Loss of RE-1 silencing transcription factor accelerates exocrine damage from pancreatic injury.

Regulation of pancreas plasticity is critical for preventing injury and promoting regeneration upon tissue damage. The intricate process of pancreatic differentiation is governed by an orchestrated network of positive and negative transcription factors for appropriate gene expression. While the transcriptional repressor REST is well characterized as a silencer of neuronal genes in non-neuronal cells, the role of REST in regulating exocrine pancreas cell identity remains largely unexplored. Rest expression is increased upon injury in the mouse pancreas, such as induced acute and chronic pancreatitis and ductal adenocarcinoma. At the cellular level, Rest expression is lower in mature acinar cells compared with pancreas progenitor and ductal cells. To investigate the role of REST activity in pancreatic transdifferentiation and homeostasis, we developed a novel mouse model (Cre/RESTfl/fl) with conditional knockout (KO) of Rest expression within pancreas cells. The high Cre-mediated excision efficiency of Rest exon two KO caused decreased Rest expression and activity within the pancreas. Short-term organoid cultures of pancreatic acini to undergo acinar-to-ductal metaplasia (ADM) showed that loss of REST impedes induced ADM, while overexpression of REST increases ADM. Interestingly, REST ablation accelerated acute pancreatitis in mice treated with the cholecystokinin analog caerulein, as indicated by cellular morphology, elevated serum amylase levels and pancreatic edema. Furthermore, Cre/RESTfl/fl mice were more sensitive to acute pancreatitis injury and displayed augmented tissue damage and cellular lesions. These results suggest REST has a novel protective role against pancreatic tissue damage by acting as a regulator of exocrine cell identity.

Relative Pancreas Volume Is Reduced in First-Degree Relatives of Patients With Type 1 Diabetes.

OBJECTIVE: Pancreas size is reduced in patients at type 1 diabetes onset and in autoantibody (AAB)-positive donors without diabetes. We sought to determine whether pancreas volume (PV) imaging could improve understanding of the loss of pancreas size in first-degree relatives (FDRs) of patients with type 1 diabetes. We also examined relationships among PV, AAB status, and endocrine and exocrine functions. RESEARCH DESIGN AND METHODS: We conducted a cross-sectional study that included five groups: AAB- control subjects (no diabetes and no first- or second-degree relatives with type 1 diabetes) (N = 49), AAB- FDRs (N = 61), AAB+ FDRs (N = 67 total: n = 31 with a single positive AAB [AAB+ single] and n = 36 with multiple positive AABs [AAB+ multiple]), and patients with recent-onset type 1 diabetes (<1 year) (N = 52). Fasting subjects underwent 1.5T pancreatic MRI, and PV and relative PV (RPV) (PV-to-BMI ratio) were analyzed between groups and for correlations with HbA1c, C-peptide, glucose, and trypsinogen. RESULTS: All FDR groups had significantly lower RPV adjusted for BMI (RPVBMI) than control subjects (all P < 0.05). Patients with type 1 diabetes had lower RPVBMI than AAB- FDR (P < 0.0001) and AAB+ multiple (P ≤ 0.013) subjects. Transformed data indicated that trypsinogen levels were lowest in patients with type 1 diabetes. CONCLUSIONS: This study demonstrates, for the first time, all FDRs having significantly smaller RPVBMI compared with AAB- control subjects. Furthermore, RPVBMI was significantly lower in patients with recent-onset type 1 diabetes than in the AAB- FDR and AAB+ multiple groups. As such, RPVBMI may be a novel noninvasive biomarker for predicting progression through stages of type 1 diabetes risk. This study highlights the potential paracrine relationships between the exocrine and endocrine pancreas in progression to type 1 diabetes in subjects at risk.

The influence of type 1 diabetes on pancreatic weight.

AIMS/HYPOTHESIS: Previous studies of pancreases obtained at autopsy or by radiography note reduced pancreas weight (PW) and size, respectively, in type 1 diabetes; this finding is widely considered to be the result of chronic insulinopenia. This literature is, however, limited with respect to the influence of age, sex, anthropometric factors and disease duration on these observations. Moreover, data are sparse for young children, a group of particular interest for type 1 diabetes. We hypothesised that the pancreas-to-body weight ratio would normalise confounding inter-subject factors, thereby permitting better characterisation of PW in type 1 diabetes. METHODS: Transplant-grade pancreases were recovered from 216 organ donors with type 1 diabetes (n = 90), type 2 diabetes (n = 40) and no diabetes (n = 86). Whole-organ and head, body and tail weights were determined. The relative PW (RPW; PW [g] / body weight [kg]) was calculated and tested for normalisation of potential differences due to age, sex and BMI. RESULTS: PW significantly correlated with body weight in control donors (R (2) = 0.76, p < 0.001) while RPW (1.03 ± 0.36, mean ± SD) did not significantly differ across ages (0-58 years). Donors with type 1 diabetes (0.57 ± 0.18, p < 0.001), but not those with type 2 diabetes (0.93 ± 0.30), had significantly lower RPW. The relative weights of each pancreatic region from donors with type 1 diabetes were significantly smaller than those of regions from control donors and donors with type 2 diabetes (p < 0.001). Perhaps most interestingly, the RPW was not significantly associated with duration of type 1 diabetes or type 2 diabetes. CONCLUSIONS/INTERPRETATION: RPW allows for comparisons across a wide range of donor ages by eliminating confounding variables. These data validate an interesting feature of the type 1 diabetes pancreas and underscore the need for additional studies to identify the mechanistic basis for this finding, including those beyond the chronic loss of endogenous insulin secretion.

Collection protocol for human pancreas.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation. Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing ß-cells followed by lower proportions of glucagon-containing α-cells and somatostatin-containing δ-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes. Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

Staining protocols for human pancreatic islets.

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region. The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&E and IHC stains. The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

The pancreas in human type 1 diabetes.

Type 1 diabetes (T1D) is considered a disorder whose pathogenesis is autoimmune in origin, a notion drawn in large part from studies of human pancreata performed as far back as the 1960s. While studies of the genetics, epidemiology, and peripheral immunity in T1D have been subject to widespread analysis over the ensuing decades, efforts to understand the disorder through analysis of human pancreata have been far more limited. We have reviewed the published literature pertaining to the pathology of the human pancreas throughout all stages in the natural history of T1D. This effort uncovered a series of findings that challenge many dogmas ascribed to T1D and revealed data suggesting the marked heterogeneity in terms of its pathology. An improved understanding and appreciation for pancreatic pathology in T1D could lead to improved disease classification, an understanding of why the disorder occurs, and better therapies for disease prevention and management.

Immune depletion with cellular mobilization imparts immunoregulation and reverses autoimmune diabetes in nonobese diabetic mice.

OBJECTIVE: The autoimmune destruction of beta-cells in type 1 diabetes results in a loss of insulin production and glucose homeostasis. As such, an immense interest exists for the development of therapies capable of attenuating this destructive process through restoration of proper immune recognition. Therefore, we investigated the ability of the immune-depleting agent antithymocyte globulin (ATG), as well as the mobilization agent granulocyte colony-stimulating factor (GCSF), to reverse overt hyperglycemia in the nonobese diabetic (NOD) mouse model of type 1 diabetes. RESEARCH DESIGN AND METHODS: Effects of each therapy were tested in pre-diabetic and diabetic female NOD mice using measurements of glycemia, regulatory T-cell (CD4+CD25+Foxp3+) frequency, insulitis, and/or beta-cell area. RESULTS: Here, we show that combination therapy of murine ATG and GCSF was remarkably effective at reversing new-onset diabetes in NOD mice and more efficacious than either agent alone. This combination also afforded durable reversal from disease (>180 days postonset) in animals having pronounced hyperglycemia (i.e., up to 500 mg/dl). Additionally, glucose control improved over time in mice subject to remission from type 1 diabetes. Mechanistically, this combination therapy resulted in both immunological (increases in CD4-to-CD8 ratios and splenic regulatory T-cell frequencies) and physiological (increase in the pancreatic beta-cell area, attenuation of pancreatic inflammation) benefits. CONCLUSIONS: In addition to lending further credence to the notion that combination therapies can enhance efficacy in addressing autoimmune disease, these studies also support the concept for utilizing agents designed for other clinical applications as a means to expedite efforts involving therapeutic translation.

Cystic fibrosis transmembrane conductance regulator deficiency exacerbates islet cell dysfunction after beta-cell injury.

The cause of cystic fibrosis-related diabetes (CFRD) remains unknown, but cystic fibrosis transmembrane conductance regulator (CFTR) mutations contribute directly to multiple aspects of the cystic fibrosis phenotype. We hypothesized that susceptibility to islet dysfunction in cystic fibrosis is determined by the lack of functional CFTR. To address this, glycemia was assessed in CFTR null (CFTR(-/-)), C57BL/6J, and FVB/NJ mice after streptozotocin (STZ)-induced beta-cell injury. Fasting blood glucose levels were similar among age-matched non-STZ-administered animals, but they were significantly higher in CFTR(-/-) mice 4 weeks after STZ administration (288.4 +/- 97.4, 168.4 +/- 35.9, and 188.0 +/- 42.3 mg/dl for CFTR(-/-), C57BL/6J, and FVB/NJ, respectively; P < 0.05). After intraperitoneal glucose administration, elevated blood glucose levels were also observed in STZ-administered CFTR(-/-) mice. STZ reduced islets among all strains; however, only CFTR(-/-) mice demonstrated a negative correlation between islet number and fasting blood glucose (P = 0.02). To determine whether a second alteration associated with cystic fibrosis (i.e., airway inflammation) could impact glucose control, animals were challenged with Aspergillus fumigatus. The A. fumigatus-sensitized CFTR(-/-) mice demonstrated similar fasting and stimulated glucose responses in comparison to nonsensitized animals. These studies suggest metabolic derangements in CFRD originate from an islet dysfunction inherent to the CFTR(-/-) state.

Localized gene expression following administration of adeno-associated viral vectors via pancreatic ducts.

Gene transfer into pancreatic cells in vivo could be of immense therapeutic benefit in cases of type 1 diabetes (T1D) through the production of molecules capable of interrupting the progression of autoimmunity or promoting regeneration of insulin-secreting beta cells. We adapted a clinically relevant surgical technique (endoscopic retrograde cholangiopancreatography) to deliver rAAV encoding human alpha1-antitrypsin (approved gene symbol SERPINA1) to the pancreas of 3-week-old Fisher 344 rats and C57BL/6 mice. We compared natural as well as bioengineered serotypes of rAAV (rAAV1, rAAV2/Apo, rAAV8) as well as different promoters (chicken beta-actin, human insulin) for their expression in vivo. Rats injected with rAAV1 showed the highest hAAT expression (week 2, rAAV1/CB-AT, 579 +/- 457 ng/ml). In mice, rAAV8 vector delivered the highest serum concentration of hAAT (week 2, rAAV8/CB-AT, 19 +/- 6 microg/ml). The chicken beta-actin promoter provided the highest expression in both rodent experiments. Immunohistochemical staining indicated transduction primarily of pancreatic acinar cells with either the rAAV1/CB-AT vector in the rat or the rAAV8/CB-AT vector in the mouse. This study demonstrates that rAAV vectors can be designed to deliver therapeutic genes efficiently to the pancreas and achieve high levels of gene expression and may be useful in treating pancreatic disorders, including T1D.

Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion.

Early systemic treatment of nonobese diabetic mice with high doses of recombinant adeno-associated virus (rAAV) vector expressing murine IL-10 prevents type 1 diabetes. To determine the therapeutic parameters and immunological mechanisms underlying this observation, female nonobese diabetic mice at 4, 8, and 12 wk of age were given a single i.m. injection of rAAV-murine IL-10 (10(4), 10(6), 10(8), and 10(9) infectious units (IU)), rAAV-vector expressing truncated murine IL-10 fragment (10(9) IU), or saline. Transduction with rAAV-IL-10 at 10(9) IU completely prevented diabetes in all animals injected at all time points, including, surprisingly, 12-wk-old animals. Treatment with 10(8) IU provided no protection in the 12-wk-old injected mice, partial prevention in 8-wk-old mice, and full protection in all animals injected at 4 wk of age. All other treatment groups developed diabetes at a similar rate. The rAAV-IL-10 therapy attenuated pancreatic insulitis, decreased MHC II expression on CD11b+ cells, increased the population of CD11b+ cells, and modulated insulin autoantibody production. Interestingly, rAAV-IL-10 therapy dramatically increased the percentage of CD4+CD25+ regulatory T cells. Adoptive transfer studies suggest that rAAV-IL-10 treatment alters the capacity of splenocytes to impart type 1 diabetes in recipient animals. This study indicates the potential for immunomodulatory gene therapy to prevent autoimmune diseases, including type 1 diabetes, and implicates IL-10 as a molecule capable of increasing the percentages of regulatory cells in vivo.