Jose Paredes

No evidence for β cell neogenesis in murine adult pancreas.

Whether facultative β cell progenitors exist in the adult pancreas is a major unsolved question. To date, lineage-tracing studies have provided conflicting results. To track β cell neogenesis in vivo, we generated transgenic mice that transiently coexpress mTomato and GFP in a time-sensitive, nonconditional Cre-mediated manner, so that insulin-producing cells express GFP under control of the insulin promoter, while all other cells express mTomato (INSCremTmG mice). Newly differentiated β cells were detected by flow cytometry and fluorescence microscopy, taking advantage of their transient coexpression of GFP and mTomato fluorescent proteins. We found that β cell neogenesis predominantly occurs during embryogenesis, decreases dramatically shortly after birth, and is completely absent in adults across various models of β cell loss, β cell growth and regeneration, and inflammation. Moreover, we demonstrated upregulation of neurogenin 3 (NGN3) in both proliferating ducts and preexisting β cells in the ligated pancreatic tail after pancreatic ductal ligation. These results are consistent with some recent reports, but argue against the widely held belief that NGN3 marks cells undergoing endocrine neogenesis in the pancreas. Our data suggest that β cell neogenesis in the adult pancreas occurs rarely, if ever, under either normal or pathological conditions.

Amniotic fluid inhibits Toll-like receptor 4 signaling in the fetal and neonatal intestinal epithelium

The fetal intestinal mucosa is characterized by elevated Toll-like receptor 4 (TLR4) expression, which can lead to the development of necrotizing enterocolitis (NEC)—a devastating inflammatory disease of the premature intestine—upon exposure to microbes. To define endogenous strategies that could reduce TLR4 signaling, we hypothesized that amniotic fluid can inhibit TLR4 signaling within the fetal intestine and attenuate experimental NEC, and we sought to determine the mechanisms involved. We show here that microinjection of amniotic fluid into the fetal (embryonic day 18.5) gastrointestinal tract reduced LPS-mediated signaling within the fetal intestinal mucosa. Amniotic fluid is abundant in EGF, which we show is required for its inhibitory effects on TLR4 signaling via peroxisome proliferator-activated receptor, because inhibition of EGF receptor (EGFR) with cetuximab or EGF-depleted amniotic fluid blocked the inhibitory effects of amniotic fluid on TLR4, whereas amniotic fluid did not prevent TLR4 signaling in EGFR- or peroxisome proliferator-activated receptor γ–deficient enterocytes or in mice deficient in intestinal epithelial EGFR, and purified EGF attenuated the exaggerated intestinal mucosal TLR4 signaling in wild-type mice. Moreover, amniotic fluid-mediated TLR4 inhibition reduced the severity of NEC in mice through EGFR activation. Strikingly, NEC development in both mice and humans was associated with reduced EGFR expression that was restored upon the administration of amniotic fluid in mice or recovery from NEC in humans, suggesting that a lack of amniotic fluid-mediated EGFR signaling could predispose to NEC. These findings may explain the unique susceptibility of premature infants to the development of NEC and offer therapeutic approaches to this devastating disease.

TGFβ Receptor Signaling Is Essential for Inflammation-Induced but Not β-Cell Workload–Induced β-Cell Proliferation

Protection and restoration of a functional β-cell mass are fundamental strategies for prevention and treatment of diabetes. Consequently, knowledge of signals that determine the functional β-cell mass is of immense clinical relevance. Transforming growth factor β (TGFβ) superfamily signaling pathways play a critical role in development and tissue specification. Nevertheless, the role of these pathways in adult β-cell homeostasis is not well defined. Here, we ablated TGFβ receptor I and II genes in mice undergoing two surgical β-cell replication models (partial pancreatectomy or partial duct ligation), representing two triggers for β-cell proliferation, increased β-cell workload and local inflammation, respectively. Our data suggest that TGFβ receptor signaling is necessary for baseline β-cell proliferation. By either provision of excess glucose or treatment with exogenous insulin, we further demonstrated that inflammation and increased β-cell workload are both stimulants for β-cell proliferation but are TGFβ receptor signaling dependent and independent, respectively. Collectively, by using a pancreas-specific TGFβ receptor–deleted mouse model, we have identified two distinct pathways that regulate adult β-cell proliferation. Our study thus provides important information for understanding β-cell proliferation during normal growth and in pancreatic diseases.

Rapid and simplified purification of recombinant adeno-associated virus.

Preclinical gene therapy studies both in vitro and in vivo require high purity preparations of adeno-associated virus (AAV). Current methods for purification of AAV entail the use of centrifugation over either a CsCl or iodixanol gradient, or the use of chromatography. These methods can be cumbersome and expensive, necessitating ultrahigh speed gradient centrifugation or, for chromatography the use of other expensive equipment. In addition, these methods are time consuming, and the viral yield is not high. Currently no commercial purification kits are available for other than AAV serotype 2. A simplified method was used for the purification of AAV, with a viral yield that is able to be used effectively in adult and embryo mice. The method does not require ultrahigh speed gradient centrifugation nor chromatography. Instead, polyethylene glycol (PEG)/aqueous two-phase partitioning is used to remove soluble proteins from the PEG8000 precipitated virus-protein mixture. The procedure obtained rapidly up to 95% recovery of high quality purified AAV. The entire purification process, including HEK293 cell transfection, can be completed readily within one week, with purity seemingly higher than that obtained after one round of CsCl gradient purification.

Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation

The mammalian embryo represents a fundamental paradox in biology. Its location within the uterus, especially early during development when embryonic cardiovascular development and placental blood flow are not well-established, leads to an obligate hypoxic environment. Despite this hypoxia, the embryonic cells are able to undergo remarkable growth, morphogenesis, and differentiation. Recent evidence suggests that embryonic organ differentiation, including pancreatic β-cells, is tightly regulated by oxygen levels. Since a major determinant of oxygen tension in mammalian embryos after implantation is embryonic blood flow, here we used a novel survivable in utero intracardiac injection technique to deliver a vascular tracer to living mouse embryos. Once injected, the embryonic heart could be visualized to continue contracting normally, thereby distributing the tracer specifically only to those regions where embryonic blood was flowing. We found that the embryonic pancreas early in development shows a remarkable paucity of blood flow and that the presence of blood flow correlates with the differentiation state of the developing pancreatic epithelial cells in the region of the blood flow.

Review of outcomes of primary liver cancers in children: our institutional experience with resection and transplantation.

BACKGROUND Operative intervention plays an important role in the management of primary liver cancers in children. Recent improvements in diagnostic modalities, pre- and postoperative chemotherapy, and operative technique have all led to improved survival in these patients. Both hepatic resection and orthotopic liver transplantation are effective operations for pediatric liver tumors; which intervention is pursued is based on preoperative extent of disease. This is a review of our institutions experience with operative management of pediatric liver cancer over an 18-year period. METHODS A retrospective chart review from 1990 to 2007 identified patients who were ≤18 years old who underwent operative intervention for primary liver cancer. Demographics, type of operation, intraoperative details, pre- and postoperative management, as well as outcomes were recorded for all patients. RESULTS Fifty-four patients underwent 57 operations for primary liver cancer, 30 of whom underwent resection; the remaining 27 underwent orthotopic liver transplantation. The mean age at diagnosis was 41 months. Twenty patients had stage 1 or 2 disease and 34 patients had stage 3 or 4 disease. Forty-eight (89%) patients received preoperative chemotherapy. Postoperative chemotherapy was given to 92% of patients. Mean overall and intensive care unit duration of stay were 18 and 6 days, respectively. About 45% of patients had a postoperative complication, including hepatic artery thrombosis (n = 8), line sepsis (n = 6), mild acute rejection (n = 3), biliary stricture (n = 2), pneumothorax (n = 2), incarcerated omentum (n = 1), Horners syndrome (n = 1), and urosepsis (n = 1). Only 6 patients had a recurrence of their cancer, 5 after liver resection, 3 of whom later received a transplant. There was only 1 recurrence after liver transplantation. There was 1 perioperative mortality from cardiac arrest. Overall survival was 93%. CONCLUSION Operative intervention plays a critical role in the management of primary liver cancer in the pediatric population. Neoadjuvant chemotherapy can be given if the tumor seems unresectable at diagnosis. If chemotherapy is unable to sufficiently downstage the tumor, orthotopic liver transplantation becomes the patients best option. Our institution has had considerable experience with both resection and liver transplantation in the treatment of pediatric primary liver cancer, with good long-term outcomes.

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.

Three‐Dimensional Analysis of the Islet Vasculature

The pancreatic islets of Langerhans are highly vascularized structures scattered throughout the pancreas that contain a capillary network 5–10 times denser than that of the exocrine pancreas. A simple method for three‐dimensional (3D) analysis of this intricate intraislet vasculature has been difficult because of the intrinsic opacity of the pancreas. We developed a whole‐mount imaging technique that allows relatively easy visualization of the islet vasculature. In combination with confocal microscopy and the use of 3D imaging software, we were able to readily reconstruct the 3D architecture of an islet, allowing delineation of the islet volume, length of the intraislet vessels, and the number of vessel branch‐points. This technique allows for straightforward 3D image analysis that may help toward understanding islet function. Anat Rec, 2012.

The Expression and Function of Glucose-Dependent Insulinotropic Polypeptide in the Embryonic Mouse Pancreas

OBJECTIVE Glucose-dependent insulinotropic polypeptide (GIP) is a member of a structurally related group of hormones that also includes glucagon, glucagon-like peptides, and secretin. GIP is an incretin, known to modulate glucose-induced insulin secretion. Recent studies have shown that glucagon is necessary for early insulin-positive differentiation, and a similar role for incretins in regulating embryonic insulin-positive differentiation seems probable. Here we studied the role of GIP signaling in insulin-positive differentiation in the embryonic mouse pancreas. RESEARCH DESIGN AND METHODS The ontogeny of the GIP ligand and GIP receptor in the embryonic pancreas was investigated by immunohistochemistry and RT-PCR. GIP signaling was inhibited in cultured embryonic pancreata using morpholine-ring antisense against GIP ligand and receptor, or small interfering RNA (siRNA) for GIP ligand and receptor. Markers of endocrine cells and their progenitors were studied by immunohistochemistry and RT-PCR. RESULTS GIP and GIP receptor mRNA were both detected in the embryonic pancreas by embryonic day 9.5 and then persisted throughout gestation. GIP was generally coexpressed with glucagon by immunostaining. The GIP receptor was typically coexpressed with insulin. Morpholine-ring antisense or siRNA against either GIP ligand or GIP receptor both inhibited the differentiation of insulin-positive cells. Inhibition of GIP or its receptor also led to a decrease in the number of Pdx-1–positive and sox9-positive cells in the cultured embryonic pancreas. The number of Pax6- and Nkx2.2-positive cells, representative of developing pancreatic endocrine cells and β-cells, respectively, was also decreased. CONCLUSIONS GIP signaling may play a role in early embryonic pancreas differentiation to form insulin-positive cells or β-cells.

A simplified purification method for AAV variant by polyethylene glycol aqueous two-phase partitioning.

Adeno-Associated Virus (AAV) has been widely used for in vivo study and preclinical therapy due to its ability to mediate long-term transgene expression, its lack of pathogenicity and low immunogenicity. It has been found that AAV has more than ten serotypes, which each transfect certain types of cells in the viral infected organ. Current methods for purification of different AAV serotypes utilize CsCl or Iodixanol ultrahigh speed density gradient centrifugation, which is expensive and time consuming. We recently developed a simplified method, PEG/(NH4)2SO4 aqueous two phase partitioning, for purification of AAV serotype 2 and 8. The method does not require ultrahigh speed gradient centrifugation or chromatography. Here we further explore the simplified method for purification of other serotypes of AAV, serotype 6 and 9. This simplified method not only can be used to purify serotype 2 and 8, but also serotype 6 and 9, indicating that a variety of AAV serotypes can be purified by this method.