Stacy Neumayer, and brother Travis Hurd

Stacy Neumayer loves to travel and hike — not easy things to do when you're on dialysis. She has suffered kidney failure since she was 15. By 2001, at age 36, she had undergone four kidney transplants and had been told she would not be eligible for another one because she had antibodies against all of her potential living donors. The news meant her life would be restricted by three, four-hour dialysis treatments every week for the rest of her life.

Neumayer accepted her prognosis. Determined to enjoy whatever time she had left, she found a dialysis center in Colorado where she loves to hike, and set about living her life to the fullest. However, her youngest brother, Travis Hurd, who had been tested and told he did not match with Neumayer, still passionately believed he would one day donate a kidney to his sister. He monitored developments in the kidney transplant field and discovered an innovative program at Mayo Clinic that seemed to be custom-designed for them.

Pulling down Antibody Barriers

In fact, it was individuals like Stacy Neumayer that Mark Stegall, M.D. had in mind.

Dr. Stegall is the Mayo kidney transplant surgeon and researcher who put together a multidisciplinary clinical team to offer patients treatments that overcome antibody barriers to kidney transplant.

Attempts to overcome barriers to kidney transplants are not new. In the 1980s some patients were successfully transplanted following a procedure called plasmapheresis, a blood-filtering process that replaces antibody-carrying plasma with albumin and a saline solution. But it was not embraced by the transplant community because it was expensive and labor-intensive. In Japan , however, where deceased donor kidney transplantation is unpopular, scientists saw the procedure as the only way to save many lives. In 1998, they surprised the transplant community when they reported an eight-year follow-up study on 61 patients who had had ABO incompatible transplants. Its publication coincided with Dr. Stegall's move to Mayo Clinic.

Mark Stegall, M.D.

"Mayo is an institution that firmly believes the patient comes first and there are more than 7,000 people in the United States in this difficult-to-transplant group that we could help," says Dr. Stegall. "ABO incompatibility or a positive crossmatch due to anti-HLA (Human Leukocyte Antigen) antibodies was once an absolute roadblock to kidney transplantation. Most of these people die while waiting for a negative cross-match deceased donor kidney, yet most of them would be candidates for living donor kidney transplantation if we could overcome the destructive effect of antibodies. In addition, these procedures are cost-effective when compared to maintenance dialysis therapy."

ABO Incompatible Kidney Transplant

ABO incompatibility refers to the immune reaction that occurs when different blood types are mixed together. The presence or lack of molecules on the surface of the blood cells defines your blood type. Type O has no molecules. Types A and B each have a combination of two molecules, which result in type A (AA or AO molecules), type B (BB or BO molecules), or type AB. When blood types are mixed, the molecules act as antigens that trigger an ABO incompatibility reaction. That's why blood types must be matched.

Dr. Stegall's multidisciplinary team included transplant nephrologist James Gloor, M.D., transplantation biologist Jeffrey Platt, Ph.D., histo(blood)compatibility expert Breanndan Moore, M.D., renal pathologist Donna Lager, M.D. and Alvaro Pineda, M.D., who directed Mayo's apheresis laboratory.

"We modified the Japanese protocol for ABO incompatibility kidney transplants and began performing them in May, 1999," says Dr. Gloor. "Our success rates almost equaled those performed with blood-type-compatible recipients."

The type of preconditioning done to cleanse the blood of antibodies depends on the patient's blood type and the amount of antibodies present. In general, it involves some combination of the following therapies before transplantation:

• Plasmapheresis — to physically remove antibodies.
• Immunoglobulin — also called gamma globulin, which appears to decrease antibody activity destructive to the graft. The mechanism is poorly understood since gamma globulin are themselves antibodies that disarm foreign antigens by binding to them.
• Splenectomy — the spleen concentrates B lymphocytes around its blood vessels to fight infection so removing it in a person with very high levels of antibodies wards off graft rejection.
• Anti-CD20 antibody (rituximab) — depletes the CD20 protein, which is found on the wall of most B cells.

James Gloor, M.D.

Recipient antibodies are monitored during the first two weeks following transplantation and treated by plasmapheresis if they rise above a predetermined level. Like all transplant recipients, the patients must remain on drugs that suppress the immune system, such as thymoglobulin, tacrolimus, mycophenolate mofetil, and prednisone, for as long as the grafted kidney survives.

Positive Crossmatch Transplant

The HLA major histocompatibility complex (MHC) is a large group of genes, located on a single chromosome, which influences the ability of different cell types to collaborate and produce or suppress immune responses. A positive crossmatch to the histocompatibility antigens blood test means you have antibodies against donor HLA antigens—a decidedly negative result for anyone waiting for transplant. The antibodies can develop as a result of blood transfusion, pregnancy or organ transplant. Specialized plasma cells, called memory B-cells, are long-lived and can quickly recognize and respond if the same antigen invades the body again. Neumayer was told she could not have any more transplants because her immune system developed anti-HLA antibodies to each of her four previous transplants.

"The protocols from Johns Hopkins and the University of Maryland were very similar to our ABO protocol, so we tried it for patients with positive crossmatch," says Dr. Gloor. "The success of these strategies has allowed individuals with kidney failure to avoid lengthy or indefinite waiting periods for deceased donor transplants."

Mayo initiated its clinical trial in January 2000. The following January, Neumayer's blood was successfully preconditioned and she received a donor kidney from her brother on January 9, 2001. When she turned 40 recently, there was none of the standard milestone birthday celebration.

"January 9th is the day I celebrate," says Neumayer. "That's the day I started to live."

From Bedside to Bench

Mayo's Kidney and Pancreas Transplant Program is one of only three performing positive crossmatch and ABO incompatible kidney transplants. Mayo has performed the most —more than 150. While Dr. Stegall is encouraged by the team's early experiences, he is aware that many of the mechanisms of transplant are not well understood and anticipates that furthering knowledge in the field will lead to improvements for his patients.

"This is a totally new area," says Dr. Stegall. "Our studies were rationally designed to remove the antibody or kill the cells that were making them and we have identified and stopped using some therapies as not helpful. But we have very poor control over the antibody-producing cells."

Dr. Stegall says it was research that enticed him to study medicine in the first place. Later, he was mentored by a scientist who regretted that his research was not more clinically applicable. It's an acknowledgment that has influenced Dr. Stegall throughout his career; his lab projects have all been dictated by the challenges presented in his clinical practice. Currently, his lab is focusing on three main areas: antibody-producing plasma cells; accommodation—how the kidney transplant acquires resistance to antibodies; and improving long-term survival of all kidney transplants.

Antibody Production Factories

Antibodies at work

Plasma cells make up an important part of your immune system. They are mature B lymphocytes (white blood cells), often referred to as B cells by scientists. They are produced and mature in the bone marrow and concentrate in the spleen and lymph nodes. Your body produces millions of types of B cells every day. Each type has a unique receptor protein, called the B cell receptor (BCR) on its membrane that is designed to fit one specific antigen.

Antigens are substances, such as bacteria, viruses and pollen, which produce an immune response because they are foreign to the body. When the BCR binds to the antigen molecule, the B cell engulfs it and breaks it up. The molecules that result are called histocompatibility molecules. Each of us has a set of histocompatibility molecules that are like fingerprints in that probably no other human being has an identical set. Normally, they protect us from infection and disease. In the case of transplants, however, they are responsible for antibody-mediated rejection of a transplanted organ because they act as antigens when introduced into a different person.

Dr. Stegall thinks that research into the process of how antibodies are produced may lead to novel therapies that interfere with antibody production.

"One of our major goals is to identify and characterize the plasma cells that make the antibodies against the donor HLA antigens or the donor blood group," says Dr. Stegall. "We perform bone marrow aspirates on our transplant patients at the time of transplant. We've also studied the plasma cells from spleens that we removed from people who have more difficult problems with rejection."

Dr. Stegall collaborates with industry to study how certain drugs influence plasma cells. One study is testing a drug that will keep the antibodies from damaging the kidneys.

"One of the ironic things we've discovered is that drugs that kill plasma cells in the test tube have no effect when we repeat the test in animal models," says Dr. Stegall. "We have also discovered that a certain kind of long-lived plasma cell is a particular problem in antibody-mediated rejection."

Accommodation

Interestingly, some grafts are known to survive despite the presence of circulating anti-donor antibodies. This acquired resistance to antibody-mediated damage, termed "accommodation" is poorly understood. Dr. Stegall's lab is studying well-functioning ABO-incompatible grafts that meet the criteria for accommodation to find out why they survive while others do not.

"We have demonstrated that the accommodated kidneys express hundreds of different protective genes when compared with ABO-compatible grafts treated with the same immunosuppressive regimen," says Dr. Stegall. "But we have found no alteration of singular genes associated with accommodation. So what's going on here? Do the plasma cells die? Does the kidney have some mechanism that allows it to tolerate high level of antibodies? Or is the patient's body producing a blocking mechanism? Our research is focused on answering these questions."

Both positive-crossmatch and ABO-incompatible living-donor kidney transplantations can be performed with survival rates approaching those of negative-crossmatch and ABO-compatible transplantations. However, for them to become more successful, additional methods to prevent antibody-mediated damage need to be developed. These include therapies that result in prolonged suppression of antibody formation.

The lab is currently evaluating the necessity of splenectomy versus intensive therapy with plasmapheresis and anti-CD20 monoclonal antibody. The next phase is to test therapies that may decrease antibodies.

What makes kidney grafts wear out?

In addition to his work to overcome antibody barriers, Dr. Stegall's laboratory is also interested in improving the long-term survival of all transplanted kidneys. "Our one-year graft survival rate for conventional kidney transplants is more than 90 percent but five-year grafts drop to 70 percent, and 10-years to 50 percent, so chronic graft deterioration is a problem," says Dr. Stegall. "The mechanisms are unclear but a likely candidate is ongoing inflammation leading to either scarring of the graft or progressive glomerular disease."

Scarring, or fibrosis, develops in the majority of living donor grafts and is associated with reduced function and survival, even when mild. In a recent published study, nephrologist Fernando Cosio, M.D., collaborated with Dr. Stegall's group and demonstrated three early histologic "markers" of subsequent kidney graft failure—interstitial fibrosis, mild cellular infiltration of the graft and glomerulopathy. Early identification of patients at risk for chronic graft loss could mean extended kidney graft survival if researchers can develop therapies to halt graft deterioration.

"My lab is focused on trying to figure out the mechanisms of chronic graft failure at the molecular level. We believe that there is a pro-inflammatory intragraft environment that encourages scarring," says Dr. Stegall. "We're trying to trace the process back to where the clinical biopsy looks normal, but where there is a gene expression profile that predicts the development of fibrosis."

"In gene expression studies, we have found that there is evidence of ongoing inflammation even in otherwise normal kidney transplant biopsies," says Dr. Stegall. "Some of the gene expression changes may not be deleterious but are there as a result of the act of transplantation."

Keeping the passion for further improvements

Dr. Stegall has had a lab ever since he has been in clinical practice and is passionate about research. He says you have to be when much of your time is taken up with clinical practice and administrative duties. Dr. Stegall is surgical director of the Kidney/Pancreas program, Chair of the Division of Transplant Surgery and Chair of the UNOS (United Network for Organ Sharing) Kidney and Pancreas Transplant Committee.

"They don't page you to come and do research," he jokes. "But it's easy to be passionate about it. Our research also has helped to grow our transplant program — we are now the fifth or sixth largest kidney transplant program in the U.S. and the largest living donor program. And the laboratory also serves an educational role. It's a great place for residents and fellows to develop their ideas and increase their understanding of the transplant process."

Dr. Stegall is resolute that both educational and research efforts remain focused on helping patients.

"It is our expectation that future trials will lead to a greater understanding of the mechanisms underlying antibody-mediated rejection, provide patients greater access to living-donor kidney transplantation, and improve the survival of kidney transplantation recipients."