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The pancreas is composed of both exocrine glands and intermingled endocrine cells to execute its diverse functions, including enzyme production for digestion of nutrients and hormone secretion for regulation of blood glucose levels. To define the molecular constituents with elevated expression in the human pancreas, we employed a genome-wide RNA sequencing analysis of the human transcriptome to identify genes with elevated expression in the human pancreas. This quantitative transcriptomics data was combined with immunohistochemistry-based protein profiling to allow mapping of the corresponding proteins to different compartments and specific cell types within the pancreas down to the single cell level. Analysis of whole pancreas identified 146 genes with elevated expression levels, of which 47 revealed a particular higher expression as compared to the other analyzed tissue types, thus termed pancreas enriched. Extended analysis of in vitro isolated endocrine islets identified an additional set of 42 genes with elevated expression in these specialized cells. Although only 0.7% of all genes showed an elevated expression level in the pancreas, this fraction of transcripts, in most cases encoding secreted proteins, constituted 68% of the total mRNA in pancreas. This demonstrates the extreme specialization of the pancreas for production of secreted proteins. Among the elevated expression profiles, several previously not described proteins were identified, both in endocrine cells (CFC1, FAM159B, RBPJL and RGS9) and exocrine glandular cells (AQP12A, DPEP1, GATM and ERP27). In summary, we provide a global analysis of the pancreas transcriptome and proteome with a comprehensive list of genes and proteins with elevated expression in pancreas. This list represents an important starting point for further studies of the molecular repertoire of pancreatic cells and their relation to disease states or treatment effects.

Pretreatment culture before islet transplantation represents a window of opportunity to ameliorate the pro-inflammatory profile expressed by human beta-cells in duress. Analcinra (IL-1 receptor antagonist) and tocilizumab (monoclonal IL-6 receptor antibody) are two known anti-inflammatory agents successfully used in the treatment of inflammatory states like rheumatoid arthritis. Both compounds have also been shown to reduce blood glucose and glycosylated hemoglobin in diabetic patients. We therefore sought to evaluate the impact of anakinra and tocilizumab on human p-cells. The islets were precultured with or without anakinra or tocilizumab and then transplanted in a marginal mass model using human islets in immunodeficient mice. Islet viability was evaluated in an in vitro model. The pretreatment culture led to a significantly improved engraftment in treated islets compared to the vehicle. Anakinra and tocilizumab are not toxic to human islets and significantly reduce markers of inflammation and cell death. These results strongly support a pretreatment culture with anakinra and tocilizumab prior to human islet transplantation.

Context: Insulinomas are the most common cause of endogenous hyperinsulinaemic hypoglycaemia in non-diabetic adult patients. They are usually benign and curative surgery is the "gold standard" treatment if they can be localized. Malignant insulinomas are seen in less than 10% and their prognosis is poor. The Glucagon Like Peptide-1 receptor (GLP-1R) is markedly upregulated in insulinomas - especially benign lesions which are difficult to localize with current imaging techniques. Objective: To assess the possibility of the detection of primary and metastatic insulinoma by PET using [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 ([(68)Ga]Exendin-4) in a patient with severe hypoglycemia. Design: Dynamic and static PET/CT examination of a patient using [68Ga]Exendin-4. Setting: Uppsala University Hospital, Uppsala, Sweden. Patients: A patient presented with hypoglycemia requiring continuous intravenous glucose infusions. A pancreatic insulinoma was suspected and an exploratory laparotomy was urgently performed. At surgery, a tumor in the pancreatic tail with an adjacent metastasis was found and a distal pancreatic resection (plus splenectomy) and removal of lymph node was performed. Histopathology showed a WHO grade II insulinoma. Postoperatively hypoglycemia persisted but a PET/CT examination using the neuroendocrine marker [(11)C]-5-hydroxy-L-tryptophan was negative. Interventions: The patient was administered with [(68)Ga]Exendin-4 and examined by dynamic PET over the liver and pancreas. Main Outcome Measures: N/A Results: The stable GLP-1 analogue Exendin-4 was labeled with (68)Ga for PET imaging of GLP-1R expressing tumors. The patient was examined by [(68)Ga]Exendin-4-PET/CT which confirmed several small GLP-1R positive lesions in the liver and a lymph node that could not be conclusively identified by other imaging techniques. The results obtained from the [(68)Ga]Exendin-4-PET/CT examination provided the basis for continued systemic treatment. Conclusion: The results of th

BACKGROUND: Enteroviruses have been implicated in the etiology of type 1 diabetes, supported by immunoreactivity of enteroviral protein in islets, but presence of enteroviral genome has rarely been reported. Failure to detect enterovirus with RT-PCR has been attributed to the possible presence of PCR inhibitors and that only few cells are infected. OBJECTIVES: The aim of this study was to evaluate strategies for detection of enterovirus in human islets. STUDY DESIGN: A scenario was modeled with defined infected islets among a large number of uninfected pancreatic cells and the sensitivity of immunohistochemistry and PCR for detection of enterovirus was evaluated. RESULTS: Enterovirus was detected with PCR when only one single human islet, infected in vitro with a low dose of virus, was mixed with an uninfected pancreatic biopsy. Enterovirus could not be detected by immunohistochemistry under the same conditions, demonstrating the superior sensitivity of PCR also in pancreatic tissue with only a small fraction of infected cells. In addition, we demonstrate that pancreatic cell culture supernatant does not cause degradation of enterovirus at 37°C, indicating that under normal culture conditions released virus is readily detectable. Utilizing PCR, the pancreases of two organ donors that died at onset of type 1 diabetes were found negative for enterovirus genome despite islet cells being positive using immunohistochemistry. CONCLUSIONS: These data suggest that PCR should be the preferred screening method for enterovirus in the pancreas and suggest cautious interpretation of immunostaining for enterovirus that cannot be confirmed with PCR.

Type 1 diabetes mellitus is believed to be triggered, in part, by one or more environmental factors and human enteroviruses (HEVs) are among the candidates. Therefore, this study has examined whether two strains of HEV may differentially affect the induction of genes involved in pathways leading to the synthesis of islet hormones, chemokines and cytokines in isolated, highly purified, human islets. Isolated, purified human pancreatic islets were infected with strains of Coxsackievirus B1. Viral replication and the degree of CPE/islet dissociation were monitored. The expression of insulin, glucagon, CXCL10, TLR3, IF1H1, CCL5, OAS-1, IFN beta, and DDX58 was analyzed. Both strains replicated in islets but only one of strain caused rapid islet dissociation/CPE. Expression of the insulin gene was reduced during infection of islets with either viral strain but the gene encoding glucagon was unaffected. All genes analyzed which are involved in viral sensing and the development of innate immunity were induced by Coxsackie B viruses, with the notable exception of TLR3. There was no qualitative difference in the expression pattern between each strain but the magnitude of the response varied between donors. The lack of virus induced expression of TLR3, together with the differential regulation of IF1H1, OAS1 and IFN beta, (each of which has polymorphic variants influence the predisposition to type 1 diabetes), that might result in defective clearance of virus from islet cells. The reduced expression of the insulin gene and the unaffected expression of the gene encoding glucagon by Coxsackie B1 infection is consistent with the preferential beta-cell tropism of the virus.

Purpose The glucagon-like peptide-1 receptor (GLP-1R) has been proposed as a target for molecular imaging of beta cells. The feasibility of non-invasive imaging and quantification of GLP-1R in pancreas using the positron emission tomography (PET) tracer [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 in non-diabetic and streptozotocin (STZ)–induced diabetic pigs treated with insulin was investigated. Methods Non-diabetic (n = 4) and STZ-induced diabetic pigs (n = 3) from the same litter were examined. Development of diabetes was confirmed by blood glucose values, clinical examinations and insulin staining of pancreatic sections post mortem. Tissue perfusion in the pancreas and kidneys was evaluated by [15O]water PET/computed tomography (CT) scans. The in vivo receptor specificity of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 was assessed by administration of either tracer alone or by competition with 3–6.5 μg/kg of Exendin-4. Volume of distribution and occupancy in the pancreas were quantified with a single tissue compartment model. Results [15O]water PET/CT examinations showed reduced perfusion in the pancreas and kidneys in diabetic pigs. [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 uptake in the pancreas of both non-diabetic and diabetic pigs was almost completely abolished by co-injection of unlabeled Exendin-4 peptide. [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 uptake did not differ between non-diabetic and diabetic pigs. In all animals, administration of the tracer resulted in an immediate increase in the heart rate (HR). Conclusion Pancreatic uptake of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 was not reduced by destruction of beta cells in STZ-induced diabetic pigs.

Insulin-producing cells are transplanted by portal vein injection as an alternative to pancreas transplantation in both clinical and preclinical trials. Two of the main limitations of portal vein transplantation are the prompt activation of the innate immunity and concomitant loss of islets and a small but significant risk of portal vein thrombosis. Furthermore, to mimic physiological release, the insulin-producing cells should instead be located in the pancreas. The trans-vessel wall approach is an endovascular method for penetrating the vessel wall from the inside. In essence, a working channel is established to the parenchyma of organs that are difficult to access by percutaneous technique. In this experiment, we accessed the extra-vascular pancreatic parenchyma in swine by microendovascular technique and injected methylene blue, contrast fluids and insulin-producing cells without acute adverse events. Further, we evaluated the procedure itself by a 1-year angiographical follow-up, without adverse events. This study shows that the novel approach utilizing endovascular minimal invasiveness coupled to accurate trans-vessel wall placement of an injection in the pancreatic parenchyma with insulin-producing cells is possible. In clinical practice, the potential benefits compared to portal vein cell transplantation should significantly improve endocrine function of the graft and potentially reduce adverse events. This study presents one-year follow-up safety data on the microendovascular trans-vessel wall technique and shows that the technique can be used to transplant insulin-producing cells to the swine pancreas parenchyma.

INTRODUCTION: Insulinoma is the most common form of pancreatic endocrine tumors responsible for hyperinsulinism in adults. These tumors overexpress glucagon like peptide-1 (GLP-1) receptor, and biologically stable GLP-1 analogs have therefore been proposed as potential imaging agents. Here, we evaluate the potential of a positron emission tomography (PET) tracer, [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4, for imaging and quantification of GLP-1 receptors (GLP-1R) in insulinoma. METHODS: [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 was evaluated for binding to GLP-1R by in vitro autoradiography binding studies in INS-1 tumor from xenografts. In vivo biodistribution was investigated in healthy control mice, INS-1 xenografted and PANC1 xenografted immunodeficient mice at two different doses of peptide: 2.5μg/kg (baseline) and 100μg/kg (block). In vivo imaging of [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 in xenografted mice was evaluated by small animal PET/CT using a direct comparison with the clinically established insulinoma marker [(11)C]5-hydroxy-tryptophan ([(11)C]5-HTP). RESULTS: GLP-1 receptor density could be quantified in INS-1 tumor biopsies. [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 showed significant uptake (p≤0.05) in GLP1-R positive tissues such as INS-1 tumor, lungs and pancreas upon comparison between baseline and blocking studies. In vivo imaging showed concordant results with higher tumor-to-muscle ratio in INS-1 xenografted mice compared with [(11)C]5-HTP. CONCLUSION: [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 has high affinity and specificity for GLP-1R expressed on insulinoma in vitro and in vivo.

Cytomegalovirus (CMV) has been suggested as a contributing force behind the impaired immune responsiveness in the elderly, with decreased numbers of naive T-cells and an increased proportion of effector T-cells. Immunological impairment is also implicated as a part of the pathogenesis in Alzheimer's disease (AD). The aim of this study was to investigate whether AD patients present with a different CMV-specific CD8 immune profile compared to non-demented controls. Blood samples from 50 AD patients and 50 age-matched controls were analysed for HLA-type, CMV serostatus and systemic inflammatory biomarkers. Using multi-colour flow cytometry, lymphocytes from peripheral blood mononuclear cells were analysed for CMV-specific CD8 immunity with MHC-I tetramers A01, A02, A24, B07, B08 and B35 and further classified using CD27, CD28, CD45RA and CCR7 antibodies. Among CMV seropositive subjects, patients with AD had significantly lower proportions of CMV-specific CD8 T-cells compared to controls, 1.16 % vs. 4.13 % (p=0.0057). Regardless of dementia status, CMV seropositive subjects presented with a lower proportion of naive CD8 cells and a higher proportion of effector CD8 cells compared to seronegative subjects. Interestingly, patients with AD showed a decreased proportion of CMV-specific CD8 cells but no difference in general CD8 differentiation.

Congenital cytomegalovirus (CMV) infection is the most common congenital infection causing childhood morbidity. The pathogenetic mechanisms behind long-term sequelae are unclear, but long-standing viremia as a consequence of the inability to convert the virus to a latent state has been suggested to be involved. Whereas primary CMV infection in adults is typically rapidly controlled by the immune system, children have been shown to excrete virus for years. Here, we compare T-cell responses in children with congenital CMV infection, children with postnatal CMV infection and adults with symptomatic primary CMV infection. The study groups included 24 children with congenital CMV infection, 19 children with postnatal CMV infection and 8 adults with primary CMV infection. Among the infants with congenital CMV infection, 13 were symptomatic. T-cell responses were determined by analysis of interferon gamma-production after stimulation with CMV antigen. Our results show that whereas adults display high CMV-specific CD4 T-cell responses in the initial phase of the infection, children younger than 2 years have low or undetectable responses that appear to increase with time. There were no differences between groups with regard to CD8 T-cell function. In conclusion, inadequate CD 4 T-cell function seem to be involved in the failure to get immune control of the CMV infection in children younger than 2 years of age with congenital as well as postnatal CMV infection.

High levels of donor-derived high-mobility group box 1 (HMGB1) protein have been associated with poor islet graft outcome in mouse models. The aim of our work was to determine whether HMGB1 released by human islets had independent proinflammatory effects that influence engraftment in humans. Human islet preparations contained and released HMGB1 in different amounts, as determined by Western blot and ELISA (median 17 pg/ml/IEQ/24 h; min-max 0-211, n=74). HMGB1 release directly correlated with brain death, donor hyper-amilasemia, and factors related to the pancreas digestion procedure (collagenase and digestion time). HMGB1 release was significantly positively associated with the release of other cytokines/chemokines, particularly with the highly released "proinflammatory" CXCL8/IL-8, CXCL1/GRO-alpha, and the IFN-gamma-inducible chemokines CXCL10/IP-10 and CXCL9/MIG. HMGB1 release was not modulated by Toll-like receptor 2,3,4,5, and 9 agonists or by exposure to IL-1 beta. When evaluated after islet transplantation, pretransplant HMGB1 release was weakly associated with the activation of the coagulation cascade (evaluated as serum cross-linked fibrin products), but not with the immediate posttransplant inflammatory response. Concordantly, HMGB1 did not affect short-term human islet function. Our data show that human islet HMGB1 release is a sign of "damaged" islets, although without any independent direct role in graft failure.

Adoptive transfer of regulatory T cells (Tregs) has been proposed for use as a cellular therapy to induce transplantation tolerance. Preclinical data are encouraging, and clinical trials with Treg therapy are anticipated. In this study, we investigate different strategies for the isolation and expansion of CD4+CD25highCD127low Tregs from uraemic patients. We use allogeneic dendritic cells (DCs) as feeder cells for the expansion and compare Treg preparations isolated by either fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) that have been expanded subsequently with either mature or tolerogenic DCs. Expanded Treg preparations have been characterized by their purity, cytokine production and in-vitro suppressive ability. The results show that Treg preparations can be isolated from uraemic patients by both FACS and MACS. Also, the type of feeder cells used in the expansion affects both the purity and the functional properties of the Treg preparations. In particular, FACS-sorted Treg preparations expanded with mature DCs secrete more interleukin (IL)-10 and granzyme B than FACS-sorted Treg preparations expanded with tolerogenic DCs. This is a direct comparison between different isolation techniques and expansion protocols with Tregs from uraemic patients that may guide future efforts to produce clinical-grade Tregs for use in kidney transplantation.

Background: Proper revascularization after transplantation is assumed to be crucial forappropriate islet graft function. Methods: We developed a novel non-invasive imagingmethod, based on adenoviral transduction of islets with a hypoxia responsive reporter gene,for continuous in vivo monitoring of hypoxia in islet grafts in a mouse model. In addition,morphological data was obtained from a deceased patient previously subject to intraportaltransplantation. Results: We detected only transient hypoxia in a minority of the animalstransplanted. Importantly, a clear response to hypoxia was observed in vitro after removal ofthe islet-grafts on day twenty-eight after transplantation. Also, the morphological data fromthe deceased patient demonstrated an extensive revascularization of the transplanted islets. Infact, no differences could be seen between native, in pancreas biopsies taken prior to isletisolation, and transplanted islets regarding the number, distribution and shape of the bloodvessels. However, fewer small islets (diameter <39μm) were found in the liver compared to those found in native pancreases. Notably, an absolute majority of the transplanted islets were found remaining within the venous lumen, in direct contact with the vessel wall. Conclusions: In conclusion results presented show less pronounced islet graft hypoxia after subcapsulartransplantation than previously reported using more invasive methods. Also, formation of anextensive intra-islet capillary network, similar to that seen in native islets in the pancreas, wasseen after clinical islet transplantation.

Recombinant adenovirus serotype 5 (Ad5) vectors represent one of the most efficient gene delivery vectors in life sciences. However, Ad5 is dependent on expression of the coxsackievirus-adenovirus- receptor (CAR) on the surface of target cell for efficient transduction, which limits it's utility for certain cell types. Herein we present a new vector, Ad5PTDf35, which is an Ad5 vector having serotype 35 fiber-specificity and Tat-PTD hexon-modification. This vector shows dramatically increased transduction capacity of primary human cell cultures including T cells, monocytes, macrophages, dendritic cells, pancreatic islets and exocrine cells, mesenchymal stem cells and tumor initiating cells. Biodistribution in mice following systemic administration (tail-vein injection) show significantly reduced uptake in the liver and spleen of Ad5PTDf35 compared to unmodified Ad5. Therefore, replication-competent viruses with these modifications may be further developed as oncolytic agents for cancer therapy. User-friendly backbone plasmids containing these modifications were developed for compatibility to the AdEasy-system to facilitate the development of surface-modified adenoviruses for gene delivery to difficult-to-transduce cells in basic, pre-clinical and clinical research.

Current or recent enteroviral infections show an association with type 1 diabetes. However, evidence for this has mainly been generated using a particular mouse monoclonal antibody (clone 5-D8/1) which binds the viral capsid protein VP1. Difficulty in confirming these findings using other independent methods has led to the concern that this might be artefactual. To address this, we examined the potential cross-reactivity of clone 5-D8/1 with normal islet proteins. Western blotting, two-dimensional gel electrophoresis, and mass spectrometry were used to identify human islet proteins bound by the clone 5-D8/1. We found a distinct reactivity with two mitochondrial proteins, creatine kinase B-type and ATP synthase beta subunit. Immunohistochemistry using the clone 5-D8/1 revealed a granular cytoplasmic staining pattern in mitochondria-rich cells, ie hepatocytes, ductal epithelial cells, vascular endothelial cells, skeletal muscle cells, and the neoplastic salivary gland oncocytoma cells, whereas connective tissue and infiltrating immune cells were negative. Staining on islets of Langerhans from subjects with recent-onset type 1 diabetes, but not on isolated human islets infected in vitro with enteroviruses, could be blocked after mixing the clone 5-D8/1 with the mitochondrial proteins. Collectively, our data show that the clone 5-D8/1 detects two human mitochondrial enzymes in addition to enteroviral VP1. The notion that the previously reported VP1 positivity in islets of recent-onset type 1 diabetes patients could reflect cross-reactivity to native islet proteins and not the presence of EV is supported by difficulties in demonstrating EV infection by independent techniques such as PCR or in situ hybridization. These findings call for revisiting the presence of enteroviruses in pancreatic islets of patients with type 1 diabetes.