Volume 16 Number 4, SUMMER 1999

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A QUICK LOOK AT THE LATEST DEVELOPMENTS FROM STANFORD UNIVERSITY MEDICAL CENTER

KEY TO PREDICTING EFFECTIVENESS OF PROSTATE CANCER SURGERY

One of the great dilemmas in treating men with prostate cancer is the problem of how to predict which patients are likely to respond well to surgery and which patients are destined to fail surgical treatment and would be better off seeking alternative options. In the past, doctors have considered several different tumor characteristics to help them make these critical treatment decisions.

Stanford researchers have now found that among nine variables, only two tumor characteristics are really critical -- the size of the tumor and the presence of grade 4 cancer, which is a more aggressive pattern of disease, says professor of urology, Thomas Stamey, MD. Other factors once thought to be important, such as whether the tumor had penetrated the outer covering of the prostate or whether there were positive surgical margins, did not prove to be significant in predicting whether surgery will cure the patient or not, says Stamey.

"Only two factors out of the nine are actually driving this cancer. Everything else paled in significance," says Stamey, chief author of the study published in the April 21, 1999, issue of the Journal of the American Medical Association.

Over five years, the researchers tracked the progress of 379 cancer patients who had had their prostates surgically removed between August 1983 and July 1992 at Stanford University Medical Center. Two key factors -- tumor size and the presence of grade 4 cancer -- emerged as the primary determinants of surgical failure.

Stamey says that the findings contradict traditional thinking about the disease and could change the way physicians manage prostate cancer patients in the future. "The study defines who can be cured much better than we were able to do before," he says. "In terms of research, it's a quantum leap forward. If you know what part of the cancer drives the disease, you know where to focus your research."-- ED.

NERVE-SPARING TOOL PROTECTS SEXUAL AND BLADDER FUNCTIONS DURING PROSTATE SURGERY

Tony Fenner is a thoughtful sort of man, one who seriously contemplates his options before making a life-changing decision. So when the 54-year-old Camarillo, Calif., resident was diagnosed last year with prostate cancer, he scoured the Internet and other resources for information on the treatment choices that lay before him.

He learned from his research that surgical removal of the prostate, the standard treatment for localized disease, can severely impact a man's quality of life. As a result of surgery, he found, a patient may be rendered impotent, unable to carry on a normal sex life, and he can lose full or partial control of his bladder.

"Those were the two things I was concerned about. That's why I searched around as much as I could," says Fenner, who is a sheriff's deputy.

His research first took him to Johns Hopkins University Medical Center, where Patrick Walsh, MD, had developed a surgical technique to preserve the nerves that control erectile and bladder function. Walsh referred Fenner to one of his protégés, James Brooks, MD, an assistant professor of urology at Stanford.

Brooks operated on Fenner in September using a specialized surgical tool that helps doctors pinpoint the key nerves so they can avoid damaging them during prostate removal. It was the first time Brooks had used the new tool, called the CaverMap Surgical Aid, with a patient. The device, which Brooks has since used with 14 other patients, consists of a probe placed at various points alongside the prostate; it delivers a mild electrical stimulus to the surrounding tissue. A fluid-filled ring placed around the patient's penis detects the slightest erectile swelling in response to the stimulus enabling doctors to create a map of the critical nerves, which are otherwise difficult to locate, Brooks says. Surgeons can then avoid these nerves when extracting the diseased organ.

According to Brooks, the cure rates for localized prostate cancer are the same whether doctors perform traditional surgery or the nerve-sparing approach. Men with cancers that have spread beyond the prostate, however, are not candidates for the nerve-sparing procedure, he notes.

Fenner's surgery proved successful. His catheter was removed three weeks after the procedure, and he regained full bladder control within a few days. He began to experience erections shortly thereafter, he says.

"It was better than I expected in the first few weeks after the catheter came out," he says. "I was surprised."

Brooks agrees. "He is doing great. I hope all my patients do this well ­ continent after a few days and potent, having intercourse," he says. "Whenever I talk to him, he seems very happy."

Brooks commented that the nerves recovered surprisingly quickly in Fenner's case. For most, it takes at least six months to one year to regain erectile function and a patient's potency can continue to improve for as much as two years following nerve-sparing surgery, Brooks says. Age is also a key factor, he adds.

Shortly after the surgery last fall, Fenner and his wife, Alex, took off for a vacation in New Zealand. Around that time, Alex penned a thank-you note to Brooks, telling him that her husband's disease had had little impact on the couple's quality of life. "And most importantly," she wrote, "we got the cancer." -- RR

BALLOON ANGIOPLASTY MOVES TO THE BRAIN

A technique traditionally used to prevent heart attacks may provide new hope for people suffering from repeated, debilitating pre-stroke symptoms, say Stanford researchers. By inserting miniscule inflatable devices into partially blocked arteries in the brain, the researchers are able to gently stretch the vessel walls and increase blood flow to oxygen-deprived regions. The procedure can alleviate transient ischemic attacks, which frequently signal an impending stroke, and decrease the likelihood of permanent neurological damage.

Strokes -- caused by a blockage in blood flow to the brain, either by a blood clot that has migrated from another part of the body, or by plaque that builds up gradually on the inside of an artery -- are the leading cause of disability and the third leading cause of death in the United States.

Warning signs of an impending stroke can include muscle weakness, difficulty speaking, dizziness or double vision. These symptoms, known as transient ischemic attacks, or TIAs, resolve themselves within minutes or hours and can be controlled by anti-coagulant or anti-platelet therapy to inhibit further clotting in the artery. However, patients who are severely affected or who do not respond well to medication can suffer several attacks per day and are unable to function normally.

In the current study, a group of patients whose symptoms were not alleviated by medication were selected for the new angioplasty procedure. The researchers used catheters and guide wires to direct the uninflated balloons from the femoral artery in the hip to the site of the blockage in the brain. Once in place, the balloons were slowly inflated over a period of about 30 to 45 seconds to about two to four millimeters, a diameter somewhat smaller than the surrounding, unblocked regions. The balloons were then deflated and withdrawn.

"The procedure is designed to basically crack or injure the atherosclerotic plaque and have it heal in a more open fashion," says Michael Marks, MD, associate professor of radiology at Stanford and lead author of the study that appeared in the May 1999 issue of the journal Stroke.

The degree of blockage was reduced from about 91 percent to about 40 percent in 22 of the 23 patients studied following the angioplasty. The procedure also resulted in a marked decrease in the group's annual rate of strokes over a three-year period, according to Marks.

"One of the most rewarding experiences was that a group of patients that were having repeated symptoms despite medication, some of whom were literally bedridden, experienced complete relief from symptoms of transient ischemic attacks," says Marks.

He believes that the results of the new intracranial angioplasty treatment are promising enough to warrant a controlled study comparing its success with traditional medication for stroke-prone patients. -- KC

LOST GENES MAY BE CAUSING TB VACCINE'S FAILURE

Nearly 80 years old, the tuberculosis vaccine is showing its age. This stalwart of the global campaign against TB may no longer work, and Stanford researchers have discovered a possible reason why: The bacteria used to make the vaccine have jettisoned some of their genes. The discovery could guide the development of an up-to-date vaccine as well as a more accurate diagnostic test for the disease.

The TB vaccine, known as BCG, is one of the most widely used vaccines in the world (though it is not used in the United States). More than 1 billion people have received it, and an additional 100 million are vaccinated each year. "The vaccine is given to virtually every child in the developing world," says Marcel M. Behr, MD, first author of the study published in the May 28, 1999, issue of Science.

Behr, an associate professor of infectious diseases and medical microbiology at McGill University in Montreal, was a member of the research team while a postdoctoral fellow at Stanford.

BCG contains live bacteria that must grow inside the body to stimulate a protective reaction from the immune system. Scientists at the Pasteur Institute in Paris created the vaccine in the early part of this century by "domesticating" a cattle bacterium that is very similar to the TB bacterium. The French scientists grew the cattle germ for more than 200 generations in a nutritious broth, a procedure that sapped the bacterium's disease-causing tendency without reducing its similarity to TB.

As far as the immune system is concerned, this cattle bacterium is a dead ringer for the TB bacterium. The early versions of the vaccine were probably quite good at stimulating the immune system to make defenses against TB, says Peter M. Small, MD, assistant professor of medicine at Stanford and leader of the research effort.

The investigators suspected that genetic changes in the bacteria used in BCG might be behind the vaccine's deterioration. To test their idea, they used a newly developed technology called a DNA microarray, which allows the quick analysis of an organism's full complement of genes.

The researchers found that the bacteria used in BCG were missing 38 genes that occur in normal strains of the cattle bacterium. Though the bacteria do just fine without the genes in the "cush" laboratory environment, they seem to need them to survive within the human body long enough to trigger the immune system, Small says.

Knowing what's missing from BCG may help scientists craft a better TB vaccine. But the first application from these results will likely be a better test for TB infection -- one that will make it possible to distinguish people who have TB from those who have merely been vaccinated against it. Now that researchers have found 91 genes carried by the TB bacterium but not its close relative in cattle, they could synthesize these genes' products and include them in a diagnostic test that would enable doctors to pinpoint TB infection. -- ML

GENE THERAPY CLEARS CLOTS IN LEG ARTERIES

Stanford researchers have devised a new way to deliver a clot-busting gene to blocked leg arteries in animals, effectively restoring blood flow to the damaged vessels.

The researchers inserted the gene for human tPA (tissue plasminogen activator), a common clot-busting agent, into a healthy leg vein in rabbits. The vein, which began pumping out large quantities of tPA, then was used as a bypass for an adjacent artery constricted by a blood clot. The procedure reduced clotting by 75 percent and effectively restored normal blood flow in the legs of treated rabbits, says Michael Kuo, MD, a radiology resident at Stanford University Medical Center.

"We're taking a vein and making it into a super structure that is resistant to clots, which is the number one killer in this country," says Kuo, who led the study and presented the results in March at the meeting of the Society of Cardiovascular and Interventional Radiology.

Kuo says the technique could benefit the millions of patients with peripheral vascular disease (PVD), in which plaque builds up in the leg arteries, sometimes causing debilitating pain and great difficulty in walking. As many as 5 percent of men and 2.5 percent of women in the United States who are age 60 or older have symptoms of PVD.

A current treatment for PVD involves injecting tPA-like clot-busting drugs into the patient's bloodstream. However, a major drawback of this generalized treatment is that it can induce serious bleeding in the body. The new therapy would sidestep this problem by delivering the gene directly to the site of the blockage.

The new technique also has potential to greatly improve the results of coronary artery bypass sur-geries, which often fail as a result of arterial clotting, Kuo says. The artery is particularly prone to clotting within a month after surgery; within 10 years, half of all bypass grafts fail as a result of accelerated plaque buildup that is believed to be related to clotting, he says.

He says surgeons could easily adapt the technique in coronary bypass procedures. Theoretically, all they would have to do is inject the tPA gene into the patient's saphenous vein -- the vessel typically used for bypasses -- before using it to channel blood past the diseased artery, he says.

Kuo says the researchers are preparing to test the clot-busting gene in human patients. -- RR

STANFORD RESEARCHERS UNCOVER HIDING PLACE OF CHOLERA BACTERIA BETWEEN OUTBREAKS

Cholera outbreaks come and go, popping up suddenly and then abruptly disappearing again. Where the cholera bacterium hides between outbreaks has puzzled scientists for some time. But a new study by Stanford researchers may help resolve this mystery. The researchers have discovered evidence that the bacterium hunkers down within a durable slime layer, known as a biofilm, that is resistant to the chlorine used to disinfect municipal water systems.

Colonies of cholera bacteria come in two forms: rugose -- named for its wrinkly texture -- and smooth, says Fitnat Yildiz, PhD, lead author of the study in the March 30, 1999, Proceedings of the National Academy of Sciences. Yildiz is a postdoctoral researcher in the lab of Gary Schoolnik, MD, professor of medicine.

Scientists have known for almost a decade that rugose colonies are resistant to chlorine disinfectants. But the basis for this elusiveness remained a mystery, Yildiz says, until she and Schoolnik discovered that the rugose colonies extrude a protective slime composed of polysaccharides, or long chains of sugar molecules. Smooth colonies do not produce this material.

The researchers saw a marked difference in how chlorine affected smooth and rugose colonies. Smooth colonies were annihilated. But snug within their slime, rugose colonies withstood chlorine concentrations 10 to 20 times higher than those found in water treatment facilities. Confirming this result, Yildiz and Schoolnik found that adding slime to smooth colonies imparted chlorine resistance.

From the bacterium's perspective, this ooze is vital for another reason -- it allows cells to band together and stick to a surface, forming a biofilm. More than a cluttered mass, a biofilm is a confederation of bacteria that may show an almost organismal degree of order, with tiny channels that transport food and waste running between the cells.

Yildiz and Schoolnik identified 25 genes that participate in the production of bacterial slime. The genes are present in both kinds of colonies but apparently are expressed only in the rugose colonies, Yildiz says. The researchers are trying to find out what causes these genes to be expressed.

Yildiz and Schoolnik hope to confirm that cholera-containing biofilms form not only in the lab but also in nature. They are collaborating with scientists from Peru and Bangladesh -- two places where cholera is endemic -- to see if the rugose form of the bacteria is found there and if it is part of natural biofilms in aquatic sediments. -- ML

STUDY OF NATURAL RESISTANCE TO HEPATITIS C REVEALS CLUES FOR TREATMENT

It stopped Evel Knievel in his tracks and threatened to silence David Crosby of the rock band Crosby, Stills and Nash. But they are only two of the 4 million Americans with hepatitis C -- a disease for which there is no easy treatment, no vaccine and no cure. For most sufferers, a liver transplant is the only option when the disease progresses unchecked and the organ ultimately fails.

Stanford scientists, collaborating with researchers at Chiron Corp., of Emeryville, Calif., may have found the first clue as to how the body can conquer infection by the hepatitis C virus: By studying chimpanzees that have overcome infection naturally, the researchers have found that it is critical for the cells of the immune system to wage a broad assault at the earliest stages of the infection.

The team studied six chimpanzees infected with hepatitis C. Four of the animals developed a long-term infection similar to that seen in humans; however, two of the chimps showed no signs of the infection beyond three months, indicating their immune systems had successfully thwarted the virus. The researchers found that T cells taken from the livers of these two chimps had gone to work early in the infection, mounting an all-out assault on the virus. In contrast, T cells from the chimps that developed chronic hepatitis C responded later and with less punch.

"These results suggest that unless a certain threshold of T-cell response is achieved during acute hepatitis, infection is likely to persist," says team leader, Stewart Cooper, MD, a research fellow in the laboratory of Peter Parham, PhD, professor of microbiology and immunology.

In addition to discovering that a certain level of T-cell response is required to fight off infection, the study, reported in the April 1999 issue of Immunity, highlighted the importance of a robust T-cell response over an antibody response, at the time of initial infection.

Until now, scientists have not known which part of the human immune system is most important in combating the hepatitis C virus. Conventional thinking held that B-cell-produced antibodies played an important role, but the Cooper study suggests that it is the T cells that are critical in combating the virus. Knowing which system is paramount in fighting off the disease is crucial for efforts to develop a vaccine against it.

Cooper now plans to identify humans who have overcome hepatitis-C infection naturally. Because chimps and humans have almost identical immune systems and react similarly to the virus, Cooper believes that his findings in chimps are likely to hold true in their primate cousins. -- KW

THE CASE OF THE FAT FROGS

With their swollen limbs and bloated bellies, the first to get sick looked more like victims of overindulgence. Yet their slimy skin was speckled with scarlet where tiny hemorrhages had erupted just beneath the surface, and blood tests revealed a plummeting white cell count -- telltale signs of an overwhelming bacterial infection. The bug was swift and lethal, sometimes killing within hours. Despite antibiotic treatment, five died the first month, followed by 14 the second month and 58 the third.

This outbreak didn't happen in some distant, disease-ridden part of the globe but here at Stanford, in the research animal facility, which houses animals used by Stanford's biomedical researchers. All of the 93 victims were frogs. But there is a happy ending to what could have been a disastrous disease outbreak. The staff at the animal facility nabbed the bacterial wrongdoer and counter- attacked it energetically. Their efforts prevented a repeat of what happened at Harvard, where some 2,000 frogs died or had to be euthanized after a similar outbreak swept through the animal facility.

WHY THE FUSS OVER A FEW DEAD FROGS?

Nearly all the victims were South African clawed frogs, one of the species so informative for science that it's called a model organism. These permanently aquatic frogs, which grow as large as your palm, have taught scientists more about the events of vertebrate development than any other organism.

Typically, between 500 and 1,000 clawed frogs live in the animal facility, according to Sherril Green, DVM, PhD, an assistant professor in the Department of Comparative Medicine and a veterinarian for the animal facility. "One of the first symptoms of illness in a frog," Green says, "is that they lose the ability to dive and will stay floating at the water's surface." As the disease progresses, the body and limbs inflate with fluid and the tiny hemorrhages called petechia spread across the skin.

When frogs began to fall sick in May of 1996, Green and her colleagues applied the same diagnostic techniques and treatments that would be used for a human illness. Besides running blood counts, they grew the bacterium from blood samples and performed autopsies on dead frogs, removing specimens of various organs to culture.

In these cultures the veterinarians found a species of bacterium known as Flavobacterium meningosepticum. A hardy, widespread germ that shrugs off most disinfectants and anti-biotics, Flavobacterium lives in food, water, and the soil and sometimes invades hospitals, even flourishing on bottles of disinfectant. Usually harmless, it occasionally infects humans --most often newborns or patients with suppressed immune systems -- though Stanford Hospital has never seen a case.

How did the microbe enter the animal facility? The veterinarians never could answer that question. Testing potential sources, from the frogs' water and food to the moss bedding in which they arrive from the supplier, yielded negative results. However, the bacterium was living in the large "airing" barrel in the animal facility, where water sat to allow chlorine to evaporate. It was also found on a few buckets, a surgical tray, and a frog tank in a research lab where the frogs were used.

However Flavobacterium established itself in the facility, Green thinks several factors probably triggered the outbreak. One was the doubling of the frog population shortly before the first case appeared. That influx may have increased the stress on the frogs or brought in an infected individual. A second factor was a doubling of the number of graduate students working on the frogs, something that also happened shortly before the first case. The combination of these two factors could have helped spread the bacterium from frog to frog, Green thinks.

Even if the bacterium couldn't be killed with antibiotics or disinfectants, its growth could be thwarted by making the research labs and the animal facility an inhospitable environment. To achieve this, the staff began an aggressive monitoring and cleanup program that included quickly culling sick frogs and scrubbing and sterilizing all frog equipment in the facility and the researchers' labs. Equally important, all who handled the frogs were instructed to regularly wash their hands.

The frogs housed at Stanford have recently moved upscale from small aquariums into black, bathtub-sized tanks with flow-through drainage, which might keep the habitat cleaner. Frogs prefer these tony digs, which can hold 100 or more animals, because they can huddle in the shadows at the bottom of the tank, much as they would in their native habitat of brackish, murky pools, Green says.

Diligent surveillance and extra cleanup efforts brought the outbreak to a halt. Fifty-eight frogs died in July of 1996, but only three in September and none in the following months. Green and colleagues have recently published an article on the outbreak in the Journal of the American Veterinary Medical Association. However, Green notes, just this spring two more frogs fell ill. Both were quickly isolated, and no other cases appeared. "I think we have the problem under control," Green says, "but the day-to-day business of animal care certainly keeps us hopping." -- ML