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• W2058262020 abstract "Assuming that neogenesis contributes to long-term function of islet grafts, it is important to study the effects of immunosuppressive drugs on precursor cell proliferation and differentiation. We examined the effects of low-dose immunosuppressive drugs on these processes in vitro Immunosuppressive drugs, including sirolimus, tacrolimus, mycophenolate mofetil (MMF), daclizumab and their combinations were tested in parallel culture wells through either the expansion phase (5–7 days) or the entire culture period (4–5 weeks). MMF, alone or in combination with sirolimus or tacrolimus, severely hampered duct-cell proliferation by 8-fold during the expansion period, and significantly reduced the total DNA content by about 40% after 5-week culture. After 4–5 week exposure to different drugs, only sirolimus and daclizumab showed no adverse effects on insulin content, whereas significant reductions of 30–60% in insulin content were seen in all other experimental groups. Only tacrolimus decreased the insulin content per DNA, as well as the proportion of insulin-positive cells. In conclusion, MMF has a potent inhibitory effect on neogenesis primarily through an antiproliferative effect on the precursors, whereas tacrolimus mainly affects beta-cell differentiation. Sirolimus and daclizumab have no adverse effects on these parameters. The immunosuppressive protocol may be an important determinant of long-term clinical islet graft function. Assuming that neogenesis contributes to long-term function of islet grafts, it is important to study the effects of immunosuppressive drugs on precursor cell proliferation and differentiation. We examined the effects of low-dose immunosuppressive drugs on these processes in vitro Immunosuppressive drugs, including sirolimus, tacrolimus, mycophenolate mofetil (MMF), daclizumab and their combinations were tested in parallel culture wells through either the expansion phase (5–7 days) or the entire culture period (4–5 weeks). MMF, alone or in combination with sirolimus or tacrolimus, severely hampered duct-cell proliferation by 8-fold during the expansion period, and significantly reduced the total DNA content by about 40% after 5-week culture. After 4–5 week exposure to different drugs, only sirolimus and daclizumab showed no adverse effects on insulin content, whereas significant reductions of 30–60% in insulin content were seen in all other experimental groups. Only tacrolimus decreased the insulin content per DNA, as well as the proportion of insulin-positive cells. In conclusion, MMF has a potent inhibitory effect on neogenesis primarily through an antiproliferative effect on the precursors, whereas tacrolimus mainly affects beta-cell differentiation. Sirolimus and daclizumab have no adverse effects on these parameters. The immunosuppressive protocol may be an important determinant of long-term clinical islet graft function. Introduction of glucocorticoid-free immunosuppressive protocols has been one of the main reasons for the improved success of clinical islet transplantation in type 1 diabetes (1Shapiro AM Lakey JR Ryan EA et al.Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen [see comments].N Engl J Med. 2000; 343: 230-238Crossref PubMed Scopus (4429) Google Scholar). Although immunosuppressive medication is still mandatory to prevent islet graft rejection, the beta-cell toxicity of these drugs may be an important issue for long-term outcome. This has been studied experimentally both in vivo and in vitro (2Ricordi C Zeng YJ Alejandro R et al.In vivo effect of FK506 on human pancreatic islets.Transplantation. 1991; 52: 519-522Crossref PubMed Scopus (97) Google Scholar, 3Fabian MC Lakey JRT Rajotte RV Kneteman NM The efficacy and toxicity of rapamycin in murine islet transplantation: In vitro and in vivo studies.Transplantation. 1993; 56: 1137-1142Crossref PubMed Scopus (57) Google Scholar, 4Paty BW Harmon JS Marsh CL Robertson RP Inhibitory effects of immunosuppressive drugs on insulin secretion from HIT-T15 cells and Wistar rat islets.Transplantation. 2002; 73: 353-357Crossref PubMed Scopus (107) Google Scholar, 5Polastri L Galbiati F Bertuzzi F et al.Secretory defects induced by immunosuppressive agents on human pancreatic beta-cells.Acta Diabetol. 2002; 39: 229-233Crossref PubMed Scopus (55) Google Scholar, 6Bell E Cao X Moibi JA et al.Rapamycin has a deleterious effect on MIN-6 cells and rat and human islets.Diabetes. 2003; 52: 2731-2739Crossref PubMed Scopus (153) Google Scholar, 7Fotiadis C Xekouki P Papalois AE et al.Effects of mycophenolate mofetil vs cyclosporine administration on graft survival and function after islet allotransplantation in diabetic rats.World J Gastroenterol. 2005; 11: 2733-2738Crossref PubMed Scopus (11) Google Scholar). It appears that many of the drugs commonly used, including sirolimus, tacrolimus and mycophenolate, do have negative effects on beta-cell function and viability in vitro (4Paty BW Harmon JS Marsh CL Robertson RP Inhibitory effects of immunosuppressive drugs on insulin secretion from HIT-T15 cells and Wistar rat islets.Transplantation. 2002; 73: 353-357Crossref PubMed Scopus (107) Google Scholar). In particular, tacrolimus has been shown to cause serious islet cell damage and abnormal glucose metabolism in experimental animal models as well as in diabetic patients receiving pancreas allograft (2Ricordi C Zeng YJ Alejandro R et al.In vivo effect of FK506 on human pancreatic islets.Transplantation. 1991; 52: 519-522Crossref PubMed Scopus (97) Google Scholar,8Drachenberg CB Klassen DK Weir MR et al.Islet cell damage associated with tacrolimus and cyclosporine: Morphological features in pancreas allograft biopsies and clinical correlation.Transplantation. 1999; 68: 396-402Crossref PubMed Scopus (295) Google Scholar). The studies published so far have mainly focused on the direct drug effects on differentiated beta cells. However, it is likely that long-term function of islet grafts is dependent on mechanisms maintaining a functional beta-cell mass through neogenesis from islet precursors. A recent report has shown a positive correlation between the transplanted ductal cells and the outcome of clinical islet transplantation (9Street CN Lakey JR Shapiro AM et al.Islet graft assessment in the Edmonton protocol: Implications for predicting long-term clinical outcome.Diabetes. 2004; 53: 3107-3114Crossref PubMed Scopus (189) Google Scholar). A study by the same group further showed that sirolimus decreased the number of cultured human ductal cells (10Bussiere CT Lakey JR Shapiro AM Korbutt GS The impact of the mTOR inhibitor sirolimus on the proliferation and function of pancreatic islets and ductal cells.Diabetologia. 2006; 49: 2341-2349Crossref PubMed Scopus (98) Google Scholar). Using cultures of adult human pancreatic cells, we have shown previously that after expansion in monolayer, duct-like epithelial cells can be induced to form three-dimensional cysts with budding islet cells in serum-free medium with overlaid Matrigel. These cell clusters are called ‘cultivated human islet buds’ (CHIBs). This process of islet neogenesis also involves the dedifferentiation of preexisting islet cells (11Gao R Ustinov J Pulkkinen MA Lundin K Korsgren O Otonkoski T Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture.Diabetes. 2003; 52: 2007-2015Crossref PubMed Scopus (234) Google Scholar,12Gao R Ustinov J Korsgren O Otonkoski T In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells.Diabetologia. 2005; 48: 2296-2304Crossref PubMed Scopus (75) Google Scholar). We have now used this in vitro model of human islet neogenesis to identify potential deleterious effects of some immunosuppressive drugs commonly used in clinical islet transplantation, on the proliferation and differentiation of human islet precursor cells in vitro Human islets were isolated according to previously described methods (13Ricordi C Lacy PE Finke EH Olack BJ Scharp DW Automated method for isolation of human pancreatic islets.Diabetes. 1988; 37: 413-420Crossref PubMed Scopus (1174) Google Scholar) in Uppsala, Sweden. After Ficoll gradient purification, the mixed fractions rich in ductal fragments and poor in islets were collected and shipped on ice to Biomedicum Helsinki for subsequent culture. Pancreatic tissues from eight donors were used in this study. Based on insulin immunostaining, the beta-cell proportions in the starting materials ranged from 1% to 17% (8%± 2%, n = 8). All procedures were approved by Institutional Ethical Committees in Sweden and Finland. As described before (11Gao R Ustinov J Pulkkinen MA Lundin K Korsgren O Otonkoski T Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture.Diabetes. 2003; 52: 2007-2015Crossref PubMed Scopus (234) Google Scholar), the fresh pancreatic cell clusters (∼400 clusters per well, approximately 106 cells) were added in six-well plates (Corning, Corning, NY) with CMRL 1066 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (PromoCell, Heidelberg, Germany). Only about 25% of the starting tissue attached and grew into monolayer. After 5–7-day expansion, the monolayers mainly consisted of CK19-positive ductal epithelial cells (11Gao R Ustinov J Pulkkinen MA Lundin K Korsgren O Otonkoski T Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture.Diabetes. 2003; 52: 2007-2015Crossref PubMed Scopus (234) Google Scholar). Then the media were changed into serum-free DMEM/F12 (National Public Health Institute, Helsinki, Finland) supplemented with ITS (5 mg/L insulin + 5 mg/L transferrin + 5 μg/L sodium selenite; Sigma, St. Louis, MO), 2 g/L bovine serum albumin, 8 mmol/L glucose, 10 mmol/L nicotinamide (Sigma). The cells were then overlaid with Matrigel, a basement membrane preparation from Engelbreth-Holm-Swarm mouse tumor cells (BD Biosciences, Bedford, MA) according to manufacturer’s instructions with the exception of dilution (1:10) and overnight gelling time at 37°C. Within 2–3 weeks after gel overlay, the cells were collected for further analyses. The drugs and their concentrations were selected based on plasma drug levels in protocols currently used in clinical islet transplantation. As described above, control condition was the culture in CMRL 1066 medium plus 10% FCS for monolayer proliferation and then in serum-free DMEM/F12 medium with the supplements for differentiation. Drug tests were performed in parallel culture wells either for the first 3 days of culture to observe the effect on ductal cell proliferation or through both proliferation and differentiation phases (4–5 weeks) in the same medium as the control culture but with the addition of one or more of the following agents: 10 ng/mL sirolimus (dilution 1:100:000 from 1 mg/mL stock, Wyeth Europa Ltd, Berkshire, UK), 5 ng/mL tacrolimus (dilution 1:1000:000 from 5 mg/mL stock, Fujisama GmbH, Munich, Germany), 20 μg/mL mycophenolate mofetil (MMF, dilution 1:1000 from 20 mg/mL solution made in sterile water, Roche, Basel, Switzerland), 10 μg/mL daclizumab (dilution 1:500 from 5 mg/mL stock, Roche). All drugs were diluted to final concentrations in culture medium from original stock of solution before each experiment, and administrated with the medium changes every 2–3 days. To compare endocrine cell proportions in different culture conditions, the recovered cell clusters were dissociated with 0.05% trypsin and 0.02% EDTA, washed in PBS and spun to microscope slides (SuperFrost®Plus, Menzel, Braunschweig, Germany) by centrifugation at 700 rpm for 8 min. The cytospin slides were fixed in 4% PFA for 15 min and rinsed in PBS. For the observation of drug effects on cell proliferation, the monolayer cells were incubated with different drugs for 3 days, then collected and fixed in 4% PFA for 30 min. After rinsing with PBS, the cells were suspended in 2% agarose-PBS solution, and centrifuged to form compact pellets, which were further embedded in paraffin for sectioning. Immunostaining was carried out using the following primary antibodies: guinea pig anti-porcine insulin 1:100; rabbit anti-human glucagon 1:500; mouse anti-human cytokeratin 19 (CK19) 1:100 (DAKO, Carpinteria, CA); rabbit anti-human Ki67 1:5000 (Novocastra, Newcastle, UK). Nonspecific binding was blocked by preincubation in 3% normal goat serum (ZYMED, San Francisco, CA) followed by incubation of primary antibodies for 1 h or overnight at room temperature. Microwave treatment at 300 W in 1 mM EDTA for 30 min was necessary to retrieve the antigenicity of Ki67. Biotinylated goat anti-rabbit IgG (ZYMED, 1:200) was used as secondary antibody. Peroxidase conjugated streptavidin (ZYMED, 1:200) was used by developing the substrate of 3-amino-9-ethylcarbazole. Light counterstaining was performed with hematoxylin. For double immunofluorescence staining of Ki67 and CK19, the conjugated secondary antibodies, FITC-conjugated donkey anti-rabbit and TRITC-conjugated donkey anti-mouse IgGs (1:50; Jackson ImmunoResearch, West Grove, PA), were used. The cell proliferation under different culture conditions was examined by Ki67 staining. The sections were photographed and morphometrically analyzed using Image-Pro Plus 4.5 software (Media Cybernetics Inc, Silver Spring, MD). The results were expressed as the percentage of Ki67-positive nuclei from total area covered by cells including cytoplasm and nuclei. For the measurement of insulin and DNA contents, the collected cells from different experimental conditions were washed 3–4 times in PBS, resuspended in 300 μL distilled cold water and homogenized by sonication on ice. An aliquot of the homogenates in duplicate was analyzed enzymatically for the DNA content, and another extracted with acid ethanol overnight and measured for insulin content using a solid-phase RIA kit (DPC, Los Angeles, CA). The differences of cell proliferation, insulin and DNA contents, proportions of endocrine cells and CHIB numbers at various experimental conditions were analyzed with one-way analysis of variance and the Fischer’s PLSD test at 95% significance level for multiple comparisons (Statview 4.1, Abacus, Berkeley, CA). During the first 3 days in culture, more than 90% of the proliferating cells were CK19-positive ductal epithelial cells (Figure 1A). After 3-day incubation with different drugs, MMF and its combination with sirolimus or tacrolimus induced a 90% reduction in the cell proliferation in comparison with the control condition, as measured by the relative cell surface area positive for the Ki67 antigen. In contrast to the significant negative effect of MMF, sirolimus and tacrolimus showed a small positive effect on cell proliferation (Figure 1B). The number of CHIBs formed during 4–5 weeks of culture was significantly reduced by MMF and its combinations with sirolimus or tacrolimus (Figure 2A). In addition, the CHIBs were smaller and less well organized in the presence of MMF (Figure 2B). In order to observe the drug effects on both cell proliferation and differentiation, the immunosuppressive drugs were administered from the beginning of the cultures. At the end of the entire experiment, implying 4–5-week exposure to the drugs, only sirolimus showed no adverse effect on insulin content, whereas significant reductions in insulin content were seen in all other experimental groups when expressed as a relative change from the control condition (Figure 3A). Both tacrolimus and MMF had an about 50% effect, and in combination they induced a 70% reduction in the insulin content. The differences in absolute amounts of insulin were not statistically significant due to considerable variation in cell compositions among different batches of starting tissue (Figure 3A). Daclizumab was not tested alone, and when added to the combination of tacrolimus plus sirolimus, it did not show an additive effect. As shown in Figure 3B only MMF reduced the total DNA content at the end of the experiment, thus reflecting a negative effect on cell proliferation or increased cell death. Tacrolimus and sirolimus did not affect the DNA content, and in combination with MMF they did not provide additional effects. The ratio of insulin to DNA can be taken as a measurement of the overall level of beta-cell differentiation induced during culture (Figure 3C). Using this parameter, it is evident that the negative effect of MMF on insulin content is a reflection of its overall negative effect on cell number, whereas there was no specific effect on insulin containing cells and the insulin/DNA ratio remained unaltered. In contrast, the effect of tacrolimus was more specific for the beta cells. In fact, only tacrolimus, alone or in combination with sirolimus, induced a significant 50% decrease in the insulin/DNA ratio of the cultured cells (Figure 3C). The proportions of insulin- and glucagon-positive cells were studied in parallel aliquots of the cultured cells. In accordance with the results of insulin and DNA quantitation, tacrolimus was the only drug which consistently decreased the percentage of insulin-positive beta cells (Figure 4A). In the presence of MMF, the percentage of beta cells was even increased as compared with control, in accordance with the idea that this drug inhibited the growth of non-beta cells but did not reduce the number of beta cells. Again, these differences were statistically significant only in relative units. The proportion of alpha-cells was not affected by any of the drugs or their combinations (Figure 4B). The neogenesis of islets from ductal cells has been suggested by many in vivo (14Bonner-Weir S Baxter LA Schuppin GT Smith FE A second pathway for regeneration of adult exocrine and endocrine pancreas: A possible recapitulation of embryonic development.Diabetes. 1993; 42: 1715-1720Crossref PubMed Scopus (612) Google Scholar, 15Gu D Sarvetnick N Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice.Development. 1993; 118: 33-46Crossref PubMed Google Scholar, 16Vinik A Pittenger G Rafaeloff R Rosenberg L Duguid WP Determinants of pancreatic islet cell mass: A balance between neogenesis and senescence/apoptosis.Diabetes Rev. 1996; 4: 235-263Google Scholar, 17Bogdani M Lefebvre V Buelens N et al.Formation of insulin-positive cells in implants of human pancreatic duct cell preparations from young donors.Diabetologia. 2003; 46: 830-838Crossref PubMed Scopus (43) Google Scholar) and in vitro studies (11Gao R Ustinov J Pulkkinen MA Lundin K Korsgren O Otonkoski T Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture.Diabetes. 2003; 52: 2007-2015Crossref PubMed Scopus (234) Google Scholar,18Bonner-Weir S Taneja M Weir GC et al.In vitro cultivation of human islets from expanded ductal tissue.Proc Natl Acad Sci U S A. 2000; 97: 7999-8004Crossref PubMed Scopus (913) Google Scholar,19Ramiya VK Maraist M Arfors KE Schatz DA Peck AB Cornelius JG Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells [see comments].Nat Med. 2000; 6: 278-282Crossref PubMed Scopus (674) Google Scholar). In clinical islet transplantation, islet grafts have been shown to be composed of a large proportion of ductal cells, and the turnover of these putative precursor cells to beta cells could contribute to the long-term function of the grafts (9Street CN Lakey JR Shapiro AM et al.Islet graft assessment in the Edmonton protocol: Implications for predicting long-term clinical outcome.Diabetes. 2004; 53: 3107-3114Crossref PubMed Scopus (189) Google Scholar). Although many experimental studies have observed the effects of immunosuppressive drugs on islets both in vivo and in vitro (2Ricordi C Zeng YJ Alejandro R et al.In vivo effect of FK506 on human pancreatic islets.Transplantation. 1991; 52: 519-522Crossref PubMed Scopus (97) Google Scholar, 3Fabian MC Lakey JRT Rajotte RV Kneteman NM The efficacy and toxicity of rapamycin in murine islet transplantation: In vitro and in vivo studies.Transplantation. 1993; 56: 1137-1142Crossref PubMed Scopus (57) Google Scholar, 4Paty BW Harmon JS Marsh CL Robertson RP Inhibitory effects of immunosuppressive drugs on insulin secretion from HIT-T15 cells and Wistar rat islets.Transplantation. 2002; 73: 353-357Crossref PubMed Scopus (107) Google Scholar, 5Polastri L Galbiati F Bertuzzi F et al.Secretory defects induced by immunosuppressive agents on human pancreatic beta-cells.Acta Diabetol. 2002; 39: 229-233Crossref PubMed Scopus (55) Google Scholar, 6Bell E Cao X Moibi JA et al.Rapamycin has a deleterious effect on MIN-6 cells and rat and human islets.Diabetes. 2003; 52: 2731-2739Crossref PubMed Scopus (153) Google Scholar, 7Fotiadis C Xekouki P Papalois AE et al.Effects of mycophenolate mofetil vs cyclosporine administration on graft survival and function after islet allotransplantation in diabetic rats.World J Gastroenterol. 2005; 11: 2733-2738Crossref PubMed Scopus (11) Google Scholar), few have focused on islet precursor cells. We have shown previously the in vitro generation of endocrine islet cells from proliferative duct-like epithelial cells, and that this also involves the dedifferentiation of preexisting islet cells (11Gao R Ustinov J Pulkkinen MA Lundin K Korsgren O Otonkoski T Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture.Diabetes. 2003; 52: 2007-2015Crossref PubMed Scopus (234) Google Scholar,12Gao R Ustinov J Korsgren O Otonkoski T In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells.Diabetologia. 2005; 48: 2296-2304Crossref PubMed Scopus (75) Google Scholar). By BrdU ce:labeling, we also found that less than 1% of endocrine cells proliferated during monolayer expansion period (12Gao R Ustinov J Korsgren O Otonkoski T In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells.Diabetologia. 2005; 48: 2296-2304Crossref PubMed Scopus (75) Google Scholar). Our results are consistent with previous studies demonstrating that the replication of human beta cells is extremely low (20Tyrberg B Eizirik DL Hellerström C Pipeleers DG Andersson A Human pancreatic β-cell deoxyribonucleic acid-synthesis in islet grafts decreases with increasing organ donor age but increases in response to glucose stimulation in vitro.Endocrinology. 1996; 137: 5694-5699Crossref PubMed Scopus (0) Google Scholar,21Lefebvre VH Otonkoski T Ustinov J Huotari MA Pipeleers DG Bouwens L Culture of adult human islet preparations with hepatocyte growth factor and 804G matrix is mitogenic for duct cells but not for beta-cells.Diabetes. 1998; 47: 134-137Crossref PubMed Google Scholar). In the present study, using this culture model we tested the effects of immunosuppressive drugs currently used in clinical islet transplantation on the proliferation and differentiation of ductal cells. Since in clinical practice they are more often used in combination, we also examined their possible synergistic toxic effects on in vitro-generated islets. Our findings indicate that MMF severely hampers duct-cell proliferation while tacrolimus has the most deleterious effects on differentiated beta cells. Sirolimus and daclizumab show no adverse effects on either proliferation or differentiation. The potentially toxic effects of immunosuppressive drugs are a major threat for the long-term function of islet grafts (22Hering BJ Kandaswamy R Ansite JD et al.Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes.JAMA. 2005; 293: 830-835Crossref PubMed Scopus (492) Google Scholar). Our results, showing that low-dose tacrolimus significantly reduces the insulin content and the number of beta cells in the neoformed islets, are consistent with previous reports of negative effects of tacrolimus on islet cell function and survival (4Paty BW Harmon JS Marsh CL Robertson RP Inhibitory effects of immunosuppressive drugs on insulin secretion from HIT-T15 cells and Wistar rat islets.Transplantation. 2002; 73: 353-357Crossref PubMed Scopus (107) Google Scholar,23Hui H Khoury N Zhao X et al.Adenovirus-mediated XIAP gene transfer reverses the negative effects of immunosuppressive drugs on insulin secretion and cell viability of isolated human islets.Diabetes. 2005; 54: 424-433Crossref PubMed Scopus (56) Google Scholar). Also in the current experiments, these effects are most likely caused by direct effects on the differentiated beta cells, for example through inhibition of insulin gene transcription (24Oetjen E Baun D Beimesche S et al.Inhibition of human insulin gene transcription by the immunosuppressive drugs cyclosporin A and tacrolimus in primary, mature islets of transgenic mice.Mol Pharmacol. 2003; 63: 1289-1295Crossref PubMed Scopus (82) Google Scholar). It is possible that some of the effects of tacrolimus could also be due to altered differentiation of beta cells from duct-like cells. However, the lack of any inhibitory effects by tacrolimus on the proliferation of ductal cells or their aggregation during the differentiation phase suggests that the major mechanism is targeted on the differentiated endocrine cells. In a recent report, MMF was used to replace tacrolimus for the maintenance of immunosuppression of patients with type 1 diabetes who had received islet grafts from a single donor (22Hering BJ Kandaswamy R Ansite JD et al.Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes.JAMA. 2005; 293: 830-835Crossref PubMed Scopus (492) Google Scholar). In addition to tacrolimus, also MMF has been shown to have an inhibitory effect on insulin secretion of cultured human and rat islets (4Paty BW Harmon JS Marsh CL Robertson RP Inhibitory effects of immunosuppressive drugs on insulin secretion from HIT-T15 cells and Wistar rat islets.Transplantation. 2002; 73: 353-357Crossref PubMed Scopus (107) Google Scholar,23Hui H Khoury N Zhao X et al.Adenovirus-mediated XIAP gene transfer reverses the negative effects of immunosuppressive drugs on insulin secretion and cell viability of isolated human islets.Diabetes. 2005; 54: 424-433Crossref PubMed Scopus (56) Google Scholar). The reduction of insulin secretion by MMF was supposed to occur through inhibition of voltage-dependent calcium channels (25Li GD Luo RH Metz SA Effects of inhibitors of guanine nucleotide synthesis on membrane potential and cytosolic free Ca2+ levels in insulin-secreting cells.Biochem Pharmacol. 2000; 59: 545-556Crossref PubMed Scopus (20) Google Scholar). However, in our experimental setup, the major effect of MMF was very clearly targeted on the proliferation of islet precursor cells. MMF is a selective inhibitor of inosine monophosphate dehydrogenase that is crucial for proliferation of T and B lymphocytes (26Borger P Kauffman HF Timmerman JA Scholma J Van Den Berg JW Koeter GH Cyclosporine, FK506, mycophenolate mofetil, and prednisolone differentially modulate cytokine gene expression in human airway-derived epithelial cells.Transplantation. 2000; 69: 1408-1413Crossref PubMed Scopus (30) Google Scholar). However, recent studies suggest that MMF also inhibits proliferation of tissue-forming cells by an undefined signaling pathway. An antiproliferative effect of MMF was reported in cultures of human lung fibroblasts and it was hypothesized that the inhibition of guanosine-dependent inducible nitric oxide synthase might be involved (27Azzola A Havryk A Chhajed P et al.Everolimus and mycophenolate mofetil are potent inhibitors of fibroblast proliferation after lung transplantation.Transplantation. 2004; 77: 275-280Crossref PubMed Scopus (100) Google Scholar). Most relevant to the current study, MMF inhibits the proliferation of hepatocytes after partial hepatectomy in rats (28Kirimlioglu H Kirimlioglu V Yilmaz S Coban S Turkmen E Ara C Liver pathology and cell proliferation after calcineurin inhibitors and antiproliferative drugs following partial hepatectomy in rats.Transplant Proc. 2006; 38: 622-626Crossref PubMed Scopus (13) Google Scholar) as well as the proliferation of human intrahepatic biliary CK19-positive epithelial cells in vitro (29Liu C Schreiter T Frilling A et al.Cyclosporine A FK-506, 40-0-[2-hydroxyethyl]rapamycin and mycophenolate mofetil inhibit proliferation of human intrahepatic biliary epithelial cells in vitro.World J Gastroenterol. 2005; 11: 7602-7605Crossref PubMed Scopus (27) Google Scholar). The antiproliferative effect of MMF on the pancreatic CK19-positive ductal cells in our cultures is consistent with the latter study. Furthermore, MMF may have affected islet neogenesis in our study through inhibition of cell adhesion molecule expression. Decreases of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by MMF have been shown in the interactions of monocytes and endothelial cells (30Glomsda BA Blaheta RA Hailer NP Inhibition of monocyte/endothelial cell interactions and monocyte adhesion molecule expression by the immunosuppressant mycophenolate mofetil.Spinal Cord. 2003; 41: 610-619Crossref PubMed Scopus (39) Google Scholar). MMF has also been shown to reduce neural cell adhesion molecule (NCAM) expression in tumor cell cultures (31Blaheta RA Bogossian H Beecken WD et al.Mycophenolate mofetil increases adhesion capacity of tumor cells in vitro.Transplantation. 2003; 76: 1735-1741Crossref PubMed Scopus (18) Google Scholar). As described previously (12Gao R Ustinov J Korsgren O Otonkoski T In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells.Diabetologia. 2005; 48: 2296-2304Crossref PubMed Scopus (75) Google Scholar,32Cirulli V Baetens D Rutishauser U Halban PA Orci L Rouiller DG Expression of neural cell adhesion molecule (N-CAM) in rat islets and its role in islet cell type segregation.J Cell Sci. 1994; 107: 1429-1436Crossref PubMed Google Scholar), NCAM is specifically expressed in islet cells and plays a significant role in the regulation of intercellular cluster formation and aggregation. Therefore, MMF-induced impairment of CHIB formation, which involves the aggregation of cells into three-dimensional structures, could be secondary to reduced NCAM expression accompanied by a loss of cell-cell contacts (Figure 2B). Sirolimus has been reported to reduce the viability of rat and human islets at supratherapeutic concentrations (6Bell E Cao X Moibi JA et al.Rapamycin has a deleterious effect on MIN-6 cells and rat and human islets.Diabetes. 2003; 52: 2731-2739Crossref PubMed Scopus (153) Google Scholar). In contrast, several other studies have demonstrated little or no adverse effects of sirolimus on islet function (3Fabian MC Lakey JRT Rajotte RV Kneteman NM The efficacy and toxicity of rapamycin in murine islet transplantation: In vitro and in vivo studies.Transplantation. 1993; 56: 1137-1142Crossref PubMed Scopus (57) Google Scholar,33Kneteman NM Lakey JRT Wagner T Finegood D The metabolic impact of rapamycin (sirolimus) in chronic canine islet graft recipients.Transplantation. 1996; 61: 1206-1210Crossref PubMed Scopus (50) Google Scholar). Our results show that 10 ng/mL of sirolimus, a concentration within the therapeutic range of plasma drug concentrations, had a marginal stimulatory effect on duct cell proliferation, but did not affect the final cell number. Nor did it have any effect on islet neogenesis. In contrast, Bussiere et al. recently found that culture of human ductal cells with 10 or 20 ng/mL sirolimus decreased cell numbers by 50% (10Bussiere CT Lakey JR Shapiro AM Korbutt GS The impact of the mTOR inhibitor sirolimus on the proliferation and function of pancreatic islets and ductal cells.Diabetologia. 2006; 49: 2341-2349Crossref PubMed Scopus (98) Google Scholar). One potentially important difference between the two studies is the higher proportion of beta cells in our study (8% vs. 1%). Moreover, unlike the present study, Bussiere and colleagues did not directly examine the rate of cell proliferation. We did not test daclizumab separately. However, it is clear that when combined with sirolimus and tacrolimus as in the clinically used protocols, daclizumab has no additional toxic effects on proliferation or differentiation. Overall, our data suggest that sirolimus and daclizumab are amenable for use in clinical islet transplantation. However, the concentrations of both sirolimus and tacrolimus have been reported to be up to 3-fold higher in the portal than systematic venous circulation in islet transplant recipients (34Desai NM Goss JA Deng S et al.Elevated portal vein drug levels of sirolimus and tacrolimus in islet transplant recipients: Local immunosuppression or islet toxicity?.Transplantation. 2003; 76: 1623-1625Crossref PubMed Scopus (138) Google Scholar). The intraportally transplanted islets may thus be subjected to somewhat higher concentrations of the drugs during the early postransplant period, possibly contributing to the early vulnerability of islet grafts. In general, our results indicate that MMF has a potent inhibitory effect on human islet neogenesis primarily through an antiproliferative effect on the precursors, whereas tacrolimus mainly affects differentiated beta cells. Sirolimus and daclizumab do not show any individual or synergistic side effects on these parameters. The immunosuppressive protocol may be an important determinant of long-term clinical islet graft function. The authors thank Dr. Kaija Salmela and the Nordic network for clinical islet transplantation for the procurement of human pancreatic cells. This work was supported by grants from the Juvenile Diabetes Research Foundation, the Sigrid Juselius Foundation, the Finnish Diabetes Research Foundation, the Academy of Finland and the Helsinki University Hospital Research Funds (EVO)." @default.
• W2058262020 created "2016-06-24" @default.
• W2058262020 creator A5033356745 @default.
• W2058262020 creator A5034283186 @default.
• W2058262020 creator A5055673489 @default.
• W2058262020 creator A5081137938 @default.
• W2058262020 date "2007-04-01" @default.
• W2058262020 modified "2023-10-14" @default.
• W2058262020 title "Effects of Immunosuppressive Drugs on In Vitro Neogenesis of Human Islets: Mycophenolate Mofetil Inhibits the Proliferation of Ductal Cells" @default.
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