ognizant Communication Corporation

CELL TRANSPLANTATION
The Regenerative Medicine Journal

ABSTRACTS
VOLUME 14, NUMBER 5, 2005

Cell Transplantation, Vol. 14, pp. 241-248, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Delivery of TAT/PTD-Fused Proteins/Peptides to Islets via Pancreatic Duct

Dagmar Klein, Valeska Mendoza, Antonello Pileggi, R. Damaris Molano, Florencia M. Barbé-Tuana, Luca Inverardi, Camillo Ricordi, and Ricardo L. Pastori

Diabetes Research Institute, University of Miami School of Medicine, Miami, FL

Delivering cytoprotective proteins/peptides into pancreata prior to islet isolation through protein transduction (PT) is a novel strategy to enhance the yield of viable transplantable islets. Previous work has shown that the protein transduction domain PTD-5 efficiently transduced islets via the pancreatic duct. TAT/PTD is a well-characterized PTD with the capability to cross even the hemato-encephalic barrier. In this study, we investigated the utilization of the 11-aa TAT protein transduction domain (TAT/PTD) to deliver peptides or proteins of different sizes ranging from 1.2 to 120 kDa, as the TAT/PTD and TAT/PTD-BH4 peptide, or the TAT/PTD-b-galactosidase fusion protein, into islets through the pancreatic duct. Using flow cytometry analysis we found that TAT/PTD derivatives transduced practically 100% of the islet cell population. Moreover, confocal laser scanning microscopy in live, nonfixed islets confirmed these results assessing transduction of TAT/PTD molecules into intact nondisaggregated islets. TAT-b-galactosidase peptide conjugated to FITC was not compartment selective, as both cytoplasmic and nucleic cellular compartments were positively stained. Furthermore, TAT-b-galactosidase peptide delivery was highly effective, as even cells located in the inner core region of the islets were transduced. Finally, transduced TAT-b-galactosidase fusion protein was biologically active after islet isolation and manipulation, and islet insulin secretion capability was not compromised by peptide transduction. These findings suggest that the transduction of chimeric TAT/PTD proteins can represent an efficient tool of molecular delivery independent of the size, to enhance or modify a specific phenotype at the nuclei or cytoplasmic level.

Key words: Islet; Transplantation; Protein transduction; Pancreatic duct

Address correspondence to Ricardo L. Pastori, Ph.D., Diabetes Research Institute, University of Miami School of Medicine, 1450 NW 10th Avenue, Miami, FL 33136. Tel: (305) 243-5349; Fax: (305) 243-4404; E-mail: rpastori@med.miami.edu




Cell Transplantation, Vol. 14, pp. 249-261, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Accelerated Functional Maturation of Isolated Neonatal Porcine Cell Clusters: In Vitro and In Vivo Results in NOD Mice

Giovanni Luca,1 Claudio Nastruzzi,2 Mario Calvitti,3 Ennio Becchetti,3 Tiziano Baroni,3 Luca M. Neri,4 Silvano Capitani,4 Giuseppe Basta,1 Paolo Brunetti,1 and Riccardo Calafiore1

1Department of Internal Medicine (Di.M.I.), Section of Internal Medicine and Endocrine and Metabolic Sciences, University of Perugia, Via E. Dal Pozzo, Perugia 06126, Italy
2Department of Chemistry and Technology of the Drugs, Faculty of Pharmacy, University of Perugia, Via del Liceo 1, Perugia 06123, Italy
3Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, Perugia 06123, Italy
4Department of Morphology and Embryology, Section of Human Anatomy, University of Ferrara, Via Fossato di Mortara 66, Ferrara 44100, Italy

Neonatal porcine cell clusters (NPCCs) might replace human for transplant in patients with type 1 diabetes mellitus (T1DM). However, these islets are not immediately functional, due to their incomplete maturation/differentiation. We then have addressed: 1) to assess whether in vitro coculture of islets with homologous Sertoli cells (SC) would shorten NPCCs' functional time lag, by accelerating the b-cell biological maturation/differentiation; 2) to evaluate metabolic outcome of the SC preincubated, and microencapsulated NPCCs, upon graft into spontaneously diabetic NOD mice. The islets, isolated from <3 day piglets, were examined in terms of morphology/viability/function and final yield. SC effects on the islet maturation pathways, both in vitro and in vivo, upon microencapsulation in alginate/poly-L-ornithine, and intraperitoneal graft into spontaneously diabetic NOD mice were deter-ed. Double fluorescence immunolabeling showed increase in b-cell mass for SC+ neonatal porcine islets versus islets alone. In vitro insulin release in response to glucose, as well as mRNA insulin expression, were significantly higher for SC+ neonatal porcine islets compared with control, thereby confirming SC-induced increase in viable and functional b-cell mass. Graft of microencapsulated SC+ neonatal porcine islets versus encapsulated islets alone resulted in significantly longer remission of hyperglycemia in NOD mice. We have preliminarily shown that the in vitro NPCCs' maturation time lag can dramatically be curtailed by coincubating these islets with SC. Graft of microencapsulated neonatal porcine islets, precultured in Sertoli cells, has been proven successful in correcting hyperglycemia in stringent animal model of spontaneous diabetes.

Key words: Diabetes; Insulin; Therapy; Transplantation; Immunity; Animal models

Address correspondence to Riccardo Calafiore, Department of Internal Medicine (Di.M.I.), University of Perugia, Via E. Dal Pozzo, Perugia 06126, Italy. Tel: +39 075 5783682; Fax: +39 075 5730855; E-mail: islet@unipg.it




Cell Transplantation, Vol. 14, pp. 263-275, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Adenovirus-Mediated CTLA4Ig or CD40Ig Gene Transfer Delays Pancreatic Islet Rejection in a Rat-to-Mouse Xenotransplantation Model After Systemic But Not Local Expression

Nicolas Potiron, Carine Chagneau, Françoise Boeffard, Jean-Paul Soulillou, Ignacio Anegon, and Brigitte Le Mauff

Institut National de la Santé et de la Recherche Médicale (INSERM) UMR643 and Institut de Transplantation et de Recherche en Transplantation, Centre Hospitalo-Universitaire, 30 boulevard Jean Monnet, 44093 Nantes Cedex 01, France

Transient costixation signal blockade of either CD28/CD80-86 interactions and/or CD40/CD154 interactions can prevent islet rejection in some models of both allo- and xenotransplantation. We have used adenoviruses coding for CTLA4Ig or CD40Ig and compared the efficacy of genetic modification of islets to systemic production through either intramuscular (IM) or intravenous (IV) injection of these vectors in a rat-to-mouse islet transplantation model. When gene transfer was performed into islets, a high level of primary nonfunction was induced. Furthermore, transduced functional grafts were rejected with the same kinetics as nontransduced islets. In contrast, IM AdCTLA4Ig and IV AdCD40Ig significantly delayed rejection (mean survival time of 54 ± 26.9 and 67.6 ± 44.9 days, respectively, vs. 24.3 ± 9.7 days for unmodified islets, p < 0.05). Combination of ex vivo AdCTLA4Ig islet transduction and IV AdCD40Ig did not improve graft survival further. In conclusion, islet graft transduction with adenoviruses coding for costixation inhibitors resulted in local expression with low serum concentrations of CTLA4Ig or CD40Ig and was unable to protect islet xenografts from rejection. In contrast, IM or IV gene transfer resulted in high serum concentrations of these molecules and was highly efficient in prolonging xenograft survival. These results contrast with the efficacy of AdCTLA4Ig we observed in a rat islet allotransplantation model and suggest that islet xenograft rejection might be more difficult to control.

Key words: Islet transplantation; Xenotransplantation; Gene transfer; Adenovirus; Costixation; CTLA4Ig; CD40Ig

Address correspondence to Brigitte Le Mauff, INSERM U643, ITERT-CHU, 30 boulevard Jean Monnet, 44093 Nantes cedex 01, France. Tel: 33 240 08 74 12; Fax: 33 240 08 74 10; E-mail: brigitte.lemauff@nantes.inserm.fr




Cell Transplantation, Vol. 14, pp. 277-290, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Experimental Models of Acute and Chronic Liver Failure in Nude Mice to Study Hepatocyte Transplantation

Arnaud Gandillet,1 Isabelle Vidal,1,6 Eliane Alexandre,1 Maxime Audet,1,2 Marie-Pierre Chenard-Neu,3 Jeanne Stutzmann,4 Bruno Heyd,5 Daniel Jaeck,1,2 and Lysiane Richert1,6

1Laboratoire de Chirurgie Expérimentale, Fondation Transplantation, 67200 Strasbourg, France
2Centre de Chirurgie Viscérale et de Transplantation, Hôpital de Hautepierre, 67098 Strasbourg, France
3Service d'Anatomo-Pathologie, Hôpital de Hautepierre, 67098 Strasbourg, France
4INSERM U381, 67200 Strasbourg, France
5Service de Chirurgie Digestive et Vasculaire, Hôpital Jean Minjoz, 25000, Besançon, France
6Laboratoire de Biologie Cellulaire, Faculté de Médecine et de Pharmacie, 25030 Besançon, France

Although hepatocyte transplantation is a promising therapy for acute liver failure in human, there is still a lack of animal models suffering from hepatic injury in which the benefits of hepatocyte transplantation could be evaluated solely, without the bias caused by immunosuppression. As a consequence, the aim of the study was first to develop reproducible models of partial hepatectomy and of thioacetamide (TA)- or Jo2-induced acute liver failure in nude mice. Chronic liver disease was also investigated by repeated injections of sublethal doses of thioacetamide. Survival rates, routine histologic observations, alanin aminotransferase sera content, Ki67, and caspase 3 immunodetection were investigated both after 40% partial hepatectomy and after toxic-induced damages. Liver injuries were more severe and/or precocious in nude mice than in Balb/c mice for a given treatment with a maximum of acute injury obtained 24 h after single toxic injection, and were found to be transitory and reversible within 10 days. Toxics induced apoptosis followed by necrosis, confirming recent published data. Onset of fibrosis leading to reproducible chronic cirrhosis in nude mice correlated with increasing number of Ki67-positive cells, indicating that high levels of cell proliferation occurred. Chronic cirrhosis progressively reversed to fibrosis when the treatment ceased. Preliminary results demonstrated that engrafted xenogeneic hepatocytes could be detected in the host liver by anti-MHC class I immunohistochemistry. Fractions enriched in 2 or 4 hepatocytes by cell sorting using a flow cytometer were equivalent to the unpurified fraction in terms of engraftment in control nude mice or in nude mice subjected to PH. However, in mice suffering from liver injury 24 h after Jo2 or TA treatment, the engraftment of 2 hepatocytes was about twice that of an unpurified hepatocyte population or of a population enriched in 4 hepatocytes.

Key words: Nude mice; Partial hepatectomy; Regeneration; Toxics; Apoptosis; Necrosis; Hepatocyte transplantation

Address correspondence to Eliane Alexandre, Laboratoire de Chirurgie Expérimentale, Fondation Transplantation, 5, Avenue Molière, 67200 Strasbourg, France. Tel: +33 3 88 26 06 26; Fax: +33 3 88 26 12 26; E-mail: eliane_alexandre@yahoo.fr




Cell Transplantation, Vol. 14, pp. 291-299, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

A Large-Scale Automated Method for Hepatocyte Isolation: Effects on Proliferation in Culture

M. J. Nieuwoudt,1,2 E. Kreft,1 B. Olivier,1 S. Malfeld,3 J. Vosloo,4 F. Stegman,5 R. Kunneke,1 A. J. Van Wyk,6 and S. W. Van der Merwe1

1Department of Internal Medicine, University of Pretoria, Pretoria, South Africa
2Department of Bioengineering, University of Pretoria, Pretoria, South Africa
3Department of Immunology, National Health Laboratory Service, Tswane Academic Hospital, Pretoria, South Africa.
4Department of Surgery, Pretoria Academic Hospital, University of Pretoria, Pretoria, South Africa
5Department of Anaesthesiology, Onderstepoort Veterinary Hospital, University of Pretoria, Pretoria, South Africa
6Department of Mechanical and Aeronautical Engineering, University of Pretoria, Pretoria, South Africa

Large-scale sterile methods for isolating hepatocytes are desirable for the development of bioartificial liver support systems. In this study the traditional centrifuge method was compared with the use of a Baylor Rapid Autologous Transfusion (BRAT) machine for isolating large quantities of porcine hepatocytes. After isolating hepatocytes, the methods were evaluated in terms of cell viability and yield per liver, proliferation over 7 days, and the effects on the cell cycle using the trypan blue exclusion test, conventional phase-contrast light microscopy, the lactate to pyruvate ratio, the leakage of lactate dehydrogenase (LD) and aspartate aminotransferase (AST), lidocaine clearance, albumin production, and flow cytometry. With the centrifuge method the mean cell viability was 92.5%, while with the BRAT method the viability was 95.9%. The minimal cell yields with the BRAT procedure were 7.3 x 109 for 250-ml centrifuge bowls and 2.8 x 109 for 165-ml bowls, which compares well with that found by other authors. Because the same initial procedures were employed in both methods the total hepatocyte yield per liver was comparable. Flow cytometry confirmed that the proliferation of hepatocytes was facilitated by oxygenation during the isolation procedure. The recovery of hepatocytes in culture following isolation was similar after either method. Daily microscopic investigation indicated that cytoplasmic vacuolization and granularities were present after either procedure and these disappeared following 3-4 days of culturing. Flow cytometry indicated that the hepatocyte cell cycle was similar after either method; at 7 days the profile indicated that the cells were still proliferating. Trends in the lactate to pyruvate ratio and the leakage of LD and AST indicated that the functional polarity of hepatocytes was regained after approximately 3 days. Lidocaine clearance at 4 days indicated that the cytochrome P450 system was active, while significant albumin production was apparent at day 5. The benefit of using BRAT technology in hepatocyte isolation lies in guaranteed sterility, convenience, speed, and the ability to oxygenate media and cell suspensions during the procedure.

Key words: Bioartificial liver; Isolated hepatocytes; Liver cell isolation; Collagenase liver perfusion

Address correspondence to M. J. Nieuwoudt, Laboratory 2-75, Pathology Building, Dr Savage Rd, Prinshof Campus, University of Pretoria, Pretoria, South Africa. Tel: +27 12 319 2329; Fax: +27 12 328 3600; E-mail: martin@postino.up.ac.za




Cell Transplantation, Vol. 14, pp. 301-309, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Combining Neuroprotective Treatment of Embryonic Nigral Donor Tissue With Mild Hypothermia of the Graft Recipient

Jenny Karlsson,1* Åsa Petersén,1 Gunilla Gidö,2 Tadeusz Wieloch,2 and Patrik Brundin1

1Neuronal Survival Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Sciences, Lund University, SE-221 84 Lund, Sweden
2Laboratory for Experimental Brain Research, Wallenberg Neuroscience Center, Lund University Hospital, SE-221 84 Lund, Sweden
Around 80-95% of the immature dopaminergic neurons die when embryonic ventral mesencephalic tissue is transplanted. Cell death occurs both during the preparation of donor tissue and after graft implantation, but the effect of combining successful neuroprotective treatments before and after transplantation has not been extensively investigated. We therefore treated embryonic rat mesencephalic tissue with a combination of the lipid peroxidation inhibitor tirilazad mesylate (3 mM) and the caspase inhibitor Ac.YVAD.cmk (500 mM) and transplanted the tissue into hemiparkinsonian rats kept hypothermic (32-33°C) or normothermic (37°C) during, and 90 min following, graft surgery. Suspension cell number did not differ between untreated or tirilazad/YVAD-treated preparations prior to transplantation. When graft survival was evaluated 6 weeks after implantation, both tirilazad/YVAD pretreatment and mild hypothermia increased the survival of transplanted dopaminergic neurons. Approximately 50-57% of the embryonic dopaminergic neurons survived the dissociation and grafting procedure in rats rendered hypothermic, but there was no significant additive effect on graft survival with a combined treatment. All groups of rats exhibited behavioral recovery in the amphetamine-induced rotation test. There was a significantly enhanced functional capacity of grafts placed in hypothermic as compared to normothermic rats. However, tirilazad/YVAD pretreated implants did not afford greater behavioral improvement than control-treated grafts. Our results suggest that neuroprotective treatments administered prior to and immediately after neural graft implantation may under certain conditions rescue, at least in part, the same subset of dopaminergic neurons. The study also emphasizes the importance of the immediate time after grafting for transplant survival, with relevance both for primary mesencephalic implants and stem cell grafts.

Key words: Ac.YVAD.cmk; Dopamine; Lazaroid; Mesencephalic transplant; Tirilazad mesylate; Parkinson's disease

Address correspondence to Patrik Brundin, Neuronal Survival Unit, Wallenberg Neuroscience Center, Lund University, BMC A10, SE-221 84 Lund, Sweden. Tel: +46-46-2220529; Fax: +46-46-2220531; E-mail: Patrik.Brundin@medl.lu.se

*Current affiliation: Community Environmental Health Program, University of New Mexico Health Sciences Center, Albuquerque, NM 87131.




Cell Transplantation, Vol. 14, pp. 311-321, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Characterization and Neural Differentiation of Fetal Lung Mesenchymal Stem Cells

Cun Gang Fan,1,2* Feng Wu Tang,1* Qing Jun Zhang,2 Shi Hong Lu,1 Hai Ying Liu,2 Zong Mao Zhao,2 Bin Liu,1 Zhi Bo Han,1 and Zhong Chao Han1

1National Research Center for Stem Cell Engineering & Technology, State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
2Department of Neurosurgery, the Second Hospital of Hebei Medical University, Shijiazhuang, People's Republic of China

Mesenchymal stem cells (MSCs) have been successfully isolated from a broad range of adult, fetal, and other nonembryonic tissues. Fetal lung has been identified as a rich source of MSCs. However, the biological characteristics and differentiation potential of fetal lung MSCs remain to be explored. In this study, we established a series of methods for isolation and expansion of fetal lung MSCs. These MSCs could withstand more than 40 passages without obvious decline in proliferation ability, significant changes in morphology, and expression of cell markers. Flow cytometric analysis showed that fetal lung MSCs expressed CD13, CD29, CD44, CD90, CD105, CD166, and HLA-ABC, but not CD14, CD31, CD34, CD38, CD41a, CD42b, CD45, CD49d, CD61, CD106, CD133, and HLA-DR. Cell cycle analysis revealed that when the MSCs reached their log phase of growth, more than 90% of the cells were in G0/G1 phase while the proportion of cells in S phase and G2/M phase were about 5.56% and 2.08% cells, respectively. These MSCs could differentiate into neural cells in addition to their mesenchymal differentiation potential. Our data suggest that the fetal lung MSC population is an alternative source of stem cells for cell-based therapy of neurological defects or mesenchymal-originating diseases.

Key words: Mesenchymal stem cell; Bone marrow; Fetal lung; Stem cell plasticity; Neural differentiation

Address correspondence to Dr. Zhong Chao Han, Professor, Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin 300020, PR China. Tel: 86 22 27317276; Fax: 86 22 27317273; E-mail: tihzch@public.tpt.tj.cn

*Cun Gang Fan and Feng Wu Tang contributed equally in this study, and should be considered as co-first authors.




Cell Transplantation, Vol. 14, pp. 323-330, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Survival Analysis of Escherichia coli Encapsulated in a Hollow Fiber Membrane In Vitro and In Vivo: Preliminary Report

L. H. Granicka,1 M. Wdowiak,2 A. Kosek,2 S. Swiezewski,3 D. Wasilewska,2 E. Jankowska,4 A. Werynski,1 and J. Kawiak1,2

1PAS, Institute of Biocybernetics and Biomedical Engineering, Warsaw
2Medical Center of Postgraduate Education, Department of Clinical Cytology, Warsaw
3PAS, Institute of Biochemistry and Biophysics, Warsaw
4University Medical Academy, Department of Electron Microscopy Institute of Biostructure, Warsaw

The purpose of the observations was the viability and quality evaluation of E. coli bacteria encapsulated in hollow fiber membranes (HF) in short in vivo and in vitro experiments. A polypropylene, surface-modified hollow fiber was applied for immunoisolation of E. coli bacteria transfected with a green fluorescent protein (E. coli GFPI). The presence of GFP fluorescence of organisms was assessed with the use of flow cytometry. The E. coli GFPIs were then observed for the period of 5 days in in vitro experiments in the culture medium. A single IPTG (isopropyl b-D-1-thiogalactopyranoside) induction of GFP gene appeared to be adequate for an expression of GFP protein for 5 days. The GFP expression values observed for E. coli GFPs encapsulated in HF during culture in different culture media were comparable. The survival of E. coli GFPIs encapsulated in HF after 1, 2, 4, or 5 days of subcutaneous implantation into mice was evaluated. The explanted E. coli GFPIs exhibited mean expression 603 ± 17 (n = 32) units of fluorescence during the implantation period. The values obtained were comparable for selected days of observation. It was observed that the membranes applied ensured the bacteria growth within the HF's space only.

Key words: Hollow fiber; Encapsulation; Escherichia coli bacteria; Implantation

Address correspondence to J. Kawiak, Medical Center of Postgraduate Education, Department of Clinical Cytology, ul. Marymoncka 99, Warsaw, Poland. Tel: +48(22) 834-03-44; Fax: +48(22) 864-08-34; E-mail: jkawiak@cmkp.edu.pl




Cell Transplantation, Vol. 14, pp. 331-337, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Adult-Fetal Fibroblast Interactions: Effects on Cell Migration and Implications for Cell Transplantation

Vlad C. Sandulache,1,2,4,5 Joseph E. Dohar,1,2,5 and Patricia A. Hebda1,2,3,4,5

1Department of Pediatric Otolaryngology, Children's Hospital of Pittsburgh
2Department of Otolaryngology and 3Department of Cell Biology and Physiology, University of Pittsburgh
4Cellular and Molecular Pathology Program, University of Pittsburgh School of Medicine
5McGowan Institute for Regenerative Medicine

Wound healing is a complex process involving close cooperation between xtiple cell types. During wound healing, fibroblasts are primarily responsible for synthesis of the replacement extracellular matrix. Fibroblast therapy is under investigation in this and other laboratories for its potential use to modulate the final outcome of the wound-healing process. This study addresses the potential interactions between transplanted and host fibroblasts, using a two-dimensional mixed culture model. Our results show that fibroblasts of two different phenotypes, fetal and adult, exhibit different speeds of in vitro migration. These migration speeds are conserved in mixed cocultures, suggesting that the migratory response is an intrinsic property of the fibroblast rather than a response to juxtacrine or paracrine signals. These results have relevance for cell-based therapies in that they demonstrate that donor fibroblasts of a different phenotype may at least partially retain that phenotype in the host environment and in the presence of endogenous fibroblasts.

Key words: Fetal fibroblasts; Wound healing; Migration

Address correspondence to Patricia A. Hebda, Ph.D., Department of Pediatric Otolaryngology/Rangos Research Center, Children's Hospital of Pittsburgh, 3460 Fifth Avenue, Pittsburgh, PA 15213. Tel: (412) 692-6217; Fax: (412) 692-5075; E-mail: hebda@pitt.edu