Islet-Kidney Allografts Correct the Diabetic Hyperglycemia...

Vascularized Islet Cell Transplantation in MiniatureSwineIslet-Kidney Allografts Correct the Diabetic HyperglycemiaInduced by Total PancreatectomyNaoki Kumagai,

1John C. LaMattina,

1Chisako Kamano,

1Parsia A. Vagefi,

1Rolf N. Barth,

1

John J. O’Neil,2

Shin Yamamoto,1

Shannon G. Moran,1

Ryu Utsugi,3

David H. Sachs,1

and Kazuhiko Yamada1

We have previously reported the preparation of vascu-larized islet-kidneys (IKs) by transplantation of isletsunder the autologous kidney capsule. Here, we comparethe efficacy of transplanting vascularized versus non-vascularized islets into diabetic allogeneic swine recip-ients. In the vascularized islet transplantation (5,000islet equivalents [IE]/kg), recipients received minor-mismatched (n � 4) or fully-mismatched (n � 2) IKsafter pancreatectomy, with a 12-day course of cyclo-sporine A (CyA) or FK506, respectively. For the non-vascularized islet transplantation (7,000 IE/kg), threerecipients received minor-mismatched islets alone andtwo recipients received minor-mismatched donor isletsplaced in a donor kidney on the day of transplantation.All recipients of nonvascularized islets were treatedwith a 12-day course of CyA. With vascularized islettransplantation, pancreatectomized recipients weremarkedly hyperglycemic pretransplant (fasting bloodglucose >300 mg/dl). After composite IK transplanta-tion, all recipients developed and maintained normogly-cemia (<120 mg/dl) and stable renal functionindefinitely (>3 months), and insulin therapy was notrequired. Major histocompatibility complex–mismatchedrecipients demonstrated in vitro donor-specific unre-sponsiveness. In contrast, recipients of nonvascularizedislets remained hyperglycemic. In conclusion, IK allo-grafts cured surgically induced diabetes across alloge-neic barriers, whereas nonvascularized islet transplantsdid not. These data indicate that prevascularization ofislet allografts is crucial for their subsequent engraft-ment and that composite IKs may provide a strategy forsuccessful islet transplantation. Diabetes 51:3220–3228, 2002

Replacement of the islets of Langerhans is cur-rently the only treatment for type 1 diabetes thatachieves an insulin-independent, normoglyce-mic state. Recent data using the Edmonton

Protocol (1) have demonstrated a much improved survivalof islets after transplantation, with initial normalization ofblood glucose levels in patients; however, this protocolrequired �9,000 islet equivalents (IE)/kg, necessitating theuse of two to four pancreas donors for a single recipient.In addition, by 1-year posttransplantation, 3 of the 12patients were diabetic, and two required insulin (2). Thus,these data, while much improved over previous islettransplantation data, are limited in clinical applicability bythe small number of donor organs currently available andby the requirement of islets from multiple donors for asingle diabetic recipient. Therefore, methods for trans-planting islets from single donors that approach the resultsof whole-organ pancreas transplantation, for which an�80% 1-year graft survival and function has been reported(3–7), must continue to be sought. In addition to theproblems of engraftment of transplanted islets, currentclinical regimens for islet transplantation require indefinitedaily administration of immunosuppressive drugs. There-fore, strategies aimed at the induction of tolerance to isletgrafts, using a short-course immunosuppression, wouldnot only improve the efficacy of islet transplantation, butwould also obviate the need for chronic immunosuppres-sion.

One of the most likely reasons for the poor success thusfar in islet cell transplantation is that these tissue graftsmust establish new vasculature from the host to survive.During the time required for such revascularization, thereis a much increased susceptibility to loss from ischemicinjury (e.g., lack of oxygen or nutrients), as well aspossible immunologic impairment as a consequence of theallogeneic environment. We have hypothesized thatgreater success in islet transplantation may be achievedthrough the use of primarily vascularized islets, eliminat-ing the period required for vascularization after allogeneictransplantation. Therefore, we recently described thepreparation of composite islet-kidney (IK) grafts contain-ing vascularized autologous islets (31). The transplanta-

From the 1Transplantation Biology Research Center, Massachusetts GeneralHospital, Boston, Massachusetts; the 2Department of Islet Transplantation andCell Biology, Joslin Diabetes Center, Boston, Massachusetts; and the 3Depart-ment of Urology, Niigata University School of Medicine, Niigata, Japan.

Address correspondence and reprint requests to Kazuhiko Yamada, MD,PhD, Transplantation Biology Research Center, Massachusetts General Hos-pital, MGH-East, Building 149-9019, 13th St., Boston, MA 02129. E-mail:[email protected].

Received for publication 8 April 2002 and accepted in revised form 8 August2002.

CML, cell-mediated lymphocytotoxicity; CyA, cyclosporine A; FBG, fastingblood glucose; H&E, hematoxylin and eosin; HBSS, Hank’s balanced saltsolution; IE, islet equivalents; IK, islet-kidney; MHC, major histocompatibilitycomplex; MLR, mixed lymphocyte response; PBMC, peripheral blood mono-nuclear cells; POD, postoperative day.

3220 DIABETES, VOL. 51, NOVEMBER 2002

tion of prevascularized islets enables a comparisonbetween transplanting vascularized versus nonvascular-ized islets in our miniature swine.

Our initial study demonstrated that autologous isletsengraft and function when implanted beneath the renalcapsule, whereas minor-mismatched islets are rejectedwhen similarly implanted (31). In the present study, weexamine the ability of IKs to cure surgically induceddiabetes with a short course of immunosuppression, ascompared with that of nonvascularized direct islet trans-plants across the same allogeneic barriers. The transplan-tation of IKs could have significant relevance as a strategyfor the induction of tolerance to allogeneic islets.

RESEARCH DESIGN AND METHODS

Animals. Minor and fully major histocompatibility complex (MHC)-mis-matched transplant donors (�1.5 years of age) and recipients (3–5 months ofage) were selected from our herd of partially inbred miniature swine (8,9).Islet harvest and transplantation. Islet donors were adult miniature swine,subjected to either total or partial pancreatectomy, as described elsewhere(31). For partial pancreatectomy, �30% of the pancreas (the head and theremainder of the body) was left intact. For total pancreatectomy, the pancreaswas carefully dissected so as to not damage the duodenum or the pancreas.The excised pancreas was placed in crushed ice immediately, and modifiedUniversity of Wisconsin solution was infused through the pancreatic duct.Pancreatic tissue was processed at the Joslin Diabetes Center (Boston, MA)(27). After islet isolation, the purity of the preparation was evaluated withdithizone staining. Islet cell viability was assessed using dual fluorescencestaining with acridine orange (AO; Sigma) and propidium iodide (PI; Sigma).The islets were cultured for 3 days, counted, and then transplanted eitherbeneath the renal capsule or via the superior mesenteric vein.IK preparation. IKs were prepared in donors as previously described (31).Briefly, islets were suspended in 1.0 ml saline and injected beneath the renalcapsule using a 20G angiocatheter. IKs were prepared 2–3 months before theintended date of allogeneic transplantationInduction of type 1 diabetes in recipients. Type 1 diabetes was inducedsurgically by total pancreatectomy 3–7 days before either composite IK ordirect islet transplantation. Total pancreatectomy was performed using asimilar procedure to that described above for islet donors. Blood supply toduodenum and bile ducts was well preserved, and no blood transfusions wererequired (Fig. 1). Thereafter, these animals received pancreatase (Pancre-zyme; Daniels Pharmaceuticals, St. Petersburg, FL) supplementation in theirdiet to replace exocrine pancreatic enzymes.IK transplantation. Allogeneic transplantation of composite IKs was per-formed by the same technique as described previously for renal transplanta-tion (10,11). Briefly, recipients underwent bilateral nephrectomy. The IK washarvested from the donor, flushed immediately with cold perfusion solution,and then placed on ice until transplantation. The IK was transplantedorthotopically into the recipient, using end-to-side aortocaval anastomoses.Urinary drainage was accomplished through a vesicoureteral anastomosis.Two semipermanent indwelling silastic central venous catheters were placedinto the external jugular veins of the recipient. One catheter was used for theadministration immunosuppression and the other to obtain blood samples.Total cold ischemic time was kept to �60 min. No blood transfusions wererequired.Immunosuppression. Cyclosporine A (CyA) (Sandimmune) was generouslyprovided by Novartis Pharmaceutical (East Hanover, NJ) and was mixed andadministered as an intravenous suspension, according to specifications of themanufacturer. CyA was given daily as a single infusion (10 mg � kg�1 � day�1)for 12 consecutive days, beginning on postoperative day (POD) zero. Whole-blood trough levels were determined by a fluorescence polarization immuno-assay (Abbott Laboratories, Dallas, TX), which measured the parentcompound but not metabolites, and the results were expressed in nanogramsper milliliter. FK506 was generously provided by Fujisawa Healthcare (Deer-field, IL) and was administered continuously through an infusor pump (BaxterHealthcare, Deerfield, IL) for 12 consecutive days, beginning on POD zero.FK506 doses were started at 0.15 mg � kg�1 � day�1 and adjusted to maintainblood levels between 30 and 40 ng/ml. Whole-blood levels were determined bymicroparticle enzyme immunoassay (Tacrolimus II, IMX System; AbbottLaboratories).Monitoring of blood glucose. Islet function was assessed through fastingblood glucose (FBG) levels, glucose tolerance tests, and histological exami-nation of biopsies. Glucose tolerance testing was performed by intravenous

infusion of a glucose load (0.5 g/kg body wt) after a 24-h fasting period. Bloodsamples were obtained at 0, 5, 15, 30, 60, and 120 min postinfusion andanalyzed for glucose and insulin concentrations.Biopsies. Biopsies were obtained on PODs 30, 60, and �100. Biopsies wereperformed through a 2-inch flank incision. Subcutaneous tissue was laterallydisplaced to permit both macroscopic examination and wedge biopsy. Biop-sies that included islet and renal tissue were divided into sections for staining(hematoxylin and eosin [H&E]) and immunohistochemistry.Monitoring of rejection. Rejection was monitored through measurement ofplasma creatinine and FBG levels, as well as histological assessment of biopsytissue. The clinical end points used in this study were either graft survival�100 days or a moribund animal with uremia or hyperglycemia. Biopsiedtissues were examined by a pathologist, and rejection of kidneys wasdiagnosed according to a standardized grading system (12).Histological features. Tangential sections were taken to include islet andrenal tissue. Formaldehyde-processed specimens were examined using H&Estaining and insulin-specific antibodies. Insulin staining was performed on4.0-�m sections cut from a paraffin-embedded block. Sections were incubatedat room temperature in a humidified chamber for 20 min with 10% normal goatserum in PBS (pH 7.4) and then for 60 min with guinea pig anti-porcine insulinantibody (Dako, Carpinteria, CA) diluted 1:10 in PBS. They were nextincubated with a 1:200 dilution of biotinylated goat anti–guinea pig antibody(Vector Laboratories, Burlingame, CA) for 60 min. The tissue-bound primaryantibodies were detected by an avidin-biotin-peroxidase complex (Dako) thatwas visualized by staining with 0.02% hydrogen peroxide containing 0.3 mg/ml3,3�-diaminobenzidine in 0.05 mol/l Tris buffer. Sections were counterstainedwith Gill’s single-strength hematoxylin.Preparation of peripheral blood lymphocytes. Blood was drawn from theexternal jugular vein on PODs 30, 60, and �100. For separation of peripheralblood lymphocytes, freshly heparinized whole blood was diluted �1:2 withHank’s balanced salt solution (HBSS; Gibco BRL, Grand Island, NY), and themononuclear cells were obtained by gradient centrifugation using lymphocyteseparation medium (Organon, Teknika, Durham, NC). The mononuclear cellswere washed once with HBSS, and contaminating red cells were lysed withammonium chloride potassium buffer (B&B Research Laboratory, Fiskeville,RI). Cells were then washed with HBSS and resuspended in tissue culturemedium. All cell suspensions were kept at 4°C until used in flow cytometryassays.Cell-mediated lymphocytotoxicity assay. Cell-mediated lymphocytotoxic-ity (CML) assays were performed as previously described using our standard

FIG. 1. Intra-operative images obtained during total pancreatectomy ina recipient demonstrate the gross appearance of the harvested porcinepancreas (A) and the peritoneal cavity after removal of the pancreas(B).

N. KUMAGAI AND ASSOCIATES

DIABETES, VOL. 51, NOVEMBER 2002 3221

CML tissue culture medium (10). Briefly, lymphocyte cultures containing 4 �106 responder and 4 � 106 stimulator peripheral blood mononuclear cells(PBMCs) (irradiated with 2,500 cGy) were incubated for 6 days at 37°C in 7.5%CO2 and 100% humidity. Bulk cultures were harvested, and effectors weretested for cytotoxic activity on 51Cr (Amersham, Arlington Heights, IL)-labeledlymphoblast targets. Effector cells were incubated for 5.5 h with target cells atE/T ratios of 100:1, 50:1, 25:1, and 12.5:1. Three target cells were tested in eachassay: swine leukocyte antigen (SLA)-matched PBMCs to the effectors,donor-matched PBMCs, and third-party PBMCs. Supernatants were thenharvested using the Skatron collection system (Skatron, Sterling, VA), and51Cr release was determined on a gamma counter (Micromedics, Huntsville,AL). The results were expressed as PSL (percent specific lysis), calculated as:

PSL �experimental release (cpm � spontaneous release (cpm)

maximum release (cpm) � spontaneous release (cpm)� 100%

(1)

Mixed lymphocyte response assays. Mixed lymphocyte response (MLR)assays were performed by plating 4 � 105 responder PBMCs in triplicate in96-well flat-bottom plates (Costar, Cambridge, MA). Cells were stimulatedwith 4 � 105 stimulator PBMCs irradiated with 2,500 cGy. Cultures wereincubated for 5 days at 37°C in 6% CO2 and 100% humidity in our standard MLRculture medium (13). 3H-thymidine was added for an additional 6 h of culture.Wells were then harvested onto Mash II glass fibers and counted for emission.

RESULTS

IKs reverse the hyperglycemia of surgically induced

diabetes across a minor-mismatched barrier. Fouranimals were completely pancreactectomized within 1week before receiving a minor-mismatched composite IK.After total pancreatectomy, the FBG of the recipientsmarkedly increased, reaching levels �280 mg/dl (Fig. 2A).No insulin therapy was given.

Donors were selected by weight to achieve the requirednumber of IE per kilogram for the intended recipient. IKscontaining �5,000 IE/kg recipient body wt were trans-planted, with a 12-day course of CyA at 10 mg � kg�1 �day�1. The recipients’ native kidneys were removed on theday of IK transplantation. After IK transplantation, FBGlevels fell immediately to normal levels (�120 mg/dl) byPOD 1. IK grafts were permanently accepted, and recipi-ents maintained normoglycemia for at least 90 days inthree of four cases (Fig. 2A, C, and D), without the needfor insulin therapy. The fourth animal had to be sacrificedbecause of complications from a catheter infection on

FIG. 2. A– D: Totally pancreatectomized recipients of minor-mismatched IKs maintained stable FBG and plasma creatinine levels after IKtransplantation. Both FBG and creatinine levels sharply increased after IK nephrectomy (IK Gx). B: Animal was killed secondary to complicationsarising from a catheter infection (#). E: Representative data demonstrating a 0.5-g i.v. glucose tolerance test in an IK recipient are compared withboth a naı̈ve animal and a totally pancreatectomized (total Px) animal. Glucose tolerance testing reveals normal responses to intravenous glucose2 months after IK transplantation. Representative biopsy obtained on POD 128 demonstrates minimal cellular infiltrate (F, H&E) and numerousinsulin-producing �-cells (G, insulin staining).

VASCULARIZED ISLET TRANSPLANTATION


POD 67; however, this animal had also maintained normo-glycemia after IK transplantation (Fig. 2B).

Glucose tolerance testing revealed normal responses tointravenous glucose loads of 0.5 g at 2 months after IKtransplantation (Fig. 2E, representative data shown). Incontrast, glucose levels in pancreatectomized control an-imals reached �450 mg/dl (Fig. 2E). Upon gross examina-tion of the IK allografts, no edema or spot hemorrhageswere visible. As seen in Fig. 2F and G, a biopsy on POD 128in a representative animal revealed numerous insulin-producing -cells with minimal cellular infiltrate (Fig. 2F,H&E staining; Fig. 2G, insulin staining). Renal structureand function were followed throughout the transplantperiod. Creatinine levels remained �1.5 mg/dl throughoutthe clinical course (Fig. 2A– D), and biopsies revealedintact kidney parenchyma with minimal cellular infiltrate(renal parenchyma in Fig. 2F). The recipient animalssurvived long term (�3 months) and demonstrated asignificant increase in weight during the experiment (40,63, and 8% weight gain on the day of IK graftectomycompared with day of IK transplant in each animal). TheIKs were explanted on PODs 67, 90, 128, and 150, andfollow-up blood glucose levels were monitored to assurethe completeness of pancreatectomies. The blood glucoselevels rose immediately after IK explantation in eachanimal, indicating that the total pancreatectomies werecomplete in all cases.Nonvascularized minor-mismatched islets did not re-

verse the hyperglycemia of surgically induced diabe-

tes. Three pancreatectomized animals received directminor-mismatched islets transplanted beneath the renalcapsule (n � 1) or injected via the portal vein (90% ofislets) and beneath the renal capsule (10% of islets) (n �2). A total of 7,000 IE/kg recipient body wt were injectedinto each animal, followed by a 12-day course of CyA (10mg � kg�1 � day�1). This increased number of islets was�40% more than that used in the preparation of IKs tocompensate for any islet cell proliferation that might have

occurred during the 2- to 3-month period after IK prepa-ration. Recipient FBG decreased on POD 1; however, thelevels subsequently increased and the islets were rejectedby histological criteria by POD 23 (Figs. 3 and 4A and B) inthe two animals that underwent direct islet transplanta-tion. The third animal was maintained on exogenousinsulin during postoperative weeks 2–4 to determinewhether prolonged survival of the animal by insulin ther-apy would provide direct islet allografts additional time toengraft. This animal’s FBG was maintained between 180and 250 mg/dl with insulin therapy; however, blood glu-cose levels immediately increased after cessation of insu-lin at week 5 (Fig. 3). Autopsy results revealedmononuclear cell infiltrates and fibrosis beneath the renalcapsule (the site of islet implantation) (Fig. 4C). Nofunctioning islets were found on histological evaluation(Fig. 4D).Simultaneous transplantation of nonvascularized

minor-mismatched islets and a renal allograft did not

reverse the hyperglycemia of surgically induced dia-

betes. Two pancreatectomized animals received directminor-mismatched islets and renal allografts, isolatedfrom the same donor and transplanted on the same day,with a standard 12-day course of CyA. Three days beforetransplantation, donor pancreata were partially removedthrough a 70% pancreatectomy, and islets were harvestedfor a 3-day culture. On the day of transplantation, renalharvest was performed on the islet donor and recipientsunderwent bilateral nephrectomies before renal transplan-tation. Immediately after revascularization of the donorrenal allograft, cultured donor islets (7,000 IE/kg recipientbody wt) were injected beneath the renal capsule. In bothanimals, FBG levels decreased on POD 1 (Fig. 5A). How-ever, thereafter, both animals became markedly hypergly-cemic. One moribund animal was killed on POD 14 (Fig.5A), while the other animal was maintained with exoge-nous insulin from POD 5 until POD 29 (Fig. 5B). The targetFBG level in this period was established between 180 and

FIG. 2—Continued



250 mg/dl. Regular and/or lente insulin was titrated be-tween 4 and 10 units/day to maintain target FBG levels.The animal required less insulin (from 10 units/day to 6units/day) to maintain target FBG levels during week 4,and insulin therapy was discontinued on POD 29. How-ever, FBG levels never fell below 180 mg/dl after thecessation of insulin and levels increased markedly to 600mg/dl on POD 31 (Fig. 5B). Creatinine levels remainednormal in the animal that died on POD 14 (3 days aftercessation of CyA); however, creatinine levels increasedslightly in the animal that demonstrated a prolongedsurvival on insulin therapy (Fig. 5B). Histological analysisof the autopsy specimen on POD 31 revealed few remain-ing islets and a diffuse mononuclear cell infiltrate in thesite of islet transplantation (Fig. 5C). Furthermore, aninterstitial mononuclear cellular infiltrate was seen in therenal parenchyma (Fig. 5D), a finding not noted in our

previous experience with minor-mismatched renal trans-plantation alone in miniature swine.IKs cured surgically induced diabetes across a two-

haplotype fully-mismatched barrier. Two pancreatecto-mized animals received fully-mismatched IKs containing�5,000 IE/kg recipient body wt, with a 12-day course ofFK506, which has been shown to facilitate the induction oftolerance to fully mismatched renal allografts in our min-iature swine (13). Native kidneys were removed on the dayof IK transplantation. In each case, FBG levels rose to�280 mg/dl after pancreatectomy. The composite IKs wereaccepted, and normoglycemia was immediately restored.However, both animals exhibited a transient period ofhyperglycemia that lasted, approximately, from POD 5 toPOD 18. These episodes resolved spontaneously within aweek after the cessation of FK506, suggesting that thetransient rise in blood glucose may have been due toFK506-associated islet toxicity (14,15). The clinical, patho-logical, and histological outcome in these animals wasotherwise similar to that seen in minor-mismatched IKtransplantation with a 12-day course of CyA. All IKs werepermanently accepted, and recipients maintained normo-glycemia for at least 120 days without insulin therapy (Fig.6A and B). Glucose tolerance testing of IK recipients wasnormal 2 months after IK transplantation (Fig. 6C). Grossexamination of the grafts revealed no edema, spot hemor-rhages, or vasculopathy. Biopsy results revealed numerousinsulin-producing -cells with a minimal cellular infiltrate(Fig. 6D). Additional immunosuppression or insulin ther-apy required was not required in either case, and creatininelevels were maintained below 1.5 mg/dl throughout theexperiment (Fig. 6A). The blood glucose levels rose imme-diately after IK explantation in each animal.IKs induced donor-specific tolerance in vitro when

transplanted across fully-mismatched barriers. IK re-cipients developed in vitro evidence of donor-specific

FIG. 3. Direct islet transplantation across minor-mismatched barrierswas unable to achieve stable FBG levels after total pancreatectomywhen nonvascularized islets were injected beneath the recipient’srenal capsule alone (K) or when nonvascularized islets were injectedinto the recipient’s liver via the superior mesenteric vein and beneaththe recipient’s renal capsule (K�L).

FIG. 4. Histological analysis of islets trans-planted directly beneath the renal capsule ofminor-mismatched pancreatectomized recip-ients revealed a subcapsular mononuclearcell infiltrate and fibrosis, and few function-ing islets were noted (A and C, H&E; B andD, insulin staining). A and B: Transplantedislets in autopsy specimens obtained on POD23. C and D: Transplanted islets in autopsyspecimens obtained on POD 33 in an addi-tional animal.



tolerance within 30 days after IK transplantation. Figure7A demonstrates the CML responses on POD 30 in bothanimals. Strong anti–third party reactivity, but no anti-donor reactivity, was observed. MLR analysis also re-vealed anti-donor specific unresponsiveness (Fig. 7B),which was maintained for the duration of the experiment,confirming that these animals were tolerant.

DISCUSSION

We report here that vascularized islet cells, when trans-planted as part of a composite IK, cured surgically induceddiabetes across minor- and fully MHC–mismatched barri-ers. The present study demonstrates that 1) nonvascular-ized islet transplants isolated from a single donor are notcapable of curing surgically induced diabetes and that 2) asimilar number of prevascularized islets, within compositeIK grafts, can cure surgically induced diabetes, as well asinduce tolerance, after a short course of immunosuppres-sion. Recipient animals that were hyperglycemic aftercomplete pancreatectomy maintained normoglycemia in-definitely after IK transplantation. Upon removal of the IK,these animals returned to their pretransplant, hyperglyce-mic state, indicating that the pancreatectomies were in-deed complete and that the transplanted vascularizedislets within the composite IK were responsible for the

normal regulation of blood glucose levels. An additionalimportant note is the fact that each IK contained 5,000IE/recipient body wt on the day of the IK transplantation.These recipients gained body weight, which effectivelydiluted the ratio of IE per kilogram recipient body weightafter IK transplantation. Therefore, by the conclusion ofthe experiment, the recipients maintained normoglycemiawith �3 � 103 IE/kg recipient body wt.

Our previous studies of allogeneic and xenogeneic thy-mic tissue transplantation have demonstrated the role ofprevascularization on graft survival (18,19), a principlethat has already been noted for transplants of kidneytissues in a rodent model (28). The present studies providefurther evidence of the critical nature of a graft’s vascularsupply. Nonvascularized islets neither engrafted nor con-trolled blood glucose levels in transplanted recipients.While nonvascularized islet transplants transiently de-creased FBG levels during the first 2 days after transplan-tation, this decrease was most likely secondary to therelease of insulin from damaged ischemic islet -cells.Histological and physiological data indicated that suchgrafts were rapidly rejected within the first weeks aftertransplantation. Vascularized grafts appeared less suscep-tible to this nonspecific graft loss and did not sensitize thehost to donor antigen—processes that are likely to have

FIG. 5. A and B: Animals that underwent simultaneous transplantation of nonvascularized minor-mismatched islets and a renal allograft acrossminor-mismatched barriers were unable to maintain stable fasting blood glucose levels after total pancreatectomy. The recipient (B), otherwiseidentical to A, was maintained with exogenous insulin from POD 5 until POD 29. Regular and/or lente insulin was titrated between 4 and 10units/day to maintain target FBG levels between 180 and 250 mg/dl. Autopsy specimens obtained from the animal depicted in B on POD 31demonstrate few remaining islets and a diffuse mononuclear cell infiltrate at the site of islet transplantation (C) and an interstitial mononuclearcellular infiltrate in the renal parenchyma (D).



limited the success of nonvascularized islet grafts. In fact,it has been shown that transplants of composite kidney-islet grafts were more successful in avoiding autoimmunedestruction in BB rats than freely grafted islets (30).

While previous studies have reported increased islet cellsurvival when injected via the portal vein (2,20), ourstudies in miniature swine were unable to confirm thisfinding.

This study also demonstrated that the success of the IKgraft is not due to antigen load or specific help by a donor

kidney, as cotransplantation of a donor kidney did notfacilitate engraftment of nonvascularized islets. The im-proved survival of composite IK grafts appears to be basedalmost entirely on the prevascularization of the islettissue. Creation of autologous IKs allowed for ischemia,reperfusion, and revascularization to all occur in a non-allogeneic setting, whereas directly transplanted isletsmust encounter all of these barriers to engraftment in anallogeneic setting. Autologous IK creation permitted notonly enhanced islet engraftment by avoidance of typical

FIG. 6. A and B: Recipients of two-haplotype fullyMHC–mismatched IKs maintained stable FBG andplasma creatinine levels after total pancreatectomy(Px). C: Both recipients demonstrated a normalresponse to a 0.5-g i.v. glucose tolerance test. D:Representative biopsy obtained on POD 143 dem-onstrates numerous insulin-producing �-cells.



rejection responses observed during this period, but alsofacilitated the long-term induction of tolerance by mini-mizing the presentation of allogeneic antigens after trans-plantation. When renal transplantation was immediatelyfollowed by the subcapsular implantation of cultured isletsisolated from the same donor, the composite grafts wereunable to cure diabetes in pancreatectomized recipients.Furthermore, the renal parenchyma in these grafts dem-onstrated an increased infiltrate as compared with histor-ical renal allograft controls performed across similarimmunological barriers (21). This indicated that 1) theprocess of renal transplantation itself does not lead toenhanced islet survival, 2) nonvascularized islets may bemore immunogenic than a kidney transplanted alone, and3) the presence of nonvascularized islets may actuallysensitize the host to donor antigen.

The present studies also indicated that immunosuppres-sive regimens that induced tolerance to renal allograftsalso induced tolerance to associated vascularized isletswithin the IK graft. However, such regimens are notcapable of inducing tolerance to islets directly trans-planted into recipients. Our previous studies have demon-strated that a 12-day course of CyA (10) or FK506 inducestolerance to partial MHC– and fully MHC–mismatchedrenal allografts, respectively (13). These same regimenswere capable of inducing tolerance to vascularized isletsacross similar barriers in the present study. Althoughshort-term high-dose FK506 has not been used for toler-ance induction clinically, we have used it successfully forthis purpose recently in baboons (P.A.V., R.N.B., S.Y.,J.M.L., J.C.L., C.K., D.H.S., K.Y., manuscript in prepara-tion), and similar doses have been used clinically for thetreatment of ABO-incompatible renal transplantation (16)and GvHD (17).

We have suggested that the transient rise in blood

glucose in our fully MHC–mismatched recipients may havebeen due to FK506-associated islet toxicity (14,15). Thislevel has never induced diabetes in our miniature swinerecipients of kidneys (13), and FK506 blood levels werecontrolled between 25 and 35 ng/ml in both cases. Thus,transplanted islets could be sensitive to this drug, espe-cially in the induction period after transplantation. Eventhough the regimen of FK506 needed to induce tolerancemight have a slightly diabetogenic effect, the benefit ofusing a short course of immunosuppression for the estab-lishment of long-term tolerance is very attractive. In fact,the hyperglycemic period, which was insulin free, wasonly transient and easily reversed upon the discontinua-tion of FK506. In the future, strategies that are currentlydemonstrating success in preclinical and early clinicalmodels of renal allograft transplantation, such as mixedchimerism (22,29), costimulatory blockade (23), and FN18immunotoxin (24,25), may be used to induce tolerance tovascularized islet allografts and avoid any secondary islettoxicity.

Although the feasibility of islet transplantation haspreviously been demonstrated (1,2), to our knowledge, thesuccessful transplantation of islets isolated from a singleliving donor has not achieved the success reported fromthe use of multiple islet donors. The IK strategy permittedthe isolation of a sufficient number of islets, while leavingthe donor normoglycemic and with intact exocrine func-tion. Such a strategy may eventually permit successfulliving-related islet donation for diabetic patients withend-stage diabetic nephropathy. We can envision the lapo-roscopic preparation of IK grafts (26) several months inadvance of kidney transplantation.

While we have chosen to initiate our studies in anallogeneic model to assess cellular responses withoutattendant complications from natural antibodies, this

FIG. 7. Recipients of two-haplotype fullyMHC–mismatched IKs demonstrate donor-specific unresponsiveness on both CML (A)and MLR assays on POD 30 (B, left column)as compared with naı̈ve controls (B, rightcolumn).



model may be even more applicable for IK transplantsacross xenogeneic barriers. In xenotransplantation, thepreparation of IKs could provide an unlimited source oforgans for transplantation into diabetic recipients requir-ing kidneys.

ACKNOWLEGEMENTS

This work was supported by the following grants/grantors:NIH R01 AI 31046, the Juvenile Diabetes Foundation, andFujisawa Health Care.

The authors would like to thank Drs. Paul S. Russell,James S. Allan, and Douglas R. Johnston for their helpfulreview of the manuscript; Scott Arn for herd managementand quality control typing; and Lisa A. Bernardo for help inmanuscript preparation. The authors would also like tothank Fujisawa Healthcare (Deerfield, IL) for generouslyproviding FK506, Novartis Pharmaceutical (East Hanover,NJ) for providing CyA, and Schering-Plough Animal Healthfor providing Flunixamine.

REFERENCES

1. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL,Kneteman NM, Rajotte RV: Islet transplantation in seven patients with type1 diabetes mellitus using a glucocorticoid-free immunosuppressive regi-men. N Engl J Med 343:230–238, 2000

2. Ryan EA, Lakey JR, Rajotte RV, Korbutt GS, Kin T, Imes S, Rabinovitch A,Elliott JF, Bigam D, Kneteman NM, Warnock GL, Larsen I, Shapiro AM:Clinical outcomes and insulin secretion after islet transplantation with theEdmonton protocol. Diabetes 50:710–719, 2001

3. Stratta RJ, Taylor RJ, Wahl TO, Duckworth WC, Gallagher TF, Fischer JL,Neumann TV, Miller S, Langnas AN: Recipient selection and evaluation forvascularized pancreas transplantation. Transplantation 55:1090–1096,1993

4. Gruessner RW: Tacrolimus in pancreas transplantation: a multicenteranalysis. Tacrolimus Pancreas Transplant Study Group. Clin Transplant

11:299–312, 19975. Alejandro R: Transplantation of islets of Langerhans in patients with

insulin-dependent diabetes mellitus. Scand J Gastroenterol Suppl 208:125–128, 1995

6. Bretzel RG, Hering BJ, Federlin KF: Islet cell transplantation in diabetesmellitus: from bench to bedside. Exp Clin Endocrinol Diabetes 103 (Suppl2):143–159, 1995

7. Alejandro R, Lehmann R, Ricordi C, Kenyon NS, Angelico MC, Burke G,Esquenazi V, Nery J, Betancourt AE, Kong SS, Miller J, Mintz DH:Long-term function (6 years) of islet allografts in type 1 diabetes. Diabetes

46:1983–1989, 19978. Sachs DH, Leight G, Cone J, Schwartz S, Stuart L, Rosenberg S: Transplan-

tation in miniature swine. I. Fixation of the major histocompatibilitycomplex. Transplantation 22:559–567, 1976

9. Sachs DH: MHC homozygous miniature swine. In Swine as Models in

Biomedical Research. Swindle MM, Moody DC, Phillips LD, Eds. Ames,Iowa, Iowa State University Press, 1992, p. 3–15

10. Rosengard BR, Ojikutu CA, Guzzetta PC, Smith CV, Sundt TM III, NakajimaK, Boorstein SM, Hill GS, Fishbein JM, Sachs DH: Induction of specifictolerance to class I disparate renal allografts in miniature swine withcyclosporine. Transplantation 54:490–497, 1992

11. Yamada K, Gianello PR, Ierino FL, Lorf T, Shimizu A, Meehan S, Colvin RB,Sachs DH: Role of the thymus in transplantation tolerance in miniatureswine. I. Requirement of the thymus for rapid and stable induction oftolerance to class I-mismatched renal allografts. J Exp Med 186:497–506,1997

12. Colvin RB: The renal allograft biopsy. Kidney Int 50:1069–1082, 199613. Utsugi R, Barth RN, Lee RS, Kitamura H, LaMattina JC, Ambroz J, Sachs

DH, Yamada K: Induction of transplantation tolerance with a short course

of tacrolimus (FK506). I. Rapid and stable tolerance to two-haplotype fullymhc-mismatched kidney allografts in miniature swine. Transplantation

71:1368–1379, 200114. Drachenberg CB, Klassen DK, Weir MR, Wiland A, Fink JC, Bartlett ST,

Cangro CB, Blahut S, Papadimitriou JC: Islet cell damage associated withtacrolimus and cyclosporine: morphological features in pancreas allograftbiopsies and clinical correlation. Transplantation 68:396–402, 1999

15. Ueki M, Yasunami Y, Ina K, Ryu S, Funakoshi A, Kamei T, Ikeda S:Diabetogenic effects of FK506 on renal subcapsular islet isografts in rat.Diabetes Res Clin Pract 20:11–19, 1993

16. Takahashi K, Saito K, Sonda K, Nakagawa Y, Katagiri A, Tomita Y,Tanigawa T, Takeda M: Tacrolimus therapy for ABO-incompatible kidneytransplantation. Transplant Proc 30:1219–1220, 1998

17. Ohashi Y, Minegishi M, Fujie H, Tsuchiya S, Konno T: Successful treatmentof steroid-resistant severe acute GVHD with 24-h continuous infusion ofFK506. Bone Marrow Transplant 19:625–627, 1997

18. Yamada K, Shimizu A, Ierino FL, Utsugi R, Barth RN, Esnaola N, Colvin RB,Sachs DH: Thymic transplantation in miniature swine. I. Development andfunction of the “thymokidney.” Transplantation 68:1684–1692, 1999

19. Yamada K, Shimizu A, Utsugi R, Ierino FL, Gargollo P, Haller GW, ColvinRB, Sachs DH: Thymic transplantation in miniature swine. II. Induction oftolerance by transplantation of composite thymokidneys to thymecto-mized recipients. J Immunol 64:3079–3086, 2000

20. Farney AC, Najarian JS, Nakhleh RE, Lloveras G, Field MJ, Gores PF,Sutherland DE: Autotransplantation of dispersed pancreatic islet tissuecombined with total or near-total pancreatectomy for treatment of chronicpancreatitis. Surgery 110:427–437, 1991

21. Fuchimoto Y, Yamada K, Shimizu A, Yasumoto A, Sawada T, Huang CA,Sachs DH: Relationship between chimerism and tolerance in a kidneytransplantation model. J Immunol 162:5704–5711, 1999

22. Kawai T, Cosimi AB, Colvin RB, Powelson J, Eason J, Kozlowski T, SykesM, Monroy R, Tanaka M, Sachs DH: Mixed allogeneic chimerism and renalallograft tolerance in cynomolgus monkeys. Transplantation 59:256–262,1995

23. Kirk AD, Harlan DM, Armstrong NN, Davis TA, Dong Y, Gray GS, Hong X,Thomas D, Fechner JH Jr, Knechtle SJ: CTLA4-Ig and anti-CD40 ligandprevent renal allograft rejection in primates. Proc Natl Acad Sci U S A

94:8789–8794, 199724. Knechtle SJ, Vargo D, Fechner J, Zhai Y, Wang J, Hanaway MJ, Scharff J,

Hu HZ, Knapp L, Watkins D, Neville DM Jr: FN18-CRM9 immunotoxinpromotes tolerance in primate renal allografts. Transplantation 63:1–6,1997

25. Thomas JM, Contreras JL, Jiang XL, Eckhoff DE, Wang PX, Hubbard WJ,Lobashevsky AL, Wang W, Asiedu C, Stavrou S, Cook WJ, Robbin ML,Thomas FT, Neville DMJ: Peritransplant tolerance induction in macaques:early events reflecting the unique synergy between immunotoxin anddeoxyspergualin. Transplantation 68:1660–1673, 1999

26. Gruessner RW, Kandaswamy R, Denny R: Laparoscopic simultaneousnephrectomy and distal pancreatectomy from a live donor. J Am Coll Surg

193:333–337, 200127. O’Neil JJ, Stegemann JP, Nicholson DT, Gagnon KA, Mullon CJP, Soloman

BA: The isolation and function of porcine islets from market weight pigs.Cell Transplant 10:235–246, 2001

28. Russell PS, Chase CM, Colvin RB, Plate JMD: Kidney transplants. In Mice:

An Analysis of the Immune Status of Mice Bearing Long-Term, H-2

Incompatible Transplants. J Exp Med 147:1449–1468, 197829. Spitzer TR, Delmonico F, Tolkoff-Rubin N, McAfee S, Sackstein R, Saidman

S, Colby C, Sykes M, Sachs DH, Cosimi AB: Combined histocompatibilityleukocyte antigen-matched donor bone marrow and renal transplantationfor multiple myeloma with end stage renal disease: the induction ofallograft tolerance through mixed lymphohematopoietic chimerism.Transplantation 68:480–484, 1999

30. Bartlett ST, Hadley GA, Dirden B, Schweitzer EJ, Eans S, Pham D, SheffieldC: Composite kidney-islet transplantation prevents recurrent autoimmunebeta-cell destruction. Surgery 114:211–217, 1993

31. Kumagai N, O’Neil JJ, Barth RN, LaMattina JC, Utsugi R, Moran SG,Yumamoto S, Vagefi PA, Kitamura H, Kamano C, Sachs DH, Yamada K:Vascularized islet cell transplantation in miniature swine. I. Preparation ofvascularized islet-kidneys. Transplantation. In press



Islet-Kidney Allografts Correct the Diabetic Hyperglycemia...

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