With knowledge gained, surgeons and researchers in Pittsburgh proceed with ambitious plans for xenotransplantation
"It's certain to go forward," says Thomas E. Starzl in discussing xenotransplantation. Starzl is a professor of surgery at Pittsburgh and director of the Pittsburgh Transplantation Institute there. "But everything that has ever happened in this field has been very tough and paid for with tears. This isn't going to be any different. I don't actually relish the prospect, but I think it'll get done, probably rather quickly, this decade."
The widely publicized transplantation--a bold first--was the culminationof an unusually well-coordinated effort involving basic researchers and clinicians from such specialties as immunology, pathology, genetics, molecular biology, pharmacology, gastroenterology, hematology, and surgery.
The June 28 experiment ended with the death of the patient--a 35-year-old man--on September 6. A massive stroke, unexplained as of press time, was given as the cause of death. But the baboon liver itself had nearly tripled in size to meet the metabolic needs of its new host, and the transplant team saw little in their preliminary analysis of the case to preclude their plans. "The state of the liver after more than 70 days was really remarkably good," says Starzl. "The hallmarks of antibody-mediated rejection were not present." Antibody-mediated rejection is the form of organ rejection that was most feared in this transplantation between species.
For more than two months after the operation, the patient, whose identity was withheld from the public at his request, made steady recovery, experiencing only mild, treatable rejection. His recovery was described as comparable to that of an ordinary human-to-human transplant recipient. After an August 28 diagnostic procedure, however, the patient developed sepsis, a dangerous systemic infection, and reduced liver function. Although his condition was initially stabilized with antibiotics, he suffered his fatal stroke just over a week after incurring the infection.
Prior to the transplant, the patient's liver was destroyed by the virus hepatitis B. Because a transplanted human liver would likely have become infected by the virus also, he was not considered a good candidate for human-to-human transplantation--or allotransplantation. But, even putting the specific indications of this patient's case aside, there are also far too few human donor organs available to meet demand overall.
| Research leading to the first baboon-to-human liver transplantation represents only one thread of the scientific effort under way at the Pittsburgh Transplantation Institute. In addition, recent discoveries concerning the permanent exchange of cells between organ grafts and their hosts are confounding some basic immunological tenets of transplantation. The exchange is called cell migration and leads to a state called chimerism--that is, an individual becomes a chimera.|
"A chimera is a very explicit term," explains Thomas E. Starzl, the institute's director. "It means there is a genetic component from two different [sources]--a donor and a recipient. The fundamental discovery is that chimer-ism can be found in every case of long-surviving graft, no matter what the graft."
The findings stem from studies of a number of long-term liver transplant survivors, including some who had discontinued their antirejection medications--drugs that often have severe side effects--without consulting their doctors. Of 206 cases between 1963 and 1981, 43 patients have survived, says Starzl. Of these, seven stopped their medications one to six years after transplantation and have been off the drugs for six to 18 years.
According to immunological understandings about the acceptance of grafts, without their immunosuppressive drugs these patients should have begun to reject their transplanted organs--a potentially fatal process. But they survived. "The word got around in the transplant community that you could get away with it," says Starzl. "Now, we're probing and trying to get people off drugs, because being on these drugs limits their survival."
Using monoclonal antibodies as markers in biopsies, Starzl and the Pittsburgh researchers have been able to determine that individual dendritic cells with the genetic makeup of the donors take up residence at locations throughout the bodies of the hosts. Polymerase chain reaction tests also showed genetic material from donors present in host tissues. In turn, biopsies of the transplanted organs found that host cells had also become part of the grafts. And the cells were thriving everywhere, contrary to immunological theories. In fact, in the past, dendritic cells have been thought responsible for initiating the immune response, and thereby rejection, as well as being important in the development of graft-versus-host disease, an often-fatal complication in some transplantations. Now, in a complete departure from previous thinking, the researchers believe that these cells may play an important role in preventing rather than promoting rejection and graft-versus-host disease.
"They're like the soldiers who go out to kill the natives and end up being missionaries," says Starzl. "In other words, they're killers, but then they can become tolerogenic, also."
In a recent article (Lancet, 339:1579-82, 1992), Starzl and his associates write, "We believe that cell migration takes place to some degree with all successful transplantation, irrespective of the organ, with rapid seeding through the blood stream." Under the protection of the immunosuppressive drugs, a two-way traffic in cells occurs between the graft organs and their hosts. "There is a bilateral, or mutual, engagement that occurs, which is variable," Starzl says. "This state of chimerism can be drug-independent, or it may be unstable without ongoing drugs.
"You look at the drugs as if they're traffic cops," he adds, "letting the traffic go on, but not allowing the motorists to carry out their lethal intentions."
Starzl also thinks that chimerism may begin to explain why human leukocyte antigen matching between donors and hosts, done prior to transplantations in the hopes of minimizing the rejection response, has not been strongly predictive of success. Chimerism may, in fact, be the more important factor.
A related observation is that the liver is apparently more read-ily tolerated in transplantation than other organs. Starzl suggests that organs may differ in their ability to be accepted as grafts in direct relationship to the numbers of these dendritic cells they carry. The cells are dense in the liver, and less so in other organs.
The mechanisms underlying chimerism are not yet clearly understood, but the researchers are already looking for ways to advance the process, suspecting it to be the key to tolerance. Experiments are being conducted in which bone marrow, rich in dendritic cells, is transplanted along with organs.
"We believe that you can induce tolerance and probably do it fairly easily," Starzl says, "if you use bone marrow augmentation with these various solid organ grafts.
"This really changes the whole foundation of transplantation."
"I think there will be a place for xenotransplantation in the field of transplantation," says one prominent liver transplant surgeon, speaking on condition of anonymity. "But I think the Pittsburgh group may have been a little premature. I'm not impressed that they have published or spoken much about success in large animals using the protocol they're using, although I understand they've had success using a smaller animal model. They really have not released much information to the community at all, which is a little bit distressing." Key to Pittsburgh's ability to press forward in xenotransplantation and other areas of transplantation is the multidisciplinary nature of the team assembled by pioneering transplant surgeon and researcher Starzl. Since his 1981 arrival from the University of Colorado at Boulder, where he performed the first successful human-to-human liver transplant in 1967, Pittsburgh has grown to be the largest transplant center in the country.
Starzl's view of how best to make progress in transplantation, by focusing various biomedical specialties on the task, crystallized in his founding of the Pittsburgh Transplantation Institute in 1990, with himself as director. Even in the short time since then, the pace of research productivity there has accelerated.
Most of the offices and laboratories of the institute are located on the 15th floor of the university's new Biomedical Science Tower, a $100 million facility. Transplant immunopathologist Jake Demetris, who was involved in both clinical and laboratory projects leading to the xenotransplantation, says the proximity of the transplantation researchers to each other encourages cross- cutting approaches to problems.
"In most other institutions, surgeons will have their offices and research labs with surgeons, biochemists with biochemists, pathologists with pathologists," Demetris says. "It breaks down along discipline lines. Here, we're all housed together. There's a pathologist over there, a molecular biologist, an immunologist, a surgeon, people from internal medicine, basic science and clinical people. If I have a problem, there's somebody on the floor who can solve it."
"Most of the people here have bilateral interests, in clinical care and also investigation," adds Starzl, who holds Ph.D. and M.D. degrees himself, with training in surgery. "There are plenty of examples around."
Members of Starzl's team emphasize that his personal leadership has been instrumental in the progress made at Pittsburgh in transplantation. "Starzl's the driving force," says John Fung, chief of transplantation and one of the three surgeons who performed the baboon-to-human liver transplantation. "He's very aggressive. And that's helped to push things along here. It's not Starzl's philosophy to sit back and watch other people do it."
The primary obstacle to transplantation is rejection of the donor graft by its host, as the host's immune system reacts to the presence of the foreign organ by attacking it. With xenotransplantation, this response is more complex and more virulent than with allotransplantation.
When an organ is transplanted between one human and another, the organ is confronted by what is called cellular rejection, a response mediated by T cells--a type of lymphocyte, or white blood cell. But when an organ is transplanted between species, other white blood cells, B cells, are stimulated to produce antibodies to attack the organ in what is called humoral rejection.
Controlling humoral rejection, also called the antibody barrier, is the special problem to be surmounted in xenotransplantation. Toward this end, microsurgeon Noriko Murase conducted crucial experiments at Pittsburgh, transplanting hearts and livers from hamsters to rats in the presence of different immunosuppressive drugs to determine which would be most effective. In particular, Murase was looking for drugs to use in combination with the experimental antirejection drug FK506 (Liz Marshall, The Scientist, Feb. 5, 1990, page 24).
Starzl and Satoru Todo, another of the surgeons who would later perform the baboon-to-human liver transplantation, secured a small vial of FK506 in 1986 from the Japanese company developing it, Fujisawa Pharmaceutical Co. Ltd., Osaka, and began the first tests of the drug. "It was a pretty small amount," says Fung. "But it was enough to do a number of small animal studies in our microvascular lab, looking at heart and liver transplant survival. A small amount also went to the in vitro lab and the pharmacology group."
The studies showed FK506 to be an effective new immunosuppressive agent, perhaps 50 to 100 times as powerful as the most widely used antirejection drug today, cyclosporine, although the new drug's toxicity is said by some to be a problem. Since February 1989, transplant patients at the University of Pittsburgh have been taking FK506, and trials are now under way at a dozen medical centers around the world. "We know FK506 is a very potent drug," says Murase, "but it is not enough to treat a xenotransplantation. FK506 inhibits T cell proliferation, T cell immune activity, completely. But it has almost no effect on B cells."
So, she says, they began screening for drugs that would work well with FK506 and effectively suppress B cell activity. She and other microsurgeons transplanted hearts and livers from hamsters to rats and then treated the rats for rejection with combinations of drugs. A group of antiproliferative drugs, drugs that inhibit cell division and are used to combat cancer, emerged as likely candidates. Several of these drugs supported long-term survival in combination with FK506, although not long enough or consistently enough to suggest use in humans. But one drug called cyclophosphamide, when used with FK506, made a dramatic impression on the researchers. "All animals treated with this drug [and FK506] survived 100 days," which was the experimental target, says Murase. "Because these are small animals, their life spans are only a couple of years, so 100 days is almost the same as 10 or 20 years for a human being." In contrast, rats receiving transplanted organs and no drug treatment at all survived only about three days.
Cyclophosphamide had the added advantage of being commercially available, as an anticancer drug. Also, Starzl had tested the drug for use in transplantation some years earlier, before settling on cyclosporine, so he was already familiar with it. These two drugs are being used to spearhead the immunosuppressive effort in the baboon-to-human xenotransplantations. In addition, two other drugs, prostaglandin and a steroid called prednisone, are playing supporting roles in suppressing immune response to the xenografts. Prostaglandin helps control inflammation connected with humoral, or antibody-mediated, rejection. Pred-nisone interferes with cells that recognize foreign substances to begin immune response chain reactions.
Once they had achieved successful xenotransplantation between hamsters and rats with these drugs, the researchers began to study what effects the hamster livers had on their rat hosts. The liver, the body's largest gland, is responsible for creating many metabolically important substances, raising the question of metabolic compatibility. Similar questions will also be important in follow-up studies on the baboon-to-human transplantation, says Starzl. The implications are far from clear.
"There are some strange things," Starzl said, several weeks before the patient's death. "For example, the baboon has no uric acid, because the baboon liver has enzymes that metabolize uric acid. This patient was actually above normal as far as uric acid [before the transplantation]. Now, he doesn't have any uric acid anymore. So it's going to take years to work out all these details. He's baboonized his whole metabolism."
Members of the animal rights community, nationally and locally, have objected to the Pittsburgh baboon-to-human liver transplantation, criticizing what they see as the immoral use of animals, but also questioning what is viewed as the extravagant use of health care resources for "glory medicine" when basic care remains unavailable to many.
"We're entirely opposed to using animals as a source of spare parts," says Sue Brebner, education director for People for the Ethical Treatment of Animals (PETA) in Washington, D.C., and herself a registered nurse and hepatitis B survivor. "But we don't blame this individual patient for having made this choice--we blame the medical system for having given him no other option. It's very important to realize you get answers where you invest your money. If you pour all your time and energy and money into animal experiments and animal transplants, that's where your results will be obtained. If [medical researchers] had chosen a different path, maybe they would have had something to offer this man."
"It's a very expensive and slow way to kill someone," says Ingrid Newkirk, PETA's director. "We shouldn't call it therapy, because every single person who has received an animal organ has died. But, were it to work, I would still be morally opposed to it." After the operation in June, a number of animal rights organizations picketed the hospital, including some groups from the Pittsburgh area.
Ray Tesi, a liver transplant surgeon at Ohio State University Hospital, Columbus, suggests that society will not find easy answers to questions of animal rights and the appropriate use of medical resources.
"I don't know how you differentiate between a cow going to slaughter for your steak tonight--or your liver and onions, let's say--and a baboon going to slaughter for `Joe Smith's' liver, so he can live," says Tesi. "In fact, in some ways, you're better off sending the baboon to slaughter. The animal ethics issues are really muddled because we're a meat-eating society. "And when people develop end-stage liver disease," he adds, "they don't get diagnosed and put on a park bench to wait there until they die. They consume a lot of medical resources during those days, months, or years of progression of their disease. "The cheapest thing to do is let them die, but we don't do that in the United States."
The group at Pittsburgh also understands that important ethical questions are raised by what they are doing. John McMichael is a biologist in the department of transplantation who, at Starzl's request, helped coordinate activities between the operating room where the baboon's liver was removed and the human operating room where the patient was prepared to receive the transplant. He recalls the scene as surgeon Todo and his team "harvested" the liver from the anesthetized baboon.
"For every person standing in there, it was an emotional moment," McMichael says. "But what are we supposed to do? People are dying. I don't know what the answer is. [Xenotransplantation] gets around a big problem that we have. The organ shortage problem has gotten to the point now where it's got to be addressed."
"We all love animals," says surgeon Tzakis. "But we have a disagreement [with animal rights activists] in the value that we place on the life of the animals. We think that, if a human life is to be saved by the sacrifice of an animal, the sacrifice of the animal is justified." "I think we've created need for reflection," says Starzl. "I respect their points of view. I don't think they're nuts at all."
Even with the careful laboratory work in preparation, the Pittsburgh xenotransplantations remain a gamble. Can they be made to work as therapy? Was this the right time to move ahead with xenotransplantation? "It will remain experimental for some time to come," says Pittsburgh's Tzakis. "The possibility is there that this might be a very useful procedure in the future, but, for now, we need to go through the stage of learning everything there is to learn." "I think you could qualify this as an unequivocal success," said Ohio State's Tesi shortly before the patient's death. "Nothing in any science works perfectly the first go-round. Starzl, true to the medical scientist that he is, has been very careful to get as much information from the various angles this patient presents as he can, while he can. There's a hell of a lot more knowledge that will be accrued from this."