pitt study examined six types of disabilities: sensory disability (blindness, deafness, or a severe vision or hearing impairment); physical disability (a condition that substantially limits one or more basic physical activities, such as walking, climbing stairs, reaching, lifting, or carrying); mental disability (learning, remembering, or concentrating); self-care disability (dressing, bathing, or getting around inside the home); going outside the home disability (going outside the home alone to shop or visit a doctor’s office); and employment disability (working at a job or business).

Among the researchers’ main findings:

-Fayette and Greene counties and the city of Pittsburgh have the highest rates of disability in the region. Nearly half of the region’s disabled population lives in Allegheny County.

-The city of Pittsburgh has much higher rates of people with disabilities living in poverty than any other area. The poverty rate among disabled children in Pittsburgh is almost double the national average.

-The disabled population in the Southwestern Pennsylvania region, and particularly in Pittsburgh, has lower employment rates than those at the state and national level.

-The most commonly reported types of disabilities were physical, employment, and “going outside the home.”

-Working-age adults (ages 21-64) and seniors (ages 65 and older) were the two age groups with the highest rates of disabilities.

-African Americans and Native Americans have the highest rates of disability among ethnic groups.

-School enrollment and education attainment are much lower among persons with disabilities than among nondisabled people and contribute to lower employment and higher poverty among disabled people.

-Disabled children have the highest rates of poverty than any other age group of disabled people.

“Among working-age individuals, disability is associated with lower education, minority status, and poverty, suggesting that these individuals represent a highly vulnerable population,” noted Richard Schulz, director of UCSUR. “These data should be of great value in helping us direct resources to this group.”

In the report, the researchers call for a “new approach” with “a positive vision, one that sees the future of the population with disabilities not as a burden but as a large underutilized human capital pool with the potential to greatly contribute to the economic and social development of the region.”

Further research could include investigation into why Pittsburgh has higher rates of unemployment and poverty among disabled people than the rest of the region, state, and country. Argueta said one of the questions remaining is: “Is this a trend characteristic of large cities, or is it particular to Pittsburgh?” The FISA Foundation provided funding for the report.

Anxiety is a key risk trait for eating disorders

The prevalence of childhood anxiety in individuals who later developed anorexia nervosa or bulimia nervosa shows it may be a vulnerability factor for these eating disorders, according to a study in the December issue of the American Journal of Psychiatry.

Eating disorders most often strike young women in their teens and 20s, but the discovery of anxiety disorders as a childhood prelude may give doctors the ability to get an early start on prevention and treatment, according to the authors.

“We identified a strong link between anxiety disorders and eating disorders that shows they not only share many of the same personality traits, but also likely share a genetic pathway,” said Walter H. Kaye, professor of psychiatry at Pitt’s School of Medicine and primary author. “This study shows that to help young women recover from these devastating illnesses, doctors need to develop strategies to treat both the eating disorder and the underlying anxiety. It also is possible that treating the anxiety disorder early on may provide some preventative effect against eating disorders.”

The study of 672 individuals is the largest ever seeking to clarify the relationship between eating and anxiety disorders. The researchers found that two-thirds of people with eating disorders experienced some sort of clinical anxiety, such as obsessive compulsive disorder or social phobia, at some point in their lives. A significant number of them – 42 percent – developed their anxiety disorder when they were children, years before their eating disorder.

The researchers also found that anxiety remains pervasive even after women had recovered from an eating disorder. The normal rate of anxiety in otherwise healthy women is between 13 and 31 percent.

The strength of the bond between anxiety and eating disorders is bolstered by the fact that nearly all women with eating disorders report having certain anxiety traits, such as harm avoidance, generalized anxiety and perfectionism, even if they do not have a diagnosable illness.

The researchers used standard psychiatric tests to assess anxiety. They discovered that rates of anxiety were similar for each of the three subtypes of eating disorders – anorexia nervosa, bulimia nervosa and anorexia nervosa comorbid with bulimia nervosa. Two anxiety disorders appeared more often than the others – 41 percent of the participants had a history of obsessive compulsive disorder (OCD) and 20 percent had social phobia.

A striking finding was that 23 percent of the patients with childhood anxiety reported a history of obsessive compulsive disorder, compared with a rate of 2 to 3 percent in other children. Because the normal age of onset of OCD is the 20s, childhood OCD could be a significant predictor of a future eating disorder.

This collaborative group is currently conducting a new study of the genetics of anorexia nervosa, for which they need families with at least two relatives with that disorder.

For more information about this study, call 1-888-895-3886, email edresearch@upmc.edu or go to www.angenetics.org.

The Price Foundation supported the research. Other authors are: Laura Thornton, Nicole Barbarich, Kim Masters, Katherine Plotinicov, Christine Pollice and Bernie Devlin, all of the department of psychiatry, Pitt’s School of Medicine, as well as researchers from around the world.

 

Racial disparities noted in immune system genes

Specific variants in genes that encode proteins regulating inflammation may hold a key to explaining a host of disease processes known to cause increased risk of illness and death among African Americans, according to a study from Pitt’s Graduate School of Public Health (GSPH). The study, “Differential Distribution of Allelic Variants in Cytokine Genes Among African Americans and White Americans,” appeared in the Dec. 1 issue of the American Journal of Epidemiology.

“We found that African Americans were significantly more likely to carry genetic variants known to stimulate the inflammatory response,” said Roberta B. Ness, professor and chair of the department of epidemiology at GSPH and the study’s primary author. “At the same time, genotypes known to dampen the release of anti-inflammatory proteins were more common among African Americans. This is kind of a double whammy.”

Researchers examined the race-specific distribution of allelic variants in cytokine genes known to promote inflammation. Chromosomes and genes occur in alternative forms, and these alternative genetic forms are called alleles. Cytokines are proteins that are secreted by immune system cells that regulate the body’s immune response to injury and illness.

Inflammation is believed to be a fundamental component of heart attack, stroke, diabetes and kidney disease, all of which strike African Americans in higher proportions than whites. Other disorders associated with the inflammatory response include premature labor, transplant rejection and autoimmune disorders such as multiple sclerosis and scleroderma – again, all more common among African Americans.

Other factors also play an important role in the well-documented health disparities between African Americans and whites, said Ness. “Socioeconomic status, access to health care, racism, community-based issues and health behaviors are critical components of racial disparities in health,” she said. Among study participants, black women were younger, heavier, less likely to smoke and of lower socioeconomic status.

A copy of the study is available at http://aje.oupjournals.org/cgi/content/full/160/11/1033.

In addition to Ness, other study authors are Catherine L. Haggerty, Gail Harger and Robert Ferrell, all of GSPH. The study was funded by grants from the National Institute of Allergy and Infectious Diseases and the National Institute of Child Health and Development, both at the National Institutes of Health; and from the federal Agency for Healthcare Research and Quality, a program of the U.S. Department of Health and Human Services.

 

New Pitt Study Aimed at Reducing Recidivism at Allegheny County Jail

When a man is released from the Allegheny County Jail, he may find his newfound freedom just as unsettling and even as terrifying as life behind bars. Research studies have shown there is an eight-in-10 chance the man has a drug or alcohol problem-and that there is a high probability that he will be rearrested and back in the county jail within three years.

Hide Yamatani, associate dean for research in Pitt’s School of Social Work, has launched a new three-year study in Pitt’s Center on Race and Social Problems that will track, interview, and assess 300 male inmates within 30 days after their release from jail and, subsequently, every six-to-eight months.

The project, called the Evaluation of the Allegheny County Jail Collaborative, will reveal which social services and networks the men are using, which ones are working for them, and which are not. The answers could shed some light on why Allegheny County’s prisoner recidivism rate is so high and how it could be reduced. Initial funding for the project comes from a $330,000 grant from the Human Services Integration Fund, a group of foundations throughout Pittsburgh and Allegheny County.

“It is critical that we reach the men in that first 30 to 45 days,” Yamatani says. “If they can survive that period, they have a slightly better chance of not returning to jail.”

As the group of 300 former prisoners, 150 Black and 150 White males, is tracked and interviewed, another simultaneous study will be under way-a thorough survey of the county’s social service agencies and how well they perform collaboratively to meet the men’s needs.

Yamatani adds that women encounter their own specific problems upon release from prison, and he hopes to replicate the study at some point with female subjects.

Other study participants include Allegheny County’s Department of Human Services, Health Department, and Bureau of Corrections, as well as dozens of agencies that help former inmates with issues ranging from housing to anger management.