Twenty Years of Clinical Islet Transplantation at the Diabetes Research Institute

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CHAPTER Twenty Years of Clinical Islet Transplantation at the Diabetes Research Institute – University of Miami

A. Pileggi, C. Ricordi, N.S. Kenyon, T. Froud, D.A. Baidal, A. Kahn, G. Selvaggi, and R. Alejandro

Clinical Islet Transplant Program and Cell Transplant Center, Diabetes Research Institute, University of Miami - Leonard Miller School of Medicine, Miami, Florida

Intensive insulin therapy with the goal of maintain- lowing pancreatectomy due to pain associated with ing blood glucose concentrations close to the normal chronic pancreatitis, benign neoplasm, or trauma)(9-15). range can delay or prevent the onset of diabetes compli- Transplantation of allogeneic islets is mainly performed cations in patients with type 1 diabetes mellitus in patients with T1DM: allogeneic islets have been trans- (T1DM)(1). This goal is difficult to achieve in the major- planted in patients with end-stage renal disease (ESRD) ity of patients and is associated with a three-fold increase together with the kidney (simultaneous islet and kidney; in severe hypoglycemia. Transplantation of pancreatic SIK) and in patients with a stable kidney allograft (islet

islets in patients with T1DM can restore β–cell function after kidney; IAK)(16-22). In recent years, transplanta- and provide a more physiological glycemic control than tion of allogeneic islets has also been performed as islet ISLETS - MIAMI, FLORIDA exogenous insulin administration (2). transplantation alone (ITA) in patients with brittle diabe- Steady progress in the field of β–cell replacement tes characterized by hypoglycemia unawareness and has been observed in recent years (2, 3), starting from progressive diabetes complications for which the risk of the seminal work by Lacy, et al (4, 5) in the late 1960s life-threatening hypoglycemia overweighed those of the demonstrating reversal of hyperglycemia by islet trans- transplantation procedure and chronic immunosuppres- plantation in diabetic rodents. Numerous challenges sion (23-31). In a smaller number of cases, transplanta- have been overcome, while many still remain unsolved tion of allogeneic islets has also been performed for dis- (2, 3). Technological advances in organ procurement eases of the exocrine pancreas in patients with pancre- and preservation techniques together with improvements atectomy-induced diabetes (32-38), metabolic disorders in pancreatic islet isolation, purification and culture have such as cystic fibrosis, hemochromatosis, and liver cir- allowed for the increasing success rate of islet transplan- rhosis (39-44) in combination with lung or liver transplantation worldwide. Implementation of safer transplanta- tation, respectively, and in few patients with type 2 DM tion procedures has also contributed to the increasing (T2DM) (45, 46). numbers of transplants performed in the recent years. A It has been twenty years since the first islet trans- total of 493 allogeneic islet transplants (including histori- plant was performed by the Clinical Islet Transplant Pro- cal cases) and 240 autologous grafts from 51 centers gram (CITP) at the Diabetes Research Institute / Univer- (18 North American, 30 European, and 6 elsewhere) were sity of Miami School of Medicine. One hundred-one islet published by the International Islet Transplant Registry infusions have been performed in 73 patients during this in 2001 (6). In light of the increasing number of centers period. Here we present an overview of the activity of performing islet transplantation, the National Institute of our CITP and of the progress in the islet transplantation Health sponsored a North American Collaborative Islet field in recent years. Transplant Registry (CITR) that was established in 2001 METHODS (7, 8). Transplantation of autologous islets is currently a Human Cell Processing Facility. The Diabetes Re- therapeutic option for a number of diseases of the exo- search Institute - University of Miami School of Medicine crine pancreas leading to DM (iatrogenic diabetes fol- was the first academic research center to house a cur-

Clinical Transplants 2004, Cecka and Terasaki, Eds. UCLA Immunogenetics Center, Los Angeles, California 2 PILEGGI, RICORDI, KENYON, ET AL rent Good Manufacturing Practice (cGMP) facility dedi- gies, National Disease Research Interchange, Interna- cated exclusively to human islet cell isolation. This facil- tional Bioresearch Solutions, and International Institute ity operates following cGMP and Food and Drug Admin- for the Advancement of Medicine. istration regulations to ensure the highest standards of Organ harvesting is performed using standard tech- quality, purity, potency and safety for medical products niques by the surgical team (either local or distant), gen- destined for human use (47, 48). In addition to the isola- erally consisting of the removal of the gland along with tion of human islets for research or clinical trials, the duodenum and spleen (49). The pancreas is collected cGMP facility is also utilized for manufacturing human and stored in University of Wisconsin (UW) solution bone marrow cells (BMC). The Cell Processing and Dis- (DuPont-Pharma; Wilmington, Delaware) on ice and tribution Center of our cGMP facility currently provides shipped to our processing facility within 12 hours. In the human islets, as well as other cells, to more than 50 col- 2000’s, the use of oxygen carrier moieties laborating scientists worldwide. The cGMP facility in- (Perfluorodecalin; FluoroMEd, L.P.) was introduced in cludes clean room suites, and various laboratory sup- order to reduce ischemic injury during preservation (50, port areas over approximately 16,000 square feet. The 51). Briefly, the gland is immersed in preservation solu- access to the facility is restricted to authorized cGMP tion and maintained at the interface between oxygen- personnel only. The clean room suites meet ISO Class ated perfluorocarbon and UW (the two-layer method; 7 (ISO 14644) requirements with HEPA-filtered air con- TLM), as described by Kuroda, et al (52), and transported ditioning. Procedures pertaining to required dirty/clean to the cell processing laboratory within 24 hours. The flow patterns and directions are enforced by standard TLM is currently utilized in most of the pancreata ob- operating procedures, training, management and facility tained at our center, allowing us to improve islet yields design. All procedures, processes and equipment are even from marginal donors (51), and therefore improv- periodically validated, and testing ensures that they op- ing the utilization of islet preparations processed for trans- erate according to the pre-established performance plantation. specifications. Islet Isolation. Islets are obtained using the auto- Pancreas Procurement and Preservation. Human mated method (53) consisting of a mechanically-en- pancreatic glands are obtained from multi-organ cadav- hanced enzymatic digestion of the pancreas. Briefly, the eric donors through the University of Miami Organ Pro- pancreatic gland is dissected-free from surrounding tis- curement Organization (OPO) part of UNOS Region 3. sue. After dividing the pancreas through its neck, retro- Other OPO’s offering pancreata for clinical and research grade injection of dissociation buffer in the pancreatic to our facility include: Gift of Life, LifeGift, LifeQuest, duct is performed. Since the 1990’s, we use the en- LifePoint, The Sharing Network, LifeNet, Arkansas Re- zyme blend Liberase HI ® (Boehringer Mannheim-Roche) gional Organ Recovery Agency, Center for Donation and characterized by reduced endotoxin contamination and Transplantation, Translife, Washington Regional Trans- higher reproducibility, when compared to other collage- plant Consortium, Life Connection of Ohio, LifeSource, nase blends; this has significantly contributed to improved LifeCenter NorthWest, SouthWest Transplant Alliance, islet yields from human pancreata (54-57). New Mexico Organ Donor Network, New England Or- After distension, the pancreatic gland is divided into gan Bank, Hawaii, Life Legacy, Carolina Donor Services, 9-11 pieces and placed into the dissociation chamber. and other organizations like Tissue Transfer Technolo- The chamber (Fig. 1A) consists of two portions divided

Figure 1. Human islet isolation. The dissociation chamber (A) is a key element of the isolation process. It allows the efficient dissociation of the pancreatic tissue and enhances the effects of the enzymatic digestion. A peristaltic pump permits the circulation of the enzyme through the chamber via a circuit of tubes (B). Agitation of the dissociation chamber enhances the enzyme action, and the automated mechanical shaker (C) allows one to standardize the force applied to the chamber. After digestion, the pancreatic slurry is collected (D) and concentrated by centrifugation. Close monitoring of multiple variables may be of assistance to improve the outcome of the isolation (E). Samples of the pancreatic digest are collected (F) in order to assess islet number and appearance (G). Islets can be easily identified by the means of dithizone staining that confers red color to endocrine cells. Purification of

3 ISLETS - MIAMI, FLORIDA

the islets from the non-endocrine pancreatic tissue is performed using a computerized COBE centrifuge (H) by sedimentation on continuous or discontinuous gradients (I). The purified fractions are collected and assessed for purity and counted (J-K) before being cultured (L). All procedures are performed under cGMP standard following SOP and maintaining documentation (M). 4 PILEGGI, RICORDI, KENYON, ET AL by a mesh: the lower cylindrical portion contains the pan- continuous density gradients are utilized as first choice creatic tissue and marbles and has two inlets on its base; for the purification of all human islet preparations ob- the upper portion is conical with an outlet at its apex. tained at our center. Separate fractions are collected Digestion of the pancreatic tissue is obtained by the com- and assessed for purity (Fig. 1F-K); the fractions with bination of enzymatic activity at 37°C and the mechani- lower exocrine contamination are generally pooled for cal action of the marbles in the chamber that is shaken transplantation, paying attention that overall tissue vol- throughout the dissociation phase. A system of tubes umes do not exceed 10 mls. In order to maximize islet connected to the chamber and a peristaltic pump allow recovery, we have implemented an additional purifica- the circulation of the enzyme from the base of the cham- tion step on discontinuous Euro-Ficoll gradients that is ber through the mesh and toward its apex (Fig. 1B). The performed on the less pure fractions obtained with the hydrostatic pressure applied contributes to the removal standard continuous step (Fig. 1I). This ‘rescue’ purifi- of small pancreatic fragments from the chamber as they cation step has allowed us to increase the yields of hu- can be pushed through the mesh, preventing excessive man islet preparations and to meet the requirements for damage from mechanical injury. Numerous improve- transplantation (>5,000 IEQ/kg of recipient’s body weight) ments have been introduced in the dissociation cham- in a higher number of preparations, therefore maximiz- ber over the years, with the more recent transparent dis- ing the overall utilization of pancreata processed at our posable models allowing for real-time monitoring of the facility (64). digestion process (Fig. 1A-C), and the use of motorized Islet Assessment. Assessment of isolated islets is mechanical arms to better standardize the procedure a prerequisite to evaluate the efficiency of the procedure (Fig. 1C). The digestion circuit allows collecting samples and the quality of the final product before its release for and assessing islet integrity to decide when to stop the transplantation. Product release criteria following islet enzymatic digestion. This is obtained by collecting the isolation and culture include assessment of a number of tissue into cold media supplemented with serum, while variables to prevent the administration of adventitious continuing with the mechanical digestion until all tissue agents to the recipient, and to ascertain that viable and has been obtained from the chamber. The pancreatic potent islet cells are being transplanted (Table 1). In the digest is then concentrated by centrifugation (Fig. 1D), 1980’s, we described the use of dithizone staining and prepared for the subsequent purification step. Sev- (Fig. 1G and K) that binds to the zinc of secretory gran- eral variables may contribute to the outcome of islet iso- ules conferring a characteristic red color to endocrine lation. We are currently utilizing a continuous multi-para- cells (65). This quick method is now the gold standard metric monitoring system (Fig. 1E) to assess selected utilized for the assessment of islet integrity and of the physiological parameters (e.g., temperature, pH, pCO2, purity of islet preparations (Fig. 1G and K)(65, 66). Islet etc.) during the critical phases of isolation and culture in enumeration allows assessing the islet yields after iso- order to identify and correct variations that could nega- lation and culture. Islets are counted and scored by size tively influence the overall outcome of the procedure (58). using an algorithm to calculate islet equivalents (IEQ; Islet Purification. After the digestion phase, the the ‘ideal’ spherical islet with diameter of 150µm)(67, 68). pancreatic slurry is purified on density gradients. Nu- Routinely performed sterility tests should be negative and merous modifications of the purification process have include cultures for aerobic and anaerobic microbes, been introduced during the years. In the late 1980’s, mycoplasma, and fungi. Pyrogenicity is also assessed based on the promising results reported by Lake, et al by measuring endotoxin concentrations in the reagents (59), we introduced the use of the COBE computerized used for islet isolation and culture, which should be cell separation system (60) that allows for more efficient <5EU/kg. Assessment of islet viability is generally per- and time-effective purification of large volumes of pan- formed by the use of membrane dye exclusion protocols creatic tissue (Fig. 1H-I). Separation of islet clusters from that allow for the discrimination of viable and dead cells non-endocrine tissue was initially obtained by the means in sampled islets (64, 69-71). Product release criteria of discontinuous density gradients made in human se- require a viability >70% using such methods. rum albumin (60-62), and later in Ficoll dissolved in Euro- Potency tests are utilized to assess the ability of iso- Collins preservation solution (Euro-Ficoll; Mediatech, lated islets to produce and secrete insulin in a regulated Herindon, VA)(63). Since the late 1990’s, Ficoll-based fashion in response to glucose stimulation (69, 72). 5

Table 1. Product release criteria for human islet preparations for transplantation.

Test Method Required outcome Islet Yields Count of IEQ on aliquots of the final preparation >5,000 IEQ/Kg recipient’s b.w. Purity Dithizone staining >30% final preparation Islet Morphology Dithizone staining Preserved shape and granularity Sterility Aerobic/Anaerobic cultures Negative Mycoplasma Negative Fungal cultures Negative Pyrogenicity Endotoxin <5 EU/kg Viability Membrane dye exclusion >70% Potency In vitro Stimulation Index >1 Glucose-stimulated insulin release

Sequential or parallel incubation of isolated islets in vitro tocol (30, 31, 49, 82). Islets are assessed for product in the presence of increasing concentrations of glucose release criteria (quality, viability, potency and sterility) at results in insulin release that is quantified in the super- our facility and at the remote transplant site prior to trans- natants by enzyme-linked immunosorbent assay. The plantation. Similar collaborations are being established stimulation index of glucose-stimulated insulin over with additional distant CITP’s. baseline is generally used as a surrogate endpoint to Transplantation Techniques. Pancreatic islets are

assess islet potency, and it is generally accepted that embolized into the recipient’s liver by cannulation of the ISLETS - MIAMI, FLORIDA indices >1 are required in order to release an islet prepa- portal vein. Most of the initial patients were transplanted ration for transplantation. Dynamic tests (i.e., perifusion) by performing a laparotomy (SIK and IAK)(83, 84), an are also utilized to determine insulin release from islet approach that is currently limited to a few cases at risk of aliquots in response to glucose challenge (64, 73). Trans- bleeding. More recently, recipients of ITA and IAK are plantation of islets into chemically-induced diabetic im- transplanted using a minimally-invasive interventional munodeficient mice was introduced in the late 1980’s radiology approach consisting of percutaneous catheter- (74-76). This allows for the in vivo assessment of hu- ization of the portal vein (62, 85), that can be performed man islet preparations by measuring the ability of trans- under ultrasound (Fig. 2A) and fluoroscopic (Fig. 2B) planted islets to reverse diabetes (64, 72, 74-76). By guidance under conscious sedation (82, 86-89). To re- transplanting decreasing islet numbers in diabetic mice duce the risk of thrombosis due to the islet infusion, he- and comparing both (i) time to diabetes reversal and (ii) parin (35U/kg) is added to the islet preparation immedi- proportion of animals reversing diabetes, it is possible ately before infusion. Portal pressure is constantly moni- to determine differences in potency between islet prepa- tored during the transplant procedure, and in order to rations. Notably, although this in vivo assay is predictive limit the risk of portal pressure increase consequent to of graft function, it requires relatively long periods of fol- islet implant (90, 91), islets are infused by gravity using low-up. This approach and the use of glucose tolerance a closed gravity transfusion bag system (Fig. 2C-E) uti- tests in the transplanted animals allow assessing islet lized at our institution since 1990 (33, 86). D-Stat™ (Vas- engraftment and may be of assistance toward the vali- cular Solutions, MN) is used for closure of the needle dation of quicker and more predictive in vitro tests for tract to prevent the occurrence of bleedings after trans- human islet quality (64, 77-81). hepatic percutaneous access (Fig. 2F)(87). Shipping of Human Islets to Distant Transplant Bone Marrow Cell Isolation. Since the early Centers. Since 2001 our Human Cell Processing facil- 1990’s, the Human Cell Processing cGMP Facility per- ity has processed human pancreata procured from our forms isolation of BMC that are utilized in numerous clini- partner CITP at the Baylor College in Houston, TX. After cal trials aiming at the induction of hematopoietic chi- isolation, the islet preparations were shipped to Houston merism in combination with solid organ and cellular grafts where they were transplanted (ITA) into patients with (42, 92-115). The vertebral column (VC) is harvested brittle T1DM using a modification of the Edmonton pro- from the same multi-organ cadaveric donor of the islets 6 PILEGGI, RICORDI, KENYON, ET AL 7

Figure 2. Islet transplantation procedure. Islets are implanted into the recipient’s liver by cannulation of the portal vein. The use of interventional radiology techniques allows performing the transplant by a percutaneous transhepatic approach (A) without the need for general anesthesia and surgery. The use of ultrasound guidance (B) is of assistance to reduce the number of passages to access the portal vein. Injecting contrast media reveals the portal tract by fluoroscopy, confirming the correct positioning of the catheter (C). Islets are infused by gravity using a close system consisting of a transfusion bag (D): this technique allows maintaining the islets in suspension and preventing increase of portal pressure, which is continuously monitored during the transplant (E). Sealing of the catheter tract is performed at the end of the procedure in order to prevent bleeding (F).

(116). Vertebral bone marrow cells (VBMC) are obtained trials. Most of the early trials included a limited number by subjecting bone fragments to gentle shaking in me- of patients and were aimed at assessing both safety and dia followed by filtration through stainless steel meshes, efficacy (Phase I-II) of allogeneic islet transplantation in in order to separate bone chips from free VBMC (104- the treatment of patients with T1DM. These clinical trials 107, 117-124). Assessment of VBMC yields is performed have contributed substantially to the acquisition of criti- using a cell counter (Coulter) to obtain a nucleated cell cal experience in the management of recipients of islet count, and trypan blue exclusion is employed to deter- grafts and of immunosuppression, allowing for the steady mine viability (125-127). In selected protocols, enrich- progress in this field of investigation. We have estab- ment of CD34+ stem cells is obtained by positive selec- lished a multidisciplinary team composed of endocrinolo-

tion using magnetic beads (128). Whole bone marrow gists, transplant surgeons, immunologists, and basic ISLETS - MIAMI, FLORIDA cells or CD34+ BMC are cryopreserved into a transfu- scientists that are involved in our center’s clinical research sion bag and stored in liquid nitrogen until use (104-107, efforts. Early trials included only IAK and SIK recipients 129). Just before infusion, the bag containing the donor for whom immunosuppression was implemented to sus- marrow inoculum is thawed and infused intravenously in tain kidney function. In 1998 we started our clinical ITA the recipient (42, 44-46, 130, 131). trials in patients with brittle diabetes, for whom the risks Selection of Study Subjects. Our study subjects associated with the transplant procedure and immuno- come predominantly from the diabetes clinics at the suppression were justified by the frequency of life-threat- University of Miami, referrals from local and outside phy- ening hypoglycemic events (3, 132, 133). Two major sicians, and self-referred patients from all over the United categories of clinical trials are being pursued at our cen- States. In the current islet transplantation trials, only in- ter: chronic immunosuppression (Table 2) and protocols dividuals with T1DM, with documented absence of C- that attempt to induce tolerance (Table 3). The latter peptide response to mixed meal challenge (<0.3 ng/ml), protocols include the use of bone marrow cells (BMC), who fulfill the selection criteria were selected as candi- either as whole BMC or fractioned CD34+ cells obtained dates for islet transplantation. Patients with advanced from the same islet donor to induce hematopoietic chi- macroangiopathy, including symptomatic ischemic and merism (134), and are designed to wean immunosup- non-ischemic heart disease and patients that are unable pression at selected time points after transplantation (42, to follow the rigorous requirements for follow-up, includ- 44-46, 130). ing those who did not pass a preliminary psychological Assessment of Graft Function. Graft function is evaluation were not considered for inclusion. Alloge- monitored using finger-stick blood glucose (FBG), mean neic islet transplantation has also been performed in se- amplitude of glycemic excursions (MAGE), basal C-pep- lected patients with DM due to diseases of the exocrine tide, glycosylated hemoglobin (HbA1c), and pancreas (insulin-requiring diabetes mellitus; IRDM) and fructosamine. We have recently implemented the use liver cirrhosis that received orthotopic allogeneic liver (24, of a continuous subcutaneous glucose monitoring sys- 25) or in patients with ESRD and T2DM. tem (18, 83, 84, 135-137) for a more accurate assess- Treatments. Different therapeutic protocols have ment of glycemic control throughout the day after trans- been utilized over the years in patients with T1DM that plantation. Stimulation testing includes mixed meal tol- were dictated by the availability of new reagents and the erance test (MMTT), intravenous glucose tolerance test promising results reported in solid organ transplantation (IVGTT), and arginine stimulation test. An oral glucose 8 PILEGGI, RICORDI, KENYON, ET AL

Table 2. Clinical islet transplantation trials – Chronic immunosuppression protocols. Period Type of transplant No. Induction Immunosuppression Indication 1985-89 IAK 4 None Cyclosporine T1DM SIK 1 Azathioprine Methylprednisolone 1990-93 IAK 7 OKT3 Cyclosporine T1DM SIK 1 Azathioprine Methylprednisolone 1995 SIL 1 None Tacrolimus T1DM (Mycophenolate Mofetil) Methylprednisolone 1998 SIK 1 Daclizumab Tacrolimus T1DM Mycophenolate Mofetil Methylprednisolone 2001-03 ITA 16 Daclizumab Sirolimus T1DM ±Infliximab Tacrolimus 2002-04 ITA 5 Daclizumab Sirolimus T1DM Tacrolimus 2003-05 IAK 7 Daclizumab Sirolimus T1DM ± Infliximab Tacrolimus (Mycophenolate Mofetil) Methylprednisolone

Table 3. Clinical islet transplantation trials – Immune tolerance induction protocols. Period Type of transplant No. Induction Immunosuppression Indication 1994-97 SIL + BMC 5 None Tacrolimus IRDM SIL 3 (Mycophenolate Mofetil) Methylprednisolone 1994-96 IAK + BMC 1 ALG (n=3) Tacrolimus T1DM Azathioprine SIK + BMC 7 OKT3 (n=5) (Mycophenolate Mofetil) Methylprednisolone 1996-1998 SIK + BMC 1 OKT3 Tacrolimus T2DM SIK 2 Azathioprine (Mycophenolate Mofetil) Methylprednisolone 1998-99 ITA + CD34+ BMC 5 Daclizumab Tacrolimus T1DM Mycophenolate Mofetil Methylprednisolone ATG Cyclosporine Mycophenolate Mofetil 2000 ITA + CD34+ BMC 6 Daclizumab Sirolimus ±Infliximab Tacrolimus T1DM ALG: Anti-Lymphocyte Globulins; ATG: Anti-Thymo-Globulin; AZA: Azathioprine; BMC: Bone Marrow Cells; IAK: Islet After Kidney; IRDM: Insulin Requiring Diabetes Mellitus; ITA: Islet Transplantation Alone; MMF: mycophenolate mofetil; SLI: Simultaneous Islet and Liver transplantation; T1DM: Type 1 Diabetes Mellitus. 9 tolerance test (OGTT) is also performed yearly after Permanent Immunosuppression transplantion on those patients who are insulin indepen- 1985-1989. In our first clinical trial, patients received dent (62). single donor allogeneic islet infusions as either IAK (n=3) Clinical Trials or SIK (n=1) and were treated with cyclosporine (CyA; Neoral, Novartis; 8-12 mg/kg; target trough levels 100- Since 1985, a total of 101 allogeneic islet transplants 300 ng/ml) and methylprednisolone (1.0 mg/kg tapered have been performed in 73 patients at the CITP of the within one week to maintenance 0.1-0.2 mg/kg). Pa- Diabetes Research Institute–University of Miami tients under azathioprine (AZA; Imuran, Prometheus (Tables 2-3). Lab.; 2.0 mg/kg) for previous kidney transplant were con- Type 1 Diabetes Mellitus - Protocols Requiring verted to CyA before islet implant. In this trial, cadaveric

1.00 A B 1985-89 1994-96 0.75 1990-93 1998-99a 1998 1998-99b 2001-03 2000

0.50 ISLETS - MIAMI, FLORIDA 0.25

0.00 0 500 1,000 1,500 2,000 0 500 1,000

Cumulative Proportion Graft Function Graft Proportion Cumulative Time Posttransplant (days) Figure 3. Islet allograft function (measurable C-peptide) in patients with T1DM recipients per era. The outcome of islet transplantation protocols requiring chronic immunosuppression has steadily improved (A). In the first trial (1985-89, ), early graft loss was observed possibly because the patients received a small islet mass and therapeutic levels of the immunosuppression were not always achieved. One of the patients received sequential islet infusions and maintained measurable C-peptide for 13.3 months, but lost graft function following modification of the immunosuppression treatment. In the 1990-93 trial ( ), graft function was observed in all patients, and two of them had measurable C-peptide for over 12 and 14 years, respectively. Introduction of daclizumab (1998, ) resulted in sustained graft function (2.5 years) in a recipient of SIK with a marginal islet graft. Introduction of a steroid-free immunosuppression regimen with daclizumab and infliximab in ITA recipients dramatically improved graft function (2001-03, ). Trials aiming at the induction of hematopoietic chimerism and donor-specific unresponsiveness were performed (B). No primary nonfunction was observed in the first trial that included the use of donor-derived whole BMC, calcineurin inhibitors and induction with ALG or OKT3 (1994-96, ), but islet graft function was limited. In the first ITA trial, daclizumab induction with conventional levels of tacrolimus and MMF with CD34+ BMC were given (1988-99a, ): one patient had primary nonfunction, while one other had sustained graft function until immunosuppression was discontinued. The use of CyA and MMF plus induction with ATG (1998-99b, ) showed only transient graft function. The use of the Edmonton style immunosuppression with infliximab and CD34+ BMC (2000, ) resulted in measurable graft function in all patients. Two patients lost their graft due to sub-therapeutic sirolimus levels, while 4 patients had sustained graft function at one year, despite having received a single donor graft. However, discontinuation of immunosuppression resulted in graft loss within 3 months. 10 PILEGGI, RICORDI, KENYON, ET AL donor pancreata were either immersed in Ringer's Lac- sistently obtained in these patients, a factor that may tate solution at 4°C for up to 6 hours, or preserved by have contributed to the relative early loss of graft func- hypothermic pulsatile perfusion with cryoprecipitated tion in this trial. Subsequent to the loss of islet function, silica-treated human plasma for up to 12 hours prior to all patients returned to their pre-transplant insulin require- isolation (85, 138, 139). Islets were isolated by intra- ments. Survival of the kidney grafts persisted in all pa- ductal instillation of collagenase solution and mechani- tients, despite loss of islet function. cal dispersion by passages of the digest through a se- 1990-1993. A subsequent trial included eight pa- ries of graded needles (62). Purification was obtained tients (IAK, n=7; SIK, n=1) receiving allogeneic islets by repeated gravity sedimentation yielding final prepa- obtained from multiple donors to provide a large endo- rations comprising 20-40% islets in a volume of 5-6 mls. crine cells mass (infusion of 9,092-21,185 IEQ/kg, iso- Prior to transplantation, isolated islets were cultured for lated from a total of 2-5 donor pancreata). Treatment <48 hours and pre-treated in vitro with anti-MHC class II consisted of induction with OKT3 antibody (Orthoclone monoclonal antibody in order to reduce immunogenicity. OKT3, Orthobiotech) and maintenance immunosuppres- In this trial the percutaneous transhepatic catheteriza- sion with CyA, methylprednisolone, and AZA. Measur- tion technique for islet implant was utilized for the first able C-peptide was observed after transplantation in all time (62, 85). After transplantation, all patients showed patients. Insulin independence was achieved in two pa- measurable graft function assessed by basal and stimu- tients for a period of 36 and 38 days, respectively. Six lated C-peptide levels (Fig. 3A). patients had measurable basal C-peptide for 1 (n=2), 4, One patient received two sequential islet infusions. 5, 11.8, and 25.2 months, respectively, while two patients The first graft led to measurable function with a 30% re- had sustained graft function for >12 and >14 years, reduction in insulin requirements and decreased HbA1c spectively (Fig. 3A). (although not reaching normal ranges) from pre-trans- One of the patients with longest graft function main- plant levels. A second islet infusion was performed 6 tained good glycemic control with normalization of HbA1c months after the first, and led to normalization of HbA1c (Fig. 4) and significantly reduced insulin requirements, levels, while insulin requirements remained stable. Graft when compared to pre-transplant doses. Remarkably, function in this patient was sustained for 13.3 months this patient did not experience any hypoglycemic epi- from the first islet infusion. Three weeks after the sec- sodes for >12 years, regardless of the need for exog- ond infusion, conversion from CyA to AZA was performed enous insulin, while 3-4 severe hypoglycemic episodes due to diagnosis of breast adenocarcinoma. Decreased a year were common prior to islet transplantation (18, basal and stimulated C-peptide were observed after the 140). In spite of persistent basal C-peptide, the patient change in therapy, although insulin requirements re- developed ESRD 10.9 years after receiving IAK. Immu- mained lower than pre-transplant until C-peptide was nosuppression was discontinued and dialysis initiated. undetectable. The patient developed intra-cerebral hemorrhage and One recipient of SIK showed basal C-peptide levels died at 12.5 years of follow-up. rising after transplant (0.64 pmol/ml at 3 weeks) although The other patient with long-term function showed with blunted responses to Sustacal. This patient experi- normalization of HbA1c paralleled by sustained basal C- enced two kidney rejection episodes (serum creatinine peptide for the first two years after islet infusion (1). Al- >1.5 mg/dl) 3 weeks after transplantation possibly re- though C-peptide was detectable for several years after lated to inadequate trough levels of CyA that were treated IAK, the patient developed insulin resistance and diffi- with external irradiation of the kidney and two cycles of culty to maintain HbA1c within the levels suggested by high doses of methylprednisolone (1,000 mg/day for 3 the DCCT (Fig. 4C)(18, 83, 84, 140). Detectable C-pep- days). While serum creatinine improved after treatment, tide with sustained kidney graft function was present for basal C-peptide declined after the first and second cycle >14 years from IAK in this patient. Deterioration of the of steroids (0.38 and 0.05 pmol/ml, respectively) and it general health status at the long follow-up was noted. was undetectable by 7 weeks post-transplant. The patient died 14.4 years after islet transplantation of The other two patients lost function after 2 and 4 an unknown cause. weeks from transplantation, respectively. It is notewor- 1998. In April 1998 we performed a SIK transplant thy that therapeutic levels of cyclosporine were incon- in a 59-year old male patient with T1DM and ESRD. This 11

6 12 Pt.4 ABC Pt.6

4 Percent HbA1c C-peptide (ng/mL) C-peptide

0 0 Pre 2 4 6 8 10 12 14 Pre 2 4 6 8 10 12 Pre 2 4 6 8 10 12 Year Figure 4. Trial 1990-93: Long-term graft function in IAK recipients. Transplantation of allogeneic islets results in improved metabolic control with reduction of insulin requirements, improved HbA1c, and absence of severe hypoglycemia even when exogenous insulin is required to maintain blood glucose in a normal range. Two patients transplanted in the 1990-1993 trial maintained sustained basal

C-peptide for >12 and >14 years after IAK (A). This resulted in improved metabolic control and good HbA1c in patient 4 (B), with HbA1c close to the DCCT recommendations and absence of severe hypoglycemia for >12 ISLETS - MIAMI, FLORIDA years. Patient 6 (C) developed insulin resistance and was unable to maintain HbA1c to target levels, despite persistence of C-peptide. was the first islet transplantation trial to include an in- monoclonal antibody infliximab (Remicade, Centocor duction therapy with the anti-interleukin (IL)-2 receptor Inc.) was introduced in this study in order to reduce the antibody daclizumab (1mg/kg; Zenapax, Roche). Main- level of inflammation generated early after intra-hepatic tenance immunosuppression included tacrolimus implant and to favor islet engraftment (146-150). The (Prograf, Fujisawa), mycophenolate mofetil (MMF; recipients were therefore randomized to receive a single Cellcept, Roche) and methylprednisolone. The patient dose of infliximab: five patients received 5 mg/kg, and received both islet and kidney from the same allogeneic three 10 mg/kg given 2 hours prior to their first islet infu- cadaveric donor. Measurable islet graft function was sion. Maintenance therapy consisted of tacrolimus (tar- observed for up to 2.5 years (Fig. 3A), while the kidney get trough level 4-6ng/ml), and sirolimus (Rapamune, graft continued functioning longer. Notably, the recipi- Wyeth Pharmaceuticals Inc.; target trough level 12-15 ent had a BMI of 34 and received a suboptimal islet graft ng/ml for three months and 10-12ng/ml thereafter). mass (2,464 IEQ/kg). Similar to the Edmonton protocol, sequential islet infu- 2001-2005. In light of the promising results of the sions were performed in order to achieve insulin inde- Edmonton trial (141) and our own preliminary data with pendence (146-150). the Edmonton-style immunosuppression combined with Sixteen patients (7 males, 9 females; 41±10 year BMC (see below), we initiated a clinical ITA trial in pa- old; diabetes duration 27±13; BMI 25±1.7) were enrolled tients with brittle T1DM. At variance to the Edmonton in the study. All patients showed a substantial reduction protocol, isolated islets were cultured in CMRL-based of exogenous insulin requirements and improved glyce- supplemented medium (142-145) for <72 hours prior to mic control after receiving the first islet graft (Fig. 5A). transplant (Fig. 4). Induction therapy included a 5-dose Two patients did not receive additional islet infusions due course of daclizumab (1mg/kg) given biweekly from the to adverse events (patient 1: aspiration pneumonia; pa- day of transplant. Our protocol also included monthly tient 11: parvovirus infection). These patients were dis- (for the first year) and bimonthly (for the second year) continued from immunosuppression and withdrawn from administrations of daclizumab in 12 of the 16 patients. the study. Insulin independence was achieved in all 14 In addition, the anti-Tumor Necrosis Factor (TNF)-α patients that remained in the study following a single (n=1) 12 PILEGGI, RICORDI, KENYON, ET AL

1.0 A B 6 0.8

4 0.6

0.4 2 0.2 Proportion Off Insulin Insulin (units/day) (x10)

0 0.0 1 3 5 7 9 11 13 15 0 180 360 Patient Number Days Posttransplant Figure 5. The Miami ITA trial using steroid-free immunosuppression. The protocol consisted of sequential infusions of cultured islets in patients with T1DM. Induction therapy with daclizumab was started on day 0, and followed by biweekly injections until week eight, monthly injections for the first year, and bimonthly injections for the second year. In selected patients, the anti-TNFα antibody infliximab was given on the day of transplantation. Sirolimus was started on day -1 targeting trough levels of 12-15ng/ml for the first 3 months, and 10-12 ng/ml thereafter. Tacrolimus was started on day -1 to achieve trough levels of 4-6ng/ml. Insulin requirements in 16 recipients of ITA (A) before and after allogeneic islet infusions. All patients showed reduced insulin requirements after the first islet infusion. One patient (#9) achieved insulin-independence with a single infusion, and 13 patients were off insulin after a second infusion. Two patients were not completed and withdraw from the study due to adverse events (patients 1 and 11). Insulin independence at one year was maintained in 79% of the 14 completed patients (B). Graft function (measurable C-peptide) was maintained also in the patients that required reintroduction of insulin therapy, however at lower doses than pre-transplantation. or two (n=13) islet infusions (Fig. 5A-B). Insulin inde- islets (ITA), induction with daclizumab, and maintenance pendence was maintained in 79% (11/14) of the recipi- immunosuppression based on sirolimus and tacrolimus. ents at one year follow-up (Fig. 5B), while patients that Sequential islet infusions were performed to achieve in- required reintroduction of exogenous insulin (at lower sulin independence in these patients. This ITA trial has doses than baseline) showed sustained graft function shown that the Edmonton protocol could be success- (measurable basal and stimulated C-peptide, and nor- fully reproduced (27, 28, 151). Notably, centers with the mal HbA1c), with no hypoglycemia (Fig. 3A)(27, 28, 151). longest experience in islet isolation and transplantation Assessment of cytotoxic lymphocyte gene expression in reported higher success rates than younger, inexperi- patients that required reintroduction of insulin showed a enced centers, highlighting the importance of acquiring clear elevation (particularly of granzyme B) 25-203 days the critical experience in islet cell processing, transplan- before hyperglycemia onset (152). Additionally, antidonor tation, and patient management in order to achieve con- mixed lymphocyte reactions showed sensitization in pa- sistently successful outcomes (154). tients who experienced partial graft loss (152). No cy- 2003-2005. In 2003 we started an IAK transplanta- tomegalovirus infections or reactivation were observed tion trial in patients with T1DM consisting of sequential even in the case of donor/recipient mismatch (153). infusions of cultured allogeneic islets and an immuno- 2002-2004. Our institution is part of the multi-cen- suppressive protocol similar to the one described above ter clinical trial sponsored by the Immune Tolerance Net- for ITA (3, 61, 103, 126, 132, 133, 155-175): induction work (NIH – National Institute of Diabetes and Digestive treatment with daclizumab with infliximab, sirolimus, and and Kidney Diseases, NIDDK) to reproduce the tacrolimus. Two patients also received maintenance ste- Edmonton Protocol (27, 28, 151). Five patients were roids. All patients bearing a kidney graft and previously enrolled at our site to receive intra-hepatic allogeneic receiving different immunosuppressive protocols were 13 converted to sirolimus and tacrolimus prior to or at the the underlying autoimmunity (3, 144, 145). Based on time of islet transplantation. Seven IAK transplants us- these premises, we have performed a number of trials in ing this protocol have been performed to date. Similar which donor-specific, non-fractioned or CD34+ BMC were to what was observed in the ITA trial, all patients showed infused in the recipients aiming at the induction of he- improved glycemic control with reduced insulin require- matopoietic chimerism and acceptance of islet grafts. ments after islet infusion. Insulin independence was 1994-1996. The first trial including donor-specific achieved after a single infusion in two patients, after two BMC infusion was started in 1994 and included a total of sequential islet infusions in four patients, and one pa- eight patients. The protocol consisted of SIK (n=7) or tient is currently waiting for a second infusion. Creati- IAK (n=1) transplantation. Islet grafts (14,800±7,152 IEQ/ nine clearance remained stable in all patients, with the kg) were infused on day 0 (2 patients received islets from longest follow-up of over two years. Our preliminary data 2 donors), and non-fractioned donor-specific BMC suggests that the Edmonton style of immunosuppres- (1x109/kg) was infused on days 5 and 11. Immunosup- sion may be a viable option for patients with T1DM re- pression consisted of an induction with either OKT3 (n=5) cipients of kidney grafts. or ALG (n=3) and maintenance with triple immunosuppression (tacrolimus, methylprednisolone and AZA or Type 1 Diabetes - Tolerance-Induction Protocols MMF). Chimerism levels were detected by PCR-flow assay (97) in peripheral blood (7.1±1.4% at one month, The need for chronic immunosuppression in recipi- and ~1% at 6 and 12 months). Loss of graft function ents of allogeneic tissues represents one of the major was observed at a mean time of 142±53 days after im-

hurdles to the widespread use of organ and cellular transplantation (Fig. 3B). One patient rejected both kidney ISLETS - MIAMI, FLORIDA plantation. Most of the drugs currently utilized to pre- and islet grafts after treatment with interferon (IFN)-α due vent allograft rejection are not selective enough and are to diagnosis of acute hepatitis C virus infection and died associated with untoward side effects (e.g., infections, of complications secondary to the viral infection. Only organ toxicity, and neoplasm)(176) that limit their use to the recipient of IAK had a kidney rejection episode (crea- selected categories of patients in which the risk is justi- tinine 2.5 mg/dl) 3 months after islet implant (4 years fied. Definition of therapeutic protocols able to induce after kidney transplant), while all other patients main- permanent acceptance of allogeneic grafts is a highly tained kidney function in the absence of rejection epi- desirable goal pursued by the transplant community. sodes. Six patients developed cytotoxic anti-donor MHC Increasing evidence from experimental transplantation antibodies, but their presence did not correlate with loss suggests that indefinite survival of allogeneic tissues in- of kidney function. cluding pancreatic islets can be obtained under selected 1998. The first ITA trial at our Center was started in treatments in animal models (92-94, 177). Furthermore, September 1998. Transplantation of allogeneic islets on clinical evidence of indefinite graft survival in recipients day 0 and infusion of donor-derived CD34+ BMC on days of solid organ allografts associated with measurable do- 5 and 11, was performed in patients with T1DM (n=5). nor leukocytes (microchimerism)(45, 46, 94-129, 132- Three patients received induction therapy with 134, 178-199) has opened the way to a number of clini- daclizumab (biweekly for the first 5 doses and monthly cal trials aiming at the induction of hematopoietic chi- for the first year) and maintenance therapy with merism to favor acceptance of donor-specific tissues by tacrolimus, MMF, and methylprednisolone (Fig. 3B). One the administration of BMC. Our Center has been involved patient received 6,922 IEQ/kg and achieved stable graft in several clinical trials of solid organ (multi-visceral, liver, function for over one year with measurable C-peptide intestine, kidney, and heart) and islet transplantation with (0.63±0.14 at one year vs. 0.2 ng/ml pre-transplant), BMC (3, 132, 133). Induction of tolerance in recipients ~40% reduction of insulin requirements and improved of islet allograft represents an appealing therapeutic ap- HbA1c levels (8.1±0.6% pre-transplant vs. 6.9±0.6%) at proach (200-204). Notably, calcineurin-inhibitors and one year. Chimerism levels in this patient were 7.7% at steroids are toxic to islet cells (3, 132, 133), and their one month, 3.6% ant 6 months, and <1% at one year. chronic use may lead to loss of islet graft function. In- Weaning from immunosuppression as per protocol in this duction of hematopoietic chimerism in patients with T1DM patient resulted in loss of graft function. The second may also be beneficial to restore self tolerance and treat patient in this group received 10,536 IEQ/kg and showed 14 PILEGGI, RICORDI, KENYON, ET AL good function (C-peptide: 0.72±0.21ng/ml), but did not respectively, possibly due to the sub-therapeutic sirolimus tolerate immunosuppressive drugs leading to graft loss trough levels achieved. Four patients (9,029±2,589 IEQ/ 45 days after transplantation. The third patient showed kg) maintained sustained islet graft function for up to one no measurable C-peptide (i.e., primary non-function or year, showing maximal mean C-peptide levels at 3 graft failure) after transplantation of 5,774 IEQ/kg. Pri- months (1.06±0.23ng/ml), and 68±26% reduction in in- mary non function was associated with high basal se- sulin requirements from baseline. Transient insulin in- rum TNF-a levels, which may have contributed to the dependence was achieved in three patients (5, 17 and failure to engraft (42, 44-46, 130, 131). 50 days, respectively). These four patients with sustained Two patients received induction with thymoglobulin graft function at one year underwent weaning from im- (rabbit anti-human thymocyte globulin, ATG), and main- munosuppression (Fig. 3B), as per protocol. Only a tran- tenance with CyA and MMF. One patient received 10,669 sient hematopoietic chimerism was observed in these IEQ/kg and showed normalization of HbA1c to 6.4% and patients in peripheral blood, dropping from 4.6±0.5% at good islet graft function after transplantation (C-peptide: one month after ITA (n=6) to 0.14±0.05% at one year. 0.92±0.26) for approximately 130 days, when loss of graft After discontinuation of immunosuppression, all four pa- function occurred, possibly related to lack of therapeutic tients invariably lost islet graft function with a mean time trough levels of the immunosuppressive drugs. Another of 95±25 days (range, 74-131). patient in this group received 7,981 IEQ/kg and showed Allogeneic Islet Transplantation for rapid reduction of insulin requirements and good graft function (C-peptide: 0.52±0.46). Following the second Insulin Requiring Diabetes BMC infusion the patient developed serum sickness syn- 1994-1997. A trial of simultaneous allogeneic islet drome secondary to ALG treatment. Hospitalization and and liver (SIL) transplantation was performed in eight discontinuation of immunosuppression was required, patients with IRDM consequent to hepatic cirrhosis (42, which resulted in complete recovery from symptoms but 44-46, 130, 131). Five out of eight patients also received loss of graft function by day 21. donor-specific BMC. Immunosuppression consisted of Microchimerism was detected in all patients in this tacrolimus and steroids. A significant reduction of insu- study in both treatment groups. No episodes of graft lin requirements was observed in most patients with nor- versus host disease were observed. All patients returned malization of HbA1c and insulin independence in three to pre-transplant insulin requirements after discontinu- patients. One patient resumed insulin treatment 5 months ing immunosuppression. after transplant, while the other 2 remained insulin inde- 2000. In 2000 an ITA trial with Edmonton style im- pendent for over 17 months (42, 44). Three patients munosuppression and donor-specific BMC was started. experienced mild liver rejection episodes that resolved Patients with brittle T1DM and hypoglycemia unaware- after treatment with MMF and steroids. The proportion ness received a single intra-hepatic islet infusion on day of rejection episodes observed was comparable to that 0, followed by two intravenous donor-specific CD34+ of patients receiving orthotopic liver grafts and BMC with- BMC inoculum on days 5 and 11 post-transplant (n=5). out islets. Importantly, islet transplantation did not have One patient received a single infusion of non-fractioned any negative effects on liver function in this series. Fur- BMC. Induction treatment consisted of daclizumab and thermore, the results of this pilot trial demonstrated the α a single dose of the anti-tumor necrosis factor (TNF)- safety of intra-hepatic islet cell transplantation in recipi- monoclonal antibody infliximab, followed by tacrolimus ents of orthotopic liver for cirrhosis, and that inclusion of and sirolimus maintenance. Endpoints of the study in- pancreatic islets can improve metabolic control and re- cluded (i) induction of hematopoietic chimerism that was duce insulin requirements in patients with liver cirrhosis assessed at monthly intervals, and (ii) acceptance of (42, 44). donor-specific islets after discontinuation of immunosup- 1996-1998. A pilot trial of SIK transplantation in pressive drugs that were gradually tapered in the pa- patients with T2DM has been performed at our center in tients with a functional islet graft at one year. All patients 3 patients. The characteristics of this category of pa- showed measurable islet graft function after implant tients with residual C-peptide pose some concerns re- (8,629±2,102 IEQ/kg)(Fig. 3B). Two patients lost islet garding enrollment in islet transplantation trials, and care graft function at 141 and 143 days post-transplantation, should be paid to the recipient body weight, age, and 15 insulin resistance in order to assess the potential ben- mia despite the use of exogenous insulin (26). efits of an islet graft (205). In 1998 we introduced the anti-IL-2R antibody daclizumab as induction therapy in combination with DISCUSSION tacrolimus and MMF in a SIK recipient that showed im- Unprecedented results have been reported in the proved metabolic control and measurable C-peptide for field of islet transplantation in recent years, which are 2.5 years despite receiving a marginal islet mass. The the fruit of a steep learning curve and steady progress results of this pilot trial demonstrated the promising prop- (Fig. 6). Each clinical trial performed at our center since erties of daclizumab as an inducing agent for islet cell 1985 has been instrumental to acquire critical knowledge transplantation, although the combination of maintenance and to help to design subsequent protocols (Fig. 3). therapy drugs including steroids and the marginal size Our first clinical trial of allogeneic islet transplanta- of the graft may have limited the overall success. tion into patients with T1DM showed that competent is- While exploring protocols aiming at chronic immu- lets could be obtained from human pancreata that had nosuppression in patients with T1DM recipients of islet been preserved in cold slush or hypothermic pulsatile allografts, we have been also strongly pursuing alterna- perfusion ex vivo and demonstrated for the first time the tive avenues for the induction of indefinite graft survival feasibility and safety of the percutaneous transhepatic based on the use of donor-derived BMC. Our first proto- catheterization of the portal vein for islet infusion without col showed that microchimerism levels could be obtained the need for general anesthesia (62, 85). The islet mass after infusion of donor BMC, but that the use of standard transplanted may have contributed to the partial success immunosuppression, dictated by the combination with a

observed in this series, possibly consequent to relative kidney graft, did not suffice to sustain long-term islet sur- ISLETS - MIAMI, FLORIDA inefficiency of isolation and purification procedures, and vival, while kidney function was maintained after islet to the lack of standardized methods for the assessment rejection. In a subsequent trial, we performed ITA with of islet quality and numbers. Indeed, the use of sequen- CD34+ BMC using daclizumab or ALG as induction, and tial infusions, similar to what observed in recent trials calcineurin inhibitors with MMF as maintenance therapy. (26, 29-31), resulted beneficial in one patient and lead Only one patient maintained a functional graft for over to substantial improvement in glycemic control as long one year, and weaning from immunosuppression resulted as therapeutic levels of the immunosuppressive drugs in graft loss to rejection. The use of the immunosup- were maintained. pressive protocol in this trial appeared inadequate to fa- In the subsequent trial, islets were isolated using vor islet graft survival and BMC engraftment. the automated method (53), and each patient received The availability of more potent immunosuppressive islet preparations obtained from multiple donors in order drugs with potential immunomodulatory effects permis- to provide a sufficient islet mass (18, 84). In this proto- sive to tolerance induction such as sirolimus (206-208), col, induction therapy with the T-cell depleting OKT3 and the success of the Edmonton style of immunosup- antibody was given. The overall outcome in this trial pression in ITA (26, 29-31) prompted us to test this ap- was better than the previous one, as it permitted the proach with a protocol aiming at the induction of toler- achievement of sustained graft function (6 patients at 4 ance. Patients in this trial received a single donor ITA months, 4 patients at 11 months, 3 at 25 months, and with donor-specific CD34+ BMC, induction with two patients with measurable C-peptide for >14 years). daclizumab and maintenance with sirolimus and The results observed in the 2 patients with extended graft tacrolimus. The use of the anti-TNF-α antibody infliximab function demonstrated that therapies able to prevent re- was introduced in this trial based on the observation that jection and (possibly) recurrence of autoimmunity in pa- primary non-function of grafted islets in a patient from a tients with T1DM may allow for indefinite survival and previous trial was associated with high serum levels of function of allogeneic islets in an ectopic site. An impor- this pro-inflammatory cytokine. Interestingly, primary non- tant message from our data is that the beneficial impact function was not observed in this series, and all grafts of restoring β-cell function can be achieved in patients functioned after implantation. Graft function was lost with T1DM even when partial function of the implanted within 5 months in 2 patients due to sub-therapeutic islets is obtained, as it resulted in sustained improved trough levels of sirolimus. Four patients showed graft glycemic control and prevention of severe hypoglyce- function at one year after transplantion and were there- 16 PILEGGI, RICORDI, KENYON, ET AL 17

Figure 6. Steady progress leading to improved success rate of islet isolation and transplantation. Numerous technological improvements have been introduced at our center in the recent years that have contributed to the steadily increasing success rate of islet transplantation. Percutaneous transhepatic catheterization of the portal vein (65), utilized for the first time in 1985, allowed performing the transplant procedure without the need for general anesthesia and surgery. The automated method for islet isolation introduced in 1988 (86) utilizes a dissociation chamber that allows for the recovery of higher islet yields from donor pancreas. The use of dithizone, a dye that stains red the secretory granules in islet cells was introduced in 1988 for the assessment of islet preparations (purity, granularity, and enumeration)(59, 60). In 1990 we started using a close system (the ‘bag method’) for the infusion of islets by gravity, which allows for controlled rate of infusion and reduced portal pressure rise (54, 55). The introduction of the semiautomatic COBE system in 1990 has allowed improving the efficiency of purification of large volumes of tissue from human pancreata (51). The use of the new enzyme blend Liberase (1997) with reduced endotoxin contamination and more consistent characteristics has contributed to further increase islet yields (152). In 1998 the anti-IL-2 receptor antibody daclizumab was utilized at our center for the first time as induction therapy in a recipient of pancreatic islets. In 2000 we implemented the use of anti-TNFα antibody treatment of islet recipients to favor engraftment. In 2001 we showed that the use of the two layer method for pancreas preservation results in improved islet yields even from marginal donors (64) which may be of assistance to increase the donor pool. Detection of cytotoxic lymphocyte markers correlates with graft dysfunction in islet graft

recipients (2004) and may be of assistance to implement anti-rejection treatments to preserve islet mass (81). Utilization of an additional purification step for the less pure fractions obtained after isolation allowed us to maxi- ISLETS - MIAMI, FLORIDA mize the utilization of islet preparations for transplantation at our center (2005), as more viable and potent islets could be recovered to meet the requirements for transplantation . Development of more sensitive tests for the assessment of human islets and predictive of the quality and potency may be of assistance to identify suboptimal preparations that should not be transplanted . fore weaned from immunosuppression. After discontinu- A prerequisite for the achievement of insulin-inde- ation of the immunosuppressive drugs they showed graft pendence after islet transplantation is that a sufficient function for an additional 3 months. The results obtained islet mass is implanted (145), which is generally obtained in this trial showed that improved and sustained meta- by the infusions of more than one islet preparation (se- bolic control can be obtained with a single islet infusion quential or pooled). Improvements in organ harvesting using this protocol, although without achieving perma- and preservation techniques have contributed substan- nent graft survival. This trial also demonstrated the effi- tially to render available a higher number of human cacy of sirolimus administration for the success of the pancreata suitable for islet isolation and transplantation, treatment, and the importance of closely monitoring and although underutilization of cadaveric donor pancreata maintaining therapeutic trough levels of this drug. Fu- for transplantation has been recognized (2, 6). ture trials using alternative drug combinations are cur- It is noteworthy that, despite the improved efficiency rently being evaluated in order to improve the success of isolation procedures and the introduction of cGMP of tolerance-inducing protocols for islet transplantation. standards and rigorous quality controls for product re- The Edmonton style of immunosuppression has al- lease criteria, islet preparations with comparable quality lowed for the achievement of unprecedented success and numbers may result in different outcomes after trans- rates in ITA for patients with T1DM, with a high propor- plantation, with partial function and primary non-function tion of recipients achieving insulin independence at one being not an uncommon problem (81). Multiple factors year after sequential islet infusions, or maintaining good may contribute to this phenomenon and to the require- metabolic control with insulin requirements much lower ment of high islet numbers observed in islet recipients than baseline. We have confirmed such results in our that are not explained solely by immunological mecha- ITA series and extended it to patients with IAK grafts. nisms (144). Islet cells are particularly vulnerable to hypoxia and oxidative stress to which they are exposed as Current Challenges and Future Directions 18 PILEGGI, RICORDI, KENYON, ET AL early as the time of donor brain death (209). They are ing, and after isolation)(141, 221, 222, 230-239), and subjected to hypoxia and oxidative stress throughout implementation of treatments targeting coagulation and organ procurement and preservation, islet isolation, pu- non-specific inflammation during the peri-transplant pe- rification and culture, possibly reducing the number of riod (27, 28, 240). Notably, prevention of early inflam- viable and/or functional islet cells at the time of trans- mation in the peri-transplant period may also be benefi- plantation (70, 210). We appreciate the intrinsic limita- cial to reduce triggering of specific allogeneic and au- tions of the current methods utilized for the assessment toimmune responses that affect the overall survival of of islet quality and potency prior to transplantation that islet grafts (176). Alternative sites for islet implantation may not allow for the identification of suboptimal islet and bioartificial devices are currently under evaluation preparations that should not be transplanted (80, 81). as they may provide better engraftment and easier ac- There is an urgent need for the development of sensi- cess for biopsies (241). tive tests to assess the quality of donor pancreata and of The introduction of more effective immunosuppres- the islet preparations. To this aim, several tests are cur- sive protocols has been a key component of the recent rently under evaluation, including flow cytometry quanti- success of allogeneic islet transplantation for the treat- fication of mitochondrial membrane potential in islet cell ment of T1DM. However, the long-term effects of such subsets (81, 211), measurement of oxygen consump- drugs have not yet been elucidated in the islet trans- tion rates of islets under selected stimuli (212), signal plantation setting. The recipients of ITA have brittle T1DM transduction (80, 210), and quantification of cell subsets and are otherwise in good general health and, therefore, in the islet preparations (81, 213). In order to address a careful assessment of the risk/benefit ratio should be these issues and improve the quality of human islets for considered before enrollment in clinical trials. Increas- transplantation, the National Center for Research Re- ing evidence coming from the recent ITA trials suggests sources and NIDDK from the NIH, the Department of that achievement of relatively high trough levels of Health and Human Resources and the Juvenile Diabe- sirolimus is a prerequisite for sustained islet graft sur- tes Research Foundation International have sponsored vival (242) posing some concerns for lipid homeostasis a consortium of 10 Islet Cell Resources (ICR) Centers and other possible untoward side effects of the use of established in 2002 that promote the interaction between this drug (176, 243, 244). The use of calcineurin-inhibi- islet processing centers (144, 145). tors in the ongoing clinical trials is problematic due to The liver is currently considered the site of choice the well-known nephrotoxicity (203, 245, 246) that may for islet grafts. The availability of interventional radiol- be enhanced by their combination with sirolimus (160, ogy techniques to perform intra-portal islet infusion by a 161, 247, 248). The hepatic site for implantation of islet minimally-invasive, outpatient procedure has made pos- grafts may expose the islets to high concentrations of sible repeated infusions without the need for open sur- drug metabolites (249) that may exacerbate their toxic gery (85, 86, 139). Non-specific inflammation generated effects to islet cells (3, 249, 250) and lead to gradual in the hepatic microenvironment upon islet implant has loss of function. Interestingly, preclinical allogeneic islet been recognized as one of the major contributing fac- transplantation studies in pancreatectomized non-human tors to the loss of viable and functional islet mass (214, primates have shown improved islet function with time 215). Islets produce mediators of inflammation (CD40, using a non-diabetogenic therapy based on anti-CD154 cytokines and chemokines)(143, 216) and coagulation antibody, suggesting that selective targeting of the im- (e.g., tissue factor)(144, 217-223) that may lead to he- mune response with non-toxic reagents is a desirable patic microenvironment and macrophage activation, en- goal to pursue (3, 132, 133, 160, 161, 163, 250-253). As dothelial cell dysfunction, and release of reactive oxy- new reagents with higher selectivity for immune cells and gen species. All these factors may contribute to reduce lower toxicity are becoming available for clinical use, we islet engraftment (77, 219, 224-229). anticipate improved results in the near future (3, 152, Strategies that may be of assistance to reduce the 254-256). The emphasis on this issue has prompted needs for large islet numbers to reverse diabetes after important initiatives to promote the study of novel immu- transplantation include delivery of cytoprotective mol- nosuppressive protocols for clinical islet transplantation ecules to islet cells during all critical steps (before, dur- including the Immune Tolerance Network (47, 48), and 19 the more recent Consortium of Islet Transplant Centers yields and potency of islets that can be recovered after sponsored by the NIH, the NIDDK and the National Insti- processing. Unfortunately, availability of suboptimal tute of Allergy and Infectious Diseases. In order to ben- pancreata to the CITP’s has limited substantially the suc- efit the large number of patients with T1DM awaiting re- cess rate of islet cell processing (close to ~50% in expe- placement of islet cells, the ultimate goal remains the rienced centers) with remarkable economic and resource induction of permanent acceptance of the grafted islets burden. Implementation of fair organ allocation rules without the need for chronic immunosuppression. Pre- between whole pancreas and islet cell transplant pro- vious clinical trials aiming at the induction of donor-spe- grams will contribute to a better utilization of both de- cific tolerance by the use of BMC transplantation have ceased donor pancreata and resources. Alternative failed so far to provide indefinite islet graft survival after sources for insulin-producing tissue for transplantation discontinuation of immunosuppression, possibly due to (xenogeneic and stem cell-derived) are potential avenues the inadequacy of the drugs utilized in these protocols to to overcome the shortage of deceased donor pancreata favor stable hematopoietic chimerism. Very promising in the years to come (53). data are emerging from the translational research focus- The FDA regulates islet transplantation under inves- ing on the definition of novel protocols for the induction tigational new drug (IND) application toward a Biologics of tolerance (to self and allogeneic antigens) that justify License Application for pancreatic islets. For this rea- a cautious optimism (257, 258). son, specific infrastructures and dedicated personnel are Development of sensitive analytical tests for the post- required along with the implementation of cGMP and SOP transplant immune monitoring of our patients will pro- to warrant high quality standard of cell manufacturing for

vide a better understanding of the complex phenomena transplantation. These requirements impose a remark- ISLETS - MIAMI, FLORIDA involved in the immune response to islet allografts in able economic burden to the CITP, and the elevated costs patients with T1DM (85). These tests are of paramount of establishing a functional new facility have contributed importance not only to help us recognize rejection and to the emerging concept of creating ‘regional’ Human recurrence of autoimmunity early enough to intervene Islet Cell Processing Facilities that provide support to and prevent islet graft loss, but also to identify patients remote CITP’s as a viable option to grant consistent qual- eligible for discontinuation of immunosuppression in pro- ity of the final product and contain the costs (30, 31, 49, tocols aiming at the induction of tolerance. 82). While future trials will allow us to define optimal pro- Monitoring of graft function is currently obtained by tocols for islet grafts, the shortage of deceased donor the means of metabolic tests and assessment of glyce- pancreata available for islet transplantation will be a lim- mic control, C-peptide and insulin secretion during meta- iting factor to a wider application of this procedure in clini- bolic challenge. There is no consensus in the islet trans- cal practice. Introduction of better organ procurement plant community on which test should represent the gold- and preservation techniques has been instrumental to standard for assessing islet mass and function, possibly improve the utilization of the available pancreata, and because most of the proposed tests have been designed the use of marginal and non-heart beating donors has for other purposes. There is, therefore, an increasing been also proposed to enlarge the donor pool. How- need for the definition of metabolic tests specific for islet ever, there is still a window of opportunity to improve the grafts that will be essential for a better assessment of utilization of cadaveric pancreata for transplantation. The graft performance. quality of the deceased donor pancreas influences the 20 PILEGGI, RICORDI, KENYON, ET AL

SUMMARY

Transplantation of allogeneic pancreatic islets for betes Research Institute – University of Miami has the treatment of patients with Type 1 diabetes mellitus contributed to the progress in the field with a 20-year (T1DM) is now a reality. The steady progress that has track record. It has been a long journey and despite allowed for the recent successful clinical trials world- the intermediate success, more work is needed in or- wide follows a steep learning curve and the persever- der to achieve the ultimate goal of a safe and long- ance of the international islet transplantation commu- lasting treatment for patients with T1DM. nity. The Clinical Islet Transplant Program at the Dia-

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ACKNOWLEDGEMENTS

Support to the Clinical Islet Transplant Program Thanks to the members of the Human Cell Process- come from the following agencies: ing facility and Translational Research Laboratory of the National Institute of Health (2R01 DK25802-20, M01 Cell Transplant Center, of the Clinical Islet Transplant RR0528, SU42 RR016603-02, NO1AI-15416, Program, General Clinical Research Center, and Admin- NO1AI95380-03); NCRR Islet Cell Resources istrative Offices at the Diabetes Research Institute, and (5U42RR016603) and General Clinical Research Cen- of the Organ Procurement Organizations for the continu- ter (MO1RR16587, 1R01-DK55347-IU42RR016603-02); ous enthusiasm and support to our Program. The Au- NIDDK (5 R01 DK55347, 5R01 DK56953-03) Immune thors are grateful to Drs. Raquel Faradji, Luca Inverardi, Tolerance Network; JDRFI (4-2000-946, and 4-2004- R. Damaris Molano, Mitra Zehtab and Hirohito Ichii for 361); American Diabetes Association; Department of their critical reading of this manuscript. Defense; State of Florida; National Aeronautics and Space Administration; and Diabetes Research Institute Foundation (www.drinet.org).