ognizant Communication Corporation


VOLUME 9, NUMBER 6, 2000

Cell Transplantation, Vol. 9, pp. 743-749, 2000
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Hibernated Human Fetal Striatal Tissue: Successful Transplantation in a Rat Model of Huntington's Disease

Carrie B. Hurelbrink, Richard J. E. Armstrong, Roger A. Barker, Stephen B. Dunnett, and Anne E. Rosser

Cambridge Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK

The use of fresh human fetal tissue in neural transplantation presents considerable logistical difficulties and limits the clinical applicability of this promising therapy. This study compared the survival of human fetal striatal tissue that had been stored for 24 h in a defined hibernation medium with that of fresh human fetal striatal tissue following xenotransplantation in a rat model of Huntington's disease (HD). Six to 7 weeks postgrafting, there was no significant difference between fresh and hibernated grafts in volume or in various striatal phenotypic markers, although there was a trend towards decreased graft volume. We conclude that short-term hibernation of this tissue is without significant adverse effects on the survival of grafted human fetal striatal tissue. This has important implications for the practical implementation of clinical neural transplant programs in HD.

Key words: Neural transplantation; Tissue storage; Xenograft; Graft survival

Address correspondence to Carrie B. Hurelbrink, Cambridge Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK. Tel: +44 1223 331160; Fax: +44 1223 331174; E-mail: cbh20@cam.ac.uk

Cell Transplantation, Vol. 9, pp. 751-758, 2000
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The Effects of Taurine on hNT Neurons Transplanted in Adult Rat Striatum

Selva Rivas-Arancibia,1,3 Alison E. Willing,1 Tanja Zigova,1 Alba I. Rodriguez,1,2 David W. Cahill,1 and Paul R. Sanberg1

1Center for Aging and Brain Repair, Department of Neurosurgery and The Neuroscience Program, College of Medicine and 2Department of Psychology, University of South Florida, Tampa, FL 33612
3Dept. de Fisiología Facultad de Medicina, Universidad Nacional Autónoma de México, México, D.F. 04510

Taurine acts as an antioxidant able to protect neurons from free radical-mediated cellular damage. Moreover, it modulates the immune response of astrocytes that participate in neurodegenerative processes. The objective of this study was to examine whether taurine can prevent or attenuate the host inflammatory response induced by the xenotransplantation of neurons derived from the human teratocarcinoma cell line (hNT neurons). Male Sprague-Dawley rats were treated IP with either saline or taurine. Animals from both groups were perfused on the 4th or 11th day and the saline or taurine was administered from the start of the study until the day prior to sacrifice. The brains were processed immunohistochemically using antibodies against glial fibrillary acidic protein (GFAP), microglia (OX42), and human nuclear matrix antigen (NuMA). In the saline group, NuMA labeling revealed small grafts on the 4th day and no surviving cells on the 11th day. However, in the group that received taurine there were surviving grafts at both time points. Strong immunoreactivity for GFAP and OX42 was detected in the saline group surrounding the transplant. These effects were reduced in animals receiving taurine. Taken together, these results demonstrated that taurine was able to facilitate graft survival and attenuate the immune response generated by the xenograft.

Key words: Taurine; Striatum; Xenograft; Oxidative stress; Antioxidant effects

Address correspondence to Dr. Selva Rivas-Arancibia, Dept. de Fisiología, Facultad de Medicina, UNAM, A.P. 70-250. México, D. F. 04510. Fax: (525) 6232241; E-mail: srivas@servidor.unam.mx

Cell Transplantation, Vol. 9, pp. 759-772, 2000
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A New Type of Biocompatible Bridging Structure Supports Axon Regrowth After Implantation Into the Lesioned Rat Optic Tract

Giles W. Plant and Alan R. Harvey

Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Perth, WA 6907, Australia

We have developed a new type of polymer/cell/matrix implant and tested whether it can promote the regrowth of retinal ganglion cell (RGC) and other axons across surgically induced tissue defects in the CNS. The constructs, which consisted of 2-2.5-mm-long polycarbonate tubes filled with lens capsule-derived extracellular matrix coated with cultured neonatal Schwann cells, were implanted into lesion cavities made in the left optic tract (OT) of 18-21-day-old rats. In one group, to promote Schwann cell proliferation and perhaps also to stimulate axon regrowth, basic fibroblast growth factor (bFGF) was added to the lens capsule matrix prior to implantation. In another group, to determine whether application of growth factors to the somata of cells enhances the regrowth of distally injured axons, the neurotrophin NT-4/5 was injected into the eye contralateral to the OT lesion. NT-4/5 and bFGF treatments were combined in some rats. After medium-term (4-10 weeks) or long-term (15-20 weeks) survivals, axon growth into implants was assessed immunohistochemically using a neurofilament (RT97) antibody. RGC axons were visualized after injection of WGA/HRP into the right eye. Viable Schwann cells were present in implants at all times after transplantation. Large numbers of RT97+ axons were consistently found within the bridging implants, often associated with the peripheral glia. Axons were traced up to 1.7 mm from the nearest CNS neuropil and there was immunohistochemical evidence of myelination by Schwann cells and by host oligodendrocytes. There were fewer RGC axons in the implants, fibers growing up to 1.6 mm from the thalamus. Neither NT-4/5 nor bFGF, alone or in combination, significantly increased the extent of RGC axon growth within the implants. A group of OT-lesioned rats was implanted with polymer tubes filled with 2-2.5-mm-long pieces of predegenerate peripheral nerve. Surprisingly, polymer/cell/matrix constructs contained comparatively greater numbers of RGC and other axons and supported more extensive axon elongation. Thus, implants of this type may potentially be useful in bridging large tissue defects in the CNS.

Key words: Schwann cells; Extracellular matrix; Retinal ganglion cells; Regeneration; Biopolymers

Address correspondence to Giles W. Plant, Ph.D., Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Perth, WA 6907, Australia. Tel: 61 8 9380 8642; Fax: 61 8 9380 1051; E-mail: gplant@anhb.uwa.edu.au

Cell Transplantation, Vol. 9, pp. 773-783, 2000
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Alginate-Encapsulated Producer Cells: A Potential New Approach for the Treatment of Malignant Brain Tumors

Frits Thorsen,1 Tracy-Ann Read,1 Morten Lund-Johansen,2 Berit Bølge Tysnes,1 and Rolf Bjerkvig1

1Department of Anatomy and Cell Biology, University of Bergen, Aarstadvn. 19, 5019 Bergen, Norway
2Department of Neurosurgery, Haukeland University Hospital, 5021 Bergen, Norway

In recent years gene therapy has evolved as a new treatment for brain tumors, where genetically engineered cells can be used to deliver specific substances to target cells. However, clinical success has been limited due to insufficient gene transfer, lack of prolonged gene expression, and immunorejection of producer cells. These obstacles may be overcome by encapsulating producer cells into immunoisolating substances such as alginate. This may provide a stable in situ delivery system of specific proteins, which can interfere with tumor growth and differentiation. This article represents a fundamental study describing the in vitro and the in vivo behavior of alginate-encapsulated producer cells. The viability and cell cycle distribution of encapsulated NIH 3T3 cells was studied by confocal laser scanning microscopy (CLSM) and by flow cytometry. The CLSM study showed a high viability of the encapsulated NIH 3T3 cells during 9 weeks in culture. The flow cytometric analysis revealed a change in cellular ploidy after 1 week in culture, with normalization in ploidy after 3 and 9 weeks. The production of the bacterial E. coli b-galactosidase in alginate-encapsulated BT4CnVlacZ cells was studied by x-gal staining, and the cells expressed prolonged b-galactosidase activity. H528 hybridoma cells producing monoclonal antibodies (mAbs) against the human epidermal growth factor receptor (EGFR) were encapsulated in alginate, and the mAb release was determined. The release of mAbs stabilized around 400 ng/ml/h after 12 days in vitro. To actually demonstrate that alginate-encapsulated H528 cells potentially inhibit a heterogeneous glioma cell population, cell migration from human GaMg glioma spheroids was studied during stimulation with EGF in the presence of encapsulated H528 cells. The migration in vitro was totally inhibited in the presence of H528 encapsulated cells. Alginate beads with H528 cells were also implanted into rat brains, and after 9 weeks the distribution of mAbs within the brain was studied by immunohistochemistry. It is shown that the alginate entrapped H528 cells produce mAbs inside the brain for prolonged periods and that the mAbs are distributed within all CSF compartments. Encapsulated producer cells represent a potential delivery system for specific proteins to brain tumors. Different producer cells may be encapsulated in alginate to target phenotypic features and microenvironmental factors, which may influence the progressive growth of brain tumors.

Key words: Sodium alginate; Gene therapy; Brain tumors; Producer cells

Address correspondence to Frits Thorsen, Ph.D., M.Sc., Department of Radiophysics, Haukeland University Hospital, 5021 Bergen, Norway. Tel: +47 55 97 35 94; Fax: +47 55 97 35 99; E-mail: frits.thorsen@haukeland.no

Cell Transplantation, Vol. 9, pp. 785-796, 2000
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Agarose Enhances the Viability of Intraperitoneally Implanted Microencapsulated L929 Fibroblasts

Shahab Lahooti and Michael V. Sefton

Department of Chemical Engineering and Applied Chemistry, and Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

To achieve immunoisolation, mouse L929 fibroblasts were encapsulated in ~400 mm poly(hydroxyethyl methacrylate-co-methyl methacrylate) (HEMA-MMA) microcapsules and were subsequently implanted in the peritoneal cavity of syngeneic C3H mice. As a baseline for the use of genetically engineered cells in cell encapsulation therapy, the L929 cells were transfected to express a secreted form of human alkaline phosphatase (SEAP). Implantation of empty microcapsules in a PBS suspension resulted in deformation, aggregation, and poor retrievability of the microcapsules. Incubation of microcapsules with medium containing xenogeneic horse serum prior to implantation increased the thickness of the fibrous tissue surrounding the microcapsules. However, immobilization of the microcapsules in a 4% (w/v) SeaPlaque® agarose gel prior to implantation allowed complete recovery of the microcapsules and prevented their aggregation and deformation. As a result, ~50% of the encapsulated cells remained viable 21 days postimplantation. Moreover, once the viable cells were released from retrieved microcapsules and regrown as monolayers, they expressed SEAP at a level similar to their encapsulated but nonimplanted counterparts.

Key words: Microencapsulation; L929; Secreted form of human alkaline phosphatase (SEAP); Agarose; Cell transplantation

Address correspondence to Michael V. Sefton, Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College, Street, Toronto, Ontario, Canada. Tel: (416) 978-3088; E-mail: sefton@chem-eng.utoronto.ca

Cell Transplantation, Vol. 9, pp. 797-804, 2000
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Acutely Sodded Expanded Polytetrafluoroethylene Grafts Produce Only Prostacyclin: A Qualitative Difference From Saphenous Veins

Sudhen B. Desai,* David G. Cable, Michael R. Phillips, and Hartzell V. Schaff

Section of Cardiovascular Surgery, Mayo Clinic and Mayo Foundation, Rochester, MN 55905

Select subsets of patients require prosthetic graft material for revascularization. Although arterial prosthetic grafts of large caliber perform acceptably, grafts of <6 mm exhibit a high attrition rate. Microvessel endothelial sodding, a method resulting in the lining of prosthetic grafts with autologous endothelium, improves graft patency; however, aggressive antiplatelet therapy is still required, because terminating an antiplatelet regimen accelerates graft attrition. The present investigation was designed to address the acute production of vasoactive substances in microvessel endothelial cell sodded expanded polytetrafluoroethylene (ePTFE) grafts in an attempt to delineate a possible mechanism behind the continued requirement for antiplatelet therapy. Equal lengths of acutely sodded ePTFE grafts (canine falciform ligament source) and saphenous veins (SV) (canine source) were evaluated by superfusion bioassay. Basal secretion from ePTFE grafts relaxed the biodetector ring 1 ± 3%, whereas SV relaxed the ring 10 ± 3% (p < 0.05, ePTFE vs. SV). Relaxation with acetylcholine stimulation was 49 ± 7% in grafts and 50 ± 10% in veins (p = NS). Calcium ionophore stimulation produced relaxation of 37 ± 9% from ePTFE grafts and 100 ± 23% from SV (p < 0.05). Indomethacin added to perfusate reduced relaxations from sodded ePTFE grafts to 20.2 ± 9.2% with acetylcholine stimulation and 12.5 ± 4.3% with calcium ionophore (p < 0.05 vs. control); addition of NG-monomethyl-L-arginine (L-NMMA) had no effect on the release of vasoactive substances from ePTFE grafts. In contrast, relaxations of effluent from SV stimulated by acetylcholine and calcium ionophore were significantly attenuated with indomethacin and L-NMMA (p < 0.05 vs. control). Scanning electron microscopy demonstrated confluent endothelium in SV and a nonconfluent endothelial cell layer in grafts. Acutely sodded ePTFE grafts produce vasoactive substances that quantitatively and qualitatively differ from those produced by canine SV. The ePTFE grafts produce mainly prostanoids, whereas SV produce both nitric oxide and prostanoids. The endothelial cell isolation procedure and absence of immediate graft luminal confluence may contribute to the observed differences.

Key words: Antiplatelet therapy; Prostacyclin; Saphenous vein; Sodded prosthetic graft

Address correspondence to Hartzell V. Schaff, M.D., Section of Cardiovascular Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Tel: (507) 255-7068; Fax: (507) 255-7378; E-mail: schaff@mayo.edu

*Medical Student, Albany Medical College.

Cell Transplantation, Vol. 9, pp. 805-815, 2000
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The Cre-loxP System: A Versatile Tool for Targeting Genes in a Cell- and Stage-Specific Manner

Manas K. Ray, Shawn P. Fagan, and F. Charles Brunicardi

Michael E DeBakey Department of Surgery, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030

Gene-targeted mice, derived from embryonic stem cells, are useful tools to study gene function during development. However, if the inactivation of the target gene results in embryonic lethality, the postdevelopmental function of the gene cannot be further studied. The Cre recombinase-loxP (Cre-loxP) system was developed to overcome this limitation as well as to confine the inactivation of the target gene in a cell- or tissue-specific manner. This system allows for the inactivation of the target gene in a single cell type, thereby allowing the analysis of physiological and pathophysiological consequences of the genetic alteration in mature animals. A unique property of the insulin gene to be expressed only in pancreatic beta cells has allowed using the beta-cell-specific rat insulin promoter (RIP) for Cre recombinase expression to inactivate genes in beta cells. The RIP has been used to inactivate genes in beta cells and analysis of these genetically altered mice has provided important information regarding the role of potential transcription factors and the receptors in vivo, for regulation of insulin gene transcription and in the development of beta cells. The Cre-loxP system is at a relatively early stage of development, and the ability of this technique to virtually target any gene in any tissue at any stage of development makes the study of gene function in a single cell type in vivo an attainable goal. It is anticipated that the continued experience with this system will provide an important tool to determine the role of the transcription factors involved in insulin gene regulation and islet cell differentiation and ultimately provide the basis for novel therapy to treat diabetes.

Key words: Cre recombinase (Cre); loxP sequence; Rat insulin promoter (RIP); Pancreatic beta cells; Gene inactivation and transgenic mice

Address correspondence to F. Charles Brunicardi, M.D., Department of Surgery, Baylor College of Medicine, Houston, TX 77030. Tel: (713) 798-8070; Fax: (713) 798-8460; E-mail: cbrunica@bcm.tmc.edu

Cell Transplantation, Vol. 9, pp. 817-828, 2000
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ICAM-1 Antisense Oligodeoxynucleotide Improves Islet Allograft Survival and Function

Stephen M. Katz,1 Frank Bennett,2 Kim Stecker,2 James H. Clark,3 Tommy Phan,1 Mou-er Wang,1 Barry D. Kahan,1 and Stanislaw M. Stepkowski1

1Division of Immunology and Organ Transplantation, Department of Surgery, The University of Texas Medical School at Houston, Houston, TX 77030
2ISIS Pharmaceuticals, Carlsbad, CA 92008
3Department of Pathology, The University of Texas Medical School at Houston, Houston, TX 77030

Expression of intercellular adhesion molecule-1 (ICAM-1) and its ligand, leukocyte function antigen-1 (LFA-1), after pancreatic islet transplantation may affect both nonspecific and alloantigen-specific phases of graft destruction. We examined the effects of ICAM-1/LFA-1 blockade on the survival of islet allografts. Fresh C57BL/10 (H2b) pancreatic islets were transplanted under the renal subcapsular space (KC) or embolized into the liver after portal vein (PV) injection to C3H (H2k) mice. Recipients remained untreated or were treated for 7 days by IP administration of: ICAM-1 antisense phosphorothioate oligodeoxynucleotide (oligo) alone; anti-ICAM-1 (aICAM-1) monoclonal antibody (mAb) alone; aLFA-1 mAb alone; ICAM-1 oligo/aLFA mAb combination; aICAM-1 mAb/aLFA-1 mAb combination; or control oligo IP-8997 or IP-1082. In some experiments, donors were pretreated with ICAM-1 oligo. Inhibition of single ligand with 5.0 mg/kg ICAM-1 oligo (25.1 ± 10.3), 100 mg/daily aICAM-1 mAb (24.2 ± 8.0 days), or 50 mg/daily aLFA-1 mAb (42.8 ± 25.9 days) prolonged the survivals of KC islet allografts in comparison with untreated controls (11.9 ± 1.0 days; all p < 0.01). However, dual ICAM-1/LFA-1 blockade with either ICAM-1 oligo/aLFA-1 mAb (78.3 ± 16.5 days) or aICAM-1 mAb/aLFA-1 mAb (65.2 ± 31.3 days) was the most effective therapy. Although pretreatment of donors with ICAM-1 oligo alone was ineffective (12.2 ± 0.8 days; NS), a combination of donor pretreatment and recipient treatment started 1 day prior to grafting with ICAM-1 oligo (39.2 ± 14.0 days) was more effective than the recipient treatment alone (24.6 ± 8.8 days). Furthermore, ICAM-1/LFA-1 blockade improved islet function as evaluated by glucose tolerance test, and decreased inflammation in comparison with untreated controls. Similar in vivo results were obtained following PV administration of islet allografts. Thus, ICAM-1/LFA-1 blockade prolongs the survival of pancreatic islet allografts and improves their early function.

Key words: Intracellular adhesion molecule-1 (ICAM-1); Leukocyte function antigen-1 (LFA-1); Allograft survival; Allograft function

Address correspondence to Stephen M. Katz, M.D., Division of Immunology and Organ Transplantation, The University of Texas Medical School-Houston, 6431 Fannin, Suite 6.246, Houston, TX 77030. Tel: (713) 500-7408; Fax: (713) 500-0784; E-mail: stephen.katz@uth.tmc.edu

Cell Transplantation, Vol. 9, pp. 829-840, 2000
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Development and Characterization of Pituitary GH3 Cell Clones Stably Transfected With a Human Proinsulin cDNA

Cesare Meoni,1 Federico Bertuzzi,1 Antonio E. Pontiroli,3 Luca Falqui,2 Lucia Monaco,4 Marco Soria,4 Cinzia Arcelloni,5 Rita Paroni,5 Chiara Foglieni,4 Luca Polastri,1 Francesca Galbiati,1 Franco Folli,3 and Alberto M. Davalli1

1Cattedra di Clinica Medica, Università Vita-Salute, H San Raffaele, Milan, Italy
2Telethon Institute for Gene Therapy (TIGET), 3Unit for Metabolic Diseases, 4Unit of Biotechnology and 5Department of Laboratory Medicine, Istituto Scientifico San Raffaele, Milan, Italy

Successful b-cell replacement therapy in insulin-dependent (type I) diabetes is hindered by the scarcity of human donor tissue and by the recurrence of autoimmune destruction of transplanted \GK\b cells. Availability of non-b cells, capable of releasing insulin and escaping autoimmune recognition, would therefore be important for diabetes cell therapy. We developed rat pituitary GH3 cells stably transfected with a furin-cleavable human proinsulin cDNA linked to the rat PRL promoter. Two clones (InsGH3/clone 1 and 7) were characterized in vitro with regard to basal and stimulated insulin release and proinsulin transgene expression. Mature insulin secretion was obtained in both clones, accounting for about 40% of total released (pro)insulin-like products. Immunocytochemistry of InsGH3 cells showed a cytoplasmic granular insulin staining that colocalized with secretogranin II (SGII) immunoreactivity. InsGH3 cells/clone 7 contained and released in vitro significantly more insulin than clone 1. Secretagogue-stimulated insulin secretion was observed in both InsGH3 clones either under static or dynamic conditions, indicating that insulin was targeted also to the regulated secretory pathway. Proinsulin mRNA levels were elevated in InsGH3 cells, being significantly higher than in bTC3 cells. Moreover, proinsulin gene expression increased in response to various stimuli, thereby showing the regulation of the transfected gene at the transcriptional level. In conclusion, these data point to InsGH3 cells as a potential b-cell surrogate even though additional engineering is required to instruct them to release insulin in response to physiologic stimulations.

Key words: Bioengineering; Pituitary cells; Insulin secretion

Address correspondence to Alberto M. Davalli, M.D., Cattedra di Clinica Medica, Università Vita-Salute, H San Raffaele, Via Olgettina 60, 20132 Milan, Italy. E-mail: alberto.davalli@hsr.it

Cell Transplantation, Vol. 9, pp. 841-851, 2000
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Insulin-Secreting Pituitary GH3 Cells: A Potential b-Cell Surrogate for Diabetes Cell Therapy

Alberto M. Davalli,1 Francesca Galbiati,1 Federico Bertuzzi,1 Luca Polastri,1 Antonio E. Pontiroli,2 Lucia Perego,2 Massimo Freschi,3 Guido Pozza,1 Franco Folli,2 and Cesare Meoni1

1Cattedra di Clinica Medica, Università Vita-Salute, H San Raffaele, Milan, Italy
2Unit for Metabolic Diseases and 3Department of Pathology, Istituto Scientifico San Raffaele, Milan, Italy

In a companion article, we describe the engineering and characterization of pituitary GH3 cell clones stably transfected with a furin-cleavable human insulin cDNA (InsGH3 cells). This article describes the performance of InsGH3 (clones 1 and 7) cell grafts into streptozotocin (STZ)-induced diabetic nude mice. Subcutaneous implantation of 2 x 106 InsGH3 cells resulted in the progressive reversal of hyperglycemia and diabetic symptoms, even though the progressive growth of the transplanted cells (clone 7) eventually led to glycemic levels below the normal mouse range. Proinsulin transgene expression was maintained in harvested InsGH3 grafts that, conversely, lose the expression of the prolactin (PRL) gene. Elevated concentrations of circulating mature human insulin were detected in graft recipients, demonstrating that proinsulin processing by InsGH3 cells did occur in vivo. Histologic analysis showed that transplanted InsGH3 grew in forms of encapsulated tumors composed of cells with small cytoplasms weakly stained for the presence of insulin. Conversely, intense insulin immunoreactivity was detected in graft-draining venules. Compared to pancreatic bTC3 cells, InsGH3 cells showed in vitro a higher rate of replication, an elevate resistance to apoptosis induced by serum deprivation and proinflammatory cytokines, and significantly higher antiapoptotic Bcl-2 protein levels. Moreover, InsGH3 cells were resistant to the streptozotocin toxicity that, in contrast, reduced bTC3 cell viability to 50-60% of controls. In conclusion, proinsulin gene expression and mature insulin secretion persisted in transplanted InsGH3 cells that reversed hyperglycemia in vivo. InsGH3 cells might represent a potential b-cell surrogate because they are more resistant than pancreatic b cells to different apoptotic insults and might therefore be particularly suitable for encapsulation.

Key words: Pituitary cells; Bioengineering; Insulin gene; Cell therapy; Diabetes

Address correspondence to Alberto M. Davalli, M.D., Cattedra di Clinica Medica, Università Vita-Salute, H San Raffaele, Via Olgettina 60, 20132 Milan, Italy. E-mail: alberto.davalli@hsr.it

Cell Transplantation, Vol. 9, pp. 857-865, 2000
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Human Pancreatic Islets Transfected to Produce an Inhibitor of TNF Are Protected Against Destruction by Human Leukocytes

Trey Dobson,1 Dan Fraga,2 Corey Saba,1 Michael Bryer-Ash,1,3 A. Osama Gaber,2 and Ivan C. Gerling1,3

1Department of Medicine and 2Department of Surgery, The University of Tennessee at Memphis, Memphis, TN 38104
33Research Service, Veterans Administration Medical Center, Memphis, Memphis, TN 38104

The objective of this study was to determine whether transfection of human islets with an adenovirus construct encoding an inhibitor of tumor necrosis factor (TNFi) was effective at limiting damage to beta cells induced by human peripheral blood leukocytes (huPBL). Human islets transfected with TNFi or control islets were transplanted under the kidney capsule of NOD-scid mice. After a 15-day engraftment period, half of the mice received injections of activated huPBL and half received buffer injections. Islet graft function was assessed by two different methods, both of which use a species-specific radioimmunoassay to determine human insulin. In some mice, insulin production following intraperitoneal glucose injection was determined in serum. In other mice, total graft insulin content was determined by acid ethanol extraction. Histochemical stains were performed on some kidneys at the termination of the experiment to evaluate graft presence, transgene expression, and huPBL infiltration. In huPBL injected mice, graft performance was maintained in mice whose grafts were transfected with TNFi but declined substantially in control groups with sham transfected or b-galactosidase transfected islet grafts. Similar results were obtained using either glucose-stimulated insulin release or graft insulin content as a measure of graft survival. There was no significant difference in graft function between control groups receiving buffer injections, regardless of whether the islets had been transfected. Human leukocytes were found in all huPBL groups regardless of islet transfection status. We conclude that transfection of human islets with an adenovirus encoding TNFi protects beta cells from destruction induced by human leukocytes. The local production of TNFi does not prevent graft infiltration by leukocytes, only the destruction of grafts by the infiltrating leukocytes. These results raise the possibility that local expression of an inhibitor of the proinflammatory cytokine TNF-a may also prevent graft failure in clinical islet transplantation.

Key words: Cytokines; Islets; Transfection; Graft survival

Address correspondence to Ivan C. Gerling, Ph.D., Department of Medicine, The University of Tennessee, VAMC Research 151, 1030 Jefferson Avenue, Memphis, TN 38104. Tel: (901) 523-8990, x5088; Fax: (901) 577-7273; E-mail: igerling@utmem1.utmem.edu

Cell Transplantation, Vol. 9, pp. 867-875, 2000
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A Large-Animal Model to Evaluate the Clinical Potential of Fetal Pig Pancreas Fragment Transplantation

Wayne J. Hawthorne, Adrian R. Cachia, Stacey N. Walters, Anita T. Patel, Janelle E. Clarke, Philip J. O'Connell, Jeremy R. Chapman, and Richard D. M. Allen

National Pancreas Transplant Unit, Westmead Hospital, Westmead, NSW, Australia

The long-term goal of this study is to assess the feasibility of using fetal pig pancreas fragment (FPPF) transplantation to treat patients with type I diabetes. Using the highly inbred Westran Pigs, our initial aim was to establish a rejection-free transplant model of FPPF grafted into sibling recipient pigs without immunosuppression. FPPFs were isolated from 80-100-day-old fetuses of either Westran Pigs or outbred pigs and transplanted into the thymus, spleen, liver, or kidney of the recipient Westran pig. Biopsies were taken from each transplant site at set time points and assessed histologically for islet viability, rejection, and endocrine function. Fifty-eight fetal donors were used to transplant 16 recipient pigs. A nonspecific inflammation was seen for both outbred and inbred FPPF donor tissue at day 3 and was considered a response to ischemic necrosis. However, all the transplanted outbred FPPF donor tissue was acutely rejected and lost by day 10-14. In contrast, inbred FPPF tissue showed little evidence of graft necrosis after 3 days, and growth and formation of epithelial islet cell nest-like structures were seen to 28 days after transplantation. With time after transplantation, increasing amounts of insulin immunoperoxidase staining was seen together with chromogranin and somatostatin staining. In summary, this study confirms the potential of the Westran pig to answer the unproven ability of fetal pancreatic tissue to reverse type I diabetes in a large animal model.

Key words: Allografts; Diabetes; Fetal pig pancreas fragment transplantation; Inbred pigs

Address correspondence to Wayne J. Hawthorne, Department of Surgery, The University of Sydney, Westmead Hospital, Westmead, NSW, 2145, Australia. Tel: 61-2-9845 7365; Fax: 61-2-9893 7440; E-mail: wayneh@med.usyd.edu.au

Cell Transplantation, Vol. 9, pp. 877-887, 2000
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Induction of Allogeneic Islet Tolerance in a Large-Animal Model

Peter J. Horton,1* Wayne J. Hawthorne,1,2 Stacey N. Walters,1 Anita T. Patel,1 Philip J. O'Connell,1,2 Jeremy R. Chapman,1,2 and Richard D. M. Allen1,2

1National Pancreas Transplant Unit, Westmead Hospital, Westmead, NSW, 2145, Australia
2The University of Sydney, Departments of Surgery and Medicine, Westmead Hospital, Westmead, NSW 2145, Australia

For islet allotransplantation to become a therapy widely applicable to patients with insulin-dependent diabetes, it will be important to avoid conventional immunosuppression and yet maintain long-term rejection-free islet survival. This possibility was tested in a large-animal model using mixed allogeneic chimeras established using total lymphoid irradiation (TLI) and donor-specific bone marrow transplantation (BMTX). Four recipient sex-mismatched and DLA class II-matched English springer spaniels became chimeric after TLI and donor-specific BMTX. Subsequent donor-specific renal allografts survived for more than a year. Acceptance of a donor-specific skin graft and rejection of a third-party graft demonstrated tolerance with maintenance of immunocompetence. Pancreatic microfragments containing islets were refluxed into the splenic vein of the recipient. Purified islets were placed under the capsule of spleen and liver. After 75 days, recipients underwent total native pancreatectomy. All four chimeric pancreatectomized dogs had functioning islet grafts 75 days after transplantation, evidenced by a prompt rise in serum insulin levels following an IVGTT and histological demonstration of islet tissue at the site of transplantation. After removal of the transplanted islet tissue, no insulin was released after IVGTT. In summary, intrasplenic allogeneic canine islets transplanted into chimeric dogs rendered tolerant to donor MHC survive and function for greater than 75 days in the absence of immunosuppression. This study represents proof of the concept that allogeneic islet transplants have the potential to reverse diabetes without the use of conventional immunosuppression.

Key words: Allograft; Canine; Islet transplantation; Kidney transplantation; Tolerance

Address correspondence to Wayne J. Hawthorne, The University of Sydney, Department of Surgery, Westmead Hospital, Westmead, NSW, 2145, Australia. Tel: 61-2-98457365; Fax: 61-2-98937440; E-mail:wayneh@med.usyd.edu.au

*Current address: The Nuffield Department of Surgery, The John Radcliffe Hospital, Headley Way, Oxford, OX3 9DU, UK.

Cell Transplantation, Vol. 9, pp. 889-893, 2000
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Simultaneous Pancreas-Kidney (SPK) Transplantation From Controlled Non-Heart-Beating Donors (NHBDs)

Anthony M. D'Alessandro, Jon S. Odorico, Stuart J. Knechtle, Yolanda T. Becker, Robert M. Hoffmann, Munci Kalayoglu, and Hans W. Sollinger

Division of Organ Transplantation, University of Wisconsin Medical School, Madison, WI 53792

From January 1993 through June 1999, 18 simultaneous pancreas-kidney transplants (SPKs) were performed from controlled non-heart-beating donors (NHBDs) and 339 SPKs were performed from heart-beating donors (HBDs). No difference in donor characteristics was noted except for warm ischemic time, which was 14.8 min (range 4-46 min) for NHBDs. Following transplantation, no difference in pancreatic function was noted; however, a higher rate of enteric conversions was seen in pancreas transplants from NHBDs (32% vs. 13%; p < 0.01). Hemodialysis for acute tubular necrosis (ATN) was higher in kidney transplants from NHBDs (22.2% vs. 4.1%; p = 0.009) as was discharge serum creatinine (1.7 mg/dl vs. 1.5 mg/dl; p < 0.05). Also, the number of patients remaining rejection free was lower for NHBDs and approached significance (33.3% vs. 50.1%; p = 0.07). However, no difference in patient survival (100% vs. 95.4%) or pancreatic (87.4% vs. 86.5%) and renal (86.3% vs. 86.3%) allograft survival was noted during the study period. Our results indicate that SPK transplantation from controlled NHBDs is a viable method for increasing the number of pancreas and kidney transplants available for transplantation.

Key words: Pancreas-kidney transplantation; Non-heart-beating donors

Address correspondence to Anthony M. D'Alessandro, M.D., Department of Surgery, University of Wisconsin Hospital & Clinics, 600 Highland Avenue, Madison, WI 53792-7375. Tel: (608) 263-2527; Fax: (608) 263-7652; E-mail: tony@tx.surgery.wisc.edu

Cell Transplantation, Vol. 9, pp. 895-901, 2000
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No Evidence of Infection With Porcine Endogenous Retrovirus in Recipients of Encapsulated Porcine Islet Xenografts

R. B. Elliott,1 L. Escobar,1 O. Garkavenko,2 M. C. Croxson,2 B. A. Schroeder,2 M. McGregor,1 G. Ferguson,1 N. Beckman,1 and S. Ferguson3

1Diatranz Ltd, PO Box 23-566, Papatoetoe, Auckland, New Zealand
2Department of Virology, Auckland Hospital, Private Bag 92-024, Auckland, New Zealand
3Genesis Research Development & Corporation Lt, PO Box 50, Auckland, New Zealand

Transplantation of pig tissues into humans has the potential for cotransferring pig infections. Knowledge of the epidemiology of pig infections transmissible to humans allows the development of risk limitation strategies at the source herd level, but potentially infectious pig endogenous retrovirus (PERV) is ubiquitous in all domestic pigs and therefore is not avoidable. Using a specific and sensitive RT-PCR and nested PCR for PERV nucleic acids with primers, the screening of pigs from New Zealand herds for the presence and expression of the PERV was conducted. The presence of PERV proviral DNA (pol and env region) and viral RNA was demonstrated in all tested pig tissues including pancreas, liver, spleen, brain, heart, and PBMC. Using the same assays it was established that different tissues (liver, spleen, and heart) of nude and nonobese diabetic (NOD) mice previously transplanted with nonencapsulated pig islets were PERV DNA and RNA negative. Alginate polylysine capsules prepared with encapsulated pig islets were tested for possible leakage of viral particles or viral nucleic acids. RNA was extracted from the supernatant of viable encapsulated pig islet cells grown in culture for 2 months. No evidence of PERV RNA or of cellular nucleic acids could be found. Two adult type I diabetic subjects were transplanted with 1 x 106 neonatal pig islets encased in alginate capsules into the peritoneal cavity. One patient was immunosuppressed. Both showed evidence of graft function (up to 34% reduction in insulin dose, corresponding increase in serum pig C-peptide) for up to 2 years. DNA and RNA were extracted from PBMC and blood plasma of both patients at 19 months posttransplant. No evidence of PERV proviral DNA or RNA could be detected. Piglet islets contain PERV DNA and RNA, but this does not traverse the capsules used or produce any evidence of infection in nude and nonobese diabetic (NOD) mice or humans.

Key words: Xenotransplantation; Safety; Pig islets; Diabetes

Address correspondence to R. B. Elliott, Diatranz Ltd, PO Box 23-566, Papatoetoe, Auckland, New Zealand. Tel: (+64 9) 276-2690; Fax: (+64 9) 276-2691; E-mail: elliott@diatranz.co.nz

Cell Transplantation, Vol. 9, pp. 903-908, 2000
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Beneficial Effect of Pancreas and Kidney Transplantation on Advanced Diabetic Retinopathy

R. Koznarová F. Saudek, T. Sosna, M. Adamec, T. Jedináková, P. Boucek, V. Bartos, and V. Lánská

Institute for Clinical and Experimental Medicine, Diabetes Center, Vídenská 1958/9, 140 21 Prague 4, Czech Republic

In pancreas recipients with advanced diabetic eye disease, conflicting ophthalmologic results over different follow-up periods have been reported. In the present prospective study we performed ophthalmologic evaluation groups of type I diabetic patients: 1) normoglycemic recipients of pancreas and kidney grafts (group SPK, n = 43, follow-up 44.9 ± 35.1 months), 2) pancreas and kidney graft recipients with nonfunctioning pancreatic graft, and recipients of isolated kidney graft (group K, n = 45, follow-up 60.3 ± 34.2 months). The examinations were performed before transplantation, at the end of follow-up (at least 1 year), and in 63 recipients also at 3 years posttransplant. Visual acuity results at baseline and at the end of follow-up were 0.48 ± 0.39 vs. 0.50 ± 0.39 in the SPK group, and 0.46 ± 0.38 vs. 0.40 ± 0.39 in the K group. While intragroup changes were not significant, the changes were significantly different between the groups (p < 0.05). Fundoscopic findings at the end of follow-up were improved, stabilized, or deteriorated in the SPK group in 21.3%, 61.7%, and 17.0%, respectively. The respective figures for the K group were 6.1%, 48.8%, and 45.1% (p < 0.001). Similar results were obtained when evaluating findings at 3 years posttransplant. Before transplantation, 78% of the SPK group and 81% of the K group had been treated by laser. The need for additional posttransplant laser therapy was significantly lower in the SPK (31%) than in the K group (58%; p < 0.001). In conclusion, pancreas transplant exerts a beneficial effect on the course of diabetic retinopathy even in its late stage.

Key words: Pancreas and kidney transplantation; Diabetic retinopathy

Address correspondence to Radomíra Koznarová, M.D., Institute for Clinical and Experimental Medicine, Diabetes Center, Vídenská; 1958/9, 140 21 Prague 4, Czech Republic. Tel: 420-2-61362250; Fax: 420-2-61362820; E-mail: radomira.koznarova@medicon.cz

Cell Transplantation, Vol. 9, pp. 909-912, 2000
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Successful Suppression of the Early Rejection of Pig Islets in Monkeys

Josephine K. R. A. Rijkelijkhuizen,1 Eelco Bouwman,1 Michel P. M. van der Burg,1 Jan Ringers,1 Miriam A. Ossevoort,2 Eva M. Kuhn,2 Pat Frost,2 and Margreet Jonker2

1Department of Surgery, Leiden University Medical Centre, 2300 RC Leiden, The Netherlands
2Biomedical Primate Research Centre, 2280 GH Rijswijk, The Netherlands

Primary nonfunction (PNF) is seen very frequently after xenogeneic transplantation of islets of Langerhans. In a pig-to-rat model we recently observed that no PNF occurs when the islets are kept in culture at 37°C for 1-2 weeks prior to transplantation. In order to investigate the rejection mechanisms in a preclinical model, we transplanted cultured porcine islets under the capsule of both kidneys in four cynomolgous monkeys. Islets were isolated from adult sows by means of digestion with Liberase in University of Wisconsin solution (UWS). The digest was purified by a density gradient of OptiPrep in UWS. Highly purified (>95%) islets were cultured 1-2 weeks in RPMI. All monkeys showed significant titers of preformed anti-pig antibodies. The immunosuppression of the monkeys consisted of cyclophosphamide (Cy) (2 days), cyclosporin A (CsA), and prednisolone. Anticipating a fast rejection we carried out nephrectomies at different time points within 2 weeks after transplantation. Following unilateral nephrectomy, well-preserved islets with no signs of rejection were observed between 3 and 7 days posttransplant. Later, between days 11 and 15 posttransplant, histology in the first three animals demonstrated no islets. In the fourth monkey histology on day 11 showed islets with excellent morphology and some small focal infiltrates. The highest CsA blood levels (around 1000 ng/ml) were found in animals with the best graft survival. We conclude that cultured porcine islets can be grafted without hyperacute rejection in monkeys with preformed anti-pig antibodies. In the presence of high levels of CsA only marginal signs of a cellular immune response were observed 11 days after transplantation.

Key words: Islet of Langerhans; Xenotransplantation; Pig; Monkey

Address correspondence to Dr. M. Jonker, Biomedical Primate Research Centre, PO Box 3306, 2280 GM Rijswijk, The Netherlands. Tel: +31 152842687; Fax: +31 152843999; E-mail: jonker@bprc.nl

Cell Transplantation, Vol. 9, pp. 913-918, 2000
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Sustained Improvements in Cardiac Geometry and Function Following Kidney-Pancreas Transplantation

A. Osama Gaber,1 Mona N. Wicks,2 Donna K. Hathaway,1,2 and Brad S. Burlew1

1College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38169
2College of Nursing, University of Tennessee Health Science Center, Memphis, TN 38163

Kidney-pancreas (KP) transplantation has been shown to improve left ventricular (LV) geometry and function 6-24 months after the procedure, yet whether these improvements are sustained in long-term survivors has not been demonstrated. This study examined whether early improvements in LV geometry and function were sustained 3-5 years after KP transplantation. Left ventricular function and geometry were prospectively evaluated prior to, and at 1, 2, and 3-5 years posttransplant using two-dimensional, M-mode, echocardiography with Doppler interrogation in the parasternal and apical views. The sample included 21 KP and a comparison group of 12 diabetic kidney-alone (KA) recipients. Long-term (3-5 years) data were obtained for KP recipients only. Although KA recipients had a longer duration of dialysis and worse diastolic function pretransplant, the groups were similar on other baseline measures. KA recipients experienced minimal improvements while KP recipients had significant improvements in cardiac function and geometry, both in terms of mean values and the percentage of KP recipients who experienced normalization posttransplant (p < 0.05). KP recipient improvements were also sustained at 3-5 years posttransplant on three of five measures, with 75% of long-term KP recipients achieving normal LV mass posttransplant compared with 31% pretransplant. Data indicate that significant impairments in cardiac geometry and function occur in diabetic KA and KP recipients. Though both groups experienced early improvements posttransplant, KP recipients achieved more dramatic and clinically significant improvements at 1, 2, and 3-5 years posttransplant. Additional studies are needed to examine the relevance of these findings with regard to the cardiac morbidity and mortality of these patients.

Key words: Diabetes; Cardiac; Kidney-pancreas transplant; End-stage renal disease

Address correspondence to Mona N. Wicks, Ph.D., University of Tennessee Health Science Center, 877 Madison Avenue, Memphis, TN 38163. Tel: (901) 448-6135; Fax: (901) 448-4121; E-mail: mwicks@utmem.edu

Cell Transplantation, Vol. 9, pp. 919-927, 2000
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Improved Solitary Pancreas Transplant Graft Survival in the Modern mmunosuppressive Era

Jon S. Odorico, Yolanda T. Becker, Marilyn Groshek, Cathy Werwinski, Bryan N. Becker, John D. Pirsch, and Hans W. Sollinger

Division of Organ Transplantation, Department of Surgery, University of Wisconsin Medical School, Madison, WI 53792

The results of solitary pancreas (SP) transplantation have traditionally lagged behind those of simultaneous pancreas-kidney (SPK) transplantation. This is one of the chief factors that has limited the wide-scale application of SP transplantation in nonuremic type I diabetic patients. The purpose of this study is to report our present experience with SP transplantation and compare it to a prior experience. Twenty-three SP transplants (14 PAK, 4 PTA, and 5 PASPK) performed since January 1997 were compared to 56 SP transplants (53 PAK, 1 PTA, and 2 PASPK) performed before 1994. Between 1993 and 1997, SP transplants were not performed because of high morbidity in the early experience. Early SP transplants were performed using bladder drainage of exocrine secretions, and enteric drainage without a Roux-en-Y was used in the recent series. In the early era, immunosuppressive therapy included cyclosporine (CsA), azathioprine (AZA), corticosteroids, and in half of the patients, ALG or OKT3. Recent SP transplants received tacrolimus (TAC), mycophenolate mofetil (MMF), corticosteroids, and induction with either anti-thymocyte globulin (n = 9), OKT3 (n = 1), daclizumab (n = 5), or basiliximab (n = 8). The 1-year Kaplan-Meier patient survival was 85% in the early era and 100% in the recent group of patients (p = 0.08). In the previous era, four patients suffered significant decrement in renal function, necessitating dialysis or kidney transplantation following pancreas transplantation. All patients transplanted since 1997 maintain near prepancreas transplant levels of renal function [mean pretransplant serum creatinine (Cr) 1.3 ± 0.3 mg/dl vs. mean current Cr 1.4 ± 0.4 mg/dl, p = NS). The 1-year Kaplan-Meier graft survival (insulin independence) of recent SP transplants was 87%, whereas for prior SP transplants it was 19% (p = 0.0001). The rate of acute pancreas rejection was significantly different between the two groups. Of early SP transplants, 76% experienced at least one rejection episode within the first year. In contrast, 35% of recent SP transplants suffered acute rejection during the same time period (p = 0.04). Current experience with SP transplantation demonstrates improved graft survival and reduced rejection rates with the use of newer immunosuppressive agents.

Key words: Pancreas transplantation; Immunosuppression; Pancreas transplant biopsy; Enteric drainage

Address correspondence to Jon S. Odorico, M.D., Division of Organ Transplantation, Department of Surgery, University of Wisconsin Medical School, 600 Highland Avenue, Madison, WI 53792-7375. Tel (608) 263-0388; Fax (608) 263-7652.

Cell Transplantation, Vol. 9, pp. 929-932, 2000
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Patient Survival and Cardiovascular Events After Kidney-Pancreas Transplantation: Comparison With Kidney Transplantation Alone in Uremic IDDM Patients

Ennio La Rocca,1 Paolo Fiorina,1 Ettore Astorri,6 Claudio Rossetti,3 Giovanni Lucignani,3 Ferruccio Fazio,3 Daniela Giudici,4 Renato Castoldi,2 Giuseppe Bianchi,5 Valerio Di Carlo,2 Guido Pozza,1 and Antonio Secchi1

Departments of 1Internal Medicine, 2General Surgery, 3Nuclear Medicine, 4Anesthesiology, and 5Nephrology, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, Italy
6Department of Cardiology, University of Parma, Via Gramsci 14, 43100 Parma, Italy

In diabetic patients cardiovascular morbidity and mortality is still a major problem. Our aim was to study the effect of kidney-pancreas transplantation on survival, cardiovascular events, and causes of death in diabetic type I uremic patients. Three hundred and thirty-three uremic IDDM patients were enrolled in our waiting list for kidney-pancreas transplantation: 107 underwent kidney-pancreas transplantation (KP), 34 underwent kidney transplantation alone (KA), whereas 192 patients remained on dialysis (WL). Actuarial survival and causes of death were recorded over a period of 7 years. Seven-year survival rate was 75% for the KP group, 63% for the KA group, and 37% for the WL group (p = 0.001). Cardiovascular death rate was 9.8% in the KP group, 17.6% in the KA group, and 18.1% in the WL group (KP vs. WL, p = 0.05). Rate of acute myocardial infarction in the KP group was lower than in the KA group (2.4% vs. 17.6%, p = 0.005) as well as rate of acute pulmonary edema (0.8% vs. 23.5%, p = 0.0001) and rate of hypertensive patients at 1 (40.9% vs. 85.0%, p = 0.0001) and at 2 years (57.6% vs. 80%, p = 0.03). Kidney-pancreas transplant helped to obtain euglycemia with positive effects on survival and cardiovascular events.

Key words: Pancreas transplantation; Kidney transplantation; IDDM; Cardiovascular disease

Address correspondence to Antonio Secchi, M.D., Medicina I, San Raffaele Scientific Institute, Via Olgettina 60, 20132, Milano, Italy. Tel: 39-02-26432805; Fax: 39-02-26437788; E-mail: Secchi.Antonio@hsr.it