Stomach & Intestine

* ENTEROPATHOGENICESCHERICHIA COLIINFECTION INDUCES TRANSMIGRATION OF NEUTROPHILS ACROSS CULTURED INTESTINAL EPITHELIAL CELLSS K Michail and F Abernathy, Pediatrics, Wright State University; and The Children’s Medical Center, Dayton, OH, United States. Enteropathogenic Escherichia coli (EPEC) has been known as a noninvasive organism. EPEC attaches to the intestinal epithelial cells producing the known “attachment and effacement lesions”, where the microvilli are lost and a “pedestal” is formed. These morphological changes, though interesting, still do not account for the development of severe diarrhea. The intestine after EPEC infection displays an inflammatory infiltrate. It is not clear whether EPEC influences the migration of those neutrophils that constitute the inflammatory reaction. The goal of the study was to determine whether EPEC is capable of inducing neutrophil migration across the intestinal epithelial monolayer. To perform the study, intestinal epithelial monolayers (Caco2 and T-84 cells) were rapidly infected with EPEC strain E2348/69 and 1.5 million Indium-labeled (Induim111) neutrophils were allowed to incubate for 150 minutes on the serosal side of the monolayer, separated by a 12 micron cell culture insert membrane. The radioactivity of the Indium was then assayed on the apical side, both in the bathing solution as well as in the monolayer. The number of migrated neutrophils was then calculated and a paired t-test analysis was made. Neutrophil migration in response to EPEC was significantly higher that the control (2.5 X 105 ± 1.5 X 105 versus 0.4 X105 ± 0.5 X 105, p=0.002). EPEC supernatant (EPEC growth media centrifuged and filtered to remove all bacteria) failed to induce neutrophil migration. The data indicate that EPEC, which is considered a noninvasive enteric pathogen, is indeed capable of inducing polymorphonuclear leukocyte transmigration across the intestinal epithelial cells. This capability requires the presence of the bacteria rather than a released bacterial factor. EPEC induced neutrophil migration can explain the intestinal inflammatory infiltrate seen with the infection and may help elucidate the pathophysiology behind the development of severe diarrhea in the host. * ESCHERICHIA COLIK1 IBE A IS REQUIRED FOR EFFICIENT INTESTINAL EPITHELIAL INVASION IN VITRO AND IN VIVO IN NEONATAL RATSM M Pietzak, J Badger, S H Huang, D W Thomas, H Shimada, K S Kim and C Inderlied, Gastroenterology and Nutrition, Infectious Diseases and Pathology, Childrens Hospital Los Angeles, Los Angeles, CA, United States; and Keck School of Medicine, University of Southern California, Los Angeles, CA, United States. Introduction: E. coli K1 is an important cause of sepsis and meningitis in pediatrics. E. coli K1 originates from the gastrointestinal tract, but it is unclear how and what factors mediate its intestinal translocation. Aims: To examine whether: (1) E. coli strain E44, a K1 encapsulated isolate from the CSF of an infant with meningitis, can invade intestinal epithelial cells in vitro (2) invasion is enhanced by microaerophilic growth conditions (3) E44 can colonize and cause bacteremia in neonatal rats (4) E44-derived TnphoA mutant 10A-23, with an insertion in gene ibeA, would be less invasive in vitro and in vivo. Methods: Invasion assays were performed on human intestinal epithelial cell lines Caco-2 and C2BBe-1 with bacteria grown under aerobic and microaerophilic conditions. Transmission electron microscopy visualized the interactions between bacteria and epithelia. Rat pups were fed E44 and 10A-23, and blood and stool were cultured. Results: E44 was 120 to 1000-fold more invasive than 10A-23 and a laboratory E. coli K12 strain, HB101, in both cell lines. E44 grown under microaerophilic conditions demonstrated a consistent 10-fold increase in invasion in both cells lines. TEM demonstrated that E44 disrupted the microvillus architecture and invaded through the apical membrane of the Caco-2 cell in an area dense with mitochondria. 10A-23 was significantly less successful at colonizing the gastrointestinal tract and causing bacteremia (p<.005) in the neonatal rat as compared to E44. Conclusions: E. coli K1 isolate E44 is capable of intestinal epithelial cell invasion in vitro. This invasion is enhanced under a microaerophilic environment, which mimics the gastrointestinal milieu. Electron microscopy suggests that intestinal epithelial cell invasion in vitro requires metabolically active enterocytes, and may occur via a receptor-mediated process. E. coli gene ibeA contributes to this organism’s ability to colonize, invade, and translocate across the intestinal epithelial barrier in a neonatal rat model. Future studies will focus on identification of the receptor for the Ibe protein and its physiologic role in the human gastrointestinal tract. * BIOCHEMICAL AND FUNCTIONAL INTERACTION OF THE EPITHELIAL CHLORIDE CHANNEL, ClC-2 WITH THE ACTIN CYTOSKELETON AND THE MOLECULAR MOTOR, DYNEINN N Ahmed, M Ramjeesingh, C Li, R Mohammad-Panah, S Dhani and C E Bear, Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada. ClC-2 is a member of the ClC family of chloride channels and its role in physiology is yet to be determined. It has been localized to the epithelia of the lung and gastrointestinal tract and it has therefore been suggested that ClC-2 may be well suited to functionally compensate for CFTR, and as such may consequently be a potential therapeutic target in cystic fibrosis. This channel has been shown to be activated by hyperpolarization and by cell swelling. However, the mechanisms underlying the regulation of ClC-2 activity remain unclear. Purified, reconstituted ClC-2 exhibits constitutive channel activity, suggesting that the purification process may remove regulatory proteins. It is our hypothesis that ClC-2 interacts with and is regulated by cytoskeletal proteins. In order to search for interacting proteins, purified ClC-2 protein was conjugated to an agarose column and we eluted interacting proteins from mouse brain lysate for analysis. Western blotting of the eluant confirmed the presence of actin. MALDI-tof analysis identified the presence of the molecular motor, dynein. Functional studies in Xenopus oocytes indicate that both these biochemical interactions were functionally important. Using the two-electrode voltage clamp technique, Xenopus oocytes expressing ClC-2 were treated with cytochalasin D to disrupt the actin cytoskeleton or EHNA, a dynein inhibitor, to evaluate the effects on channel function. Disruption of the actin cytoskeleton, or inhibition of dynein resulted in ClC-2 activation. Further characterization of the biochemical interaction between ClC-2 and actin, indicates that actin associates directly with ClC-2 in vitro, and that this interaction is sensitive to ionic strength, consistent with an electrostatic interaction. Subcellular fractionation studies suggest that ClC-2 colocalizes with markers of the early endosomal compartment and the plasma membrane. Furthermore, dynein inhibition with EHNA, appears to cause redistribution of ClC-2 to the plasma membrane compartment. These studies suggest that ClC-2 channel interacts with the actin cytoskeleton and that dynein plays a role in the regulation of this channel possibly by affecting movement between cellular compartments. * HYPERAGGRESSIVE ADENOMATOUS POLYPOSIS COLI - ARE CURRENT SCREENING RECOMMENDATIONS ADEQUATE?T M Attard, R Hijazeen, N Babouk, L Fayyad, S Tinley, H T Lynch and C Cuffari, Pediatrics, Division of Gastroentrology, Johns Hopkins Hospital, Baltimore, MD, United States; Pediatrics, University of Medical Center, United of Pediatrics, The Medical Center, and University School of Medicine, United States. and Aims: for at These are on the that of in to are of of the in with as early as of may occur in the Gastroenterology we a of with coli and a family of or early were with or of the of that to the Gastroenterology at The Medical in with growth and in was in and all and are and and are from of and Conclusions: This of early The mechanisms are unclear but severe of or in with for may be in THE OF IS J K T A J and A University of Baltimore, MD, United States. - is by and its effects do not to be by or is regulated by the role of in the of purified was in of and mouse in potential and were to The role of in was by of with or of and analysis. were performed on both and to the of in induced a increase in and of in with and significantly the and of in induced a in in the but not in the and analysis confirmed regulation to and which intestinal in data suggest that may be an important diarrhea to enteric and EPITHELIAL IN J A C A M and T and Pediatrics, MD, United States. are seen in with an to function. The of in epithelial is To determine the effects of on Methods: rats to induce or of were in to and to to and To evaluate the of enteric were compared in the and presence of the Results: rats less than ± ± 12 and significantly ± ± The in both that not In to and were in the presence and of that was of In of the response to was to were in in response to and and induced a than 10-fold in the In the of to and and that the effects were directly on the epithelial In the in the response to was not in a on enteric by and effects on epithelial cells. induced a in the that would in the These effects may to the in A M E T W and S M Division of Gastroenterology and United States. is an of and that exhibits an In this we and protein and in is a protein localized in the apical of the is the of in vitro culture of suggesting a role in a characterization of the of in and where it at the A of the protein gene that are protein in the and are of the protein and is a member of the of proteins. Functional of across and suggests that and are and that and may all to the of in A M J E and D W Thomas, Division of of and Division of and Childrens Hospital Los Angeles, Los Angeles, CA, United States. with on and and been well oocytes were on stool to and was on was to the with and diarrhea. gastrointestinal in the a active and confirmed active with was treated with and for and was by the of and of and confirmed and is a and is with a to that the epithelia of and is by the of infection from to diarrhea. from infection are and This the of is a with and and and shown to be in the of and to and of been for and with and of and for and IN THE IN A M D and and Introduction: of the and migration such as and can be to has the of in To determine the after with to by and with between and from of to search effects in to the The we and blood these in the and blood to In we stool in the and for analysis was by its analysis of and on the through the been a The and this Results: The cause of was of in and the of in in not in to cause of and in the after of with we the of to by In was of in after the were presence of effects in of the The after in with do not in with and in effects in OF IN R J D and J A Gastroentrology, University of Medical Center, United States. intestinal the of of to a the of the identified were by and molecular analysis. of the after and at the of were and all demonstrated of at of been at of and to be for has been for to be with a stool culture infant a culture for after were not identified in at of may with by at the of The of this is but we demonstrated more in these compared to of by of in in in of a the effects of a in this are to be IN J S A D D H J D H and R Pediatrics, University of Canada. is the gastrointestinal in Methods: In a to determine whether with the of all of in with and were to or with the of and plasma and were in all at and and at the of of Results: in and neonatal plasma and were between the and plasma were in both at study and in A at and the of in in A compared to in The at which was in A and in were in both at of of in the of SCREENING FOR IN WITH H A and D Pediatrics, Hospital for Sick Children, Toronto, ON, Canada. and with may The of the on the of in is unclear. was to study the between of by of and the of and A search of and to was to was for analysis from studies which studies were for a from the on or Results: and a and of and and and of in from is control was by the studies for and for in are or with a from of of of of is with with would be This would and of severe at the of to was that of with a will and However, the of the of the on in is therefore for or IN M D D and T and Hospital Introduction: markers with or growth not growth of the and after with Methods: with between and were at with for as and to of our were to for H at A and was to both Results: was significantly in to in less growth in the and after the the H was significantly we with with for the and for the in H in the by in is still to In the of H at of all of at the an increase with compared to with less H at with growth the of is to explain mechanisms in these A M C and L S Children’s Medical of Medical Center, United States. with may or gastrointestinal The and and is the for that in with To determine the of consistent with in to with and to determine how a of was made. were between where the was to the or of the in all of the number of for the then performed a analysis of the from the of the the of those with and of Results: in of all from In the were - - - - such as inflammatory changes, and a with in the in it was In the a from was made. of and a with a of the are in for gastrointestinal with of these consistent with a of In with was in the of the the of in this IN and A of Canada. Introduction: is a of that can to and of and is a to that an in It can be with However, it has been with a with was to To our this is the in the of in a with with a of was or not in the The of was at of with severe was on and and of for the The family was for of but was A blood and was was at of the The and a inflammatory cell infiltrate in the were seen in the or The was The and the in Conclusions: can occur in the of this unclear. be a in with has gastrointestinal INDUCES M R K A and F Pediatrics, Hospital for Children, University School of Medicine, United States. a of is known to gastrointestinal is known to induce in the intestine of on has not been the of on in between and at the for in were a of of at the of the was then for a of minutes the and to for of intestinal were in the all of the which were were to minutes of of the from the and at the the in the in this of the as in the may be a to in is in may a therapeutic role in with Further studies are in a number of to determine the role of in and in the of OF WITH C T D K C S and E United States. data in pediatrics. A study in with a therapeutic for the of was the in in the Methods: the for were a where via an of or stool stool and of were with a or between and a stool of and for Results: were The was were were was stool was was of was The was with in with a in stool in and with a in stool in at in stool and and of in were by the as and as the the that was by Conclusions: The of in this study is to in In these or as as well as for stool and stool OF OF IN INTESTINAL A E M and Division of United States; and Division of Gastroenterology and Medical United States. A with the of intestinal was for of and severe from and the of was on the and the in and in and was to the for for a in the and and and studies of activity and of a and The a of the to be to and from the and from the was by and and by for and The and analysis for the of a of these cells in the and cells were in the and of the cells of are the cells in the gastrointestinal studies shown that of these cell in and Disruption of the in of and of the To our this is the of the of the The of these cells with the of and with the of the and on This is in to the and of where of is in as our FUNCTIONAL IN WITH THE OF AND IN WITH A S and R M Pediatrics, OH, United States. and are for of and of these cause in on is to to and and The of this in is to examine as potential of the between of and in with are with to been for as a of To and in from to been on of and are via and are of and suggest of more than but the data suggest a those with more has not been with and not a on this The are consistent with of gastrointestinal and the of to factors and and A WITH IN A K D E D and A Pediatrics, Wright State University; and The Children’s Medical Center, Dayton, OH, United States. cell are an in are in the and to our been in the a of cell in a The with and was for a on and studies the from the from the of the demonstrated a cell consistent with an gastrointestinal cell were The a at the of through was The well and has 12 after of are in this the of a in these AND IN WITH AND J F and S and Childrens Hospital, OH, United States. is a in and not an with and and an in A with an of with and The was for of the area was and the was for with was for an with at from the The demonstrated and of a was treated with and stool A with and on stool for with blood in the stool studies were with the of an The in in the The of The of stool was in of and are seen in and are by to which may for or both of a from in is to stool and with the for is from that for it is important to in with and which not to IN WITH A F T L J M and N N of University of School of Medicine, United States; Children’s Hospital of United States; and Hospital for Children, United States. been to in with or but not been in was to the of in with severe to a AND the we identified 12 of a and by were and been to of was a to a of from the of with of to of in all number of for number of and number of were all significantly after of and solution and on an in or in and development of in of solution in and of the in been to the of a of after with of a and for with severe not to OF WITH S C C S D K and E United States. in of in is with and has a on the suggests that of these not been well Methods: from to were for an study with a therapeutic for of were and of conditions and was a to and the on via an response Results: the were and the for all was The of was for and a of and were with and on the growth the for and was and and and were to and the were and The of were and of the or less of or the to the not Conclusions: in compared to is with to conditions and In is this study not a of as compared to studies are IN IN A L R C and C of The Childrens Hospital, United States. The of this study was to determine the of by in with Methods: with a of for to were and and for H and were from the and of the for were from in the and for electron of the electron was performed in a Results: was on in and the were by were than or to in in 12 and less than or to in from the were by of the with or of protein was in of and of membrane or were seen in of and of in of with are activated by in a of and this can occur on IN AND D and M Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, United States. with with and with known consistent with gastrointestinal is known the of to the of is made. To the of with are with Methods: the of all between and an and been to a at the Johns Hopkins Children’s was by in blood or increase in in the of Results: were and and The were and of for more than and for than the of of at of the with or of been to a or number of and studies to was the in which was of with of of and by or in Conclusions: These data suggest that a of with studies are to the of a of of or in to IS A IN WITH FUNCTIONAL of Pediatrics, Southern Medical CA, United States. of and is a for both and This study therefore to determine the and of in with Methods: data were for from to from all were by the in in and and were on the and the were as and on the data for the were calculated for all were of were compared between that were on and with versus Results: were seen in the study of the were of and of these was in the study the or the that the However, the a that was higher than that of the versus and of the that the and for In and of the that were less than the and for those with to of the of with that the is are for of and OF EPITHELIAL AND INTESTINAL EPITHELIAL IN AND W S R and J Pediatrics, Medical of United States. and Aims: through the intestinal barrier is important in of and The development of the of epithelial cells has not been The ability of to a protein was and with that of intestinal epithelial cells Methods: and epithelial cells of by and from or was by and The of cytoskeleton and activity of on was to was compared between of from to of Results: cells in a is not at as as requires cells to be metabolically active and is by or of the from with significantly more than from in in is not to of but to to are more at both in and to with of the in the between the and in with is less in with as compared to Conclusions: are to compared to are more in to with INTESTINAL AND and OF FOR University of Toronto, Toronto, Canada. The is in and is a and can be or The of is but can the intestinal tract, and of the intestinal is by of the to and of is and to and and may be of in which the and failed to to a was to control of well as with and and and from after and and of failed to to and resulted in of of 12 with known the gastrointestinal tract, and was for of that failed to to and been with with to to our therapeutic with was on with a in in The the was for of to a role as in both with of will be in more THE OF IN M Attard, S and C Cuffari, The Johns Hopkins Children’s Center, Baltimore, MD, United States; and Pediatrics, University of Medical Center, United States. are on are more in with known and In Pediatrics, the of not been we the and of in Methods: analysis - was on all the of with on The were and the data was in the Results: of demonstrated The was with a at of were The at the of gastrointestinal The are in Conclusions: are in pediatrics. are as a of or where potential may with from intestinal or gastrointestinal tract of IN A WITH OF AND D and Pediatrics, The United States. is a by of the gastrointestinal tract. is by with and may be number of or of invasion is for and may be in early inflammatory markers of are and been in the The in not been a and with for