Immune signalling responses in intestinal epithelial cells exposed to pathogenic Escherichia coli and lactic acid-producing probiotics.

Enterohaemorrhagic Escherichia coli O157:H7 and adherent-invasive Escherichia coli are two groups of enteric bacterial pathogens associated with haemorrhagic colitis and Crohn's Disease, respectively. Bacterial contact with host epithelial cells stimulates an immediate innate immune response designed to combat infection. In this study, immune responses of human epithelial cells to pathogens, either alone or in combination with probiotic bacteria were studied. Industrially prepared Lactobacillus helveticus strain R0052 was first examined by microarray analysis and then compared to broth-grown strains of R0052 and Lactobacillus rhamnosus strain GG using quantitative realt-time polymerase chain reaction. Results showed host immune activation responses increased following pathogen exposure, which were differentially ameliorated using probiotics depending on both the preparation of probiotics employed and conditions of exposure. These findings provide additional support for the concept that specific probiotic strains serve as a promising option for use in preventing the risk of enteric bacterial infections.

Relationship between fractional anisotropy of cerebral white matter and metabolite concentrations measured using (1)H magnetic resonance spectroscopy in healthy adults.

Fractional anisotropy (FA) of water diffusion in cerebral white matter (WM), derived from diffusion tensor imaging (DTI), is a sensitive index of microscopic WM integrity. Physiological and metabolic factors that explain intersubject variability in FA values were evaluated in two cohorts of healthy adults of different age spans (N=65, range: 28-50years; and N=25, age=66.6±6.2, range: 57-80years). Single voxel magnetic resonance spectroscopy (MRS) was used to measure N-acetylaspartate (NAA), total choline-containing compounds, and total creatine, bilaterally in an associative WM tract: anterior corona radiata (ACR). FA values were calculated for the underlying, proximal and two distal WM regions. Two-stage regression analysis was used to calculate the proportion of variability in FA values explained by spectroscopy measurements, at the first stage, and subject's age, at the second stage. WM NAA concentration explained 23% and 66% of intersubject variability (p<0.001) in the FA of the underlying WM in the younger and older cohorts, respectively. WM NAA concentration also explained a significant proportion of variability in FA of the genu of corpus callosum (CC), a proximal WM tract where some of the fibers contained within the spectroscopic voxel decussate. NAA concentrations also explained a significant proportion of variability in the FA values in the splenium of CC, a distal WM tract that also carries associative fibers, in both cohorts. These results suggest that MRS measurements explained a significant proportion of variability in FA values in both proximal and distal WM tracts that carry similar fiber-types.

Lactobacillus rhamnosus strain GG prevents enterohemorrhagic Escherichia coli O157:H7-induced changes in epithelial barrier function.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 intimately attaches to intestinal epithelial monolayers and produces attaching and effacing (A/E) lesions. In addition, EHEC infection causes disruptions of intercellular tight junctions, leading to clinical sequelae that include acute diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome. Current therapy remains supportive since antibiotic therapy increases the risk of systemic complications. This study focused on the potential therapeutic effect of an alternative form of therapy, probiotic Lactobacillus rhamnosus strain GG, to attenuate EHEC-induced changes in paracellular permeability in polarized MDCK-I and T84 epithelial cell monolayers. Changes in epithelial cell morphology, electrical resistance, dextran permeability, and distribution and expression of claudin-1 and ZO-1 were assessed using phase-contrast, immunofluorescence, and transmission electron microscopy and macromolecular flux. This study demonstrated that pretreatment of polarized MDCK-I and T84 cells with the probiotic L. rhamnosus GG reduced morphological changes and diminished the number of A/E lesions induced in response to EHEC O157:H7 infection. With probiotic pretreatment there was corresponding attenuation of the EHEC-induced drop in electrical resistance and the increase in barrier permeability assays. In addition, L. rhamnosus GG protected epithelial monolayers against EHEC-induced redistribution of the claudin-1 and ZO-1 tight junction proteins. In contrast to the effects seen with the live probiotic, heat-inactivated L. rhamnosus GG had no effect on EHEC binding and A/E lesion formation or on disruption of the barrier function. Collectively, these findings provide in vitro evidence that treatment with the probiotic L. rhamnosus strain GG could prove to be an effective management treatment for preventing injury of the epithelial cell barrier induced by A/E bacterial enteropathogens.

Corticotropin-releasing hormone (CRH) regulates macromolecular permeability via mast cells in normal human colonic biopsies in vitro.

OBJECTIVE: Persistent stress and life events affect the course of ulcerative colitis and irritable bowel syndrome by largely unknown mechanisms. Corticotropin-releasing hormone (CRH) has been implicated as an important mediator of stress-induced abnormalities in intestinal mucosal function in animal models, but to date no studies in human colon have been reported. The aim was to examine the effects of CRH on mucosal barrier function in the human colon and to elucidate the mechanisms involved in CRH-induced hyper-permeability. DESIGN: Biopsies from 39 volunteers were assessed for macromolecular permeability (horseradish peroxidase (HRP), (51)Cr-EDTA), and electrophysiology after CRH challenge in Ussing chambers. The biopsies were examined by electron and confocal microscopy for HRP and CRH receptor localisation, respectively. Moreover, CRH receptor mRNA and protein expression were examined in the human mast cell line, HMC-1. RESULTS: Mucosal permeability to HRP was increased by CRH (2.8+/-0.5 pmol/cm(2)/h) compared to vehicle exposure (1.5+/-0.4 pmol/cm(2)/h), p = 0.032, whereas permeability to (51)Cr-EDTA and transmucosal electrical resistance were unchanged. The increased permeability to HRP was abolished by alpha-helical CRH (9-41) (1.3+/-0.6 pmol/cm(2)/h) and the mast cell stabilizer, lodoxamide (1.6+/-0.6 pmol/cm(2)/h). Electron microscopy showed transcellular passage of HRP through colonocytes. CRH receptor subtypes R1 and R2 were detected in the HMC-1 cell line and in lamina propria mast cells in human colon. CONCLUSIONS: Our results suggest that CRH mediates transcellular uptake of HRP in human colonic mucosa via CRH receptor subtypes R1 and R2 on subepithelial mast cells. CRH-induced macromolecular uptake in human colon mucosa may have implications for stress-related intestinal disorders.

The natural history and significance of ultrasonographically defined polypoid lesions of the gallbladder in children.

BACKGROUND/PURPOSE: Gallbladder polyps are relatively common in adults and may increase the risk of gallbladder cancer. The increased use of high-quality abdominal ultrasonography has likely led to the increase detection of these lesions in the pediatric population. The aim of the present study was to address the significance of the ultrasonographic finding of a gallbladder polyp in children. METHODS: This prospective case series of 4 children with ultrasonographically defined gallbladder polyps outlines their presentation and clinical course. A detailed review of the literature and a management approach has been provided. RESULTS: In the 4 cases, polyps were followed for 2, 5, 72, and 84 months with a clinical follow-up of 7 to 11 years. Three underwent cholecystectomy and only 1 of 3 had a polypoid gallbladder lesion. One child, followed for 7 years, had no change in size, shape, or location of the polyp. This case series adds to the small group of children in the literature with polypoid gallbladder lesions and, to our knowledge, none have developed malignant transformation. CONCLUSIONS: Clearly more studies on such children are required to further add to our knowledge on the natural history and the appropriate management of these lesions.

Combined challenge of mice with Citrobacter rodentium and ionizing radiation promotes bacterial translocation.

PURPOSE: Both enteric infection and exposure to ionizing radiation are associated with increased intestinal permeability. However, the combined effect of irradiation and enteric infection has not been described. We combined infection of mice with the enteric pathogen, Citrobacter rodentium, with exposure to ionizing radiation and assessed the impact on colonic epithelial ion transport, permeability and bacterial translocation. MATERIALS AND METHODS: Mice were infected with C. rodentium and then received whole-body exposure to 5 Gray gamma-radiation 7 days later. Three days post-irradiation, mice were euthanized and colons removed. Control groups included sham-infected mice that were irradiated and mice that were infected, but not irradiated. RESULTS: Macroscopic damage score and colonic wall thickness were increased by C. rodentium infection, but these parameters were not exacerbated by irradiation. Infection caused an increase in myeloperoxidase activity that was reduced by irradiation. Irradiation reduced the secretory response to electrical field stimulation, forskolin and carbachol; these changes were not altered by infection with C. rodentium. None of the treatments caused an increase in permeability to 51Cr-ethylenediaminetetraacetic acid (EDTA). However, combined infection and irradiation synergistically increased bacterial translocation to mesenteric lymph nodes, liver, spleen and blood. CONCLUSIONS: Although the combination of irradiation and infection did not exacerbate the individual effects of these challenges on ion secretion and mucosal permeability to 51Cr-EDTA, it dramatically increased susceptibility to bacterial translocation and bacteremia. These results have important implications for patients who develop an enteric infection during the course of abdominopelvic radiotherapy.

Meta-analysis: Helicobacter pylori eradication treatment efficacy in children.

BACKGROUND: Several meta-analyses assessing the efficacy of anti-Helicobacter pylori treatment in adults have been published but a comparable meta-analysis in children is lacking. AIMS: To summarize the efficacy of treatments aimed at eradicating H. pylori in children and to identify sources of variation in treatment efficacy across studies. METHODS: We searched Medline, reference lists from published study reports, and conference proceedings for anti-H. pylori treatment trials in children. Weighted meta-regression models were used to find sources of variation in efficacy. RESULTS: Eighty studies (127 treatment arms) with 4436 children were included. Overall, methodological quality of these studies was poor with small sample sizes and few randomized-controlled trials. The efficacy of therapies varied across treatment arms, treatment duration, method of post-treatment assessment and geographic location. Among the regimens tested, 2-6 weeks of nitroimidazole and amoxicillin, 1-2 weeks of clarithromycin, amoxicillin and a proton pump inhibitor, and 2 weeks of a macrolide, a nitroimidazole and a proton pump inhibitor or bismuth, amoxicillin and metronidazole were the most efficacious in developed countries. CONCLUSIONS: Before worldwide treatment recommendations are given for eradication of H. pylori, additional well-designed randomized placebo-controlled paediatric trials are needed, especially in developing countries where both drug resistance and disease burden is high.

Probiotics prevent bacterial translocation and improve intestinal barrier function in rats following chronic psychological stress.

BACKGROUND AND AIMS: Chronic psychological stress, including water avoidance stress (WAS), induces intestinal mucosal barrier dysfunction and impairs mucosal defences against luminal bacteria. The aim of this study was to determine the ability of a defined probiotic regimen to prevent WAS induced intestinal pathophysiology. METHODS: Male rats were subjected to either WAS or sham stress for one hour per day for 10 consecutive days. Additional animals received seven days of Lactobacillus helveticus and L rhamnosus in the drinking water prior to stress and remained on these probiotics for the duration of the study. Rats were then sacrificed, intestinal segments assessed in Ussing chambers, and mesenteric lymph nodes cultured to determine bacterial translocation. RESULTS: All animals remained healthy for the duration of the study. Chronic WAS induced excess ion secretion (elevated baseline short circuit current) and barrier dysfunction (increased conductance) in both the ileum and colon, associated with increased bacterial adhesion and penetration into surface epithelial cells. Approximately 70% of rats subjected to WAS had bacterial translocation to mesenteric lymph nodes while there was no bacterial translocation in controls. Probiotic pretreatment alone had no effect on intestinal barrier function. However, WAS induced increased ileal short circuit current was reduced with probiotics whereas there was no impact on altered conductance. Pretreatment of animals with probiotics also completely abrogated WAS induced bacterial adhesion and prevented translocation of bacteria to mesenteric lymph nodes. CONCLUSION: These findings indicate that probiotics can prevent chronic stress induced intestinal abnormalities and, thereby, exert beneficial effects in the intestinal tract.

Anastomotic ulceration following small bowel transplantation.

We describe, for the first time, anastomotic ulcers (AU) following bowel transplantation at the Hospital for Sick Children. Two children presented with rectal bleeding, 6 and 9 months, following the transplantation. Isolated ulcers were identified at the ileo-colonic and the colo-colonic anastomosis site. The ulcers resolved, but recurred 6 and 7 months after the initial presentation. Both patients were positive for adenovirus in the stool and were treated with rapamycin. The histology revealed granulation tissue formation with mild inflammation in the adjacent mucosa, without evidence of rejection or infection. A literature search revealed 10 studies reporting 29 patients who developed AU following various surgical etiologies, none of which was bowel transplant. Numerous factors that are unique to the post-transplant period may predispose to such ulcer and are discussed in detail. Physicians and surgeons should be aware of this multifactorial complication, among other etiologies, as a cause of anemia or rectal bleeding following intestinal transplantation.

Development of a cavum septi pellucidi after Ommaya reservoir placement: report of an unusual complication.

Objective and importance. We present a complication of Ommaya reservoir placement that has not been previously reported. Following injection of a seemingly appropriately placed catheter, the patient developed seizures. Imaging studies showed the development and resolution of a cavum septi pellucidi. This case illustrates that the septum pellucidum is made of two layers and that a potential space exists between these layers. Caution is recommended when injecting a single-hole ventricular catheter if the tip is against the septum pellucidum.

Increased epithelial uptake of protein antigens in the ileum of Crohn's disease mediated by tumour necrosis factor alpha.

BACKGROUND AND AIMS: The exact nature of the epithelial barrier defect in Crohn's disease remains to be elucidated. Previously we showed increased permeability to proteins in ileal Crohn's disease. Our aims were to study if this barrier defect (a) involves endocytotic uptake of antigens and (b) is related to low grade inflammation not detectable by histology. METHODS: Macroscopically normal segments of distal ileum of Crohn's disease patients (n = 10) were subgrouped into non-inflamed (histologically unaffected) and slightly inflamed tissues and studied in Ussing chambers, with normal ileal specimens from colon cancer patients (n = 9) as controls. Endocytotic uptake into enterocytes of the protein antigen horseradish peroxidase was assessed by measuring the area of horseradish peroxidase containing endosomes in electron photomicrographs. Mucosal tumour necrosis factor alpha (TNF-alpha) mRNA was quantified using real time polymerase chain reaction. For comparison, the effects of low doses of TNF-alpha on endosomal uptake of horseradish peroxidase were studied in cultured T84 cells grown on filter supports. RESULTS: The area of horseradish peroxidase containing endosomes was increased (p<0.001) in enterocytes of non-inflamed ileum of Crohn's disease (2.8 (0.7) mum(2)/300 mum(2)) compared with control ileum (0.6 (0.06)). In non-inflamed mucosa, a significant association between endosomal uptake and mucosal expression of TNF-alpha mRNA (p = 0.03) was found. Low concentrations of TNF-alpha (0.25-1.0 ng/ml) enhanced the endosomal uptake of horseradish peroxidase in polarised T84 cells, without affecting transepithelial electrical resistance. CONCLUSIONS: Our findings suggest increased endosomal uptake of antigens in ileal Crohn's disease that may be mediated by TNF-alpha. These data highlight the transcellular route of antigen uptake in barrier dysfunction and implicate the interaction between epithelial cells and the innate immune system in the development of mucosal inflammation.

In vivo evaluation of the effects of gravitational force (+Gz) on over-the-wire stainless steel Greenfield inferior vena cava filter in swine.

This study was done to determine the effect of exposure to gravitational force (acceleration stress) on in vivo over-the-wire stainless steel Greenfield inferior vena cava filters. Fifteen pigs underwent venous cut down and placement of a stainless steel Greenfield filter. A 4-week observation period simulated realistic convalescence and allowed sufficient time for epithelialization. Ten pigs were exposed to acceleration stress in a centrifuge (3G run for 15 sec followed by rest until return to baseline heart rate, then a 9G run for 15 sec), with inertial loading in a head-to-tail direction (+Gz). Fluoroscopy during acceleration stress allowed assessment for filter migration. Five pigs were not exposed to acceleration stress. AP and lateral abdominal radiographs were obtained at post-filter placement, convalescence, and centrifuge exposure to determine the position and integrity of the filter. All 15 IVCs were resected and evaluated for gross or histological injury to the vessel wall. IVC filter placement was technically successful in all 15 pigs. Radiographic measurements were limited secondary to differences in pig positioning. Fluoroscopy showed no filter migration. All filters were securely attached to the vena cava by the hooks without gross evidence of perforation or hemorrhage. There were varying degrees of fibroplasia involving the hooks and tip of the filters in both the control and experimental groups. Histologically, there was evidence of prior hemorrhage at the level of the hooks, which was similar between the control and experimental groups. It is concluded that Greenfield filter position and vena caval integrity at the implantation site is unaffected by high acceleration stress.

Acetabular paralabral cyst: an uncommon cause of sciatica.

The association between tears of the acetabular labrum and paralabral cysts has been well documented, and magnetic resonance imaging (MRI) has been shown to be the most accurate noninvasive method of depicting not only the normal anatomic structures of the hip, but also the common pathologic processes such as labral tears and paralabral cysts. We present the case of an acetabular paralabral cyst that resulted in clinically symptomatic compression of the sciatic nerve.

Current controversies associated with Helicobacter pylori infection in the pediatric population.

Helicobacter pylori is a human bacterial gastric pathogen, fulfilling each of Koch's postulates for causal inference for ulceration in children and adults. In addition many reports purport to show that the organism causes a variety of extra-intestinal manifestations in children. This review of the English language literature provides evidence that H. pylori is likely a cause of unexplained iron deficiency (sideropenic) anemia in children, even in the absence of gastrointestinal bleeding. Much stronger evidence is required however, before H. pylori infection can be considered as an etiologic agent in recurrent abdominal pain of childhood, unexplained short stature, protracted diarrhea in pre-schoolers and sudden infant death syndrome.

Screening for celiac disease in children with recurrent abdominal pain.

BACKGROUND: The clinical presentation of celiac disease--a life-long gluten intolerance--may be characterized by chronic abdominal pain. The objective of this study was to determine if children with recurrent abdominal pain had a higher prevalence of antiendomysial antibodies (a serologic marker of celiac disease) compared with healthy children. METHODS: Children with recurrent abdominal pain and healthy control participants were recruited from the offices of community pediatricians. Serum samples were drawn and antiendomysial antibodies were measured in both groups. Demographic data included age, gender, height, and weight. RESULTS: A total of 200 children were recruited, of whom 173 (87%) had serum samples drawn. Of these, 92 were children with recurrent abdominal pain and 81 were control participants. Only 2 of the 173 samples (1.2%) were positive for antiendomysial antibody. The frequency of antiendomysial antibody positivity in children with recurrent abdominal pain was 1 in 92 (1%; 95% confidence interval, 0-6%) compared with 1 in 81 (1%; 95% confidence interval, 0-7%) in control participants. CONCLUSIONS: This community-based case-control study found no association between recurrent abdominal pain and the prevalence of antiendomysial antibody. Therefore, these data do not support screening for celiac disease in the child with classic recurrent abdominal pain in the primary care setting.

Inhibition of attaching and effacing lesion formation following enteropathogenic Escherichia coli and Shiga toxin-producing E. coli infection.

Enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) induce cytoskeletal changes in infected epithelial cells. To further characterize host cytosolic responses to infection, a series of specific cell-signaling inhibitors were employed. Initial bacterial adhesion to HEp-2 epithelial cells was not reduced, whereas alpha-actinin accumulation in infected cells was blocked by a phosphoinositide-specific phospholipase C inhibitor (ET-18-OCH3), phosphoinositide 3-kinase inhibitors (wortmannin and LY294002), and a 5-lipoxygenase inhibitor, nordihydroguaretic acid. A cyclooxygenase-2 inhibitor (NS-398), however, did not block alpha-actinin reorganization in response to EPEC and STEC infections. Understanding signal transduction responses to enteric pathogens could provide the basis for the development of novel therapeutic strategies.

Characterization of virulence factors of mouse-adapted Helicobacter pylori strain SS1 and effects on gastric hydrophobicity.

Gastric infection with Helicobacter pylori results in chronic active gastritis and in some individuals is associated with complications such as peptic ulceration and gastric cancers. A balance between bacterial factors and host responses may determine disease outcome. The mouse-adapted H. pylori strain SS1 has been utilized as a model to study disease pathogenesis. Although chronic gastritis is observed in this murine model of H. pylori infection, other complications of disease seen in the human host (such as peptic ulceration) are not identified. The objectives of this study were to characterize virulence factors of the mouse-adapted H. pylori strain SS1 and determine host responses to infection. Vacuolating cytotoxin activity of H. pylori strain SS1 was determined after incubation of HEp-2 cells with culture supernatant for 24 hr. Polymerase chain reaction was performed to detect the presence of the cagA and cagE genes. Chemokine responses from human gastric epithelial cells infected with H. pylori SS1 were assessed by measurement of the concentration of interleukin-8 in cell-free supernatants. C57BL/6 and gld mice were infected with strain SS1 or sham-infected. Eight weeks following infection, gastric tissues were obtained for histological analysis and surface hydrophobicity was measured by axisymmetric drop-shape analysis. H. pylori strain SS1 was cytotoxin negative, cagA positive, and cagE positive, but induced only a modest interleukin-8 response (684 +/- 140 pg/ml) from AGS gastric epithelial cells in comparison to a clinical isolate (4170 +/- 410 pg/ml, P < 0.0005). Increased inflammation was observed in the stomachs of H. pylori strain SS1-infected animals compared to uninfected controls. Gastritis was not associated with any disease complications. Despite mucosal inflammation, infected mice did not demonstrate alterations in gastric surface hydrophobicity (42.2 degrees +/- 2.2 degrees and 41.4 degrees +/- 3.2 degrees for C57BL/6 and gld, respectively) compared to uninfected mice (43.2 degrees +/- 2.3 degrees and 39.5 degrees +/- 1.6 degrees, respectively). In conclusion, murine infection with H. pylori SS1, which contains putative bacterial virulence factors, results in gastric inflammation. However, the mucosal changes are not associated with alterations in surface hydrophobicity. Therefore, the mouse model of infection with H. pylori, strain SS1 may not serve as an entirely appropriate model to study host factors associated with disease complications.

MR imaging of the posterior cruciate ligament graft: initial experience in 15 patients with clinical correlation.

PURPOSE: To describe the magnetic resonance (MR) appearance of the posterior cruciate ligament (PCL) graft in the 1st postoperative year and to correlate the MR findings with clinical examination findings. MATERIALS AND METHODS: Nineteen MR examinations were performed in 15 patients (with 15 grafts) 1-33 months after PCL reconstruction. Results of clinical follow-up were available in 14 patients. Graft shape and thickness were recorded, and intrasubstance signal intensity at T2-weighted imaging was graded. The MR findings were correlated with the time between surgery and clinical examination. RESULTS: At MR imaging, 13 grafts appeared intact, one could not be assessed owing to hardware artifact, and one initially appeared disrupted. Graft thickness was 7-19 mm. There was no significant difference between graft thickness versus time since surgery and signal intensity versus time since surgery (P =.14). In two of three patients who underwent sequential MR examinations, graft thickness and intrasubstance signal intensity decreased as the time between reconstruction and MR imaging increased. Two of 14 patients who underwent physical examination had a posterior drawer, and one also had an anterior cruciate ligament graft tear. Both patients with knee instability demonstrated intact PCL graft fibers at MR imaging. There was no correlation between knee stability and graft thickness, signal intensity, or shape. CONCLUSION: After PCL reconstruction, MR imaging in the 1st year depicts a thickened graft with increased signal intensity. There does not appear to be a relationship between clinical stability and findings at MR imaging.