Behboo, R

Fontes, P

Rao, AS

Rastellini, C

Ricordi, C

Rilo, HLR

Starzl, TE

Transplantation Proceedings

English

PubMed

Rastellini, C

Behboo, R

Rilo, H L

Fontes, P

Ricordi, C

Starzl, T E

Rao, A S

University of Miami: Scholarship Miami

Functional evaluation of isolated islets from baboon pancreata

Name not available

D-Scholarship@Pitt

Functional Evaluation of Isolated Islets from Baboon Pancreata C. Rasteliini, R. Behboe, H.L.R. Rilo, P. Fontes, C. Ricordi, T.E. Starzl, and A.S. Rae ALLOTRANSPLANTATION of pancreatic islets has been used with variable success to treat insulin-dependent Type I diabetes mellitus (IDDM).I The recent reports of long-term insulin-free existence following encap-sulated2 or delayed3 islet cell transplantation in diabetic recipients have paved the way for its successful Qinical application. However, its global implementation will be impeded by the acute shortage of cadaveric donors, which has fomented our interest in the search for alternative sources of pancreata. Nonhuman primates (chimpanzees, baboons, etc.) by sharing many physiological and genetic characteristics with Homo Sapiens, are a reasonable alter-native as donors for xenogeneic islet cell transplantation. The present study was designed to investigate the feasibility of using baboon pancreata to obtain viable and functional islets for future xenotransplantation. MATERIALS AND METHODS Animals Nine healthy juvenile baboons (Pappio annllbis) screened for all known viral and microbial infections were obtained from the Southwest Foundation for Biomedical Research, San Antonio, Texas. They were housed at the University of Pittsburgh, Monte-fiore Hospital Animal Facilities. Male athymic nude (nu/nu) mice were obtained from Harlan Sprague-Dawley. Indianapolis, Ind, maintained in the pathogen-free facility al the University of Pittsburgh, and used at 6 to 8 weeks of age. Isolation of Pancreatic Islets Islets were isolated by a modification of the automated method described elsewhere.· They were subsequently purified by density centrifugation. The methods of dithizone staining and trypan blue dye exclusion were used to assess islet purity. viability, and yield. Dynamic perifusion assays were used to further assess their viability and in vitro functional capacity. Reversal of Diabetes in Nude Mice Hyperglycemia was induced in nude mice by a single IV injection of streptozolocm (STZ; 185 mg/kg/animal). The animals were consid-ered diabetic when their blood glucose levels were found to be >250 mg;dL. Freshly isolated baboon islets (600 IEq) were trans-planted 2 10 3 days post-STZ treatment beneath the left renal capsule. A reduction in blood glucose levels to < 150 mg/dL was considered as an indication of reversal of diabetes. An intraperi-toneal glucose tolerance test (IPGTT) was performed approxi-mately 5 weeks after islet cell transplantation to evaluate their function under stress. Unilateral nephrectomy of the graft-bearing kidney was eventually carried out to verify the function of the transplanted islets and the chemical destruction of the native pancreas. Insulin and glucagon content of the transplanted islets within the nephrectomized specimens were assessedby-i~ histochemistry and by Northern blotting. RESULTS AND DISCUSSION Islets were isolated from the pancreata of nine baboons who, based on their age, have been arbitrarily divided into two groups; group I: donors <2 years of age; group II: donors >4 years of age (Table 1). Pancreatic islet cell yields from baboons in both groups were comparable to those obtained form human cadaveric donors.3 When tested in in vitro dynamic perifusion assays, islets isolated from the pancreata of animals in group II responded efficiently to both glucose and caffeine stimulation. On the contrary, islets isolated from the pancreata of group I animals responded only to caffeine but not to glucose challenge in in vitro functional assays. This observation is similar to that made previously in the islets obtained from human pediat-ric donors. Implantation of baboon islets under the renal capsule of diabetic athymic nude mice led to restoration of eugJycemic status in 36 of 40 (90%) of mice, with prompted reversal of hyperglycemia following intraperitoneal glucose challenge (lPGTI). The functional status of transplanted islets was further confirmed by unilateral nephrectomy of From the Pittsburgh Transplantation Institute and the Depart-ments of Surgery (C.R., A.B., H.L.A.A., P.F., T.E.S., A.S.A.) and Pathology (A.S.A.), University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, and the Diabetes Research Institute (C.R.), University of Miami, Miami, Florida. Aided by Project Grant No. OK 29961 from the National Institute of Health, Bethesda, Maryland. Address reprint requests to Abdul S. Rao, MD, DPhil, Pitts-burgh Transplantation Institute, E1545 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15213. © 1995 by Appleton & Lange 0041-1345/95/$3.00/ +0 Table 1. Cell Yield and Purity of Islets Isolated From Baboon Pancreata Donor age Groups (range: yr) I 1-2 1/ 4-18 Results are expressed as x '" SO. 'Cold Ischemia time Pancreas n weight (g) 7 31.2::: 16.2 2 11.1 :': 0.76 1 Estimated number of Islets With an average diameter of 150 1'. Clr(h) 4.0:': 2.0 5.5:': 3.4 Transplantation Proceedings, Vol 27. No 6 (December), 1995: pp 3385-3386 Islets (IEq)t Islets/gram Purity (X10') (X103) (%) 1.46:,: 0.5 5.3:': 1.3 85.0:,: 10.0 0.80:': 0.1 6.9:': 0.4 86.4 :': 2.8 3385 1" .. 3386 the graft-bearing kidney, which precipitated reappearance of diabetes. Examination of transplanted islets in the ne-phrectomized specimen revealed the presence of granu-lated f3 cells that stained positively for insulin and glucagon. Furthermore, RNA obtained from transplanted islets, when immobilized onto a PVDF membrane and hybridized with insulin-specific peroxidase-based chemiluminescent probe, showed positive bands suggesting the presence of insulin mRNA. Because viable islets with acceptable purity and yield can be obtained from baboon pancreata, it is tempting to speculate that barring immunologic impedance, they may RASTELLINI. BEHBOO. RILO ET AL be considered for xenotransplantation to treat humans with IDDM. REFERENCES I. Ricordi C. Tzakis A, Carroll P. et al: Transplantation 53:407, 1992 2. Soon-Shiong P. Heintz RE, Merident N, et al: Lancet 343: 950,1994 3. Ricordi C. Murase N, Rastellini C. et al: Transplant Proc 26:3358. 1994 4. Ricordi C, Lacy PE. Finke EH, et al: Diabetes 37:413, 1988 

http://d-scholarship.pitt.edu/5111/1/31735062125335.pdf

Functional evaluation of isolated islets from baboon pancreata

Abstract

Similar works

Full text

Available Versions

University of Miami: Scholarship Miami

Name not available

D-Scholarship@Pitt

Name not available