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Accumulating evidence implicates defective innate immunity in the pathogenesis of Crohn's disease (CD). Ineffectual NOD2 (nucleotide-binding oligomerization domain 2) is the most common susceptibility gene contributing to CD. Vitamin D (vD), a potent modulator of innate and adaptive immunity, induces NOD2 gene expression and its downstream function. We hypothesized that the hormonal form of vD (1,25D) could beneficially modulate innate immune function in CD. Using peripheral mononuclear cells and monocyte-derived dendritic cells (Mo-DCs) from CD, it was found that 1,25D decreased Toll-like receptor (TLR)-induced cytokine production and enhanced cytokine levels induced by muramyl dipeptide (MDP), the NOD2 ligand. 1,25D increased the synergistic effect provided by NOD2 and TLR co-activation on interleukin (IL)-10, IL-23, and tumor necrosis factor-alpha (TNF-α). Whereas 1,25D inhibits Mo-DC TLR-induced cytokines, co-stimulation of NOD2 results in increased IL-10 and IL-23. IL-12p70 was completely abrogated by 1,25D. 1,25D similarly modulated cytokine production by immune cells in ulcerative colitis patients and healthy controls. Mo-DCs from CD patients heterozygous for NOD2 mutations had a response similar to those from patients without NOD2 mutations. Immune cells from patients homozygous for the 1007 fs mutation were unresponsive to MDP and 1,25D. Our in vitro data support 1,25D as a potential modulator of immunity. However, these results cannot be extrapolated to CD patients without further controlled studies.

Objectives: Thiopurines (azathioprine, 6-mercaptopurine) are a mainstay of treatment in Crohn disease (CD). Monitoring intracellular metabolite (6-thioguanine nucleotides [6-TGN] and 6-methylmercaptopurine [6-MMP]) levels can help optimize therapeutic efficacy and minimize potential toxicity. Determination of 6-MMP/6-TGN ratios may provide additional useful information, such as the identification of individuals with excessive thiopurine methyltransferase activity and disadvantageous 6-MMP overproduction. These patients are at increased risk of therapeutic failure and hepatotoxicity. The aim of the study was to evaluate the correlation of 6-MMP/6-TGN ratios with therapeutic efficacy and risk of hepatotoxicity in CD., Methods: The present study was a single-center cross-sectional study including pediatric patients with CD studied prospectively with clinical and laboratory assessments along with serial measurements of 6-MMP and 6-TGN. Clinical response was determined using established clinical indices., Results: The study included 238 pediatric patients with CD with a total of 1648 evaluation points. The patients were in steroid-free remission at 59.1% of the evaluation points. 6-MMP/6-TGN ratios of 4 to 24 were protective against relapse (odds ratio [OR] 0.52, 95% confidence interval [CI] -0.39 to 0.69, P = 0.001). Hepatotoxicity was associated with high 6-MMP levels (>3919  pmol/8 × 10 red blood cell count: OR 7.65, 95% CI 3.7-15.9, P = 0.001) and high 6-MMP/6-TGN ratios (>24: OR 5.35, 95% CI -3.43 to 8.43, P = 0.001)., Conclusions: We observed significant associations between 6-MMP/6-TGN ratios and clinical response, and risk of hepatotoxicity. Our results suggest that determination of thiopurine metabolite ratios is a valuable tool for identification of patients at increased risk of therapeutic failure and hepatotoxicity.

Background: Azathioprine (AZA), a pro-drug metabolised to the active metabolites 6-tioguanine nucleotides (6TGN), is a steroid-sparing therapy for Crohn's disease (CD)., Aim: To investigate whether AZA therapy is optimised by individualised dosing based on thiopurine methyltransferase (TPMT) activity and 6TGN concentrations., Methods: This multicentre, double-blind, randomised controlled trial compared the efficacy and safety of weight-based vs. individualised AZA dosing in inducing and maintaining remission in adults and children with steroid-treated CD. The primary outcome was clinical remission (CR) at 16 weeks. In the weight-based arm, subjects received 2.5 mg/kg/day. In the individualised dosing arm, the initial AZA dose was 1.0 mg/kg/day (if intermediate TPMT) or 2.5 mg/kg/day (if normal TPMT). Starting at week 5, the dose was adjusted to target 6TGN concentrations of 250-400 pmol/8 × 10(8) red blood cells (RBC), or to a maximal dose of 4 mg/kg/day., Results: After randomising 50 subjects, the trial was stopped prematurely due to insufficient enrolment. In intention-to-treat analysis, CR rates at week 16 were 40% in the individualised arm vs. 16% in the weight-based arm (P = 0.11). In per-protocol (PP) analysis, week 16 CR rates were 60% in the individualised arm and 25% in the weight-based arm (P = 0.12). At week 16, median 6TGN concentrations in PP remitters and nonremitters were 216 and 149 pmol/8 × 10(8) RBC respectively (P = 0.07)., Conclusions: Despite trends favouring individualised over weight-based AZA dosing, there were no statistically significant differences in efficacy, likely due to low statistical power and inability to achieve the target 6TGN concentrations in the individualised arm. [Clinicaltrials.Gov Identifier Nct00113503]., (© 2013 John Wiley & Sons Ltd.)

Background: Tumour necrosis factor (TNF)-antagonists have an established role in the treatment of inflammatory bowel diseases (IBDs), however, subtherapeutic drug levels and the formation of anti-drug antibodies (ADAs) may decrease their efficacy., Aim: The evidence supporting the use of therapeutic drug monitoring (TDM) based clinical algorithms for infliximab (IFX) and their role in clinical practice will be discussed., Methods: The literature was reviewed to identify relevant articles on the measurement of IFX levels and antibodies-to-infliximab., Results: Treatment algorithms for IBD have evolved from episodic monotherapy used in patients refractory to all other treatments, to long-term combination therapy initiated early in the disease course. Improved remission rates have been observed with this paradigm shift, nevertheless many patients ultimately lose response to therapy. Although empiric dose optimization or switching agents constitute the current standard of care for secondary failure, these interventions have not been applied in an evidence-based manner and are probably not cost-effective. Multiple TDM-based algorithms have been developed to identify patients that may benefit from measurement of IFX and ADA levels to guide adjustments to therapy., Conclusions: Therapeutic drug monitoring offers a rational approach to the management of secondary failure to IFX. This concept has gained momentum based on evidence from case series, cohort studies and post-hoc analyses of randomised controlled trials., (© 2013 John Wiley & Sons Ltd.)

Background: Recent genome-wide association studies based on adult and paediatric populations have implicated >30 genes/loci as susceptibility loci for Crohn's disease (CD)., Aims: To investigate whether reported genes/loci were also associated with CD in Canadian children., Design and Methods: A case-control design was implemented at three paediatric gastroenterology clinics in Canada. Children < or =18 years of age with a confirmed diagnosis of CD were recruited along with controls. Single nucleotide polymorphisms (SNPs) in five genome-wide association studies reported genes/loci were genotyped. Associations between individual SNPs and CD were examined., Results: A total of 406 cases and 415 controls were studied. The mean (+/-s.d.) age of the cases was 12.3 (+/-3.2) years. Most cases were male (56.6%), had ileo-colonic disease (L3 +/- L4, 52.0%) and inflammatory behaviour (B1 +/- p, 86.9%) at diagnosis. Allelic association analysis (two-tailed) showed that three of the five targeted SNPs were significantly associated with overall susceptibility for CD (ZNF365, r10995271, P = 0.001; PTPN2, rs1893217, P = 0.005; STAT3, rs744166, P = 0.01). Associations with SNP rs4613763 in the PTGER4 locus were marginally nonsignificant (P = 0.07). The ZNF365 and STAT3 SNPs were predominantly associated with ileal disease with or without colonic involvement., Conclusion: The identified susceptibility genes/loci for adult-onset CD also confer risk for paediatric-onset CD.

Background: A recent genome-wide association study in adult patients with ulcerative colitis (UC) has implicated the interleukin 10 (IL-10) gene as an important candidate gene. Moreover, a UC-associated single nucleotide polymorphism (SNP) rs3024405 was also significantly associated with adult Crohn's disease (CD)., Aims: To examine whether IL-10-CD associations extended to paediatric-onset CD., Methods: We implemented the case-control design at three paediatric gastroenterology clinics in Canada. CD patients (

Pacemaker location in the abdominal wall is considered a contraindication to videocapsule endoscopy (VCE). The aim of this study was to review our experience on the use of VCE in patients with a pacemaker located in the abdominal wall. VCE was carried out with monitoring of cardiac rhythm. This was a retrospective review of VCE case studies performed at two tertiary care university medical centers (pediatric and adult). The main outcome measures were adverse events and quality of VCE images. No adverse events were experienced in any of the five patients with implanted cardiac pacemakers, including the two with abdominal pacemaker. No interference with the VCE recording was observed during the studies, although the capsule was observed to be briefly inactivated by the pacemaker in one case. The present study, though small, suggests that VCE is safe in adult and pediatric patients who are fitted with cardiac pacemakers, even when implanted in the abdominal wall. The VCE exam can be carried out successfully under close supervision. Dysfunction of the capsule appears to be more likely than problems with cardiac pacing.

Mannan-binding lectin (MBL) triggers complement activation upon binding to microbial surfaces. MBL deficiency has been associated with increased susceptibility to severe bacterial infections. We hypothesized that MBL deficiency may predispose children to Shiga toxin-producing Escherichia coli (STEC) O157:H7 infections and the associated haemolytic uraemic syndrome (HUS). We compared circulating levels of MBL among children with uncomplicated O157:H7 haemorrhagic colitis (HC), patients with O157:H7 HUS, normal and diseases control groups. Circulating MBL concentrations on admission were as follows: 3.22 +/- 2.43 micro g/ml among normal controls (n = 23); 2.90 +/- 2.44 micro g/ml in patients with rotavirus enteritis (n = 10); 2.78 +/- 1.65 micro g/ml in children with HC due to non-STEC bacterial pathogen (n = 15); 2.67 +/- 2.44 micro g/ml in patients with uncomplicated O157:H7 HC (n = 27); 2.80 +/- 2.97 micro g/ml in children with O157:H7 HUS (n = 15); 6.70 +/- 4.49 micro g/ml in patients with chronic renal failure unrelated to O157:H7 infection (n = 6). Higher MBL levels were found in patients with chronic renal failure compared to O157:H7 HC (P < 0.047). However, MBL concentrations <0.5 micro g/ml, which have been associated with MBL deficiency in relation to increased susceptibility to infections, were noted at comparable rates between the different groups (P = NS). Our data does not support that MBL deficiency may predispose to O157:H7 infections nor than the development of diarrhoea associated HUS.

The intestinal flora play an important role in experimental colitis and inflammatory bowel disease (IBD). Using colonic explant cultures from 132 IBD and control subjects, we examined tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 and interleukin-1 receptor antagonist (IL-1RA) production in vitro in response to bacterial activators. Unstimulated TNF-alpha release was increased significantly in rectal biopsies from involved IBD tissue, correlating with inflammation severity. Whereas lipopolysaccharide (LPS) only moderately stimulated TNF-alpha production from inflamed tissue, pokeweed mitogen (PWM) induced its release in all groups, with a stronger response in involved IBD tissue. Superantigen staphylococcal enterotoxin A (SEA) had a similar, but weaker effect. SEB was observed to be the strongest inducer of TNF-alpha for all groups, again with a more marked response in inflamed tissue. Stimulated release of IL-1 was considerably less than for TNF-alpha. The superantigens' superior potency over LPS was not as marked for IL-1 as it was for TNF-alpha. In addition to IL-1, IL-1RA release was also triggered by the bacterial products. The net effect of activation on the IL-1RA/IL-1 ratio was relatively modest. Release of the proinflammatory cytokines TNF-alpha and IL-1, as well as that of the anti-inflammatory cytokine IL-1RA was increased by incubation of colonic tissue with bacterial factors. TNF-alpha production and release was increased significantly in involved colonic explants from IBD. SEB was even capable of inducing TNF-alpha release from uninvolved colonic tissue.

Background: During the process of tumorigenesis most colon cancer cells acquire resistance to apoptosis. The short chain fatty acid butyrate is well established as an antitumour agent which selectively induces apoptosis in colon cancer cells but not in normal intestinal epithelial cells., Aims: To analyse the signalling pathway of butyrate induced apoptosis., Methods: Using Caco-2 cells we focused on the bcl family of proteins, mitochondrial pathway, and caspase signalling cascade involved in butyrate induced apoptosis. Techniques employed included western blots, immunofluorescence, as well as experiments with peptide inhibitors of specific caspases., Results: Butyrate induced a clear shift of the mitochondrial bcl rheostat towards a proapoptotic constellation, as demonstrated by upregulation of proapoptotic bak accompanied by reduced antiapoptotic bcl-x(L) levels. This was associated with translocation of cytochrome-c from the mitochondria to the cytosol, resulting in activation of the caspase cascade via caspase-9. Key executioner enzymes were caspases-3 and -1. No effect of butyrate on regulatory proteins of the inhibitor of apoptosis family was observed., Conclusions: Butyrate induced Caco-2 cell apoptosis via the mitochondrial pathway. Upregulation of bak and translocation of cytochrome-c were upstream of the caspase cascade. Subsequently, this cascade was activated via the formation of an apoptosome.

Background: Circulating levels of endotoxin (or lipopolysaccharide (LPS)) and anti-endotoxin antibodies are increased in patients with inflammatory bowel disease, supporting the hypothesis of a role for endogenous bacterial products in the pathogenesis of these disorders., Aim: The aim of this study was to analyse the direct effects of LPS on intestinal epithelial cell turnover., Methods and Results: LPS significantly inhibited growth of the human non-transformed immature crypt cell line (HIEC), whereas IEC-6 cell proliferation was stimulated by LPS. As LPS is a physiological inducer of tumour necrosis factor alpha (TNFalpha) in various cell systems and this cytokine exerted similar anti-proliferative (HIEC) or growth stimulatory (IEC-6 cells) effects, the study thus tested the hypothesis that endogenously produced TNFalpha in response to LPS mediates this growth modulatory effect in an autoparacrine/paracrine way. Therefore, during LPS stimulation, the biological activity of TNFalpha was blocked using neutralising anti-TNFalpha antibodies, as well as inhibitory, antagonistic antibodies directed against the p55 TNF receptor, signalling the antimitotic TNFalpha effect in HIEC. Both experimental approaches completely abolished the growth modulatory effects of LPS in HIEC/IEC-6 cells. Production and secretion of TNFalpha by HIEC/IEC-6 cells in response to LPS was confirmed on mRNA and protein level by reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay. LPS signalling was independent of CD14 in HIEC, as these cells lack this receptor. However, HIEC expressed TLR4 and MD2 resulting in a fully functional signalling complex as demonstrated by RT-PCR, western blot, and immunofluorescence analyses., Conclusion: These results support the hypothesis that LPS induced changes of intestinal epithelial cell turnover may directly contribute to the pathogenesis of inflammatory epithelial cell lesions by endogenous TNFalpha production by enterocytes.

Various members of the tumor necrosis factor receptor superfamily are implicated in the regulation of enterocyte apoptosis. Previously, we observed that human intestinal epithelial cells are rather unsusceptible to Fas-induced apoptosis. In the present study we analyzed these protective mechanisms in the human intestinal epithelial cell line HIEC, focusing on antiapoptotic molecules of the IAP family which block apoptosis at the level of the caspase cascade. HIEC expressed all key molecules required to trigger Fas-induced apoptosis. However, no apoptosis occurred after activation of Fas, whereas an upregulation of antiapoptotic cIAP1 and 2 was observed. Suppression of this upregulation with the proteasome inhibitor MG132 or the protein synthesis inhibitor cycloheximide highly sensitized HIEC toward Fas-induced apoptosis. Western blot analyses revealed that both inhibitors potently suppressed endogenously produced cIAP1 and 2. No effect was observed on XIAP expression. These data indicate that enterocytes are particularly protected against Fas-induced apoptosis on the level of executionary caspases., (©2002 Elsevier Science (USA).)

Congenital immunodeficiencies are relatively rare disorders that result in a marked predisposition to infection and to a variety of gastrointestinal problems. Increasingly recognized in both children and adults, accumulating worldwide experience with these disorders reveals that the long-term prognosis is poor in many cases, despite available treatments. This review highlights the past year's advances concerning the molecular basis of several of these disorders, new diagnostic considerations, and novel therapeutic approaches concerning their gastrointestinal complications. The availability of molecular diagnostic tools increases the options for earlier definitive treatments such as bone marrow transplantation and somatic gene therapy.

Since its introduction into clinical use, flexible fiberoptic endoscopy progressively has become an indispensable tool to diagnose and treat gastrointestinal disorders. Few innovations have had a greater impact on the clinical practice of gastroenterology. In effect, diagnostic endoscopy has become an extension of physical diagnosis. This article reviews the expanding use of endoscopy in inflammatory bowel disease in the pediatric age group.

Background and Aims: A substantial number of patients with inflammatory bowel disease (IBD) fail to achieve a complete clinical response with 6-mercaptopurine (6-MP) and azathioprine (AZA). Inability to achieve therapeutic 6-thioguanine nucleotide (6-TGN) levels due to the preferential overproduction of 6-methylmercaptopurine ribonucleotides (6-MMPR) upon dose escalation characterizes a newly described subgroup of IBD patients resistant to 6-MP/AZA therapy. Treatment with 6-thioguanine (6-TG), a related thiopurine, which forms 6-TGNs more directly may be beneficial in such patients. This pilot study evaluated the safety, tolerance, and efficacy of 6-TG in the subgroup of Crohn's disease (CD) patients failing to attain adequate disease control with traditional 6-MP/AZA therapy., Methods: Ten CD patients with preferential 6-MMPR production upon 6-MP/AZA dose escalation were enrolled in an open-label pilot study. Seven of 10 patients had experienced dose-related 6-MP toxicities., Results: Seventy percent of the patients (7 of 10) responded or were in remission at week 16. Clinical response was evident by week 4 in most. 6-TGN levels were nine-fold higher with 6-TG treatment than with 6-MP, whereas 6-MMPR levels were undetectable. No patient developed a recurrence of hepatic or hematological toxicity., Conclusions: 6-TG was a safer and more efficacious thiopurine in this subgroup of IBD patients resistant to 6-MP therapy. Larger controlled trials are warranted to further evaluate both the short- and long-term safety and efficacy in this subgroup of patients as well as a broader spectrum of IBD patients.

The aim of this review is to examine recent advances in experimental and clinical research relevant to the pathogenesis of diarrhea-associated hemolytic uremic syndrome with special reference to histopathologic findings, virulence factors of Shiga toxin-producing Escherichia coli, the host response, and the prothrombotic state. Despite significant advances during the past decade, the exact mechanism by which Shiga toxin-producing E. coli leads to hemolytic uremic syndrome remains unclear. Factors such as Shiga toxin, lipopolysaccharide, the adhesins intimin and E. coli-secreted proteins A, B, and D, the 60-MD plasmid, and enterohemolysin likely contribute to the pathogenesis. Data on the inflammatory response of the host, including leukocytes and inflammatory mediators, are updated. The pathogenesis of the prothrombotic state leading to thrombocytopenia secondary to endothelial cell damage and platelet activation is also discussed. A hypothetical sequence of events from ingestion of the bacteria to the development of full-blown hemolytic uremic syndrome is proposed.

To examine the multiple stages of lipoprotein packaging during development, we studied localization, ontogeny, and regulation of microsomal transfer protein (MTP), a crucial protein for lipid transport. With the use of immunofluorescence, MTP was identified in villus and crypt epithelial cells in different regions of human fetal intestine, including colon. Staining was detected as early as the 13th wk of gestation in all gut segments and was almost entirely confined to the columnar epithelial cells of the jejunum and colon. Unlike immunofluorescence, which provides qualitative but not quantitative information on MTP signal, enzymatic assays revealed a decreasing gradient from proximal small intestine to distal, as confirmed by immunoblot. Activity of MTP in small intestinal explants cultured for different incubation periods (0, 4, 8, and 24 h) peaked at 4 h but remained insensitive to different concentrations of oleic acid. Also, a trend toward increasing MTP activity was observed at 20-22 wk of gestation. Finally, in strong contrast to jejunal efficiency, colonic explants displayed impaired lipid production, apolipoprotein biogenesis, and lipoprotein assembly, in association with poor expression of MTP. These findings provide the first evidence that human fetal gut is able to express MTP and emphasize the distinct regional distribution, regulation by oleic acid, and ontogeny of MTP.

Objectives: Confronted with nonspecific symptoms, accurate screening tests would be useful to clinicians to distinguish between functional childhood disorders and inflammatory bowel disease (IBD), thus avoiding invasive diagnostic testing. Traditional ulcerative colitis-specific perinuclear antineutrophil cytoplasmic antibody (pANCA) and Crohn's disease-specific anti-Saccharomyces cerevisiae antibody (ASCA) serodiagnostic assays have recently been modified, with ELISA cut-off values recalculated to maximize sensitivity. The aim of this study was to determine whether the combination of these serodiagnostic tests could maximize diagnostic accuracy and minimize invasive investigations in pediatric patients presenting with nonspecific symptoms suggestive of IBD., Methods: With investigators blinded to clinical diagnoses, ASCA, ANCA, and pANCA profiles were obtained prospectively from 128 patients undergoing complete diagnostic evaluation for IBD. In phase I, diagnostic accuracy and predictive values of the modified and traditional assays were compared for the IBD (n = 54) and non-IBD groups (n = 74). In phase II, the overall accuracy of a novel sequential diagnostic testing strategy was determined. Additionally, the potential number of invasive investigations avoided with the hypothetical application of this strategy to the cohort was determined., Results: For phase I, the modified serodiagnostic assay was more sensitive (81 vs 69%), whereas the traditional assay had a higher specificity (96 vs 72%) for IBD (p < 0.05) For phase II, false-positive diagnoses would have been reduced by 81%, yielding an overall sequential testing strategy accuracy of 84%., Conclusions: The incorporation of sequential noninvasive testing into a diagnostic strategy may avoid unnecessary and costly evaluations and facilitate clinical decision making when the diagnosis of IBD in children is initially uncertain.

Purpose: To determine if neovascularization associated with Crohn disease, as detected with Doppler ultrasonography (US), reflects clinical disease activity., Materials and Methods: A devised measurement, vessel density, was estimated with color Doppler US. Patients with Crohn disease underwent clinical and laboratory assessment in which the Crohn disease activity index was measured; patients underwent abdominal US the same week. Color Doppler US was performed by using a 7.5-10.0- or 8.0-12.0-MHz transducer, the lowest possible pulse repetition frequency without aliasing, a low wall filter, and high Doppler gain settings. The length and thickness of the affected loops were measured, and the number of color Doppler signals per square centimeter in the bowel loop was counted. Pulsed Doppler US was used to confirm that the signals originated from arteries or veins and not from movement artifacts., Results: Ninety-two patients (aged 7-20 years; mean, 14.85 years; 44 female, 48 male) underwent 119 examinations; 85 were performed in patients with active disease. Affected loops were thicker (10.6 vs 4. 6 mm; P: <.001) and had a higher vessel density with disease (69 of 119 examinations) than during remission (two of 34 examinations; P: <.001)., Conclusion: Vessel density in affected bowel loops, as estimated with Doppler US, and bowel wall thickness (>5 mm) reflect disease activity in patients with Crohn disease.

Background: Despite its pivotal role in mucosal inflammation, data on TNFalpha levels in inflammatory bowel diseases have been contradictory., Aim: To examine TNFalpha production in relation to the type and severity of inflammation and therapy, using colonic explant cultures., Materials and Methods: Rectal mucosal biopsies from 271 paediatric patients (178 inflammatory bowel disease, 27 inflammatory controls, 66 normal) were cultured for 4 or 18 h. Basal TNFalpha tissue content and release into the medium were measured by ELISA and compared to histological severity and clinical parameters., Results: TNFalpha release as well as tissue-associated TNFalpha levels were significantly increased in rectal biopsies from involved inflammatory bowel disease tissue. The amount of TNFalpha correlated with inflammation severity scores. TNFalpha levels were higher at 18 compared to 4 h in all groups, whether inflamed or not. TNFalpha released from rectal biopsies was lower among treated patients at 18 h. The presence of proximal colonic involvement was associated with higher TNFalpha release by uninvolved Crohn's disease rectal biopsies compared to patients with ileitis alone., Conclusions: TNFalpha production and release is increased in involved rectal explants from inflammatory bowel disease. Anti-inflammatory treatment diminishes this response.

We measured soluble Fas-ligand (sFas-L) and soluble Fas (sFas) levels by sandwich enzyme-linked immunosorbeny assay and compared them among (1) healthy controls (n = 11), (2) children with hemorrhagic colitis (HC) caused by a non-verotoxin-producing pathogen (n = 23), (3) patients with uncomplicated Escherichia coli O157:H7 HC (n = 14), and (4) children with O157:H7-associated hemolytic uremic syndrome (HUS) (n = 24). Children with uncomplicated E coli O157:H7 HC and HUS were matched for duration of enteric prodrome before blood sample collection. We also compared sFas-L and sFas levels among patients with HUS according to severity of renal dysfunction; abnormally increased sFas-L levels were noted in only 4% of the children (n = 3). Abnormally high concentrations of sFas were noted in 9% of the children with HC caused by a non-verotoxin-producing pathogen, 29% of the patients with uncomplicated E coli O157:H7 HC, and 69% of the children with O157:H7-associated HUS. Compared with healthy controls, patients with HUS had twofold greater concentrations of sFas (P: < 0.0001). Levels of sFas were not statistically different between 14 patients with uncomplicated O157:H7 HC and 14 children with HUS (8.2 +/- 4.7 versus 11.0 +/- 4.6 U/mL, respectively; P: < 0.07) when matched for time after onset of enteritis (7.0 +/- 3.7 versus 7.3 +/- 3.8 days, respectively). Greater concentrations of sFas were noted in patients with HUS who developed oligoanuria (n = 10; P: < 0.007), required peritoneal dialysis (n = 10; P: < 0.007), or had a decreased glomerular filtration rate (n = 5; P: < 0.002) 1 year later. Our data show that plasma concentrations of sFas but not sFas-L are abnormally increased in children with O157:H7 infections. Levels of sFas are associated with severity of renal dysfunction during HUS. Further studies are needed to clearly determine the role and origin of circulating sFas among children with infections caused by E coli O157:H7.

Intestinal epithelial cells participate in an acute phase response (APR) by responding to cytokines and by expressing acute phase protein genes. We hypothesized that butyrate, a fermentation product of the bacterial intestinal flora with deacetylase activity, affects the APR in intestinal epithelial cells. Sodium butyrate (NaBu) and Trichostatin A (TSA) induced alkaline phosphatase activity and histone H4 acetylation in IEC-6 rat intestinal epithelial cells treated with or without interleukin-1beta (IL-1). In contrast, both NaBu and TSA attenuated the IL-1-dependent induction of the acute phase protein gene haptoglobin, as well as C/EBPbeta and C/EBPdelta transcription factors mRNAs. Gel shift and supershift assays showed a strong decrease in the IL-1-induced C/EBPbeta and C/EBPdelta containing complexes binding to the HaptoA C/EBP DNA-binding site of the haptoglobin promoter, by NaBu and TSA. Furthermore, site-specific mutation of the HaptoA site abolished the NaBu- and TSA-dependent inhibition of haptoglobin, as determined by transient transfection assays. These results suggest that deacetylase inhibitors may regulate the IL-1 dependent induction of haptoglobin by down-regulating C/EBP isoforms, and that C/EBPs represent a target for the action of butyrate in the control of the APR of intestinal epithelial cells., (Copyright 2000 Academic Press.)

The diagnosis of inflammatory bowel disease is usually straightforward, based on a detailed history and physical examination, along with standard radiographic and endoscopic investigations, biopsies, and laboratory parameters. More challenging is the search for clinically useful, noninvasive markers for Crohn disease and ulcerative colitis to accurately screen cases with nonspecific and indolent symptoms. Equally required are diagnostic markers that discriminate between these two disorders in cases with indeterminate colitis. Another dilemma for clinicians is that there are no simple measures to observe disease activity and predict relapses. This review describes the recent advances in diagnostic markers that afford the ability to screen for inflammatory bowel disease, discriminate between its types, and monitor disease activity. These include serological, fecal, and tissue markers; permeability tests; and diagnostic imaging using color Doppler ultrasonography.

The diagnosis and management of inflammatory bowel disease (IBD) in children and adolescents is a challenge to patients, their families, and to the healthcare team. This review highlights recent advances in the epidemiology, diagnosis, and management of pediatric IBD. Among the most prominent advances are the new diagnostic serologic assays that can help screen for IBD in the absence of physical signs of disease and help discriminate between ulcerative and Crohn's colitis. Other tests have been identified as potential noninvasive markers of disease activity, including color Doppler abdominal ultrasound and sugar permeability tests. Recent advances in pharmacogenetics afford clinicians the ability to optimize and individualize therapy using azathioprine or 6-mercaptopurine. Finally, bone health has come forth as a major issue in the complete management of pediatric IBD.

Background & Aims: The effects of 6-mercaptopurine (6-MP) are mediated via its intracellular conversion to 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP) nucleotide metabolites, the latter genetically controlled by thiopurine methyltransferase (TPMT). We sought to determine optimal therapeutic 6-MP metabolite levels and their correlation with medication-induced toxicity and TPMT genotype., Methods: Therapeutic response was determined in 92 pediatric patients with inflammatory bowel disease coincidentally with hematologic, pancreatic, and hepatic laboratory parameters, and compared with erythrocyte metabolite levels and TPMT genotype., Results: Clinical response was highly correlated with 6-TG levels (P < 0.0001) but not with any other variable, including 6-MMP levels, drug dose, gender, and concurrent medications. The frequency of therapeutic response increased at 6-TG levels > 235 pmol/8 x 10(8) erythrocytes (P < 0.001). Hepatotoxicity correlated with elevated 6-MMP levels (>5700 pmol/8 x 10(8) erythrocytes; P < 0.05). Although leukopenia was associated with higher 6-TG levels (P < 0.03), it was observed in only 8% of responders. Patients heterozygous for TPMT (8/92) had higher 6-TG levels (P < 0.0001), and all responded to therapy., Conclusions: 6-MP metabolite levels and TPMT genotyping may assist clinicians in optimizing therapeutic response to 6-MP and identifying individuals at increased risk for drug-induced toxicity.

Mammalian sterol regulatory enzymes are integral membrane proteins of the endoplasmic reticulum. They play a critical role in liver cholesterol homeostasis and the maintenance of overall cholesterol balance in different species. Because lipid peroxidation has been implicated in hepatic dysfunction and atherosclerosis, we hypothesized that its occurrence could alter the composition and properties of the bilayer lipid environment, and thereby affect the functions of these membrane proteins. Preincubation of rat liver microsomes with iron (Fe)/ascorbate (50 microM/200 microM), known to induce peroxidation, resulted in a significant inhibition of (i) the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase (46%, p < .01), (ii) the crucial enzyme controlling the conversion of cholesterol in bile acids, cholesterol 7alpha-hydroxylase (48%, p < .001), and (iii) the central enzyme for cholesterol esterification: Acyl-CoA:cholesterol acyltransferase (ACAT, 80%, p < .0001). The disturbances of these key enzymes took place concomitantly with the high production of malondialdehyde (350%, p < .007) and the loss of polyunsaturated fatty acids (36.19 +/- 1.06% vs. 44.24 +/- 0.41 in controls, p < .0008). While alpha-tocopherol simultaneously neutralized lipid peroxidation, preserved microsomal fatty acid status, and restored ACAT activity, it was not effective in preventing Fe/ascorbate-induced inactivation of both HMG-CoA reductase (44%, p < .01) and cholesterol 7alpha-hydroxylase (71%, p < .0001). These results indicate that Fe/ascorbate alters the activity of the rate-determining steps in liver cholesterol metabolism, either directly or via lipid peroxidation, capable of modifying their membrane environment. The present data also suggest that the three regulatory enzymes respond differently when exposed to Fe/ascorbate or antioxidants, which may be due to dissimilar mechanisms.

Recent advances in the understanding of the pathogenesis of immune-mediated hepatic and intestinal diseases have led to major therapeutic advances. The introduction of genetically engineered biologic agents specifically designed to target inflammatory mediators responsible for the perpetuation of chronic inflammatory processes is a novel example. Although corticosteroids remain important as a first-line therapeutic option for active inflammatory bowel disease, approximately one third and one fifth of patients develop steroid dependence and resistance, respectively. From a pediatric perspective, a major advance has thus been the advocation of prolonged immunosuppressive therapy with 6-mercaptopurine or azathioprine for children with inflammatory bowel disease. The introduction of effective steroid-sparing agents for the induction and maintenance of remission is a key management issue. The past year has also witnessed the increased utilization of powerful immunosuppressive agents with rapid onset of action, such as cyclosporine and tacrolimus, in patients resistant to conventional therapies. This review will afford pediatricians a sense of what to expect for the management of hepatic and intestinal disorders with immunosuppression as we advance into the new millenium.

FAS-FAS ligand interaction has been implicated in increased enterocyte apoptosis seen in immune-mediated bowel injury. However, scant information exists on the role of FAS in physiological enterocyte turnover. In the present study, the regulation of enterocyte FAS and FAS ligand expression by cytokines and its functional role in human intestinal epithelial cell apoptosis and proliferation were analyzed with two different models: a nontransformed human intestinal epithelial cell line (HIEC) and normal colonic explant cultures. HIEC constitutively expressed FAS, as analyzed by flow cytometry. However, stimulation with agonistic anti-FAS antibody (1-500 ng/ml) did not induce HIEC apoptosis. In contrast, in the presence of tumor necrosis factor alpha (TNFalpha) and/or interferon gamma (IFNgamma), HIEC became highly susceptible to FAS-induced apoptosis. The sensitizing effect to FAS-induced apoptosis was mediated via TNFalpha- and IFNgamma-induced upregulation of FAS expression (maximally 348%). Receptor studies showed that the effect of TNFalpha on FAS was mediated via the p55 TNF receptor. In colonic organ cultures, IFNgamma and TNFalpha also enhanced colonocyte FAS expression, resulting in a markedly increased apoptotic response to stimulation of this receptor, as shown by in situ terminal deosyuridine triphosphate nick-end staining. Neither FAS ligand expression nor its induction by cytokines was observed in HIEC or colonic explants. Proliferation studies showed that FAS is not implicated in regulating HIEC growth. These findings suggest that, despite the fact that normal human enterocytes express FAS, costimulatory factors, such as TNFalpha or IFNgamma, abundantly secreted under inflammatory conditions, are necessary to sensitize intestinal epithelial cells to FAS-induced apoptosis by upregulating this receptor., (Copyright 1999 Wiley-Liss, Inc.)

Caco-2 cells and human colonic explants were compared for their ability to esterify lipid classes, synthesize apolipoproteins and assemble lipoproteins. Highly differentiated cells and colonic explants were incubated with [(14)C]oleic acid or [(35)S]methionine for 48 h. Caco-2 cells demonstrated a higher ability to incorporate [(14)C]oleic acid into cellular phospholipids (13-fold, P<0.005), triglycerides (28-fold, P<0.005) and cholesteryl ester (2-fold, P<0. 01). However, their medium/cell lipid ratio was 11 times lower, indicating a limited capacity to export newly synthesized lipids. De novo synthesis of apo B-48 and apo B-100 was markedly increased (7%0 and 240%, respectively), whereas the biogenesis of apo A-I was decreased (60%) in Caco-2 cells. The calculated apo B-48/apo B-100 ratio was substantially diminished (107%), suggesting less efficient mRNA editing in Caco-2 cells. When lipoprotein distribution was examined, it displayed a prevalence of VLDL and LDL, accompanied along with a lower proportion of chylomicron and HDL. In addition, differences in lipoprotein composition were evidenced between colonic explants and Caco-2 cells. Therefore, our findings stress the variance in the magnitude of lipid, apolipoprotein and lipoprotein synthesis and secretion between the two intestinal models. This may be due to various factors, including the origin of Caco-2 cell line, i.e., colon carcinoma.

Butyrate exerts potent anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. Using the Caco-2 cell line, a well established model of colon cancer cells, our data show that butyrate induced apoptosis (maximum 79%) is mediated via activation of the caspase-cascade. A key event was the proteolytic activation of caspase-3, triggering degradation of poly-(ADP-ribose) polymerase (PARP). Inactivation of caspase-3 with the tetrapeptide zDEVD-FMK completely inhibited the apoptotic response to butyrate. In parallel, butyrate potently up-regulated the expression of the pro-apoptotic protein bak, without changing Caco-2 cell bcl-2 expression. Butyrate-induced Caco-2 cell apoptosis was completely blocked by the addition of cycloheximide, indicating the necessity of protein synthesis. However, when this inhibitor was added at a time point where bak expression was already enhanced (12 - 16 h after butyrate stimulation), it failed to protect Caco-2 cells against apoptosis. Taken together, these data provide evidence that the molecular events involved in butyrate induced colon cancer cell apoptosis include the caspase-cascade and the mitochondrial bcl-pathway.

The management of inflammatory bowel disease in children remains a major challenge to patients, their families, and the health care team. This review highlights the past year's advances concerning epidemiologic studies, new diagnostic considerations, and novel therapeutic approaches. Epidemiologic studies confirmed that Crohn's disease is being increasingly diagnosed in children, whereas the incidence of ulcerative colitis has remained stable. African-American children are as likely to have inflammatory bowel disease as other children. Serologic assays specific for Crohn's disease and ulcerative colitis can assist clinicians in screening for inflammatory bowel disease, as well as discriminating between the two diseases. Studies have revealed that osteopenia and antioxidant vitamin deficiency are common and require screening in pediatric inflammatory bowel disease. Increasing experience with immunosuppressive drugs (6-mercaptopurine, azathioprine) point to their efficacy and generally good toxicity profile in pediatric patients.

Tumor necrosis factor (TNF)alpha is considered to play a key pathogenetic role in inflammatory bowel diseases. In this study we analyzed the mechanisms by which TNFalpha induces intestinal epithelial cell apoptosis. TNFalpha alone, and more potently in combination with IFNgamma, induced a high degree of IEC-6 cell apoptosis. This effect was more than 100-fold stronger if both of the TNF-R were stimulated, compared to stimulation of the p55-TNF-R alone, indicating an important apoptosis enhancing effect of the p75-TNF-R. TNFalpha-induced apoptosis required activation of ICE caspases and was completely abolished by its inhibitor, zVAD-fmk. Specific inhibition of caspase-3 with zDEVD-fmk did not alter the effect of TNFalpha. Western blot analyses confirmed that caspase-3 was not activated in response to TNFalpha. In the presence of complete inhibition of the caspase cascade with zVAD-fmk (>/=50 microM), TNFalpha induced cell necrosis rather than apoptosis. Our data reveal that TNFalpha can trigger enterocyte cell death via apoptosis or necrosis, depending upon the activation or blockade of specific caspases., (Copyright 1999 Academic Press.)

Background: In the presence of inflammation, an increased expression of enterocyte MHC class II is observed, leading to altered mucosal antigen handling. Corticosteroids are potent anti-inflammatory drugs, widely used in treating inflammatory bowel disorders. However, their diverse mechanisms of action are only partially understood., Aim: To evaluate effect and mechanisms of corticosteroids on intestinal crypt epithelial cell MHC class II., Methods: The effect of dexamethasone treatment on cytokine-induced MHC class II expression was measured in IEC-6 cells by immunofluorescence and flow cytometry. To determine the role of the TGF-beta1 regulatory pathway in mediating the effects of dexamethasone, neutralizing anti-TGF-beta antibodies were used. Additionally, endogenous and dexamethasone-stimulated IEC-6 cell TGF-beta1 production was measured by ELISA., Results: Dexamethasone potently down-regulated IFNgamma-induced class II expression on IEC-6 cells, in a dose-dependent manner. TGF-beta1 had a similar inhibitory effect on class II expression. However, neutralizing anti-TGF-beta antibodies did not alter the effect of dexamethasone. Furthermore, dexamethasone reduced endogenous TGF-beta1 synthesis., Conclusions: Corticosteroids inhibit cytokine-induced MHC class II expression on IEC-6 cells in a TGF-beta1 independent way. This effect may markedly alter enterocytic antigen presentation, reducing the aberrant state of activation of mucosal immune cells.

Autoimmune enteropathy (AIE) is an entity reported primarily in infancy, resulting in intractable diarrhea and associated with small bowel villous atrophy and the presence of circulating anti-enterocyte (AEA) antibodies. It is a multisystem disorder with a response, in many cases, to immunosuppressive therapy.

Patients with Crohn's disease (CD) often have low bone mineral density (BMD) for their chronological age (CA). However, pediatric cases frequently have growth failure and delayed bone age (BA) and height age (HA). Do they really have the amount of osteoporosis as measured by BMD and calculated for their CA? The aim was to compare z-scores for BMD in relation to CA and z-scores corrected for BA or height age in pediatric patients. A group of 43 pediatric patients (mean age, 12 years; 14 girls, 29 boys) with CD in remission, prospectively had BMD (measured by dual energy x-ray absorptiometry) and BA. Abnormally low z-score for BMD (below 2 standard deviation [SD] for CA was found in 19 patients (44%). Among these, 9 patients (21% overall) had a BA of more than 2 SD lower than their CA and 12 had height age at least 2 years below CA. When the BMD z-score in these patients was corrected for their BA and height age, 6 of 9 and 8 of 12 patients had a normal BMD. In conclusion, when corrected for BA or CA delay, BMD was abnormal in 26 to 30% rather than 44% of cases. Correct assessment of BMD in Crohn's disease patients with bone age delay requires interpretation in terms of BA or HA, rather than CA.

Structured lipids have been proposed as efficient vehicles for the supplementation of essential fatty acids (EFA) to patients with malabsorption. We investigated how a novel structured triglyceride (STG), containing purely octanoic acid in the sn-1/sn-3 and [14C]linoleic acid in the sn-2 positions, was incorporated into different lipid classes in Caco-2 cells. We also evaluated the contribution of gastric lipase in the uptake and metabolism of [14C]linoleic acid from the STG. We furthermore determined the potential of the STG to correct EFA deficiency induced in Caco-2 cells. The absorption of STG by Caco-2 cells was significantly greater compared with that of triolein. The addition of human gastric lipase significantly enhanced cellular uptake of the labeled substrate, reflecting the stereoselectivity of gastric lipase to hydrolyze medium chain FA. Analysis of the intracellular lipids synthesized revealed a predominance of phospholipids-monoglycerides. Most of the radioactivity in the lipoproteins isolated from Caco-2 cells was recovered in TG-rich lipoproteins (45%) and to a lesser extent in the high-density lipoprotein (36%) and low-density lipoprotein (17%) fractions. The administration of STG to Caco-2 cells rendered EFA deficient produced a marked increase of the cellular level of linoleic and arachidonic acids. This resulted in a lower ratio of 20:3(n-9) to 20:4(n-6), reflecting the correction of EFA deficiency in Caco-2 cells. Our data demonstrate that STG, in the presence of gastric lipase, have beneficial effects on lipid incorporation, lipoprotein production, and EFA status, utilizing Caco-2 cells as a model of EFA deficiency.

Background & Aims: Accurate serological assays are desirable for the diagnosis of inflammatory bowel disease (IBD) types in the pediatric age group. The aim of this study was to test the diagnostic accuracy of modified assays for perinuclear (p) antineutrophil cytoplasmic antibodies (ANCAs) and anti-Saccharomyces cerevisiae antibodies (ASCAs) in patients with pediatric ulcerative colitis (UC) and Crohn's disease (CD) and in those without IBD., Methods: With observers blinded to patients' diagnoses, serum specimens were analyzed for immunoglobulin (Ig) A and IgG ASCAs and ANCAs by enzyme-linked immunosorbent assay. The perinuclear location of ANCAs visualized by indirect immunofluorescence was confirmed by its disappearance after administration of deoxyribonuclease., Results: IgA and IgG ASCA titers were significantly greater and highly specific for CD (95% for either, 100% if both positive). pANCA was 92% specific for UC and absent in all non-IBD controls. The majority of patients with CD positive for pANCA had a UC-like presentation. Disease location, duration, activity, complications, and treatment with immunosuppressive drugs did not have an impact on the ASCA or pANCA assay results. After resection, UC patients remained pANCA positive, in contrast to patients with CD, in whom ASCA titers decreased toward normal values postoperatively., Conclusions: ASCA and pANCA assays are highly disease specific for CD and UC, respectively. These serological tests can assist clinicians in diagnosing and categorizing patients with IBD and may be useful in making therapeutic decisions.

Dietary triglycerides, the major precursors of long chain fatty acids (FA), require hydrolysis by pancreatic enzymes prior to their absorption by the small intestine. Although Caco-2 cells are frequently employed for the study of enterocyte lipid metabolism, the presence of an endogenous lipase activity has never been previously reported. The major goal of this investigation was to determine the presence of endogenous Caco-2 cell lipase activity, to examine its capacity to hydrolyze triglycerides, and to define its intracellular location. Caco-2 cells were found to have an endogenous lipase activity, capable of hydrolyzing [1-14C]triolein from the apical cell compartment. A time and concentration dependence of lipase activity was observed, with hydrolysis of triolein into free fatty acids and monoglyceride. The majority of the lipase activity was found in the cytosolic cell fraction and, to a lesser extent, in the apical brush border membrane and other organelles. Protamine sulfate markedly reduced the Caco-2 cell lipase activity, yet it remained relatively insensitive to high concentrations of NaCl, taurocholate, calcium, heparin and chloroquine. The addition of exogenous human gastric lipase to the medium of the apical compartment resulted in a significant increased rate of hydrolysis of triolein, followed by enhanced Caco-2 cell fatty acid uptake and basolateral lipid secretion. The major esterified intracellular lipids were triglycerides and phospholipids. We conclude that Caco-2 cells possess an endogenous lipase capable of hydrolyzing cytosolic triglycerides. Furthermore, activity present on the apical membrane and secreted into the apical medium, though quantitatively less important than the cytosolic lipase, may permit an additional route for energy uptake. The addition of gastric lipase to the Caco-2 cell cultures greatly enhanced FA uptake above that seen with the endogenous lipase alone.

Intestinal epithelial cells and the mucosal immune cells in close proximity are thought to interact very closely. One well-established mechanism of this intercellular cross-talk is via the production of cytokines such as interferon gamma (IFNgamma). The aim of this study was to analyze the effects of IFNgamma on intestinal crypt epithelial cells. IEC-6 cells were cultured in the presence or absence of IFNgamma to measure its effects on proliferation, cell cycle, apoptosis, and major histocompatibility complex (MHC) class II antigen expression. Even at very low doses (0.01 U/ml), IFNgamma significantly inhibited IEC-6 cell proliferation, as demonstrated by reduced 3H-thymidine uptake, stable cell count, and complete arrest in the quiescent G0/G1 phase of the cell cycle. Incubation with supraphysiological doses of IFNgamma (100-1,000 U/ml) did not induce apoptosis, as assessed by morphology and the TUNEL assay. IFNgamma significantly induced de novo IEC-6 class II antigen expression. Tumor necrosis factor alpha (TNF alpha), which alone had no effect, synergistically enhanced this effect of IFNgamma. MHC class II antigen expression was observed to be independent of cell cycle phase. Our results indicate that IFNgamma alters immature crypt epithelial cell turnover and upregulates MHC class II expression. These alterations may be important in the pathogenesis of immune-mediated bowel disorders.

IBD is associated with an increased activation of intestinal immune cells, which causes overproduction of proinflammatory cytokines such as IL-1beta. IL-1beta is implicated in mediating the sustained inflammatory response. IL-1 receptor antagonist (IL-1Ra), the naturally occurring inhibitor of IL-1, has been shown to have beneficial effects in experimental models of colitis. In this study we investigated the hypothesis that an imbalance between IL-1 and IL-1Ra exists in IBD by measuring their secretion by explant cultures of colonic biopsies. Freshly homogenized biopsies from involved tissue in IBD patients exhibited significantly lower IL-1Ra/IL-1beta ratios than control and uninvolved IBD mucosal tissue. Using explant cultures, in vitro production of IL-1beta and IL-1Ra increased progressively during the 4-18-h culture periods. IL-1beta secretion was higher in supernatants from involved Crohn's disease (CD) and ulcerative colitis tissue compared with control tissue, and IL-1beta levels increased with severity of inflammation. IL-1Ra secretion was not elevated in involved IBD samples, but significantly higher levels were released when moderate to severely involved tissue samples were compared with noninflammatory controls. Similar to freshly homogenized tissue, explant studies showed that the IL-1Ra/IL-1beta ratios were significantly decreased in involved IBD tissue, but not in uninvolved CD or inflammatory control specimens. These data support the hypothesis of an imbalance between IL-1beta and IL-1Ra in IBD.

The role of TNF-alpha in modulating intestinal crypt cell growth was examined, in comparison with EGF. Both significantly increased IEC-6 cell proliferation. Neither EGF nor TNF-alpha overcame the inhibitory effect on growth exerted by the tyrosine kinase inhibitor genistein. Immunoblots with phosphotyrosine antibodies showed increased tyrosine phosphorylation of IEC-6 cell proteins in response to EGF and TNF-alpha stimulation. TNF-alpha increased ERK1 and ERK2 MAPK phosphorylation. A MAPK assay confirmed the increased activity upon TNF-alpha stimulation. Selective inhibition of MAPK activation by PD98059 resulted in a dose dependent inhibition of TNF-alpha or EGF-induced IEC-6 cell growth. These findings suggest a role for TNF-alpha in the regulation of intestinal epithelial cell growth and that the mitogenic effect of TNF-alpha requires protein tyrosine phosphorylation and MAPK activation.

The intestinal epithelial cell population is comprised of a dynamic continuum, ranging from undifferentiated, actively proliferating crypt cells, to mature absorptive villus enterocytes, lacking mitotic capacity. Under normal conditions, the constant loss of differentiated villus tip cells via apoptosis leads to a complete renewal of the epithelial cell population every few days. The physiological factors regulating enterocyte proliferation, maturation and apoptosis in health, as well as those that modulate these events in disease states remain largely unknown. It has been demonstrated in vitro that immature crypt cell proliferation is stimulated by factors such as TGF alpha and TNF alpha, whereas TFG beta and IFN gamma inhibit mitotic activity. Further studies showed that intestinal epithelial cells are able to produce and secrete several cytokines such as IL6, IL8, TNF alpha, TGF alpha and TGF beta, indicating the potential for autocrine and paracrine responses. A variety of immune mediated bowel disorders, including celiac disease, Crohn's disease and ulcerative colitis, are characterized by accelerated epithelial cell turnover and apoptosis, leading to altered crypt/villus morphology. There is increasing evidence that these changes, and the accompanying functional alterations of the bowel epithelium, are mediated by the cytokines released from infiltrating inflammatory cells, as well as from enterocytes themselves in an autocrine fashion.

Background: Dientamoeba fragilis is a rare cause of chronic infectious diarrhea and colitis in children., Methods: Review of the clinical manifestations, diagnostic methods, and clinical course of D. fragilis infection in our hospital., Results: Eleven pediatric patients are discussed, seven of whom had a history of recent travel. Clinical manifestations of infectious diarrhea included anorexia, intermittent vomiting, abdominal pain, and diarrhea, ranging from 1 to 100 weeks in duration. Peripheral eosinophilia was present in seven patients. One patient with well-documented bovine protein allergy had intermittent episodes of diarrhea and abdominal pain, despite an appropriate elimination diet. Eosinophilic colitis documented by colonoscopy, was due to D. fragilis. Metronidazole was effective in treating five patients, and iodoquinol was effective in treating four others., Conclusions: D. fragilis should be included in the differential diagnosis of chronic diarrhea and eosinophilic colitis. The identification of this pathogen requires clinical awareness of epidemiologic risk factors and presenting complaints, as well as the laboratory staining procedures essential to its proper identification.