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The Road Worth Traveling

August 1, 2004

Fran Simon
fsimon@tulane.edu
Michael DeMocker

Snow blanketed the wooded 500 acres north of Lake Pontchartrain back in 1963, a rare occurrence in Southeast Louisiana, during construction of a new research facility for Tulane University. The pristine, white powder was an auspicious sign to set the stage for a new era in Tulane research and a clean slate for the old woods, once the site of a rice plantation that also manufactured bricks.

tulaniansum04_primate1_1Originally opened in November 1964 as the Delta Regional Primate Research Center, now the Tulane National Primate Research Center is the largest of the eight centers in the National Primate Research Center program funded by the National Institutes of Health.

The Tulane center houses approximately 5,000 primates of several different species, most of which live in outdoor breeding colonies.

"Nonhuman primates are limited and precious resources, so scientists at Tulane and collaborating institutions only use them when other research models cannot be used," says Andrew Lackner, who became director of the center in 2001. "Without research in primates there would be no vaccines for polio or Hepatitis B and advances in our understanding of AIDS would be greatly delayed. A variety of animal models are used in research but the primates are particularly useful because of their close genetic and physiologic relatedness to humans. This is particularly true for studies of infectious disease where nonhuman primates may be the only species other than humans susceptible to a particular infectious diseases."

Located 35 miles north of New Orleans in Covington, the primate center currently employs 240 people, including 30 doctoral-level scientists. Many of the staff would not consider working anywhere else--33 employees have been at the center for at least 20 years. In 2003, the center supported research being conducted by 335 scientists from 32 states and 13 countries. This research resulted in 162 publications with an additional 30 in press.

The primate center's grant portfolio of funding for research projects has tripled since Lackner joined Tulane, and total funding (including construction) has increased fourfold. In the current fiscal year, total funding is approaching $40 million. The primary areas of focus today at the Tulane National Primate Research Center are infectious diseases, including biodefenserelated work, gene therapy, reproductive biology and neuroscience.

THE MISSING LINK IN BIODEFENSE

The Tulane primate center is playing a key role in the federal strategic plan for biodefense research. The National Institutes of Health has established a nationwide group of multidisciplinary centers as Regional Centers of Excellence for Biodefense and Emerging Infectious Diseases with about $350 million over five years. The Tulane primate center is collaborating with scientists at two of the eight biodefense centers, located at the University of Texas Medical Branch at Galveston and at Duke University.

In addition, Tulane received a $13.6 million competitive grant from the National Institute of Allergy and Infectious Diseases to build a new regional biocontainment laboratory. The focus of research in the facility will be on the development of diagnostics, therapeutics and vaccines for protection from potential bioterrorist attacks and new, naturally occurring "emerging" infectious disease. The biocontainment lab will be a biosafety level-three facility adhering to the most stringent standards.

The Tulane primate center has safely operated a biosafety level-three laboratory for more than a decade for work on a variety of infectious diseases. The center's expertise with infectious disease research using primates was a significant factor in winning the grant. The Tulane primate center was the only primate center in the nation to receive funding for construction of a regional biocontainment laboratory. Tulane University will invest about $5 million in the project.

"Tulane has a long history of studying infectious diseases at the primate center," Lackner says. "Our scientists are among those at the forefront of this area of research. The infectious diseases program focuses on HIV/AIDS, malaria, Lyme disease, West Nile virus, tuberculosis, microsporidiosis and other diseases. These are multidisciplinary studies involving investigators in multiple divisions at the primate center and collaborators from elsewhere at Tulane and other institutions."

A BREED APART

"Veterinary medicine is the foundation of everything we do at the primate center, and Tulane follows the strictest procedures of animal care," says Rudolf Bohm, chair of veterinary medicine.

The research program at the Tulane primate center has been awarded continuous full accreditation since 1983 by the Association for Assessment and Accreditation of Laboratory Animal Care International, a private, nonprofit organization that promotes the responsible treatment of animals in science through a voluntary accreditation program. To earn and maintain accreditation, a research program undergoes periodic, extensive assessment and rigorous evaluations by independent panels of experts from around the world.

tulaniansum04_primate2_1"An accredited program must demonstrate that it is going beyond the minimal requirements to exhibit excellence in animal care and use," says Bohm, who supervises nearly 100 veterinarians and support staff.

"We develop improved techniques of animal care and strive to continually improve the veterinary care for animals here and at other primate centers. We have derived about 1,000 animals that are free of viruses that are dangerous to monkeys and which might infect humans, with an $8 million National Institutes of Health grant.

"In addition to developing better characterized populations of primates, the veterinary facility is involved in refining the use of animal models by developing the use of state-of-the-art diagnostics and surgical procedures," Bohm says. "In the past five years, we have utilized minimally invasive surgery widely for a number of infectious disease and reproductive biology studies. And we commonly utilize more advanced imaging modalities, including MRI, for both clinical and research use."

A NEW APPROACH

While researchers around the country and at Tulane continue their quest for an effective AIDS vaccine, scientists at the Tulane primate center are making strides in the fight against HIV infection, hoping to take a new approach to halting the HIV epidemic. Since the epidemic began, 60 million people have been infected with HIV worldwide and an estimated 45 million more people will be infected by 2010, according to the Joint United Nations Programme on HIV/AIDS.

Once considered a disease that spreads primarily through homosexual sex and intravenous drug use, AIDS increasingly is striking heterosexuals. Until recently, most HIV vaccine research concentrated on the body's immune system as a whole and HIV transmission through blood. The Tulane team has shown that major sites for HIV transmission and early viral replication are the mucosal linings of the body--particularly the gastrointestinal tract and vagina. Their research focusing on the mucosal immune system is supported by nine grants, primarily from the National Institutes of Health, totaling more than $3.5 million for the current year.

"Women are much more vulnerable to HIV infection than men during sex, partly because mucosal surfaces contain many of the cells that the virus targets, and women have larger mucosal surface areas that can tear to further facilitate viral infection," says comparative pathologist Ronald Veazey.

In addition to Veazey, the key members of the Tulane team that have received these HIVrelated grants are Preston Marx, Andrew Lackner, Louis Martin and Karol Sestak. They work together on a variety of projects involving mechanisms of HIV transmission, pathogenesis and vaccine development. Veazey and Marx, who has made major contributions to our understanding about the origins of HIV, are conducting research into a topical microbicide that may reduce the risk of transmitting HIV infection from men to women.

"We're working on a molecular barrier, a coating on the cells that would prevent the virus from 'seeing' the cells," Veazey says. Veazey and Marx have shown that such a barrier known as a topical microbicide--a germ-fighting barrier that would be placed in the vagina before sexual intercourse--prevented SIV infection, the monkey correlate of HIV, in most of the monkeys in their study. They co-authored a paper about this "proof of principle" discovery in the prestigious journal Nature Medicine (2003).

This has led to a tremendous increase in interest in commercial development of a topical microbicide to prevent HIV transmission. The National Institute of Allergy and Infectious Diseases has awarded Veazey and his team a five-year, $2 million grant for the project "Mechanisms of CD4 Depletion and Proliferation in SIV" (the monkey form of HIV). Veazey hopes this project will provide greater understanding of how the virus overcomes the body's immune system. The team also received an additional four-year grant of more than $1.6 million to study the pathogenesis of neonatal SIV infection.

In their work to perfect an effective HIV vaccine, Marx and other scientists at the primate center are focusing on new strategies to incorporate their knowledge of the mucosal immune system. Sestak also received a twoyear, $500,000 grant from the National Institutes of Health to collaborate with a vaccine company in Sweden and John Clements, professor and chair of microbiology and immunology at Tulane, who holds a patent on a mucosal adjuvant that has been shown to boost the immune system.

"So far, the vaccines that are being tested in humans do not target the mucosal immune system," Lackner says. "These traditional vaccines have not proven effective. By targeting the mucosal tissues, our hope is to block HIV infection at an earlier stage."

AN EMERGING DISEASE

Of great concern to the National Institutes of Health, the Centers for Disease Control and Prevention, and scientists alike are emerging infectious diseases--new diseases that will require development of new or improved treatments and diagnostic procedures. Lyme disease is one to watch. While the number of humans infected with Lyme disease each year is relatively small--about 20,000 in the United States--the number of new cases of Lyme disease has been increasing steadily during the last 10 years.

tulaniansum04_primate3_1Lyme disease is a bacterial infection spread by tick bite that can lead to arthritis, heart rhythm irregularities and nervous system abnormalities, including numbness, pain, paralysis of the facial muscles, meningitis and neurocognitive symptoms.

Rare deaths from Lyme disease have been reported. In addition to human infection, each year hundreds of thousands of dogs are bitten by ticks harboring the spirochete that causes Lyme disease.

Bacteriologist Mario Philipp and colleagues were the first to develop a monkey model for Lyme disease that led to the development of a new diagnostic test, now patented by Tulane. The U.S. Food and Drug Administration and Department of Agriculture approved the test, which is more sensitive and specific than previous tests. It is now the most widely used test for Lyme disease in veterinary practice.

Philipp and his team recently received a four-year Centers for Disease Control grant of more than $1 million and a five-year, $2.25 million grant from the National Institute of Neurological Disorders and Stroke to continue studying the pathogenesis of the Lyme disease spirochete in the rhesus monkey.

"Our goal is to advance understanding of the effects of Lyme disease on the central nervous system by determining the mechanisms of neural injury using primate brain cells in culture as well as experiments in vivo," Philipp says. He believes the spirochetes that cause Lyme disease produce inflammation that leads to degeneration and death of neurons in the brain.

This research may have broader applications for diseases such as AIDS dementia, Parkinson's disease and Alzheimer's disease by explaining the role of inflammation in killing neurons in the brain. Lyme disease is important as an emerging infectious disease as well as a model for other neurologic disorders. Scientists have demonstrated that the ticks found in Louisiana are fully competent to transmit Lyme disease infection, although they appear not to be infected with the Lyme disease spirochete.

"In doing this work, we owe a lot to our graduate students and postdoctoral fellows," says Philipp, who has trained 17 young scientists in the last 10 years. "The infrastructure of the primate center, along with highly skilled veterinary support, enables this multidisciplinary research."

A UNIQUE OPPORTUNITY

While many degenerative illnesses of the brain are caused by infectious diseases and are related to aging, others are genetic disorders that afflict babies who are born with a defective chromosome. Several years ago, veterinarians at the Tulane primate center began to notice that animals born in a particular group of rhesus monkeys in the breeding colony had a devastating neurological disorder. Some of the afflicted rhesus newborns died within a few days of birth, while others died before adolescence.

Tulane identified the first and only monkey model in the world of a human genetic disorder, Krabbe's disease. Though a rare genetic disease, the monkeys with Krabbe's may one day provide the key to curing many other inborn errors of metabolism, such as Tay-Sachs disease, in which children lack vital enzymes to break down their food and eliminate waste. About one child in every 7,000 born has a lysosomal storage disorder like Tay-Sachs disease and Krabbe's disease. There are about 40 such disorders.

Bruce Bunnell heads up a team that is using various approaches of gene therapy and regenerative medicine to explore potential methods that might cure Krabbe's disease in the monkeys. If they find a solution, they may help countless children born every year with these devastating genetic diseases. In one approach, Bunnell and his team are collaborating with Chromos, a Canadian company exploring a proprietary synthetic chromosome technology it has developed. Their sights are on the possibility of replacing the damaged chromosome in the monkeys with Krabbe's disease. But many questions must be asked--and answered--before attempting gene therapy in the monkeys.

"The Krabbe's disease animals are a rare and valuable resource," Bunnell says. The team is testing different strategies that might be used to deliver chromosomes into stem cells, tracking expression of the chromosomes to determine if the systems work. The chromosomes in question are marked with a chemical that glows green with fluorescence under a microscope. The team has been able to show that the chromosomes have been introduced successfully into the target cells. The next hurdles in the race for a genetic cure are laid out clearly in Bunnell's mind.

"This summer, we need to continue our bench studies to see if we can make any enzyme, if it can be expressed at high levels over time, and if the stem cells can maintain their ability to differentiate into other types of cells such as brain cells."

All this before the team begins to work with normal animals, to determine if stem cells can be injected into the monkeys' brains where, the scientists hope, the cells will become brain cells that glow green with the synthetic chromosome. "We're in a unique situation here, with the models, the necessary skills and tools to explore methods of cellular transplantation, bone-marrow transplantation and other techniques compared with traditional, drugbased therapies," Bunnell says. "Ultimately, the solutions probably will involve combination therapy. It will be many years before human trials with gene therapy, but right now there is no available therapy whatsoever for any of the lysosomal storage diseases."

People used to fret that there was no way to prevent polio, but now polio has been eradicated. And HIV infection once was considered a death sentence, while now it is considered a chronic disease that can be managed with a variety of medications. Much as the Lyme disease work may one day benefit patients with Parkinson's disease, Bunnell hopes that the gene-therapy technology his team is building will help Parkinson's patients re-grow normal brain cells. Hopefully, within his own lifetime.
 

A SELF-SUSTAINING POPULATION

The eight centers in the national primate research program house about 25,000 primates, including about 15,000 rhesus monkeys. About half of the primate center monkeys in the national program are kept for breeding. Yet, a government study in 2003 found that it will take up to 10 years to produce a self-sustaining rhesus population capable of meeting the demand for research monkeys. Rhesus monkeys, the species most commonly used in biomedical research, mate only during a certain period each year.

tulaniansum04_primate4_1Each female that becomes pregnant produces only one offspring, which the rhesus mother nurses for up to a year. Nature has many ways to foil scientists who are trying to increase the rhesus population.

Enter embryologist Hans-Michael Kubisch and the team of scientists who are using assisted reproductive technology. After coming to Tulane four years ago, Kubisch has established an in vitro fertilization system for the rhesus monkeys at the primate center.

Kubisch's team gives carefully chosen female monkeys hormones, to "superovulate" them, the precious eggs are collected, the scientists place the eggs in a dish with sperm retrieved from male monkeys, from 70 to 80 percent of the eggs become fertilized, and the resulting embryos are frozen in liquid nitrogen when they are two days old. Then, at the opportune moment, the reproductive technology team implants two embryos in a rhesus that plays surrogate mother.

So far, the team has seen eight healthy, normal monkeys born from six pregnancies using these techniques-- including two rare sets of twins. Now, Kubisch is shifting focus to more complicated challenges. For the first time anywhere in the world, a pigtail monkey surrogate mother at the Tulane primate center gave birth to a rhesus.

"This represents the first cross-species surrogate newborn produced in such evolutionarily divergent monkeys," Kubisch says. "The rhesus monkey and the pigtail monkey are thought to be several million years apart on the evolutionary tree."

The advantage? While rhesus monkeys are ready to carry offspring only at one time each year, pigtail monkeys breed all year. If the scientists can use pigtail monkeys as surrogate mothers for rhesus monkeys all year long, the window of opportunity for producing more rhesus offspring opens wider. In the future, Kubisch's team plans to begin work with the monkeys in the Krabbe's disease colony, with the goal of producing more monkeys with the rare genetic mutation that might serve as models of Krabbe's disease in humans.

"We can select each female only four or five times for super-ovulation. So if we are able to obtain 20-30 eggs with each attempt, we might obtain 100 eggs total from each female with the Krabbe's trait," Kubisch says. While it might seem that female monkeys are more valuable than the males that are born, Kubisch says, "I still get excited about every birth that results from embryo transfer, and I can't help thinking, 'I knew you when you were just a single cell in a petri dish!'"

Just as each precious monkey in the national primate research program begins with a single cell, every research project at the Tulane National Primate Research Center--the success of which depends on these very monkeys--takes baby steps on the paths to new discoveries.

Fran Simon is director of public relations for the Tulane University Health Sciences Center.

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