Proc. Nati. Acad. Sci. USAVol. 88, pp. 11100-11104, December 1991Medical Sciences

Xenotransplantation of canine, bovine, and porcine islets in diabeticrats without immunosuppression

(diabetes/artfia Pancreas/m/munobolatlo)

ROBERT P. LANZA*tt, DEBORAH H. BUTLER*, KERMIT M. BORLAND*, JAMES E. STARUK*,DENISE L. FAUSTMAN*§, BARRY A. SOLOMON¶, THOMAS E. MULLER¶, RANDALL G. RUPP*,TAKASHI MAKIt, ANTHONY P. MONACOt, AND WILLIAM L. CHICK**BioHybrid Technologies, Inc., Shrewsbury, MA 01545; Research Division, W. R. Grace & Co.-Conn., Lexington, MA 02173; and tHarvard Medical Schooland New England Deaconess Hospital, Division of Organ Transplantation, Boston, MA 02212

Communicated by Susan E. Leeman, September 16, 1991

ABSTRACT Permselective acrylic membranes were em-ployed to prevent une rejection of discordant islet xe-nografts isolated from various large animals. Canine, porcine,and bovine islets were seeded into tubular diffusio abersand transplanted into the peritoneum of 27 nmmunosuppressed streptozotocin-induced diabetic Lewis rats. Si recip-ients received islet grafts from bovine calves, 7 receIved graftsfrom pigs, and 14 received grafts from dogs. Four of the latterwere removed at 1 month. In the control group of 10 diabeticrats, 4 received nonencapsulted canine islets, 3 receivednonencapsulated bovine islets, and 3 received nonencapatedporcine islets. Recipients of encapsulated Islets promptlydropped from a pretranpaotation plasma glucose level of 487± 36 (mean ± SEM) to 84 ± 2 (canine), 81 ± 4 (bovine), and81 ± 3 mg/dl (porcine) during the first week. 411 of the animalssustained theqe levels for at least 1 month. One rat tane-ously reverted to diabetes at 54 days taton; 4other rats became hyperglycemic (glucose, >600 mg/di) aftermembrane removal on day 30. The rem 22 rats main-tained fasting euglycema for >10 weeks. In contrast, rats thatreceived Encapsulated Islets became hyperglycemic in <7days. Intravenous glucose tolerance test K values (decline inglucose levels, %/min) at 1 month for the canine and bovineencapsulatedislet tr ant group were 3.5 ± 0.3 and 3.3 ±0.1 compared with 3.3 ± 0.1 (P = 0.63) and 0.91 ± 0.1 (P <0.0001) for normal (n = 4) and diabetic (n = 4) control groups.Morphologic studies of long-term functioning grafts (30-130days) revealed well-preserved a, (3, and 8 cells, with varyingdegrees ofgranuatio. These results demonstrate that immunesolation of Islet tisse using permselective afcial membranescan protect discordant Ilet xenografts from i rejectionin the absence of any immunosuppressive drugs.

Diabetes afflicts an estimated 80 million people worldwide (1).Even with improved procurement ofhuman organs, the supplyof donor tissue would remain quite inadequate if pancreaticislet transplantation were to be developed as an effectivetherapy. One potential solution to this problem is the use ofnon-human donor islets. However, the transplantation ofxenogeneic tissues of widely divergent species is difficult dueto vigorous humoral and cellular immune responses. The mostencouraging experience involves the encapsulation of donorislets in artificial membranes' that are impermeable to immu-nocytes and immunoglobulins but that permit crossover oflower molecular weight substances such as glucose and insulin(2, 3). Preliminary studies in rodents have demonstrated thatxenografted encapsulated islets can function forup to 210 days(4). Although these experiments have proven Successful for

closely related islet xenografts (concordant, rodent-to-rodentcombinations), islet xenografts between discordant speciesadd another dimension to the rejection response, in that mostdiscordant species have natural cytotoxic antibodies againsteach other (5). The development of successful islet isolationtechniques for animals such as the cow (6) and pig (7) couldenable the use of readily available supplies of large-animaldonor tissue for islet grafting into human diabetics. Unfortu-nately, there is currently no clinically applicable immunologicmethod available that prevents the destruction of discordantislet xenografts (8-11).Encouraged by the results obtained with the encapsulation

of allogeneic and xenogeneic rodent islets in capillary hollowfibers (4, 12-14), we sought to determine whether largertubular membrane diffusion chambers (fabricated fromXM-50 acrylic copolymer) can protect xenografts of canine,bovine, and porcine islets from rejection in the absence ofanyimmunosuppressive drugs. Because the formation of fibroustissue around the membrane has been a cause offailure in thepast, a smooth external skin was used to minimize this hostreaction.

MATERIALS AND METHODSAnimals. Adult male Lewis rats (Charles River Breeding

Laboratories) weighing 250-300 g were used'as transplantrecipients. Animals were fed ad libitum with a standardpelleted diet (Agway, no. 3000 RHM-Prolab) and allowed freeaccess to water. Diabetes was induced by a single injectionof streptozotocin (42 mg/kg of body weight in 0.01 M citratebuffer, pH 4.5; Sigma) into the tail vein 10-14 days prior tosurgery. Only rats with plasma glucose levels >400 mg/dlwere used in these studies. Fasting plasma glucose concen-trations were measured by tail bleedings using -the glucoseoxidase method (Beckman glucose analyzer 2; Fullerton,CA); determinations were performed three times weekly for30 days and then weekly for the duration of each study. Theanimals were not fasted the day of or the day after transplantsurgery. Failure was considered to have occurred whenplasma glucose concentrations exceeded 200 mg/dl on twoconsecutive determinations.

Islet Isolation and Islets were prepared fromeither adult mongrel dogs, pigs, or bovine calves (0-2 weeksold) by a modification of the method ofWarnock and Rajotte(15). Pancreatic tissue was dissociated using a collagenasedigestion procedure, and the islets were separated from

Abbreviation: IVG1T, intravenous glucose tolerance test.*To whom reprint requests should be addressed at: BioHybridTechnologies, Inc., Park Nine West, 910 Turnpike Road, Shrews-bury, MA 01545.1Present address: Diabetes Unit, Massachusetts General Hospital,Boston, MA 02129.

11100

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 88 (1991) 11101

exocrine tissue on a discontinuous Ficoll density gradient.Isolated islets were then cultured for 1 day either in M199/Earle's medium supplemented with 10%6 (vol/vol) fetal bo-vine serum, 20 mM Hepes, 100 mg of glucose per dl, and 400international units of penicillin per ml (canine) or in aminimum essential medium plus 10% heat-inactivated horseserum (bovine and porcine) in a humidified atmosphere of5%C02/95% air at 370C. After preculturing, the islets wereplaced in semipermeable tubular membranes (2-3 cm long,1.77mm i.d., 69-Am wall thickness) with a nominal molecularweight cutoff of 50,000-80,000 (W. R. Grace & Co.). Isletswere suspended in 1.2% (wt/vol) Pronova LVG sodiumalginate (Protan, Drammen, Norway) at a concentration of4-6 islet equivalentsll per mm3 (200-400 islets per mem-brane). The cells were seeded into the tubular membranechambers using a 16-gauge angiocatheter. After gelation in asolution of 1.5% CaCl2, the chambers were washed in culturemedium, and the ends were sealed by heat followed bydipping in a solution of acrylic copolymer (XM castingsolution; W. R. Grace & Co.) similar to that used to fabricatethe tubular membranes.In Vitro Glucose-Insulin Kinetics. In vitro perifusion was

carried out to test the insulin secretory activity from the isletsseeded within the capsules and to evaluate the kinetic per-formance of this type of diffusion-based biohybrid artificialpancreas as an insulin delivery device. Encapsulated andnonencapsulated islets (381 + 48 equivalents) were perifusedwith glucose concentrations of 100, 300, and 100 mg/dl for 60min at each concentration as described (16). The flow ratewas 0.5 ml/min and the perifusate was collected with amicrofraction collector (Gilson model 203). The sampleswere frozen for subsequent insulin assay using a standardequilibrium radioimmunoassay protocol as previously de-scribed (17). The limit of assay sensitivity was 25 microunits(1 ng)/ml and data reduction was done by a log-logit trans-formation of the standard curve by COBRA y counter(Packard Instruments).

Experimental Design. Recipient rats were anesthetizedwith ether inhalation. Free or macroencapsulated islets (1-2x 104 islet equivalents) were introduced into the peritonealcavity through a small (1-2 cm) midline incision. The woundwas closed in two layers with 4-0 silk suture. In the experi-mental group of 27 rats, 6 received islet grafts from bovinecalves, 7 received grafts from pigs, and the remaining 14received grafts from dogs. Four of the latter were removed at1 month. In the control group of 10 diabetic rats implantedwith nonencapsulated islets, 4 received canine islets, 3 re-ceived bovine islets, and the remaining 3 received porcineislets. Intravenous glucose tolerance tests (IVGTTs) wereperformed at 1 month after transplantation and comparedwith normal (n = 4) and untreated diabetic (n - 4) rats. Fiftypercent (wt/vol) glucose (0.5 g/kg of body weight) wasinjected into the tail vein, and plasma glucose concentrationswere measured at 0, 1, 5, 10, 20, 30, 40, 50, and 60 min afterthe glucose injection. K values (decline in glucose levels,%/min) were calculated according to standard methods (18).

Histology. The islet implants and the pancreas of recipientanimals were fixed in Bouin's solution, dehydrated, andembedded in paraffin. Five-micron sections were preparedand stained with hematoxylin/eosin and for the presence ofinsulin, glucagon, and somatostatin using immunoperoxidasetechniques (19). Two representative longitudinal sections ofeach native pancreas were examined.

Statistical Analysis. Data are presented as mean (Q) ± SEMand compared by using the unpaired Student's t test orone-way analysis of variance (ANOVA). In conjunction with

100 mg/dl 300 mg/dl 100 mg/dl 300 mg/dIII I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

0.5

0.4 F--

E

D

cc-

0.3 F

0.2 F

0.1

0.5

I--

E0E1-

(Aa

0.4

0.3

0.2

0. 12

300

250

200 E

8)150 a)00

100 0

50

30000

250'-

50U)

100

50 o

0O 40 60 80 100 120 140 160 180 200

Time (minutes)

FIG. 1. Insulin (e) secretory response to glucose (o) by perifusednonencapsulated (Upper) and macroencapsulated (Lower) canineislets. The increase in insulin release from encapsulated islets wasonly slightly delayed in comparison to nonencapsulated islets. mU,milliunits.

ANOVA, multiple comparison methods of either Tukey orScheffe were used, with a significance level of 0.05. Corre-lation was analyzed by univariate linear regression with theSTATVIEW ii program (Abacus Concepts, Berkeley, CA).

RESULTSIn Vitro Glucose-Insulin Kinetics. The in vitro insulin secre-

tory response of macroencapsulated and nonencapsulatedislets is shown in Fig. 1. An =4-fold average increase from thebasal insulin secretion was observed in both groups. Thesecretory response of the macroencapsulated islets was sus-tained for 60 min of glucose stimulation (300 mg/dl) andreturned to basal levels after perifusion with the low-glucosesolution (100 mg/dl). Five separate experiments in which thisprotocol was used indicated a delay of 7 ± 1 min beforeinsulin concentration in the perfusate began to increase. Thisfinding indicates that the islets encapsulated by this proce-dure were functionally intact and could respond rapidly to anincrease in glucose concentration.

700

-0U)600-

a-) 500-coU 400

CDID 300-E-0 200-

0-

0Ol 100

0 10 20 30 40 50 60 70

Days Posttransplantation

FIG. 2. Fasting plasma glucose levels (x ± SEM) in 10 diabeticrats that received intraperitoneal implants of macroencapsulatedcanine islets. One graft failed at 54 days posttransplantation. (Inset)Nonencapsulated canine islets (n = 4).

600

mo0 M e _~-2 0 2 4 6 8 10 12 14

,L..~~,. i, s ^ - F~E30

CL200.' 100

-I

IA unit introduced to standardize islet number, equivalent to thevolume of an average islet of 150 ILm in diameter.

Medical Sciences: Lanza et al.

11102 Medical Sciences: Lanza et al.

600I--m 500

a)O 4000(-ao 3000c

O 200

0

Ino'

I300 N_

-2 0 2 4 6 8 10 12

_ -I _~~~~~~~~~~0 10 20 30 40 50 60 70

Days Posttransplantation

FIG. 3. Fasting plasma glucose levels C + SEM) in six diabeticrats that received intraperitoneal implants of macroencapsulatedbovine islets. (Inset) Nonencapsulated bovine islets (n = 3).

Transplantation. Intraperitoneal transplants of 1-2 x 104encapsulated islets reversed the diabetic state of all 27recipient rats within 24 hr (Figs. 2-4). Plasma glucose levels(Q ± SEM) dropped from a pretransplantation level of 487 +36 to 84 ± 2 (canine), 81 ± 4 (bovine), and 81 ± 3 mg/dl(porcine) during the first week. All of the animals sustainedthese levels for at least 1 month. One rat subsequentlyreverted to diabetes at 54 days posttransplantation and fourcanine islet xenograft recipients reverted to diabetes aftermembrane removal on day 30. The remaining 22 rats main-tained fasting euglycemia for >10 weeks. In contrast, non-encapsulated islet transplants only partially reversed thediabetic state ofthe recipients, with-hyperglycemia returningto pretransplantation levels after 6-7 postoperative days(Figs. 2-4 Insets).

Metabolic Studies. The results of IVGTTs on normal con-trol and transplanted rats are shown in Table 1. Glucoseclearance (K) rates of the animals transplanted with encap-sulated islets were not statistically different from those ofnormal rats after the administration of- glucose (P = 0.63).However, the IVGTT profiles were not totally normal, al-though plasma glucose declined promptly, reaching near-normal levels after 60 min and resulting in K values of 3.5 +0.3 (canine islets), 3.3 ± 0.1 (bovine islets), and 3.3 ± 0.1(normal). By contrast, the untreated streptozotocin diabeticrats remained significantly hyperglycemic after glucose chal-lenge, with K values imipaired at 0.91 ± 0.1 (P < 0.0001).Histogy and Memborae Removal. To check for evidence

against spontaneous recovery by (3 cells, pancreata from theexperimental and control groups were routinely fixed andprocessed. The histological appearance is shown in Fig. 5 for

600

V -A\T /

Table 1. Fasting plasma glucose concentrations Q- ± SEM) andIVGTT K values at 1 month in transplanted, normal, anduntreated diabetic rats

Transplanted islets

Parameter Canine Bovine Normal DiabeticPlasma glucose,mg/dl 82 ± 7 101 ± 1 94 ± 1 347 ± 32

Kvalue, %/min 3.5 ± 0.3 3.3 ± 0.1 3.3 ± 0.1 0.91 ± 0.1Number of animals 4 3 4 4

the pancreas recovered from a normal control animal (Fig.SA) and a streptozotocin-treated animal sacrificed 93 daysafter transplantation with encapsulated canine islets (Fig.5B), respectively. The latter pancreas exhibited signs ofstreptozotocin toxicity with loss ofnormal islet architecture.Virtually no granulated (3 cells were seen. Histology of themembranes retrieved from this animal showed well-preserved islets, interspersed with solid,' basophilic alginate,much as in freshly seeded chambers. Overall, perhaps 75-80% of the seeded endocrine tissue remained viable. Cellsstaining for glucagon and somatostatin were also foundamong the (3 cells in most section planes.To confirm further that the euglycemic state of the trans-

plant recipients resulted from the transplantation of mac-roencapsulated islets, membranes containing encapsulatedcanine islets were removed 30 days after implantation in fourrats. Hyperglycemia was subsequently observed in all ani-mals, with plasma glucose levels rising to >600 mg/dl within3 days (Fig. 6). The implanted membranes were generally freefloating and free of cellular overgrowth. The external sur-faces were free of fibrosis and host cell adherence. Onmicroscopic examination, the membranes contained up to85-900% viable islets. Immunohistochemical staining of theislets revealed hormone-producing cells, with varying de-grees of a, A, and 8 granulation (Fig. 7).

DISCUSSIONTransplantation of islets across a wide species barrier hasbeen difficult to achieve. Islets isolated from large mammals

1.1

x..;.n i

7IV V 'MIX 1 ,s

J.+Xt ||t B&VP-

700

600

500

400

300

200

100

00 1 0 20 30 40 50 60 70

Days Posttransplantation

FIG. 4. Fasting plasnma glucose levels (Q SEM) in seven

diabetic rats that. receifaed intraperitoneal implants of macroencap-sulated porcine islets. (Inset) Nonencapsulated porcine islets (n = 3).

4~~~~~~~~~~4

FIG. 5. Immunoperoxidase stain for insulin of the pancreasrecovered from normal control rat (A) and streptozotocin-treatedanimal sacrificed 93 days after transplantation with encapsulatedcanine islets (B). (x230.) The latter pancreas exhibits signs ofsteptozotocin toxicity, with loss of normal islet architecture. Virtu-ally no granulated ,3 cells are seen. The background stained withhematoxylin.

I-,~0Ea)Un0u

1:30

c)0~

C

0Ul-

Proc. Nad. Acad. Sci. USA 88 (1991)

Proc. Natl. Acad. Sci. USA 88 (1991) 11103

700

600E

500Q)

0 400U-3 300o 200EE 1000

u0 5 10 15 20 25

Days Posttransplantation

5 4035 40

FIG. 6. Random plasma glucose levels Q- ± SEM) in four diabeticrats that received intraperitoneal implants of macroencapsulatedcanine islets. The membranes were removed at 1 month.

and humans and transplanted into nonimmunosuppresseddiabetic rats are rapidly rejected (20, 21). In the presentstudy, prolongation ofdiscordant xenograft survival has beenachieved in diabetic rats using artificial permselective mem-branes. Tubular membranes containing canine, bovine, orporcine islets routinely normalized fasting plasma glucoselevels after transplantation into streptozotocin-induced dia-betic rats. All of the grafts functioned for 1 month, and for atleast 10 weeks in bovine and porcine islet xenograft recipi-ents.** In comparison, nonencapsulated islet transplantssustained euglycemia for <1 week.Although prolongation of survival of islets from discordant

species has been achieved by microencapsulation, these stud-ies have been performed mainly in mice and have usuallyrequired adjunctive treatment with immunosuppressive agents

A

(22-24). Weber et al. (8) found that alginate/poly(lysine)microcapsules containing 4 x 103 to 1.2 x 104 canine isletsttfunctioned for only 11.5 + 3 days (range, 5-17 days) innonobese diabetic mice. With anti-CD4 monoclonal antibodytreatment, however, long-term functional survival was ob-served for 4/8 (50%) recipients. Histologic examination ofthemicrocapsules at the time ofrejection revealed the presence ofmacrophages, multinucleate giant cells, granulocytes, andlymphocytes, consistent with an inflammatory reaction. It islikely that, once recruited to the surroundings of the micro-capsule, lymphokine-secreting cells injured the xenogeneicislets via soluble factors. This is in keeping with reports thatthe microcapsule membrane is permeable to the immunomod-ulatory polypeptide products of lymphocytes, macrophages/monocytes, and natural killer cells of the immune system (2,25) and that anumber ofcytokines (interferon 'yand interleukin2) and monokines (interleukin 1 and tumor necrosis factor)induce the degeneration of islet cells (26-28).

Recently, Zekorn et al. (29) have shown that rat islets areprotected from interleukin 1 toxicity by hollow fiber mem-branes. A high dose of recombinant human interleukin 1(3resulted in an inhibition of the glucose-stimulated insulinrelease of free islets, whereas encapsulated islets showed apreserved insulin release. Therefore, the use of hollow fiberand tubular membrane devices might have potential advan-tages, especially when engraftment is carried out across awide species barrier. Indeed, Altman et al. (30) reported thatintraperitoneal transplants ofhuman islets and human insuli-noma tissue into nonobese diabetic mice had a survival timeof <2 weeks, and the survival was increased to >6 months(two-thirds of the animals in each group) when encapsulatedin hollow fiber membranes. However, this type of fiber wasobserved to elicit an inflammatory pericapsular response inthe rat (30, 31) and the large-animal (porcine) model (32). Thefibrotic reaction in the rats was qualitatively similar to thatseen in the pig, except for lymphoid clusters with giant andpseudo-epithelioid cells that were observed only in pigs. Thereaction consisted of a layer (<50 um) of fibroblasts andcollagen with polymorphonuclear leucocytes, macrophages,histiocytes, and small lymphocytes. The fiber wall wasalways infiltrated by collagen, fibroblasts, and macrophages.Experiments performed in our laboratory corroborate

these findings. We compared the use of tubular diffusionchambers with a smooth skin versus a rough skin-i.e., anopen trabecular or honeycombed surface-on the outsidesurface. Membranes with rough external surfaces elicited asignificant fibrotic response in streptozotocin-induced dia-betic rats in <1 week. In contrast, smooth membranesretrieved >10 weeks after implantation showed no evidenceof an inflammatory response. The external membrane sur-faces were generally free of fibrotic encapsulation and ex-hibited only occasional host-cell adherence (Fig. 8). Theresponse was not influenced by seeding or nonseeding of thechambers or by the type of tissue inside (canine, bovine, orporcine islets). Protection of the xenogeneic tissue by themembrane is demonstrated by the preservation of the struc-ture of the islets residing for >6 months in the peritonealcavity (Fig. 9) as well as by the presence of hormone-producing cells containing varying degrees of a, (3, and 8granulation. Occasionally the chambers were broken or ex-

FIG. 7. Encapsulated canine islets retrieved from the peritonealcavity of diabetic rats 30 days after xenotransplantation. Immuno-peroxidase stained for insulin (A), glucagon (B), and somatostatin(C), revealing multiple, viable, hormone-containing endocrine cells.(x570.)

**Most of the animals (9/16) have continued to maintain fastingeuglycemia, with functional survival now >200 days.

ttAlthough hyperglycemia in rodents has been reversed by inter-portal or subrenal capsule transplantation using fewer islets, thesestudies have been performed mainly with free, nonencapsulatedislets. Dose-response curves performed in our laboratory usingencapsulated islets indicate that larger numbers of islets arenecessary to achieve long-term normoglycemia in the large-animal-to-rodent encapsulated islet xenograft model.

Medical Sciences: Lanza et al.

.0N.,p

0

..s::.;,:

f::..

i..

..-.l

11104 Medical Sciences: Lanza et al.

'A A4

FIG. 8. Encapsulated porcine islets retrieved from the peritonealcavity of a diabetic rat 82 days after xenotransplantation. (Hema-toxylin/eosin; x570.) Note the absence of fibrosis and host celladherence on the external surface (arrowheads).

hibited bends that resulted in fracture of the external andinternal membrane walls. Such complete fractures permittedxenograft rejection within the interior characterized by thepresence of fibroproliferative connective tissue and immunecell elements. Islet cell survival within the alginate matrixwas markedly reduced within these damaged membranes.Although our results with survival of islet xenografts in

streptozotocin-induced rats are encouraging, it will be im-portant to utilize this encapsulation technology in spontane-ously diabetic animals such as BB rats or nonobese diabeticmice in which diabetes is believed to result from autoimmune,B-cell damage. Preliminary experiments in diabetic BB/Worrats suggest that canine islet implants can restore euglycemiafor >150 days without immunosuppression. In addition, thisshould eliminate the possibility of (-cell regeneration that hasbeen reported for the streptozotocin model of diabetes (33).In related studies in our laboratory we have also observedthat removal of islet implants after periods of several weeksdid not always result in subsequent large rises in plasmaglucose concentrations.Our results demonstrate the feasibility of long-term immu-

noisolation of discordant islet xenografts by artificial mem-branes and the long-term biocompatibility of the membraneversus the graft and versus the recipient. However, tofunction as an implantable biohybrid artificial pancreas, it isnecessary that the islets placed in the device respond appro-priately to glucose within a time frame compatible withclosed-loop insulin delivery, which requires a response timeof <15 min (34, 35); pharmacokinetic modeling of glucosehomeostasis in man suggests that the lag time of the increasein insulin delivery by an artificial pancreas must be shorterthan this to avoid the overexcursion of postprandial bloodglucose (35). Indeed, the increase in insulin release in vitro inresponse to glucose from perifused macroencapsulated isletswas delayed by only 7 min in comparison to nonencapsulatedislets. Furthermore, glucose clearance during intravenous

FIG. 9. Encapsulated canine islets retrieved from the peritonealcavity of a diabetic rat 220 days after xenotransplantation. (Hema-toxylin/eosin; x270.) Note the smooth skin on the external surface(arrowheads) versus the rough-i.e., the open trabecular or honey-combed surface-on the luminal side.

glucose tolerance testing was also not delayed. Since there isincreasing evidence that the incidence and severity of themicrovascular complications of diabetes are linked to thedegree of metabolic control attained, the ability of the en-capsulated islets to achieve rapid glucose-insulin kineticsmay have important implications in assessing the potentialrole for this type of diff-usion-based biohybrid artificial pan-creas as therapy for human insulin-dependent diabetes.

We thank S. Sullivan, J. Harvey, S. Sherman, D. Moore, S.Teman, and E. Krumpholz (BioHybrid Technologies), J. Wolfrum,K. Dunleavy, and E. Doherty (W. R. Grace & Co.-Conn.), and K.Andrutis and K. Lightbaum (Tufts Veterinary School).

1. Lanza, R. P. & Soon-Shiong, P. (1991) in Xenotransplantation: TheTransplantation of Organs and Tissues Between Species, eds. Cooper,D. K. C., Kemp, E., Reemtsma, K. & White, D. J. G. (Springer, Heidel-berg), pp. 297-312.

2. Soon-Shiong, P., Lu, Z. N., Grewal, I., Lanza, R. P. & Clark, W. (1990)Transplant Proc. 22, 754-755.

3. Darquy, S. & Reach, G. (1985) Diabetologia 28, 776-780.4. Archer, J., Kaye, R. & Mutter, G. (1980) J. Surg. Res. 28, 77-85.5. Terasaki, P. I., Esail, M., Cannon, J. A. & Longmire, W. P. (1961) J.

Immunol. 87, 383-395.6. Borland, K., Harvey, J., Harvey, M., Sherman, S., Sullivan, S., Rupp,

R. & Chick, W. Transplant Proc., in press.7. Ricordi, C., Socci, C., Davalli, A. M., Staudacher, C., Vertova, A.,

Baro, P., Sassi, I., Braghi, S., Giuzzi, N., Pozza, G. & DiCarlo, V. (1990)Transplant Proc. 22, 784-785.

8. Weber, C. J., Zabinski, S., Koschitzky, T., Wicker, L., Rajotte, R.,D'Agati, V. D., Peterson, L., Norton, J. & Reemtsma, K. (1990) Trans-plantation 49, 396-404.

9. Auchincloss, H. (1988) Transplantation 46, 1-20.10. Lafferty, K. (1988) in Transplantation of the Endocrine Pancreas in

Diabetes Mellitus, eds. Van Schilfgaarde, R. & Hardy, M. (Elsevier,Amsterdam), p. 279.

11. Reemtsma, K. & Weber, C. (1986) in The Sabiston Textbook ofSurgery,ed. Sabiston, D. (Saunders, Philadelphia), p. 469.

12. Altman, J. J., Manoux, A., Callard, P., Houlbert, D., Desplanque, N.,Bruzzo, F. & Galletti, P. M. (1981) Artif. Organs 5, Suppl., 776-779.

13. Altman, J. J., Houlbert, D., Bruzzo, F., Desplanque, N., Manoux, A. &Galletti, P. M. (1982) in Islet-Pancreas-Transplantation and ArtificialPancreas, eds. Federlin, K., Pfeiffer, E. & Raptis, S. (Thieme-Stratton,New York), pp. 43-45.

14. Zekorn, T., Siebers, U., Filip, L., Mauer, K., Schmitt, U., Bretzel, R. G.& Federlin, K. (1989) Transplant Proc. 21, 2748-2750.

15. Warnock, G. L. & Rajotte, R. V. (1988) Diabetes 37, 467-470.16. Scharp, D. W., Lacy, P. E., Finke, E. H. & Olack, B. J. (1987) Surgery

102, 869-879.17. Sullivan, S. J., Maki, T., Borland, K. M., Mahoney, M. D., Solomon,

B. A., Muller, T. E., Monaco, A. P. & Chick, W. L. (1991) Science 252,718-721.

18. Moorhouse, J. A., Grahame, G. R. & Rosen, N. J. (1964) J. Clin.Endocrinol. 24, 145-159.

19. Warnke, R. & Levy, R. (1980) J. Histochem. Cytochem. 28, 771-776.20. Tze, W. J., Tai, J. & Cheung, S. (1990) Transplantation 49, 502-505.21. Tze, W. J., Tai, J. & Cheung, S. (1989) Transplant Proc. 21, 2741-2743.22. Wang, Y., Hao, L., Gill, R. & Lafferty, K. (1987) Diabetes 36, 535-538.23. Calaflore, R., Janjic, D., Koh, N. & Alejandro, R. (1987) Clin. Res. 35,

499A (abstr.).24. Ricker, A., Stockberger, S. & Halban, P. (1989) in The Immunology of

Diabetes Mellitus, ed. Jaworski, M. (Elsevier, Amsterdam), p. 1193.25. Cole, D. R., Waterfall, M., McIntyre, M., Smith, W. & Baird, J. D.

(1989) Diabetologia 32, 477A (abstr.).26. Comens, P. G., Wolf, B. A., Unanue, E. R., Lacy, P. E. & McDaniel,

M. L. (1987) Diabetes 36, 963-970.27. Miyajima, E., Yamada, K. & Nonaka, K. (1990) Diabetes 39, Suppl. 1,

242A (abstr.).28. Pukel, C., Baquerizo, H. & Rabinovitch, A. (1988) Diabetes 27, 133-136.29. Zekorn, T., Siebers, U., Bretzel, R. G., Renardy, M., Planck, H.,

Zschocke, P. & Federlin, K. (1990) Transplantation 50, 391-394.30. Altman, J. J., Penfornis, A., Boillot, J. & Maletti, M. (1988) Am. Soc.

Artif. Intern. Organs 34, 247-249.31. Altman, J. J., Houlbert, D., Callard, P., McMillan, P., Sol-

omon, B., Rosen, J. & Galletti, P. M. (1986) Diabetes 35, 625-633.32. Icard, P., Penfornis, F., Gotheil, C., Boillot, J., Cornec, C., Barrat, F.

& Altman, J. J. (1990) Transplant Proc. 22, 724-726.33. Chicheportiche, D., Darquy, S., Lepeintre, J., Capron, F., Halban, P. A.

& Reach, G. (1990) Diabetologia 33, 457-461.34. Sorensen, J., Colton, C. K., Hillman, R. S. & Soeldner, J. S. (1982)

Diabetes Care 5, 148-156.35. Kraegen, E. W., Chisholm, D. J. & MacNamara, M. E. (1981) Horm.

Metab. Res. 13, 365-367.

Proc. Natl. Acad. Sci. USA 88 (1991)