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Introduction: The study of pancreatic regeneration would benefit greatly from the design and validation of robust human-based models. Human Pancreatic Slices (HPSs) are thin organotypic sections of live pancreatic tissue. The sectioning method preserves the overall histological structure of the organ, maintaining the integrity of the extracellular matrix and the natural interaction between the endocrine and exocrine compartments, as well as the local neural, vascular and immune milieu. Conditions for the long-term culture of HPSs, recently reported by our team, have enabled the real-time analysis of beta-cell neogenesis using adenoviral (AV) co-transduction of a red-green reporter and an insulin tracer in human pancreatic slices. However, the ability of these cells to respond to glucose was not established at that time. Methods: To determine whether new INS+ cells respond to glucose, we have designed an AV in which INS-dependent recombination leads to the expression of a blue marker (moxBFP) and a Calcium Imaging Reporter (gcAMP6s, green) whose intensity is proportional to glucose-dependent INS secretion. This allowed us to monitor glucose-stimulated calcium influx in the newly created cells. To track the generation of new beta cells with an even higher degree of resolution we subsequently placed the transduced slices in a custom-made microfluid chip. Conclusions: This setting further allows for longitudinal functional analyses in precise and highly controlled experimental conditions. Our ability to study regeneration in a clinically meaningful model represents a groundbreaking advance that may fast-track the screening and preclinical development of therapeutic agents. Disclosure S. Alvarez-cubela: n/a. A. Pugliese: Advisory Panel; Provention Bio, Inc., Consultant; Quell Tx. C. Ricordi: Advisory Panel; Vertex Pharmaceuticals Incorporated. E. Oltra: None. A. Pugliese: Advisory Panel; Provention Bio, Inc., Consultant; Quell Tx. A. G. Agarwal: None. R. Pastori: None. J. Domínguez-bendala: None. D. Klein: None. C. G. Alver: None. M. Qadir: None. L. Mateus goncalves: None. F. Qureshi: None. J. Almaca: None. C. Fraker: None. A. Caicedo: None. Funding National Institutes of Health (RO1DK130846UO1DK12393)

Background Vericiguat, a stimulator of soluble guanylate cyclase, has been developed as a first-in-class therapy for worsening chronic heart failure in adults with left ventricular ejection fraction < 45%. Objective The objective of this article was to characterize the pharmacokinetics and pharmacokinetic variability of vericiguat combined with guideline-directed medical therapy (standard of care), and identify exposure–response relationships for safety (hemodynamics) and pharmacodynamic markers of efficacy (N-terminal pro-B-type natriuretic peptide concentration [NT-proBNP]) in patients with heart failure and left ventricular ejection fraction < 45% in the SOCRATES-REDUCED study (NCT01951625). Methods Vericiguat and NT-proBNP plasma concentrations in 454 and 432 patients in SOCRATES-REDUCED, respectively, were analyzed using nonlinear mixed-effects modeling. Results Vericiguat pharmacokinetics were well described by a one-compartment model with apparent clearance, apparent volume of distribution, and absorption rate constant. Age, bodyweight, plasma bilirubin, and creatinine clearance were identified as significant covariates on apparent clearance; sex and bodyweight on apparent volume of distribution; and bodyweight and plasma albumin level on absorption rate constant. Pharmacokinetic/pharmacodynamic analysis showed initial minor and transient effects of vericiguat on blood pressure with low clinical impact. There were no changes in heart rate following initial or repeated vericiguat administration. An exposure-dependent and time-dependent turnover pharmacokinetic/pharmacodynamic model for NT-proBNP described production and elimination rates and an demonstrated exposure-dependent reduction in [NT-proBNP] by vericiguat plus standard of care compared with placebo plus standard of care. This effect was dependent on baseline [NT-proBNP]. Conclusions Vericiguat has predictable pharmacokinetics, with no long-term effects on blood pressure in patients with heart failure and left ventricular ejection fraction < 45%. A pharmacokinetic/pharmacodynamic model described a vericiguat exposure-dependent reduction of NT-proBNP. Clinical Trial Identifier NCT01951625. Supplementary Information The online version contains supplementary material available at 10.1007/s40262-021-01024-y.

Significance The existence of progenitors within pancreatic ducts has been studied for decades, but the hypothesis that they may help regenerate the adult endocrine compartment (chiefly insulin-producing β-cells) remains contentious. Here, we examine the single-cell transcriptome of the human ductal tree. Our data confirm the paradigm-shifting notion that specific lineages, long thought to be cast in stone, are in fact in a state of flux between differentiation stages. In addition to pro-ductal and pro-acinar transcriptomic gradients, our analysis suggests the existence of a third (ducto-endocrine) differentiation axis. Such prediction was experimentally validated by transplanting sorted progenitor-like cells, which revealed their tri-lineage differentiation potential. Our findings further indicate that progenitors might be activated in situ for therapeutic purposes., We have described multipotent progenitor-like cells within the major pancreatic ducts (MPDs) of the human pancreas. They express PDX1, its surrogate surface marker P2RY1, and the bone morphogenetic protein (BMP) receptor 1A (BMPR1A)/activin-like kinase 3 (ALK3), but not carbonic anhydrase II (CAII). Here we report the single-cell RNA sequencing (scRNA-seq) of ALK3bright+-sorted ductal cells, a fraction that harbors BMP-responsive progenitor-like cells. Our analysis unveiled the existence of multiple subpopulations along two major axes, one that encompasses a gradient of ductal cell differentiation stages, and another featuring cells with transitional phenotypes toward acinar tissue. A third potential ducto-endocrine axis is revealed upon integration of the ALK3bright+ dataset with a single-cell whole-pancreas transcriptome. When transplanted into immunodeficient mice, P2RY1+/ALK3bright+ populations (enriched in PDX1+/ALK3+/CAII− cells) differentiate into all pancreatic lineages, including functional β-cells. This process is accelerated when hosts are treated systemically with an ALK3 agonist. We found PDX1+/ALK3+/CAII− progenitor-like cells in the MPDs of types 1 and 2 diabetes donors, regardless of the duration of the disease. Our findings open the door to the pharmacological activation of progenitor cells in situ.

The culture of live pancreatic tissue slices is a powerful tool for the interrogation of physiology and pathology in an in vitro setting that retains near-intact cytoarchitecture. However, current culture conditions for human pancreatic slices (HPSs) have only been tested for short-term applications, which are not permissive for the long-term, longitudinal study of pancreatic endocrine regeneration. Using a culture system designed to mimic the physiological oxygenation of the pancreas, we demonstrate high viability and preserved endocrine and exocrine function in HPS for at least 10 days after sectioning. This extended lifespan allowed us to dynamically lineage trace and quantify the formation of insulin-producing cells in HPS from both non-diabetic and type 2 diabetic donors. This technology is expected to be of great impact for the conduct of real-time regeneration/developmental studies in the human pancreas., The ability to culture live pancreatic tissue slices for long periods of time would enable longitudinal studies ex vivo. Here the authors culture human and mouse pancreatic slices in a perfluorocarbon-based culture system and show stable endocrine and exocrine function for up to ten days in culture.

We have described multipotent progenitor-like cells within the major pancreatic ducts (MPDs) of the human pancreas. They express PDX1, its surrogate surface marker P2RY1, and the bone morphogenetic protein (BMP) receptor 1A (BMPR1A)/activin-like kinase 3 (ALK3), but not carbonic anhydrase II (CAII). Here we report the single-cell RNA sequencing (scRNA-seq) of ALK3 bright+ -sorted ductal cells, a fraction that harbors BMP-responsive progenitor-like cells. Our analysis unveiled the existence of multiple subpopulations along two major axes, one that encompasses a gradient of ductal cell differentiation stages, and another featuring cells with transitional phenotypes toward acinar tissue. A third potential ducto-endocrine axis is revealed upon integration of the ALK3 bright+ dataset with a single-cell whole-pancreas transcriptome. When transplanted into immunodeficient mice, P2RY1 + /ALK3 bright+ populations (enriched in PDX1 + /ALK3 + /CAII − cells) differentiate into all pancreatic lineages, including functional β-cells. This process is accelerated when hosts are treated systemically with an ALK3 agonist. We found PDX1 + /ALK3 + /CAII − progenitor-like cells in the MPDs of types 1 and 2 diabetes donors, regardless of the duration of the disease. Our findings open the door to the pharmacological activation of progenitor cells in situ.

The culture of live pancreatic tissue slices is a powerful tool for the interrogation of physiology and pathology in an in vitro setting that retains near-intact cytoarchitecture. However, current culture conditions for human pancreatic slices (HPSs) have only been tested for short-term applications, which are not permissive for the long-term, longitudinal study of pancreatic endocrine regeneration. Using a culture system designed to mimic the physiological oxygenation of the pancreas, we demonstrate high viability and preserved endocrine and exocrine function in HPS for at least 10 days after sectioning. This extended lifespan allowed us to dynamically lineage trace and quantify the formation of insulin-producing cells in HPS from both non-diabetic and type 2 diabetic donors. This technology is expected to be of great impact for the conduct of real-time regeneration/developmental studies in the human pancreas. The ability to culture live pancreatic tissue slices for long periods of time would enable longitudinal studies ex vivo. Here the authors culture human and mouse pancreatic slices in a perfluorocarbon-based culture system and show stable endocrine and exocrine function for up to ten days in culture.

Cellular stress, combined with dysfunctional, inadequate mitochondrial phosphorylation, produces an excessive amount of reactive oxygen species (ROS) and an increased level of ROS in cells, which leads to oxidation and subsequent cellular damage. Because of its cell damaging action, an association between anomalous ROS production and disease such as Type 1 (T1D) and Type 2 (T2D) diabetes, as well as their complications, has been well established. However, there is a lack of understanding about genome-driven responses to ROS-mediated cellular stress. Over the last decade, multiple studies have suggested a link between oxidative stress and microRNAs (miRNAs). The miRNAs are small non-coding RNAs that mostly suppress expression of the target gene by interaction with its 3’untranslated region (3′UTR). In this paper, we review the recent progress in the field, focusing on the association between miRNAs and oxidative stress during the progression of diabetes.

Introduction: The exocrine compartment of the pancreas has been hypothesized to harbor progenitor cells. While their existence remains the subject of debate, the widespread consensus is that any such putative progenitors should express the pancreatic-duodenal homeobox 1 protein (PDX1). Progenitor cell stimulation often depends on concurrent TGFb inhibition and BMP activation. We further hypothesized that PDX1-expressing putative b-cell progenitors may respond to BMP-7. Our data (1) indicate that extrainsular PDX1+/ALK3+ cells do so by proliferating and subsequently, upon BMP-7 withdrawal, differentiating in a multilineage fashion. Here we present additional mechanistic data on b-cell regeneration using a novel human pancreatic slice culture system that allows for the observation of pancreatic tissue for 2 weeks. We further show single cell RNAseq analyses of sorted ALK3+ from the human pancreas, confirming the heterogeneity of the ductal compartment. Methods: HPS: Low melting point agarose-embedded, vibratome-sectioned live pancreatic slices were generated from human donors. Transduction with a CMV-[loxP]-dsRed-[loxP]-EGFP adenovirus + insulin promoter-Cre adenovirus was done in slices cultured atop optically clear perfluorcarbon (PFC)-based membranes. scRNAseq of ALK3+ sorted cells from human non-endocrine tissue (hNEPT, the leftover of islet isolations) was done on 1,000 cells/sample (250K reads/cell). Conclusions: We report the use of live HPS as a novel tool for the study of pancreatic regeneration. Our preliminary results confirm the feasibility of tracing and monitoring discrete transitional events in response to BMP signalling stimulation, as well as the existence of multiple ductal populations at various degrees of differentiation in healthy donor pancreata. References: 1. Qadir MMF, et al. P2RY1/ALK3-Expressing Cells within the Adult Human Exocrine Pancreas Are BMP-7 Expandable and Exhibit Progenitor-like Characteristics. Cell Reports. 2018 Disclosure M. Qadir: None. S. Alvarez-Cubela: None. J. van Dijk: None. J. Weitz: None. S. Cechin: None. D. Klein: None. A.M. Tamayo: None. J. Almaca: None. A. Caicedo: None. I. Kusmartseva: None. H. Hiller: None. M. Beery: None. K.B. Johnson: None. Y.B. Moreno Hernandez: None. M.B. Navarro Rubio: None. S. Sadiq: None. C. Ricordi: Advisory Panel; Self; Zone Labs. A. Pugliese: None. C. Fraker: None. R. Pastori: Other Relationship; Self; Ophysio Inc. J. Domínguez-Bendala: None. Funding Diabetes Research Institute Foundation; Inserra Family Foundation; Fred and Mabel R. Parks Foundation; Foundation for Diabetes Research; Tonkinson Foundation; Michael J. and Katherine E. Franco Foundation; Frank Strick Foundation; Mildred Graff; National Institutes of Health (R43DK105655-01, R44DK105655-02, U01DK120393-01); Fulbright Scholarship Board/International Institute of Education (to M.M.F.Q.)

Islet transplantation is an effective cell therapy for type 1 diabetes (T1D) but its clinical application is limited due to shortage of donors. After a decade-long period of exploration of potential alternative cell sources, the field has only recently zeroed in on two of them as the most likely to replace islets. These are pluripotent stem cells (PSCs) (through directed differentiation) and pancreatic non-endocrine cells (through directed differentiation or reprogramming). Here we review progress in both areas, including the initiation of Phase I/II clinical trials using human embryonic stem cell (hESc)-derived progenitors, advances in hESc differentiation in vitro, novel insights on the developmental plasticity of the pancreas, and groundbreaking new approaches to induce β cell conversion from the non-endocrine compartment without genetic manipulation.

Introduction: Bone morphogenetic protein 7 (BMP-7), a protein with BMP-activating and TGF-β inactivating properties, induces the proliferation of a unique population of progenitor-like cells characterized by the expression of PDX1 and the BMP receptor activin-like kinase 3 (ALK3/BMPR1A). These cells can be sorted using ALK3 and the purinergic receptor P2Y1 (P2RY1) as a novel surrogate surface marker for PDX1. Sorted P2RY1+/ALK3bright+ cells form BMP-7 expandable colonies. Upon BMP-7 withdrawal, they differentiate into cells of all three lineages (endocrine, acinar and ductal) of the adult pancreas. PDX1+/ALK3+ cells absent from islets are represented in the major pancreatic ducts and pancreatic duct glands. Preliminary evidence indicates that these cells are also present in the murine pancreas. We hypothesized that islet β-cell regeneration could be studied in real time using live murine pancreatic slices transduced with a loxP-dsRed-loxP-EGFP reporter + insulin promoter-Cre adenovirus. If new β-cells arise from preexisting insulin-expressing-cells they would remain green. However, if neogenesis occurs from non-β-cells, there is a window of time during which the degrading red and the new green protein co-localize in the cell. Allowing us to discriminate between neogenesis and β-cell proliferation. Methods: Low melting point (LMP) agarose-embedded, vibratome-sectioned live pancreatic slices were generated from CD-1 mice. Transduction with a loxP-dsRed-loxP-EGFP reporter + insulin promoter-Cre adenovirus was done in slices cultured atop optically clear membranes. Slices were treated for 4 days with an ALK3 agonist (THR-123) or saline at the same time of the co-transduction. Bright field and fluorescence images of them were taken every 24h for 7 days. Conclusions: This is the first report on the use of live pancreatic slices as a novel tool for the study of pancreatic regeneration. Our preliminary results suggest that there are quantifiable β-cell neogenic events in response to BMP-7-like agents. Disclosure F. Qadir: None. J. Weitz: None. S. Alvarez-Cubela: None. D. Klein: None. G. Lanzoni: None. C.A. Garcia-Santana: Employee; Self; Ophysio Incorporated. A. Montalvo: None. C. Ricordi: None. L. Inverardi: None. A. Caicedo: None. R. Pastori: None. J. Domínguez-Bendala: Stock/Shareholder; Self; Ophysio.

Summary The transplantation of human embryonic stem cell (hESC)-derived insulin-producing β cells for the treatment of diabetes is finally approaching the clinical stage. However, even with state-of-the-art differentiation protocols, a significant percentage of undefined non-endocrine cell types are still generated. Most importantly, there is the potential for carry-over of non-differentiated cell types that may produce teratomas. We sought to modify hESCs so that their differentiated progeny could be selectively devoid of tumorigenic cells and enriched for cells of the desired phenotype (in this case, β cells). Here we report the generation of a modified hESC line harboring two suicide gene cassettes, whose expression results in cell death in the presence of specific pro-drugs. We show the efficacy of this system at enriching for β cells and eliminating tumorigenic ones both in vitro and in vivo. Our approach is innovative inasmuch as it allows for the preservation of the desired cells while eliminating those with the potential to develop teratomas., Graphical Abstract, Highlights • hESCs were engineered with suicide genes for safety and differentiation efficiency • One cassette is exclusively expressed in teratogenic cells (safety) • Another is selectively excised out in hESC-derived pancreatic β cells (selectivity) • Our strategy allows for hESC-derived tumors to be prevented or ablated in vivo, In this article, Domínguez-Bendala and colleagues present a strategy to modify pluripotent stem cells to allow for the selective destruction of tumorigenic escapees and/or fully developed teratomas, as well as for the specific selection of differentiated insulin-producing β-like cells. Cells engineered in this manner feature a double fail-safe mechanism designed to minimize the risks associated with their clinical transplantation.

The exocrine pancreas can give rise to endocrine insulin-producing cells upon ectopic expression of key transcription factors. However, the need for genetic manipulation remains a translational hurdle for diabetes therapy. Here we report the conversion of adult human nonendocrine pancreatic tissue into endocrine cell types by exposure to bone morphogenetic protein 7. The use of this U.S. Food and Drug Administration–approved agent, without any genetic manipulation, results in the neogenesis of clusters that exhibit high insulin content and glucose responsiveness both in vitro and in vivo. In vitro lineage tracing confirmed that BMP-7–induced insulin-expressing cells arise mainly from extrainsular PDX-1+, carbonic anhydrase II− (mature ductal), elastase 3a (acinar)−, and insulin− subpopulations. The nongenetic conversion of human pancreatic exocrine cells to endocrine cells is novel and represents a safer and simpler alternative to genetic reprogramming.

The chemical behavior of various oligoenes 2 has been studied. The catalytic hydrogenation of diene 3 yielded monoene 4. Triene 7 was hydrogenated to diene 8, monoene 9 and saturated hydrocarbon 10. Bromine addition to 3 and 7 yielded the dibromides 17 and 18, respectively, i.e., the oligoene system has been attacked at its terminal olefinic carbon atoms. Analogously, the higher vinylogs 19 and 20 yielded the 1,8- and 1,10-bromine adduts 23 and 24, respectively, when less than 1 equivalent of bromine was employed. Treatment of tetraene 19 with excess bromine provided tetrabromide 25. In epoxidation reactions, both with meta-chloroperbenzoic acid (MCPBA) and dimethyldioxirane (DMDO) two model oligoenes were studied: triene 7 and tetraene 19. Whereas 7 furnished the rearrangement product 31 with MCPBA, it yielded the symmetrical epoxide 32 with DMDO. Analogously, 19 was converted to mono-epoxide 33 with MCPBA and to 34 with DMDO. Diels–Alder addition of 7 with N-phenyltriazolinedione (PTAD) did not take place. Extension of the conjugated π-system to the next higher vinylog, 19, caused NPTD-addition to the symmetrical adduct 37 in good yield. Comparable results were observed on adding NPTD (equivalent amount) to pentaene 20 and hexaene 21. Using 36 in excess provided the 2:1-adduct 40 from 21 and led to a complex mixture of adducts from heptaene 22. With tetracyanoethylene (TCNE) as the dienophile, tetraolefin 19 yielded the symmetrical adduct 43, although the reaction temperature had to be increased. Pentaene 20 and hexaene 21 led to corresponding results, adducts 44 and 45 being produced in acceptable yields. With nonaene 42 and TCNE the 2:1-adduct 48 was generated according to its spectroscopic data. Exploratory photochemical studies were carried out with tetraene 19 as the model compound. On irradiation this reacted with oxygen to the stable endo-peroxide 52.

MicroRNAs regulate gene expression by inhibiting translation or inducing target mRNA degradation. MicroRNAs regulate organ differentiation and embryonic development, including pancreatic specification and islet function. We showed previously that miR-7 is highly expressed in human pancreatic fetal and adult endocrine cells. Here we determined the expression profile of miR-7 in the mouse-developing pancreas by RT-PCR and in situ hybridization. MiR-7 expression was low between embryonic days e10.5 and e11.5, then began to increase at e13.5 through e14.5, and eventually decreased by e18. In situ hybridization and immunostaining analysis showed that miR-7 colocalizes with endocrine marker Isl1, suggesting that miR-7 is expressed preferentially in endocrine cells. Whole-mount in situ hybridization shows miR-7 highly expressed in the embryonic neural tube. To investigate the role of miR-7 in development of the mouse endocrine pancreas, antisense miR-7 morpholinos (MO) were delivered to the embryo at an early developmental stage (e10.5 days) via intrauterine fetal heart injection. Inhibition of miR-7 during early embryonic life results in an overall downregulation of insulin production, decreased β-cell numbers, and glucose intolerance in the postnatal period. This phenomenon is specific for miR-7 and possibly due to a systemic effect on pancreatic development. On the other hand, the in vitro inhibition of miR-7 in explanted pancreatic buds leads to β-cell death and generation of β-cells expressing less insulin than those in MO control. Therefore, in addition to the potential indirect effects on pancreatic differentiation derived from its systemic downregulation, the knockdown of miR-7 appears to have a β-cell-specific effect as well. These findings suggest that modulation of miR-7 expression could be utilized in the development of stem cell therapies to cure diabetes.

Nonspecific inflammation in the transplant microenvironment results inβ-cell dysfunction and death influencing negatively graft outcome. MicroRNA (miRNA) expression and gene target regulation in transplanted islets are not yet well characterized. We evaluated the impact of inflammation on miRNA expression in transplanted rat islets. Islets exposedin vitroto proinflammatory cytokines and explanted syngeneic islet grafts were evaluated by miRNA arrays. A subset of 26 islet miRNAs was affected by inflammation bothin vivoandin vitro. Induction of miRNAs was dependent on NF-κB, a pathway linked with cytokine-mediated islet cell death. RT-PCR confirmed expression of 8 miRNAs. The association between these miRNAs and mRNA target-predicting algorithms in genome-wide RNA studies ofβ-cell inflammation identified 238 potential miRNA gene targets. Several genes were ontologically associated with regulation of insulin signaling and secretion, diabetes, and islet physiology. One of the most activated miRNAs was miR-21. Overexpression of miR-21 in insulin-secreting MIN6 cells downregulated endogenous expression of the tumor suppressor Pdcd4 and of Pclo, a Ca2+sensor protein involved in insulin secretion. Bioinformatics identified both as potential targets. The integrated analysis of miRNA and mRNA expression profiles revealed potential targets that may identify molecular targets for therapeutic interventions.

Substantial amounts of nonendocrine cells are implanted as part of human islet grafts, and a possible influence of nonendocrine cells on clinical islet transplantation outcome has been postulated. There are currently no product release criteria specific for nonendocrine cells due to lack of available methods. The aims of this study were to develop a method for the evaluation of pancreatic ductal cells (PDCs) for clinical islet transplantation and to characterize them regarding phenotype, viability, and function. We assessed 161 human islet preparations using laser scanning cytometry (LSC/iCys) for phenotypic analysis of nonendocrine cells and flow cytometry (FACS) for PDC viability. PDC and beta-cells obtained from different density fractions during the islet cell purification were compared in terms of viability. Furthermore, we examined PDC ability to produce proinflammatory cytokines/chemokines, vascular endothelial growth factor (VEGF) and tissue factor (TF) relevant to islet graft outcome. Phenotypic analysis by LSC/iCys indicated that single staining for CK19 or CA19-9 was not enough for identifying PDCs, and that double staining for amylase and CK19 or CA19-9 allowed for quantitative evaluation of acinar cells and PDC content in human islet preparation. PDC showed a significantly higher viability than beta-cells (PDC vs beta-cell: 75.5+/-13.9 and 62.7+/-18.7%; P

As shown in this study, the solvatochromic behavior of polyenes depends exclusively on the polarizability of the medium and, even more interestingly, their solvatochromism increases markedly with increasing length of the polyene chain. By virtue of the electronic nature of the interaction of polyenes with the medium, their solvatochromic response to a polarizability change is instantaneous, making these compounds extremely effective polarizability probes for molecular environments. The extreme sensitivity of polyenes to the polarizability of their environment is consistent with the fact that changes in molecular architecture such as those occurring in photosynthetic systems can give rise to polarizability gradients resulting in red shifts in the 1Ag --> 1Bu transition, thereby opening up new channels directing the energy transfer involved to energy trapping sites in such systems.

Hepatic homeostasis is essential for survival in critically ill and burned patients. Insulin administration improves survival and decreases infections in these patients. To determine the molecular mechanisms, the aim of the present study was to establish a stress model using primary human hepatocytes (PHHs) and to study the effects of insulin on the hepatic inflammatory signaling cascade. Liver tissue was obtained from general surgical patients, and PHHs were isolated and maintained in culture. Primary hepatocyte cultures were challenged with various doses of lipopolysaccharide (LPS), and the inflammatory signal transcription cascade was determined by real-time PCR. In subsequent experiments, primary hepatocyte cultures were challenged with LPS and insulin was added in various doses. Glucose was determined by colorimetric assays. PHHs treated with 100 microg/mL LPS showed a profound inflammatory reaction with increased expression of interleukin (IL)-6, IL-10, IL-1beta, tumor necrosis factor (TNF), and signal transducer and activator of transcription 5 (STAT-5). Insulin at 10 IU/mL significantly decreased IL-6, TNF, and IL-1beta at pretranslational levels, an effect associated with decreased STAT-5 mRNA expression (P0.05). Glucose concentration and cellular metabolic activity were not different between controls and insulin-treated cells. Based on our results, we suggest that primary hepatocyte cultures can be used to study the effect of LPS on the inflammatory cascade. Insulin decreases hepatic cytokine expression, which is associated with decreased STAT-5 expression.

The mammalian target of rapamycin (mTOR) has become an interesting target for cancer therapy through its influence on oncogenic signals, which involve phosphatidylinositol-3-kinase and hypoxia-inducible factor-1α (HIF-1α). Since mTOR is an upstream regulator of HIF-1α, a key mediator of gastric cancer growth and angiogenesis, we investigated mTOR activation in human gastric adenocarcinoma specimens and determined whether rapamycin could inhibit gastric cancer growth in mice. Expression of phospho-mTOR was assessed by immunohistochemical analyses of human tissues. For in vitro studies, human gastric cancer cell lines were used to determine S6K1, 4E-BP-1 and HIF-1α activation and cancer cell motility upon rapamycin treatment. Effects of rapamycin on tumor growth and angiogenesis in vivo were assessed in both a subcutaneous tumor model and in an experimental model with orthotopically grown tumors. Mice received either rapamycin (0.5 mg/kg/day or 1.5 mg/kg/day) or diluent per intra-peritoneal injections. In addition, antiangiogenic effects were monitored in vivo using a dorsal-skin-fold chamber model. Immunohistochemical analyses showed strong expression of phospho-mTOR in 60% of intestinal- and 64% of diffuse-type human gastric adenocarcinomas. In vitro, rapamycin-treatment effectively blocked S6K1, 4E-BP-1 and HIF-1α activation, and significantly impaired tumor cell migration. In vivo, rapamycin-treatment led to significant inhibition of subcutaneous tumor growth, decreased CD31-positive vessel area and reduced tumor cell proliferation. Similar significant results were obtained in an orthotopic model of gastric cancer. In the dorsal-skin-fold chamber model, rapamycin-treatment significantly inhibited tumor vascularization in vivo. In conclusion, mTOR is frequently activated in human gastric cancer and represents a promising new molecular target for therapy. © 2007 Wiley-Liss, Inc.

MicroRNAs (miRNAs) control pancreatic and islet development, as well as β cell physiology. For this reason they have an important role in the regulation of gene expression in health and in disease, including diabetes. Each phase of human pancreatic development exhibits a specific miRNA profile. The expression of multiple miRNAs is inversely correlated with the expression of involved critical genes, suggesting miRNA-mediated regulation. Modulation of miRNAs to restore β cells is a potential strategy in diabetes treatment. However, current therapeutic approaches based on manipulation of miRNA expression are more feasible in vitro than in vivo because of difficulties with specific delivery. This chapter discusses the miRNAs involved in islet and pancreas development in the context of diabetes. It also looks at their potential role in the generation of surrogate β cells for transplantation.

The absorption, fluorescence and fluorescence excitation spectra for 3,20-di(tert-butyl)-2,2,21,21-tetramethyl-all-trans-3,5,7,9,11,13,15,17,19-docosanonaen (ttbP9) in dilute solutions of 2-methylbutane were recorded at temperatures over the range 120-280 K. The high photostability of this nonaene allows us to assert that it exhibits a single fluorescence and that this can be unequivocally assigned to emission from its 1(1)B(u) excited state, it being the first excited electronic state. Available photophysical data for this polyene and the wealth of information reported for shorter all-trans polyenes allow us to conclude that if the first excited electronic state for the chromophore possessed 2(1)A(g) symmetry, then the energy of such a state might have been so close to that of the 1(1)B(u) state that: 1) the radiationless internal conversion mechanism would preclude the observation of the emission from the 1(1)B(u) state reported in this work and 2) the 2(1)A(g) state reached through internal conversion would be vibrationally coupled to 1(1)B(u) and would facilitate the detection of the emission from 2(1)A(g), which was not observed in any of the solvents used in this work. The spectroscopic and photochemical implications of these findings for other polyenes are discussed.

Rapid healing of acute wounds, e.g., in burned patients, can be essential for survival. Oxidized regenerated cellulose/collagen (ORC/collagen) has been shown to improve wound healing of chronic wounds. The aim of the present study was to determine the effect of ORC/collagen on dermal and epidermal healing as well as growth factor concentration in acute wounds. Rats received a full-thickness excision wound and were treated with either ORC/collagen plus a hydrocolloid dressing or a hydrocolloid dressing alone. Planimetry, immunological assays, histological and immunohistochemical techniques were used to determine dermal and epidermal regeneration, protein concentration, and growth factor concentration. In addition, dermal vascularization and structure were determined. Wounds treated with ORC/collagen showed a significantly faster reepithelization than those treated with hydrocolloid alone, p < 0.05. This accelerated wound healing rate may be explained by significantly higher levels of platelet-derived growth factor, keratinocyte growth factor, insulin-like growth factor-I, and insulin-like growth factor binding protein-3 in the ORC/collagen group leading to antiapoptotic effects of skin cells, p < 0.05. There were no significant differences in collagen morphology or deposition, neo-angiogenesis, or vascular endothelial growth factor concentration between both treatment groups. We conclude that ORC/collagen matrix accelerates epidermal regeneration and locally increases growth factor concentrations. Increased reepithelization was associated with decreased skin cell apoptosis. Based on our data we hypothesize that the ORC/collagen matrix may also have beneficial effects on acute wounds in a clinical setting.

Despite differences in function and embryonic origin, pancreatic islet cells and neurons express proteins belonging to the tumour necrosis factor receptor superfamily. While neurons express the CD40 receptor, it is unknown whether islet cells also express it. We investigated CD40 expression in human and mouse pancreatic islets as well as in NIT-1 insulinoma cells.CD40 expression was studied by reverse transcriptase polymerase chain reaction, flow cytometry, immunohistochemistry and western blot. Responses mediated by CD40 were assessed by a luciferase gene reporter assay following stimulation with a CD40 agonist antibody.We found that CD40 is expressed in mouse and human pancreatic islet cells. CD40 is expressed by beta cells, and its expression is upregulated by proinflammatory cytokines (IL-1beta, IFN-gamma and TNF-alpha). CD40 signalling in NIT-1 insulinoma cells activates nuclear factor kappa-B, demonstrating that CD40 is functional.We present evidence that, in addition to immune cell types, mouse and human pancreatic beta cells express CD40. Its expression is upregulated by proinflammatory stimuli, and signalling through this receptor activates NF-kappaB. We suggest that the effects of inflammatory stimuli that affect beta cell function and survival may be also mediated by signalling through the CD40 receptor. Thus, CD40 may have a role in processes associated with islet autoimmunity and transplantation.

Type 1 diabetes mellitus is an autoimmune disorder in which the insulin-producing beta-cells of the pancreatic islets of Langerhans are selectively destroyed. Transplantation of allogeneic islets offers a novel therapeutic approach for type 1 diabetic patients. Primary obstacles to the successful outcome of this treatment are loss of the islets occurring first during the isolation procedure and then immediately following transplantation. The genetic make up of beta-cells contributes to making them particularly vulnerable to apoptosis and necrosis-induced cell death caused by the trauma of the isolation procedure and by non-specific inflammatory events at the transplantation site. In this review we present description of chemical and molecular biology based strategies to confer cytoprotection to beta-cells.

The purpose of the present study was to examine whether exogenous liposomal cDNA gene transfer is recognized by the cell and causes endogenous cellular and physiological responses. When administered as a protein, IGF-I is known to cause adverse side effects due to lack of cellular responses. Therefore, we used IGF-I cDNA as a vector to study cellular and physiological effects after liposomal administration to wounded skin. Sprague-Dawley rats were given a scald burn to inflict an acute wound and were divided into two groups to receive weekly subcutaneous injections of liposomes plus the Lac-Z gene (0.2 μg vehicle) or liposomes plus the IGF-I cDNA (2.2 μg) and Lac Z gene (0.22 μg). Transfection was confirmed by histochemical assays for β-galactosidase. Planimetry, immunological assays, and histological and immunohistochemical techniques were used to determine molecular mechanisms after gene transfer, protein expression, and dermal and epidermal regeneration. IGF-I cDNA transfer increased IGF-I protein expression and caused concomitant cellular responses by increasing IGF binding protein (IGFBP)-3 and decreasing IGFBP-1. IGF-I cDNA gene transfer increased keratinocyte growth factor expression and exerted promitogenic antiapoptotic effects on basal keratinocytes, thus improving epidermal regeneration. IGF-I cDNA improved dermal regeneration by an increased collagen deposition and morphology. IGF-I cDNA increased VEGF concentrations and thus neovascularization. Exogenous-administered IGF-I cDNA is recognized by the cell and leads to similar intracellular responses as the endogenous gene. Liposomal IGF-I gene transfer further leads to improved dermal and epidermal regeneration by interacting with other growth factors.

Determine the effect of insulin on the systemic inflammatory response, pro- and anti-inflammatory cytokines and hepatic acute-phase-response in severely burned pediatric patients.The systemic inflammatory and hepatic acute-phase-response contribute to hypermetabolism, multi-organ failure, and mortality. Insulin has been recently shown to decrease mortality and to prevent the incidence of multi-organ failure in critically ill patients; however, the underlying mechanisms have not been defined.Thirteen thermally injured children received insulin to maintain blood glucose at a range from 120 to 180 mg/dl, 15 children received no insulin with blood glucose levels also at range from 120 to 180 mg/dl and served as controls. Our outcome measures encompassed the effect of insulin on pro-inflammatory mediators, the hepatic acute-phase-response, fat, and the IGF-I system.Insulin administration decreased pro-inflammatory cytokines and proteins, while increasing constitutive-hepatic proteins (P0.05). Burned children receiving insulin required significantly less albumin substitution to maintain normal levels compared with control (P0.05). Insulin decreased free fatty acids and serum triglycerides when compared with controls (P0.05). Serum IGF-I and IGFBP-3 significantly increased with insulin administration (P0.05).Insulin attenuates the inflammatory response by decreasing the pro-inflammatory and increasing the anti-inflammatory cascade, thus restoring systemic homeostasis, which has been shown critical for organ function and survival in critically ill patients.

Inflammation and catabolism in response to trauma, surgery, critical illness or bacteria lead to a compromise of essential organs, which can lead to prolonged clinical stay and even death. Mediators responsible for catabolism were thought to be proinflammatory cytokines, but recently the focus has shifted to signal transduction. The purpose of the present study was to determine differences between two pathophysiologic states, sepsis and thermal injury, in signal transduction and cytokine expression and thus define the importance of the signal transcription pathway. Rats were randomly divided to either receive lipopolysaccharide (3 mg/kg body weight or a 30% total body surface area burn) or they received no treatment and served as controls. Animals were sacrificed 1, 2, 5, and 7 days postinsult and serum and liver harvested for analysis. A thermal injury appeared to have a slow release and expression of signal transcription factors and cytokines and a sepsis showed a rapid increase of mediators and also a fast decrease. The changes in cytokine profiles after burn, particularly interleukin-1beta and macrophage inhibitory factor, appear to be mediated by C/EBP-beta and STAT-3, whereas after the induction of a sepsis, tumor necrosis factor and interleukin-6 are mainly mediated by STAT-5. Based on our findings we suggest that the pathophysiologic state of a thermal injury is not comparable with sepsis in association with signal transcription factors and the differences in intracellular and extracellular signaling therefore opens new ideas for therapeutic options.

The careful assessment of microchimerism is essential to investigate the effects of donor bone marrow-derived cells in transplantation. We have developed a protocol to assess microchimerism based on the HLA mismatch between the recipient and the donor. Our approach combines real-time polymerase chain reaction (PCR) with sequence-specific primer PCR (SSP-PCR) to selectively amplify and measure the abundance of donor HLA alleles in DNA samples extracted from the recipient after transplant. To optimize and validate the reliability of this method at different levels of microchimerism, we tested serial dilutions of donor DNA into recipient DNA. We demonstrate that donor alleles can be readily detected and reliably measured at concentrations as low as 0.1%. This method is simple and rapid and could find practical application in the assessment of microchimerism in patients receiving organ or cellular transplants in conjunction with donor bone marrow cells infusion.

In this study we have designed and constructed an anti-Fas ribozyme and show that it can specifically cleave the Fas mRNA in vitro. Moreover, to test its efficacy ex vivo, we transfected the anti-Fas ribozyme into betaTC-3 insulinoma cells, using a RNA polymerase III promoter to drive its expression. Like pancreatic beta cells, betaTC-3 cells do not constitutively express Fas, but Fas expression can be induced with IL-1 and IFN-gamma. Transfected cells expressed an average of 5000 copies of anti-Fas ribozyme transcript per cell as assessed by reverse transcriptase-real-time PCR. After IL-1/IFN-gamma treatment, betaTC-3 cells transfected with the anti-Fas ribozyme expressed 80% less Fas compared with mock-transfected cells. In addition, the anti-Fas ribozyme also inhibited Fas expression in NIT-1 insulinoma cells and in primary cultures of dispersed pancreatic islet cells. Inhibition of de novo Fas expression in betaTC-3 cells expressing the anti-Fas ribozyme correlated with resistance to Fas-mediated apoptosis as determined by the number of cells exhibiting caspase 3 proteolytic activity. Hence, we have engineered a ribozyme capable of preventing Fas expression in the betaTC-3 pancreatic insulinoma cell line and conferring resistance to Fas-mediated apoptosis. We suggest that ribozymes may be potentially useful to engineer resistance to apoptosis in transplantable beta cells, a feature that may significantly improve the survival of islet cell grafts.

Cichlid fishes of the East African Rift Valley lakes constitute an important model of adaptive radiation. Explosive speciation in the Great Lakes, in some cases as recently as 12 400 years ago, generated large species flocks that have been the focus of evolutionary studies for some time. The studies have, however, been hampered by the paucity of biochemical markers for phylogenetic reconstruction. Here, we describe a set of markers which should help to alleviate this problem. They are the class I genes of the major histocompatibility complex. We provide evidence for the existence of at least 17 class I loci in cichlid fishes, and for extensive polymorphism of three of these loci. Since the polymorphism has a trans-species character, it will be possible to use it in investigating the founding events of the individual species. The sequences of the cichlid class I fishes support the monophyly of actinopterygian fish on the one hand, and of tetrapods on the other.