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Gap junctions are required for enterocyte migration in vitro, and are Inhibited in experimental NEC
PEDIATRIC SURGERY inner leaflet of plasma membranes in live cells and externalized upon apoptosis. We examined the effects of PS on DCs maturation.
Coelomic epithelium is necessary for containing organ-specific diffusible signals Barry Preuett BS, Krishna Prasadan PhD, Christina Cantu BA, Christopher McFall BS, Kok-Hooi Yew MA, Amanda Crowley BA, Mark Hembree BS, Sidhartha Tulachan MD, PhD, Susan Sharp PhD, Charles Snyder MD, FACS, George Gittes MD, FACS Kansas University Medical Center Kansas City, KS
METHODS: Bone marrow derived mouse DCs were generated in the presence of granulocyte macrophage stimulating factor (GM-CSF), exposed to PS or phosphotidylcholine (PC) containing liposomes on day 5, and treated with lipopolisaccharide (LPS) on day 6. On day 7, DCs were analyzed by flow cytometry. RESULTS: DCs exposed to media or PC and treated with LPS to induce maturation expressed significantly high levels of MHCII, CD80, CD86 and CD40 compared to iDCs. DCs exposed to PS expressed significantly less CD40 in response to LPS than control DCs.
INTRODUCTION: During embryogenesis, developing organs are in direct apposition to one another. The coelomic epithelium (peritoneal anlage) has barrier properties and its removal adversely affects development. We hypothesize that coelomic epithelium contains signals within a developing organ to prevent inappropriate attenuation of signals.
iDC mDC PC-DC PS-DC
METHODS: Pancreas was harvested from day 11 mouse embryos. Several experimental systems were used: apposition of organs with and without enveloping coelomic epithelium; transfilter experiments; cultures to allow regrowth of coelomic epithelium in the absence of underlying mesenchyme.
MHC II 17 (⫾ 5.33) 54 (⫾ 28.2) 55 (⫾ 27.0) 49 (⫾ 29.5)
CD80 43 (⫾ 24.3) 100 (⫾ 44.1) 105 (⫾ 46.2) 102.6 (⫾ 49.3)
CD86 14 (⫾ 4.8) 70 (⫾ 28.6)* 73 (⫾ 24.4) 56 (⫾ 8.6)†
CD40 8 (⫾ 7.4) 57 (⫾ 16.5)* 63 (⫾ 17.5)* 41 (⫾ 14.2)*
*Statistically significant (p ⬍ 0.05). Trends toward significance.
†
RESULTS: Differentiation of embryonic pancreas in the presence of mesenchyme, but absence of coelomic epithelium, led to decreased acinar tissue and overabundance of endocrine differentiation. These results suggest a loss of acinar-specific mesenchymal factors, and support coelomic epithelium as a barrier that contains signals. Isolated pancreatic epithelium was unable to differentiate significantly if coelomic epithelium-enveloped mesenchyme on the opposite side of a filter. Importantly, however, endocrine differentiation occurred if the coelomic epithelium had been removed from the mesenchyme on the opposite side of the filter, confirming a role for coelomic epithelium in containing signals. An instructive role for coelomic epithelium was ruled out using a culture system in which coelomic epithelium regrows over epithelium in the absence of mesenchyme. Here, no differentiation of the underlying epithelium was seen.
CONCLUSIONS: We demonstrated that PS modulates mouse DC maturation, specifically reducing CD40 expression. Diminished CD40 signaling is likely to lead to reduced IL-12 production and inhibition of Th1 responses. These findings suggest that PS, externalized by apoptotic cells and some tumor cells, could prevent autoimmunity and contribute to immune evasion by tumor.
Gap junctions are required for enterocyte migration in vitro, and are Inhibited in experimental NEC Faisal G Qureshi MBBS, Selma Cetin MD, Catherine Batey PhD, Jun Li BS, Patricia Boyle BS, Jeffrey Upperman MD, Orkan Ergun MD, Caterina Wong BS, Henri Ford MD, FACS, David Hackam MD, PhD Children’s Hospital of Pittsburgh Pittsburgh, PA
CONCLUSIONS: These results support a novel concept in developmental biology, wherein coelomic epithelium serves to compartmentalize organs during growth, preventing diffusion out of critical morphogenic signals.
Rezal Aziz MD, Andrew Lodge PhD, Xiao Chen PhD, Sonia Sugg MD, FACS, Joel Shilyansky MD, FACS Medical College of Wisconsin Milwaukee, WI
INTRODUCTION: Necrotizing enterocolitis (NEC) is characterized by inadequate mucosal healing due to impaired enterocyte migration along the crypt villus axis (Surgical Forum 2003). Because adjacent enterocytes migrate together, inter-enterocyte communication may be required for mucosal healing. Many cells communicate via gap junctions (GJ) comprised of the protein connexin-43. We therefore hypothesize that enterocyte migration requires inter-enterocyte communication, and that impaired healing during NEC is associated with impaired connexin-43 expression.
INTRODUCTION: Dentritic cells (DCs) are the most potent professional antigen presenters and are required to initiate adaptive immunity. Immature DCs (iDC) capture antigen efficiently, but express low levels of MHC II and costimulatory molecules. DCs mature in response to “danger” signals, migrate into secondary lymphoid organs and activate T cells. Maturation is characterized by increase in MHC II and costimulatory molecule expression. Apoptotic cells inhibit DCs maturation. Phosphotidylserine (PS) is an anionic phospholipid that is limited to the
METHODS: Connexin-43 expression was assessed by SDS-PAGE and immunofluorescence in IEC-6 enterocytes. GJ function was determined by the selective inter-enterocyte passage of lucifer yellow microinjected into IEC-6 cells with impermeant rhodamine-dextran ⫾GJ inhibitor oleamide (25M).Migration was measured by time-lapse microscopy of IEC-6 cells moving into a scraped wound ⫾oleamide. NEC was induced in newborn rats by formula gavage ⫹ hypoxia and terminal ileal mucosal scrapings were harvested for SDS-PAGE.
Phosphotidylserine modulates the phenotype of mouse dentritic cells
© 2004 by the American College of Surgeons Published by Elsevier Inc.
ISSN 1072-7515/04/$30.00
S51
S52
Pediatric Surgery
RESULTS: The GJ protein connexin-43 was expressed between adjacent IEC-6 cells and in mucosal scrapings. Lucifer yellow but not Rd-dextran passed between adjoining cells, indicating functional gap junctions. Oleamide significantly impaired GJs in enterocytes (communicating cells: control 3 ⫾ 1 vs. 0.5 ⫾ .2, p ⬍ 0.05). Gap junction inhibition prevented migration of IEC-6 cells into a scraped wound (control: 5 m/hr vs. oleamide 0.5 m/hr, p ⬍ 0.05), without affecting viability or proliferation. Strikingly, ileal mucosa from NEC rats showed significantly reduced expression of connexin-43 compared to controls (connexin/actin control:1 ⫾ 0.2 vs. NEC 0.2 ⫾ 0.05, p ⬍ 0.05), implying impaired gap junction function in NEC. CONCLUSIONS: Enterocyte migration requires intercellular communication via gap junctions, which are reduced in NEC. These findings provide insights into the mechanisms leading to impaired mucosal healing in this disease.
Effect of esophageal ligation (EL) on small intestinal (SI) development in a rabbit model of intrauterine growth retardation (IUGR) Christina Cellini MD, Jian Xu MD, Terry L Buchmiller-Crair MD, FACS Weill Cornell Medical College New York, NY INTRODUCTION: Uninterrupted passage of amniotic fluid (AF) through the GI tract appears necessary for normal fetal growth. Previous work suggests that this is related to defective nutrient transport when AF passage is eliminated by EL in normal weight animals. The rabbit provides a naturally occurring model of IUGR based on fetal position within the bicornuate uterus. The effect of in-utero EL on SI development in normal and IUGR fetuses was evaluated. METHODS: Thirteen pregnant rabbits underwent operation on day 24 of their 31-day gestation. Ipsilateral normal and IUGR fetuses underwent EL; contralateral fetuses underwent cervical exploration only, forming 4 study groups (Cont-Nl, Cont-IUGR; EL-Nl, ELIUGR). Fetuses were sacrificed on day 31. The SI was analyzed for: villus height, epithelial cell proliferation by PCNA staining, sodiumglucose cotransporter-1 (SGLT-1) expression by PCR against internal controls and SGLT-1 localization by immunohistochemistry. Statistical analysis was performed using the paired Student’s t-test. RESULTS: Fetal survival was 92%. IUGR fetuses had decreased weight vs. normal (p ⬍ 0.0004) which was further decreased by EL. Villus height and SI epithelial proliferation were significantly decreased in IUGR fetuses in both groups yet not affected by EL. IUGR increased SGLT-1 mRNA production (Cont-IUGR ⫽ 1.3 vs. ContNl ⫽ 1.2; p ⫽ 0.03). EL virtually eliminated villus enterocyte SGLT-1 protein expression in both groups. CONCLUSIONS: AF exclusion by in-utero EL decreased fetal weight and SGLT-1 protein expression. SI villus height and enterocyte proliferation is depressed in IUGR fetuses, but remains independent of AF passage. Exposure of the fetal GI tract to AF appears necessary for proper expression of nutrient transporter proteins.
J Am Coll Surg
Hepatocyte growth factor (HGF) increases glucagon immunoreactivity in jejunal cells during intestinal adaptation Shaheen Timmapuri MD, David Otterburn MD, Marshall Z Schwartz MD, FACS, Hwyda Arafat MD, PhD Thomas Jefferson University Philadelphia, PA INTRODUCTION: The administration of HGF during intestinal adaptation is known to enhance intestinal adaptation. Glucagon has been implicated as a potential mediator of intestinal adaptation. Previous studies have shown that HGF and glucagon synergistically increase the proliferation of hepatocytes. This study was designed to determine if HGF stimulation influenced glucagon expression in the small intestine during intestinal adaptation. METHODS: Adult male Sprague-Dawley rats were randomized to either a 70% massive small bowel resection group (MSBR) or an HGF treated MSBR group (MSBR-HGF). Seven days after surgery HGF was administered intravenously at 150ug/kg/day for 14 days. At day 21 ileal and jejunal mucosa was harvested. The RAE 230A GeneChip and MAS5 software were used to determine alterations in gene expression in the small intestine mucosa. Glucagon immunoreactivity was semiquantified in the ileum and jejunum using rabbit antiglucagon antibody. RESULTS: The MSBR-HGF group had significantly greater protein and DNA content (p ⬍ 0.05) than the MSBR group. However, glucagon gene expression in the MSBR-HGF group was decreased. Immunohistostaining for glucagon in the ileum revealed no difference in intensity between the two groups. However, the MSBR-HGF group demonstrated significantly greater jejunal glucagon immunoreactivity. CONCLUSIONS: Our data suggest that the HGF induced increase in glucagon availability is disassociated from glucagon gene up-regulation. Thus, HGF may not only enhance intestinal adaptation directly, but also indirectly by increasing the “local” availability of other growth factors in the absence of their gene up-regulation.
The role of vasculogenesis in the pathogenesis of intestinal atresia Timothy J Fairbanks MD, Robert Kanard MD, Y Robert Kim MD, Chrissy Lopez BS, Frederic Sala MS, Pierre Del Moral MS, Stijn De Langhe MS, Jacqueline Veltmaat PhD, Saverio Bellusci PhD, Kathryn Anderson MD, FACS, R. Cartland Burns MD, FACS Childrens Hospital Los Angeles Los Angeles, CA INTRODUCTION: Invalidation of the Fibroblast growth factor 10 (Fgf10) pathway results in colonic atresia. Fetal liver kinase (Flk-1), a receptor for vascular endothelial growth factor, is critical for vasculogenesis. The goal of the current study is to evaluate the role of vasculogenesis (via Flk-1 expression), as a pathogenic factor in the colonic atresia caused by the deletion of Fgf10. METHODS: Fgf10⫺/⫺, Fgf10nlacZ/⫹ & Flk-1nlacZ/⫹ mutant mouse strains were crossed, creating mutant embryos with colonic atresia expressing a lacZ reporter gene under to the control of the
Coelomic epithelium is necessary for containing organ-specific diffusible signals Barry Preuett BS, Krishna Prasadan PhD, Christina Cantu BA, Christopher McFall BS, Kok-Hooi Yew MA, Amanda Crowley BA, Mark Hembree BS, Sidhartha Tulachan MD, PhD, Susan Sharp PhD, Charles Snyder MD, FACS, George Gittes MD, FACS Kansas University Medical Center Kansas City, KS
METHODS: Bone marrow derived mouse DCs were generated in the presence of granulocyte macrophage stimulating factor (GM-CSF), exposed to PS or phosphotidylcholine (PC) containing liposomes on day 5, and treated with lipopolisaccharide (LPS) on day 6. On day 7, DCs were analyzed by flow cytometry. RESULTS: DCs exposed to media or PC and treated with LPS to induce maturation expressed significantly high levels of MHCII, CD80, CD86 and CD40 compared to iDCs. DCs exposed to PS expressed significantly less CD40 in response to LPS than control DCs.
INTRODUCTION: During embryogenesis, developing organs are in direct apposition to one another. The coelomic epithelium (peritoneal anlage) has barrier properties and its removal adversely affects development. We hypothesize that coelomic epithelium contains signals within a developing organ to prevent inappropriate attenuation of signals.
iDC mDC PC-DC PS-DC
METHODS: Pancreas was harvested from day 11 mouse embryos. Several experimental systems were used: apposition of organs with and without enveloping coelomic epithelium; transfilter experiments; cultures to allow regrowth of coelomic epithelium in the absence of underlying mesenchyme.
MHC II 17 (⫾ 5.33) 54 (⫾ 28.2) 55 (⫾ 27.0) 49 (⫾ 29.5)
CD80 43 (⫾ 24.3) 100 (⫾ 44.1) 105 (⫾ 46.2) 102.6 (⫾ 49.3)
CD86 14 (⫾ 4.8) 70 (⫾ 28.6)* 73 (⫾ 24.4) 56 (⫾ 8.6)†
CD40 8 (⫾ 7.4) 57 (⫾ 16.5)* 63 (⫾ 17.5)* 41 (⫾ 14.2)*
*Statistically significant (p ⬍ 0.05). Trends toward significance.
†
RESULTS: Differentiation of embryonic pancreas in the presence of mesenchyme, but absence of coelomic epithelium, led to decreased acinar tissue and overabundance of endocrine differentiation. These results suggest a loss of acinar-specific mesenchymal factors, and support coelomic epithelium as a barrier that contains signals. Isolated pancreatic epithelium was unable to differentiate significantly if coelomic epithelium-enveloped mesenchyme on the opposite side of a filter. Importantly, however, endocrine differentiation occurred if the coelomic epithelium had been removed from the mesenchyme on the opposite side of the filter, confirming a role for coelomic epithelium in containing signals. An instructive role for coelomic epithelium was ruled out using a culture system in which coelomic epithelium regrows over epithelium in the absence of mesenchyme. Here, no differentiation of the underlying epithelium was seen.
CONCLUSIONS: We demonstrated that PS modulates mouse DC maturation, specifically reducing CD40 expression. Diminished CD40 signaling is likely to lead to reduced IL-12 production and inhibition of Th1 responses. These findings suggest that PS, externalized by apoptotic cells and some tumor cells, could prevent autoimmunity and contribute to immune evasion by tumor.
Gap junctions are required for enterocyte migration in vitro, and are Inhibited in experimental NEC Faisal G Qureshi MBBS, Selma Cetin MD, Catherine Batey PhD, Jun Li BS, Patricia Boyle BS, Jeffrey Upperman MD, Orkan Ergun MD, Caterina Wong BS, Henri Ford MD, FACS, David Hackam MD, PhD Children’s Hospital of Pittsburgh Pittsburgh, PA
CONCLUSIONS: These results support a novel concept in developmental biology, wherein coelomic epithelium serves to compartmentalize organs during growth, preventing diffusion out of critical morphogenic signals.
Rezal Aziz MD, Andrew Lodge PhD, Xiao Chen PhD, Sonia Sugg MD, FACS, Joel Shilyansky MD, FACS Medical College of Wisconsin Milwaukee, WI
INTRODUCTION: Necrotizing enterocolitis (NEC) is characterized by inadequate mucosal healing due to impaired enterocyte migration along the crypt villus axis (Surgical Forum 2003). Because adjacent enterocytes migrate together, inter-enterocyte communication may be required for mucosal healing. Many cells communicate via gap junctions (GJ) comprised of the protein connexin-43. We therefore hypothesize that enterocyte migration requires inter-enterocyte communication, and that impaired healing during NEC is associated with impaired connexin-43 expression.
INTRODUCTION: Dentritic cells (DCs) are the most potent professional antigen presenters and are required to initiate adaptive immunity. Immature DCs (iDC) capture antigen efficiently, but express low levels of MHC II and costimulatory molecules. DCs mature in response to “danger” signals, migrate into secondary lymphoid organs and activate T cells. Maturation is characterized by increase in MHC II and costimulatory molecule expression. Apoptotic cells inhibit DCs maturation. Phosphotidylserine (PS) is an anionic phospholipid that is limited to the
METHODS: Connexin-43 expression was assessed by SDS-PAGE and immunofluorescence in IEC-6 enterocytes. GJ function was determined by the selective inter-enterocyte passage of lucifer yellow microinjected into IEC-6 cells with impermeant rhodamine-dextran ⫾GJ inhibitor oleamide (25M).Migration was measured by time-lapse microscopy of IEC-6 cells moving into a scraped wound ⫾oleamide. NEC was induced in newborn rats by formula gavage ⫹ hypoxia and terminal ileal mucosal scrapings were harvested for SDS-PAGE.
Phosphotidylserine modulates the phenotype of mouse dentritic cells
© 2004 by the American College of Surgeons Published by Elsevier Inc.
ISSN 1072-7515/04/$30.00
S51
S52
Pediatric Surgery
RESULTS: The GJ protein connexin-43 was expressed between adjacent IEC-6 cells and in mucosal scrapings. Lucifer yellow but not Rd-dextran passed between adjoining cells, indicating functional gap junctions. Oleamide significantly impaired GJs in enterocytes (communicating cells: control 3 ⫾ 1 vs. 0.5 ⫾ .2, p ⬍ 0.05). Gap junction inhibition prevented migration of IEC-6 cells into a scraped wound (control: 5 m/hr vs. oleamide 0.5 m/hr, p ⬍ 0.05), without affecting viability or proliferation. Strikingly, ileal mucosa from NEC rats showed significantly reduced expression of connexin-43 compared to controls (connexin/actin control:1 ⫾ 0.2 vs. NEC 0.2 ⫾ 0.05, p ⬍ 0.05), implying impaired gap junction function in NEC. CONCLUSIONS: Enterocyte migration requires intercellular communication via gap junctions, which are reduced in NEC. These findings provide insights into the mechanisms leading to impaired mucosal healing in this disease.
Effect of esophageal ligation (EL) on small intestinal (SI) development in a rabbit model of intrauterine growth retardation (IUGR) Christina Cellini MD, Jian Xu MD, Terry L Buchmiller-Crair MD, FACS Weill Cornell Medical College New York, NY INTRODUCTION: Uninterrupted passage of amniotic fluid (AF) through the GI tract appears necessary for normal fetal growth. Previous work suggests that this is related to defective nutrient transport when AF passage is eliminated by EL in normal weight animals. The rabbit provides a naturally occurring model of IUGR based on fetal position within the bicornuate uterus. The effect of in-utero EL on SI development in normal and IUGR fetuses was evaluated. METHODS: Thirteen pregnant rabbits underwent operation on day 24 of their 31-day gestation. Ipsilateral normal and IUGR fetuses underwent EL; contralateral fetuses underwent cervical exploration only, forming 4 study groups (Cont-Nl, Cont-IUGR; EL-Nl, ELIUGR). Fetuses were sacrificed on day 31. The SI was analyzed for: villus height, epithelial cell proliferation by PCNA staining, sodiumglucose cotransporter-1 (SGLT-1) expression by PCR against internal controls and SGLT-1 localization by immunohistochemistry. Statistical analysis was performed using the paired Student’s t-test. RESULTS: Fetal survival was 92%. IUGR fetuses had decreased weight vs. normal (p ⬍ 0.0004) which was further decreased by EL. Villus height and SI epithelial proliferation were significantly decreased in IUGR fetuses in both groups yet not affected by EL. IUGR increased SGLT-1 mRNA production (Cont-IUGR ⫽ 1.3 vs. ContNl ⫽ 1.2; p ⫽ 0.03). EL virtually eliminated villus enterocyte SGLT-1 protein expression in both groups. CONCLUSIONS: AF exclusion by in-utero EL decreased fetal weight and SGLT-1 protein expression. SI villus height and enterocyte proliferation is depressed in IUGR fetuses, but remains independent of AF passage. Exposure of the fetal GI tract to AF appears necessary for proper expression of nutrient transporter proteins.
J Am Coll Surg
Hepatocyte growth factor (HGF) increases glucagon immunoreactivity in jejunal cells during intestinal adaptation Shaheen Timmapuri MD, David Otterburn MD, Marshall Z Schwartz MD, FACS, Hwyda Arafat MD, PhD Thomas Jefferson University Philadelphia, PA INTRODUCTION: The administration of HGF during intestinal adaptation is known to enhance intestinal adaptation. Glucagon has been implicated as a potential mediator of intestinal adaptation. Previous studies have shown that HGF and glucagon synergistically increase the proliferation of hepatocytes. This study was designed to determine if HGF stimulation influenced glucagon expression in the small intestine during intestinal adaptation. METHODS: Adult male Sprague-Dawley rats were randomized to either a 70% massive small bowel resection group (MSBR) or an HGF treated MSBR group (MSBR-HGF). Seven days after surgery HGF was administered intravenously at 150ug/kg/day for 14 days. At day 21 ileal and jejunal mucosa was harvested. The RAE 230A GeneChip and MAS5 software were used to determine alterations in gene expression in the small intestine mucosa. Glucagon immunoreactivity was semiquantified in the ileum and jejunum using rabbit antiglucagon antibody. RESULTS: The MSBR-HGF group had significantly greater protein and DNA content (p ⬍ 0.05) than the MSBR group. However, glucagon gene expression in the MSBR-HGF group was decreased. Immunohistostaining for glucagon in the ileum revealed no difference in intensity between the two groups. However, the MSBR-HGF group demonstrated significantly greater jejunal glucagon immunoreactivity. CONCLUSIONS: Our data suggest that the HGF induced increase in glucagon availability is disassociated from glucagon gene up-regulation. Thus, HGF may not only enhance intestinal adaptation directly, but also indirectly by increasing the “local” availability of other growth factors in the absence of their gene up-regulation.
The role of vasculogenesis in the pathogenesis of intestinal atresia Timothy J Fairbanks MD, Robert Kanard MD, Y Robert Kim MD, Chrissy Lopez BS, Frederic Sala MS, Pierre Del Moral MS, Stijn De Langhe MS, Jacqueline Veltmaat PhD, Saverio Bellusci PhD, Kathryn Anderson MD, FACS, R. Cartland Burns MD, FACS Childrens Hospital Los Angeles Los Angeles, CA INTRODUCTION: Invalidation of the Fibroblast growth factor 10 (Fgf10) pathway results in colonic atresia. Fetal liver kinase (Flk-1), a receptor for vascular endothelial growth factor, is critical for vasculogenesis. The goal of the current study is to evaluate the role of vasculogenesis (via Flk-1 expression), as a pathogenic factor in the colonic atresia caused by the deletion of Fgf10. METHODS: Fgf10⫺/⫺, Fgf10nlacZ/⫹ & Flk-1nlacZ/⫹ mutant mouse strains were crossed, creating mutant embryos with colonic atresia expressing a lacZ reporter gene under to the control of the