New flow cytometer to
aid immunosuppressant studies
BY WILLIAM A. WELLS
It's still in a box, but
one of the new generation of flow cytometers soon will be
available for use in a wide variety of basic and clinical
research projects at the School of Medicine.
Dr. Randall Morris,
director of transplantation immunology and research
professor of cardiothoracic surgery, purchased his first
flow cytometer in early 1996 and is now upgrading to a
more powerful model for use in determining the right
doses of experimental immunosuppressant drugs. The new
machine is likely to appeal to other researchers as well,
he said.
Flow cytometers pluck
fluorescently labeled cells out of a mixture of cells.
Unlike Morris' older machine, which merely counted the
labeled cells, the new one separates and sorts cells at
the end of the analysis, so these purified batches can be
analyzed further. In addition, it can simultaneously sort
through more cell subcategories than the older one.
Both models were purchased
through grants from the Oakland-based Hedco Foundation,
which supports the purchase of medical research
equipment. Morris has also used the Hedco grant funds,
which totaled $400,000, to purchase complementary
equipment, including microscopes coupled with hardware
and software to allow digital imaging of tissue sections.
"Instead of just describing the histology on slides
in words, we can now use numbers to quantitate
changes," he said.
Among other uses, flow
cytometers can detect the effects of drugs on cells from
treated animals and patients. Morris' team plans to use
the equipment to optimize complex immunosuppressive
drug-dosing regimens for people receiving organ
transplants.
"Currently, it is
often very unclear whether doses of immunosuppressive
drugs are within the narrow range needed to ensure that
the drugs are both effective and safe," explained
senior scientist Steven Sherwood, who is directing the
flow cytometry effort in Morris' group. "Lower
levels lead to rejection, while higher levels result in
infection."
In animal studies over the
past two years, Morris' group has used flow cytometry to
establish that the effects of new drugs on immune cells
in the bloodstream correlate well with the drugs' ability
to stave off tissue rejection. To do these studies, the
researchers first stimulate blood cells from the graft
recipient by a process that mimics exposure to a donor
organ. Then, using new methods developed by the group,
they use the flow cytometer to find out whether the cells
rise to the challenge or the drug has successfully
subdued them.
Broad appeal
Morris thinks the
expanding flow cytometry facilities and capabilities are
likely to interest other Stanford researchers, especially
those seeking to trace which cells are involved in a
disease or to learn how cells respond to anticancer
agents or other drugs. Dr. John Cooke, associate
professor of medicine (cardiovascular), has already used
flow cytometry to study atherosclerosis. With the older
machine, he identified the blood cells that stick to the
inner lining of blood vessels when cholesterol levels are
elevated. Cooke is now trying to learn whether the high
glucose levels in diabetics another risk factor for
atherosclerosis cause blood cells to stick to the
vessel lining, blocking the flow of blood. He is using
the flow cytometer to test the cells lining vessels for
abnormal production of certain proteins that attract
other blood cells.
The world's first flow
cytometer was developed at Stanford in the early 1970s by
a team led by genetics professor Dr. Leonard Herzenberg.
Flow cytometry centers currently available for use at
Stanford include a shared facility in the Beckman Center
for Molecular and Genetic Medicine, which is used
primarily for basic research, and the Zambon Cell Biology
Diagnosis Laboratory, which is used for clinical
applications.
Now in the planning
stages, said Morris, is a flow cytometry facility that
would complement these existing centers. The new center
would be devoted primarily to basic research on
immunosuppression and clinical research on new
immunosuppressive drugs, he said.
"Flow cytometry
allows you to do molecular biology without opening the
cell up," said Morris. "It's a much more
realistic version of what the cell is really doing."
For an experiment using
the flow cytometer, cells are mixed with a fluorescent
antibody that sticks only to cells that have a particular
protein. That protein may indicate what function the cell
has, or it may denote that the cell is actively
multiplying or committing suicide. The machine also
reveals how much DNA the cells have, another indicator of
cell multiplication.
The labeled cells fly
through a tiny nozzle at a speed of more than 30
kilometers per hour. The nozzle, vibrating approximately
40,000 times a second, breaks the cell suspension into
droplets. The droplets pass a laser that causes any
labeled cells to emit fluorescent light. That light is
detected and translated into an electric pulse, which
charges the droplet. Finally, an electric field deflects
the charged droplets into a collector.
In the past, researchers
have used this technology to define different types of
blood cells, to sort fetal cells from maternal blood
cells for prenatal diagnosis, to distinguish among
leukemias that need different drug regimens, and to
measure T-cell subsets to monitor disease progression in
HIV patients.
New drugs
With the new equipment in
place, Morris and his colleagues will extend their
current work using flow cytometry to study several new
drugs. These drugs include RAD, a relative of rapamycin,
and several relatives of leflunomide, called
malononitrilamides.
In 1989, Morris discovered
that the drug rapamycin could be used to control graft
rejection in animals. In initial trials in kidney
transplant patients, rapamycin has reduced rejection by
75 percent, he said. Phase III trials with rapamycin are
under way for kidney transplants, but Morris is now
interested in lung transplants.
Approximately 80 percent
of patients who receive lung transplants develop acute
rejection in their first year, and almost half develop
chronic rejection within two years, he noted.
"Chronic rejection is a scourge to the whole
lung-transplant program," said Morris, because once
it sets in, "there is an inevitable downward
spiral."
Several years ago, his
group tested a variety of immunosuppressant drugs on his
animal model for chronic rejection, and rapamycin was the
winner. It stopped the two events that obliterate the
airways of transplant patients: invasion by the
recipient's blood cells, and the growth of scar tissue.
It is probably because rapamycin works on a protein that
is needed for all sorts of cells to multiply, said
Morris, that it can keep both blood cells and cells
lining the airways in check.
This past April, Morris
and his co-workers received a $3 million grant from
Novartis Corp. to study the effect of RAD, their modified
version of rapamycin, on lung transplants. The award also
covers the establishment of a database of lung-transplant
treatments and their relative success rates, Morris said.
In May, his group started a large monkey trial and a
phase I human trial for patients whose lung transplants
have been stable for six months. The researchers will use
the new flow cytometer to study the effects of RAD on
immune cells in both trials. If the trials are
successful, a phase II/III trial for patients with newly
transplanted lungs will be started, Morris said.
The other set of drugs,
the malononitrilamides, will not enter phase I human
trials (for kidney transplants) until at least the end of
the year, he noted. His group, in collaboration with Dr.
Clare Gregory, professor of veterinary surgery at UC
Davis, has studied the predecessor of these drugs,
leflunomide, for the last four years. The researchers
have demonstrated that leflunomide is effective for heart
and kidney transplants in several animal species, Morris
said.
"There hasn't been
the toxicity as with other immunosuppressants," he
said. "It's been a very benign drug and the most
effective ever in the most challenging dog kidney graft
model."
Hoechst Marion Roussel, a
pharmaceutical company based in Frankfurt, Germany,
awarded Morris' group $1.5 million last year to establish
transplant models for these new drugs, whose blood levels
are easier to control. The flow cytometer will be used in
the animal studies and in the later human trials, Morris
said.
The malononitrilamides are
important, he noted, because they can shut down both B
and T cells, the two main armies of the immune system.
"We simply have not had safe and highly effective
drugs against the antibody-producing B cells in the
past," Morris said.
And now he can explain why
the drugs have this advantage. Earlier this year, Morris
and his colleagues, including postdoctoral fellow Dr.
Helio Silva, found that this type of drug jams the
production of a particular building block of DNA. When
the researchers gave this building block, called a
pyrimidine nucleotide, to rats that had received a graft,
or added it to cells in culture, they observed that the
immunosuppressive effects of the drug were reversed.
"The Achilles heel of
B and T cells is their need to produce and use these
pyrimidine nucleotides when stimulated by foreign graft
tissue," said Morris. Most other cells use a
"salvage" pathway, scavenging secondhand
nucleotides from their environment. But the needs of B
and T cells are too great, so they have to make the
nucleotides from scratch.
Morris' collaborators
include Dr. James Theodore, associate professor of
medicine (pulmonary); Dr. Robert Robbins, assistant
professor of cardiothoracic surgery, Dr. Ramona Doyle,
assistant professor of medicine (pulmonary medicine); Dr.
Bruce Reitz, Shumway Professor, chair of cardiothoracic
surgery and acting chair of surgery; postdoctoral fellows
Drs. Bernard Hausen, Tuija Ikonen and Norman Briffa; and
Dr. Charles Poirier, clinical fellow in heart and lung
transplantation. Medical student Tim Brazelton and
registered nurse Julie Altinger participated in the RAD
project; postdoctoral fellow Dr. Ying Wang took part in
the malononitrilamide project.
In addition to the funding
from Novartis, Hoechst Marion Roussel and the Hedco
Foundation, further support for Morris' research comes
from the Ralph and Marian C. Falk Medical Research Trust.
SR
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