Acta Chir. A u s t r i a c a . Vol. 33 - Supplement No 174 • 2001

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tiate indicates that over expression of PDX-I may increase [~-cell mass. Methods: NPI were transduced with a PDX-1 adenoviral vector, microencapsulated with alginate and cultured for 7 days in Hams FI0 + 10% autologous serum. Samples were stained for PDX-I and endocrine hormones. Hormone content and viability assays were performed as well as Western blot analysis to determine infection efficiency. Results: Insulin secretory activity was not altered after infection, indicating that transduction did not reduce NPI viability. Infection efficiency indicated a MOI dependent increase of PDX- i expression shown both by immunohistochemical staining (46% to 51% positive cells) as well as by Western blot. The proportion of [3- and aceils increased from 20.7 _+ 6.3 to 3t.4- _+ 7.6 and 21.1 _+ 3.8 to 30.2 _ 11.8, respectively (p<0.05). In transduced and control NPI. CK7/insulin double positive cells increased from <1% to 25%, indicating differentiation of duct cells to [3-ceils. Conclusions: These data demonstrate that in our in vitro NPI expansion model, PDX-1 over expression increases islet endocrine cell mass. Moreover, this model has the potential to induce the differentiation of human ductal cells in vitro in order to increase the transplantable mass of islet cells.

cein (FITC) conjugated mouse anti-BrdU MoAb (green) and rhodamine (TRITC) -conjugated mouse anti-insulin Ab (red). The samples were then imaged on laser confocal microscopy (LCM). Glucose-stimulated insulin release (GSIR) from the different conditions also was determined. Finally, I and I+SC clusters were enveloped in our alginate/poly-L-ornithine microcapsules, and, respectively, grafted into two groups of CD-I mice with streptozotocin-induced diabetes. Results: Unlike I and I+AA, I+SC or I+SC S were associated with very bright fluorescence patterns. Mitotic nuclei coincided with the [3-cells (LCM), and [3-cell mitotic rate was significantly higher for I+SC (8.1%) and I+SC S (7.4%) as compared to I and I+AA (1%). GS1R was significantly greater from I+SC and I+SC S than for I and I+AA (p<0.0t). While normogtycemia was restored in all transplanted mice, it was sustained through 120 d of followup in 3/5 mice grafted with encapsulated I+SC, against none of the encapsulated I group. Conclusions: [3-ceil-induced mitogenicity is a SC-specific and likely humoral event, that may positively affect I TX outcome.

03-73. I~ Cell Proliferation in Cultured Human Islets Using Pancreatic Regenerating (REG) Protein

P3-01. Is the Mode of Preoperative Renal Replacement Therapy Related to the Occurence of Early Postoperative Complications after Simultaneuos Pancreas Kidney Transplantation (SPK)?

Wang Z.Q., Ta&tsawa S., Okamoto H.. Omori K., Valiente L., Todorov I.. Smith C.V., Mu/len E (Department of Surgery, UCLAVA Islet Transplant Program, Los Angeles. USA) Background: The shortage of donor islets is a major obstacle to pancreatic islet transplantation. Generation of human islets ex vivo would be one approach to alleviating this problem. For this purpose, we examined the effects of purified recombinant human REG protein on the replication of [3 cells in cultured human islets. Methods: Human islets from 5 donors were cultured for 7 days in RPMI1640 medium with or without 10 nM REG protein. BrdU was added to the medium for the final 24 hr of culture and cells were harvested on day 7. Immunocytochemistry and insulin measurements were used to assess results. Results: BrdU uptake was detected in 2.9 + 0.6% and 0.5 _+0.3% of cells in REG- and control cultures, respectively (p<0.01). Among BrdU-positive cells, 28.0 - 8.51% in REG- and 1.5 _+ 0.8% in control cultures were also stained for insulin (p<0.01). Among insulinpositive cells, 5.3 _ 1.75% in REG cultures were also BrdU-positive, but essentially none in controls. At the end of culture, islets cultured with REG contained higher amount of insulin than controls: 87.3 _+ 14.4% vs. 60.7 _+ 10.5% of the amount measured in the original islets on day 0 (p = 0.02). Insulin released into the culture medium during the last 24 hours was significantly higher in REGcultures than controls (11.5 +_. 1.73 vs. 9.3 + 1.42 ~tU/IEQ; p<0.01). Static incubation tests on day 7 also revealed significantly higher stimulation indices in REG-cultures than controls. Conclusions: Results clearly demonstrated that human REG protein promotes proliferation of [3 ceils in cultured human islets. REG protein may provide a new approach for generation of new islets ex vivo.

03-74. Mechanisms of Sertoli's Cell (SC)-Derived Mitogenic Effects on Adult Rat Pancreatic Islet (I) B-Cells: Implications for Transplant (TX) in Diabetes Mellitus Calafiore R., Lucca G., Calvitti M., Basta G., Neri L.NI., Becchetti E., Capitani S., Brunetti P. (Department of Internal Medicine, University of Perugia, Italy) Background: The low mitotic capacity of adult I [3-cells impairs long-term I TX functional longevity. We had observed that in vitro co-incubation of rat SC with homologous I induced significant [3cell mitogenesis. We then aimed to clarify nature and specificity of the SC-related mitogenic effects, as well as impact on TX. Methods: Adult Spague-Dawley (SD) rat 1 were incubated with either isolated pre-pubertai SC, or SC-supernatant (S). Astrocytes (AA) were separately co-incubated with I. At 12 days, the preparations were incubated with BrdU, and double-stained with fluores-

Poster Presentations

Kahl A., Bechstein W O , Sauer l., Miiller A.R., Settmacher U., Lepenies J.. Oppert M., Venz S., Neuhaus P., Frei U. (Department of Nephrology, Humboldt-University, Charitd, Virchow-Klinikum, Berlin, Germany) Background: Intraabdominal complications after SPK such as transplant pan-creatitis, fluid retention, infections and abscess formation are not only due to the transplant itselL but also to organ receiver specific factors like uremia or decreased peritoneal defense after peritoneal dialysis (PD). We investigated a possible relationship between the preoperative renal replacement therapy and the occurrence of early postoperative (< 3 months) surgical complications. Methods: Between April 1995 and June 2000, 100 type I diabetics (59 males. 41 females, age 41 + 8 years) received 99 simultaneous pancreas and kidney transplants and 1 pancreas after kidney transplant. Preoperative 74 patients were on hemodialysis (HD), 11 on peritoneal dialysis (PD) and 15 still without dialysis (WD). Complications requiring surgical revision within the first 3 months (except for the kidney transplant) were monitored. Results: Such complications appeared in 18 out of 100 patients. 11 of the 74 HD-group patients (15%), 5 of the 11 PD-group patients (45%) and 2 of the 15 WD-group patients (13%) underwent surgical revision. The indications for revision are given in table 1. Table l. Indication

HD-pat. (11/74)

PD-pat.(5/11) WD-pat.(~15)

Pancreatitis/lnfection

4

2

Bleeding

2

l

Leakage of Gt-anastomosis

2

1

1

Vascularthrombosis

2

Other (i.e. lleus)

l

1

l

Conclusions: Early postoperative complications occurred predominately in the PD-group and were less likely in patients without preoperative renal replacement therapy. For this reason early SPK can be recommended.

Acta Chirurgica Austriaca Blackwell Wissenschafts-Verlag Berlin • Wien

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P3-02. Comparison Between The Inhibitory Effects Of Short-Term And Long-Term Glucose Toxicity On Growth Hormone Induced Potentiation Of Insulin Secretion From Isolated Human Fetal Islets Lalic N.M., Djordevic P.B., Jotic A., Pazmovic l., Lalic K., Petkovic S., Raketic N., Zamaklar M., Rajkovic N.. Lukic Lj. (Institute of Endocrinology, Belgrade, Yugoslavia)

Background: It has been previously shown that ambient glucose levels influences the growth hormone (GHI -induced increase in insulin secretion from isolated human fetal islets. The aim of this study was to compare the effects of short-term (24 hr) and longterm (14 days) preincubation of isolated islets with high glucose concentration (22 mmol/l) on the GH-stimutated insulin secretion capacity. Methods: The islets were isolated b5 collagenase digestion. Protocol A (24 h preincubation,) and protocol B (14 d preincubation,) were divided into 4 subsets based on glucose concentration during preincubation (22 mmol/l: AI and A2. B1 and B2; 5 mmol/l: A3 and A4, B3 and B4). After preincubation, islets from subsets A1, A3. B1 and B3 were incubated for 3 days with 1000 mg/l GH (Genotropin, Kabi Pharmacia). The insulin response was evaluated in vitro after lhr incubation with low (1.67 mmol/l) and high-glucose (16.7 mmol/l) + 5 mmol/l theoph~lline sequentially, and expressed as a percentage of the increase in insulin levels after the stimulation. Results: We found that insulin response was moderately but not significantly decreased after 24 h preincubation with 22 mmol/l glucose (AI: 703.6 ± 66.3%; A2:184.6 ± 29.4%; A3:758.3 ± 71.7%; A4:336.5 _+ 39.4%; A1 vs. A3, p = NS). However, after 14 d preincubation with 22 mmol/1 glucose, the insulin response was significantly diminished, becoming similar to that in control islets (B l: 176.5 ± 32.3%; B 2 : 1 5 9 . 2 ± 31.5c~: B 3 : 7 1 2 . 7 ± 39.6%: B4: 317.3 ± 29.3%; B1 vs. B3 p<0.01:B1 vs. B2 p = NS). Conclusions: Our results have shox~n that short-term glucose toxicity diminished GH-stimulated insulin response only moderately, while long-term glucose toxici b completely inhibited the GH-induced improvement of insulin secretion from isolated human fetal islets.

P3-03. Successful Islet Transplantation Does Not Prevent the Development of Neuropathy in AIIoxan-lnduced Diabetic Rats Spadella C.T., Machado J.L.M., Caramori C.A., Greg6rio E.A. (Departamento de Cirurgia, School of Medicine of Botucatu, UNES, Faculdade de Medicina, Sao Paulo, Brazil)

Background: Previous studies in our laboratory have shown that good metabolic control of diabetes has been achieved by islet transplantation (IT) in alloxan-induced diabetic rats for a long-term period. On the hypothesis that if perfect glucose homeostasis is possible, we investigated if the development of somatic nerve lesions could also be prevented by IT. Methods: One-hundred fifty inbred male Lewis rats were randomly assigned to 3 experimental groups: group NC included 50 non-diabetic control rats, group DC included 50 untreated-diabetic control rats, and group IT included 50 diabetic rats that received pancreatic islet transplantation prepared by collagenase from normal donor Lewis rats and injected into the portal vein. Each group was further divided into 5 subgroups of 10 rats and killed after 1, 3, 6, 9, and 12 months of follow-up, respectively. Clinical and laboratory parameters during these periods were documented. The right sciatic nerve of 5 rats in each subgroup was removed and routinely processed for examination under the light and electron microscope. Micrographs were obtained from each tissue block for the morphometric study which was performed by Digital Image Analysis System using an Image-Pro Plus Program. Results: There were no significant changes in the number of myelinated fibers and of axons with gbcogen deposits in the three experimental groups at 1, 3, and 6 months of follow-up. However, DC rats presented a significant decrease of myelinated fibers, with increase of the number of axons with small diameter and severe demyelination when compared with NC rats (P<0.05) at 9 and 12 months. Intraaxonal glycogen deposits in diabetic rats were also more evident than observed in NC rats in these periods. Despite

Acta Chir. Austriaca • Vol. 3 3 . Supplement No 174 • 2001

good metabolic control achieved by IT, the severity of these lesions in transplanted rats was not statistically different trom DC rats, even in rats with sustained normoglycemia, all over the study. Conclusions: We concluded that IT failed to prevent somatic neuropathy in alloxan-induced diabetic rats despite glucose homeostasis. It suggests that other factors might be participating in their genesis. (Research supported by FAPESP)

P3-04. Purification of Porcine Pancreatic Islets Using Hydroxyethyl Starch (HES)-Collins Solution Kenmochi T., Asano T.. Sakamoto K.. Jingu K., Maruyama M., Akutsu N., Matsui Y., Yamada K., Ochiai T. (Department of Surgery. Chiba University, Chiba City, Japan)

Background: Adult pig has been considered to be the most suitable donor that will provide the xenogenic pancreatic islets to human. However, the isolation and culture of the adult porcine islets are extremely difficult because of the lack of capsules and their fragility. In this paper, we examined the efficiency of HES-Collins solution as a purification media on porcine islet isolation. Methods: Twenty-eight NIH miniature swine were used for the islet isolation. Islets were digested using our automated digestion apparatus. Then. the digests were purified by discontinuous gradient technique on HES (MW: 400,000)-Collins solution (group 1: n = 6) or on Euro-Ficoll solution (group 2: n = 22) using COBE2991 cell processor. Yield, purity, static incubation and perifusion study were performed. Results: Islet yield and purity were 2919 + 596IEQ/g and >90% in group 1. While. those were 2132 _ 1974 and >90 in group 2. Although those data did not provide significant differences, islets were larger and well-formed in group 1 as compared to group 2. Stimulation index of static incubation was 2.2 __. 0.4 in group 1, which was significantly higher than the 1.2 _+ 0.8 in group 2 (p<.05). Perifusion study showed the prompt insulin release against glucose challenge in group 1 (S.I. = 3.5) indicating the good function of the islets. Conclusions: HES-Collins solution is recommended for islet isolation from the adult porcine pancreas according to the preservation of morphology as well as the function of the islets.

P3-05. Low-Speed isopycnic Islet Separation Is Effective and Yields Islets with Superior Quantity and Quality Shibata S., Sageshima J., Hiraoka K., Zhang H., Koyama K., Sutherland D.E.R., Hering B.J. (Department of Surgery, University of Minnesota, Minneapolis, USA)

Background: The inconsistency of islet processing remains the major obstacle to the application of islet transplantation. This study addresses the impact of centrifugal force (CF) applied during isopycnic islet separation. Methods: Islets were purified in an alternate fashion using low (100G) and standard (800 G) CF on a Cobe2991 (n = 18). Continuous iodixanol gradients with low viscosity were used in both groups. The low and standard CF-purified islet were compared with respect to islet enumeration, purity, islet equivalent/islet count (IE/IC), and glucose-stimulated insulin release in vitro. The posttransplant function of low CF-purified islets was evaluated in diabetic pigs using the small bowel intramuscular implantation site. Results: The%recovery and purity of low CF-purified islets were significantly higher compared to islets separated at standard CF (118 vs. 100%, 90.3 vs. 88.8%). The insulin secretory response to glucose was higher for islets separated at low vs. standard CF (2.2 vs. 1.2). At standard CE the IE/IC after purification was significantly lower compared to that before purification (0.73 vs. 0.84), which is suggestive of islet fragmentation. In contrast, there was no significant difference in IE/tC before and after islet separation at low CF (0.73 vs. 0.73). Low CF-purified islets consistently reversed diabetes in the pig allotransplant model with a follow-up greater than 30days in all immunosuppressed pigs. Conclusions: Low CF-purified islet separation yields islets with superior results compared to standard method with respect to both islet recovery and quality. Low CF islet separation represents an improvement and warrants evaluation for human islet separation.

Acta Chir. Austriaca • Voi. 33 • S u ~ o l e m e n t No 1 7 4 . 2001

P3-06. Effect of Air Transportation on Islet Tissue Survival Fraga D.W., Sabek 0., Clark S, Koth M. E, Gaber A.O. (Department of Surgery. University of Tennessee. Memphis, USA) Background: As the prospect for succebsful human islet transplantation improves, there is interest in sharing islet preparations among centers. However, the impact of air transport on islet tissue has not been well studied. Methods: We evaluated the effect of long distance air transportation on the survival of islets. Ten parallel aliquots from each of 6 sequential human islet preparations x~ere placed in 6 ml of media in individual culture flasks. Five flasks from each preparation (n = 30) were packaged for roundtrip air transport and delivered within 12 hours of shipment. The remaining flasks (n = 30) were maintained in standard culture (27C, 5% CO2). On receipt of the transported tissue, all islet preparations were assessed for islet equivalent number (IE). apoptosis and histology with the goal of obtaining a differential comparison of the shipped and unshipped islets. Count data was evaluated using ANOVA and t-test comparison of IE recoveries. Apoptosis studies were performed using flow cytometric analysis. Results: Islet recovery was highly ~ariable between the different islet preparations (range 53.1-97.3%i aith a significant loss of tissue in the air transported flasks (p<0.001). This variability was not randomly distributed as might be expected if due to purely mechanical disruptio nof tissue as the pre and post transport IE counts were highly correlated (r = 0.94). Apoptosis evaluation of tissue by flow cytometry showed no elevation in apoptotic tissue for the pre and post transport groups (3.2% vs. 3.6g apoptotic cells respectively). Conclusions: These results suggest the need for continued study of the issue of islet transportation, particularly the feasibility of mmsporting marginal preparations, and determination of optimal conditions tbr islet transportation.

P3-07. Islet Quantity and Functional Quality Are Donor Strain Dependent de Groot M., Kei=er P. P. M., de Haan B. J., Leuvenink H. G. D., Schuurs T. A., van Schilj'gaarde R. (Department of Surgery, Groninden University Hospital, The Netherlands)

Background: Donor factors influence the quality of the islet graft. We hypothesize that the donor strain is one of these factors. In this study, four different rat strains were compared with regard to islet yield and function in vitro. Methods: Lewis, Sprague Dawley tSD), Wistar and Albino Oxford (AO) rats (290-310 gram) were used as donors. Islets were isolated using a two times 10 minutes incubation step with collagenase P and purified using a Dextran gradient followed by handpicking. Islets were randomly divided over three portions and tested by a static glucose (16.5 mM + 0.1% IBMX) challenge test, immediately after isolation and after two and seven days of culture. Results: AO rats yielded 614 - 16 islets (number of islets _ sem, n = 6) per pancreas, which is significantly (p<0.05) more than Wistar (352 _+ 9, n = 6), Lewis (303 _+ 7. n = 4) and SD (346 +_ 13, n = 4). The glucose challenge test results show that Wistar islets respond best to glucose immediately after isolation, but the glucose stimulated insulin secretion clearly decreases during culture time. AO islets, however, show a significant improvement of their insulin secretion during 7 days of culture. Conclusions: We conclude that AO rats are superior donors, because they donate significantly more islets than Wistar, Lewis and SD rats and show the ability to recover from the isolation procedure.

P3-08. The Outcome of Graft Survival and Function after Syngeneic Islet Transplantation into the Omental Pouch Kin T., Korbutt G.S., Rajotte R.V. (Surgical-Medical Research Institute, University of Alberta, Edmonton. Canada)

Background: The aim of this study was to evaluate the omental pouch as a site for islet transplantation.

63

Methnds: We transplanted syngeneic islets into the omental pouch or renal subcapsular space of streptozotocin-induced diabetic Wistar-Furth rats. Results: All animals receiving 2000 islets in the omental pouch or under the kidney capsule returned to normoglycemia within 4 and 2 weeks after transplantation, respectively. The time to achieve normoglycemia was significantly longer in rats with omental pouch grafts than in those with islets placed under the capsule, When oral glucose tolerance tests were performed at 4 weeks post-transplant. recipients with islets placed in the omentum showed impaired glucose tolerance when compared to those transplanted renal subcapsularily. However, this glucose intolerance was ameliorated at 8 weeks after transplantation, where no differences were observed between the two groups. Prompt hyperglycemia was observed in all rats after removal of the islet grafts. No significant differences were found in insulin contents of the harvested grafts, irrespective of the transplantation site. When the number of islets in the omental pouch was increased to 4000, all animals returned to normoglycemia within one week after transplantation, and exhibited normal glucose tolerance. Conclusions: The results indicate that the omentat pouch is a viable site to transplant islets, however more islets are needed to cure diabetes compared to the kidney capsule. Nevertheless, the maintenance of viable and functional islets in the omental pouch makes this model attractive for further studies as a transplantation site for impure islet preparations which are excessive in volume.

P3-09. Regulated Expression of Hypoxia-lnducible Factor1Alpha in Isolated Pancreatic Islets: Implications for Primary Non-Function in Transplanted Islets? Morit= W., Stroka D.M., Meier F., Nett Ph., Lehmann R., Gassmann M., Weber M. (Clinic for Visceral- and Transplantation Surgery. University Hospital. Z[irich. Switzerland) Background: In spite of recent success in islet transplantation, the demand of islet tissue requires at least two donor pancreata, implying that a substantial part of the transplanted islets mass is nonfunctional. Hypoxia is suspected to play a major role in cell damage and apoptosis. An important mediator of oxygen-dependent gene expression is the heterodimeric hypoxia-inducible factor-I (HIF-I). We aimed to investigate the presence and regulation of HlF-lalpha. the oxygen dependent subunit of HIF-1 in pancreatic islets. Methods: HIF-lalpha mRNA and protein levels were studied in insulinoma cell lines and isolated human and rat islets under normoxic (20% 02) or hypoxic (1% 02) conditions by means of RTPCR, Northern Blot and Western Blot technique as well as by immunocytochemistry and electromobility shift assays (EMSA). Results: RT-PCR analysis confirmed the presence of HIF-lalpha mRNA in three rat and mouse insulinoma cell lines and in isolated rat and human islets. When exposed to hypoxia, HIF-latpha mRNA levels in MIN6 cells were unchanged. Protein levels, however, were upregulated after I hour and maximally increased after 8 hrs of exposure to hypoxia. Nuclear extracts of MIN6 cells and human islets revealed a hypoxia-dependent binding activity to an oligonucleotide harboring a 8 bp hypoxia response element (HRE). Conclusions: Pancreatic islets do produce HIF-lalpha, that is regulated by hypoxia at a post-transcriptional level. Elucidation of the interrelationship of HIF-lalpha expression and islet primary non-function or the revascularization process will provide important information for future strategies to improve the survival of isolated and transplanted islets.

P3-10. The Stimulatory Effect of IL-1Beta on the Insulin Secretion of Rat Pancreatic Islet Is Not Related with iNOS Pathway Jeong I.K., Oh S.H., Yang TY., Chung, J.H., Min Y.K., Lee M.S., Lee M. K., Kim K.W. (Department of Endocrinology, Internal Medicine, Samsung Medical Center, Seoul, Korea)

Background: IL-lbeta has been implicated in the beta cell dysfunction in the state of islet transplantation or autoimmune disease. IL-lbeta not only inhibits insulin secretion but also stimulates insulin secretion from rat islets. The mechanisms by which IL-lbeta stimulates insulin secretion have been reported. However. whether NO mediates IL-lbeta-induced stimulation of glucose-stimulated insulin secretion remain unclear.

64

Methods: To examine the rote of NO in stimulatory effect of ILl beta, rat islets were treated with different concentrations (0, 0.5, 5, 50, 500 pmmol/L) of IL- I beta tbr 2 or 6 hours. Static stimulation of insulin to glucose, nitrate concentration, iNOS mRNA expression were measured in islets exposed to IL-lbeta alone or IL-lbeta plus NG-monomethyl-L-arginine (a competitive inhibitor of nitirc oxide synthase: NMMA). Results: 1) Insulin secretion was stimulated in islets treated with 5, 50, and 500 pmol/L of IL-lbeta for 2 hours and 0.5 pmol/L for 6 hours. 2) Nitrate concentration was increased in a time and concentration-dependent manner. Nitrate production was inhibited by NMMA. 3) iNOS mRNA expression was increased above 5 pmol/L of IL-Ibeta in dose dependent manner. It was detectable after 2 hours in the presence of IL-lbeta, peaks at 6 hour and decreased after 24hours. 4) The stimulatory effect of IL-lbeta on the insulin secretion of rat islets was not prevented by NMMA. Conclusions: The stimulatory effect of IL-lbeta on the insulin secretion of rat islets is independent on NO production of IL-lbeta and the enzyme activity of nitric oxide synthesis.

P3-11. Survival Identification and Monitoring Pancreatic Islet Grafts Michalska IV., Szymafiska K., Garnuszek P.. Ma~urek A.P., Fiedor P. (Department of General and Transplantation Surgery, Transplantation Institute, Medical University of Warsaw. Poland)

Background: Our previous study with radiotabeled of I-DTZ confirmed the possibility in vivo localization of pancreatic islets. Goal of this study was to create a reliable and easy to monitor experimental model of pancreatic islet grafting in rats and to apply original method of accumulation of synthetic radiolabeled 13tI derivative of dithizone (I-DTZ) for monitoring of survival of grafted cells. Methods: Syngeneic pancreatic islets transplantation in n = 6 streptozotocin induced diabetes WAG rats was performed. Approximately 3000 isolated islets were transplanted between layers of the colon mesentery. Three weeks after transplantation in animals with normal blood sugar intravenous glucose tolerance test [IVGTT, glucose of 2.5 g/kg/b.w, iv injected] and assessment of insulin concentration [RIA] were performed. At the same at 20 ~tl [activity ca. 1.9 GBq/~tmol] of 131I-DTZ solution was i.v. administered. Animals were sacrificed [n = 3] and the grafted islets were examined. Pathology showed viable grafted tissue. Results: All animals became normoglycemic within 2 to 3 days. I V G T r was in normal range [120-140 mg%] 24 h after glucose challenge normoglycemia returned. ~31I-DTZ activity in grafted islets was measured after 30 minutes in counter [per gram of tissue] in vitro and compared to the background [20 control vs. 25%ID/g experimental group]. Injected DTZ solution stained confirming their viability [n = 3]. Staining with antibody against glucagon and insulin confirmed that grafted cells produced these hormones. Conclusions: This site was especially useful for I-DTZ monitoring of the islets since the background activity in this area was much lower than in the liver. The use of 131I-DTZ in the future may be of extreme value for patients with human islets transplantation.

P3-12. The Islet Vascularization: A Bimodal Process of Vasculogenesis and Angiogenesis Madureira M.L.C. (Oporto School of Medicine, Porto, Portugal)

Background: The clinical practice of islet transplantation is confronted with problems of islet mass rentability and one of the variables that can contribute is the loss of the core b cells by isquemic hipoxya, worsened by retarded or deficient vascularization specially in a immunossupressed receptor with advanced degenerative diabetic vasculopathy. A better knowledge of the vascularization mechanisms, cell phenotypes gene expression and factors involved is crucial to optimize this complex process. Methods: Adult pancreatic fragments autotransplanted into the spleen of totally pancreatectomized dogs, whose fate was studied in fasting normoglycemic dogs at weeks 4th. 8th, 24th and 36th by Light and Electronic Microscopy. Results: The adult pancreatic tissue fate was characterized by a regenerative process, exocrine and endocrine, sustained at all times in study, being outlined the ultrastructural details of the endocrine component from the discrete islet cell to islet structures with vari-

Acta Chir. Austriaca • Vol. 33 • Supplement No 174. 2001

able volume, composition and maturity, being identified all islet cell types, reinnervated by the adrenergic network of the spleen. A bimodal mechanism of vasculogenesis and angiogenesis was characterized integrated in a complex regenerative morphogenetic process, being tentatively defined the sequential steps of the early islet core permeabilization, the cooperative production and deposition of matrix products by the intervenient players in the process: islet, schwann, endothelial, mesenchimal, blood cells and neurons.The migration/ remodelation of endothelial cells, their transitory intimate contacts with islet cells and their high production of ECM appeared particularly meaningful. Conclusions: The islet cells appeared to have an intrinsic vasculogenic program and. contrarily to the single cel~ layered exocrine tubes, are strong inducers of angiogenesis with construction of functional peripheral and core capillaries progressively taking the place of the naked channels of the early permeabilization process.

P3-13. Insulin Secretory Reserve of Porcine Islets after Intraportal Transplantation Pattou F, Lamb~in A., N'Guyen H.D., Mulliez E., Franc. C., Gmyr V., Amrouni H., D'herbomez M., Vandewalle A.B., Kerr-Conte J. (ERM 106 Inserm, Facult6 de M6decine, Lille, France)

Background: Using autotransplantation to avoid confounding immunological factors, we studied the metabolic function of isolated porcine islets transplanted intraportally. Methods: Islets were isolated after total pancreatectomy in adult minipigs (37 _+ 3 kg, n = 9) and autotransplanted in the portal vein through a percutaneous catheter.). Functional insulin secretory reserve was evaluated during an IVGTT (0.5g of glucose&g) at 3 months after Tx (T) by the first phase acute insulin response (AIR). Results were compared to those of non diabetic (ND, n = 14), pancreatectomized (P, n = 8). and hemi-pancreatectomized (HP, n = 5) minipigs. Results: (mean _ SEM. *: P<0.05 vs. P). All pancreatectomized controls and three animals that received 1582 _+ 437 150 gin-islet equivalents (IE) /kg became hyperglycemic (fasting blood gtc >250 mg/dl at 1 week) with progressive acidocetosis and cachexia within respectively 14 +_ 3 days, and 38 _+ 12 days. Six animals which received 4557 +_ 1185 IE/kg became euglycemic (fasting blood gucose: 106 _ 11 mg/dl at 3 months for T*, vs. 259 _ 12 mg/dl for P, 83 _.+ 3 mg/dl for HP*, and 84 _ 3 mg/dl for ND*). AIR was 11 _+ 5 mU/L for T*, vs. -1 _+ 1 mU/L for P, 18 -+ 3 mU/L for HP* and 27 _.*4 mU/L for ND*. Both AIR and K values were significantly correlated (P<0.001) with the islet mass. Intrahepatic islets were detected by immunohistochemistry in 4 euglycemic animals, and by intraoperative simultaneous sampling in portal and suprahepatic veins after arginine injection in two chronic experiments (> one year). Conclusions: The intraportal Tx of 5000 IE/kg adult porcine islets can achieve long term control of fasting glycemia but only 10,000 IEQ/kg restore normal functional insulin secretory reserve and glucose tolerance.

P3-14. Ultra-Structural Changes to Adult Rat Beta Cells Following Growth Factor Induced Proliferation in Vitro Yoon T.W., Kim J.H., Shin J.S., Choi S.J., Yoon K.S., Kim I.S., Kim B.J., Kwon 0.0., Seo S.O.. James R. (Korea Islet Transplantation Institute, Kyungkido, South Korea)

Background: Using a combination of recombinant growth factors, we have shown that purified adult rat islets can be induced to proliferate in vitro. Within a period of 5 days the beta cell mass can be increased by 3-4 times. To assess whether the treatment may be associated with cell transformation, ER, and insulin granules were examined of normal [3-cells. HIT cells, proliferated [3-cells as well as the expression of p53, k-Ras, c-Jun, c-Fos, and Bcl-2 was performed in parallel with fresh normal islets as a control. Methods: Purified adult rat islets were cultured in RPMI1640 medium supplemented with 10% FBS, and various growth factors (EGF, IGF-1, etc). Following recombinant growth factor induced proliferation, islets were fixed for 1.5-2 hrs at 4 ° in 2% paraformaldehyde, and embedded in Epon-Araldite. Sectioned tissue was stained with lCk toluidin-borax solution.

Acta Chir. A u s t r i a c a . Vol. 33 • S u p p l e m e n t No 174 • 2001

Results: Freshly isolated islets sho~ the distinctive presence of many secretory granules in the beta cells plus abundant smooth endoplasmic reticulum (SER) and free ribosomes. In contrast, there is generally little rough endoplasmic reticulum (RER) detectable. Transformed beta cell lines, such as HIT cells, have VSER XRER but few insulin granules. Following recombinant growth factor induced proliferation (RGFIP), adult islets show beta cells with greatly increased amounts of RER and reduced SER but with the decreased expression of insulin granules. When transplanted under the kidney capsule of streptozotocin-induced diabetic syngeneic rats following (RGFIP). and removed after 30 days, such islets have "matured" in vivo and reverted to the normal phenoype of VSER XRER. Islets exposed to RGRIP reverse diabetes and maintain normal gtycaemia until the graft is removed by nephrectomy. Longer term culture (8-14 days) of RGFIP treated islets also allows "maturation" to occur in vitro with reversion to the normal VSER XRER phenotype, and Vinsulin granules. No expression of the oncogenes tested was observed in the RGFIP treated islets. Conclusions: RGFIP treatment of isolated islets leads to greatly increased islet mass in vitro with concomitant changes to islet ultrastructure, which reverse with time both in vitro and in vivo allowing such islets to function normally and reverse diabetes in an animal model.

P3-15. Effect of Growth Hormone on Apoptosis of Islet Cells During Isolation Process

65

and washed three times with Hanks" solution. Aliquots obtained after incubation confirmed islet take-up of DAPI. Islets were transplanted into the liver of isogeneic routine recipients by puncture of the portal vein with a 27G needle. Animals were sacrificed at different time points (days 1, 7. and 14 posttransplant) and livers snap frozen in embedding compound for storage at - 7 0 ~ C until section in a cryostat. Frozen sections of 5~m without further treatment were observed under fluorescence microscopy (excitation wave length. 350-372 nm: emission wave length, 420-454 nm). Results: Islets were distinctively identified for their blue fluorescence in a dark background up to day 14 posttransplant. No evidence of portal vein thrombosis was observed upon histologic examination. Conclusions: Pretransplant islet staining with the fluorescent dye DAPI in a simple laboratory protocol may facilitate posttransplant cell identification for further histologic, functional, and immunologic studies.

P3-17. Three Dimensional Cell Culture of Human Islets Is Associated with Increased TNF-Alpha Leeper-Woodford S.K., Uchakin P.N.. Smith S.M., Lakey J.R.T., Rothenberg M.R.. Tobin B.V~ (Mercer University Schoot of Medicine, Macon, Georgia. USA)

sis following isolation and purification. Most islet failures are known to occur early after transplantation. We hypothesized that the loss of graft function following transplantation were related to apoptosis in islet isolation process. This study was designed to determine the growth hormone involved in cell death of pancreatic islets during isolation procedure. Methods: Islets were isolated from Wistar rats (age 5 weeks, weight 110-130 g) using a standard surgical pancreas harvest followed by collagenase digestion, mechanical dissociation and Ficoll density gradient centrifugation. Pancreas were treated in HBSS containing collagenase, Ficoll in the presence of growth hormone (I07 M) during isolation. In addition, islets were washed in HBSS solution containing growth hormone. Control pancreases were isolated in conventional HBSS solution without growth hormone. Islets were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum. A ELISA kit was used which detects cytoplasmic oligonucleosomes as a measure of apoptotic activity. Results: Addition of growth hormone during isolation had no significant effect on the apoptosis of islets collected immediately isolation, and collected after 1 day in culture. However, the apoptosis of islets was decreased during the 2 and 3 days in culture (35% and 31% that of control), Conclusions: We conclude from these data that cell death of pancreatic islets can be inhibited by growth hormone treatment during isolation.

Background: Tumor necrosis factor-alpha (TNF), an inflammatory cytokine, may be involved in pancreatic islet function. Production of TNF in an endocrine or paracrine manner has been implicated in both protective and cytotoxic actions on islet beta-cell function. The present studies were designed to determine effects of a three-dimensional in vitro culture system upon lipopolysaccharide (LPS) stimulated islet TNF activity. Methods: Human islets from pancreas donors (n = 4) were incubated (37 °C, 5% CO2) in: 1) high aspect ratio vessel (HARV) cell culture, 2) HARV plus LPS, 3) static culture, and 4) static culture plus LPS. The L929 cytotoxicity assay was used to specifically measure islet medium TNF unit values (means _ SE). Results: There were no significant differences (p>0.05, ANOVA) in TNF baseline values for any treatment groups. After LPS, there were no significant increases (p>0.05) in TNF of static-cultured islets. There were significant increases (p<0.05) in TNF of LPSstimulated HARV-cultured islets of 6, 12, and 24h samples (TNF Units/ml, LPS vs. CONTROL, 17.0 _ 5.8 vs. 2.6 + 1.3; 15.8 + 5.0 vs. 3.3 + 1.2:6.3 + 1.7 vs. 2.3 + 1.2, at 6, 12 and 24 h, respectively). HARV-cultures with LPS retained a significantly greater (p<0.05) TNF concentration than LPS-stimulated static cultures at the 6 and 12 h time periods (TNF Units/ml, HARV vs. static, 14.4 _ 5.0 vs. 2.5 + 0.8; 12.5 +_ 5.3 vs. 4.3 + 2.0, at 6 and 12 h, respectively). There were no significant changes (p>0.05) in TNF among non-LPS-stimulated control cultures. Conclusions: These studies demonstrate that alterations in islet TNF production favor greater activity in the HARV cultures. These data may have implications for culturing human islets, and for development of countermeasures for microgravity-induced biological alterations of manned spaceflight. (Support from the National Space Biomedical Research Institute through Cooperative Agreement NCC 9-58 with NASA)

P3-16. Intrahepatic Identification of Transplanted Islets Tagged With the Fluorescent Dye 4',6-Diamidino-2Phenylindol (DAPI)

P3-18. Successful AIIogenic Islet Transplantation without lmmunosuppression Through Co-Grafting with Placental Tissues in STZ-lnduced Diabetic Mice

Hyon S. H., Ceballos C., I,Teiro M., Argibay P. (Surgery Department, Hospital Italiano de Buenos Aires. Buenos Aires, Argentina)

Suzuki K., Ueda H., Tanioka Y, Deal T., Takahashi T., Kuroda E, Yokono K., Taniguchi H. (Department of Metabolism, Kobe Mahoshi Hospital, Japan)

Suh K.S., Kim B.J., Park C.Y., Yang 1.M., Kim S.W., Kim ES., Kim J.W., Choi YK. (Department of Internal Medicine, Endocrine R. 1, Kyunghee University, Seoul, Korea)

Background: Pancreatic islet cells are induced to undergo apopto-

Background: Animal models of intrahepatic islet transplantation may be more suitable for posttransplant functional studies than kidney subcapsular space models. However, the difficulty to identify islets within the extensive liver parenchyma may result a drawback for posttransplant histologic assessment. Here we describe a simple method for fluorescent tagging of islets before intrahepatic transplantation followed by posttransplant direct cell identification. Methods: The nuclear DNA staining fluorescent dye 4',6-diamidino-2-phenylindol (DAPI) was prepared at 0.02% containing 100 mM ClNa, 5 mM HEPES buffer, pH 7.0. C57BL/6 mice were used as islet donors. A suspension of 150 islets in 500 ~tL of Hanks' solution was incubated with 25 ~tL of DAPI at 37 ~ C for 30 min

Background: Placenta (trophoblast layer) is the diaphragm between the fetus and its mother, and their direct blood contact is blocked, and considered to protect the rejection of the fetus by the mother's immune system. During a period of gestation, the fetus is nested in the uterus not only as semi-allograft but also as allograft. However, the fetus is not rejected during the whole period of pregnancy. Methods: We have studied allogenic transplantation of islets together with placental tissues (trophoblasts) in STZ-induced diabetic mice without any immunosuppressive agents. Placentae from mice 14 days pregnant were harvested by peeling each one off carefully,

66

leaving the maternal decidua behind, and were cut into small pieces. 500 freshly isolated islets together with placental tissues from ICR mice were placed under the left kidney capsule of diabetic C57BL/6J mice. Results: Blood glucose level was reduced from 500 _.+.26 mg/dl at the pre-transplant to the normal range ~149 ± 18 mg/dl) soon atter the grafting, and did not return to the pre-transplant level before the 14th post-transplant day. Glucose level of the diabetic mice transplanted with islets only as well as islets with liver tissues was not normalized. Conclusions: This is the first successful achievement of islet allo-transplantation without immunosuppressive agents through cografting of allogenic placental tissues. The underIying mechanism is now under investigation.

Acta Chir. Austriaca • Vol. 33 • Supplement No 174. 2001

Free HI IR (% of ctrl)

400 G

0.5 FFA

3.3G

+34__.11

+7-*2

16.7G

- 5 7 ~ 13

-31 ± 8

mc H1 1.0 FFA

400 G

0.5 FFA

1.0 FFA

~

+30±15

-2+0.4

-18±

- 6 5 ± 19

- 5 3 ± 19

~-,-2.0

- 4 9 ± 14

6

The values underlined are statistically different (p < 0.05 or less) vs. ctrl. Conclusions: This suggests that microencapsulation can partially prevent the phenomenon of lipotoxicity, whereas it does not seem to protect the islets from glucotoxicity. Supported by the European Community, project "Bioartifical Pancreas", contract BMH4-CT98-9516.

P3-19. Transartery Intrahepatic Xenotransplantation of Encapsulized Newborn Porcine Islets (NPI) - A n Experimental Therapy for Type I Diabetes

P3-21. Autologous Chondrocyte Matrix as a New Immunoisolation Material for Encapsulated Islets

Wang ~ , Liu S.. Ye B., Luo X., Zhu R. (Department Radiology, The Third Affiliated Hospital of Central-So. Changsha, RR.China)

Maruvama M., Kenmochi T., Asano T., Matsui Y., Akutsu N., Saito T., Nakagawa K., Yamashita T., Wada Y, Ochiai T. (Department Surgery II, Cbiba University. Japan)

Background: Biocapsule can protect islets against the rejection from host. Our study was to evaluate the biocompatibility, immunology and physiologic features of encapsulated NPI in the liver of recipient dogs with Type I diabetes. Methods: Type I diabetic dogs were perfused with 400000600000 unencapsulated NPI (group A, n = 15) or encapsulated NPI (group B, n = 15) through hepatic arter'~ without immunosuppressant treatment. Liver function and CD4/(2D8 in the recipients were measured before and after transplantation. The livers from all NPT recipient dogs were also analyzed by histopathology 6 months after Tx. Results: While insulin dose administrated to group A animals was reduced gradually within one week after Tx (from 22U before Tx to 5U after Tx), exogenous insulin required for group B animals was decreased from 24u to 10u. However. after 2 to 3 weeks post Tx. the insulin dose given to grorap A recipients was reversed to the original level used before Tx. In contrast, the amount of insulin administrated to group B recipients was continually reduced till to 8u. Moreover, CD4 in blood of group A recipients was higher than that before Tx, whereas no significant alteration of CD4 and CD8 in blood of group B animals after Tx. All NPI recipient dogs demonstrated a normal function and structure of livers after Tx. Conclusions: Microcapsulated NPI has a good biocompatibility in recipients livers. Microcapsule can prolong xenograft survival and xenotransplantation of microcapsulized NPI can correct the hyperglycemia of diabetic canines.

Background: Transplantation of encapsulated islets is an ideal method to prevent grafts from host immune attack without immunosuppression. However an artificial material such as alginate or agarose, induce unspecific inflammatory reaction of macrophage and fibroblast and deteriorate the graft function, even if they are ultra-purified. Autologous materials do not have immunogenesity and do not induce an inflammatory reaction and can maintain the good graft function. In this study we report the application of autologous chondrocyte matrix for encapsulated allogeneic islets in vitro. Methods: Islets were isolated from DA rats (250-300 g) by a collagenase ductal digestion method and ficoll gradient separation. Chondrocytes were obtained from the cartilage of the knee of Lewis rats (6--7 weeks) using a collagenase. 100 islets and 9 x l0 s chondrocytes were co-cultured using RPMI culture medium containing 15% fetal bovine serum and 50 mmg/ml ascorbic acid at 37 °C in 95% air/5% CO2. The structure of islets and chondrocytes were observed every other day using phase contrast microscope and insulin levels in the culture medium were measured 2-3 times a week. Results: Islets structures were maintained morphologically during observation period. Chondrocytes were proliferated very well and embedded islets. The insulin levels in the culture medium kept the same level as the islet alone group. Conclusions: Autologous cartilage may be an optimal material to prevent the encapsulated islets of Langerhans from not only host immune system but also an unspecific inflammatory reaction.

P3-20. Effect of Microencapsulation on Gluco- and/or Lypotoxicity of Human Pancreatic Islets

P3-22. Simple Barium Alginate Capsules Protect Neonatal Porcine Islet Cells in Diabetic Mice for over 20 Weeks

Del Guerra S., De Vos P., Rossi A., Lupi R., Marselli L., Boggi U., Mosca F., Belcourt A., Del Prato S., Marchetti P. (Department of Endocrinology and Metabolism, University of Pisa. Italy)

Omer A., Duvivier-Kali V., Trivedi N., Wilmot K., Bonner-Weir S., Weir G.C. (Joslin Diabetes Center/Islet Transplantation, Harvard Medical School, Boston, USA)

Background: In the present study we investigated whether mi-

Background: The effect of barium alginate encapsulation on the

croencapsulation of human islets (HI) is able to prevent the deleterious effects of prolonged exposure to glucose (G) or free fatty acids (FFA). Methods: HI were isolated by collagenase digestion and density gradient purification, and then suspended in 2% sodium alginate; beads of approximately 600 ~tm diameter were obtained by converting this suspension into droplets by means of an air-driven droplet generator; the formation of a poly-L-lysine (PLL) membrane was successively induced by suspending the beads in a 0,1% PLL solution, and an outer alginate layer was finally applied by suspending the capsules in a 0,3% alginate solution. Within 7 days from the preparation, free or microencapsulated (mc) HI were incubated for 24 h in the presence of either 400 mg, dl G, 0.5 mmol FFA or 1.0 mmol/l FFA, and insulin release (IR) in response to acute 3.3 and 16.7 m m o l / G stimulation was assessed. Results: The results (mean ± SD of 5 experiments) are summarized in the table and show that, as expected, tree HI exposed to G or FFA had altered glucose-stimulated IR. compared to control (ctrl) HI: microencapsulation protected HI from the deleterious action of 0.5 mmol/l FFA, but not of G or 1.0 mmobl FFA.

survival, function and proliferation of porcine neonatal pancreatic cell clusters (NPCCs) after transplantation into immunocompetent diabetic mice was investigated. Methods: Pancreases from 1-2 day old pigs were digested with collagenase, and cell clusters cultured for 8 days. NPCCs were encapsulated with purified high M alginate (Monsanto) and crosslinked with BaC12, the capsules being about 900 micrometer in diameter. Approximately 10,000 islet equivalent of NPCCs were transplanted into the peritoneal cavity of streptozocin diabetic B6AF1 mice (n = 32). Body weight and blood glucose levels were measured every three days, with normoglycemia considered as less than 200 mg/dl. Capsules were removed at 2, 6 and 20 weeks and examined for cellular overgrowth, insulin content and insulin secretory responses to 16.7 mM glucose and 16.7 mM glucose with 10 mM theophylline. Control animals were transplanted with NPCCs under kidney capsule. Results: Glucose levels were normalized in 71% of the mice in 10 ± 7 days and remained normal until end of the study. A mild tissue reaction was found on all of the capsules. Insulin content of the encapsulated NPCCs increased from 310 _ 180 pg/IE to 920 +- 90

Acta Chir. Austriaca • Vol. 33 - Suc01ement No 1 7 4 . 2001

pg/IE at 2 weeks and to 1630 + 430 pg at 6 weeks. Insulin secretion stimulation index in response to glucose plus theophylline was 4 at 2 weeks and 19 at 6 weeks. Conclusions: NPCCs encapsulated ~ith simple barium alginate capsules can mature and normalize glucose levels in immunocompetent mice for more than 20 weeks,

67

micmcapsule volume may improve the survival of encapsulated islet grafts.

P3-25. JAK-3 Inhibition in Human T Cells Abrogates IL-2 Production and Early T Cell Clustering: Evidence for an Impaired Early TCR-Signalling

P3-23. Enzymatic Removal of Alginate-Poly-L-Lysine Capsules from Microencapsulated Islets

Saemann M.D., BOhmig G.A.. Diakos C., Prieschl-Strassmeier E., Baumruker T.. HOrl W.H.. Zlabinger G. (Institute Of Immunology, Vienna)

de Groot M., Keiz,er P., de Haan B.J., Leuvenink H.G.D., De Vos P., Schuurs T.A., van Schilf~aarde R. (Surgical Research Laboratory, Groningen University Hospital, The Netherlands)

Background: Considerable interest exists in the development of new immunosuppressive drugs that target the IL-2-receptor coupled signalling pathway. Recent evidence from our group as well as from SCID-animals suggests an interference of the IL-2-receptor associated tyrosine kinase JAK-3 with early IL-2 production in T-cells. Methods: The following study was designed to evaluate the impact of a new selective JAK3-inhibitor the tyrphostin AG490 on several aspects of early T-cell activation with special emphasis on IL-2 production. Results: Isolated human T-cells or unfractioned PBMC were challenged either allogeneically or with distinct monoclonal (mAbs) in the presence/absence of AG-490. The addition of AG-490 to human T-cells stimulated with allogeneic cells, OKT-3 or OKT-3 plus CD28 mAb led to a profound inhibition of IL-2 production. This was confirmed at the transcriptional level by means of luciferase assays in Jurkat T-cells. Importantly electrophoretic mobility shift assays revealed profoundly reduced nuclear NF-AT binding, which is essential for IL-2 transcription. Interestingly, AG490 supplemented cultures contained no or only a few T-cell clusters typical for early T-cell activation. In contrast, calcineurin inhibition not or only marginally interfered with T-cell clustering suggestive of an alternative mode of T-cell activation interference. Conclusions: We conclude that AG-490 not only interferes with IL-2-receptor signalling, but instead profoundly downregulates IL-2 production in human T-cells presumably by interfering with TCRtriggered signals. Due to this peculiar characteristic, which is not shared by classical immunosuppressants we anticipate a strong immunosuppressive activity of such drugs in vivo.

Background: Microencapsulation of isIets of Langerhans enables successful transplantation in the absence of immunosuppression. In order to study graft function, encapsulated islets are retrieved and qualitatively analysed. Since the presence of the alginate-poly-L-lysine capsule interferes with applications like RT-PCR and histology, removal of the capsule is necessar3. Therefore, we developed a novel method in which the encapsulated islets are enzymatically decapsulated using a mild trypsin/EDTA treatment. Methods: A protocol was designed using a combination of EDTA (to liquefy the inner Ca2+-alginate core) and trypsin (to degrade the poly-L-lysine layer covering the capsules), This protocol was optimised by using various concentrations and incubation times, and subsequently tested on encapsulated rat islets. Results: Empty capsules were effectively disrupted by a 30 min. (37 ~C) exposure to a combination of 0.25% trypsin and 0.5 mM EDTA, followed by gentle pipetting the suspension. With this approach islet containing capsules were also readily disrupted. The function and integrity of the decapsulated islets were unaffected as shown by insulin secretion capacity (glucose challenge test), vitality (acrydine-orange/propidium-iodide) staining and expression analyses of metabolic (GAPDH and 13-actinl and apoptotic genes (bcl-2 and bax). Conclusions: We developed a novel method to decapsulate encapsulated islets, using a mild trypsin/EDTA treatment. This method enables large scale decapsulation without functional impairment of the islets.

P3-24. Pravastatin Suppresses Inflammation Induced by Alginate Microcapsule Implants in Mice Mure T,, Maruyama M., Stein E., Smith C.V., Mullen Y (Department of Surgery, UCLA-VA Islet Transplant Program, Los Angeles. USA)

Background: Alginate-microencapsulation of islets has been used for preventing graft rejection. However. islet survival is limited, especially in large animals, Foreign body reactions caused by the capsules may play a role in islet failure, because inflammatory products can permeate the membrane and destroy islets. We have shown that pravastatin prevents inflammation-mediated primary islet graft nonfunction. Thus, it may also be effective for suppressing inflammation caused by microcapsules. Methods: Alginate capsules (50, 100 and 500 [xL/mouse) were intraperitoneally implanted into C57/BL6 mice. Pravastatin (40 mg/kg/day) was orally administered starting on day -2. Peritoneal-exudate cells (PEC) were harvested on days 1, 3 and 7. Cytokine release was measured in the medium by culturing PEC for 24 hrs. Results: Capsules were free of adherent cells and >80% were retrieved. PEC numbers were higher with larger capsule volumes and 2-4 fold of those in the sham operated. Macrophage percentages in the PEC were 63.4-88.4%. Pravastatin lowered the PEC numbers by 14.6-44.7%, macrophages by 15.9--45.0% and eosinophiles by 10.1-73.7%. This effect was more pronounced with capsule volumes <100 ~tl/mouse. In in vitro cultures of harvested PEC, the production of IL-I[3 (174.7 ~ } 71.1 vs. 65.4 A.} 17.1 pg/ml) and TNFet (281.6 A} 43.9 vs. 172.8 A,} 67.2 p~ml) was significantly suppressed in the treated groups with the PEC harvested on day 7. Conclusions: Inflammation caused by alginate capsules was dose-dependent. Pravastatin suppressed PEC infiltration and their cytokine production. Thus, treatment with pravastatin and reduced

P3-26. The Use of Mycophenolate Mofetil as Immunosuppressive Treatment, in an Experimental Model of Islet Transplantation Xekouki P., Fotiadis C., Papalois A., Poussios D., Karampela E., Grigoriou Th., Sfiniadakis J., Sechas M.N. (Department of Surgery, School of Medicine, University of Athens, Greece)

Background: The aim of this study was the isolation and allotransplantation (allo-Tx) of unpurified islets in diabetic rats and to evaluate the effect of Mycophenolate Mofetil as an immunosuppressive agent tbr which there are few experimental reports worldwide in islets transplantation. Methods; Totally, 72 rats were used (48 male Wistar rats as recipients and 24 Lewis rats as donors). For induction of diabetes the streptozotocin IV administration was used. Unpurified islets were isolated using the collagenase digestion technique. Immunosuppressire agents were given daily per os starting right after the islet transplantation and for the next twelve days. Glucose was measured on day 0 (Tx day) and on the 3rd, 5th, 7th, 10th and 12th day. The last one was the day in which the rats were sacrificed. The recipients were divided into 4 groups with 12 rats in each of them. Group A was the control group (no immunosuppression). Group B received Cyclosporine A 100 mg as immunosuppressive treatment. Groups C and D received 250 and 500 mg of Mycophenolate Mofetil respectively. Results: No animal in Group A became normoglycemic. The mean plasma glucose was 384 mg/dl and the mean survival time was 4 days. In group B mean plasma glucose was 160 mg/dl and the mean survival time was 8 days. In groups C and D were the Mycophenolate Mofetil was used the survival time reached almost 100% and in the last group the mean plasma glucose was closer to the normal range (~145 mg/dl). The histological findings were in accordance with the results that are mentioned above. Conclusions: The statistical comparison of the mean values for plasma glucose and survival time between groups showed a significant difference with the use of Mycophenolate Mofetil.

68

P3-27. Liposome-Encapsulated Tacrolimus Produce Better Immunosuppressive Effect in Discordant Islet Xenograft Model Yang H., McAlister V.C., Al-Jazaeri A.. Wright J.R. (Department of Pathology, IWK Grace Health Centre. Halifax. Canada)

Background: We investigated whether treatment with liposome-encapsulated tacrolimus (LTAC). prepared as we have previously described (Transplantation 67:1205-8, 19991. would achieve a better immunosuppressive effect in a discordant islet xenograft model than tacrolimus (TAC). Methods: Tilapia (fish) islets were transplanted under the left kidney capsules of Balb/c mice made diabetic (blood glucose > 350 mg/dl) with streptozotocin. Mice IGroups 1-5) were treated with: G1 - empty liposomes; G2 - TAC (2 mg/kg/d); G3 - TAC (5 mg/kg/d); G4 - LTAC (2 mg/kg/d); or G5 - LTAC (5 mg/kg/d): all treatments were for 35 days or until rejection (i.e., 2 glucose measurements >200 mg/dl). Graft-bearing kidneys were removed for histology after rejection (n.b., one mouse in G5 was killed for histology on day 36 while normoglycemic): sections were stained routinely (H&E) and for tilapia insulin. Results: Mean graft survival time (mGST) for control islets (G l) was 7.5 _+ 1.3 days (n = 4). Daily TAC treatment at 2 mg/kg/d (G2) did not prolong graft function (mGST = 7.7 _+ 1.6; n = 6) while 5 mg/kg/d (G3) produced minimal prolongation to 12.8 _+ 4.8 days (n = 12). Treatment with LTAC at 2 mg/kg/d (G4) significantly prolonged mGST to 26.6 _+ 4.9 (n = 5); however, all recipients rejected during treatment (i.e., <35 days). LTAC at 5 mglkg/d (G5) further prolonged mGST to 42.5 _+ 10 days (n = 10) with only one mouse rejecting prior to day 35. Histologically. at the time of functional rejection, grafts in Groups 1-4 were generally either totally or partially effaced by mononuclear cell infiltrates, eosinophils, and fibrosis. In G5, islet grafts removed from three mice that died while normoglycemic (i.e., days 9,18, or 21) and from a mouse terminated while normoglycemic at day 36 (excluded from mGST calculations) were viable, well-granulated and free from cellular infiltration. The G5 grafts examined at rejection (i.e., 1-2 weeks after discontinuing LTAC) were generally totally obliterated and were sometimes associated with nodular aggregates of atypical lymphocytes. Conclusions: Liposome-encapsulated tacrolimus is the most potent immunosuppressive compound we have tested in our discordant fish-to-mouse islet xenograft model: however, toxicity appears to be an issue at high doses.

P3-28. Study of Endothelial Function and Citokine Production in Diabetic and Islet Transplanted Rats Erce C., Arnado J.A., Unzueta M.T., Martine= R., Casanova D. (Universidad de Cantabria, Bergara, Spain)

Background: For years, endothelium has been considered an inert limit between blood and vascular wall. It is now considered to play a significant role in regulating vasodilation and homeostasis. Nitric oxide is produced by the endothelium and it is involved in vasodilatation or acts as a cytotoxic molecule in inflammatory response and interleukin-6, produced by endothelium and macrophages, induces hepatic synthesis of acute phase reactants in inflammatory response. Diabetes is a disease that leads to early microvascular disease that may be mediated by oxidative and inflammatory distress. We evaluated the role that nitric oxide and interleukin-6 had in diabetes and if islet transplantation could reverse these alterations. Methods: Male Wistar rats of 250 g of weight at the beginning of the work were divided in four groups: G1 (n = 13):Control rats that received iv saline injection. G2 (n = 28): Rats that received an iv streptozotocin injection. G3 (n = 8): Control rats that received an islet transplantation. G4 (n = 13): Rats with an streptozotocin induced diabetes of 7-14 days rendered normoglycemic after an islet transplantation in the subcapsular space of the kidney. Both last groups received 25 mg/kg of cyclosporine A on days 0, 1 and 2 after transplantation. All animals had free access to non-treated water and standard rat chow all the time. 7-14 days after saline or streptozotocin injection in groups G1 and G2. and 10 days after islet transplantation in G3 and G4, half of the animals in each group received an intraperitoneal dose of E. coil LPS (0.1 mg/kg) and the other half received plain saline injection. The animals were located again in the metabolic cages for 8 hours and urine and blood were collected afterwards. Nitrites and nitrates were assayed in plasma

Acta Chir. Austriaca • Vol. 33 • Supplement No 174 - 2001

and urine with Griess colorimetric reaction and interleukin-6 was assayed in blood with an ELISA kit specific for rat IL-6. Results: Nitrites and nitrates: There was a significant increase of nitrite and nitrate levels in blood and urine in diabetic animals compared to control rats. Transplanted rats had levels significantly interior to those of diabetic rats and comparable to those of control rats. IL-6: Levels of [L-6 were higher in diabetic rats compared to control rats and transplanted animals had levels similar to control rats, although these differences were not statistically significant. Nitrites/nitrates

IL-6

microM

pg/ml Control

461.5

95.73

Diabetic

692.0

196.48

Sham transpl

214.2

44.94

Transplanted

395.95

43.96

Conclusions: Nitric oxide pathway is altered by diabetes, even in early stages of diabetes, and these alterations can be reverted by islet transplantation. Although interleukin-6 shows also an elevation in diabetes and reversal after islet transplantation, these changes are not significant.

P3-29. Isolation and Culture Affect the Phenotypic Expression of Glycoconjugates on Islet Cells: Potential Implications on Primary Nonfunction Vieiro M., Hyon S.H., Hidalgo A., Ceballos C., Argibay P. (Surgery Department, Hospital Italiano de Buenos Aires, Argentina)

Background: Transplantation of pancreatic islets has a disappointingly higher rate of primary nonfunction compared to pancreas transplantation. Enzymatic processing of the pancreas for islet isolation, among other factors, may result in the modification of exposed glycoconjugates. Since these molecules may play a role in cell-to-cell interactions and subsequent immunomodulatory reactions, it was our objective to study sialylated and nonsialylated terminal modifications on islet cells upon enzymatic isolation. Methods: Maackia amurensis lectin II (MAL II) and Erythrina cristagalli lectins (ECL). which preferentially recognize alpha 2,3linked sialyl residues and galactosyl (beta-l,4) N-acetylglucosamine, respectively, were used as probes. Islets from C57BL/6 mice were isolated, purified, and cultured in standard medium. Plaques were sacrificed at 18, 24, and 48 hours, and at 3 and 6 days. Pancreatic specimens were used as controls for normal pattern of glycosylatiou. Incubation of ABC alone served as a non-specific negative control and incubation of lectin with the corresponding binding inhibitor served as a control for binding specificity. Results: In the pancreatic specimens, islets showed extensive cytoplasmic binding of both lectins. Immediately after the isolation process, intensity was moderate. At different culture timepoints, ECL showed no modification of its binding pattern while MAL II evidenced a reduction in the intensity of cytoplasmic staining (ranging between 10-50%). Conclusions: These preliminary findings suggest that isolation and culture of pancreatic islets may affect the glycosylation patterns of these cells. This approach could provide an alternative way to investigate mechanisms related with the initial poor function of transplanted islets.

P3-30. Targeting CD45RB Prevents Diabetes in NOD Mice through CTLA-4 Signaling Ariyan C.E., Salvalaggio P.R.O., Fecteau S., Deng S., Rothstein D.M., Basadonna G.P. (Department of Surgery, Organ Transplantation, Yale University, New Haven, USA)

Background: Anti-CD45RB mAb treatment prevents diabetes in >85% of NOD mice if given in the first week of life. We now examine the mechanism of anti-CD45RB mAb activity in preventing diabetic autoimmune response in this model. Methods: NOD Mice were treated with anti-CD45RB mAb (100 ug/IP) on days 0,5,10 starting at 1 week of age. Untreated littermates served as controls. Lymphocytes were analyzed by FACs and pancreata were analyzed by immunohistochemistry. In T cells, anti-

Acta Chir. A u s t r i a c a . Vol. 33 • S u p p l e m e n t No 174 • 2001

CD45RB mAb treatment resulted in 56% increase of lower Mr CD45RB isoform expression and 55% increase of CTLA-4 over controls. Activation markers were unchanged. Results: Imrnunohistocbemistry of pancreata at 12 weeks of age revealed: grade II/III insulitis with invasion by CD4+ and CD8+ cells in untreated animals (n = 2) vs. an absence of infiltrate in treated animals (n = 3). The protective effect of anti-CD45RB (DM incidence 15% treated vs. 75%untreated) was lost when combined with CTLA4-Ig (80% DM). This suggests that B7 may be required for engagement of CTLA-4, which is partially disrupted by administration of CTLA-4 Ig. Adoptive transfer of splenocytes from antiCD45RB mAb treated nondiabetic adult NODs into nafve irradiated NODs, resulted in 100% prolongation of disease free interval (17.2 vs. 8.67 weeks), when compared with transferring splenocytes from untreated animals. Conclusions: 3 doses of anti-CD45RB mAb prevent DM in the NOD mouse and cause a shift in CD45RB expression to the lower Mr isoforms. Anti-CD45RB mAb, treatment is associated with upregulation of CTLA4 on CD4+ T cells. In addition, adoptive transfer of anti-CD45RB mAb treated cells dramatically prolongs the disease free interval. Whether anti-CD45RB mAb treatment causes the onset of regulatory cells is currently under investigation.

P3-31. Prolonged Islet AIIograft Survival by Targeting CD45rb and CD154 in NOD Mice Molano R.D., Berney T., Pileggi A., Ricordi C., Rothstein D.M., Basadonna G., lnverardi L. (School of Medicine, Diabetes Research Institute, University of Miami. USA)

Background: Therapies targeting CD45RB, a transmembrane protein involved in regulation of lymphocyte activation signals, or CD154 (CD40L), a mediator of T-cell costimulation, have been effective in delaying islet allograft rejection in several models. In this study, we assessed the effect of the combination of anti-CD45RB and anti-CD154 monoclonal antibody on islet allograft survival in spontaneously diabetic NOD mice, where in addition to allorejection, autoimmunity contributes to islet destruction. Methods: C57BL/6 mice islets were transplanted into spontaneously diabetic NOD mice. Therapy in group 1 (n = 5) consisted of 100 btg anti-CD45RB mAb (MB23G2) IP and 500 ~tg anti-CD154 (MRI) IP on days -1, 0, and 5. Group 2 (n = 5), received the same induction treatment and, in addition, anti-CD45RB mAb therapy was repeated every 15 days until day 50. In group 3 (n = 5) antiCD45RB mAb therapy was maintained until rejection or until day 100, while CD154 was administered as in the other groups. Three control groups received either anti-CD45RB alone (group 4, n = 5), anti-CD154 alone (group 5, n = 5) or no treatment (group 6, n = 8). Results: Median graft survival was 47 (group 1), 63 (group 2), 67 (group 3), 17 (group 4), 18 (group 5) and 10 days (group 6). Combinatorial therapy (groups 1, 2, and 3) significantly prolonged graft survival as compared to monotherapy or control groups (groups 4, 5 and 6) (Mantel-Cox p<0.05). Conclusions: These results suggest that targeting of signals 1 and 2 of T cell activation improves islet graft survival in a model where both allorejection and autoimmunity contribute to islet graft loss.

P3-32. Reduction of MHC Class I and II on Rat Pancreatic Islets Surface by During Incubation with Calcitriol Kinasiewicz A., Juszczak M., Michalska W., Korczak-Kowalska G., Pachecka J., Mazurek A.P.., Fiedor P. (Department of General and Transplantation Surgery, Transplantation Institute, Medical University of Warsaw, Poland)

Background: Calcitriol is well known as a final active product of vitamin D that regulates calcium and phosphorus homeostasis. In the past few years calcitriol was indicated as a regulator of imuune system. New analogs of vitamin D are devoid of calcium effect and with increased cell differentiation properties. The aim of our study to evaluate immune response of isolated pancreatic islets treated with calcitriol. Methods: Pancreatic islets were isolated from WAG rats using collagenase digestion of the pancreas and Ficoll separation. Islets were incubated with calcitriol in concentration 10-s and 10-7 raM. MHC class I and II levels on the islet surface was determined by direct immunofluorescence using the monoclonal antibody (OH-6, OX-18) with FACS procedure. In each group endocrine function of

69

isolated islets was assessed by glucose challenge test with 1.67, 16.7, 1.67 mM glucose concentrations. Insulin concentration in medium was measured using radio-immuno assay (RIA). Results: Our data showed that calcitiol reduces islet cell MHC class 1 and class 1I expression. Calcitriol concentration of 10-7 mM reduces stroger MHC class I than concentration of 10-8 mM (54,4 _+ 0,75% vs. 44.4 +_ 1,1%, p<0.05). The differences between both concentrations in MHC class II expression is comparable, but much lower then control values (control [12,6 +_ 0,87%]; vs. 10-8 mM [4,6 + 1,2%], p<0.01; vs. 10-7 mM [3,6 + 0,9%], p<001. Islets exposed to calcitriol presented better insulin secretion profile and stimulation index. Conclusions: The active form of vitamin D, 1,25 (OH) 2D3, is an immunomodulator that interacts with T cells but mainly targets antigen-presenting cells. In our experiments, we revealed that islets incubation with calcitriol influence MHC Class I and II expression in in vitro study.

P3-33. Factors Influencing Islet Allograft Success in Multiple Islet AIIograft Situations Gaur L.K., Nitta E, Stone S.M., Lernmark A., Nelson K.A., Allen M., Nepom G.T. (Puget Sound Blood Center, Seattle, USA)

Background: A major task of diabetes research consists of developing new forms of treatment to delay or prevent the development of chronic complication associated with the disease. Recently the Edmonton group reported 100% cure of type l diabetes after two or more islet transplants. We addressed issues such as islet mass, tolerance induction and immunosuppression that could potentially be the contributing factors in our macaque islet transplant model. Methods: Nonhuman primate model will provide the best surrogate study for these human trials. We have established a diabetic macaque model with streptozotocin induced beta cell destruction. The insulin dependent diabetic animals received islet allografts in the liver with or without peripheral injections of stem cell fractions and no immunosuppression. If correction in blood glucose levels was not observed within two months the macaques received second transplants with tapering doses of MMF. Results: All seven animals that received the second transplant have shown initial raise in C-peptide levels, regardless of the kind of pretreatment they have received in the first transplantation. Since graft success did not correlate with number of transplanted islets, the correction of blood glucose levels toward normoglycemia after the second transplantation is suggestive of a mechanism that the allo-tolerance to second transplant is facilitated by the first islet transplantation. Conclusions: These initial observations suggest approaches to to[erize the recipient to accept the "second transplant-islets" a) through pre conditioning the animal to improve the rate of success for the first transplant or b) through tolerization to islets in first transplant to facilitate better engraftment of the "second transplant-islets'.

P3-34. Improved Survival of AIIogeneic Islets in Rats PreTreated with Bone Marrow Transplantation under Temporary Tacrolimus Therapy Kriz J., Girman P., Saudek F. (Institute for Clinical and Experimental Medicine, Prague, Czech Republic)

Background: Establishment of stable chimerism of hematopoetic blood cells may confer permanent specific transplantation tolerance for solid organ and cellular grafts. The aim of the present study was to investigate a nonmyeloablative and anti-T-cell therapy-free protocol to induce allogeneic islet tolerance using pretransplant donorspecific bone marrow infusion in combination with only a temporary immunosuppressive regimen. Methods: Male Lewis-Brown Norway and female Brown Norway rats were used as donors and recipients, respectively. In Group 1, 13 diabetic animals were treated by islet transplantation only. In Group 2, 14 animals underwent islet transplantation after previous 45-day immunosuppressive therapy (tacrolimus 0.5 mg/kg im. + hydrocortisone 2 mg/kg im.) that was stopped 5 days after islet transplantation. Recipients in Group 3 (n = 26) were treated as Group 2 and, in addition, underwent transplantation of 100 million mononuclear marrow cells I0 days after initiation of the immunosuppressive therapy. Microchimerism was documented using PCR detection

70

Acta Chir. Austriaca • Vol. 33 • SuppLement No 174. 2001

of the male SRY locus in female recipients. MHC mismatch and its ability to induce cross lymphocyte activation were tested by mixed lymphocyte cultures (MLC). Results: In Group 1, 12 grafts were rejected by 21 days. Positive MLC were detected only in 2 of 8 animals {in third-party animals MLCs were positive in 75%). In Groups 2 and 3, cumulative 120day islet survival rates were 69 and 92%. respectively (p = 0.045) and MLCs were uniformly negative. C o n c l u s i o n s : Induction of mixed lymphocyte chimerism using bone marrow transplantation prior to islet transplantation improved allogeneic islet survival. Examination of MLC failed to predict islet rejection.

P3-35. Role of the Thymus in Controlling CD45 Isoform Expression and CTLA-4 Regulation Following AntiCD45RD mAb Treatment Sah'alaggio P.R.O., Arivan C.E., Deng S., Rothstein D.M., Basadonna G.P. (Department of Surge U. Yale School of Medicine, New Haven, USA) B a c k g r o u n d : Targeting CD45RB leads to long-term graft survival and donor specific tolerance in islet atlografts. This is associated with a shift in CD45RB isoform expression and upregulation of CTLA-4 on CD4+ cells. In this study we analyze the timing of these events and the role of the thymus on both CD45RB isoform and CTLA-4 expression. M e t h o d s : BALB/c mice (6-12 group) were treated with antiCD45RB mAb (100 ug IV x 3 doses). A second group of mice underwent thymectomy prior to anti-CD45RB treatment. Lymphocytes from spleen and periphera blood were analyzed by multi-color flow cytometry. Results: Anti-CD45RB treatment causes CD45RB isotbrm expression to shift from CD45RBHi to CD45RBLo on CD4+ cells (2%Hi/98%Lo vs. 48%Hi/52%Lo in untreated mice) and doubles CTLA-4 intracellular expression (18% vs. 9% in untreated mice). These events peak 10 days after treatment and return to pre-treatment values (60%Hi/40%Lo vs.,62%Hi/38%Lo in untreated mice) by day 18. Anti-CD45RB mAb re-treatment at day 18 causes CD45RB isoform to shift again (23%Hi/77%Lo), but it does not upregulate CTLA-4 once more (11% vs. 9% in untreated mice). In treated animals, thymectomy (vs. no thymectomy) delays CD45RB isoforms from shifting back to pre-treatment values by day 18, maintaining a larger population of CD45RBLo cells in treated animals (55% vs. 42% in untreated mice) until day 21. Similarly, thymectomy upholds CTLA-4 upregulation by day 18 (17% vs. 8% in untreated mice). C o n c l u s i o n s : Anti-CD45RB mAb treatment induces long-term islet graft survival by upregulating low Mr isoforms and CTLA-4 expression. These events are temporary and quickly reverse back to pre-treatment values. They are highly regulated by the thymus: thymectomy upholds the expression of CD45RBLo isoforms and CTLA-4 upregulation.

P3-36. Induction of Tolerance to AIIotransplanted Pancreatic Islets by Nonmyeloablative Preconditioning of Recipients and UV-B Irradiated AIIogeneic Bone Marrow Transplantation Socha-Urbanek K., Socha M.W., Fiedor P.. Kwasny M. (Department Clinical Pathology, Military Medical University, Warsaw, Poland) B a c k g r o u n d : Stable bone marrow (BM) chimeras can permanently

accept donor-derived allogeneic pancreatic islets. Transplantation of mismatched BM necessitates myeloablative conditioning of the re-

cipient and T-cell depletion of donor BM to induce tolerance to allotransplants and prevent GVHD. Methods: In this study we evaluated the possibility of induction of tolerance to allogeneic islets grafts via nonmyeloablative treatment of recipients and transplantation of UVB-irradiated donor BM on rat model (WAG to Lewis). Diabetes was induced in recipients by streptozotocine injection (70 mg/kg). Myeloablation was performed by treating recipients with Busulfan (30 mg/kg) and Cyclophosphamide-CY (60 mg/kg) while selective cytoreduction of specifically activated donor-reactive host cells was obtained by donor splenocytes and blood transfusion followed by high-dose CY (200 mg/kg) treatment. Afterwards UVB-irradiated intravenous allogeneic BMT (4x108 cells) and allogeneic pancreatic islets transplantation under the kidney capsule were performed. Evaluation of acquired tolerance was confirmed by graftectomy and recurrence of diabetes (glicemia > 250 mg%) and immunohistochemical analysis. Results: Transplantation of UVB-irradiated donor BM into allogeneic recipients preconditioned with myeloablative regimen resuited in stable chimerism - recipients accepted donor-type islets grafts permanently (>100 days). Immunomodulation of recipients with CY and UVB-irradiated BMT also resulted in significant prolongation of islets allografts survival. Conclusions: These observations indicate that selective cytoreduction by CY and UVB-irradiated BMT converted recipients into stable chimeras that permanently accepted donor-type pancreatic islets grafts. Our results demonstrate that combination of selective reduction of donor-reactive host cells by CY together with donor BM T-cells depletion by UVB irradiation is a promising approach to induce tolerance to allotransplanted islets of Langerhans.

P3-37. Induction of Tolerance to Pancreatic Islet Transplantation with Different Hepatic Cell Subpopulations, in Wistar Rats, without Immunosuppressive Drugs Jara A., Soto M.L., De/Rio R., Zugasti A., Mah,etti V. (Unidad de Medicina Y Cirugia Experimental, Servicio de Endocrinologia, Hospital General Universitario "Gregorio Maranon", Madrid, Spain) B a c k g r o u n d : Looking for tolerance to pancreatic islets without us-

ing immunosuppressive drugs, we have tried to inject different hepatic cells subpopulations to search which is the best form to generate tolerance for these islets. M e t h o d s : Diabetes was induced by streptozotocine and confirmed after 3 blood glucose measures of 350 mg/dl. Islets were isolated, purified and cultured for 3--4 days. Fresh hepatic cells and hepatocytes were isolated after double perfusion . Fibroblasts were obtained after 1 month of culture. Non singeneic Wistar rats were used as donors and recipients. Three groups of Wistar rats have been studied: A) Co-Tx with a pool of hepatic cells (N = 8); B) CoTx with only (pure) hepatocytes (N = 8) and C) Co-Tx with liver fibroblasts (N = 6). Different hepatic cell subpopulations, with a ratio of 100 cells per islet (100:I), were injected via porta vein and after a period of 15 min islets were injected by the same vein. No immunosuppressive drugs have been employed. The average of islet number (IE) transplanted was 1533.2 _+ 111.31 in group A, 1625 +_ 53.2 in group B and 1717.4 _ 209.2 in group C; with purity of 90%. Blood glucose was measured during 30 days. SPSS have been applied for statistical study. Results: Reversion of diabetes (< 150 mg/dl) was observed among the three groups with some differences, for the first five-six days after Co-Tx and finally reached with high levels. 3 rats of group A showed euglycemia for more than 30 days, and one rat 18 days; while none of the group B was euglycemic for a period longer than 11 days. One rat in group C was euglycemic for 19 days (see table below).

0

1

2

3

4

5

6

7

9

16

23

30

MFB P/P

412.88 53,32

194,75 79,27

146,63 94,10

136,88 65,41

124,75 38,49

187,13 90,77

251,38 122,72

312.25 134,59

330,25 127,46

380,63 110,83

360,00 81,53

397,57 73,12

Hp p/p 100

431,00 63.68

256,60 97,69

204.60 70,57

159.80 45,75

121,60 33,25

113,20 32,59

123,00 44,64

128,80 48,70

318,80 39,47

428.20 38,29

422,20 51.55

421.40 37,55

Hc P/P 100:t

435,90 58,52

388,30 115,40

316,40 132,99

278,50 125,41

181,20 122,53

238,10 127,99

295,10 156,58

318,00 154,60

334,00 166,08

348.50 189,86

345,90 178,22

368,60 169,32

Acta Chir. Austriaca - Vol. 33 • Supulement No 174 • 2001

Conclusions: The results conlirm
P3-38. Islet Re-Transplantation after Co-Transplanation of Islets and Hepatocytes, in Rats, without Immunosuppressive Drugs Soto M.L., De/Rio R., Zugasti A., Ma£erti V, Jara A. (Unidad De Medicina Y Cirugia Experimental. Servicio De Endocrinologia, Hospital General Universitario "'Gregorio Maranon", Madrid, Spain) Background: We have observed induction of tolerance when hepatic cells are transplanted before islets with an interval of t5 minutes. Our aim was to demonstrate if a new islet re-transplantation (Re-tx), without using hepatic cells, could revert diabetes in those cases of relapsing diabetes after co-transplantation (Co-tx). Methods: Diabetes was induced bx streptozotocine and confirmed after 3 blood glucose measures of 350 mg/dl. Islets were isolated, purified and cultured for 3-4 days. Fresh hepatocytes were isolated after double perfusion. Non singeneic Wistar rats were used as donors and recipients. Seven Wistar rats were transplanted. Five of them received a ratio of 200 hepatocytes per islet (200:1 ) and the other two rats a ratio 300: 1. In all cases, hepatocytes were injected via cava inferior vein and after a period of 15 min islets were injected by porta vein. No immunosuppressive drugs were employed. After a short period of time, diabetes relapsed and then, we performed islet re-transplantation. The axerage of islet number (IE) transplanted was 1741 _+ 372 in the Co-tx and 1995 + 157 in the retx: with purity of 90%. Blood glucose ~as measured during 30 days after Co-Tx and Re-tx in all groups. The statistical analysis was done using SPSS software. Results: In four cases, a period of 12, 15, 12 and 30 days of euglycemia (< 150 mg/dl) after re-transplantation was obtained, without the use of immunasuppressive drugs. Conclusions: Results show a possible relation between the period o f time when the different transplants are performed and the tolerance's period generate by hepatoc3tes. So, in those Re-tx performed before 15 days from the first Co-tx, there are a positive response in contrast with the other Re-tx performed after 15 days. Moreover, the days of hyperglycaemia before Re-tx are also influential in the success of the transplant, having the best results with <5 days with hyperglycaemia.

P3-39. Free Fatty Acid-Induced Apoptosis of Isolated Human Pancreatic Islet Cells Lupi R., Marselli L., Dotta F., Del Guerra S., Santangelo C., Boggi U., Mosca F., Del Prato S., Marcherti P. (Department Endocrinology and Metabolism, University of Pisa. Italy) Background:. Experiments in rodents have shown that prolonged exposure of pancreatic islets to high concentrations of free fatty acids (FFA) causes reduced islet cell survival (lipotoxicity). We evaluated the phenomenon of lipotoxicity in isolated human islets (HI), and investigated some of the possible mechanisms. Methods: For the purpose of this study. HI were prepared by collagenase digestion and density gradient purification and then incubated for 24h with 1.0 mmol/1 FFA (oleate-to-palmitate, 2-to-l). Results: Compared to control islets. FFA-exposed cells exhibitied a significant increase of the amount of dead cells (see table), and electron microscopy showed the involvement o f betacells, with morphological appearance compatible with the presence o f apoptotic phenomena.

Controls

.-Mnountof dead cells FFA..exposed

TUNEL (%)

t2.3 ± 3.0

44.0 ± 5.0*

ELISA (OD)

0.6 ± 0.2

3.3 ± 0.5"

* p < 0.01 vs. Controls

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FFA-induced islet cell death was completely prevented by inhibition of up-stream caspases. RT-PCR studies revealed no major change of iNOS and Bax mRNA expression, and a marked decrease of Bcl-2 mRNA expression in the islets cultured with FFA. Conclusions: Thus, prolonged exposure to FFA has caspase-mediated, pro-apoptotic effects on human pancreatic beta-cells: these alterations are accompanied by no major change of iNOS and Bax mRNA expression, and decreased Bcl-2 expression.

P3-40. Peripheral Benzodiazepine Receptors Activation Has Cytostatic and Cytotoxic Effects on Human Pancreatic Islets Marselli L., Dotta F., Martini C., Santangelo C., Lupi R., De/ Guerra S., Boggi U., Mosca E, Del Prato S., Marchetti P. (Departmeat of Endocrinology and Metabolism, University o f Pisa, Italy) Back.ground: The peripheral benzodiazepine receptors (PBRs) are proteins located on the mitochondrial membrane where they take place in the formation of the rnitochondrial permeability transition (PT) pore. PI" pore regulates the function and integrity of mitochondria, that, in turn, have a crucial roIe on cell function and survival. Methods: We have previously shown the presence of PBRs in human pancreatic islets (HI). Hereby we describe the effects of a prolonged (12 h) exposure of purified HI to PBRs ligands (PK11195, PK, and Ro 5-4864, Ro, which have overlapping but not identical binding domains, and with the latter having a slightly higher binding affinity). Results: As shown in the table, glucose-stimulated insulin release was significantly lower after exposure to PK 0.5 gM and 1.0 ttM, than after exposure to Ro 1.0 gM, Ro 10 gM, control medium or clonazepam (CI) 600 ttM, a specific central benzodiazepine receptor ligand. Insulin Release (~.U/ml/islet/min)

Controls (n = 14)

3.3 mM Glucose (mean ± SD)

I6.7 mM Glucose (mean+ SD)

0.08 ± 0.03

0.19 ± 0.09

PK 0.5 p.M (n = I2)

0.07 ± 0.01

0.09 ± 0.02*

PK 1.0 p.M (n = 10)

0.03 -0.01#

0.07 ± 0.03*

Ro 1.0 I.tM(n = I0)

0.08 ± 0.03

0.24 ± 0.07

Ro 10.0 gM (n = 10)

0.09 ± 0.04

0.25 ± 0.07

Cl 600 ttM (n = 6)

0.10 ± 0.02

0.19 ± 0.07

# significantlylowerthan the other values at 3.3 mM glucose;significantlylower than the other ~alues at 16.7 mM glucose The amount of dead cells was evaluated by the TUNEL technique (Tt) and an ELISA method, and was significantly higher in 1.0 p.M PK-exposed HI (Tt: 42.3 + 7.9%, n = 5; OD: 2.2 _+ 0.5, n = 6) than in 10 ~M Ro-exposed (Tt: 11.1 _+ 4.1%, n = 4; OD: 1.24 +_ 0.2, n = 4) and control (Tt: 14.3 _+ 6.6%, n = 8; OD: 1.27 +_ 0.4, n = 8) HI. Electron microscopy demonstrated typical apoptotic changes (cellular shrinkage, chromatin condensation and apoptotic bodies) in PK-exposed human beta-cells. These effects were accompanied by no major change of mRNA expression of iNOS, Bax and Bcl-2, as evaluated by RT-PCR. Inhibition o f up-stream caspases, caspase3 or caspase-6 significantly reduced the amount of dead cells. Conclusions: Thus, specific and selective binding to PBRs causes human beta-cells functional damage and apoptosis, a phenomenon which occurs without any clear change of iNOS, Bax and Bcl-2 mRNA expression, and involves caspase activation. This suggests o f a possible role o f PBRs in regulating human n-cell function and survival.

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P3-41. Heme Oxygenase-1 Upregulation-Mediated Improvement of Islet Graft Function: The Role of Inducible Nitric Oxide Synthase Pileggi A., Berney T., Molano R.D., Oliver R., Pastori R.L., Bach F.H., Ricordi C., lnverardi L. (Diabetes Research Institute, School of Medicine, University of Miami, USA~

Background: Local microenvironment activation post-transplant results in loss of islet mass and function, mainly via production of cytokines, and reactive oxygen species ~ROS) including nitric-oxide- (NO), resulting in increased islet-mass needs. We reported on the beneficial effects of induction of the cytoprotective and antiapoptotic factor heme-oxygenase-I (l-IO-l) on islet graft pedbrmance. In this study the role of NO in HO- 1-mediated protection of islets was investigated. Methods: Transplantation of a sub-optimal islet mass into syngeneic chemically-induced-diabetic recipients allows to study the consequences of local inflammation on graft function, in the absence of allorecognition and autoimmunity. Time to normoglycemia (TNG) inversely correlates with transplanted islet mass. Manipulations of islets or microenvironment beneficial for graft function result in shorter TNG. Islets were incubated with iron- (Fe) -protoporphyrin (PP) to induce HO-1. Transplant combinations were: wild-type (WT) into WT; iNOS-KO into WT: iNOS-KO into iNOSKO. Recipients of FePP-induced islets received cobalt- (Co) -PP (20 mg/Kg b.w., IP) on days -1, 1, 3, 5. and 7 to induce systemically HO-I upregulation. Control animals received unmanipulated islets and vehicle injections. Results: Absence of iNOS resulted in shorter TNG in all combinations. TNG (days) was 48 + 30 in WT into WT, 25 __. 30 iNOSKO into WT, and 19 _ 21 in iNOS-KO into iNOS-KO. When HO1 induction treatment was used, further advantage was observed, TNG was 18 _ 28 in WT into WT, 12 +_ 16 iNOS-KO into WT, and 16 _ 7 in iNOS-KO into iNOS-KO. Conclusions: Our data suggests an important role for NO mediating early graft dysfunction, and that protection obtained by HO-I upregulation occurs via interfereoce with NO and other pathways.

A c t a Chir. A u s t r i a c a . Vol. 33 • S u p p l e m e n t N o 174 • 2001

P3-43. A Baboon Model of Type 1 Diabetes Kendall Jr. Vv~F., Co/lins B.H., Hobbs H.A., Bollinger R.R., Opara E.C. (Department of Surgery, Duke University Medical Center, Durham, USA)

Background: Humans and baboons do not readily develop diabetes after either subtotal pancreatectomy or streptozotocin administration alone. The purpose of the present study was to develop a reliable model of type ldiabetes in the baboon, thus providing a readily available model for islet transplantation studies. Methods: Subtotal pancreatectomy (85-90%) was performed in 3 female baboons and Streptozotocin (65 mg/kg) was administered intraoperatively via the celiac artery to the pancreatic remnant. To avoid hypoglycemic shock attributed to massive release of insulin due to streptozotocin induced beta-cell destruction, an infusion of 5% dextrose solution was provided during recovery. After one month of recuperation, several additional doses of streptozotocin were administered over a 3-month interval until the onset of diabetes. Results: Diabetes was diagnosed when non-fasting baseline blood sugar levels increased from 112.8 _+ 3.3 to 400 +_ 5.4 mg/dL (p<0.001), along with abnormal glucose tolerance during OGTT. The mean body weight decreased from a pre-operative level of t8.4 kg __. 1.6 to 12.2 kg _ 2.3 at the onset of diabetes. The postoperative baseline serum C-peptide level of 2.7 + 0.33 ng/mL decreased to 1.9 ng/ml __. 0.210 (p<0.05) when diabetes occurred. There was no evidence of restoration of secretory reserve as assessed by C-peptide levels during OGTT. The diabetic baboons were successfully treated with 70/30 recombinant human insulin. Contusions: We have developed a reliable model of type 1 diabetes in the baboon that may be utilized for islet cell transplant studies.

P3-44. Histological Findings in Chronic Pancreatitis (CP) Liu X., Foerster S., Adam U., Schmidt W., Hopt U.T. (Department of Surgery, University of Rostock, Germany)

Background: Pancreatic histology directly influences the outcome of

P3-42. Islet Cell Surface Mannose 6-PhosphateJInsulinLike Growth Factor II Receptor Binds and Internalizes Granzyme B Released from Cytotoxic T Lymphocytes Lord S.J., Mo~ka B., Gainer A., Bleacklev R.C., Korbutt G.S., Rajotte R.V. (Surgical-Medical Research Institution, University of Alberta, Edmonton, Canada)

Background: Mannose 6-phosphate/insulin-like growth factor II receptor (MPR) on the surface of Jurkat cells binds and internalizes granzyme B (grB) released from cytotoxic T lymphocytes (CTL) resuiting in apoptosis. We explored whether a similar mechanism occurs in pancreatic islets. Methods: Balb/c mouse islets were isolated and dissociated into single cells. Dissociated islets were then labelled with florescent markers for the detection of: 1) the cell surface cation independent (CI) -MPR, 2) grB binding to the CI-MPR. and 3) grB uptake into the islet cell. Labelled cells were analysed by quantitative flow cytometric analysis (FACS). Results: We have determined that islet cells express high levels of cell surface CI-MPR and that grB binding and internalization through this receptor does occur. The proportion of islet cells staining positive for CI-MPR was 64.5%, which was significantly higher than the negative control value (16.2%). Likewise, the proportion of islet cells staining for grB binding was determined to be 51.1% as compared to the negative control value (9.6%). The mean fluorescence intensity value for grB uptake into the cell was found to be 11.2 as compared to the negative control value (3.9). Conclusions: These results demonstrate that islet cell CI-MPR binds and internalizes grB. Future studies will be aimed at examining whether islet grafts are destroyed through this pathway as well as developing strategies for blocking this receptor interaction in hopes of preventing graft rejection.

islet isolation and clinical transplantation (Tx). Therefore, this study was aimed to investigate morphological changes of the islets in CP. Methods: 10 CP patients with secondary diabetes in insulin-dependent state (group 1), 6 CP patients with impaired glucose tolerance in non-insulin-dependent state (group 2), 10 CP patients with normal glucose tolerance (group 3). The pancreatic tissue were derived from surgical resection and evaluated by H&E staining and immunoenzymatic double staining for insulin and glucagon. The extent of parenchyma fibrosis was scored as mild (<30%), moderate (30-60%) and severe (>60%). Results: In group 1, all pancreatic tissue showed severe degree of fibrosis (>80%). Proliferating fibres invaded into the damaged islets. There were only little complete islets to be seen. Within the damaged islets, insulin positive cells were reduced and glucagon positive cells increased so that a-/[3-cell ratio was increased by >60%. In some damaged islets even only a-cells appeared. In group 2, the degree of fibrosis was between moderate and severe. Most islets were damaged. In one case, the a-/13-cell ratio was >1. In other 5 cases, some complete islets existed so that the a-/[3-ratio was <1. In group 3, the degree of fibrosis was between mild to moderate, a-/IS-cell ratio was <1. In addition, mononuclear cells infiltrated the islets in all three groups. Conclusions: There is only a little possibility of isolating complete islets from pancreata of CP patients with either insulin dependent diabetes or impaired glucose tolerance. The implication of transplantation of the proliferative a-cells in CP patients with secondary diabetes should be evaluated.

P3-45. Cytokine-lnduced Apoptosis of Human Islet BetaCells Is Associated with Cytochrome C Release from Mitochondria and Oxygen Free Radical Production Suarez-Pinzon W.L., Laker J.R.T., Rajotte R.V., St~'nadka K., Rabinovitch A. (Department ol( Medicine, University of Alberta, Edmonton, Canada) Background: The proinflammatory cytokines, interleukin-1 (IL-I)2, tumor necrosis factor (TNF)-K, and interferon (IFN)-3 are can-

A c t a Chir. A u s t r i a c a • Vol. 33 . S u p p l e m e n t N o 174 • 2001

didate mediators of pancreatic islet 2-cell death in autoimmune (type 1) diabetes, and apoptosis is the morphological form of cytokine-induced 2-cell death. The aim of this study was to examine further the mechanisms of cytokine-induced apoptosis of human islet 2-cells. Methods and Results: The cytokine combination of IL-I-2 (30 U/ml). TNF-K (103 U/ml), and IFN-3 (i03 U/ml) induced a tour-fold increase in release of cytochrome c. an electron transport hemeprotein. from mitochondria into the cytosol of human islet cells after only 4 to 8 h of incubation. Cytochrome c release from mitochondria is known to result in increased mitochondriat production of the oxygen free radical, superoxide. Therefore. we measured cytokine-induced production of hydrogen peroxide (H202), a product of superoxide, and the level of glutathione (GSH). a major endogenous cellular antioxidant, and its oxidized form. GSSG. We found that cytokines significantly increased H,O,_ production and decreased the redox potential (GSH/GSSG) of human islet cells. Another effect of cytochrome c release from mitochondria is cellular apoptosis; and we found that cytokines significantly increased islet 2-cell apoptosis, measured by immunohistochemistry as an increase in annexin Vpositive insulin-staining islet cells (12 ± 1% with cytokines vs. 5 _+ 1% with control medium at 8 h of incubation). Conclusions: These findings suggest that cytokine-induced apoptosis of human islet 2-cells is a rapid event that follows cytochrome c release from mitochondria and oxygen free radical production.

73

Methods: In male BB/Wor DR and DP rats, iNOS and NF-K were localized by immunoreactive staining. NF-~: binding activity was determined by EMSA. Rats received (I) NOX-700 (0.5 to 3 mg/ml in drinking water) from age 30 days or (2) 5 mg/ml of NOX-700 vs. CsA (2.5 mg/kg) from day 60 until IDDM or day 125. Results: Islets of recent-onset, hyperglycemic BBDP rats stained intensely for NF-~: and iNOS vs. normoglycemic BBDR littermate controls, iNOS and NF-~ were present in islets of prediabetic BBDP rats. NF-~; binding activity was increased by day 40-45 but not day 30 in pancreas of hyperglycemic BBDP vs. normoglycemic BBDR rats and in normoglycemic BBDP rats. NOX-700 from day 30 significantly reduced IDDM incidence (Untreated = 89%, NOX700 3 mg/ml = 40%) or prolonged time to IDDM onset with no effect on hyperglycemia, insulin concentration or insulitis. In treated normoglycemic BBDP rats, insulitis was significantly reduced. CsA or NOX-700 from day 60 significantly reduced IDDM vs. untreated BBDP rats (40% and 71% resp. vs. 89%). Conclusions: NF-K activation and iNOS gene expression occur prior to disease onset. Treatment with a NO scavenger delayed the onset and reduced the incidence of IDDM. These data suggest an important role for NF-~: and NO i n , cell destruction in IDDM.

P3-48. Cytokine Targets in Human and Murine Pancreatic Islet Cells

P3-46. Mechanisms of Pravastatin's Protective Effect on Islet Grafts

Riachy R., Vandewalle B., Belaich S., Kerr-Conte J., Lukowiak B., Gmvr V., Dubois M., Bouckenooghe T., Lefebvre J., Pattou F. (ERM 106 Laboratoire de Culture Cellulaire, Facuhe de Medecine 1, Lille, France)

Arita S., Stein E., Smith C.V., Mullen E (Department of" Surgery, UCLA-VA Islet Transplant Program, Los Angeles, USA)

Background: The inflammatory cytokines, interleukin-lb (IL-lb),

Background: Macrophages and lymphocytes play an important role in both primary nonfunction (PNF) and rejection of transplanted islets. We have shown in mice and dogs that pravastatin treatment reduces the incidence of PNF and graft rejection. To better understand the mechanisms of this effect, we examined macrophage and lymphocyte properties of pravastatin-treated mice. Methods: Macrophages were obtained as peritoneal exudates cells (PEC) from BALB/c mice injected intraperitoneally with thioglycollate and treated with either 40 mg/kg/day pravastatin or vehicle from day -1 until PEC harvest. PEC were cultured at 2x106 cells/ml. PEC were activated by combined IFN3 and LPS to measure cytokine production. Splenocyte mitogenic responses were tested with PHA. Results: Fewer PEC were harvested from pravastatin-treated mice than controls (2.53 ± 0.21x107 vs. 4.08 _ 0.25 x l 0 7, p-<0.001), but relative numbers of the different cell types were unaffected (93.8 + 3.4% vs. 92.2 ± 2.7% macrophages, experimentals vs. controls). PEC adherence to culture plates (MTT assay) was reduced in experimentals (57.8 _-+4.0% vs. 83.6 -'- 2.1%, p<0.01). IL-t~- and TNFct production were both reduced with PEC from pravastatintreated mice: IL-113, 11.8 _ 2.4 vs. 29.6 ± 5.5 at 18 hrs and 24.5 _.+3.9 vs. 53.1 _ 4.0 at 24 hrs, both p_<0.05; TNFc~, 2825 _+ 188 vs. 3987 ± 226 at 18 hrs and 2350 _ 389 vs. 3613 ± 422 at 24 hrs, p-< 0.05 at 18 hrs only. Mitogen-stimulated proliferation of splenocytes was also reduced, but not significantly: SI's of 28.3 ± 10.5 and 62.4 ± 15.1. Conclusions: Pravastatin treatment reduced migration of macrophages into the peritoneal cavity and also their adherent properties, possibly reflecting decreased maturation/activation. Reduced inflammatory activity was also indicated by their lower cytokine production.

P3-47. Pancreatic NF-K Activation, iNOS Gene Expression and Impact of NO Scavenging in IDDM

tumor necrosis factor-a (TNFa) and interferon-g (IFNg), acting individually or more potently in combination, are cytotoxic in vitro to human islet beta cells by stimulating cytotoxic mediators. Simultaneously, they induce potentially important defense and repair responses. In this study, we specifically investigated the effect of cytokines on inflammatory status, oxidative stress and death induction in human pancreatic islets and murine beta cell lines. Methods: Human pancreatic islets, isolated from heart-beating donors, were treated with the combination IL-lb (50 IU/ml), TNFa and IFNg (both at 1000 IU/ml), and compared with untreated control cells. Results: As expected, metabolic activity, assessed by insulin content was decreased by 40% after 48 h of culture. Alteration in the inflammatory status was demonstrated by a significant 2-3 fold increase of IL-6 production and a 6-7 fold increase of class I and II MHC molecules. The oxidative stress was concurrently ascertained by the significant 2-3 fold enhancement of both oxygen free radical scavenger manganese superoxide dismutase (MnSOD) and nitrite release in the culture medium, a reflection of nitric oxide (NO) synthesis. The regulatory mechanisms underlying cell death were studied in murine beta cell lines. Cytokines induced an early and clear enhancement of mitochondrial membrane permeabilization assessed by FACS using cationic redistribution dyes and a 3-4 fold stimulation of the proapoptotic enzyme caspase 3. Modalities of cell death studied by FACS using two dyes that allowed us to rapidly identify and quantify normal, apoptotic and necrotic ceils based on their cytoplasmic membrane permeability, demonstrated a clear enhancement of membrane permeability during cytokine treatment. Conclusions: Our results contribute to highlight different mechanisms implicated in beta cell death and may help develop strategies favoring beta cell survival in the early stage of the disease where there is a negative balance between beta cell damage and repair.

P3-49. Rapid Recovery of Mouse Pancreatic Beta Cell in Neonatal Pig Pancreatic Ceil Cluster Xenotransplantation

Roza A.M., Pieper G.M., Adams M.B., Lai C.S. (Transplant Surgery, Medical College of Wisconsin, Milwaukee, USA)

Hsu B.R-S., Juang J.H., Fu S.H., Kuo C.H., Lee W.C. (Division of Endocrinology and Metabolism, Chang-Gung Memorial Hospital, Tao-Yuan Hsien, Taiwan)

Background: iNOS derived nitric oxide (NO) is believed involved

Background: Using pig to mouse model to study the effect of var-

in ,-cell destruction. Transcription factor, NF-K, regulates iNOS gene expression. We examined NF-K and iNOS in BB rats and the effect of NOX-700, a NO scavenger, on IDDM.

ious combinations of hCTLA4Ig, mlL-lra and Nordihydroguaiaretic acid (NDGA) on graft survival of neonatal pig pancreatic cell clusters (NPCC).

74

Methods: After 14 days of alloxan injection, diabetic BALB/c mice were transplanted with 2000 NPCC underneath left kidney capsule. A replication defective adenovirus carrying either hCTLA41g or mlL-lra was used to infect animals and/or NPCC. NDGA was injected daily. Blood glucose (BG) and body weight were measured twice a week. Insulin contents of kidneys and pancreas were determined. Grafts were evaluated using HE and immune staining. Results: When both the recipient and graft were expressing CTLA4Ig, the BG decreased significantl? (Groups A and B). Mice with (Gr A) and without (Gr B) graft expressing IL-lra were 348 _+ 12 and 359 +_ 15. 211 _ 32 and 172 __. 31, 227 _+ 28 and 296 _ 45. 227 _+ 30 and 353 _ 39, 238 _+ 3 and 427 _ 41 mg/dl at pre-. 1 . 2 . 3 and 4 weeks post-transplantation, respectively. Mice in the control group (Gr C, received nothing) had all BG>380 mg/dl. Pancreatic insulin content was in a good correlation with days of normoglycemia (PIC in £gg = 1.55+0.86X. R2 = 0.67). Insulin content in left kidneys was higher than in right thought not significantly (0.54 _.+0.09 vs. 0.38 _+ 0.03£gg, n = 11. p = 0.1). Histological examination revealed detectable NPCC tissues in the graft from groups A and B but not C. Conclusions: In NPCC xenotransptantation, the combination of cytokine and macrophage inhibitors and single costimulatory blockade preserves more grafts. Moreover, early conversion of normoglycemia is accompanied by rapid recovery of mouse pancreatic beta cells.

P3-50. Successful Reduction of Islet Numbers for Transplantation Using in Vitro Proliferating Technique Kiln B.-J., Kim L-S., Ahn K.-J., Choi S.-Y.. Jung D.-J., Kwak B.-K., Woo Y.-C., Cho H.-P., Do S.-G., Yoon T.-W. (Division of Endocrinology/Department of Internal Medicine, Eulji University School of Medicine, Seoul, Korea)

Background: Islet transplantation has been an attractive possibility for diabetes treatment. Islet cell tJroliferation has been pursued experimentally in an effort to success islet transplantation with small islet numbers. We had reported new technique for islet cell proliferation from isolated fresh islet using various growth factors (Yoon's method). We carried out a canine islet autotransplantation, and compared the efficiency of proliferated islets with non-proliferated fresh islets. Methods: Total pancreatectomy was performed in beagle dogs. Islets were isolated by intraductal collagenase perfusion. Purified islets were proliferated by using Yoon's method. After 5-day islet proliferation, autotransplantion was performed to the liver under the ultrasound guided. Three groups were studied: Group I (control group, 5,000 non-proliferated fresh islets/kg, n = 2), Group II (3,000 proliferated islets/kg, n = 3) and Group III (1,500 proliferated islets/kg, n = 3). Before and after transplantations, IVGTT, LFT and CBC were performed. Results: Morphologically, 5-day proliferated islets were bigger in size and had larger numbers of islet cell than fresh islets. In all group, liver function (AST, ALT and ALK) and platelet count were slightly changed during peri-transplantation period, but these changes were recovered to normal within 1 week after transplantation. Insulin free euglycemia after islet transplantation was achieved in group II and I. Insulin free period was longer in group I than in group II. Conclusions: Islet proliferation using Yoon's method might reduce the islet number for transplantation. Efficiency of proliferated islets was at least twice than that of non-proliferated fresh islets. Long-term follow-up study for the transplanted animal should be performed.

P3-51. Proliferation-Regulated Genes in Human Beta-Cells Aita C.A.M., Krogh K., Lojudice F, Maria-Engler S.S., Oliveira E.M.C., Genzini T., Miranda M.P., Noronha I.L., Eliaschewitz F.G., Sogayar M.C. (Chemistry Institute, University of Sao Paulo, Brazil)

Background: Transplantation of human pancreatic islets has recently been shown to be a viable therapeutic alternative of Diabetes Mellitus, but insulin independence requires isolation and transplantion of islets obtained from 2-3 pancreas donors. An alter-

Acta Chir. A u s t r i a c a . Vol. 33 • S u p p l e m e n t No 174 • 2001

native, would be to promote beta cell proliferation in in vitro pretransplantation culture, to increase cellular availability for transplant. Methods: In order to identify genes related to the control of betacell mitogenesis, we are employing cDNA subtractive hybridization of human islets cultured in vitro under high (11.2 mM) or low (2.8 mM) glucose and of human insulinoma versus normal islets. Highly purified human islets were obtained at the Human Pancreatic Islets Unit located at the Chemistry Institute, University of $5_o Paulo, Brazil, using LiberaseTM digestion of donor pancreas and Ficoll gradient purification. Total RNA was extracted from each sample, monitored for insulin transcripts by Northern blot analysis, using an insulin-specific probe and reverse-transcribed to construct cDNA libraries. Subtracted cDNAs were cloned into a pUC vector. PCR amplified and used to generate 96-spots cDNA macroarrays that were screened for regulated sequences by hybridization with radioactive cDNA probes made from unsubtracted libraries. Results: Preliminary screening revealed 3 cDNA clones whose expression is more abundant in normal islets than in insulinoma, but a number of subtracted cDNA clones are yet to be screened and sequenced, indicating that this is a very promising approach. Conclusions: Identification of genes regulating human beta-cell proliferation is an important step towards understanding of beta cell proliferation and mass beta cell production. Support: FAPESP, ICGEB

P3-52. Long-Term Culture of Human Pancreatic Beta Cells in Matrigel: Tri-Dimensional Structure Formation and Insulin Secretion Maria-Engler S.S., Corr~a-Giannella M.L.C.. Loli D., Genzini T., Corr~a T.C.S., Mortara R.A., Mares-Guia M., Noronha I.L., Eliaschewitz FG., Sogayar M.C. (lnstituto de Qufmica, Universidade de $5.o Paulo, Brazil)

Background: Successful human islet transplantation has recently been reported (Shapiro et al. New Engl. J. Med. 343, 2000). However, the scarcity of donors and of insulin producing tissue, still hampers the widespread use of this as therapeutic approach to diabetes mellitus.The extracellular matrix (ECM) is the most important component of the islet microenvironment and the most critical and poorly controlled factor during islet isolation, compromising islet yield and function. Methods: In order to mimic the original tissue tri-dimensional structure, we are culturing isolated human pancreatic islets in plastic and Matrigel (basement membrane components) for different time periods and subjecting this material to immunohistochemical staining and confocal microscopy to discriminate between insulinproducing beta cells and ductal cells. Results: The results indicate that in 12 days cultures, cells expand in a monolayer in plastic, but, when cultured onto Matrigel, islet-like structures are formed. Confocal analysis revealed that up to 12 days culture in Matrigel, insulin-producing cells are surrounded by ductal cells. We are currently investigating gene expression, by Northern blot, for insulin, glucagon and PDX-l-beta cell marker, at different time periods in culture, to better characterize these islet-like structures. Conclusions: This study could contribute to elucidate cell differentiation and gene expression triggered by extracellular matrix components in islet cells and provide the basis for human islet transplantion and tissue engineering. Supported by: FAPESP, ICGEB, CNPq and Biobr~is S/A.

P3-53. Macrophage Blockade Induced by Repeated Gadolinium Chloride Injections Saves Human Fetal Islet Xenografts in Rats Farkas G., Szdsz Zs., Ldzdr, Jr. G., Csanddi J., Ldzdr G. (Department of Surgery, Faculty of Medicine, University of Szeged, Hungary)

Background: The immunologic consequences of xenogenic islet transplantation remain unsolved. Gadolinium chloride (GdC13) depresses the reticuloendothelial activity, selectively blocking the function of the hepatic Kupffer cells. In this work, the effects of GdCl3-induced long-term Kupffer cell blockade on the survival of human fetal islet xenografts were investigated.

Acta Chir. Austriaca - Vol. 33 • Supplement No 174. 2001

Methods: Cultured fetal human islets (18 weeks old: 800 islets) were transplanted through the portal vein into the liver of male, inbred CFY rats with streptozotocin-induced diabetes. 24 hr before grafting and 2 and 4 weeks following islet transplantation, 8 diabetic rats were treated repeatedly with l rag/100 g body weight GdC13. while the control diabetic rats were untreated (n = 6). Results: In the control. GdC13-untreated transplanted rats, the decrease in blood glucose level was only transitory. In contrast, in the repeatedly GdC13-treated rats transplanted with human fetal islets, the normoglycemic condition remained for as long as 5 weeks during the observation period (blood glucose 5.98 _+ 1.78 mmol/l). Histologically, human fetal islets were seen in the liver of the rats treated with GdCI3. without any inflammatory cell reaction. No human fetal islets were observed in the liver of the diabetic control rats not treated with GdCI3. Conclusions: These studies suggest that the Kupffer cells play significant roles in the recognition of xenoantigens and in the induction of xenograft rejection. The long-term Kupffer cell blockade promotes successful long-lasting islet xenotransplantation.

P3-54. The Swine Islets with the Downregulated Antigenicity by N-Acetylglucosaminyltransferase III (GnT-II)

75

antibodies was used to show the distribution of alpha-gal antigen. human CD46, lgG and IgM, C3. C4. and C5b-9 (MAC) binding following exposure of mouse islets to human serum. Results: Alpha-gal KO mice: All cells were negative for alphagal in alpha-gal KO islets. Transplanted mouse alpha-gal KO islets into primate recipients (n = 3) were not protected from early destruction and histology confirmed destruction by necrosis. In vitro incubation in human serum resulted in binding of lgM and IgG with C3, C4 and C5b-9 deposition equal in intensity to controls. CD46 transgenic mice: Human CD46 expression was strong on the transgenic mouse islets when compared to pancreatic acinar tissue. Histological examination after transplantation into cynomotgus monkeys (n = 3) showed that early destruction was little changed in one but markedly reduced in the other two. Incubation in human serum resulted in tgG, IgM. C3 and C4 deposition equal in intensity to controls but C5b-9 deposition was reduced. Conclusions: Our findings suggest that the a-gal epitope does not play a significant role in xenorecognition of pancreatic islets. Preliminary observations with CD46 transgenic islets are encouraging and suggest that complement plays a more significant role in islet xenograft destruction.

Miyagawa S.M., Murakami H.M, Nakai R.N., Yamada M.Y., Murase A.M., Koyota S.K., Takahagi KT., Nagashima H.N., Taniguchi N.Z, Shirakura R.S. (Biomedical Research. Osaka University Graduate

P3-56. Characteristics and Transplantation of the Porcine Neonatal Pancreatic Cell Clusters Isolated from 1to 3-Day and 1-Month Pigs

School of Medicine, Japan)

Juang J.-H., Hsu BR.-S., Kuo C.-H., Fu S.-H., Lee W.-C., Yao N.-K.

Background: The increasing problem of the worldwide shortage of

(Chang Gung Memorial Hospital, Taoyuan, Taiwan)

donor organs has led to a revival interest in xenotransplantation. We have generated several lines of transgenic pigs with human b-Dmannoside b-l,4-N-acetylglucosaminyltransferase III (GnT-III) gene, and examined the effect of overexpression of GnT-III on the transgenic tissues, especially pancreas. Methods: Gene: A cDNA for human GnT-III was subcloned into the pCAGGS (b-actin promoter). Transgenic pig: Prepubertal crossbred gilts were used as embryo donors and recipients. Transgenic pigs were produced using microinjection methods described previously. I. The enzyme activities of GnT-III were assessed by HPLC. II. Immunohistochemical staining. Each tissue was stained with normal human serum (NHS), GS-IB4 lectin and anti-the Gal al,3 Gal-bt,4 GIcNAc-R (the a-GaI epitope) mAb (M86). Double staining of pancreatic islets with anti-insulin Ab and anti-GnT-III Ab was also carried. III. Flow cytometric analysis of endothelial cells (SEC) from transgenic pigs was done. Results: I. GnT-III activity in organs of control and transgenic pig. Control: Pancreas-l.2, Heart-0.6. Lung-3.9, Kidney-0.7, Liver0.1, SEC-not detected. Transgenic: Pancreas-551.2, Heart-146.1, Lung-609.4, Kidney-732.3, Liver-270.4, SEC-3121.5. (pmol/h/mgprotein) II. The antigenicity and a-Gal expression of all these tissues were clearly downregulated. Double staining of pancreas tissue also revealed the islets with high expressed GnT-III enzyme. III. FACS analysis of the endothelial cell. Control: 10%NHS-77.7, GSIB4-832.5, M86-15.7. Transgenic: 10%NHS-44.0, GS-IB4-574.4, M86-6.8. (mean shift) Conclusions: Expression of the GnT-III in transgenic pigs modifies the pancreatic tissue glycoantigen, especially the a-Gal epitope.

Background: We h)pothesize that the porcine neonatal pancreatic cell clusters (NPCCs) isolated from 1-month-old pig would be mature and could shorten the latent period between transplantation of these cells and the reversal of hyperglycemia. Methods: NPCCs were isolated from 1- to 3-day-old (I-A) and l-month-old (I-B) pigs and were cultured for 6 days for in vitro studies, including size. insulin content and insulin secretion. Three hundred NPCCs were transplanted under kidney capsule of nondiabetic nude mice. After transplantation, the grafts were removed and their insulin content and biVcell mass were measured. Results: Soon after isolation, I-B was larger than I-A (0.211 i 0 0.006 vs. 0.189 ± 0.003 mm 2, P = 0.0003). After 6-day culture, I-B contained more insulin than I-A (6.8 - 1.4 vs. 2.3 ± 0.2 mg/150 NPCCs. P = 0.02). However, the stimulation indices of I-A and I-B during static incubation with 500 mg/dl glucose (26.5 ± 3.2 vs. 23.9 _+ 1.7) or 500 mg/dl glucose plus 50 mol/l IBMX (62.2 ± 14.0 vs. 41.9 _+ 4.4) were not significantly different. (P>0.05) Furthermore, both I-A and I-B had no first or second phase of insulin secretion during sequential perifusion with 100 and 300 mg/dl glucose. The insulin content of the graft at 1 month after transplantation was 0.331 _ 0.031 and 0.333 ± 0.133 mg, and biVcell mass of the graft at 3 months was 0.069 _+ 0.022 and 0.067 _.+0.023 mg in recipients received I-A and I-B, respectively. (P>0.05) Conclusions: These data indicate NPCCs isolated from l- to 3day-old and 1-month-old pigs have different characteristics but similar effects on transplantation.

P3-55. Effect of Donor Alpha-gal Gene Knockout and Human CD46 Transgene Expression on Hyperacute Rejection of Pancreatic Islet Xenografts (Murine to Primate) Titus T., Badet L., McShane P., Chang L.W., Handa A., Gray D.W.R. (Nuffield Department of Surgery, John Radcliffe Hospital, University of Oxford, United Kingdom)

Background: We had previously reported C57B16 mouse islets transplanted under the renal subcapsular space of cynomolgus monkeys undergo destruction within 24 hours. Now,we report the effect of donor alpha-gal gene knockout and human CD46 transgene expression. Methods: Handpicked islets from alpha-gal knockout, CD46 transgenic or normal (C57B16) mice were separately transplanted under the renal capsule of cynomolgus monkeys. The islets were retrieved after 24 hours and processed for light and (in some cases) electron microscopy. Immunoperoxidase technique using specific

P3-57. Small Bowel Intramural Site for Xenogeneic Islet Transplantation in the Pig-To-Nonhuman Primate Model Wijkstrom M., Sageshima J., Kirchhof N., Pilon K.J., Kandaswamy R., Gilmore T., Eckman E., Wahlberg J., Sutherland D.E.R., Hering B.J. (Diabetes Institute for Immunology and Transplantation, Minneapolis, USA)

Background: Our laboratory has previously reported the successful use of the small bowel intramural (SBIM) site for islet isografts (rat) and allografts (pig). We have also shown immediate function of islet xenografts and prevention of cellular rejection in the pig-to-nonhuman primate model. This experiment was conducted to evaluate the SBIM site for islet xenotransplantation in rhesus monkeys (RM). Methods: 25,000 adult islet equivalents each were transplanted to the SBIM site in three streptozotocin-diabetic rhesus monkeys (4.1-6.2 kg). Transplants were performed by creating multiple subserosal or intramuscular tunnels within the proximal portion of the small bowel. Animals received antithymocyte globulin, daclizumab, tacrolimus, rapamycin and etanercept.

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Acta Chir, Austriaca • Vol. 33 • S u p p l e m e n t No 1 7 4 . 2001

Results: RMs were sacrificed on or before day 3 posttransplant due to functional small bowel obstruction. Ultrasound demonstrated distended intestines and stomach proximal to the transplantation site. collapsed bowel distally, but preserved intestinal motility. At necropsy, mild ascites was present and a laver of fibrin adhered the bowl segments at the SBIM site. The lumen proximal to the site was significantly dilated. Histology showed well-preserved islets within a severe intramural edema and areas of hemorrhage. Few neutrophils were seen. Conclusions: Although the SBIM site has advantages for many reasons, it cannot be transferred to nonhuman primates without significant adjustments.

P3-58. Subcapsular and Intra-Portal Transplantation of Adult Islets of Langerhans to the Rat Rijkelijkhuizen J.K.R.A., TOns A., Bouwman E. (Department of Surgery, Leiden University Medical Centre. The Netherlands)

Background: Reported earlier on the successful transplantation of porcine islets under the renal capsule of the rat under cyclosporine (CsA) monotherapy. Graft survival ranged from 10 to over 134 days. Methods: First, CsA-dosing was optimized by extending the induction-period and starting before grafting. Second, the effect of the inevitable damage towards the graft upon transplantation was analyzed by deliberate addition of 50% dead islets. Third, blocking of macrophages was investigated by gadoLinium-chloride (GdCI). Fourth, intra-portal transplantation (ip) was explored as an alternative to sub-renal transplantation (src). Results: See table. Gp

graft

treatment

n

1

isl/src

CsA from d 0

8 !0:8

27,27,27,31,31,44, 97, 117

2

isl/src

CsA from d -2

5

l1

PNF

4--5

graft-survival (days)

3

isl/sre

-

7

77

4

isVsrc, mixed with dead isl

CsA from d 0

5

05

21.41, 71, >107, >108

5

isl/src, dead isl other kidney

CsA from d 0

5

if5

27, >46, 47, 67, 129

6

isl/src

GdCI d -2, -1

3

03

8, 9, 10

7

isl/src

GdCI from dO, Iwk

4

0~4

6,8,8,9

8

isl/src

GdCI from d - 2 , lwk

7

1i7

8, 8, 8,

isl/ip, heparine

CsA from d 0

4

0:.,t

7, 10, 12, 62

isl/ip, no heparine

CsA from d 0

7

17

4, 7, 7, 7, 38, >70

9 10

9,9,9

Conclusions: Extension of induction-CsA did not improve graftsurvival over earlier results, whereas starting at day-2 caused primary nonfunction (PNF). Both CsA and GdCI effectively block PNF found in untreated controls, suggesting cooperation of T-cells and macrophages in early graft-rejection. Addition of dead islets did not diminish graft-survival indicating that bystander-lysis plays no role. Preliminary data show that intra-portal transplantation is feasible.

P3-59. Hierarchy of Dependency on Host Class II MHC Molecules in Rejection of Pancreatic Islet AIIo- and Xenografts Rayat G.R., Beilke J.N., Korbutt G.S, Rajotte R.V., Gill R.G. (Health Sciences Center, Immunology Medicine, University of Colorado, Denver, USA)

Background: Understanding of the processes resulting in islet rejection is crucial for developing future strategies to circumvent these responses. The present studies were carried out in order to determine whether the strength of celt-mediated islet graft rejection is dependent on the host class II MHC molecules.

Methods: We compared the proliferative response of C57B1/6 (B6) lymph node cells against allogeneic BALB/c and xenogeneic WF rat (concordant) and neonatal porcine (discordant) cells in vitro. We also determined the fate of BALB/c. WF rat, and neonatal porcine islet (NPI) grafts in streptozotocin-induced diabetic immunocompetent B6 and MHC class II-deficient B6 (C2D) mice. Results: We found that B6 lymph node cells and purified CD4+ T cells responded robustly when stimulated with allogeneic BALB/c or xenogeneic WF rat splenocytes but not after stimulation with porcine APCs. We also found that allogeneic (n = 8) and xenogeneic (WF n = 7 and porcine n = 5) islet grafts were acutely rejected in B6 mice (n = 6) within 13 days post-transplantation. In contrast, WF rat (n = 5) islet xenografts but not BALB/c islet allografts survived longer in C2D mice (>56.8 _+ 10.5 and 12.8 +_ I.L days. respectively). Most interesting is that NPI grafts failed to reject in these mice with mean graft survival of >107 __ 2.0 days posttransplantation. Conclusions: These data suggest that the intensity of the T celldependent response to islet graft decreases as the phylogenetic disparity between the donor and recipient species increases. Further, the data also support the idea that xenograft rejection is more dependent on the host MHC class II molecules than allograft rejection.

P3-60. Pancreatic Exocrine Cell Ablation by in Vivo Targeting with Lethal Genes Trani J., Deng S., E//man P., Lerner S., Markmann J.W., Wang J., Raper S., Lee K., Markmann J.F (Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, USA) Background: A persistent obstacle to clinical application of isolated pancreatic islet transplantation is technical difficulty in the isolation and purification of large numbers of functional islets. In the present study, we examined the possibility that in vivo gene transfer that results in the expression of cytotoxic gene products can be applied to ablate exocrine tissue in vivo, without injury to adjacent islet endocrine cells. Methods: We generated adenoviral constructs utilizing the either the CMV promoter or the murine Amylase promoter to control expression of either the LacZ gene (Ad-CMV-Lacz and Ad-AmyLacZ), or the gene encoding thymidine kinase (Ad-CMV-tk) which converts Gancyclovir (GCV) into cytotoxic byproducts. Gene transfer to the pancreas was accomplished by direct injection with 1011 viral particles. Gancyclovir (GCV) was administered continuously via a subcutaneous mini-osmotic pump (0.1 ml/ 2 weeks) 125 mg/ml. Results: Ad-AMY-LacZ virus demonstrated tissue specific targeting to exocrine cells. Marked exocrine destruction with intact islet tissue was observed in mice inoculated with Ad-CMV-tk (n = 8) but not Ad-CMV-LacZ (n = 5) after GCV for t to 2 weeks. No diabetes was observed in treated mice. Conclusions: Our data demonstrate that: 1) the murine amylase promoter provides tissue specific adenoviral gene expression in vivo, and 2) that tk gene expression in conjunction with GCV is produces in vivo exocrine tissue destruction. We believe a similar approach might be used in xenogenic islet transplantation by pancreas specific expression of toxic genes in large animals. In fact, we have recently obtained very promising data in Amy-tk transgenic mice.

P3-61. A Comparison of Gene Transfer Efficiency in Monolayer Cultured Porcine Neonatal Pancreatic Cells Yoon K.-H., Suh S.-H., Hong O.-K., Lee J.-M., Ahn Y-B., Cha B.-Y, Son H.-Y., Kang S.-K., Cho H.-I., Kim T.-G. (Department of Internal Medicine, The Catholic University of Korea, Seoul, South Korea)

Background: The establishment of the efficient gene delivery in pancreatic duct cells is important not only for the study about ontogeny of the beta-cells but also for generating an genetically modified beta-cell for transplantation in vitro. Methods: Using beta-GAL and EGFP as the reporter genes, the current study evaluates the efficiency of gene transfer with adenovirus-polylysine/DNA complexes (AdpL), DNA/liposome complexs and recombinant adenoviral vector in monolayer cultured

Acta Chir. Austriaca • Vol. 33 - Supplement No 1 7 4 . 2001

porcine neonatal pancreatic duct cells. The transfection rates of adenovirus vector were measured by FACS analysis for the total cells. For the duct cells and beta-cells, double stained images Iduct cell: pancytokeratin/GFR beta-cell: insulin GFP) were made in confocal microscopy with systemic manner and positive stained cells were counted. Results: The tranfection rate and cell death rate of the total cells showed linear correlation with a range of multiplicity of infection (moi: tranfection rate/cell death rate moi = 500; 88%/62%, moi = 100; 77%/42%. moi = 50: 72% 32%, moi = 10: 33%/32%, moi = 5; 21%/35%). The transfection rate and viability were well maintained in moi 50 of the adenovirus. The transfection rate of adenovirus vector in duct cells, composed of 70% of the total cells, was very similar with that of total cells. While the transfection rate in the beta-cells were about 10%. In case of the AdpL and lipofection, the transfection rate were below 10~ of the duct cells and it is hard to find the transfected beta-cell in the whole dishes. We could obtain the good transfection rate and ~iability with appropriated concentration of the adenovirus vector in monolayer cultured neonatal porcine pancreas duct cells. Conclusions: adenoviral gene transfer could be useful for the genetic modification of porcine neonatal pancreatic cells.

P3-62. AAV-Mediated Gene Transfer into the Pancreas Leads to Long-Term and Localized Transgene Expression Lilt C., Jiang K., Deng S., Brayman K.L. (School of Medicine, Hospital of the University of Pennsylvania. Philadelphia, USA)

Background: Vascular perfusion of adenoviral vectors into pancreata results in highly efficient and localized gene transfer. Unlike adenovirus, AAV is nonpathogenic and does not elicit any ocular inflammatory response. In this study, we examined 1) pancreatic gene expression following AAV transduction and 2) the effect of AAV on pancreas endocrine function. Methods: Viral vectors were administered by systemic intravenous (IV) injection or intra-arterial perfusion of isolated whole pancreaticoduodenal grafts. Ad-LacZ (2.5x10 m pfu) was used as control. Two doses of AAV were examined (2.5xl010 and 5xl0m pfu), and duration of cold storage time analyzed. Vitally- transduced grafts were transplanted into syngeneic STZ-diabetic recipients. FBG and IVGTT were followed. Immunohistochemistry and RT-PCR were performed to examine LacZ gene expression and the morphology of grafts.

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Results: IV injection of either AAV-LacZ or AdLacZ yielded little LacZ expression in the host pancreas or the transplanted grafts. LacZ (30%) was detected in arterial AdlacZ-transduced grafts on POD1 and disappeared by POD 42. No activity was found in AAVtransduced grafts treated under the same condition. However, increase in storage time or AAV dosage or both led to increased LacZ expression. AAV transduction resulted in expression largely confined to islets and duodenal villi for more than 1 month. Conclusions: AAV-mediated gene transfer through vascular perfusion can lead to stable long-term expression of the transgene. The extent of AAV transduction depends on dosage and storage time. While AAV has lower transduction efficiency than adenovirus in this model, AAV leads to longer and more staNe transgene expression.

P3-63. Glucose Activates KATP Signaling Pathways in the Insulin Secreting Liver Cell Line HEP G2ins/g Simpson A.M., Lui G.J., Martin D.K. (Department Cell & Molecular Biology, University of Technology, Sydney, Australia)

Background: As part of our research into the liver-directed gene therapy of Type I diabetes, we have engineered a human hepatoma cell line to store and secrete insulin to a glucose stimulus-HEP G2ins/g cells. The aim of this study was to see if HEP G2ins/g cells respond to glucose via signaling pathways that depend on ATP sensitive potassium channels (K,,,Tp). Methods: We used patch-clamp electrophysiology to both characterise KATeand to identify pharmacological inhibitors and activators of KATP. The effect of the KATP inhibitors tolbutamide (100 gM) and glibenctamide (20 btM) and the K~;rp activator diazoxide (150 l-tM) on acute insulin secretion from HEP G2ins/g cells was also assessed by radioimmunoassay following 1 hr incubation. Results: In symmetrical KCI solutions the single-channel conductance of KAT p w a s 61 pS. KATp was inhibited by ATP (1 mM) or cAMP (50 l.tM) applied to the cytosolic side of the membrane. Single K,xrp channels and macroscopic whole-cell currents were inhibited by glucose (20 mM) and glibenclamide (20 gM) and activated by diazoxide (150-400 btM). Exposure of the cells to tolbutamide resulted in an increase in insulin secretion from 0.3 + .05 to 1.8 + 0.2 pmol insulin/ 10~ cells and glibenclamide from 0.4 +_ .06 to 2.1 _+ 0.3 (n = 4), similar to what is seen on glucose (20 mM) stimulation. Diazoxide completely inhibited glucose-stimulated insulin release. Conclusions: HEP G2ins/g cells respond to glucose via signaling pathways that depend on ATP-sensitive potassium channels, similar to a normal [3 cell.

LIST OF AUTHORS Abdi R. 03-43 Abecassis M.M. O 1-26 Abensur H. P1-44 Abreo K.D. O1-23 Abul-Ezz S.R. PI-18 Adam U. P2-10, P3-44 Adamec M. P1-01, PI-03, P I - l l , Pl-19, P1-34 Adams A.B. 03-33 Adams M.B. P3-47 Aerts R. P1-32 Abroad O.K. O 1-36 Ahn K.-J. P3-50 Ahn Y.-B. P3-61 Ahren B. 03-05 Aita C.A.M. P3-51 Akamaru Y. 03-52 Akutsu N. 03-49, P3-04, P3-21 AI-Abdullah I.H. 02-06 Alam N.M. P2-08 Aldrighetti L. 02-05 Alejandro R. 02-03, 02-06. 02-20, O2-21, 02-22, 03-46, P2-19, P2-26 Algranati S. PI-45 AI-Jazaeri A. P3-27

03-39, P3-33 03-02, O3-12 PI-25, O1-25, O 1-50 Amado J.A. P3-28 Amaratunga A. 03-61 Ammon H.RT. 03-22 Amrouni H. P3-13 Ancona E. PI-07 Andereggen E. P2-11 Anders-Hoepgen M. O 1-37 Anderson T.J. P2-14 Andersson A. 03-06, 03-08, 03-20 Ansite J. 02-19 Anthuber M. P 1-04 Aragona M. O1-35, P1-38 Araujo M.R. PI-44 Argibay R PI-05, PI-45, P2-23, P3-16, P3-29 Arita S. P3-46 Ari~an C.E. P3-30, P3-35 Asano T. 03-49, P3-04, P3-21 Asolati M. OI-04, PI-08 Astorri E. 02-07 Auchincloss H. 03-43, 03-53 Allen M. Allen R.D.M. Alloway R.R.

Auchincloss Jr. H.

O3-10, 03-4 l Bach EH. P3-4 l BadetL. O1-13, O1-15, O1-62, 02-08, 03-07, P3-55 Baidal D.A. 02-03, 02-20, O2-21, 02-22, P2-19, P2-26 Baker M.S. 03-70 Balakrishnan S. O1-44, PI-35 Baldan N. PI-07 Barbich M. P2-23 Barker C. 03-48 Barker C.E 03-28 Barneo L. O3-15 Barone G.W. Pl-18 Bartlett S. O 1-49 Bartlett S.T. O1-31, O1-53, OI-54, O1-58, P1-37 Barton M. O3-13 Bartoz V. P 1-34 P3-31 Basadonna G. P3-30, P3-35 Basadonna G.E 03-74 Basta G. O 1-46 Battezzati A. P3-25 Baumruker T. 02-08 Bayle E

Becchetti E. Bechstein W . O . Becker B. Becker B.N.

03-74 O1-37, P3-01 O1-39, PI-21 O1-36, PI-22, P 1-42 Becker T. O1-02, P1-20 Becket Y.T. O1-08, O1-39, P1-21, O1-36, P1-22, PI-42 Beebe T. O 1-50 Belike J.N. 03°36, P3-59 Bektas H. OI-02, P1-20 Betaich S. P2-20, P3-48 Belcourt A. P3-20 Bell RR.E P2-09 Benedetti E. O1-04, O2-17, 03-32, 03-45, P1-08 Bergsten R 03-06 Berney T. 03-25, 03-26, O3-40, P2-26, P3-31, P3-41 Bertuzzi E OI-42, 02-05 Beutner U. 03-55, 03-64 Bigam D.L. 03-29 Binette T.M. 03-72 Bland B. O1-19 Bleackley R.C. P3-42 Blinder Y. 03-33