Jeff Hitchcock also has a 
report on the DRI Conference

The mandate of the Diabetes Research Institute is simple - Just Cure It! During the 3-day A World of Hope conference in Miami, we saw a facility and a team of people focussed exclusively on one goal - curing diabetes. There was no talk of excellent science, only of excellent results. We had a chance to hear about the major research programs underway, all of which had a direct and obvious line from bench to bedside.

Hurricane Irene hit south Florida on the first day of our visit to Miami. Despite the relentless wind, rain and flooding, the conference was largely unaffected. Fortunately, most people arrived just before or just after the worst part of the storm. The people of Florida were left once again with massive damage and disruption. 

The following summary discusses some of the major research programs that we learned about through visiting the DRI labs, talking to the researchers, presentations of progress and challenges, and reports from researchers with whom DRI is co-operating to help bring their work to clinical reality.

Daniel H. Mintz, MD...
Mary Lou Held Professor of Medicine
Scientific Director Emeritus
Diabetes Research Institute

Dr. Mintz is the driving force behind the Diabetes Research Institute and, as I learned, a truly remarkable human being. DRI was originally formed in 1971 by a group of parents who wanted to create an environment in which research focused on curing diabetes could flourish. A few years later, Mrs. Miki Mele whose daughter has diabetes approached Dr. Mintz and convinced him to share their dream. Dr. Mintz then brought his legendary intellect, energy, powers of persuasion, and humanity to bear, and the rest is history. In his moving talk to the conference, Dr Mintz spoke of this meeting with Micki Mele, and said, "I gave her reason to hope. She gave me purpose".

Perhaps one of Dr. Mintz's most amazing non-scientific accomplishments was the creation of DRI's new research facility. The original funding for the magnificent DRI building came entirely from the AFL-CIO Building and Construction Trades. Thanks to the AFL-CIO who have covered all the capital and operating costs of the physical building and its infrastructure, almost 100% of DRI's donated dollars go directly to cure research.

Dr. Mintz was involved in all aspects of the DRI building, including the efficient layout of the laboratories, utility corridors, and patient clinics. The building is optimized for the rapid movement of research from inception to cure.  As researchers enter the building, they walk through the waiting area for diabetes patients visiting the clinic. The building never lets them forget their true mission. This achievement demonstrates the kind of outstanding, and all too rare, leadership that has kept DRI clearly focused on its goal of curing diabetes.

On Saturday night, there was a dinner honouring Dr. Mintz. Speaker after speaker told of their personal experiences, and a portrait emerged of extraordinary intellect, will and compassion. Throughout the building of DRI and all the years of research, Dr. Mintz has remained a diabetes doctor, never forgetting what DRI is all about.

Camillo Ricordi, MD...
Scientific Director and Chief Academic Officer
Chief, Division of Cellular Transplantation
Diabetes Research Institute

Dr. Camillo Ricordi has worked in the field of islet transplantation for many years. His method of isolating islets from pancreatic tissue has become a standard used by researchers throughout the world. Dr. Ricordi characterized DRI as follows:

• Over 95% of the effort at DRI is focused directly on finding a cure for diabetes
• Other diabetes research goals such as prevention, management and complications - while recognized as important - are not emphasized at DRI. This group has chosen to specialize in finding the cure. There is no behavioural or compliance research.
• There is no "science for the sake of science". Every research program must be defended by its potential to advance a cure for diabetes.
• No researcher is allowed to make a career out of studying mice, "learning more and more about less and less". Research must move from basic science (rodents) to pre-clinical trials (large animals) to clinical trials (people). The entire facility is build around this translational model. 
• DRI will run pre-clinical or clinical trials for any promising technology in the field of islet transplantation, while absolutely protecting the intellectual property rights of the developer. DRI is testing technology from both academic and industrial research programs. The developer retains full rights and DRI maintains confidentiality. This facility allows small research groups to take advantage of the animal and clinical testing facilities available at DRI.
• DRI has instituted cGMP (current Good Manufacturing Practice) that helps assure the repeatability of procedures and speeds up regulatory approval for trials.
• Operating funding for research at DRI come from the following sources: 40% National Institutes of Health (NIH), 5% Juvenile Diabetes Foundation (JDF), 0.5% American Diabetes Association (ADA), and the balance from the DRI Foundation (DRIF) that raises money through the generosity of private individuals and companies.
• DRI will work with anyone to accelerate finding a cure for diabetes. 

Speaking personally, I have not seen another research facility in which every program is so directly aimed at finding a cure for diabetes. The motto Just Cure It! expresses the impatience and intense focus of DRI. 

Norma S. Kenyon, PhD...
Associate Professor of Medicine
Director, Pre-Clinical Research
Diabetes Research Institute

Dr. Kenyon has achieved the remarkable milestone of reversing diabetes in monkeys by transplanting mismatched islet allografts without conventional immunosuppression. In 3 of the 6 monkeys, the animals were free of injected insulin one year after transplant, and one monkey has been insulin-free for 544 days. Dr. Kenyon used the new immunomodulating agent, anti-CD154 (anti-CD40 ligand). The monkeys have shown no adverse effects and graft survival has been maintained with a monthly injection. The islets were transplanted into the portal vein, and lodge in the liver. 

The breakthrough in Dr. Kenyon's pre-clinical trials is the use of an anti-rejection agent offering the following characteristics:

• Anti-CD154 can be administered through a monthly injection, as opposed to massive numbers of pills per day required for conventional immunosuppression.
• Anti-CD154 has shown none of the adverse effects normally associated with conventional immunosuppression.
• Anti-CD154 was able to arrest a rejection episode after it had started.
• Anti-CD154 is not diabetogenic, meaning it does not worsen diabetes. Conventional immunosuppression suppresses insulin production by about 2/3 in transplanted islets, forcing the islets to work three times harder.
• Dr. Alan Kirk, one of the original developers of anti-CD154, was quoted on the possible unknown effects of the agent: "There are bound to be some side effects, but so far we haven't seen any."  Since it is impossible to prove that anything is 100% safe (no one can prove a negative), these are very reassuring words from a scientist. 

DRI now has approval to begin a clinical trial using anti-CD154 and human islets. The results of this trial will be a very important step in finding a cure for diabetes.

Dr. Kenyon is the mother of Laura, a young child with diabetes, and agrees wholeheartedly with DRI's mandate - Just Cure It! 

Rodolfo Alejandro, MD...
Professor of Medicine
Director, Islet Transplantation
Diabetes Research Institute

Dr. Alejandro is in charge of islet transplantation trials at DRI. He described the preferred method for transplanting islets, which involves a small incision in the abdomen and a catheter inserted into the portal vein leading to the liver. A volume of islets are then dripped through the catheter and take up residency in the liver where they can receive a rich supply of blood.  During the 20-minute procedure, the patient is monitored to assure that hypertension does not develop while the islets are being introduced. The procedure is minor and safe, and avoids the cost and surgical risks associated with whole organ transplants.

Dr. Alejandro discussed some of the partial successes in islet transplantation, normally in conjunction with a kidney transplant. Some patients failed to become fully insulin-independent, yet experienced dramatic improvements in glucose control, elimination of hypoglycemic episodes, restoration of normal HbA1c, and arresting of complications. I think we would all agree that a few injections are not the problem, it's the inability to achieve anything close to normal blood glucose control.

Luca Inverardi, MD...
Associate Professor of Medicine
Director, Basic Research
Diabetes Research Institute

Dr. Luca Inverardi is specializing in xenotransplantation of pig islets. He presented the terrible statistics on organ donation in the United States:

• 60,000 people awaiting organs
• 8,000 available organs per year
• 1,000,000+ people with type 1 diabetes
• 4,000,000+ people with type 2 diabetes without endogenous insulin production
• 2-4,000 pancreases available per year

• Reasonable physiological compatibility
• Ease of breeding and raising in a specific-pathogen-free environment
• Possibility of genetic modification to reduce rejection
• Large litters
• Rapid growth from birth to maturity
• Possible avoidance of recurring autoimmunity, as pig insulin may not present the same provoking antigens as human insulin

Dr. Inverardi is working on ways to reduce the initial inflammatory response that occurs locally when pig tissue is introduced. If this inflammation can be reduced, then subsequent rejection processes are likewise weakened.

Gordon Weir, MD...
Professor of Medicine, Harvard Medical School
Associate Director of Research, Joslin Diabetes Center

Dr. Weir spoke of the state of the art in islet encapsulation. These immunobarriers are a potential method for protecting islets from an attack by the host's immune system, without any systemic effect of the immune system. An immunobarrier (whether a microcapsule or a macrocapsule) works like a screen door, allowing the small molecules of oxygen, glucose, sodium, and insulin to pass freely, while blocking the larger components of the immune system. Because of their inherent safety and lack of systemic effect, immunobarriers hold a lot of appeal in islet transplantation, especially in children.

Dr. Weir described the major challenges associate with encapsulation today:

• Different requirements for protecting allografts, xenografts and engineered cells
• Packing density of islets required to facilitate a manageable volume of transplanted material
• Survival of the islets, especially assuring an adequate oxygen supply
• Dynamics of insulin release across the semi-permeable membrane to prevent excessive slowing of insulin response to glucose, especially during periods of rising and falling glucose
• Biocompatibility of the immunobarrier material that could lead to fibrosis, thereby choking the islets even though they have been protected from a direct immune attack
• Finding the right balance between an open barrier (excellent insulin response dynamics, weaker protection) and a tight barrier (slower insulin response dynamics, better protection)
• Intrusion of small immune molecules, including cell killers such as free radicals and cytokines, that may be produced near the capsules. Even though these non-specific killers will attack all cells in the vicinity, islets are particularly sensitive to their assault.

Christopher Newgard, PhD...
Touchstone Chair in Diabetes Research
Professor of Biochemistry and Medicine
University of Texas

I have followed the work of Dr. Newgard for several years. Each year, it seems some of the problems of the year before are solved, and the goal of islet replacement comes closer to reality. Dr. Newgard has been working with Betagene (a company he founded to take his discoveries to the marketplace) and Gore Hybrid Technologies. As a result, he has not been free to discuss many of the details of his work. This time we learned more, and I like what I heard.

Dr. Newgard's approach to this problem is unique and different from most other researchers. He has been working to engineer a cell line that will behave like human islets while being better equipped genetically to survive in the transplant environment.

Cell lines are malignant tumour cells that will continue to divide indefinitely until stopped by some external condition. As such, these cells are considered "immortal". Dr. Newgard has started with insulinoma cells from a type of tumour that secretes insulin continuously, but not in response to blood glucose levels. By inserting genes into these cells, Dr. Newgard has created a cell line that is responsive to glucose. By inserting different genes, he has managed to increase the insulin output per cell 30-fold, allowing for smaller devices. Last year, the cell line had a serious remaining flaw that made it release insulin until blood glucose dropped to around 20 mg/dl (1.0 mmol/l). Obviously, such a low glucose target would leave transplant recipients in a constant hypoglycemic fog. Over the past year, Dr. Newgard's team has successfully inserted the genes that raise the target to the physiological level normally found in people.

Perhaps the biggest surprise was to finally learn about the Gore immunobarrier device, which is very clever in its simplicity. The device is a flexible U-shaped tube whose ends are blocked by removable plugs. This device is placed below the skin, possibly on the forearm, and contains no cells when first implanted. After several weeks, an extensive network of blood vessels develops and integrates itself into the wall of the device tube. Once vascularized, a small incision provides access to the plugged ends of the tube, the plugs are removed, and a flexible cartridge containing the insulin-producing cells is inserted. The ends of the tube are plugged, and the incision closed. Because of the extensive vascularization, a ready supply of oxygen and glucose is delivered to the cells, and the insulin is carried to peripheral body cells. 

Dr. Newgard has opted for a fairly open immunobarrier to assure optimal insulin release dynamics. Of course, such a barrier leaves the islets exposed to attacks from free radicals and cytokines. To overcome this problem, the cells are put through a form of Darwinian selection (nature's alternative to direct gene insertion for modifying DNA to achieve desired characteristics). Each batch of cells is exposed in vitro to an assault of free radicals and cytokines. The few surviving cells (as low as one percent) are recovered and again assaulted in vitro. After several passes, the surviving cells are, to quote Dr. Newgard, "tough as dirt". Of course, the progeny of these cells will provide a virtually limitless supply of islets that are likewise resistant to free radicals and cytokines.

What are the remaining challenges? So far Dr. Newgard has created his cells using a rat cell line. For optimum immunocompatibility, he is now working to apply the same genetic enhancements to a human insulinoma cell line. His work with the rat cells has taught him about the genes required to achieve the desired characteristics of glucose-responsiveness, insulin production, and physiological target levels.

The Gore device has a number of desirable characteristics, including:

• Ease of replacing the cell cartridge. As a new cartridge is inserted, the depleted cartridge is pushed out the other end of the U-tube. The vascularized network is not disturbed.
• Containment of the dividing cells. The device acts as a growth limiter on the cells. The cells will continue to divide until they are pressed against the inside of the U-tube wall. In such a setting, the cells reach their optimum population level, and cannot escape into surrounding tissue.
• The device is both tough and flexible, assuring reliable containment and performance despite physical abuse.

Alberto Hayek, MD...
Professor of Pediatrics / Director of Islet Research Laboratory
University of California at San Diego
Whittier Institute

With his characteristic humour and self-deprecating anecdotes, Dr. Hayek provided important insights into alternate sources of islets and the NOD mouse model of diabetes. On the NOD mouse, he described a Japanese paper presented at a recent conference that was considered interesting because the researcher had actually failed to prevent autoimmune diabetes from developing in these animals. His point is that researchers who spend years finding ways to prevent the onset of autoimmune diabetes in NOD mice are doing little for people with diabetes, as these creatures can be saved from the disease with just about any intervention.

Dr Hayek spoke of the challenges and opportunities of proliferated human islets. If this technology could be made viable, then a small supply of human islets could be expanded to meet the needs of everyone who could benefit from an islet transplant. These would be human islets, and so the immune protection challenge would be lessened.

While it is possible to take a small number of human islets and expand them hundreds or thousands of times using specific growth factors, the expanded cells will no longer secrete insulin. To overcome this problem, genetic engineering of the expanded cells is being considered. Dr Hayek sees the challenge not in engineering the cells to secrete insulin, but to assure that the insulin secretion is glucose-responsive. To drive home his point,  he joked that you could engineer fiberglass to secrete insulin.

Dr. Hayek also discussed the possible use of human fetal islets, while emphasizing the enormous scientific and ethical hurdles. He talked about a clinical trial that his group had planned to place human fetal islets subcutaneously in 10 people, but the trial was blocked for regulatory and ethical reasons.

Another possibility for creating new cells is islet neogenesis, the internal differentiation of pancreatic duct cells into functioning islet cells. While a reliable agent has not been found to promote such neogenesis, it is an appealing goal since the islets would be "self" and would be situated in the pancreas. Survival of these new cells would require suppression of autoimmunity that caused the diabetes in the first place, but no other immunosuppression or protection. Some researchers are optimistic that anti-CD154 may also have the potential to create autoimmune tolerance for islets in a person with diabetes. Until the necessary growth factors that cause pancreatic duct cells to become islet cells are identified and isolated, this goal remains elusive. It is conceivable that after several decades of curing diabetes using transplanted islets, islet neogenesis may be the next generation, just as bioengineered insulin recently replaced animal insulin after 60 years.

Elizabeth S. Fenjves, PhD...
Associate Professor of Medicine
Diabetes Research Institute

Dr. Fenjves is researching the best methods for inserting specific genes into cells in order to achieve defined characteristics. Her labs uses various vectors to carry the gene fragments into the target cells. These vectors include special viruses and lipids (lipofection). She has tested the gene insertion capability of these vectors by carrying genes that cause specific colour changes in the cell. After successful infection of the cell, the marker colours are clearly visible.

The goal of Dr. Fenjves's research is to create an efficient method for introducing genes into islet cells to cause expression of proteins that are desirable for successful transplantation, as well as knocking out genes that produce detrimental proteins.

These methods could be applied either to the donor animal germ cells to create a transgenic pig optimized for human islets, or can be performed directly on the islets after harvesting and isolation.
 

Alberto Pugliese, MD...
Research Assistant Professor of Medicine
Head, Immunogenetics Laboratory
Diabetes Research Institute

Dr. Pugliese is an expert on the immunology and genetics of diabetes. He has done a great deal of research looking at how to prevent the autoimmune attack that leads to diabetes. This research is very important both for future prevention strategies, as well as stopping autoimmune destruction of transplanted islets.

A major part of Dr. Pugliese's work has looked at the role of the thymus gland in the immune system. He described the thymus as the "school for the immune system". All immune cells are forced to pass through the thymus gland where they are exposed to the spectrum of antigens present throughout the body. Any immune cell that binds to these normal antigens is destroyed, thereby preventing the later destruction of healthy cells. If no binding occurs, then the cell is deemed to be friendly to host tissue and is released to become part of the immune system.

Even though it is generally believed that islets are the only body cells that release insulin, there are in fact other cells that release tiny amounts of insulin, but not in response to blood glucose. These cells present insulin to the visiting immune cells in the thymus, and any immune cell that binds is killed. It is believed that a low insulin output in these "decoy" cells in people who develop diabetes may be the reason that immune cells are allowed to live that will later track insulin back to its source and destroy healthy islets.

In people who have the genetic markers that protect against diabetes, these cells secrete more insulin than they do in people with genes that pre-dispose them to diabetes. The more insulin in the thymus, the more likely that insulin-specific autoreactive lymphocytes will be killed, with fewer chances of developing diabetes. 

Dr. Pugliese also spoke of the various triggering events that seem to lead to diabetes in people with a genetic predisposition, although DRI is not focusing resources on this line of investigation.

Ricardo Pastori, PhD...
Molecular Biologist
Diabetes Research Institute

Dr. Pastori is focusing on developing novel ribozyme technology to prevent the expression of proteins that lead to inflammation and death of transplanted islets. Ribozymes are presently seen as a novel, exciting tool both for research and the manipulation of gene expression for therapeutic purposes. 

Ribozymes are catalytic RNA molecules that can specifically cleave messenger RNAs and thus prevent the expression of unwanted proteins. The unique properties of ribozymes have recently been exploited to modify gene expression.  For example, ribozymes can be engineered to abrogate the expression of a specific RNA transcript and to therefore prevent the release of the corresponding molecule that promotes cell death.  Such strategies are being developed for certain types of cancer and viral illnesses to prevent or reduce the expression of RNAs coding for viral or cancer-related proteins, and several clinical trials are currently testing ribozymes for different diseases. 

Non-specific inflammation, mediated by cytokines, occurring at the site of implant is deleterious for islet cell grafts.  For instance, pro-apoptotic genes, such as those coding for inducible nitric oxide (iNOS) and Fas, are activated by pro-inflammatory cytokines and significantly impair graft survival by inducing apoptosis (programmed cell death) of transplanted islets. Dr. Pastori is using ribozyme-mediated inhibition of these and other genes to "immunoprotect" islets, rendering them less susceptible to apoptosis. 

Jackie Warren Demijohn
Islet Transplant Recipient
Diabetes Research Institute

Jackie Demijohn spoke to the conference at lunch on Saturday, and told her amazing story. On September 11, 1998, Jackie received an islet transplant at DRI. To prevent rejection, Jackie was treated with an immunomodulating agent called anti IL-2 receptor antibody and a bone marrow infusion from the donor. In addition, she is taking conventional immunosuppression and expects to be tapered off these drugs starting in a few weeks. The hope is that the bone marrow will create a state known as chimerism in which Jackie's immune system will be mostly her own, but a little bit of the donor's. The experience has been that an infusion of bone marrow creates greater tolerance of transplanted tissue and organs.

Jackie spoke eloquently of the terrible state she was in before the transplant, with uncontrollable blood glucose levels, and almost every diabetic complication. Today, she enjoys near normal blood glucose control with minimal injected insulin and a profound improvement in her general health and sense of well-being. She talked about her career as an Abuse Counsellor, something that would have been impossible in her previously uncontrollable and deteriorating condition.

Jackie's talk reminded us all of why DRI exists. Just Cure It!