Bone-marrow derived cells do not contribute to new beta-cells in the inflamed pancreas.

The contribution of bone-marrow derived cells (BMCs) to a newly formed beta-cell population in adults is controversial. Previous studies have only used models of bone marrow transplantation from sex-mismatched donors (or other models of genetic labeling) into recipient animals that had undergone irradiation. This approach suffers from the significant shortcoming of the off-target effects of irradiation. Partial pancreatic duct ligation (PDL) is a mouse model of acute pancreatitis with a modest increase in beta-cell number. However, the possibility that recruited BMCs in the inflamed pancreas may convert into beta-cells has not been examined. Here, we used an irradiation-free model to track the fate of the BMCs from the donor mice. A ROSA-mTmG red fluorescent mouse was surgically joined to an INS1Cre knock-in mouse by parabiosis to establish a mixed circulation. PDL was then performed in the INS1Cre mice 2 weeks after parabiosis, which was one week after establishment of the stable blood chimera. The contribution of red cells from ROSA-mTmG mice to beta-cells in INS1Cre mouse was evaluated based on red fluorescence, while cell fusion was evaluated by the presence of green fluorescence in beta-cells. We did not detect any red or green insulin+ cells in the INS1Cre mice, suggesting that there was no contribution of BMCs to the newly formed beta-cells, either by direct differentiation, or by cell fusion. Thus, the contribution of BMCs to beta-cells in the inflamed pancreas should be minimal, if any.

An overview of opioid usage and regional anesthesia for patients undergoing repair of anorectal malformation.

PURPOSE: The recent opioid crisis in the USA compelled us to evaluate our practice of opioid use for postoperative pain management and the influence of regional anesthesia on opioid requirement in patients undergoing repair of anorectal malformations. METHODS: A retrospective chart review was performed evaluating patients who underwent posterior sagittal anorectoplasty (PSARP) and posterior sagittal anorecto-vagino-urethroplasty (PSARVUP), with or without laparotomy, between January 2016 and March 2020. Morphine milligram equivalents per kilogram (MME/kg) were calculated. IRB approval was obtained for this study. RESULTS: A total of 105 surgical patients had either a PSARP (74 without laparotomy, 10 with laparotomy) or PSARVUP (13 without laparotomy, 8 with laparotomy). Regional anesthesia included epidurals, transversus abdominis plane block, caudal block or paravertebral catheters. Of the PSARP patients, 4 without laparotomy and 7 with laparotomy received regional anesthesia. For PSARVUP with laparotomy, 4/8 received regional. 44% of PSARP patients without laparotomy did not require opioids postoperatively. The MME/kg required exponentially increased for patients over the age of 5 who underwent PSARP. CONCLUSIONS: Regional anesthesia is a useful modality for pain control for PSARP/PSARVUP with laparotomy, decreasing the opioid usage, but it is unnecessary for the already low opioid requirements, in patients younger than 5 years of age, without laparotomy.

Simplified Purification of AAV and Delivery to the Pancreas by Intraductal Administration.

Genetic manipulation is a very powerful tool for studying diabetes, pancreatitis, and pancreatic cancer. Here we discuss the use of an adeno-associated virus (AAV) vector to modify gene expression, such as to introduce a green fluorescence protein (GFP) in wild-type mice, cre recombinase in loxP mice, or to inactivate a gene with shRNA. The use of viruses for genetic modification allows for time-specific genetic changes which have advantages over time-consuming and often complex cross-breeding strategies. Here we provide a detailed approach for this process from viral production and purification through pancreatic ductal infusion. Our protocol allows efficient delivery of AAV to mediate GFP or cre expression for cell lineage tracing in the mouse pancreas or for the delivery of transgenes under a specific promoter to these cells.

Natural History of Isolated Skull Fractures in Children.

Head injury is the most common cause of neurologic disability and mortality in children. We had hypothesized that in children with isolated skull fractures (SFs) and a normal neurological examination on presentation, the risk of neurosurgical intervention is very low. We retrospectively reviewed the medical records of all children aged six to sixteen years presenting to our Level 1 trauma center with traumatic brain injuries between January 1, 2006 and December 31, 2014. We also analyzed the National Trauma Data Bank (NTDB) research data set for the years 2012-2014 using the same metrics. During this study period, our center admitted 575 children with skull fractures, 197 of which were isolated (no associated intracranial lesions (ICLs)). Of the 197 patients with isolated SFs, 155 had a normal neurological examination at presentation. In these patients, there were no fatalities and only three (1.9%) required surgery, all for the elevation of the depressed skull fracture. Analyzing the NTDB yielded similar results. In 5,194 children with isolated SFs and a normal neurological examination on presentation, there were no fatalities and 249 (4.8%) required neurosurgical intervention, almost all involving craniotomy/craniectomy and/or elevation of the SF segments. In conclusion, children with non-depressed isolated skull fractures and a normal Glasgow coma scale (GCS) at the time of initial presentation are at extremely low risk of death or needing neurosurgical intervention.

Transient Suppression of TGFß Receptor Signaling Facilitates Human Islet Transplantation.

Although islet transplantation is an effective treatment for severe diabetes, its broad application is greatly limited due to a shortage of donor islets. Suppression of TGFß receptor signaling in ß-cells has been shown to increase ß-cell proliferation in mice, but has not been rigorously examined in humans. Here, treatment of human islets with a TGFß receptor I inhibitor, SB-431542 (SB), significantly improved C-peptide secretion by ß-cells, and significantly increased ß-cell number by increasing ß-cell proliferation. In addition, SB increased cell-cycle activators and decreased cell-cycle suppressors in human ß-cells. Transplantation of SB-treated human islets into diabetic immune-deficient mice resulted in significant improvement in blood glucose control, significantly higher serum and graft insulin content, and significantly greater increases in ß-cell proliferation in the graft, compared with controls. Thus, our data suggest that transient suppression of TGFß receptor signaling may improve the outcome of human islet transplantation, seemingly through increasing ß-cell number and function.

Intraislet Pancreatic Ducts Can Give Rise to Insulin-Positive Cells.

A key question in diabetes research is whether new ß-cells can be derived from endogenous, nonendocrine cells. The potential for pancreatic ductal cells to convert into ß-cells is a highly debated issue. To date, it remains unclear what anatomical process would result in duct-derived cells coming to exist within preexisting islets. We used a whole-mount technique to directly visualize the pancreatic ductal network in young wild-type mice, young humans, and wild-type and transgenic mice after partial pancreatectomy. Pancreatic ductal networks, originating from the main ductal tree, were found to reside deep within islets in young mice and humans but not in mature mice or humans. These networks were also not present in normal adult mice after partial pancreatectomy, but TGF-ß receptor mutant mice demonstrated formation of these intraislet duct structures after partial pancreatectomy. Genetic and viral lineage tracings were used to determine whether endocrine cells were derived from pancreatic ducts. Lineage tracing confirmed that pancreatic ductal cells can typically convert into new ß-cells in normal young developing mice as well as in adult TGF-ß signaling mutant mice after partial pancreatectomy. Here the direct visual evidence of ducts growing into islets, along with lineage tracing, not only represents strong evidence for duct cells giving rise to ß-cells in the postnatal pancreas but also importantly implicates TGF-ß signaling in this process.

Embracing Errors in Simulation-Based Training: The Effect of Error Training on Retention and Transfer of Central Venous Catheter Skills.

INTRODUCTION: Error management training is an approach that encourages exposure to errors during initial skill acquisition so that learners can be equipped with important error identification, management, and metacognitive skills. The purpose of this study was to determine how an error-focused training program affected performance, retention, and transfer of central venous catheter (CVC) placement skills when compared with traditional training methodologies. METHODS: Surgical interns (N = 30) participated in a 1-hour session featuring an instructional video and practice performing internal jugular (IJ) and subclavian (SC) CVC placement with guided instruction. All interns underwent baseline knowledge and skill assessment for IJ and SC (pretest) CVC placement; watched a "correct-only" (CO) or "correct + error" (CE) instructional video; practiced for 30 minutes; and were posttested on knowledge and IJ and SC CVC placement. Skill retention and transfer (femoral CVC placement) were assessed 30 days later. All skills tests (pretest, posttest, and transfer) were videorecorded and deidentified for evaluation by a single blinded instructor using a validated 17-item checklist. RESULTS: Both the groups exhibited significant improvements (p < 0.001) in knowledge and skills after the 1-hour training program, but the increase of items achieved on the performance checklist did not differ between conditions (CO: IJ Δ = 35%, SC Δ = 29%; CE: IJ Δ = 36%, subclavian Δ = 33%). However, 1 month later, the CO group exhibited significant declines in skill retention on IJ CVC placement (from 68% at posttraining to 44% at day 30; p < 0.05) and SC CVC placement (from 63% at posttraining to 49% at day 30; p < 0.05), whereas the CE group did not have significant decreases in performance. The CE group performed significantly better on femoral CVC placement (i.e., transfer task; 62% vs 38%; p < 0.01) and on 2 of the 3 complication scenarios (p < 0.05) when compared with the CO group. CONCLUSIONS: These data indicate that incorporating error-based activities and discussions into training programs can be beneficial for skill retention and transfer.

How do Perceptions of Autonomy Differ in General Surgery Training Between Faculty, Senior Residents, Hospital Administrators, and the General Public? A Multi-Institutional Study.

OBJECTIVE: Identify barriers to resident autonomy in today's educational environment as perceived through 4 selected groups: senior surgical residents, teaching faculty, hospital administration, and the general public. DESIGN: Anonymous surveys were created and distributed to senior residents, faculty, and hospital administrators working within 3 residency programs. The opinions of a convenience sample of the general public were also assessed using a similar survey. SETTING: Keesler Medical Center, Keesler AFB, MS; the University of Texas Health Science of San Antonio, TX; and the University of Nebraska Medical Center, Omaha, NE. PARTICIPANTS: A total of 169 responses were collected: 32 residents, 50 faculty, 20 administrators, and 67 general public. RESULTS: Faculty and residents agree that when attending staff grant more autonomy, residents' self-confidence and sense of ownership improve. Faculty felt that residents should have less autonomy than residents did (p < 0.001). When asked to reflect on the current level of autonomy at their institution, 47% of residents felt that they had too little autonomy and 38% of faculty agreed. No resident or faculty felt that residents had too much autonomy at their institution. The general public were more welcoming of resident participation than faculty (p = 0.002) and administrators (p = 0.02) predicted they would be. When the general public were asked regarding their opinions about resident participation with complex procedures, they were less welcoming than faculty, administrators, and residents thought (p < 0.001). The general public were less likely to think that resident involvement would improve their quality of care (p < 0.001). CONCLUSION: Faculty and senior residents both endorse resident autonomy as important for resident development. The general public are more receptive to resident participation than anticipated. However, with increasing procedural complexity and resident independence, they were less inclined to have residents involved. The general public also had more concerns regarding quality of care provided by residents than the other groups had.

Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes.

Genetic manipulations, with or without lineage tracing for specific pancreatic cell types, are very powerful tools for studying diabetes, pancreatitis and pancreatic cancer. Nevertheless, the use of Cre/loxP systems to conditionally activate or inactivate the expression of genes in a cell type- and/or temporal-specific manner is not applicable to cell tracing and/or gene manipulations in more than one lineage at a time. Here we report a technique that allows efficient delivery of dyes for cell tagging into the mouse pancreas through the duct system, and that also delivers viruses carrying transgenes or siRNA under a specific promoter. When this technique is applied in genetically modified mice, it enables the investigator to perform either double lineage tracing or cell lineage tracing combined with gene manipulation in a second lineage. The technique requires <40 min.

Barrier function of the coelomic epithelium in the developing pancreas.

Tight spatial regulation of extracellular morphogen signaling within the close confines of a developing embryo is critical for proper organogenesis. Given the complexity of extracellular signaling in developing organs, together with the proximity of adjacent organs that use disparate signaling pathways, we postulated that a physical barrier to signaling may exist between organs in the embryo. Here we describe a previously unrecognized role for the embryonic coelomic epithelium in providing a physical barrier to contain morphogenic signaling in the developing mouse pancreas. This layer of cells appears to function both to contain key factors required for pancreatic epithelial differentiation, and to prevent fusion of adjacent organs during critical developmental windows. During early foregut development, this barrier appears to play a role in preventing splenic anlage-derived activin signaling from inducing intestinalization of the pancreas-specified epithelium.

Renal blood flow and oxygenation drive nephron progenitor differentiation.

During kidney development, the vasculature develops via both angiogenesis (branching from major vessels) and vasculogenesis (de novo vessel formation). The formation and perfusion of renal blood vessels are vastly understudied. In the present study, we investigated the regulatory role of renal blood flow and O2 concentration on nephron progenitor differentiation during ontogeny. To elucidate the presence of blood flow, ultrasound-guided intracardiac microinjection was performed, and FITC-tagged tomato lectin was perfused through the embryo. Kidneys were costained for the vasculature, ureteric epithelium, nephron progenitors, and nephron structures. We also analyzed nephron differentiation in normoxia compared with hypoxia. At embryonic day 13.5 (E13.5), the major vascular branches were perfused; however, smaller-caliber peripheral vessels remained unperfused. By E15.5, peripheral vessels started to be perfused as well as glomeruli. While the interior kidney vessels were perfused, the peripheral vessels (nephrogenic zone) remained unperfused. Directly adjacent and internal to the nephrogenic zone, we found differentiated nephron structures surrounded and infiltrated by perfused vessels. Furthermore, we determined that at low O2 concentration, little nephron progenitor differentiation was observed; at higher O2 concentrations, more differentiation of the nephron progenitors was induced. The formation of the developing renal vessels occurs before the onset of blood flow. Furthermore, renal blood flow and oxygenation are critical for nephron progenitor differentiation.

M2 macrophages promote beta-cell proliferation by up-regulation of SMAD7.

Determination of signaling pathways that regulate beta-cell replication is critical for beta-cell therapy. Here, we show that blocking pancreatic macrophage infiltration after pancreatic duct ligation (PDL) completely inhibits beta-cell proliferation. The TGFß superfamily signaling inhibitor SMAD7 was significantly up-regulated in beta cells after PDL. Beta cells failed to proliferate in response to PDL in beta-cell-specific SMAD7 mutant mice. Forced expression of SMAD7 in beta cells by itself was sufficient to promote beta-cell proliferation in vivo. M2, rather than M1 macrophages, seem to be the inducers of SMAD7-mediated beta-cell proliferation. M2 macrophages not only release TGFß1 to directly induce up-regulation of SMAD7 in beta cells but also release EGF to activate EGF receptor signaling that inhibits TGFß1-activated SMAD2 nuclear translocation, resulting in TGFß signaling inhibition. SMAD7 promotes beta-cell proliferation by increasing CyclinD1 and CyclinD2, and by inducing nuclear exclusion of p27. Our study thus reveals a molecular pathway to potentially increase beta-cell mass through enhanced SMAD7 activity induced by extracellular stimuli.

Pancreatic duct cells as a source of VEGF in mice.

AIMS/HYPOTHESIS: Vascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking. METHODS: We analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegf (also known as Vegfa) short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo. RESULTS: Pancreatic duct cells express high levels of Vegf mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegf shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegf shRNA. CONCLUSIONS/INTERPRETATION: Human islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes.

Toll-like receptor 4-mediated endoplasmic reticulum stress in intestinal crypts induces necrotizing enterocolitis.

The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4(ΔIEC-OVER) mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4(ΔIEC) mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development.

A smad signaling network regulates islet cell proliferation.

Pancreatic ß-cell loss and dysfunction are critical components of all types of diabetes. Human and rodent ß-cells are able to proliferate, and this proliferation is an important defense against the evolution and progression of diabetes. Transforming growth factor-ß (TGF-ß) signaling has been shown to affect ß-cell development, proliferation, and function, but ß-cell proliferation is thought to be the only source of new ß-cells in the adult. Recently, ß-cell dedifferentiation has been shown to be an important contributory mechanism to ß-cell failure. In this study, we tie together these two pathways by showing that a network of intracellular TGF-ß regulators, smads 7, 2, and 3, control ß-cell proliferation after ß-cell loss, and specifically, smad7 is necessary for that ß-cell proliferation. Importantly, this smad7-mediated proliferation appears to entail passing through a transient, nonpathologic dedifferentiation of ß-cells to a pancreatic polypeptide-fold hormone-positive state. TGF-ß receptor II appears to be a receptor important for controlling the status of the smad network in ß-cells. These studies should help our understanding of properly regulated ß-cell replication.

Specific transduction and labeling of pancreatic ducts by targeted recombinant viral infusion into mouse pancreatic ducts.

Specific labeling of pancreatic ducts has proven to be quite difficult. Such labeling has been highly sought after because of the power it would confer to studies of pancreatic ductal carcinogenesis, as well as studies of the source of new insulin-producing ß-cells. Cre-loxp recombination could, in theory, lineage-tag pancreatic ducts, but results have been conflicting, mainly due to low labeling efficiencies. Here, we achieved a high pancreatic duct labeling efficiency using a recombinant adeno-associated virus (rAAV) with a duct-specific sox9 promoter infused into the mouse common biliary/pancreatic duct. We saw rapid, diffuse duct-specific labeling, with 50 and 89% labeling in the pancreatic tail and head region, respectively. This highly specific labeling of ducts should greatly enhance our ability to study the role of pancreatic ducts in numerous aspects of pancreatic growth, development and function.

α-Cells are dispensable in postnatal morphogenesis and maturation of mouse pancreatic islets.

Glucagon-producing α-cells are the second-most abundant cell type in the islet. Whereas α-cells make up less than 20% of the cells in a mature mouse islet, they occupy a much larger proportion of the pancreatic endocrine cell population during the early postnatal period, the time when morphological and functional maturation occurs to form adult islets. To determine whether α-cells have a role in postnatal islet development, a diphtheria toxin-mediated α-cell ablation mouse model was established. Rapid and persistent depletion of α-cells was achieved by daily injection of the toxin for 2 wk starting at postnatal day 1 (P1). Total pancreatic glucagon content in the α-cell-ablated mice was undetectable at P14 and still less than 0.3% of that of the control mice at 4 mo of age. Histological analyses revealed that formation of spherical islets occurred normally, and the islet size distribution was not changed despite the near-total lack of α-cells. Furthermore, there were no differences in expression of ß-cell maturation marker proteins, including urocortin 3 and glucose transporter 2, in the α-cell-ablated islets at P14. Mice lacking α-cells grew normally and appeared healthy. Both glucose and insulin tolerance tests demonstrated that the α-cell-ablated mice had normal glucose homeostasis. These results indicate that α-cells do not play a critical role in postnatal islet morphogenesis or functional maturation of ß-cells.

Neurogenin3 activation is not sufficient to direct duct-to-beta cell transdifferentiation in the adult pancreas.

It remains controversial whether adult pancreatic ducts harbor facultative beta cell progenitors. Because neurogenin3 (Ngn3) is a key determinant of pancreatic endocrine cell neogenesis during embryogenesis, many studies have also relied upon Ngn3 expression as evidence of beta cell neogenesis in adults. Recently, however, Ngn3 as a marker of adult beta cell neogenesis has been called into question by reports of Ngn3 expression in fully-developed beta cells. Nevertheless, direct evidence as to whether Ngn3 activation in adult pancreatic duct cells may lead to duct-to-beta cell transdifferentiation is lacking. Here we studied two models of Ngn3 activation in adult pancreatic duct cells (low-dose alloxan treatment and pancreatic duct ligation) and lineage-traced Ngn3-activated duct cells by labeling them through intraductal infusion with a cell-tagging dye, CFDA-SE No dye-labeled beta cells were found during the follow-up in either model, suggesting that activation of Ngn3 in duct cells is not sufficient to direct their transdifferentiation into beta cells. Therefore, Ngn3 activation in duct cells is not a signature for adult beta cell neogenesis.