HMGB1-promoted and TLR2/4-dependent NK cell maturation and activation take part in rotavirus-induced murine biliary atresia.

Recent studies show that NK cells play important roles in murine biliary atresia (BA), and a temporary immunological gap exists in this disease. In this study, we found high-mobility group box-1 (HMGB1) and TLRs were overexpressed in human and rotavirus-induced murine BA. The overexpressed HMGB1 released from the nuclei of rotavirus-infected cholangiocytes, as well as macrophages, activated hepatic NK cells via HMGB1-TLRs-MAPK signaling pathways. Immature NK cells had low cytotoxicity on rotavirus-injured cholangiocytes due to low expression of TLRs, which caused persistent rotavirus infection in bile ducts. HMGB1 up-regulated the levels of TLRs of NK cells and promoted NK cell activation in an age-dependent fashion. As NK cells gained increasing activation as mice aged, they gained increasing cytotoxicity on rotavirus-infected cholangiocytes, which finally caused BA. Adult NK cells eliminated rotavirus-infected cholangiocytes shortly after infection, which prevented persistent rotavirus infection in bile ducts. Moreover, adoptive transfer of mature NK cells prior to rotavirus infection decreased the incidence of BA in newborn mice. Thus, the dysfunction of newborn NK cells may, in part, participate in the immunological gap in the development of rotavirus induced murine BA.

An unusual case of foreign body pulmonary embolus: case report and review of penetrating trauma at a pediatric trauma center.

PURPOSE: Penetrating thoracic trauma is relatively rare in the pediatric population. Embolization of foreign bodies from penetrating trauma is very uncommon. We present a case of a 6-year-old boy with a penetrating foreign body from a projectile dislodged from a lawn mower. Imaging demonstrated a foreign body that embolized to the left pulmonary artery, which was successfully treated non-operatively. METHODS: We reviewed the penetrating thoracic trauma patients in the trauma registry at our institution between 1/1/03 and 12/31/12. Data collected included demographic data, procedures performed, complications and outcome. RESULTS: Sixty-five patients were identified with a diagnosis of penetrating thoracic trauma. Fourteen of the patients had low velocity penetrating trauma and 51 had high velocity injuries. Patients with high velocity injuries were more likely to be older and less likely to be Caucasian. There were no statistically significant differences between patients with low vs. high velocity injuries regarding severity scores or length of stay. There were no statistically significant differences in procedures required between patients with low and high velocity injuries. CONCLUSIONS: Penetrating thoracic trauma is rare in children. The case presented here represents the only report of cardiac foreign body embolus we could identify in a pediatric patient.

Heparin-binding epidermal growth factor-like growth factor protects mesenchymal stem cells.

BACKGROUND: We have previously demonstrated that mesenchymal stem cell (MSC) administration protects the intestines from injury in a mouse model of intestinal ischemia/reperfusion injury. We have also shown that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent intestinal cytoprotective agent in vivo that can protect the intestines by way of its effects on stem cells. The goal of the present study was to examine the effects of HB-EGF on both amniotic fluid (AF)- and bone marrow (BM)-derived MSCs in vitro. METHODS: MSCs were isolated from the AF and BM of pan-EGFP mice, grown in MSC-specific culture medium, and purified by sequential passages according to their adherence properties. Pluripotency was confirmed by induced differentiation. After incubation of MSCs with HB-EGF, proliferation was quantified using the CyQuant cell proliferation assay kit under normoxic and anoxic conditions. Chemotaxis was quantified using the CHEMICON QCM cell migration kit, and apoptosis was determined using caspase-3 immunohistochemistry after exposure of the MSCs to anoxic stress. RESULTS: AF-MSCs and BM-MSCs showed significantly increased proliferation and migration in response to HB-EGF. HB-EGF significantly protected AF-MSCs and BM-MSCs from anoxia-induced apoptosis. The proliferative and anti-apoptotic effects of HB-EGF were even more pronounced in AF-MSCs than in BM-MSCs. CONCLUSIONS: These results have demonstrated that HB-EGF acts as a mitogenic and chemotactic agent for MSCs that protects MSCs from injury. These findings could have important implications for future experiments designed to use MSCs to protect the intestines from injury.

HB-EGF augments the ability of mesenchymal stem cells to attenuate intestinal injury.

BACKGROUND: We have previously demonstrated that heparin-binding EGF-like growth factor (HB-EGF) and mesenchymal stem cell (MSC) administration protect the intestines from ischemia/reperfusion (I/R) injury in vivo, with amniotic fluid-derived MSC (AF-MSC) being more efficacious than bone marrow-derived MSC (BM-MSC). The goal of the current study was to determine whether the protective effects of HB-EGF were from direct effects on MSC or via alternative mechanisms. METHODS: Murine MSC were transfected with an HB-EGF plasmid or control plasmid by electroporation. Mice were subjected to segmental intestinal I/R injury and received either BM-MSC or AF-MSC either with or without exogenous HB-EGF, or BM-MSC or AF-MSC that endogenously over-expressed HB-EGF. MSC engraftment, intestinal histologic injury, and intestinal permeability were quantified. RESULTS: There was increased MSC engraftment into injured compared to uninjured intestine. HB-EGF increased AF-MSC engraftment into injured intestine. Administration of HB-EGF and MSC improved intestinal histology and intestinal permeability after I/R injury, with AF-MSC being most efficacious. The effect of HB-EGF on MSC was similar when the growth factor was administered exogenously, or when it was overexpressed endogenously. CONCLUSIONS: The effect of HB-EGF on AF-MSC was similar with both exogenous administration and endogenous overexpression of the growth factor, implying that HB-EGF has a direct effect on AF-MSC. This information may assist in guiding potential future AF-MSC-based therapies for patients at risk of intestinal ischemic injuries.

The role of the intestinal microcirculation in necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) continues to be a devastating inflammatory disease of the newborn intestine. Despite advances in management, morbidity and mortality remain high. While it is clear that intestinal ischemia plays a large role in disease pathogenesis, attempts to link NEC to intestinal macrovascular derangement have been largely unsuccessful. More recently, there has been a concerted effort to characterize the pathologic changes of the intestinal microcirculation in response to intestinal injury, including NEC. This microcirculatory regulation is controlled by a balance of vasoconstrictor and vasodilator forces. Vasoconstriction is mediated primarily by endothelin-1 (ET-1), while vasodilation is mediated primarily by nitric oxide (NO). These chemical mediators have been implicated in many aspects of intestinal ischemic injury and NEC, with the balance shifting toward increased vasoconstriction associated with intestinal injury. With a proper understanding of these antagonistic forces, potential therapeutic avenues may result from improving this pathologic microcirculatory dysregulation.

Synergistic effects of HB-EGF and mesenchymal stem cells in a murine model of intestinal ischemia/reperfusion injury.

BACKGROUND: We have previously demonstrated that heparin-binding EGF-like growth factor (HB-EGF) administration protects the intestines from ischemia/reperfusion (I/R) injury in vivo. We have also shown that HB-EGF promotes mesenchymal stem cell (MSC) proliferation and migration in vitro. The goals of the current study were to examine the effects of HB-EGF and both bone marrow (BM)- and amniotic fluid (AF)-derived MSC on intestinal I/R injury in vivo. MATERIALS AND METHODS: MSC were isolated from pan-EGFP mice, expanded, and purified. Pluripotency was confirmed by induced differentiation. Mice were subjected to terminal ileum I/R and received either: (1) no therapy; (2) HB-EGF; (3) BM-MSC; (4) HB-EGF+BM-MSC; (5) AF-MSC; or (6) HB-EGF+AF-MSC. MSC engraftment, histologic injury, and intestinal permeability were quantified. RESULTS: There was increased MSC engraftment into injured compared to uninjured intestine for all experimental groups, with significantly increased engraftment for AF-MSC+HB-EGF compared to AF-MSC alone. Administration of HB-EGF and MSC improved intestinal histology and intestinal permeability after I/R injury. The greatest improvement was with combined administration of HB-EGF+AF-MSC. CONCLUSIONS: Both HB-EGF alone and MSC alone can protect the intestines from I/R injury, with synergistic efficacy occurring when HB-EGF and AF-MSC are administered together.

Enteric nervous system abnormalities are present in human necrotizing enterocolitis: potential neurotransplantation therapy.

INTRODUCTION: Intestinal dysmotility following human necrotizing enterocolitis suggests that the enteric nervous system is injured during the disease. We examined human intestinal specimens to characterize the enteric nervous system injury that occurs in necrotizing enterocolitis, and then used an animal model of experimental necrotizing enterocolitis to determine whether transplantation of neural stem cells can protect the enteric nervous system from injury. METHODS: Human intestinal specimens resected from patients with necrotizing enterocolitis (n = 18), from control patients with bowel atresia (n = 8), and from necrotizing enterocolitis and control patients undergoing stoma closure several months later (n = 14 and n = 6 respectively) were subjected to histologic examination, immunohistochemistry, and real-time reverse-transcription polymerase chain reaction to examine the myenteric plexus structure and neurotransmitter expression. In addition, experimental necrotizing enterocolitis was induced in newborn rat pups and neurotransplantation was performed by administration of fluorescently labeled neural stem cells, with subsequent visualization of transplanted cells and determination of intestinal integrity and intestinal motility. RESULTS: There was significant enteric nervous system damage with increased enteric nervous system apoptosis, and decreased neuronal nitric oxide synthase expression in myenteric ganglia from human intestine resected for necrotizing enterocolitis compared with control intestine. Structural and functional abnormalities persisted months later at the time of stoma closure. Similar abnormalities were identified in rat pups exposed to experimental necrotizing enterocolitis. Pups receiving neural stem cell transplantation had improved enteric nervous system and intestinal integrity, differentiation of transplanted neural stem cells into functional neurons, significantly improved intestinal transit, and significantly decreased mortality compared with control pups. CONCLUSIONS: Significant injury to the enteric nervous system occurs in both human and experimental necrotizing enterocolitis. Neural stem cell transplantation may represent a novel future therapy for patients with necrotizing enterocolitis.