Immunotherapy and pancreatic transplantation

ORA-012-001 IMMUNOTHERAPY OF NOD MICE: CAN ANTI-T CELL MONOCLONAL ANTIBODY (CD4 MAb)

INDUCE TOLERANCE TO THE DIABETOGENIC ANTIGEN?

J.A. SHIZURU, C. TAYLOR-EDWARDS, A.K. GREGORY AND C.G. FATHMAN Stanford University School of Medicine

Stanford, California U.S.A. Studies from our laboratory have recently demonstrated that ~he T-helper/inducer lymphocyte subset defined by the CD4 surface molecule is central to disease pathogenesis in NOD mice. Sustained treatment with a monoclonal antibody (MAb) against the CD4 determinant initiated at the age when NOD islets demonstrate maximal mononuclear cell infiltration (90-110 days) leads to complete re- versal of the insulitis and prevents the emergence of overt hyperglycemia. In this report, we treated NOD mice with a short course of anti-CD4MAb therapy prior to the onset of insulitis. Since anti-CD4 MAbs can induce tolerance to soluble proteins, as well as transplanted allogeneic islets, we reasoned that anti-CD4 treatment administered coincidently with the emergence of the putative diabetogenic antigen may tolerize the mice to this antigen.

Litter-matched female NOD mice 19-21 days old were divided into control and treatment groups. Treated mice received an initial course of anti-CD4 MAb, GK1.5 (200 ug x 3 days) and then received weekly injections of i00 ug for 4 weeks. The controls received no treatment for the duration of the experiment. Six animals from each group were sacrificed for histologic evaluation of insuli- tis every two weeks from days 40 to 96. Correlate analysis for lymphocyte subset (CD4, CD8, Thy i) depletion was studied by fluorescence activated cell sorter (FACS) analysis. Evidence of insulitis was significantly suppressed in the treated group versus control. All control animals had severe lymphocytic infiltration into their islets by day 82, while all treated animals demon- strated only mild perivascular and periductal lymphocytic involvement. FACS analysis revealed that antibody treatment resulted in inversion of the normal CD4+/CD8+ T-lymphocyte ratios, however, by day 82, the subsets returned to normal. These data show that treatment with anti-CD4 MAb administered at the time of the primary immune response against the islets can suppress insulitis. To demonstrate that treated animals have been tolerized to a diabetogenic antigen, we are currently following these mice for evidence of delayed insulitis and overt hyperglycemia beyond 200 days of age.

ORA-012-002 SAFETY OF PROLIFERATED HUMAN PREISIET C~.T

TRANSPIANTED I]qlO DIABETIc P ~ WITH RENAL TRANSPIANTS.

A. Brewin, F. Voss, Hana Biologics, Alameda, Ca, W. Bry, G. Collins, Pacific Presbyterian Medical Center, San Francisco, I. Dawidson, U Texas, Dallas, K. Lafferty, B. Davis Center, Denver, E. Spees, AMI St. Luke's, Denver, H. Sollinger, U Wisconsin, ~,~di~=r,, USA.

Proliferated pre-islets transplanted (TX) into ~zotocin induced diabetic nude ~ce restores glycemic oontrol when from 1 to i0.0 X i0 v cells are implanted (ave 4.0 X 10v). To test the safety of this procedure, a clinical study was carried out in patients under ia~m/nosuppression for renal transplants. Proliferated pre-islet cells were prepared from fetal cadavers under 6~P guidelines using methods previously described. ~his resulted in a i0 - 20 fold increase in cells over that available when tissue is processed for transplantation without proliferation. The procedure eliminates most Class II bearing cells and fibroblasts. Incoming specimens were tested for ccmmon pathogens and all specimens were keep separate until results of these viral and microbiological tests were completed. Harvested cells were packaged into 3 d~ional, 5 lambda drops for TX. These were carried on wet ice to 4 TX sites and implanted within 48 hrs of harvest. Ten patients received implants of 5 - 15 X 106 cells, 5 received from 16 - 35 X 10Voells, and 5 received 36 -50 X 106 cells. Eleven had implants under the capsule of a stable (>6 months) renal TX as an outpatient procedure. Five of the 9 patients receiving new renal TXs underwent rejection episodes. All 20 enrolled patients have functioning renal grafts, and all remain insulin dependent at su~ssion (25-140 days post TX). Baseline and periodic c-peptide assays were done. I_nmnlnosuppression regimens were not altered, but were standard for renal TX at each site. These were variations of standard regimens of cyclosporin-A, ~ and steroids. Patients are being monitored for evidence of adverse clinical or laboratory reactions as well as for insulin usage or production. There has been no clinical or laboratory evidence of an adverse reaction related to the implants.

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ORA-012-003 PERFUSION OF SUBCAPSULARLY TRANSPLANTED MOUSE ISLETS: EFFECTS OF HYPERGLYCEMIA ON THE GRAFT FUNCTION

A. ANDERSSON, O. KORSGREN, L. JANSSON Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.

Studies of the impact of a long-term hyperglycemic stress in vivo on the islets of Langerhans have been hampered by the paucity of a suitable experimental system for such investigations. For that purpose we developed a perfusion technique for mouse kidneys bearing a subcapsular graft of syngeneic pancreatic islets. By varying the number of implanted islets and use either normoglycemic or alloxan-diabetic recipients it was possible to set the intensity of the glycemic stress. Furthermore, by the addition of intermittent insulin treatment or a second intrasplenic islet graft consisting of a sufficient number of islets to normalize the hyperglycemia,it was also possible to investigate the reversibility of an induced functional deterioration. For control purposes whole pancreas perfusions of non-diabetic mice were also performed. In some mice the islet grafts were removed in toto and analyzed for insulin content and (pro)insulin biosynthesis. The islet grafts of mice normoglycemic (non-diabetic) throughout the experiments responded with a distinct first peak of insulin release in response to both a glucose and an arginine challenge. No response to either of these substances was observed from islet grafts of mice hyperglycemic (alloxan-diabetic) for the whole observation period (4 weeks). Insulin treatment of the hyperglycemic mice partly normalized the insulin response to glucose, as did a simultaneous intrasplenic islet graft. The insulin content of the islet grafts of the grafted but still diabetic mice was markedly decreased but insulin treatment reversed this degranulation to some extent. Glucose-stimulated (pro)insulin biosynthesis was less markedly impaired. It is concluded that perfusion of graft-bearing kidneys is a valuable tool for in vivo and in vitro functional studies of long-term effects of hyperglycemia on the islets of Langerhans.

ORA-012-004 THE FUNCTIONAL DIFFERENTIATION OF A HUMAN FETAL PANCREAS IN VITRO AND IN NORMAL AND DIABETIC NUDE MICE

T. E. MANDEL and MARIA KOULMANDA Transplantation Unit, The Walter and Eliza Hall Institute of Medical Research; Parkville, 3050, Australia Experimental animal studies have suggested that fetal pancreas may be a good source of islets for transplantation in IDDM. However, clinical results with fetal islets have not been encouraging and there is little evidence to support the results of animal studies. This discrepancy between animal and clinical results may be due, in part, to differences in the rate of functional development of fetal human and fetal rodent islets. We have studied the development of an 18w gestation fetal human pancreas, obtained by hysterotomy, initially in vitro and then in vivo as a xenograft, to define its development. The pancreas was initially maintained in organ-culture under euglycemic conditions. The islet cells survived in vitro with a high yield of viable tissue whereas the exocrine cells rapidly died. After 6w in vitro, the islets were tested for insulin secretion in response to a glucose or arginine challenge before being grafted into non-diabetic athymic mice. These mice were challenged with intraperitoneal arginine to determine whether human C-peptide was being produced. The primary grafts were then removed and retransplanted into diabetic nude mice to test the capacity of the islets to reverse streptozotocin-induced diabetes. The human fetal islets secreted insulin in vitro over the entire period but did not develop normal responses to hyperglycemia. During a 4m period in vivo as a primary graft under the renal capsule in non- diabetic recipients, more growth and maturation occurred and human C-peptide was produced in increased amounts following arginine challenge. When this tissue was retransplanted into diabetic nude mice, the grafts slowly developed functional responsiveness and restored the mice to euglycemia. Thus, human fetal tissue can survive in vitro but does not develop full physiological function. After a further 4m in vivo, slow development of an adequate response occurred. Thus, fetal human islets may take a long time to function normally and this time may be even longer in diabetics, particularly when a small amount of tissue is used.

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ORA-012-005 ISOLATION AND MICROENCAPSULATION OF PORCINE ISLETS OF LANGERHANS FOR A BIOARTIFICIAL PANCREAS

C. REACH, D. CHICHEPORTICHE, S. DARQUY, P. CHENARD, 3. LEPEINTRE, C. KHOSROVANI, H. BRIANDET, F. MOUSSY, and 3. ROUCHETTE, Service de Dlab4tologie, H~tel Dieu, Parls, France

We set up a method for large scale isolation of is lets of Langerhans from the pig pancreas. The pancreas, obtained from a slaughterhouse, were distended with 200 ml of Hanks solution contai- ning 300 mg collagenase (type 11, Sigma), and incubated at 37°C for about 30 mln. Serial biopsies were used for the determination of the end-polnt of the digestion. The digested pan- creas was subsequently brushed on the surface of three successive sieves ( I , 0.8, 0.5 mm meshes), and the resulting tissue was purified on a discontinuous albumin gradient. More than 50,000 Islets were obtained from one pancreas. Insulln secretion by isolated is lets was 92 ~ 17 pU/5 ls- lets/hr when incubated in MEN medium oontalnlng 5.5 mmol/1 glucose, and 198 ~ 31 ~U/5 Islets/hr in the presence of glucose 20 mmol/1 and theophylline 5.5 mmol/1 (n=5 different isolatlons, mean ± SEN). Pig Islets were mlcroencapsulated according to the alginate-polylyslne procedure. An in v i t ro kinetic study demonstrated a f lve-fold stimulation by glucose 20 mmol/l and theophylline of insulin release by mlcroenoapsulated is lets: Incubation In basal medium: 26 ± #, 29 ± 6, #2 ± 9, and 69 ± 16 pU/ I0 islets at 2, 30, 60, and 120 mln; in stimulatory medium: 31 ± #, 76 ± 10, 167 ± 39, and 366 ± 56 pU/ i0 islets, at the same times (mean ~SEM, i0 successive expe- riments). 5olaf, a sustained (more than 50 days) normalization of blood glucose level has been obtained in two out of five diabetic rats (streptozotocln, 70 mg/kg iv) treated wlth the intra- peritoneal implantation of 6000 micreencapsulated pig islets of Langerhans.

ORA-012-006 HUMAN ISLET ISOLATION FOR TRANSPLANTATION: AN AUTOMATED METHOD

C RICORDI, D SCHARP, P LACY Departments of Surgery and Pathology, Washington University, St. Louis, MO Department of Surgery, San Raffaele Institute, Milan Italy

USA

An automated procedure for the isolation of human pancreatic islets is described. The method allows the separation and purification of large numbers of intact islets during a continuous digestion process that is minimally traumatic to the islets. 150-200 ml of Hanks' solution containing 2 mg/ml collagenase (Sigma, Type X) was injected into the pancreatic duct. The whole distended gland was loaded into a digestion chamber containing a 280 mesh screen and 7 glass marbles. The chamber was shaken (320 oscillation/min) and the colla- genase solution was maintained at 37°C by pumping it through a heating chamber. After passing through the screen, a cooling circuit inactivated the collagenase saving the released islets, while reheated solution was drawn back to the chamber to continue the digestion. An average of 276,000 islets/pancreas (3,900 islets/gm, vol. = 0.82 ml) was obtained. After purification on Fi¢oll gradients, the purity was 70-90% islets with a 60% recovery from the pre-purification total islet number. Insulin secretory response to glucose after overnight culture revealed an average five-fold increase from the basal insulin secretion. The average insulin content of the final preparation was 93.4 Units of insulin. Transplantation of an aliquot of the islet preparation beneath the renal capsule of diabetic nude mice (n=16) either itmnediately after isolation, after 7 days culture at 24°C, or after cryopreservation produced normoglycemia in all recipients. Nephrectomy of the kidney bearing the grafts 30-45 days after transplantation produced a rapid return to the diabetic state. The islets were morphologically intact with a notmml degree of beta granulation. The automated method is now being used in clinical trials of islet transplantation.

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ORA-012-007 CONSEQUENCES OF HUMAN PANCREATIC TRANSPLANTATION ON ISLET HORMONE SECRETION IN DIABETIC RECIPIENTS AND NON-DIABETIC DONORS.

RP ROBERTSON, M ABID, FC GOETZ, DER SUTHERLAND, D KENDALL AND P DIEM University of Minnesota, Minneapolis, Minnesota USA

Human pancreas transplantation typically results in a denervated, heterotopic pancreas with systemic venous drainage in immunosuppressed patients. To assess the metabolic consequences of this procedure, we examined hormone secretion and glucose kinetics during IV glucose tolerance tests (IVGTT) and insulin tolerance tests (ITT). Compared to 15 normal controls (CON), success- ful pancreas transplantation in i0 previously C-peptide negative recipients (REC) produced normo- glycemia but significant elevations of basal insulin (8±1 vs 27±5; uU/ml; p<.001) and exaggerated first phase insulin responses to IVGTT (36±7 vs 122±24; p<.001) yet normal glucose disappearance rates (1.3±.2 vs 1.6±.1; KG; %/min; p=ns). During ITT, 12/12 REC had glucagon increments over basal (81±13; pg/ml; p<.001) whereas 14/18 Type I patients matched for duration of diabetes (23±1 years) did not. However, glucose recovery in REC was sluggish compared to 5 CON (REC=52±2; CON = 67±2; p<.01; glucose level 30 min after nadir; mg/dl). To assess the impact of hemipancreatectomy on nondiabetic donors, insulin secretion during OGTT before and one year after hemipancreatectomy was examined. Although fasting glucose levels remained normal (86±3 vs 93±4; mg/dl), there was marked deterioration of insulin secretion (73±12 vs 24±5; uU/ml at 30 min; p<.01) and glucose tolerance (112±12 vs 162±7 mg/dl at 90 minutes; p<.01). Conclusions: for recipients, transplan- tation carries the consequences of hyperinsulinism and abnormal counterregulation of hypoglycemia whereas impaired insulin secretion and glucose intolerance are consequences for donors. Thus, both groups had abnormal pancreatic islet function with as yet unknown long term consequences of hyperinsulinism and defective counterregulation of hypoglycemia in the recipients and of dimin- ished islet reserve in the donors.

ORA-012-008 THE FATE OF INTRAHEPATIC ISLET ALLOGRAFTS IN DOGS WITH SPONTANEOUS DIABETES

D.H. MINTZ t R. ALEJANDRO~ A.D. BLOOM~ P. ZUCKER~ and K.S. POLONSKY t University of Miami, USA, and University of Chicago, USA.

The ultimate fate of intrahepatic islet allografts in large animals with spontaneous diabetes has not yet been defined. To address this question, we have begun to study the natural history of intrahepatic multidonor islet allografts in dogs with spontaneous insulin dependent diabetes (SIDDM). We showed previously that cyclosporine (CSA) can uniformly prevent multidonor islet allograft rejection in pancreatectomized recipients (Diabetes 34:825,1985). In the present study, continuous CSA was administered to five dogs with spontaneous diabetes (20-40mg/kg daily x 30 days, and 40-80mg/kg per day p.o. thereafter) following intrahepatic transplantation of islet allografts. CSA dosages were adjusted to sustain serum trough levels of ~500ng/ml for the first 30 days post transplant and 100-300ng/ml thereafter. Fasting euglycemia has been sustained for >208,>427, and >671 days in 3 recipients; one animal died of pneumonia with normoglycemia 186 days post transplant; hyperglycemia recurred in I animal 231 days post transplant. The c-peptide responses to i.v.glucose (0.Sgm/kg) and oral glucose (Igm/kg) have also been serially followed. Despite normal fasting blood glucose levels, C-peptide response to i.v. and oral glucose, both decline as a function of time. Conclusion: We observe progressive losses of islet secretory responses in intrahepatic islet autografts in dogs (J.Clin.lnvest. 78:1339,1986) and in the present work in intrahepatic islet allografts in dogs with SIDMM as a function of time. It appears that eventually these insulin secretory defects fall below the threshold required to sustain fasting hyperglycemia. It is still uncertain whether these findings are due to chronic rejection, a direct toxic effect of CSA on islets, or reflects a finite life span of a heterotopic islet, alone or in combination.

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