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Supplementary Materials, Tables 1-4, Figures 1-10 from Different Tumor Microenvironments Contain Functionally Distinct Subsets of Macrophages Derived from Ly6C(high) Monocytes

Tumor-associated macrophages (TAM) form a major component of the tumor stroma. However, important concepts such as TAM heterogeneity and the nature of the monocytic TAM precursors remain speculative. Here, we show for the first time that mouse mammary tumors contained functionally distinct subsets of TAMs and provide markers for their identification. Furthermore, in search of the TAM progenitors, we show that the tumor-monocyte pool almost exclusively consisted of Ly6ChiCX3CR1low monocytes, which continuously seeded tumors and renewed all nonproliferating TAM subsets. Interestingly, gene and protein profiling indicated that distinct TAM populations differed at the molecular level and could be classified based on the classic (M1) versus alternative (M2) macrophage activation paradigm. Importantly, the more M2-like TAMs were enriched in hypoxic tumor areas, had a superior proangiogenic activity in vivo, and increased in numbers as tumors progressed. Finally, it was shown that the TAM subsets were poor antigen presenters, but could suppress T-cell activation, albeit by using different suppressive mechanisms. Together, our data help to unravel the complexities of the tumor-infiltrating myeloid cell compartment and provide a rationale for targeting specialized TAM subsets, thereby optimally “re-educating” the TAM compartment. Cancer Res; 70(14); 5728–39. ©2010 AACR.

The presence of sugar in the gut causes induction of SGLT1, the sodium/glucose cotransporter in intestinal epithelial cells (enterocytes), and this is accompanied by stimulation of sugar absorption. Sugar sensing was suggested to involve a G-protein coupled receptor and cAMP - protein kinase A signalling, but the sugar receptor has remained unknown. We show strong expression and co-localization with SGLT1 of the β2-adrenergic receptor (β2-AR) at the enterocyte apical membrane and reveal its role in stimulating glucose uptake from the gut by the sodium/glucose-linked transporter, SGLT1. Upon heterologous expression in different reporter systems, the β2-AR responds to multiple sugars in the mM range, consistent with estimated gut sugar levels after a meal. Most adrenergic receptor antagonists inhibit sugar signaling, while some differentially inhibit epinephrine and sugar responses. However, sugars did not inhibit binding of I125-cyanopindolol, a β2-AR antagonist, to the ligand-binding site in cell-free membrane preparations. This suggests different but interdependent binding sites. Glucose uptake into everted sacs from rat intestine was stimulated by epinephrine and sugars in a β2-AR-dependent manner. STD-NMR confirmed direct physical binding of glucose to the β2-AR. Oral administration of glucose with a non-bioavailable β2-AR antagonist lowered the subsequent increase in blood glucose levels, confirming a role for enterocyte apical β2-ARs in stimulating gut glucose uptake, and suggesting enterocyte β2-AR as novel drug target in diabetic and obese patients. Future work will have to reveal how glucose sensing by enterocytes and neuroendocrine cells is connected, and whether β2-ARs mediate glucose sensing also in other tissues.

During mouse pregnancy placental lactogens stimulate prolactin receptors on pancreatic islet beta cells to induce expression of the tryptophan hydroxylase Tph1, resulting in the synthesis and secretion of serotonin. Presently, the functional relevance of this phenomenon is unclear. One hypothesis is that serotonin-induced activation of 5-HT2B receptors on beta cells stimulates beta cell proliferation during pregnancy. We tested this hypothesis via three different mouse models: (i) total Tph1KO mice, (ii) 129P2/OlaHsd mice, which are incompetent to upregulate islet Tph1 during pregnancy, whereas Tph1 is normally expressed in the intestine, mammary glands, and placenta, and (iii) Htr2b-deficient mice. We observed normal pregnancy-induced levels of beta cell proliferation in total Tph1KO mice, 129P2/OlaHsd mice, and in Htr2b-/- mice. The three studied mouse models indicate that islet serotonin production and its signaling via 5-HT2B receptors are not required for the wave of beta cell proliferation that occurs during normal mouse pregnancy. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:23 issue:24 ispartof: location:Switzerland status: published

Objective: The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas. Design: We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed. Results: In normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme. Conclusion: In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models., SCOPUS: ar.j, info:eu-repo/semantics/published

Autoantibodies against islet cell antigens are routinely used to identify subjects at increased risk of symptomatic type 1 diabetes, but their relation to the intra-islet pathogenetic process that leads to positivity for these markers is poorly understood. We screened 556 non-diabetic organ donors (3 months to 24 years) for five different autoantibodies and found positivity in 27 subjects, 25 single- and two double autoantibody-positive donors. Histopathological screening of pancreatic tissue samples showed lesion characteristic for recent-onset type 1 diabetes in the two organ donors with a high-risk profile, due to their positivity for multiple autoantibodies and HLA-inferred risk. Inflammatory infiltrates (insulitis) were found in a small fraction of islets (

SUMMARYObjectiveAn aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) exists, driven by ΔNp63. In other epithelia, ΔNp63+basal cells have stem cell capacity and can be at the origin of tumors. In the pancreas, basal cells have not been identified.DesignWe assessed basal cell markers in human and mouse pancreas, chronic pancreatitis and PDAC, and developed a 3D imaging protocol (FLIP-IT) to study sizeable samples at single cell resolution. We generated organoid cultures of ducts from Sox9-eGFP reporter mice.ResultsIn normal human pancreas, rare ΔNp63+cells exist in ducts that expand in chronic pancreatitis. ΔNp63+cells express KRT19 and canonical basal markers (KRT5, KRT14 and S100A2) but lack markers of duct cells such as CA19.9 and SOX9. In addition, ΔNp63+cells pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views of the ductal tree in formalin fixed paraffin embedded samples show that basal cells are localized on the basal membrane of medium to large ducts and expand as multilayer dome-like structures in chronic pancreatitis. In mice, ΔNp63 expression is induced when culturing organoids from Sox9-low ductal cells but could not be found in normal pancreas nor in models of pancreatitis or pancreatic cancer.ConclusionWe discovered a novel ductal cell population in normal human pancreas similar to basal cells in other tissues. Using FLIP-IT, we provide unprecedented 3D visualization of these cells in archival clinical specimens. ΔNp63+cells may play an important role in pancreatic tissue regeneration and cancer.SUMMARY BOXWhat is already known about this subject?ΔNp63 has a central role in determining the basal-like subtype of pancreatic ductal adenocarcinoma (PDAC).Different to other tissues with basal cancers, the normal pancreas reportedly does not contain (ΔNp63-expressing) basal cells.Current protocols face severe limitations for marker-based identification and 3D imaging of individual (rare) cells in archival pancreatic samples.What are the new findings?We report a rare and atypical pancreatic duct cell that expresses ΔNp63, other basal cell markers and g.i. stem cell markers.The number of these basal cells increases in diseases such as chronic pancreatitis and pancreatic cancer.We provide an easy to implement protocol for 3D clearing and high-resolution imaging of sizeable samples of (fresh or FFPE) human pancreas or an entire mouse pancreas.Except after culturing medium to large ducts as organoids, we fail to detect basal cells in mouse experimental pancreatic models.How might it impact on clinical practice in the foreseeable future?Extrapolating from knowledge in other organs, basal cells in the pancreas may have a stem cell/progenitor role, including in diseases such as (basal) pancreatic cancer.Use of the 3D imaging protocol in archival clinical specimens will allow unprecedented insights in pancreatic histopathology.For above mentioned diseases, we caution for findings in experimental mouse models that may not (fully) recapitulate the etiopathogenesis.

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions. ispartof: NUCLEIC ACIDS RESEARCH vol:46 issue:16 pages:8275-8298 ispartof: location:England status: published

PURPOSE OF REVIEW: The purpose of this article is to summarize recent developments in the histopathology of recent-onset type 1 diabetes. RECENT FINDINGS: Insulitis is considered to be the defining lesion in young type 1 diabetic patients, and insight into its pathogenic mechanism is crucial for the development of effective new therapies. The present overview highlights some recent developments including an international consensus guideline for the diagnosis of insulitis in type 1 diabetic patients, immunophenotyping of the insulitic lesions, evidence for a heterogeneity of the disease process and a discussion on the differences and similarities between insulitis in patients and in rodent models of the disease. SUMMARY: We have reviewed recent data, published over the last year, on the nature and mechanism of insulitis in both patients and the nonobese diabetic mouse model.

Islet intraportal transplantation is since long considered as therapy for type 1 diabetes (1). Clinical proof-of-concept has been provided since the early 1980s and initiated clinical trials worldwide (2–4). In most trials with human donor islets, the liver is still used as implant site, although there is growing awareness that this site is not optimal (5). It has been shown that several of its immunologic, anatomic, and physiologic features contribute to a significant early graft loss and β-cell dysfunction. Moreover, because this implant site is inaccessible, it can also not be considered when it comes to the use of alternative β-cell sources, such as stem cell–derived β cells or the use of xenografts (6–8). Several alternative sites have been tested in animal models to improve engraftment and long-term survival and to minimize surgical complications (6, 8). Few of these alternative sites hold the potential to be translated into clinical trials, and in general, evidence of posttransplantation functions better than those reached after intraportal infusion are lacking. The present study further assesses the omentum as implant site. This site was previously shown successful for rat islet isografts, which is in itself not particular because these preparations have functioned well in many sites (9, 10). When testing human islet cell grafts in diabetic immunodeficient rats, we found a better survival in the omentum than in the liver: omental implants exhibited little infiltration and were capable to correct hyperglycemia, whereas intraportal implants lost function after a heavy inflammatory infiltration (11). We now assess whether the omental site would also provide an adequate environment for growth of the β-cell mass as is known to occur in implants of immature pancreatic cell preparations (12–14). In a previous study, we have shown the growth potential of perinatal porcine β-cell grafts implanted under the kidney capsule of nude mice (15). The implants became structurally organized as homogenous endocrine clusters with predominantly insulin-positive cells and few other endocrine cells in the periphery; the increase in β-cell mass generated the potency to normalize diabetes (15, 16). Efficient revascularization is considered to be a prerequisite for this process (17). Within 2 days after implantation, we recognized the first endothelial cells and small blood vessels in the proximity of the implants; the revascularization process, however, proceeded beyond the 20-week study period (15, 18). It is conceivable that the rapidity and extent of this process determines initial engraftment as well as subsequent adaptive growth. Because the omentum is well vascularized and has been described as a rich source of angiogenic and neurogenic factors (19–21), we wanted to compare the growth of immature porcine β-cell grafts in omentum and kidney and relate it to the vessel density that has developed in both implants. Implants in the omentum reached vessel densities comparable with those in endogenous adult porcine islets within 10 weeks after implantation, which was not the case in the kidney subcapsular space. In parallel, implant volumes were increased reflecting a beneficial effect on β-cell proliferation and numbers as well as on cellular insulin content. Our data elaborate our prior findings with human islets implants and add further support on the potential use of the omentum as an alternative site for islet transplantation. Whether the omentum could offer an alternative to intraportal transplantation in humans remains to be evaluated.

OBJECTIVE This study assesses β-cell replication in human donor organs and examines possible influences of the preterminal clinical conditions. RESEARCH DESIGN AND METHODS β-Cell replication was quantified in a consecutive series of n = 363 human organ donors using double immunohistochemistry for Ki67 and insulin. Uni- and multivariate analysis was used to correlate replication levels to clinical donor characteristics and histopathologic findings. RESULTS β-Cell replication was virtually absent in most donors, with ≤0.1% Ki67-positive β-cells in 72% of donors. A subpopulation of donors, however, showed markedly elevated levels of replication of up to 7.0% Ki67-positive β-cells. β-Cell replication was accompanied by the increased replication of glucagon-, somatostatin-, and CA19.9-positive cells. Prolonged life support, kidney dysfunction, relatively young donor age, inflammatory infiltration, and prolonged brain death before organ retrieval were all found to be significantly associated with an increased level (≥90th percentile) of β-cell replication, with the first three risk factors being independent predictors. Increased β-cell replication was most often noted in relatively young donors (≤25 years) who received prolonged (≥3 days) life support (68%); in contrast, it was rare in donors with a short duration of life support regardless of age (1%). Prolonged life support was accompanied by increased levels of CD68+ and LCA/CD45+ infiltration in the pancreatic parenchyma. CONCLUSION These results indicate that preterminal clinical conditions in (young) organ donors can lead to increased inflammatory infiltration of the pancreas and to increased β-cell replication.

Pregnancy requires a higher functional beta cell mass and this is associated with profound changes in the gene expression profile of pancreatic islets. Taking Tph1 as a sensitive marker for pregnancy-related islet mRNA expression in female mice, we previously identified prolactin receptors and placental lactogen as key signalling molecules. Since beta cells from male mice also express prolactin receptors, the question arose whether male and female islets have the same phenotypic resilience at the mRNA level during pregnancy. We addressed this question in vitro, by stimulating cultured islets with placental lactogen and in vivo, by transplanting male or female islets into female acceptor mice. Additionally, the islet mRNA expression pattern of pregnant prolactin receptor deficient mice was compared with that of their pregnant wild-type littermates. When cultured with placental lactogen, or when transplanted in female recipients that became pregnant (day 12.5), male islets induced the ‘islet pregnancy gene signature’, which we defined as the 12 highest induced genes in non-transplanted female islets at day 12.5 of pregnancy. In addition, serotonin immunoreactivity and beta cell proliferation was also induced in these male transplanted islets at day 12.5 of pregnancy. In order to further investigate the importance of prolactin receptors in these mRNA changes we used a prolactin receptor deficient mouse model. For the 12 genes of the signature, which are highly induced in control pregnant mice, no significant induction of mRNA transcripts was found at day 9.5 of pregnancy. Together, our results support the key role of placental lactogen as a circulating factor that can trigger the pregnancy mRNA profile in both male and female beta cells. ispartof: PLoS One vol:10 issue:3 ispartof: location:United States status: published

Human islets of Langerhans are complex microorgans responsible for maintaining glucose homeostasis. Islets contain five different endocrine cell types, which react to changes in plasma nutrient levels with the release of a carefully balanced mixture of islet hormones into the portal vein. Each endocrine cell type is characterized by its own typical secretory granule morphology, different peptide hormone content, and specific endocrine, paracrine, and neuronal interactions. During development, a cascade of transcription factors determines the formation of the endocrine pancreas and its constituting islet cell types. Differences in ontogeny between the ventrally derived head section and the dorsally derived head, body, and tail section are responsible for differences in innervation, blood supply, and endocrine composition. Islet cells show a close topographical relationship to the islet vasculature and are supplied with a five- to tenfold higher blood flow than the exocrine compartment. Islet microanatomy is disturbed in patients with type 1 diabetes, with a marked reduction in beta-cell content and the presence of inflammatory infiltrates. Histopathological lesions in type 2 diabetes include a limited reduction in beta-cell content and deposition of amyloid in the islet interstitial space.

Use of sulfonylureas in diabetes treatment is based on their insulin-releasing effect on pancreatic β-cells. Prolonged action is known to degranulate β-cells, but functional consequences have not been examined at the cellular level. This study investigates influences of in vivo (48-h) and in vitro (24-h) glibenclamide treatment on the functional state of the β-cell population. Both conditions decreased cellular insulin content by >50% and caused an elevated basal insulin biosynthetic activity that was maintained for at least 24 h after drug removal. Glibenclamide stimulation of basal insulin synthesis was not achieved after a 2-h exposure; it required a calcium-dependent translational activity and involved an increase in the percent activated β-cells (50% after glibenclamide pretreatment vs. 8% in control cells). The glibenclamide-activated β-cell subpopulation corresponded to the degranulated β-cell subpopulation that was isolated by fluorescence-activated cell sorter on the basis of lower cellular sideward scatter. Glibenclamide pretreatment did not alter cellular rates of glucose oxidation but sensitized β-cells to glucose-induced changes in metabolic redox and insulin synthesis and release. In conclusion, chronic exposure to glibenclamide results in degranulation of a subpopulation of β-cells, which maintain an elevated protein and insulin synthetic activity irrespective of the presence of the drug and of glucose. Our study demonstrates that the in situ β-cell population also exhibits a functional heterogeneity that can vary with drug treatment. Glibenclamide induces degranulated β-cells with a sustained elevated basal activity that might increase the risk for hypoglycemic episodes.

A recently published study by Butler et al. concluded that incretin treatment had adverse effects on the human type 2 diabetic pancreas including 'a marked expansion of the exocrine and endocrine pancreatic compartments, the former being accompanied by increased proliferation and dysplasia and the latter by ?-cell hyperplasia with the potential for evolution into neuroendocrine tumours'. Incretin therapy has become widely used for type 2 diabetes, so these conclusions have instigated major concerns with regard to patient safety. We reassessed both the clinical case information and virtual microscopy images of the same 34 cases that were used in the Butler study as well as Network for Pancreatic Organ Donation (nPOD) cases that were not included. Whereas we would like to stress that it is important to investigate in depth any indication that incretin treatment may lead to inflammation or dysplasia in the pancreas, we find that the data presented in the Butler paper have serious methodological deficiencies thatpreclude any meaningful conclusions

Type 1 diabetes is an incurable disease associated with risk of serious acute and chronic complications. It is caused by a marked loss in insulin-producing beta cells. Immune intervention at clinical onset can transiently suppress a further decline in residual functional beta cell mass, particularly in young patients with a higher residual insulin producing capacity at start of treatment. It might achieve a higher effect during the preclinical phase when the beta cell mass is not yet severely affected. Such prevention trials can be prepared by identifying individuals at very high risk to develop diabetes within 3 years and presenting signs of declining, yet relatively preserved, functional beta cell mass. This article reviews biomarker screening strategies to select these participants and discusses developments that can facilitate rapid and straightforward conclusions from novel clinical studies.

The histopathology of type 1 diabetes is defined by a decreased β-cell mass in association with insulitis, a characteristic lymphocytic infiltration limited to the islets of Langerhans and prominent in early stage disease in children. A cytotoxic T-cell mediated destruction of insulin-producing β-cells is thought to be initiated by an unknown (auto)antigen, leading to the destruction > 75% of β-cell mass at clinical diagnosis. Although considered to be pathognomonic for recent onset disease, insulitis has only been described in approximately 150 cases over the past century. This review describes the quest for this elusive lesion and gives its incidence in various patient subpopulations stratified for age of onset and duration of the disease. It discusses recent new insights into the regenerative capacity of the β-cell mass in the pre-clinical stages of the disease and relates these findings to the inflammatory processes within the islet tissue.

OBJECTIVE: To investigate whether solid-surface vitrification (SSV) is an effective cryopreservation strategy regarding the integrity and function of prepubertal mouse testicular tissue. DESIGN: Prospective experimental study. SETTING: Academic research unit. PATIENT(S): Mice. INTERVENTION(S): Testicular tissue from 5- to 10-day-old GFP(+) mice was cryopreserved with the use of a conventional uncontrolled slow freezing (USF) technique and SSV before intratesticular grafting in busulfan-treated GFP(-) mice. MAIN OUTCOME MEASURE(S): Ultrastructural cryoinjury to spermatogonial stem cells (SSCs) and somatic cells was assessed by electron microscopy. Tubular structure was evaluated by histology, and graft survival and spermatogenic recovery by immunohistochemistry. RESULT(S): The tubular morphology and the proportion of ultrastructural cryodamage were similar between vitrified and slow-frozen testicular fragments. Allografting of tissue after both USF and SSV resulted in arecovery of spermatogenesis similar to fresh samples. CONCLUSION(S): SSV resulted in success rates similar to USF in maintaining testicular cell ultrastructure, tubular morphology, and tissue function. These data provide further evidence that vitrification, being an inexpensive and simple technique, can be considered as an alternative for cryopreservation of prepubertal testicular tissue.

The gene expression of human endometrium on the day of oocyte retrieval of pregnant and nonpregnant patients in a stimulated IVF cycle was compared in two independent groups with different GnRH-antagonist ovarian stimulation protocols. The present data suggest that increased gene expression of cyclooxygenase-2, together with the expression of other molecules in the cyclooxygenase-2 network, on the day of oocyte retrieval in GnRH-antagonist cycles coincides with a lower probability of achieving a clinical pregnancy in this cycle.

AIMS/HYPOTHESIS: Upregulation of the functional beta cell mass is required to match the physiological demands of mother and fetus during pregnancy. This increase is dependent on placental lactogens (PLs) and prolactin receptors, but the mechanisms underlying these events are only partially understood. We studied the mRNA expression profile of mouse islets during pregnancy to gain a better insight into these changes. METHODS: RNA expression was measured ex vivo via microarrays and quantitative RT-PCR. In vivo observations were extended by in vitro models in which ovine PL was added to cultured mouse islets and MIN6 cells. RESULTS: mRNA encoding both isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase (TPH), i.e. Tph1 and Tph2, were strongly induced (fold change 25- to 200-fold) during pregnancy. This induction was mimicked by exposing islets or MIN6 cells to ovine PLs for 24 h and was dependent on janus kinase 2 and signal transducer and activator of transcription 5. Parallel to Tph1 mRNA and protein induction, islet serotonin content increased to a peak level that was 200-fold higher than basal. Interestingly, only a subpopulation of the beta cells was serotonin-positive in vitro and in vivo. The stored serotonin pool in pregnant islets and PL-treated MIN6 cells was rapidly released (turnover once every 2 h). CONCLUSIONS/INTERPRETATION: A very strong lactogen-dependent upregulation of serotonin biosynthesis occurs in a subpopulation of mouse islet beta cells during pregnancy. Since the newly formed serotonin is rapidly released, this lactogen-induced beta cell function may serve local or endocrine tasks, the nature of which remains to be identified.

Human islets of Langerhans are complex micro-organs responsible for maintaining glucose homeostasis. Islets contain five different endocrine cell types, which react to changes in plasma nutrient levels with the release of a carefully balanced mixture of islet hormones into the portal vein. Each endocrine cell type is characterized by its own typical secretory granule morphology, different peptide hormone content, and specific endocrine, paracrine, and neuronal interactions. During development, a cascade of transcription factors determines the formation of the endocrine pancreas and its constituting islet cell types. Differences in ontogeny between the ventrally derived head section and the dorsally derived head, body, and tail section are responsible for differences in innervation, blood supply, and endocrine composition. Islet cells show a close topographical relationship to the islet vasculature, and are supplied with a five to tenfold higher blood flow than the exocrine compartment. Islet microanatomy is disturbed in patients with type 1 diabetes, with a marked reduction in beta-cell content and the presence of inflammatory infiltrates. Histopathological lesions in type 2 diabetes are less pathognomonic with a more limited reduction in beta-cell content and occasional deposition of amyloid in the islet interstitial space.

Type 1 diabetes is often considered as a disease where more than 90% of the beta cells have been destroyed at clinical onset and where beta cell antigen-driven autoimmune reactivities progressively destroy remaining beta cells as well as newly formed or implanted beta cells. This view will be evaluated in light of histological observations in the pancreas of type 1 diabetic patients and of antibody-positive non-diabetic organ donors.

A standardized procedure was developed for the preparation of rat islet cell grafts with selected cell number and composition. After collagenase digestion of pancreases and elutriation of tissue fragments, islets were isolated and dissociated, and cells were purified by autofluorescence-activated cell sorting. Approximately 30% of the initial beta-cell mass was lost during digestion and elimination of small mostly exocrine particles. Fifty percent was recovered in isolated islet preparations and 30% in the purified beta-cell suspensions of greater than 95% purity and viability. Sorting according to cellular flavin adenine dinucleotide content discriminated islet beta-cells from islet endocrine non-beta-cells, fibroblasts, leukocytes, and exocrine cells. Purified endocrine islet cell grafts were prepared by aggregating 10(6) pure beta-cells with or without 8 x 10(5) pure endocrine non-beta-cells. In contrast to intact islets, the purified aggregates were devoid of nonendocrine and damaged cells. Intraportal implantation of a pure beta-cell graft rapidly and permanently normalized the diabetic state of streptozocin-administered animals. The standardized preparation of purified beta-cell grafts allows us to address several metabolic and immunological questions concerning islet cell transplantation in diabetes.

Feeder-free human embryonic stem cell (hESC) culture is associated with the presence of mesenchymal-like cells appearing at the periphery of the colonies. The aim of this study was to identify this early differentiation process. Long-term feeder-free hESC cultures using matrigel and conditioned medium from mouse and from human origin revealed that the appearance of mesenchymal-like cells was similar regardless of the conditioned medium used. Standard characterization confirmed the preservation of hESC properties, but the feeder-free cultures could not be maintained longer than 37 passages. The early differentiation process was characterized in the short term after switching hESCs cultured on feeders to feeder-free conditions. Transmission electron microscopy showed an epithelium-like structure inside the hESC colonies, whereas the peripheral cells revealed the acquisition of a rather mesenchymal-like phenotype. Immunochemistry analysis showed that cells at the periphery of the colonies had a negative E-cadherin expression and a positive Vimentin expression, suggesting an epithelial-mesenchymal transition (EMT). Nuclear staining of beta-catenin, positive N-cadherin and negative Connexin 43 expression were also found in the mesenchymal-like cell population. After RT-PCR analysis, Slug and Snail, both EMT-related transcription factors, were detected as up-regulated in the mesenchymal-like cell population. Taken together, our data suggest that culturing hESCs in feeder-free conditions enhances an early differentiation process identified as an EMT

BACKGROUND: Pre-natal porcine endocrine islet cell grafts were recently shown to contain immature beta cells with a marked potential for growth and differentiation following transplantation, and hence for a progressive and long-term correction of diabetes in immune-incompetent mice. The present study investigates whether these grafts are also capable of correcting hyperglycemia in immune-competent mice receiving a short treatment with anti-CD4-CD8 antibodies. METHODS: Pure endocrine islet cell grafts with 0.5 to 1.0 million beta cells were prepared from pre-natal pigs and transplanted under the kidney capsule of alloxan-diabetic CBA/Ca mice. Survival, growth and function of implanted beta cells were followed by measuring plasma porcine C-peptide and glucose, and graft insulin content at start and at post-transplant (PT) week 35. The effect was studied of a 5-day treatment with non-depleting anti-CD4 YTS177 and depleting anti-CD8 YTS169 antibody, either without or with transient insulin injections. RESULTS: Without antibody treatment, all graft recipients remained porcine C-peptide negative and died. Antibody treatment decreased CD4-expression and percentage CD8 cells for 10 and 18 weeks respectively. It resulted in a 30 week-survival of nine out of 14 graft recipients; all nine had progressively become C-peptide positive but only one proceeded to normoglycemia. When antibody treatment was combined with transient insulin injections, 11 out of 14 graft recipients survived long-term, eight became C-peptide positive and six were normoglycemic at PT week 30. In both groups, surviving recipients exhibited a graft insulin content that was 6- to 9-fold higher than at implantation. CONCLUSIONS: Pre-natal porcine beta cells grow and differentiate when transplanted in diabetic immune-competent mice that have been transiently immune suppressed with anti-CD4 and anti-CD8 monoclonal antibodies. They develop metabolic control when recipients are also transiently treated with insulin injections.

Use of sulfonylureas in diabetes treatment is based on their insulin-releasing effect on pancreatic beta-cells. Prolonged action is known to degranulate beta-cells, but functional consequences have not been examined at the cellular level. This study investigates influences of in vivo (48-h) and in vitro (24-h) glibenclamide treatment on the functional state of the beta-cell population. Both conditions decreased cellular insulin content by50% and caused an elevated basal insulin biosynthetic activity that was maintained for at least 24 h after drug removal. Glibenclamide stimulation of basal insulin synthesis was not achieved after a 2-h exposure; it required a calcium-dependent translational activity and involved an increase in the percent activated beta-cells (50% after glibenclamide pretreatment vs. 8% in control cells). The glibenclamide-activated beta-cell subpopulation corresponded to the degranulated beta-cell subpopulation that was isolated by fluorescence-activated cell sorter on the basis of lower cellular sideward scatter. Glibenclamide pretreatment did not alter cellular rates of glucose oxidation but sensitized beta-cells to glucose-induced changes in metabolic redox and insulin synthesis and release. In conclusion, chronic exposure to glibenclamide results in degranulation of a subpopulation of beta-cells, which maintain an elevated protein and insulin synthetic activity irrespective of the presence of the drug and of glucose. Our study demonstrates that the in situ beta-cell population also exhibits a functional heterogeneity that can vary with drug treatment. Glibenclamide induces degranulated beta-cells with a sustained elevated basal activity that might increase the risk for hypoglycemic episodes.

The development of islet cell transplantation as a cure for diabetes is limited by the shortage of human donor organs. Moreover, currently used grafts exhibit a marginal beta-cell mass with an apparently low capacity for beta-cell renewal and growth. Although duct-associated nonendocrine cells have often been suggested as a potential source for beta-cell production, recent work in mice has demonstrated the role of beta-cells in postnatal growth of the pancreatic beta-cell mass. The present study investigated whether the beta-cell mass can grow in implants that are virtually devoid of nonendocrine cells. Endocrine islet cells were purified from prenatal porcine pancreases (gestation110 days) and implanted under the kidney capsule of nude mice. beta-Cells initially presented with signs of immaturity: small size, low insulin content, undetectable C-peptide release, and an inability to correct hyperglycemia. They exhibited a proliferative activity that was highest during posttransplant week 1 (2.6 and 5% bromodeoxyuridine [BrdU]-positive beta-cells 4 and 72 h posttransplant) and then decreased over 20 weeks to rates measured in the pancreas (0.2% BrdU-positive cells). beta-Cell proliferation in implants first compensated for beta-cell loss during posttransplant week 1 and then increased the beta-cell number fourfold between posttransplant weeks 1 and 20. Rates of alpha-cell proliferation were only shortly and moderately increased, which explained the shift in cellular composition of the implant (beta-cell 40 vs. 90% and alpha-cell 40 vs. 7% at the start and posttransplant week 20, respectively). beta-Cells progressively matured during the 20 weeks after transplantation, with a twofold increase in cell volume, a sixfold increase in cellular insulin content, plasma C-peptide levels of 1-2 ng/ml, and an ability to correct diabetes. They became structurally organized as homogenous clusters with their secretory vesicles polarized toward fenestrated capillaries. We concluded that the immature beta-cell phenotype provides grafts with a marked potential for beta-cell growth and differentiation and hence may have a potential role in curing diabetes. Cells with this phenotype can be isolated from prenatal organs; their presence in postnatal organs needs to be investigated.

Amyloid-containing (A+) islets are characteristic for type-2 diabetes (T2D), but their abundance seems variable among patients. It is unclear whether the distribution of A+ islets follows a certain pattern or occurs randomly throughout the pancreatic organ. We investigated the topography of A+ islets in eight pancreata of T2D patients and eight sex- and age-matched non-diabetic subjects. Transversal sections of head, body and tail segments were stained with synaptophysin combined with Congo red to map/quantify islet tissue and amyloid. In the eight T2D pancreata, the overall percentage of A+ islets varied from 4% to 85%. Further analysis in body and tail indicated that peripheral regions exhibited higher percentages of A+ islets than central regions (averages of, respectively, 30% and 17%, P

OBJECTIVE Insulitis is an important pathological feature of autoimmune diabetes; however, mechanisms governing the recruitment of diabetogenic T-cells into pancreatic islets are poorly understood. Here, we determined the importance of leukocyte integrins β2(Itgb2) and αL (ItgaL) in developing insulitis and frank diabetes. RESEARCH DESIGN AND METHODS Gene-targeted mutations of either Itgb2 or ItgaL were established on the NOD/LtJ mouse strain. Experiments were performed to measure insulitis and diabetes development. Studies were also performed measuring mutant T-cell adhesion to islet microvascular endothelial cells under hydrodynamic flow conditions. T-cell adhesion molecule profiles and adoptive transfer studies were also performed. RESULTS Genetic deficiency of either Itgb2 or ItgaL completely prevented the development of hyperglycemia and frank diabetes in NOD mice. Loss of Itgb2 or ItgaL prevented insulitis with Itgb2 deficiency conferring complete protection. In vitro hydrodynamic flow adhesion studies also showed that loss of Itgb2 completely abrogated T-cell adhesion. However, ItgaL deficiency did not alter NOD T-cell adhesion to or transmigration across islet endothelial cells. Adoptive transfer of ItgaL-deficient splenocytes into NOD/Rag-1 mice did not result in development of diabetes, suggesting a role for ItgaL in NOD/LtJ T-cell activation. CONCLUSIONS Together, these data demonstrate that genetic deficiency of Itgb2 or ItgaL confers protection against autoimmune diabetes through distinctly different mechanisms.

Pancreatic transplantation is being used to treat insulin-dependent diabetes. An intact structure of the graft is a prerequisite for preserved function and we therefore monitored the light microscopic and ultrastructural changes in 30 human donor pancreases stored in the cold in University of Wisconsin preservation solution. Twenty-three pancreases were stored for less than 24 h and 7 for more than 30 h. All glands stored longer than 30 h displayed cytoplasmic vacuolisation in a variable proportion of acinar cells. In addition, the glands stored over 40 h showed focal acinar necrosis. Endocrine tissue was only slightly affected, while duct cells showed no changes. It is concluded that cold preserved pancreases stored for less than 24 h are best for transplantation purposes and that acinar cells are more sensitive to ischaemic damage than endocrine and duct cells.

A 5-day culture of adult rat islets with human recombinant IL-1 beta (3 U/ml) resulted in the death of most alpha-cells and 50% of beta-cells. The IL-1--exposed islet tissue contained--in addition to poorly granulated beta-cells--patches of outgrowing monolayers and dispersed activated macrophages. In purified alpha- and beta-cell preparations, no cytodestructive effects of IL-1 (as high as 30 U/ml) were noticed, indicating that the cytokine is in itself not a beta-cell--selective killer. Pure beta-cells were, on the other hand, more sensitive (from 0.3 U/ml on) than intact islets to an IL-1--induced suppression of hormone synthesis. This inhibitory action was reversible and affected predominantly the production of insulin, leading to degranulated cells with modified shape and attachment. Further studies with IL-1 should take into account that isolated islet preparations do not allow distinction between its irreversible, indirect, and aspecific beta-cell toxicity and its reversible, direct, and specific suppression of beta-cell functions. It is not yet known whether IL-1--suppressed beta-cells exhibit an altered sensitivity to beta-cell--toxic conditions.