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To investigate if beta cell neoformation occurs in the transplanted pancreas in patients with type 1 diabetes who received a simultaneous pancreas-kidney (SPK) transplant and later developed recurrence of autoimmunity.
We examined pancreas transplant biopsies from 9 SPK patients with/without recurrent autoimmunity/recurrent diabetes and 16 non-diabetic organ donors. Tissues were analyzed by immunohistochemistry and immunofluorescence.
Numerous CK-19+ pancreatic ductal cells expressed insulin in 6 SPK recipients with recurrent autoimmunity, of whom 5 developed recurrence of diabetes requiring insulin therapy. These cells expressed the pancreatic-duodenal homeobox-1 transcription factor (PDX-1), implicated in pancreatic development and beta cell differentiation. The number of insulin+ ductal cells varied, being highest in the patient with the most severe beta cell loss and lowest in the normoglycemic patient. We detected insulin+CK-19+PDX-1+ cells expressing Ki-67 in the patient with the most severe beta cell loss, indicating proliferation. We could not detect Ki-67+ beta cells within the islets in any SPK patient. Some insulin+CK-19− ductal cells expressed chromogranin A, suggesting further endocrine differentiation. Insulin+ cells were rarely noted in the pancreas transplant ducts in 3 SPK patients without islet autoimmunity and in 6/16 non-diabetic organ donors; these insulin+ cells never expressed CK-19.
Insulin-expressing pancreatic ductal cells, some apparently proliferating, were found in the transplanted pancreas with recurrent islet autoimmunity/diabetes. Replicating beta cells were not detected within islets. The observed changes may represent attempts at tissue remodelling and beta cell regeneration involving ductal cells in the human transplanted pancreas, possibly stimulated by hyperglycaemia and chronic inflammation.
Type 1 diabetes is an autoimmune disease resulting in the destruction of pancreatic beta cells and insulin-dependence. However, residual insulin secretion is often detected at disease onset and marginal amounts of C-peptide are secreted by several patients even many years after diagnosis . Consistently, beta cells are not completely absent in the pancreas of patients with type 1 diabetes [2–4]. A recent meta-analysis suggested that residual beta cell mass at diagnosis is related to age of onset, with younger patients having much more significant destruction than older ones . Beta cells were demonstrated in 88% of autopsy pancreata from 42 patients with disease duration ranging between 4 and 67 years, appearing as single cells or small clusters. While this may simply reflect the survival of a few beta cells, the finding of ongoing beta cell apoptosis and the contemporary presence of non-apoptotic beta cells indirectly suggested beta cell neogenesis . However, replication may be hampered by cytokine-induced damage and apoptosis associated with chronic autoimmunity, to which newly formed beta cells are sensitive [7;8]. In mice, direct beta cell replication appears to be the main mechanism for maintaining beta cell mass  in physiologic conditions such as pregnancy  and experimentally after pancreatectomy  or beta cell depletion induced by transgenically expressed diphtheria toxin [11;12]. Other regenerative mechanisms include regeneration from pancreatic (and perhaps extra-pancreatic) precursor cells and trans-differentiation of other pancreatic (or extra-pancreatic) cell types [13–16]. Trans-differentiation and regeneration were reported in experimental conditions associated with tissue damage or beta cell loss, such as pancreatectomy , cellophane wrapping , ductal ligation [19;20], streptozotocin treatment  and the development of autoimmune diabetes in nonobese diabetic mice [21;22] and diabetes-prone rats . Pancreatic cells with features of ductal and beta cells in pancreatic ducts were originally characterized by electron microscopy . Growing evidence suggests that ductal cells or precursors in the ducts may be involved in beta cell regeneration [17;20;25–30]; for example, human ductal cells transplanted into immunodeficient mice differentiate into new beta cells . Rare insulin+ cells in pancreatic ducts were reported in the pancreas of patients with long standing type 1 diabetes , but the phenotype of those cells was not characterized further. Overall, there is growing evidence that pancreatic tissue damage may trigger several regenerative and remodelling mechanisms that may contribute to beta cell neogenesis .
Recurrence of autoimmunity and diabetes after pancreas transplantation was originally described in twins and HLA-identical siblings . Other studies contributed evidence that recurrence of autoimmunity can occur regardless of HLA sharing and despite immunosuppression . At our institution we are following approximately 275 patients who received a simultaneous pancreas-kidney (SPK) transplant several years after developing type 1 diabetes. We are monitoring these patients for recurrence of autoimmunity, defined as the reactivation of humoral and/or cellular autoimmune responses, and followed several patients to the full recurrence of type 1 diabetes requiring reinstitution of insulin therapy . Most of these patients had no clinical signs of rejection associated with recurrent diabetes and maintained normal exocrine pancreas and kidney graft function. We could obtain pancreas transplant biopsies from several of these patients, which confirmed the diagnosis of recurrent diabetes revealing variable degrees of insulitis and/or beta cell loss. In this study we investigated if beta cell neoformation occurred in the transplanted pancreas of SPK patients with recurrent autoimmunity and diabetes, by examining the frequency, distribution, localization and phenotype of insulin-expressing cells.
We obtained open pancreas transplant biopsies from nine patients identified from a cohort of SPK recipients transplanted at the University of Miami. Patients included 2 females and 7 males with a mean age of 44.1 ± SD 4.6 years (Table 1). All transplantation-related research procedures were approved by the University of Miami Institutional Review Board. Informed consent was obtained from all patients (or family members when appropriate) prior to transplantation and again prior to biopsy. We also examined 16 pancreata from non-diabetic, deceased organ donors, identified at the University of Miami and the University of Colorado. These included 7 female and 9 male donors, with a mean age of 40.8 ± SD 14.5 years (data shown in ESM Table 1). Additional features of the non-diabetic organ donors were previously reported .
As shown in Table 1, patients were assessed for recurrent autoimmunity, recurrent diabetes and rejection. We measured autoantibodies to glutamic acid decarboxylase (GAD) and the tyrosine phosphatase-like protein IA-2 using standardized radioimmunoassays . We tested for the presence of autoreactive CD4 and/or CD8 T cells using MHC class II/I tetramer assays, as described [36;37]. Recurrent type 1 diabetes was diagnosed if the following criteria were met: 1) hyperglycaemia requiring insulin therapy; 2) clinical symptoms of diabetes in the presence of unchanged pancreas transplant exocrine function and kidney transplant function; 3) autoantibodies and/or autoreactive T cells in the circulation; 4) insulitis and/or beta cell loss at biopsy; 5) reduction in C-peptide levels.
Patients #1–6 had ongoing or recent recurrence of islet autoimmunity demonstrated by disease-associated autoantibodies and autoreactive T cells; 5/6 patients had both autoantibodies and autoreactive T cells. For patient #2, autoreactive T cells could not be determined. Patients #1–5 had become diabetic again by the time of biopsy and required insulin therapy. Biopsies showed variable degrees of beta cell loss and/or insulitis. Patient #1 had the most severe beta cell loss and had very little residual insulitis. Patients #2, #3, and #4 had both insulitis and significant beta cell loss. Patient # 5 had no insulitis but several islets showed reduced cellular content and reduced insulin staining. Patient #6 was normoglycemic at the time of his accidental passing, when the pancreas transplant was retrieved with the family's consent, 2 years after transplantation. There was no evidence of beta cell destruction, truly minimal insulitis. He had autoantibodies before and at the time of his passing, when T cells were detected in his peripheral blood, pancreas transplant lymph node and pancreas transplant tissue, reacting against GAD and proinsulin . These data and other features of the biopsies are summarized in Table 1, noting that some inflammatory changes are almost invariably found in pancreas transplant biopsies even in absence of clinical rejection. For comparison, we examined open pancreas transplant biopsies available from three SPK patients (#7–9) without evidence of recurrent autoimmunity. Patients #7 and #8 were normoglycemic and had functioning grafts. Patient #8 could also be tested for GAD autoreactive T cells, which were not detected. This patient had evidence of mild acute and chronic rejection in the pancreas transplant biopsy but largely normal islets and insulin staining 9 years after transplantation. Patient #9 was hyperglycaemic and treated with insulin, although still secreting C-peptide and believed to have developed type 2 diabetes.
Pancreatic tissue from pancreas transplant biopsies and from non-diabetic organ donors was fixed in formalin and paraffin embedded. Five μm-thick sections were cut and mounted on glass slides, which were stained using immunofluorescence and immunohistochemistry standard protocols, as described below.
The following primary antibodies were used: mouse anti-insulin (1:100, clone K36AC10, Sigma, St. Louis, MO, USA), guinea pig anti-insulin (pre-diluted, Biogenex, San Ramon, CA, USA), rabbit anti-glucagon (1:50, DAKO, Carpinteria, CA, USA), goat anti-PDX-1 (1:5000, a gift from Dr. C. Wright, including an acid-eluted affinity-purified batch), mouse anti-CK-19 (1:50, Clone RCK108, Biogenex), rabbit anti-CK-19 (1:100, ProteinTech Group, Chicago, IL, USA). We used a rabbit anti-Ki-67 serum (1:50, Zymed Laboratories/Invitrogen, San Francisco, CA, USA) to assess proliferation  and both a rabbit serum (1:200, NeoMarkers, Fremont, CA, USA) and mouse monoclonal antibody (1:600, Clone LK2H10, NeoMarkers) to stain for chromogranin A (CgA), a protein found in secretory granules of endocrine cells .
For some stains, namely PDX-1, Ki-67 and CgA, epitope retrieval is necessary to successfully stain the tissues. Heat induced epitope retrieval was performed as follows: for PDX-1 staining, slides were boiled in Tris-EDTA (pH 9.0) in a hot plate for 30 minutes (95–100 °C). For Ki-67 staining, tissue sections were digested with trypsin for 10 minutes at 37 °C followed by incubation with citrate buffer (pH 6.0) in a pressure cooker for 5 minutes, 125 °C, using the protocol provided by the manufacturer (Zymed Laboratories/Invitrogen). For CgA staining, tissue sections were incubated with citrate buffer (pH 6.0) in a pressure cooker for 10 minutes, 120 °C. After all the various epitope retrieval treatments, sections were let cool at room temperature for 30 minutes.
For insulin, glucagon, CK-19 and Ki-67 staining, we used the labelled-streptavidin-biotin (LAB-SA) method and the Histostain Plus kit (Zymed Laboratories/Invitrogen) according to the manufacturer's protocol. Formalin-fixed, paraffin-embedded tissue sections were deparaffinised and dehydrated in a graded series of ethanol washes. Tissue sections were subject to epitope retrieval if required by the specific stain, as described above. After washing in phosphate buffered saline (PBS, pH 7.4), endogenous peroxidase was quenched using a 3% solution of hydrogen peroxidase and methanol. Sections were washed with PBS and incubated with protein block (serum free) reagent (Dako) to block non-specific staining. Slides were incubated with primary antibodies (1–3 hours for insulin, glucagon and CK-19, overnight for Ki-67 at 4 °C). After washing in PBS, sections were incubated with biotinylated secondary antibody, washed again in PBS and incubated with horseradish peroxidase-conjugated streptavidin. The 3-amino, 9 ethyl-carbazole (AEC) substrate revealed specific staining. Slides were counterstained with hematoxylin. For PDX-1 staining, we used the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA), which also uses the peroxidase method but 3, 3'-diaminobenzidine (DAB) as substrate. Slides were incubated with the primary antibody (goat anti-PDX-1) overnight at 4 °C. Negative control reactions included those in which the primary antibody was replaced by PBS or by the appropriate isotype-matched control antibody, those in which the secondary antibody was omitted, and those in which the streptavidin horseradish peroxidase conjugate was omitted to control for residual endogenous peroxidase activity. For Ki-67 staining, tonsil sections were used as positive control.
The following double and triple staining combinations were used to assess colocalization: insulin/CK-19, PDX-1/CK-19, insulin/PDX-1, insulin/PDX-1/CK-19, insulin/CK-19/Ki-67 and insulin/CK-19/CgA. After deparaffinization, tissue sections were subject to epitope retrieval if required by the specific stain, as described above. Tissue sections were incubated with protein block (serum free) reagent (Dako) to block non-specific staining. The first primary antibody was applied overnight at 4 °C for Ki-67, PDX-1 and insulin, for 1–2 hours at room temperature for CK-19 and CgA. After washing in PBS, the appropriate secondary antibodies were applied for 1 hour at room temperature (1:400). Double and triple-immunofluorescence stains were carried out sequentially, so the above process was repeated for the second and third primary antibodies. The choice of secondary antibodies was such that the secondary antibodies could only react with their respective primary antibody. For all experiments, control reactions included the omission of the primary antibodies, the replacement of the primary antibody with the appropriate isotype-matched irrelevant antibody, and the omission of the secondary antibody. Secondary antibodies were fluorophore-labelled conjugates: Alexa Fluor 568 goat anti-guinea pig IgG, Alexa-Fluor 488 chicken anti-goat IgG, Alexa-Fluor 647 goat anti-mouse IgG, Alexa-Fluor 488 goat anti-rabbit IgG and Alexa-Fluor 568 goat anti-mouse IgG, all from Invitrogen (Carlsbad, CA, USA).
Slides stained by immunohistochemistry were imaged using a Leica DMLB microscope connected to a Leica DFC-420C digital camera using the IM50 Image Manager software. For immunofluorescence, we used a Zeiss Axiovert 200M inverted microscope. Images were acquired digitally using a Hamamatsu ORCA-ER camera and the Zeiss Axiovision 4.6.3 software. We used the following objectives: Plan-neofluar 10×/0.3 NA, Plan-neofluar 20×/0.5 NA, F-fluar 40×/1.30 oil and Plan-apochromat 63×/1.40 oil. Confocal microscopy was performed with a Zeiss LSM-510 microscope, equipped with the same objectives, to confirm colocalization by Z-stack analysis.
We examined pancreas transplant biopsies from 9 SPK patients, of whom 6 (patients #1–6) had developed recurrent islet autoimmunity. Five of these patients (#1–5) had progressed to full diabetes recurrence with hyperglycaemia requiring reinstitution of insulin therapy by the time of biopsy. Table 1 shows the main clinical characteristics of the SPK patients and their stimulated C-peptide levels at the time of biopsy. In these five patients' biopsies, insulin staining revealed the presence of numerous insulin+ cells in the pancreatic ducts (Fig. 1A, 1B, 1D–M, Table 2). Two antibodies to insulin were used in separate experiments yielding similar results. The biopsy of patient #1, who had completely lost C-peptide secretion, showed the most severe beta cell loss and the most evident insulin expression in ductal cells (Fig. 1A, 1B, 1D–G). Indeed, most of the ducts examined (127/141; 90%) and most ductal cells (1,529/1,608; 95%) stained for insulin (Table 2). Patient #1 had two clinically justified pancreas transplant biopsies, approximately 3 years apart; the first biopsy was obtained one year after the patient had resumed insulin therapy. The presence of insulin+ ductal cells was observed in both biopsy specimens. Insulin expression in ductal cells occurred in a variable number of ducts in patients #2–5 (Fig. 1H–M, and Table 2), ranging from 33% to 85% of the ducts examined. Specifically, 65%, 33%, 82% and 85% of the ducts examined had insulin+ cells and, 35% (400/1,134), 17% (294/1,429), 75% (541/714) and 65% (393/604) of the ductal cells stained for insulin in patients #2–5, respectively. The intensity of the staining varied, but it was usually weaker than in beta cells in nearby islets, even if those beta cells were often in infiltrated (Fig. 1J–L, patient #4) or damaged islets (Fig. 3B, patient #2). There was no apparent predilection for duct type or size. In patient #2, many small ducts or ductal like structures resembling tubular complexes primarily stained for insulin (Fig. 1H). A similar pattern was also observed in patient#1 (Fig. 1G) and in patient #4 (Fig. 1J–L). Larger ducts were stained in other patients, for example patient # 5 (Fig. 1M).
Patient #6 had evidence of recurrent autoimmunity but had not developed diabetes. The examination of his pancreas transplant biopsy revealed no evidence of beta cell destruction. Only 7% of the ducts examined in his transplant biopsy contained insulin+ cells and these were rare, representing less than 1% of the ductal cells (15/2,109) (Fig. 1N, Table 2). Similarly, insulin+ cells in the ducts were rarely noted in 6/16 control pancreata from non-diabetic organ donors who also tested negative for diabetes-associated autoantibodies (Fig. 1O, Table 2). For additional comparison we examined available pancreas transplant biopsies from 3 SPK patients (#7–9, Table 2) who had no evidence of recurrent autoimmunity. Patients #7 and #8 had functioning grafts and had normal glucose tolerance. Patient #9 had apparently developed type 2 diabetes. Insulin+ cells were rarely seen in the ducts of these patients, mainly consisting of single cells like the ones seen in the non-diabetic organ donors and amounting to less than 0.5% of the ductal cells examined in each patient (range 413–798 cells). The insulin+ cells in the ducts did not co-express glucagon, and we did not identify glucagon+ cells in the ducts of our SPK patients. However, glucagon+ cells were occasionally seen in the ducts of control pancreata (not shown).
In order to better define the phenotype of the insulin+ cells in the ducts and confirm that these were ductal cells, we stained serial sections by immunohistochemistry for insulin and CK-19, a marker of ductal cells, and PDX-1, a transcription factor required for pancreatic development that is expressed in mature beta cells and in ductal cells. We used double or triple immunofluorescence to assess co-expression of insulin with CK-19 and PDX-1. In the SPK patients with recurrent autoimmunity, including patient #6, many but not all of the insulin+ cells in the ducts co-expressed CK-19 (Fig. 2, patient #1; the original image of the corresponding confocal plane from the Z-stack series is provided in ESM Fig. 1). This confirmed that those were ductal cells, in addition to their anatomical localization. The insulin+CK-19+ cells also stained for PDX-1, demonstrating features of both endocrine and ductal cells (Fig. 3). Usually, insulin+CK-19+ ductal cells expressed PDX-1 at higher levels than CK-19+insulin− cells, which could also lack PDX-1 expression (Fig. 3D). As noted, we observed rare insulin+ cells in the ducts in three additional SPK patients (#7–9) without autoimmunity and in 6/16 pancreata from non-diabetic organ donors. In contrast to the SPK patients with recurrent autoimmunity, the insulin+ cells identified in the ducts of the SPK patients without autoimmunity (not shown) and in the control pancreata (Fig. 4A; ESM Fig. 2 shows another image acquired by confocal microscopy) did not express CK-19. Consistent with an endocrine phenotype, those cells expressed PDX-1 (not shown).
We investigated if proliferation could be observed in insulin+ cells, in islets or ducts, by staining for the proliferation marker Ki-67. In patient #1, in whom beta cell destruction was the most severe, we observed insulin+CK-19+PDX-1+ cells which, albeit rarely, expressed Ki-67 in the nucleus, indicating proliferation (Fig. 4B; ESM Fig. 3 shows another image acquired by confocal microscopy). Ki-67 expression was not observed in the insulin+ ductal cells of the other 5 SPK patients with recurrent autoimmunity (not shown) and of the non-diabetic pancreas organ donors (Fig. 4A and ESM Fig. 2). We did not observe Ki-67+ beta cells in the islets of any of the pancreas transplant biopsies from the SPK patients with recurrent autoimmunity.
While most of the insulin+CK-19+ ductal cells did not express CgA, we observed ductal structures containing insulin+CgA+ cells. Importantly, these cells did not express CK-19. Fig. 5A shows two ducts from the second biopsy of patient #1, one in which cells express insulin together with CK-19 and another in which ductal cells express insulin and CgA in the absence of CK-19. Insulin staining was more intense in the latter duct. Fig. 5B shows coexistence of insulin+CK-19+CgA− cells with insulin+CK-19−CgA+ cells in the same duct (biopsy of patient #5). Fig. 5C shows ductal structures, observed in the biopsy of patient #2, staining for insulin and CgA in the absence of CK-19, which is however expressed in other small ductal structures nearby.
Our study of pancreas transplant biopsies provides evidence connecting the ductal and beta cell phenotypes in the human pancreas, further supporting the concept that ductal cells may give origin to new beta cells under certain conditions. We demonstrate the existence of insulin+ ductal cells, confirmed by CK-19 co-expression, in the ducts of the transplanted pancreas, specifically in SPK patients with recurrent autoimmunity and diabetes. These cells expressed PDX-1, a transcription factor required for pancreatic development that is expressed in mature beta cells [41;42] and in ductal cells that do not express insulin . The number of insulin+ ductal cells varied between 33 and 90% of the ductal cells examined. Similarly, 17% to 95% of the ducts had insulin+ cells, indicating that these phenomena were quite extensive. The patient with the most severe beta cell destruction and complete loss of C-peptide secretion at the time of biopsy was the one with the highest number of ducts containing insulin+ cells (patient #1). A consistent feature observed in our SPK patients with recurrent autoimmunity who had also progressed to diabetes by the time of biopsy was that most (although not all, Fig. 2C, E) ductal cells were insulin+ in those ducts that contained insulin+ cells. In contrast, SPK patient #6 who was normoglycemic and without beta cell loss, although with clear evidence of ongoing autoimmunity, had rare cells in the ducts that stained for insulin, CK-19 and PDX-1. Thus, the presence of both hyperglycaemia and autoimmunity may be critical for triggering insulin expression in ductal cells.
By comparison, we rarely observed insulin+ cells in the ducts of non-diabetic organ donors. Importantly, while those cells expressed PDX-1, they did not express CK-19. Similarly, we rarely observed insulin+ cells in the ducts when we examined pancreas transplant biopsies from SPK patients without apparent islet autoimmunity, of whom two were normoglycemic and one had developed type 2 diabetes. Mirroring the observations in non-diabetic pancreas donors, these cells did not stain for CK-19. The findings in these SPK patients without autoimmunity and in the non-diabetic organ donors are consistent with earlier reports of single insulin+ cells in close association with ductal structures in the normal human pancreas .
Insulin and PDX-1 expression in CK-19+ ductal cells, with a pattern similar to that of our SPK patients with recurrent autoimmunity and diabetes, was noted in human pancreata from patients with autoimmune chronic pancreatitis with diabetes . This condition is similar to recurrence of autoimmune diabetes in the transplanted pancreas in that both autoimmunity and hyperglycaemia are present. Thus, there are now two studies of human pancreata suggesting that hyperglycaemia and chronic autoimmunity/inflammation may stimulate pancreas remodelling pathways including insulin expression in ductal cells. A similar role for autoimmunity and inflammation in driving beta cell self-replication was suggested by mouse studies . Moreover, autoimmunity and diabetes have been associated with beta cell and islet neoformation in diabetes prone rats , with regeneration occurring in tubular complexes which resemble the small ductal structures that stained for insulin in the biopsies of patient #1 (Fig. 1G) and patient # 2 (Fig. 1H).
Another study of patients with pancreatitis of unspecified aetiology reported the presence of cells expressing insulin, or glucagon, or PDX-1 in pancreatic ducts . However, this study did not show co-expression of islet hormones with CK-19 or Ki-67, albeit Ki-67-positive cells were noted in the ducts. Here we find that insulin+CK-19+ cells can express Ki-67, suggesting that these cells are capable of replication. This was noted in the patient with the most severe beta cell destruction, again suggesting a possible link between the replication of insulin+ ductal cells and the severity of beta cell loss/hyperglycaemia. We did not observe Ki-67+ cells among the surviving beta cells in the islets of any of the SPK patients with recurrent autoimmunity. However, we must recognize the limitations associated with the study of biopsy materials, in particular when assessing direct beta cell replication. Moreover, our SPK patients were immunosuppressed with tacrolimus, which inhibits direct beta cell replication in mice . On the other hand, the formation of beta cells from ductal cells may be enhanced by chronic immunosuppression if this antagonizes chronic autoimmunity.
Three of our SPK patients with recurrent autoimmunity (patients #1, 2, and 5) also had ductal cells expressing insulin together with CgA, a protein found in the secretory granules of endocrine cells, including pancreatic beta cells . Importantly, those cells expressing CgA and insulin no longer expressed CK-19, while clearly being present in ductal structures. Various stages of this putative transition were observed in these patients, with some ducts having mixed cell composition (insulin+CgA+CK-19− and insulin+CK-19+CgA−; the latter represented the majority). This observation provides further evidence for the differentiation potential of ductal cells towards an endocrine phenotype, accompanied by an apparent loss of ductal cell features. Among these three patients, patients #1 and #2 had undetectable C-peptide levels, even after stimulation, in the days preceding the biopsy. The absence of C-peptide secretion in these patients seems consistent with the rarity of insulin+ ductal cells expressing CgA, despite a high overall proportion of insulin+ ductal cells. Based on the C-peptide data and on the rarity of this putative transition towards a more mature beta cell phenotype, we speculate that if these cells secrete insulin this may be of negligible clinical significance.
In conclusion, our findings suggest that ductal cells participate in beta cell regenerative processes occurring in the transplanted human pancreas, in the context of hyperglycaemia and recurrent autoimmunity, which may be critical stimuli to trigger pancreas remodelling mechanisms in the adult. Dissecting the mechanisms involved in these remodelling processes may potentially lead to therapeutic exploitation.
This study was supported by the Diabetes Research Institute Foundation and in part by a grant from NIDDK (R01 DK070011-01A1). We acknowledge the support of the University of Miami Analytical Imaging Core, partly funded by the Juvenile Diabetes Research Foundation (Center Grant JDRFI 4-2004-361); George McNamara, PhD, Beata Frydel, PhD and Brigitte Shaw assisted with confocal microscopy. Kevin Johnson assisted with tissue processing and histology. We thank Dr. Christopher Wright and Michael Ray, Vanderbilt University, and Dr. Helena Edlund, University of Umea, for providing PDX-1 anti-sera and for helpful discussion of the results.
Duality of interest The authors declare no duality of interest.