ognizant Communication Corporation

CELL TRANSPLANTATION

ABSTRACTS
VOLUME 10, NUMBER 3

Cell Transplantation, Vol. 10, pp. 235-246
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Isolation and Function of Porcine Islets From Market Weight Pigs

John J. O'Neil, Jan P. Stegemann, Donald T. Nicholson, Kerry A. Gagnon, Barry A. Solomon, and Claudy J.-P. Mullon

Circe Biomedical Inc., 99 Hayden Ave, Lexington, MA 02421

The efficacy of clinical islet transplantation has been demonstrated with autografts, and although islet allografts have established insulin independence in a small number of IDDM patients, the treatment is confounded by the necessity of immunosuppression, the lack of donor tissue, and recurring islet immunogenicity. These limitations underscore a need to develop therapies to serve the large population of diabetic patients. Porcine islet xenotransplantation, together with a successful immune intervention strategy, may provide the necessary clinical alternative. However, a major obstacle in evaluating this approach has been the difficulty of obtaining adequate volumes of functional islet tissue from pigs. Donors of market weight are preferable to retired breeders due to their abundance, lower animal and husbandry costs, and are more suitable to meet regulatory guidelines for donor tissue for xenotransplantation. We describe a simple isolation procedure that following purification yields a mean of 350,000 IE, corresponding to 179 units of insulin and 1.8 mg of DNA with an islet purity and viability in excess of 85% (n = 317 isolations). In both short- and long-term cell cultures, porcine islets demonstrated glucose-responsive insulin secretion. However, this secretion is density dependent, which may have significant consequences in the development of immunoisolation technologies to support porcine islet xenotransplantation. Following implantation into diabetic nude mice, porcine islets remained functional in excess of 1 year. Implantation of a bioartificial pancreas containing porcine islets into pancreatectomized dogs provided significant clinical benefit with an improved diabetic condition. Finally, secretagogue-induced insulin release was demonstrated in vitro from these devices after removal from immunocompetent recipients. Immunohistochemical staining identified well-granulated islets following long-term implantation in both the rodent and canine models. This study demonstrates the ability to isolate porcine islets in clinically relevant numbers from market animals, which survive and remain functional for prolonged periods of time in an immune-deficient or immunoprotected environment.

Key words: Porcine islets; Islet transplantation; Xenotransplantation; Bioartificial pancreas

Address correspondence to John J. O'Neil at his current address: Joslin Diabetes Center, RISLE, One Joslin Place, Boston, MA 02215. Tel: (617) 732-2580; Fax: (617) 732-2650; E-mail: jack.oneil@joslin.harvard.edu.




Cell Transplantation, Vol. 10, pp. 247-253
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Islet Cryopreservation: Improved Recovery Following Taurine Pretreatment

Anandwardhan A. Hardikar,1* Makarand V. Risbud,1 Claude Remacle,2 Brigitte Reusens,2 Joseph J. Hoet,2 and Ramesh R. Bhonde1

1Tissue Engineering and Banking Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411007, India
2WHO Collaborating Centre, Laboratoire de Biologie Cellulaire (BANI/CELL), Universite Catholique de Louvain, Place 5, Croix du Sud, B 1348, Louvain-la-Neuve, Belgium

Simple and efficient freezing methods with maximal postthawing recovery form the basis of ideal cryopreservation. Taurine (2-amino ethanesulfonic acid), an end-product of sulphur amino acid metabolism, is one of the most abundant free amino acids in the body. The membrane stabilizing, free radical scavenging, and osmoregulatory roles of taurine have been well documented. We studied the effect of physiological and supra-physiological concentrations (0.3 and 3.0 mM) of taurine on islet cryopreservation. Islet viability on cryopreservation was significantly improved in both the taurine-treated groups (91.9 ± 2.3% in 0.3 mM and 94.6 ± 1.58% in 3.0 mM group, p < 0.05) compared with the controls (85.7 ± 3.4%). Loss of peripheral islet cells was highly reduced in the taurine group, as examined under phase contrast and quantified by islet morphometric analysis (p < 0.05) using a digital image analysis system. Taurine-treated islets showed significant reduction in lipid peroxidation (0.905 and 0.848 nM MDAmg protein for 0.3 and 3.0 mM taurine, respectively, p < 0.05) compared with control (1.307 nM MDAmg protein) islets. In all, 500 islet equivalents (IE) of treated or control group islets were transplanted to BALB/c mice rendered diabetic with STZ. All animals showed a normal glucose clearance following a glucose load. Graft functionality was confirmed by normoglycemia (fasting plasma glucose: fpg < 150 mg/dl) after transplantation and reappearing hyperglycemia (fpg > 200 mg/dl) following removal of the graft. Suboptimal islet transplantation using 250 IE suggests that the grafted islet mass was inadequate for diabetes reversal. In addition, no significant differences were observed in the islet insulin content between the three groups following cryopreservation of the islets at -196°C. Our studies indicate that taurine pretreatment and its continued presence during islet cryopreservation improves the postthawing viable recovery of islets.

Key words: Islet cryopreservation; Taurine; Postthawing recovery; Transplantation

Address correspondence to Ramesh R. Bhonde, Tissue Engineering and Banking Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411007, India. Tel: 91-20-5670951; Fax: 91-20-5672259; E-mail: rrbhonde@hotmail.com

*Present address: The Pancreas Transplant Unit, Department of Endocrinology, Diabetes and Metabolism, Blacket Building, South Wing, Randwick, Prince of Wales Hospital, Sydney, NSW 2031, Australia.




Cell Transplantation, Vol. 10, pp. 255-262
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Reduction in Primary Nonfunction of Syngeneic Islet Transplants With Nordihydroguaiaretic Acid, a Lipoxygenase Inhibitor

Brend Ray-Sea Hsu, Jyuhn-Huarng Juang, Shin-Huei Fu, Chien-Hung Kuo, and Wen-Tsoung Lu

Department of Endocrinology and Metabolism, Chang-Gung Memorial Hospital, Tao-Yuan Hsien, Taiwan

To study the effectiveness of a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), in the reduction of primary nonfunction, an insufficient number of syngeneic islets were transplanted underneath the renal capsule with NDGA administered daily for 4 weeks. After transplantation of the 150 islets, the decrement of blood glucose levels was significantly faster in the mice that had received NDGA than in the mice that had received no drug at all or dimethyl sulfoxide (DMSO) (p < 0.005, p < 0.05). The mean duration of temporary posttransplant hyperglycemia was 22.3 ± 3.2 (n = 10), 35.9 ± 2.3 (n = 14), and 33.7 ± 4.1 (n = 6) days for the respective groups. The diabetic mice that received 300 islets had their blood glucose levels decrease faster than those that received 150 islets (19.7 ± 1.6 vs. 35.9 ± 2.3 days, n = 14, p < 0.0001). There was no significant difference in the blood glucose reducing effect between the mice that received 150 islets with NDGA and the mice that received 300 islets [22.3 ± 3.2 (n = 10) vs. 19.7 ± 1.6 (n = 14) days, p > 0.05]. The insulin content of the graft from the mice treated with 150 islets and NDGA (3.02 ± 0.24 mg, n = 4) was higher than that from the mice that received 150 islets but no treatment (1.10 ± 0.26 mg, n = 15, p < 0.005) or that had been treated with DMSO (1.21 ± 0.30 mg, n = 4, p < 0.05). The insulin content of the pancreas remnant had no significant differences among the three groups. The net glucose-stimulated insulin secretion was 0.82 ± 0.14 vs. 0.20 ± 0.10 mIU/islet x 60 min (n = 8, p < 0.005) and 0.59 ± 0.08 vs. 0.04 ± 0.02 mIU/islet x 60 min (n = 8, p < 0.0001) for islets cultured without NDGA vs. with NDGA at 1 and 2 weeks, respectively. However, the insulin content of the cultured islets was similar between the two groups for up to 2 weeks of incubation (at 1 week: 0.71 ± 0.01 vs. 0.67 ± 0.04 ng/islet, n = 8, p > 0.05; at 2 weeks: 0.71 ± 0.02 vs. 0.80 ± 0.07 ng/islet, n = 8, p > 0.05). Serum leukotriene B4 (LTB4) concentrations before and between the fifth and seventh days after transplantation were determined. For diabetic mice that received 150 islets, serum LTB4 levels were 25,835 ± 3335 and 27,631 ± 3136 pg/ml (n = 4, p > 0.05). For diabetic mice that received 150 islets and NDGA, the corresponding figures were 22,401 ± 2,706 pg/ml and 27,530 ± 2,190 pg/ml (n = 8, p > 0.05). The graft histology revealed viable islet cells and networks of close vascular structures around the islets and did not reveal microscopic differences among the samples of all four groups. In conclusion, our data revealed that daily administration of NDGA for 4 weeks enhanced isoislet engraftment and preserved three times more mass of the islet beta cells in the isografts. This result indicates that NDGA reduces primary nonfunction of islet syngeneic grafts in diabetic mice.

Key words: Nordihydroguaiaretic acid; Primary nonfunction; Leukotriene; Islet of Langerhans

Address correspondence to Dr. Brend Ray-Sea Hsu, Department of Endocrinology & Metabolism, Chang-Gung Memorial Hospital, Lin-Kou Medical Center, 5 Fu-Shin St. Kwei-Shan County, Tao-Yuan Hsien, Taiwan. Tel: 886-3-328-1200, Ext. 8821; Fax: 886-3-328-8257; E-mail: bh0658@adm.cgmh.org.tw




Cell Transplantation, Vol. 10, pp. 263-275
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Poly-L-Lysine Induces Fibrosis on Alginate Microcapsules via the Induction of Cytokines

Berit L. Strand,1,2 Liv Ryan,2 Peter In't Veld,3 Bård Kulseng,2 Anne Mari Rokstad,2 Gudmund Skjåk-Bræk,1 and Terje Espevik2

1Department of Biotechnology, and 2Department of Cancer Research and Molecular Biology, Norwegian University of Science and Technology, Trondheim, Norway
3Department of Metabolism and Endocrinology, Free University of Brussels, Brussels, Belgium

Alginate-poly-L-lysine (PLL) microcapsules can be used for transplantation of insulin-producing cells for treatment of type I diabetes. In this work we wanted to study the inflammatory reactions against implanted microcapsules due to PLL. We have seen that by reducing the PLL layer, less overgrowth of the capsule is obtained. By incubating different cell types with PLL and afterwards measuring cell viability with MTT, we found massive cell death at concentrations of PLL higher than 10 mg/ml. Staining with annexin V and propidium iodide showed that PLL induced necrosis but not apoptosis. The proinflammatory cytokine, tumor necrosis factor (TNF), was detected in supernatants from monocytes stimulated with PLL. The TNF response was partly inhibited with antibodies against CD14, which is a well-known receptor for lipopolysaccharide (LPS). Bactericidal permeability increasing protein (BPI) and a lipid A analogue (B-975), which both inhibit LPS, did not inhibit PLL from stimulating monocytes to TNF production. This indicates that PLL and LPS bind to different sites on monocytes, but because they both are inhibited by a p38 MAP kinase inhibitor, they seem to have a common element in the signal transducing pathway. These results suggest that PLL may provoke inflammatory responses either directly or indirectly through its necrosis-inducing abilities. By combining soluble PLL and alginate both the toxic and TNF-inducing effects of PLL were reduced. The implications of these data are to use alginate microcapsules with low amounts of PLL for transplantation purposes.

Key words: Alginate-poly-L-lysine microcapsule; Fibrosis; Cytokines; Necrosis

Address correspondence to Berit L. Strand, Department of Biotechnology, NTNU, Sem Saelandsvei 6/8, 7491 Trondheim, Norway. Tel: (47) 7359-0662; Fax: (47) 7359-1283; E-mail: Berit.Loeken.Strand@chembio.ntnu.no




Cell Transplantation, Vol. 10, pp. 277-284
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Influence of Donor Age on Mouse Islet Characteristics and Transplantation

Jyuhn-Huarng Juang,1 Brend Ray-Sea Hsu,1 Chien-Hung Kuo,1 and Nan-kuang Yao2

1Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan
2Microsystem Laboratory, Industrial Technology Research Institute, Hsinchu, Taiwan

Old donor age has been considered as a risk factor and relative contraindication for transplantation. This study was designed to investigate the influence of donor age on islet characteristics and transplantation. Islets isolated from 8 (I-A)-, 32 (I-B)-, or 64 (I-C)-week-old C57BL/6 mice were studied for number, size, insulin content, and secretion. After syngeneically transplanting 300 islets under the kidney capsule of streptozotocin-diabetic mice (R-A, R-B, and R-C, respectively), we measured recipients' metabolic parameters as well as the b-cell mass and insulin content of the graft. Eight-week-old donors had better glucose tolerance than 32- and 64-week-old donors. However, 64-week-old donors had more pancreatic insulin content than 8- and 32-week-old donors. I-B and I-C were greater in number, larger in size, and higher in insulin content than I-A. But perifusion study showed I-C secreted less insulin, albeit with a similar stimulation index compared with that of I-A and I-B. After transplantation, the fall of blood glucose in R-C was faster than that in R-A and R-B. At 12 weeks, the recipients' blood glucose, body weight, HbA1c, and the b-cell mass and insulin content of the graft were comparable in all groups. However, R-C had better glucose tolerance than R-A. During follow-up, R-A and R-B maintained lifelong normoglycemia and their glucose tolerance did not deteriorate. These data indicate that islets isolated from donors with different ages have different characteristics and effects on transplantation. The islets isolated from aged donors are functioning well and can be a potential source for transplantation; however, because we transplanted a large islet mass from the aged donors, the role of the islet dose needs to be further clarified.

Key words: Donor age; Pancreatic islet cells; Islet transplantation

Address correspondence to Dr. Jyuhn-Huarng Juang, Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, 5 Fu-Shin Street, Kweishan, Taoyuan, Taiwan. Tel: +886-3-3277976; Fax: +886-3-3277976; E-mail: jjuang@adm.cgmh.org.tw




Cell Transplantation, Vol. 10, pp. 285-293
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Role of Pancreatic Polypeptide as a Market of Transplanted Insulin-Producing Fetal Pig Cells

Bernard E. Tuch, Muhammad T. Tabiin, Frances M. Casamento, Mu Yao, Pauline Georges, Anil Amaratunga, and Angie N. Pinto

Pancreas Transplant Unit, The Prince of Wales Hospital, Sydney, New South Wales, Australia

Transplantation of insulin-producing fetal pancreatic tissue into diabetic recipients has been shown to normalize blood glucose levels after several months. This time period is required for the growth and maturation of the fetal tissue so insulin levels cannot be used as a marker of graft function while the \GK\b-cell is immature. Therefore, we have examined the use of another pancreatic endocrine hormone, pancreatic polypeptide (PP), to monitor graft function. The cell that produces this hormone has been shown to be the first mature endocrine cell in the fetal pancreas. Fetal pig pancreatic tissue, both in the form of 1 mm3 explants and islet-like cell clusters (ICCs), was transplanted into immunodeficient SCID mice and the levels of PP and insulin were measured in plasma and in the graft for up to 12 weeks. PP was detected in the untransplanted explants (0.58 pmol/mg) and ICCs (0.06 pmol/ICC) and the PP to insulin ratio was 2.7% and 5.8%, respectively. PP (but not porcine C-peptide, a marker of insulin secretion) was detectable in the plasma of SCID mice from 4 days to 3 weeks after transplantation, but not thereafter. The highest values were obtained at 4 days to 1 week. In the grafted tissue PP and insulin were present at all time points and the ratio of PP to insulin was 59%, 87%, 75%, 56%, 7%, 8%, and 7% at 4 days, 1, 2, 3, 6, 9, and 12 weeks, respectively. The decline in PP levels 3 weeks after transplantation was associated with b-cell development in the graft. PP was also secreted by fetal pig pancreatic explants transplanted into diabetic NOD/SCID mice, with plasma levels measurable in the first week after the tissue was grafted. In immunocompetent BALB/c mice transplanted with the tissue, PP was detectable in plasma for 2 days after transplantation but not at 4 days, when cellular rejection commenced, or thereafter. We conclude that plasma PP levels can be used as a marker of the viability of fetal porcine pancreatic tissue in the first 3 weeks after it is transplanted into mice. These findings may have relevance to fetal pancreatic tissue transplanted into humans if suitable techniques can be developed to separate pig from human PP.

Key words: Pancreatic polypeptide; Fetal pig pancreas; Diabetes; SCID mice; Xenotransplantation

Address correspondence to Bernard Tuch, M.D., Ph.D., Pancreas Transplant Unit, Department of Endocrinology, Diabetes and Metabolism, Prince of Wales Hospital, High Street, Randwick, NSW 2031 Australia. Tel: 61-2-9382 4814; Fax: 61-2-9382 4826; E-mail: b.tuch@unsw.edu.au




Cell Transplantation, Vol. 10, pp. 295-304
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Enhanced Survival of Porcine Neural Xenografts in Mice Lacking CD1d1, But No Effect of NK1.1 Depletion

Lena C. Larsson,1 Per Anderson,1 H&aring;kan Widner,1 and Olle Korsgren2

1Wallenberg Neuroscience Center, Lund University, S&ouml;lvegatan 17, S-223 62 Lund, Sweden
2Department of Clinical Immunology and Transfusion Medicine, Rudbeck Laboratory, Uppsala University, S-751 85 Uppsala, Sweden

Transplantation of embryonic porcine neurons may restore neurological function in patients with Parkinson's disease, if immunological rejection could be prevented. This study was performed to investigate the role of natural killer cells (NK cells) and NK1.1+ T cells (NK T cells) in the rejection of neural xenografts. A cell suspension was prepared from the ventral mesencephalon of 26-27-day-old pig embryos, and 2 ml was implanted in the right striata of mutant CD1d1 null (CD1.1-/-) mice, NK1.1-depleted mice, and controls. The CD1.1-/- mice are deficient in NK T cells and the antigen-presenting molecule CD1d1. Graft survival and host responses were determined immunohistochemically using markers for dopamine neurons, CD4-, CD8- cells, microglia, and macrophages. At 2 weeks, the grafts were significantly larger in CD1.1-/- mice, 0.09 ± 0.02 ml (mean ± SEM), compared with controls, 0.05 ± 0.01 ml. There was no significant difference between NK1.1-depleted mice, 0.02 ± 0.01 ml, and controls. At 5 weeks, two grafts were still present in the CD1-/- mice, whereas only scars remained in the controls and in the NK1.1-depleted mice. Immune reactions were strong at 2 weeks and less pronounced at 5 weeks in all groups. Microglial activation was lower in NK-depleted mice than in the controls at 2 weeks. In contrast to organ xenografting, NK1.1+ cells do not seem to be important mediators of the rejection of discordant cellular neural xenografts. However, our results suggest that the antigen-presenting molecule CD1d1 may be involved in the rejection process.

Key words: Xenotransplantation; NK cells; CD1; Parkinson's disease; Brain

Address correspondence to Lena Larsson, Section for Neuronal Survival, Wallenberg Neuroscience Center, S&ouml;lvegatan 17, S-223 62, Lund, Sweden. Tel: +46 46 222 05 63; Fax: +46 46 222 05 31; E-mail: lena.larsson@mphy.lu.se




Cell Transplantation, Vol. 10, pp. 305-315
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Remyelination of Demyelinated CNS Axons by Transplanted Human Schwann Cells: The Deleterious Effect of Contaminating Fibroblasts

C. M. H. Brierley,1 A. J. Crang,2 Y. Iwashita,2 J. M. Gilson,2 N. J. Scolding,3 D. A. S. Compston,1 and W. F. Blakemore2

1Department of Neurology and Cambridge Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK
2Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, UK
3Department of Clinical Neurosciences, Frenchay Hospital, Bristol BS16 1LE, UK

Areas of demyelination can be remyelinated by transplanting myelin-forming cells. Schwann cells are the naturally remyelinating cells of the peripheral nervous system and have a number of features that may make them attractive for cell implantation therapies in multiple sclerosis, in which spontaneous but limited Schwann cell remyelination has been well documented. Schwann cells can be expanded in vitro, potentially affording the opportunity of autologous transplantation; and they might also be spared the demyelinating process in multiple sclerosis. Although rat, cat, and monkey Schwann cells have been transplanted into rodent demyelinating lesions, the behavior of transplanted human Schwann cells has not been evaluated. In this study we examined the consequences of injecting human Schwann cells into areas of acute demyelination in the spinal cords of adult rats. We found that transplants containing significant fibroblast contamination resulted in deposition of large amounts of collagen and extensive axonal degeneration. However, Schwann cell preparations that had been purified by positive immunoselection using antibodies to human low-affinity nerve growth factor receptor containing less than 10% fibroblasts were associated with remyelination. This result indicates that fibroblast contamination of human Schwann cells represents a greater problem than would have been appreciated from previous studies.

Key words: Human Schwann cells; Schwann cell transplantation; Remyelination; Fibroblasts

Address correspondence to Professor William Blakemore, Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, UK. Tel: 01223-337639; Fax: 01223-337610; E-mail: wfb1000@hermes.cam.ac.uk




Cell Transplantation, Vol. 10, pp. 317-327
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Posterior Segment Approach for Subretinal Transplantation or Injection in the Canine Model

Maria E. Verdugo,1 Julie Alling,1 Eliot S. Lazar,2 Manuel del Cerro,2,3 Jharna Ray,1 and Gustavo Aguirre1

1James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853
Departments of 2Ophthalmology and 3Neurobiology and Anatomy, University of Rochester School of Medicine, Rochester, NY 14642

A posterior segment approach for cell transplantation or injection into the subretinal space of the dog has been developed. Controlled penetration to the subretinal space was achieved using a 29-gauge injection cannula, either blunted or with a 30° sharpened bevel, and partially ensheathed with moveable plastic tubing. Depending on the injection volume used, the retina detached, and the fluid was reabsorbed within 1-3 weeks, although for smaller volumes the retina reattached within a matter of days. The optimal injection volume used was between 100 and 150 ml, or two injections of 55 ml each. By ophthalmoscopy following the surgery, it was possible to serially monitor the injection site and retinal bleb through fundus photography. Light microscopy demonstrates the distribution of stable, viable RPE cells in the subretinal space up to 6 months. The transplantation technique developed for the dog is atraumatic and free from any major surgical or clinical complications. It can be readily used to deliver cells or fluids to localized regions of the subretinal space.

Key words: Animal model; Dog; Posterior segment; Retinal pigment epithelial transplantation; Subretinal injection; Retinitis pigmentosa

Address correspondence to Gustavo Aguirre, V.M.D., Ph.D., James A. Baker Institute of Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. Tel: (607) 256-5620; Fax: (607) 256-5689; E-mail: gda1@cornell.edu




Cell Transplantation, Vol. 10, pp. 329-342
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Probing Enhanced Cytochrome P450 2B1/2 Activity in Rat Hepatocyte Spheroids Through Confocal Laser Scanning Microscopy

Emmanouhl S. Tzanakakis,1 Chang-Chun Hsiao,1 Taku Matsushita,2 Rory P. Remmel,3 and Wei-Shou Hu1

1Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455-0132
2Department of Applied Life Science, Sojo University, Ikeda, Kumamoto 860-0082, Japan
3Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455-0132

Cytochrome P450 (CYP450) enzymes are essential for xenobiotic metabolism. Although CYP450s are found in many tissues, CYP2B1/2 are primarily expressed in the rat liver. The constitutive expression in vivo of CYP2B1/2 is low but it is induced in the presence of various drugs such as phenobarbital (PB). In this study, CYP2B1/2 activity in cultured hepatocytes was assessed in situ with the introduction of a fluorogenic substrate, pentoxyresorufin. The product of 7-pentoxyresorufin-O-dealkylation (PROD), which is catalyzed specifically by CYP2B1/2, was detected using confocal laser scanning microscopy (CLSM). Primary hepatocytes cultured as monolayers on collagen-coated surfaces exhibited background PROD activity and minimal PB inducibility after 4 days in culture. In contrast, rat hepatocytes organized in compacted aggregates, or spheroids, exhibited higher levels of PROD activity and retained their ability for PB induction. The results from the CLSM analysis were verified by RT-PCR and Western immunoblotting analysis. Furthermore, CLSM in conjunction with image processing techniques and three-dimensional reconstruction revealed the localization of enhanced PROD activity in the center of spheroids. The results support the use of CLSM as a powerful tool for investigating CYP2B1/2 activity in cultured rat hepatocytes.

Key words: Hepatocytes; Self-assembly; PROD; CLSM; Deconvolution; Three-dimensional reconstruction

Address correspondence to Wei-Shou Hu, Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis MN 55455. Tel: (612) 625-0546; Fax: (612) 626-0587; E-mail address: wshu@cems.umn.edu or Rory P. Remmel, Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455. Tel: (612)624-0472; Fax: (612)624-0139; E-mail: remme001@tc.umn.edu




Cell Transplantation, Vol. 10, pp. 343-350
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Liver-Derived Dendritic Cells Induce Donor-Specific Hyporesponsiveness: Use of Sponge Implant as a Cell Transplant Model

Yang-Jen Chiang, Lina Lu, John J. Fung, and Shiguang Qian

Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 152123

Spontaneously accepted mouse liver allografts are capable of protecting subsequently transplanted donor organs from rejection; however, the underlying mechanisms are unclear. Dendritic cells (DC) residing in liver grafts are likely important in tolerance induction. DC propagated from mouse liver with GM-CSF are phenotypically and functionally immature. They are poor allostimulators in MLR and prolong survival of pancreatic islet allografts. It has been problematic to perform mechanistic studies in an islet transplant model because of difficulties in obtaining sufficient graft infiltrating cells. In this study, we used a sponge allograft model [i.e., a subcutaneously implanted sponge matrix loaded with B10 (H2b) spleen cells]. To investigate the influence of administration of donor (B10) liver-derived DC on alloimmune reactivity of C3H (H2k) hosts, sponge graft infiltrating cells (SGIC) and recipient spleen cells were isolated, and their immunophenotype and donor-specific cytotoxic T lymphocyte (CTL) activity were examined. The results illustrate that donor-specific CTL activity of T cells are lower in recipients that had received systemic treatment with liver-derived immature DC, associated with a decrease in CD8+ cell population and an increase in Gr-1+ cells in SGIC, compared with recipients treated with mature bone marrow (BM)-derived DC. Interestingly, administration of liver DC directly into the sponge did not inhibit T cell responses. These data suggest that systemic administration of donor liver DC induces donor-specific hyporesponsiveness, probably not by direct inhibition of graft infiltrating T cells. The increased Gr-1+ cells may play immune regulatory roles in induction of host donor-specific hyporesponsiveness.

Key words: Dendritic cell; Liver; Cell transplant; T lymphocyte

Address correspondence to Shiguang Qian, M.D., Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh Medical Center, E1540 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15213. Tel: (412) 624-6611; Fax: (412) 624-6666; E-mail: qians@msx.upmc.edu