Subcutaneous xenotransplantation of bovine pancreatic islets

Transplantation of pancreatic islets in diabetes is currently limited by the need of immunosuppressive therapy. The present study was designed to test an immunoprotection planar device for subcutaneous xenotransplantation of pancreatic islets in the diabetic rat. We tested three different devices made of polyethersulfone hollow fibers. In all diabetic rats, implantation of islet-containing devices promptly normalized hyperglycemia. In vitro membrane permeability to glucose was correlated with implant function duration. These data confirm that bovine islets contained within devices and implanted subcutaneously remain functional for several days. Strategies to prolong islet function may allow achieving successful long-term islet implantation in this attractive site.     

Transplantation of islets of Langerhans.

Transplantation of insulin-secreting tissues is currently being evaluated as a possible method of treating diabetic patients. Isolation of islets of Langerhans from the rat pancreas is now routinely accomplished, and methods for islet isolation from the human pancreas are being explored. Transplantation of isolated islets into isologous rats is capable of reversing streptozotocin-induced diabetes. Unfortunately, isolated islets are not immunologically privileged and various immunosuppression regimens have not effectively obviated the rejection phenomena seen in rat allograft experiments.     

Amyloid of human islets of Langerhans

Summary Isolated amyloid from the islets of Langerhans of patients with maturity onset diabetes mellitus was compared with amyloid fibrils from patients with different types of systemic amyloidosis. It was found that systemic amyloids had in common rigid and non-branching filaments with a width of about 75 å and that these filaments sometimes were attached laterally, forming thicker fibrils. Similar filaments could also be extracted from islet amyloid but the main part of this amyloid was built up by large aggregates of very thin and often very wavy units. This structure, which has not been previously described in human amyloid, probably explains some properties of isolated islet amyloid.Supported by the Swedish Medical Research Council (Project No. 102) and Nordisk Insulinfond. Thanks are due to Anni Bedy and Cristina Bittkowski for skilled technical assistance     

Cryopreservation of rat islets of Langerhans by vitrification

Cryopreservation could be a possible means of addressing the shortage of islets of Langerhans. We investigated the effects of EDT324 solution on the vitrification of isolated rat islets of Langerhans. Rat pancreatic islets were cryopreserved in 10% DMSO by a slow-rate freezing method or were cryopreserved in EDT324 solution by vitrification. The cryopreserved islets were compared in terms of viability, stimulation index and metabolic function after transplantation. After cryopreservation, the viability and stimulation of islets stored in EDT324 were 92.4% and 6.4, respectively, and were higher than islets stored by slow freezing (72.5% and 1.5, respectively). Streptozotocin-induced diabetic rats were transplanted with islets cryopreserved in EDT324, which corrected diabetes and achieved euglycemia within 2?days after transplantation. These results indicate that EDT324 allows successful cryopreservation of rat islets for long-term storage as an alternative solution to traditionally used solutions, such as 10% DMSO. Transplantation of cryopreserved islets into diabetic rats can achieve euglycemia.     

Clinical xenotransplantation]

The growing numerical gap between the number of patients and available human donor organs have led to a revival interest in xenotransplantation. This review will mainly focus on the clinical affairs of xenotransplantation and the project of producing the gene modified pigs. Trials, designed to overcome xenogenic rejection by the expression of human complement regulatory protein (CRP), such as DAF (CD55), on the pig organ and knocking out the alpha-Gal epitope(Galalpha1-3Galbeta1-4GlcNAc-R), which is biosynthesized by the action of alpha1,3 galactosyltransferase (alpha1,3GT), were accomplished in several institutes, such as Harvard University, Pittsburgh University, Mayo Clinic, and BresaGen. We have also produced the [DAF(CD55)+GnT-III+alpha-Gal KO] pigs in last year. On the other hand, the clinical pig islets transplantation was done in many countries, such as Russia, Sweden, Mexico and China, until 2005. In addition, the new clinical trials of pig islets transplantation will be started in USA within three years. In addition, as the current studies in the xenotransplantation field, the strategies for the downregulation of the glycoantigen, complement activation, NK cell, and other immuno responces on the xenografts, are reviewed. The studies for the infectivity of porcine endogenous retrovirus (PERV) to human cells are also introduced.     

Cytoprotection of PEG-modified adult porcine pancreatic islets for improved xenotransplantation

Functional poly(ethylene glycol) (PEG) derivatives, including monosuccinimidyl PEG (MSPEG) with molecular weight (MW) of 2000 (2 kDa) as well as 5 kDa and disuccinimidyl PEG (DSPEG) with MW of 3 and 6 kDa, were synthesized and characterized. They were used to modify the surface of adult porcine islets for cytoprotection. The islets were isolated, purified and modified with functional PEG. Untreated porcine islets were used as control. An in vitro human antibody/complement-mediated cytotoxicity test based on the release of intracellular lactate dehydrogenase was used to evaluate cytotoxicity of human serum to the modified islets. In vitro cell viability was assessed using membrane-integrity straining and islet metabolism in culture. In vitro islet functionality was evaluated by glucose-stimulated insulin release of islets in static incubation with human serum. In vivo islet functionality was evaluated by monitoring non-fasting blood glucose level in streptozotocin-induced diabetic (SCID) immunocompromized mice after intraportal transplantation of porcine islets. Results show that all the PEG derivatives used in the study showed significant in vitro and in vivo cytoprotections against cytotoxic effects elicited by human serum and diabetic SCID mice, respectively, to porcine islets. DSPEG derivatives combined with human albumin exhibited a better cytoprotection, as compared to MSPEG ones, due to the capacity of the succinimidyl groups to selectively react with amino groups of the albumin under physiological conditions. The effects of both MW and concentration of the PEG derivatives on cytoprotection were significant. It appears that this novel biotechnology will be an attractive approach for improved xenotransplantation of islets.     

Quantitative assessment of islets of Langerhans encapsulated in alginate

Improved methods have recently been developed for assessing islet viability and quantity in human islet preparations for transplantation, and these measurements have proven useful for predicting transplantation outcome. The objectives of this study were to adapt these methods for use with microencapsulated islets, to verify that they provide meaningful quantitative measurements, and to test them with two model systems: (1) barium alginate and (2) barium alginate containing a 70% (w/v) perfluorocarbon (PFC) emulsion, which presents challenges to use of these assays and is of interest in its own right as a means for reducing oxygen supply limitations to encapsulated tissue. Mitochondrial function was assessed by oxygen consumption rate measurements, and the analysis of data was modified to account for the increased solubility of oxygen in the PFC-alginate capsules. Capsules were dissolved and tissue recovered for nuclei counting to measure the number of cells. Capsule volume was determined from alginate or PFC content and used to normalize measurements. After low oxygen culture for 2 days, islets in normal alginate lost substantial viable tissue and displayed necrotic cores, whereas most of the original oxygen consumption rate was recovered with PFC alginate, and little necrosis was observed. All nuclei were recovered with normal alginate, but some nuclei from nonrespiring cells were lost with PFC alginate. Biocompatibility tests revealed toxicity at the islet periphery associated with the lipid emulsion used to provide surfactants during the emulsification process. We conclude that these new assay methods can be applied to islets encapsulated in materials as complex as PFC-alginate. Measurements made with these materials revealed that enhancement of oxygen permeability of the encapsulating material with a concentrated PFC emulsion improves survival of encapsulated islets under hypoxic conditions, but reformulation of the PFC emulsion is needed to reduce toxicity.     

Fine structure of islets of Langerhans in insular amyloidosis

Summary The islets of Langerhans of diabetic and non-diabetic patients with different degrees of islet amyloidosis were studied by electron microscopy. The islet amyloid exhibited the typical fine fibrillar ultrastructure and was mainly located interstitially. Adjacent to the cells the amyloid fibrils were often highly orientated perpendicularly to the cell surface and bundles of amyloid fibrils entered in deep plasmalemmal invaginations of the cells. This was more rarely seen in other types of cell. The epithelial cells exhibited no signs of increased activity. Macrophages were common in the amyloid masses. Amyloid occurred in invaginations of these cells but usually the fibrils showed no orientation. The capillaries, the fibrocytes and the mast cells were not so closely related to the amyloid. These findings probably indicate that the amyloid of the islets of Langerhans is a product of degenerating cells even if other possibilities are not excluded.

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Zusammenfassung Die Ultrastruktur der Langerhansschen Inseln bei der Inselamyloidose.Die Langerhansschen Inseln diabetischer und nicht-diabetischer Patienten mit verschiedenen Schweregraden einer Inselamyloidose wurden elektronenmikroskopisch untersucht. Das Inselamyloid zeigte eine typische feinfibrilläre Ultrastruktur und war vorwiegend interstitiell lokalisiert. In der Nachbarschaft der -Zellen waren die Amyloidfibrillen oft senkrecht zur Zelloberfläche orientiert, wobei Bündel von Amyloidifibrillen in tiefe Invaginationen der Zellgrenzmembran hineinragten. Diese Beobachtung fand sich selten bei den anderen Inselzelltypen. Die Epithelzellen zeigten keine Hinweise auf eine gesteigerte Aktivität. Im Bereich der Amyloidmassen fanden sich in der Regel Makrophagen. Das Amyloid war in den Invaginationen dieser Zellen erkennbar, allerdings gewöhnlich nicht in paralleler Orientierung. Capillaren, Fibrocyten und Mastzellen waren dem Amyloid weniger dicht benachbart. Aus den Befunden wird der Schluß gezogen, daß das Amyloid der Langerhansschen Inseln ein Produkt degenerierender -Zellen darstellt. Andere Möglichkeiten der Entstehung lassen sich allerdings nicht ausschließen.

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Supported by the Swedish Medical Research Council (project No. B73-12X-102-09B, the Research Fund of the Swedish Diabetes Association, the Swedish Society for Medical Research and the Medical Faculty of Uppsala. Thanks are due to Ann-Charlotte Hallner, Lena Rönning and Anders Strand for their skilled technical assistance.     

Functional abnormalities of islets of Langerhans of obese hyperglycemic mouse.

Glucose-induced insulin release was studied in vitro with isolated islets of Langerhans obtained from obese hyperglycemic C57Bl/6J-ob/ob (ob/ob) and lean C57Bl/6J-+/+ (control) mice. The threshold concentrations of glucose for insulin release were determined. In addition, the effect of total fast and of chronic food restriction on in vitro insulin release were studied. The following was observed: 1) with fasting, islet volume decreased. Islets obtained from ob/ob mice were larger than control islets, except for the chronic food restricted group. 2) Ob/ob islets were more sensitive to glucose than were controls in that the threshold for glucose-induced insulin release occured at lower glucose concentrations. 3) Fasting for 48 h completely abolished glucose-induced insulin release in control islets, whereas glucose-induced insulin release was maintained in 48-h and 7-day fasted ob/ob islets. 4) The increased glucose sensitivity of the ob/ob islets was maintained despite chronic food restriction.     

抗原负载的免疫缺陷树突状细胞诱导异种胰岛移植耐受

目的:探讨负载异种MHC抗原的免疫缺陷树突状细胞(dendriticcell,DC)预处理受体对异种胰岛移植的耐受诱导作用及其机制。方法:从BALB/c小鼠骨髓干细胞分别诱导分化成熟DC及免疫缺陷DC,负载Wistar大鼠MHC抗原。将上述DC通过尾静脉回输糖尿病小鼠体内,7天后分别将Wistar或SD大鼠胰岛移植于受体鼠肾包膜下。观察移植物存活时间,检测T细胞增殖及Th1/Th2细胞因子表达。结果:对照组胰岛存活时间为8.2±1.1天;成熟DC组胰岛存活时间缩短为6.1±1.1天(P<0.05);免疫缺陷DC组胰岛存活时间显著延长,为42.3±3.5天(P<0.05)。SD大鼠胰岛移植物平均存活时间与正常受体组无差异。与正常受体鼠相比,成熟DC预处理组的T细胞增殖反应强烈,而Th1/Th2细胞因子水平无明显差异。免疫缺陷DC预处理组的T细胞增殖反应微弱,且Th1/Th2细胞因子表达明显下降。结论:负载异种MHC抗原的免疫缺陷型DC预处理受体可诱导抗原特异性T细胞无能以及Th1/Th2细胞因子的低表达,从而有效延长异种胰岛存活时间。     

Anatomy of the pancreas and Langerhans islets in snakes and lizards

The pancreas of snakes (18 species) was comparatively examined and classified into five major types, based on structure of the lobes and ducts, spatial relationships with the spleen and the gall bladder, and the disposition of islet cells. These types trend toward fusion of the pancreatic lobes and compaction of the pancreas--a progression that coincides with the phylogeny of the snakes. The more primitive pancreas of lizards (17 species) also was surveyed; that of Varanus is of special interest because its structure is intermediate between the extended, tri-lobate pancreas of lizards and the compact pancreas of snakes and may represent a transitional link in the evolution of this organ. Islet tissue is always confined to the dorsal lobe and is concentrated in its distal region adjacent to the spleen. In primitive snakes and in Varanus, a large islet mass is sequestered within a distinct juxtasplenic "islet body" distanced from the dorsal lobe and connected to it by a slender stalk. In some of the most advanced snake species, numerous islets of endocrine cells are found within the spleen. The occurrence and formation of these intrasplenic islets is described in detail. The anatomic "affinity" between spleen and the islet region of the pancreas is discussed. A hypothesis for the development of the pancreas from embryonal placodes on the mid-gut is presented; it proposes that the exocrine and the endocrine components derive from different progenitor cells, and that the endocrine progenitors are located in the center of the dorsal placode. The hypothesis combines embryological and evolutionary views about the origin of the pancreas, and offers a rationale for differences in its structure and in the disposition of the islets.     

Light- and electron-microscopic studies on isolated bovine islets of Langerhans.

While several recent studies have focussed on the critical assessment of yield, purity and function of isolated islets, fine-structural investigations of isolated islets have been the subject of only a few studies. After intraductal injection of collagenase solution, the distended bovine pancreata were processed in a continuous digestion-filtration device, after which the purification of the islet suspension was accomplished by density gradient centrifugation. Purified islets were then prepared for light- and transmission electron-microscopic analysis. In semithin sections, no evidence of either connective tissue or exocrine tissue surrounding isolated islets was found. Endocrine cells exhibiting cytoplasmic granules were packed in clusters varying in size. In ultrathin sections, A-cells containing numerous secretory granules were distinctive; their cytoplasm contained conspicuous formations of rough endoplasmatic reticulum and juxtanuclear Golgi complexes. In the B-cells, the Golgi complexes were also prominent; these elements, however, were poor in endoplasmic reticulum and displayed characteristic B-cell granules surrounded by a halo. A few ultrastructurally heterogeneous cells were scattered among the identified cellular elements.     

Hypertrophy and hyperplasia of islets of Langerhans associated with androgen therapy.

Hypertrophy and hyperplasia of the islets of Langerhans were observed in three patients treated with androgen-anabolic steroids for aplastic anemia. These patients formed a unique subset of patients with aplastic anemia in that they all had Fanconi's anemia, all demonstrated glucose intolerance following institution of androgen therapy, all had received androgen for at least 42 months prior to death, and all had coexistent benign liver cell tumors. Androgen-induced glucose intolerance may play a major role in the pathogenesis of both the pancreatic and hepatic abnormalities.