LACTOBACILLUS GG PREVENTS CROHN'S DISEASE (CD) MUCOSA ADHERENT-E. COLI INDUCED DEGRADATION OF TIGHT JUNCTION (TJ)-ASSOCIATED PROTEINS AND ACTIN CYTOSKELETAL ALTERATIONS
Zahangir Khaled, Stefano Guandalini, Barbara Kirschner, Barbara Hendrickson, Section of Pediatric Gastroenterology; Pediatric Infectious Disease, University of Chicago, Chicago, IL
Probiotics are microorganisms of human origin, which are associated with therapeutic or preventive health benefits. Impaired paracellular permeability is a known feature of CD patients. It has been suggested that LGG may ameliorate the intestinal permeability defect and disease activity in CD patients. We have identified mucosa adherent E. coli strains from CD patients that affect the paracellular permeability of human intestinal cell monolayers. The paracellular defect was associated with degradation of the TJ proteins ZO-1 and Occludin as well as disruption of the actin cytoskeletal architecture. In this study we examined the role of LGG in preventing the CD E. coli-induced loss of TJ proteins and actin cytoskeletal changes.
The human intestinal villous epithelial cell line Caco-2/bbe subclone C2 exhibits decreased transepithelial electrical resistance (TEER) when exposed to CD E. coli isolate CD1033 for 10 hr. The laboratory E. coli strain DH5a does not affect TEER. Cells were preincubated with LGG overnight before adding the E. coli. Cells not exposed to bacteria or exposed only to LGG were included as controls. TEER was measured at 0, 2, 4, 6, 8 and 10 hr. Levels of the TJ-associated proteins ZO-1 and Occludin were assessed in C2 cells using quantitative Western Blots. Actin cytoskeletal structure of the bacteria-exposed C2 cells was examined at 10 hr using staining with FITC-Phalloidin.
Baseline TEER increased by 25%–40% following overnight incubation with LGG. Decrease in TEER of CD1033-exposed cells was prevented by pre-incubation with LGG. LGG blocked CD1033-induced degradation of the TJ protein ZO-1 at 10 hr. Staining of CD1033-exposed cells revealed a widening in the intercellular bands, with noticeable beading. LGG pre-treatment prevented these effects of CD1033 on the actin cytoskeleton.
LGG increased the baseline TEER of human intestinal epithelial cell monolayers and prevented CD E. coli-mediated changes in paracellular permeability. LGG appeared to mediate these effects at least in part by preventing the degradation of the TJ protein ZO-1 and by preventing the disruption of the actin cytoskeletal architecture.
ALTERED ION TRANSPORT IN VIVO ACROSS CYSTIC FIBROSIS JEJUNUM: ATTENUATING FACTORS TO INTESTINAL INSPISSATION
Michael A Russo, Christoph Hogenauer, Carol A Santa Ana, Jack L Porter, Mark Millard, Randall L Rosenblatt, John S Fordtran, Baylor University Medical Center, Dallas, TX; Department of Pediatric Gastroenterology; Cystic Fibrosis Clinic, Children's Medical Center, Dallas, TX
In vitro jejunal studies in CF (knock-out) mice have shown defective Cl secretion and increased neutral Na absorption. These two abnormalities, acting in concert, are believed to cause net hyperabsorption of water and predispose neonatal CF mice to death from inspissated intestinal obstruction. The validity of this concept has not been tested in humans with CF in vivo. We therefore studied groups of young adults with CF, using in vivo intestinal perfusion.
ANTIOXIDANTS IMPROVE SURVIVAL IN FAT-LADEN HEPATOCYTES AFTER EXPOSURE TO HYDROPHOBIC BILE ACIDS
Gregory E Kobak, Rolf Dahl, Michael W Devereaux, Eric Gumpricht, Ronald J Sokol, Pediatric Liver Center, Pediatric Gastroenterology, Hepatology and Nutrition, University of Colorado School of Medicine and The Children's Hospital, Denver, CO
Hepatic steatosis and cholestasis are features of metabolic liver diseases, such as cystic fibrosis and tyrosinemia; the relative roles of steatosis vs. cholestasis in the pathogenesis of liver injury are not understood. Steatosis and cholestasis are capable of inducing increased oxidative stress in liver. Consequently, the aim of this study was to determine if antioxidants could reduce cell injury and oxidant stress in fat-laden hepatocytes exposed to GCDC.
8-week-old obese Zucker rats and lean littermates were sacrificed (n=4). Hepatocytes were isolated, suspended in buffer and preloaded x 30 min. with the antioxidants 250 μM alpha-tocopherol (Toc), 250 μM Trolox (Trl), 100 μM Idebenone (Idb), or vehicle. Glycochenodeoxycholic acid (GCDC;100 μM), a toxic bile acid that accumulates in the cholestatic liver, was added and hourly aliquots were removed for determination of: hydroperoxide generation measured by the florescence of dichlorofluororescin (DCF), cell necrosis measured by % LDH release, and % apoptosis measured by fluorescence microscopy of DAPI-stained nuclei.
Fat-laden hepatocytes showed significantly increased levels of cell necrosis, decreased apoptosis and similar hydroperoxide generation compared to lean cells exposed to 100 μM GCDC (Table). In fat-laden cells, antioxidants significantly reduced cell necrosis and hydroperoxide generation. In lean cells, antioxidants significantly reduced levels of apoptosis and hydroperoxide generation.
These data demonstrate that antioxidants protect fat-laden hepatocytes from oxidative stress and cellular necrosis caused by hydrophobic bile acids, suggesting that antioxidant therapy should be explored in metabolic liver diseases characterized by steatosis and cholestasis.
THE RON RECEPTOR TYROSINE KINASE DRAMATICALLY ALTERS INFLAMMATORY CYTOKINE PRODUCTION IN MURINE ENDOTOXIN-INDUCED ACUTE LIVER FAILURE
Mike A Leonis, Susan E Waltz, Pediatrics, Children's Hospital Medical Center, Cincinnati, OH, USA
We previously demonstrated that the targeted deletion of the tyrosine kinase-containing cytoplasmic domain of the Ron receptor leads to a protected liver injury phenotype (as assessed by liver histology, serum aminotransferase levels, and multiple markers of hepatocellular apoptosis) in a well-characterized model of endotoxin (LPS)-induced acute liver failure (ALF) in galactosamine (GalN)-sensitized mice. In response to endotoxin, Kupffer cells release a variety of cytokines, most importantly tumor necrosis factor alpha (TNFα), which leads to hepatocyte apoptosis. We hypothesized that the protected liver injury phenotype in Ron TK−/− mice is due to Kupffer cell dysfunction, leading to decreased secretion of TNFα and/or abnormal secretion of cytokines which modulate the hepatocyte's response to TNFα. To test this hypothesis, we measured serum levels of select cytokines known to modulate LPS-induced ALF, including the proinflammatory cytokines TNFα and interleukin (IL)-6, IL-10 (a suppressor of TNFα production) and interferon (IFN) γ(a hepatocyte TNFa sensitizer). Cytokines were assessed hourly by ELISA up to six hours post-injection of LPS/GalN. Cytokines were not detected in control or Ron TK−/−[mice treated with GalN alone, or at baseline prior to exposure to LPS/GalN. The time course for the release of all cytokines was comparable between groups, however, significant differences in peak serum levels were observed. Paradoxically, serum TNFα levels rose 3.5-fold higher over the first three hours post injection of LPS/GalN in Ron TK−/− mice compared to control mice (p< 0.001), whereas peak serum levels of IL-6 were identical between groups. Peak serum levels of IL-10 and IFNγ were significantly reduced in Ron TK−/− mice compared to control mice (p< 0.001). Thus, Ron TK−/− mice generate elevated serum TNFα levels and yet have a protected liver injury phenotype in response to LPS/GalN. This finding is associated with significant alterations in additional cytokines which either singly or collectively could modulate the hepatocyte's response to TNFα, and perhaps contribute to the protective liver injury phenotype. Further characterization of Ron TK−/− mice is likely to produce important insights into the role of the Ron receptor in modulating endotoxin-induced ALF, as well as other hepatic diseases where Kupffer cell responses to LPS play a prominent role, such as obesity-related steatohepatitis.
EARLY EXPRESSION OF HEPATIC ENOS IS HEPATOPROTECTIVE IN MICE WITH REDUCED SIZE LIVER ISCHEMIA/REPERFUSION INJURY: IMPLICATIONS IN LIVER TRANSPLANTS
Sulaiman Bharwani, Hirohisa Harada, Matthew B Grisham, Pediatrics and Molecular Physiology, LSU Health Sciences Center, Shreveport, LA
We have previously reported that female mice are protected to a much greater extent than are males in a model of reduced-size liver ischemia and reperfusion (RSL+I/R).This protective effect appears to be dependent upon endogenous estrogen,17β-Estradiol (E2). The purpose of this study was to determine if E2 receptor mediated endothelial nitric oxide synthase(eNOS) expression determines reduced hepatic injury in the females. Anesthetized male and female C57BL/6 wild type(wt.) and eNOS−/− mice were subjected to 70% liver ischemia for 45 min followed by resection of the remaining 30% non-ischemic lobes. Liver injury was assessed by quantifying serum alanine aminotransferase (ALT) levels and histopathology whereas the message levels of eNOS in the liver tissue were evaluated using RT-PCR. We found that 100% of female mice survived indefinitely whereas all male mice died within 5 days post-surgery. In addition, liver injury was significantly greater in male vs female mice as assessed by serum ALT levels at 20 hours post-surgery (6290 ± 1091 vs 2361 ± 528 U/L for male vs female, respectively; P<0.05). Histological scores (grade 0–5) of the liver specimens post-RSL+I/R revealed significant protection in females from the RSL+I/R injury compared to males (4.2 ± 0.4 vs 2.5 ± 0.4 average grade for male vs female; P<0.05). The protection from severe necrosis in female wt. correlated well with hepatic eNOS mRNA levels in the first hour after surgery (11-fold increase over male wt.). E2 treated wt. males showed reduced hepatic injury with 14-fold increase in eNOS over control wt. males. Conversely estrogen receptor antagonist (ICI-182780) treated female wt. showed >60% reduction in eNOS message levels with severe hepatic necrosis and death. Both eNOS−/− male and female mice showed closely matched histopathology with increase in neutrophil infiltration and necrosis following RSL I/R injury. Pretreatment with E2 in eNOS−/− male mice did not improve histopathology or survival. However, wt. males pretreated with pravastatin showed enhanced eNOS mRNA expression, less injury and improved survival. These data demonstrate that estrogen agonists as well as eNOS inducing agents and NO donors may protect the liver from RSL+I/R related neutrophil mediated injury.
ENTEROPATHOGENIC ESCHERICHIA COLI STIMULATE NEUTROPHIL MIGRATION ACROSS A CULTURED INTESTINAL EPITHELIUM WITHOUT ALTERING TRANSEPITHELIAL CONDUCTANCE
Sonia Michail, Dan Halm, Frank Abernathy, Pediatrics, Wright State University and The Children's Medical Center, Dayton, OH; Physiology and Biophysics, Wright State University, Dayton, OH
Migration of neutrophils across the intestinal epithelium is the hallmark of inflammatory conditions of the bowel. In cultured intestinal epithelial monolayer models, neutrophils can be induced to migrate along a chemotactic gradient such as n-formyl-methionyl-leucyl-phenylalanine (fMLP). Physical passage of the neutrophils across the epithelium could disrupt the tight-junctions possibly leading to a large increase in the transepithelial conductance (Gt). The goal of this study is to determine whether transepithelial migration of neutrophils induced by enteropathogenic Escherichia coli (EPEC) causes changes in Gt comparable to those seen with fMLP. The apical side of T84 monolayers were rapidly infected with EPEC E2348/69 or exposed to 1μM fMLP. A third group of monolayers exposed to neither EPEC nor fMLP served as control. Indium labeled neutrophils were added to the serosal side of monolayers grown on a cell culture insert membrane (12μm pores). Gt was measured at fixed intervals up to four hours. After a 150-min incubation, radioactivity of the neutrophils that migrated to the apical side was assayed and the number of migrating neutrophils was calculated. At 150 minutes, EPEC induced similar neutrophil chemotactic capability compared to fMLP (231±34x103 and 193±48x103, respectively, n=13, P>0.05). However, EPEC induced neutrophil migration was not associated with significant increase in Gt (1.13±0.16 fold of baseline Gt, in distinction with fMLP groups, 13.3±0.48 fold, n=7, P<0.05). Gt changes with EPEC were seen after 4 hours of infection, but were not different in the presence or absence of neutrophil migration (1.37±0.12 fold and 1.42±0.17 fold of baseline Gt, respectively, n=4, p>0.05). The results indicate that EPEC-induced neutrophil migration can occur without significant disruption of barrier function. In addition, the chemo-attractant recruiting neutrophils during EPEC infection is unlikely to be fMLP; and, the Gt increase seen with fMLP-driven recruitment may indicate a discretionary compromise of barrier function during neutrophil migration.
HEME UPTAKE AND TRANSPORT EXHIBITS POLARITY AND INFLUENCES ION TRANSPORT IN CACO-2 CELLS
Aliye Uc, Amani McMurray, John B Stokes, Bradley E Britigan, Pediatrics and Medicine, University of Iowa, Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA
Despite their vital functions, heme prosthetic groups can promote cellular injury by generating reactive oxygen species. Therefore, heme absorption and distribution should be carefully regulated. Dietary heme provides the majority of the body's iron, but little is known about its absorption through the intestines. Presumably, after entering the enterocyte, iron is acquired from heme via heme oxygenase-1 (HO-1).
Heme uptake and transport is actively regulated in a cultured intestinal epithelial cell line, Caco-2 cells.
Confluent monolayers of Caco-2 cells grown on filters were treated with hemin from either apical (lumen) or basolateral side. HO-1 induction was determined by immunoblot; hemin transport and uptake with spectrophotometry. Transepithelial resistance (TER) and short circuit currents (Isc) were measured in Ussing chambers. Cell viability was confirmed by using MTT assay.
Contrary to our expectations, Caco-2 cells treated with hemin from the basolateral side demonstrated a higher HO-1 induction than cells treated with hemin from the apical surface. In addition, basolateral-to-apical transport of hemin was more rapid than hemin transport in the opposite direction. Cellular hemin uptake was not different with apical or basolateral exposure. Hemin uptake and transport from the basolateral side, but not the apical side slowed at 4°C, suggesting an active metabolic process. Hemin increased Isc in Caco-2 cells, suggesting a linkage of heme metabolism with an electrolyte transport. Hemin-induced Isc was not affected by benzamyl, bumetanide or DIDS. However, removal of Cl− and exposure to the Na/K ATPase inhibitor, ouabain greatly reduced the increase in Isc, suggesting that the current was secondary to active Cl− secretion.
Hemin is acquired and transported in both directions (apical to basolateral and vice versa), which leads to HO-1 induction in Caco-2 cells. Transport of hemin from basolateral to apical side appears to occur via an active process. This process may also be linked to Cl− secretion. This bi-directional movement of heme may play a role in intestinal regulation of iron stores and influence ion transport.
MODIFIED LABILE TOXIN (mLT) ADJUVANT INDUCES STRONGER ANTIBODY RESPONSES TO A VACCINE FOR ENTEROTOXIGENIC ESCHERICHIA COLI THAN VACCINE ALONE
Arthur J de Lorimier, Wyatt Byrd, Eric R Hall, Fredrick J Cassels, Tripler Army Medical Center, Honolulu, HI, WRAIR, MD
A vaccine against enterotoxigenic Escherichia coli (ETEC) could have great utility in lowering the morbidity and mortality of infantile diarrhea in developing nations. Our aim was to compare the immunogenicity of coli surface antigen 6 (CS6), an antigen expressed by 31% of ETEC worldwide, encapsulated in biodegradable poly(DL-lactide-co-glycolide) microspheres (CS6-PLG) administered intragastrically (IG) to mice with purified unencapsulated CS6 given with or without modified heat labile toxin (LT(R192G),mLT) as an adjuvant.
Twenty-four female BALB/c mice were randomized to receive either 25 mcg CS6-PLG (Group I, n=6), 25mcg CS6 (Group II, n=6), 25mcg CS6-PLG with 25mcg mLT (Group III, n=3) 25 mcg CS6 with 25mcg mLT (Group IV, n=3), normal saline (Group V, n=3) or 25mcg mLT (Group VI, n=3) on days 0, 24, and 56. Sacrifice was day 84. Serum and mucosal secretion specimens were collected from all animals and weights were recorded within three days prior to each dose of vaccine and prior to sacrifice. Anti-CS6 antibody was measured by enzyme linked immunosorbent assay (ELISA) for serum IgG and secretory IgA (sIgA). The anti-CS6 titers for all groups were expressed as geometric mean titer values (GMT). Analysis of variance was used to assess the data.
There were no significant differences in weight gain or activity in the mice. There was a significant rise (p<0.05) in anti-CS6 IgG GMT values in mice receiving CS6-PLG and CS6 with mLT (1:457 and 1:1530, respectively) compared to mice receiving no mLT (1:10). There was a modest rise in serum anti-CS6 IgG (1:40) in mice receiving CS6-PLG alone but no response in mice receiving CS6 alone. Mice receiving mLT with either CS6-PLG or CS6 demonstrated a small rise in anti-CS6 sIgA (2.2 fold and 1.7 fold, respectively) over mice receiving either vaccine without mLT whereas mice receiving CS6-PLG or CS6 alone demonstrated no rise in anti-CS6 sIgA.
CS6-PLG and CS6 are well tolerated when administered to mice by the IG route with or without mLT. Neither IG administered CS6-PLG nor CS6 in a dose of 25 mcg to mice produce a significant antibody response whereas in the presence of mLT both of these produce a significant antibody response. mLT is a safe and effective adjuvant when given with CS6-PLG and CS6. Additional studies will provide dose optimization and preclinical data for human trials.