Single-cell resolution analysis of the human pancreatic ductal progenitor cell niche.

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 ß-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.

A Double Fail-Safe Approach to Prevent Tumorigenesis and Select Pancreatic ß Cells from Human Embryonic Stem Cells.

The transplantation of human embryonic stem cell (hESC)-derived insulin-producing ß 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, ß 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 ß 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.

The Human Endocrine Pancreas: New Insights on Replacement and Regeneration.

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 ß cell conversion from the non-endocrine compartment without genetic manipulation.

Inflammation-Mediated Regulation of MicroRNA Expression in Transplanted Pancreatic Islets.

Nonspecific inflammation in the transplant microenvironment results in ß-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 exposed in vitro to proinflammatory cytokines and explanted syngeneic islet grafts were evaluated by miRNA arrays. A subset of 26 islet miRNAs was affected by inflammation both in vivo and in 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 of ß-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 Ca(2+) 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.

Antisense miR-7 impairs insulin expression in developing pancreas and in cultured pancreatic buds.

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 ß-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 ß-cell death and generation of ß-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 ß-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.

Characterization of pancreatic ductal cells in human islet preparations.

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<0.0001). Although beta-cell viability was independent of its density, that of PDCs was higher as the density from which they were recovered increased. There was no correlation between PDCs and beta-cell viability (R(2)=0.0078). PDCs sorted from high-density fractions produced significantly higher amounts of proinflammatory mediators and VEGF, but not TF. We conclude that PDCs isolated from different fractions had different viability and functions. The precise characterization and assessment of these cells in addition to beta-cells in human islet cell products may be of assistance in understanding their contribution to islet engraftment and in developing strategies to enhance islet graft function.

Insulin decreases inflammatory signal transcription factor expression in primary human liver cells after LPS challenge.

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 (P < 0.05). 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.

Targeting heat shock protein 90 in pancreatic cancer impairs insulin-like growth factor-I receptor signaling, disrupts an interleukin-6/signal-transducer and activator of transcription 3/hypoxia-inducible factor-1alpha autocrine loop, and reduces orthotopic tumor growth.

PURPOSE: Inhibitors of heat-shock protein 90 (Hsp90) may interfere with oncogenic signaling pathways, including Erk, Akt, and hypoxia-inducible factor-1alpha (HIF-1alpha). Because insulin-like growth factor-I receptor (IGF-IR) and signal transducer and activator of transcription 3 (STAT3) signaling pathways are implicated in the progression of pancreatic cancer, we hypothesized that blocking Hsp90 with geldanamycin derivates [17-allylamino-geldanamycin (17-AAG), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG)] would impair IGF-I- and interleukin-6-mediated signaling and thus reduce pancreatic tumor growth and angiogenesis in vivo. EXPERIMENTAL DESIGN: Human pancreatic cancer cells (HPAF-II, L3.6pl) were used for experiments. Changes in signaling pathway activation upon Hsp90 blockade were investigated by Western blotting. Effects of Hsp90 inhibition (17-AAG) on vascular endothelial growth factor were determined by ELISA and real-time PCR. Effects of 17-DMAG (25 mg/kg; thrice a week; i.p.) on tumor growth and vascularization were investigated in a s.c. xenograft model and in an orthotopic model of pancreatic cancer. RESULTS: 17-AAG inhibited IGF-IR signaling by down-regulating IGF-IRbeta and directly impairing IGF-IR phosphorylation. Hypoxia- and IL-6-mediated activation of HIF-1alpha or STAT3/STAT5 were substantially inhibited by 17-AAG. Moreover, a novel IL-6/STAT3/HIF-1alpha autocrine loop was effectively disrupted by Hsp90 blockade. In vivo, 17-DMAG significantly reduced s.c. tumor growth and diminished STAT3 phosphorylation and IGF-IRbeta expression in tumor tissues. In an orthotopic model, pancreatic tumor growth and vascularization were both significantly reduced upon Hsp90 inhibition, as reflected by final tumor weights and CD31 staining, respectively. CONCLUSIONS: Blocking Hsp90 disrupts IGF-I and IL-6-induced proangiogenic signaling cascades by targeting IGF-IR and STAT3 in pancreatic cancer, leading to significant growth-inhibitory effects. Therefore, we suggest that Hsp90 inhibitors could prove to be valuable in the treatment of pancreatic cancer.

Inhibition of heat shock protein 90 impairs epidermal growth factor-mediated signaling in gastric cancer cells and reduces tumor growth and vascularization in vivo.

Oncogenic signaling through activation of epidermal growth factor receptor (EGFR), HER-2, and hypoxia inducible-factor-1alpha (HIF-1alpha) has been implicated in gastric cancer growth and angiogenesis through up-regulation of vascular endothelial growth factor (VEGF). Recently, heat shock protein 90 (Hsp90) has been identified as a critical regulator of oncogenic protein stability, including EGFR, HER-2, and HIF-1alpha. We hypothesized that inhibition of Hsp90 impairs EGF- and hypoxia-mediated angiogenic signaling in gastric cancer cells and consequently inhibits angiogenesis and tumor growth. In vitro, the geldanamycin derivate 17-allylamino-17-demethoxygeldanamycin (17-AAG) led to marked reduction in constitutive and inducible activation of extracellular signal-regulated kinase 1/2, Akt, and signal transducer and activator of transcription 3 and decreased nuclear HIF-1alpha protein. In addition, EGFR and HER-2 were down-regulated after Hsp90 inhibition. With respect to regulation of angiogenic molecules, 17-AAG significantly reduced EGF-mediated VEGF secretion. Phosphorylation of focal adhesion kinase and paxillin were both abrogated by 17-AAG, which resulted in significant impairment of cancer cell motility. Interestingly, cytotoxic effects of 17-AAG in vitro were higher on cancer cells and gastric fibroblasts than on pericytes. In vivo, the water-soluble compound 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; 25 mg/kg, thrice per week) significantly reduced s.c. xenografted tumor growth. By immunohistochemistry, 17-DMAG significantly reduced vessel area and numbers of proliferating tumor cells in sections. Furthermore, similar significant growth-inhibitory effects of 17-DMAG were achieved when administered as low-dose therapy (5 mg/kg, thrice per week). In conclusion, blocking Hsp90 disrupts multiple proangiogenic signaling pathways in gastric cancer cells and inhibits xenografted tumor growth in vivo. Hence, gastric cancer harbors attractive molecular targets for therapy with Hsp90 inhibitors, which could lead to improved efficacy of antineoplastic therapy regimens.

Mammalian target of rapamycin is activated in human gastric cancer and serves as a target for therapy in an experimental model.

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-1alpha (HIF-1alpha). Since mTOR is an upstream regulator of HIF-1alpha, 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-1alpha 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-1alpha 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.

CD40-CD40 ligand interaction activates proinflammatory pathways in pancreatic islets.

Pancreatic islet transplantation is becoming an alternative to insulin therapy in patients suffering from brittle type 1 diabetes. A major obstacle to the procedure is the early graft loss caused by nonspecific inflammation at the site of implantation. We recently discovered that CD40, a member of tumor necrosis factor (TNF) receptor family, is expressed in pancreatic beta-cells. CD40 expression in nonhematopoietic cells is generally associated with inflammation. Therefore, we investigated the potential proinflammatory role of CD40 in human and nonhuman primate islets. Islet beta-cells responded to CD40L interaction by secreting interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and macrophage inflammatory protein (MIP)-1beta, the latter a chemokine first reported to be produced by islets. Induction of IL-8 and MIP-1beta was confirmed at the transcriptional level by quantitative RT-PCR. MIP-1beta expression in beta-cells was verified by double-immunofluorescence staining. CD40-CD40L interaction activates extracellular signal-regulated kinase 1/2 and nuclear factor-kappaB pathways in insulinoma NIT-1 cells, and inhibitors of either pathway suppress cytokine/chemokine production in islets. Moreover, ligation of CD40 receptor upregulates intercellular adhesion molecule-1, associated with inflammation, at both transcriptional and translational levels. Our results in vitro indicate that the CD40 receptor expressed by beta-cells could be activated in vivo, inducing proinflammatory responses contributing to early islet graft loss after transplantation.

Effect of oxidized regenerated cellulose/collagen matrix on dermal and epidermal healing and growth factors in an acute wound.

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.

Insulin prevents liver damage and preserves liver function in lipopolysaccharide-induced endotoxemic rats.

BACKGROUND/AIMS: Liver integrity and function are crucial for survival of patients suffering from trauma, operations or infections. Insulin decreased mortality and prevented the incidence of multi organ failure and infection in critically ill patients. The aim of the present study was to determine whether insulin exerts positive effects on hepatic homeostasis and function during endotoxemia. METHODS: Endotoxemic rats received either saline or insulin. Hepatic morphology and function was determined by measuring the effect of insulin on liver proteins, enzymes, hepatocyte apoptosis and proliferation including caspases-3 and -9 and Bcl-2. Intrahepatic ATP, glucose and lactate concentration were determined by bioluminescence. To determine possible molecular changes the effect of insulin on hepatic cytokine mRNA and gene profile analysis were assessed. RESULTS: Insulin significantly improved hepatic protein synthesis by increasing albumin and decreasing c-reactive protein, P<0.05. Insulin attenuated hepatic damage by decreasing AST and ALT, P<0.05. Improved liver morphology was due to decreased hepatocyte apoptosis along with decreased caspase-3 concentration and increased hepatocyte proliferation along with Bcl-2 concentration, P<0.05. Insulin decreased hepatic IL-1beta, IL-6 and MIF mRNA and improved hepatic glucose metabolism and glycolysis, P<0.05. GeneChip analysis revealed an anti-inflammatory effect of insulin. CONCLUSIONS: Insulin improves hepatic integrity, hepatic glucose metabolism and hepatic function by increasing cell survival and attenuating the hepatic inflammatory response in endotoxemic rats.

Delivery of Bcl-XL or its BH4 domain by protein transduction inhibits apoptosis in human islets.

Viability of isolated islets is one of the main obstacles limiting islet transplantation success. It has been reported that overexpression of Bcl-2/Bcl-XL proteins enhances islet viability. To avoid potential complications associated with long-term expression of anti-apoptotic proteins, we investigated the possibility of delivering Bcl-XL or its anti-apoptotic domain BH4 to islets by protein transduction. Bcl-XL and BH4 molecules were fused to TAT/PTD, the 11-aa cell penetrating peptide from HIV-1 transactivating protein, generating TAT-Bcl-XL and TAT-BH4, respectively. Transduction efficiency was assessed by laser scanning confocal microscopy of live islets. Biological activity was tested as the ability to protect NIT-1 insulinoma cell line from death induced by staurosporine or serum deprivation. Spontaneous caspase activation in human islets and cytotoxicity caused by IL-1beta were significantly reduced in the presence of TAT-Bcl-XL and TAT-BH4. We conclude that both TAT proteins are biologically active after transduction and could be an asset in the improvement of islet viability.

Insulin treatment improves hepatic morphology and function through modulation of hepatic signals after severe trauma.

OBJECTIVE: The purpose of the present study was to determine the effect of insulin therapy on hepatic function, structure, and hepatic mRNA and protein cytokine expression during the hypermetabolic cascade post burn. SUMMARY BACKGROUND DATA: Liver function and morphology are crucial for survival of patients suffering from trauma, operations, or infections. Insulin decreased mortality and prevented the incidence of multiorgan failure in critically ill patients. METHODS: Rats received a thermal injury and were randomly divided into the insulin or control group. Our outcome measures encompassed the effect of insulin on hepatic proteins, hepatic pro- and anti-inflammatory cytokines mRNA and proteins, hepatocyte proliferation, including Bcl-2 and hepatocyte apoptosis, with caspases-3 and caspases-9. RESULTS: Insulin significantly improved hepatic protein synthesis by increasing albumin and decreasing c-reactive protein and fat (P < 0.05). Insulin decreased the hepatic inflammatory response signal cascade by decreasing hepatic pro-inflammatory cytokines mRNA and proteins IL-1beta and tumor necrosis factor at pretranslational levels. Insulin increased hepatic cytokine mRNA and protein expression of IL-2 and IL-10 at a pretranslational level when compared with controls (P < 0.05). Insulin increased hepatocyte proliferation along with Bcl-2 concentration, while decreasing hepatocyte apoptosis along with decreased caspases-3 and -9 concentration, thus improving liver morphology (P < 0.05). CONCLUSIONS: Our data provide insight that insulin attenuates the inflammatory response by decreasing the pro-inflammatory and increasing the anti-inflammatory cascade, thus restoring hepatic homeostasis, which has been shown to be critical for organ function and survival of critically ill patients.

Exogenous liposomal IGF-I cDNA gene transfer leads to endogenous cellular and physiological responses in an acute wound.

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 microg vehicle) or liposomes plus the IGF-I cDNA (2.2 microg) and Lac Z gene (0.22 microg). Transfection was confirmed by histochemical assays for beta-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.

Insulin treatment improves the systemic inflammatory reaction to severe trauma.

OBJECTIVE: 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. SUMMARY BACKGROUND DATA: 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. METHODS: 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. RESULTS: Insulin administration decreased pro-inflammatory cytokines and proteins, while increasing constitutive-hepatic proteins (P < 0.05). Burned children receiving insulin required significantly less albumin substitution to maintain normal levels compared with control (P < 0.05). Insulin decreased free fatty acids and serum triglycerides when compared with controls (P < 0.05). Serum IGF-I and IGFBP-3 significantly increased with insulin administration (P < 0.05). CONCLUSION: 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.

Differences in the hepatic signal transcription pathway and cytokine expression between thermal injury and sepsis.

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.

Heme oxygenase-1 fused to a TAT peptide transduces and protects pancreatic beta-cells.

Transplantation of islets is becoming an established method for treating type 1 diabetes. However, viability of islets is greatly affected by necrosis/apoptosis induced by oxidative stress and other insults during isolation and subsequent in vitro culture. Expression of cytoprotective proteins, such as heme oxygenase-1 (HO-1), reduces the deleterious effects of oxidative stress in transplantable islets. We have generated a fusion protein composed of HO-1 and TAT protein transduction domain (TAT/PTD), an 11-aa cell penetrating peptide from the human immunodeficiency virus TAT protein. Transduction of TAT/PTD-HO-1 to insulin-producing cells protects against TNF-alpha-mediated cytotoxicity. TAT/PTD-HO-1 transduction to islets does not impair islet physiology, as assessed by reversion of chemically induced diabetes in immunodeficient mice. Finally, we report that transduction of HO-1 fusion protein into islets improves islet viability in culture. This approach might have a positive impact on the availability of islets for transplantation.