Assessment of systemic genetic damage in pediatric inflammatory bowel disease.

The etiology of distal site cancers in inflammatory bowel disease (IBD) is not well understood and requires further study. We investigated whether pediatric IBD patients' blood cells exhibit elevated levels of genomic damage by measuring the frequency of mutant phenotype (CD59-/CD55-) reticulocytes (MUT RET) as a reporter of PIG-A mutation, and the frequency of micronucleated reticulocytes (MN-RET) as an indicator of chromosomal damage. IBD patients (n = 18 new-onset disease, 46 established disease) were compared to age-matched controls (constipation or irritable bowel syndrome patients from the same clinic, n = 30) and young healthy adults age 19-24 (n = 25). IBD patients showed no indication of elevated MUT RET relative to controls (mean ± SD = 3.1 ± 2.3 × 10-6 vs. 3.6 ± 5.6 x 10-6 , respectively). In contrast, 59 IBD patients where %MN-RET measurements were obtained, 10 exceeded the upper bound 90% tolerance interval derived from control subjects (i.e., 0.42%). Furthermore, each of the 10 IBD patients with elevated MN-RET had established disease (10/42), none were new-onset (0/17) (p = .049). Interestingly, each of the subjects with increased chromosomal damage was receiving anti-TNF based monotherapy at the time blood was collected (10/10, 100%), whereas this therapy was less common (20/32, 63%) among patients that exhibited ≤0.42% MN-RET (p = .040). The results clearly indicate the need for further work to understand whether the results presented herein are reproducible and if so, to elucidate the causative factor(s) responsible for elevated MN-RET frequencies in some IBD patients.

Human blood PIG-A mutation and micronucleated reticulocyte flow cytometric assays: Method optimization and evaluation of intra- and inter-subject variation.

We previously described flow cytometry-based methods for scoring the incidence of micronucleated reticulocytes (MN-RET) and PIG-A mutant phenotype reticulocytes (MUT RET) in rodent and human blood samples. The current report describes important methodological improvements for human blood analyses, including immunomagnetic enrichment of CD71-positive reticulocytes prior to MN-RET scoring, and procedures for storing frozen blood for later PIG-A analysis. Technical replicate variability in MN-RET and MUT RET frequencies based on blood specimens from 14 subjects, intra-subject variability based on serial blood draws from 6 subjects, and inter-subject variation based on up to 344 subjects age 0 to 73 years were quantified. Inter-subject variation explained most of the variability observed for both endpoints (≥77%), with much lower intra-subject and technical replicate variability. The relatively large degree of inter-subject variation is apparent from mean and standard deviation values for MN-RET (0.15 ± 0.10%) and MUT RET (4.7 ± 5.0 per million, after omission of two extreme outliers). The influences of age and sex on inter-subject variation were investigated, and neither factor affected MN-RET whereas both influenced MUT RET frequency. The lowest MUT RET values were observed for subjects <11 years old, and males had moderately higher frequencies than females. These results indicate that MN-RET and MUT RET are automation-compatible biomarkers of genotoxicity that bridge species of toxicological interest to include human populations. These data will be useful for appropriately designing future human studies that include these biomarkers of genotoxicity, and highlight the need for additional work aimed at identifying the sources of inter-individual variability reported herein.

Dextran sulfate sodium mouse model of inflammatory bowel disease evaluated for systemic genotoxicity via blood micronucleus and Pig-a gene mutation assays.

Inflammatory bowel disease (IBD) is an important risk factor for gastrointestinal cancers. Inflammation and other carcinogenesis-related effects at distal, tissue-specific sites require further study. In order to better understand if systemic genotoxicity is associated with IBD, we exposed mice to dextran sulfate sodium salt (DSS) and measured the incidence of micronucleated cells (MN) and Pig-a mutant phenotype cells in blood erythrocyte populations. In one study, 8-week-old male CD-1 mice were exposed to 0, 1, 2, 3 or 4% w/v DSS in drinking water. The 4-week in-life period was divided into four 1-week intervals-alternately on then off DSS treatment. Low volume blood samples were collected for MN analysis at the end of each week, and cardiac blood samples were collected at the end of the 4-week period for Pig-a analyses. The two highest doses of DSS were observed to induce significant increases in reticulocyte frequencies. Even so, no statistically significant treatment-related effects on the genotoxicity biomarkers were evident. While one high-dose mouse showed modestly elevated MN frequencies during the DSS treatment cycles, it also exhibited exceptionally high reticulocyte frequencies (e.g. 18.7% at the end of the second DSS cycle). In a second study, mice were treated with 0 or 4% DSS for 9-18 consecutive days. Exposure was continued until rectal bleeding or morbidity was evident, at which point the treatment was terminated and blood was collected for MN analysis. The Pig-a assay was conducted on samples collected 29 days after the start of treatment. The initial blood specimens showed highly elevated reticulocyte frequencies in DSS-exposed mice (mean ± SEM = 1.75 ± 0.10% vs. 13.04 ± 3.66% for 0 vs. 4% mice, respectively). Statistical analyses showed no treatment-related effect on MN or Pig-a mutant frequencies. Even so, the incidence of MN versus reticulocytes in the DSS-exposed mice were positively correlated (linear fit R2 = 0.657, P = 0.0044). Collectively, these results suggest that in the case of the DSS CD-1 mouse model, systemic effects include stress erythropoiesis but not remarkable genotoxicity. To the extent MN may have been slightly elevated in a minority of individual mice, these effects appear to be secondary, likely attributable to stimulated erythropoiesis.

Unexpectedly High Prevalence of Cystoisospora belli Infection in Acalculous Gallbladders of Immunocompetent Patients.

Objectives: Indications for cholecystectomy have changed dramatically over the past three decades. Cystoisospora belli has been reported in cholecystectomy specimens of immunocompetent patients. The present study was designed to determine the prevalence and clinical association of C belli in the gallbladder. Methods: The study included retrospective review of cholecystectomy specimens (n = 401) removed for various indications, and a prospective cohort of cholecystectomy specimens (n = 22) entirely submitted for histologic evaluation. Correlations of presence of C belli with age, sex, clinical indication, and abnormalities of preoperative laboratory values were assessed by Fisher exact test. Results: C belli was identified in 39/401 (9.7%) of the retrospective cohort, and 6/22 (27.3%) of the entirely submitted specimens. The presence of C belli showed no correlation with age, sex, clinical indication, or laboratory abnormalities. Conclusions: C belli resides in a latent state in the gallbladder and may be best considered a commensal organism.

Hepatic Issues and Complications Associated With Inflammatory Bowel Disease: A Clinical Report From the NASPGHAN Inflammatory Bowel Disease and Hepatology Committees.

Hepatobiliary disorders are common in patients with inflammatory bowel disease (IBD), and persistent abnormal liver function tests are found in approximately 20% to 30% of individuals with IBD. In most cases, the cause of these elevations will fall into 1 of 3 main categories. They can be as a result of extraintestinal manifestations of the disease process, related to medication toxicity, or the result of an underlying primary hepatic disorder unrelated to IBD. This latter possibility is beyond the scope of this review article, but does need to be considered in anyone with elevated liver function tests. This review is provided as a clinical summary of some of the major hepatic issues that may occur in patients with IBD.

Neither direct nor developmental exposure to bisphenol A alters the severity of experimental inflammatory colitis in mice.

Bisphenol A (BPA) is a high production volume endocrine disrupting chemical that is widely used in many consumer products and prevalent in human biological fluids. Recent studies suggest that BPA is active even at low levels, raising concern about its potential harm to human health. Given that the main route of exposure to BPA is oral, via the consumption of BPA-tainted foods and beverages, intestinal tissues could be particularly vulnerable to BPA-induced changes. A novel examination is reported here of whether oral exposure to BPA affects inflammatory bowel disease (IBD), an immune-mediated disease of the colon, using a mouse model of inflammatory colitis. In addition to direct exposure, the possible contribution of maternal BPA exposure to disease later in life is explored. It was found that daily oral exposure to BPA at the US Environmental Protection Agency described oral reference dose (50 µg/kg/day), either via direct oral route or through maternal sources (i.e. developmental exposure), did not significantly alter disease outcomes of body weight, survival, or colonic pathology. These observations suggest that oral BPA exposure, at this dose and for this exposure duration, has minimal influence on aspects of the inflammatory response that regulate immune mediated diseases of the gastrointestinal tract.

Mitigation effect of an FGF-2 peptide on acute gastrointestinal syndrome after high-dose ionizing radiation.

PURPOSE: Acute gastrointestinal syndrome (AGS) resulting from ionizing radiation causes death within 7 days. Currently, no satisfactory agent exists for mitigation of AGS. A peptide derived from the receptor binding domain of fibroblast growth factor 2 (FGF-P) was synthesized and its mitigation effect on AGS was examined. METHODS AND MATERIALS: A subtotal body irradiation (sub-TBI) model was created to induce gastrointestinal (GI) death while avoiding bone marrow death. After 10.5 to 16 Gy sub-TBI, mice received an intramuscular injection of FGF-P (10 mg/kg/day) or saline (0.2 ml/day) for 5 days; survival (frequency and duration) was measured. Crypt cells and their proliferation were assessed by hematoxylin, eosin, and BrdU staining. In addition, GI hemoccult score, stool formation, and plasma levels of endotoxin, insulin, amylase, interleukin (IL)-6, keratinocyte-derived chemokine (KC) monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor (TNF)-alpha were evaluated. RESULTS: Treatment with FGF-P rescued a significant fraction of four strains of mice (33-50%) exposed to a lethal dose of sub-TBI. Use of FGF-P improved crypt survival and repopulation and partially preserved or restored GI function. Furthermore, whereas sub-TBI increased plasma endotoxin levels and several pro-inflammation cytokines (IL-6, KC, MCP-1, and TNF-alpha), FGF-P reduced these adverse responses. CONCLUSIONS: The study data support pursuing FGF-P as a mitigator for AGS.

Characterization of K-ras gene mutations in association with mucinous hypersecretion in intraductal papillary-mucinous neoplasms.

BACKGROUND/PURPOSE: Intraductal papillary-mucinous neoplasms (IPMNs) of the pancreas have a favorable prognosis. However, invasive ductal carcinomas of the pancreas show a rapid progression. The aim of this study was to investigate gene mutations in pure pancreatic juice from IPMN patients and to define these genetic mutations in relation to the histopathological and clinical features of IPMNs. METHODS: Twenty-two patients with IPMN, 21 patients with ductal carcinoma, and 20 patients with normal pancreas or chronic pancreatitis were recruited for this study. We measured the main pancreatic duct's largest diameter and the maximum size of a dilated branch was assessed by ultrasonography or endoscopic ultrasonography. Pure pancreatic juice was collected and was investigated for K-ras, p16, and p53 mutations. RESULTS: Mutant K-ras gene was detected in 13 of the 22 patients (59.1%) with IPMNs. Different kinds of mutations were detected in the same patient in 4 cases. In the 13 patients with mutant K-ras gene, the diameter of the most dilated part of the main pancreatic duct was 2-8 mm (average, 4.5 mm) and in 7 patients with wild-type K-ras gene, the diameter was 2-5 mm (average, 2.7 mm). There was a significant difference in the diameter of the main pancreatic duct between patients with and without the mutant K-ras gene (P = 0.0323). CONCLUSIONS: The incidence of K-ras mutation may be associated with the hypersecretion of mucin.

Repertoire of the alpha beta T-cell receptor in the intestine.

The majority of T cells in the human and mouse intestine express the T-cell receptor (TCR) as an alphabeta heterodimer on their cell surface. As the major recognition element of antigens in the context of major histocompatibility complex-derived proteins, an examination of the structure of the alpha beta TCR in intestines has provided significant insights into the potential function of these cells and the major determinants that drive their selection. Studies in the human intestine have shown that the repertoires of intraepithelial lymphocytes (IELs), and likely lamina propria lymphocytes, are polyclonal before and shortly after birth, with the repertoire becoming oligoclonal in adults. Similarly, in adult mice the repertoire is oligoclonal, while in the newborn it is polyclonal. Investigations in mice have shown that some T cells may evade thymic selection. The population size and oligoclonality of IELs is influenced by the microbial content of the luminal microenvironment. This microenvironment probably directly determines the TCR repertoire. Studies in human inflammatory bowel disease (IBD) indicate that inflammation further skews the TCR repertoire. We speculate that dominant antigens associated with the pathogenesis of IBD are responsible for such skewing and that identifying the antigenic drivers may shed light on the environmental factors that trigger or potentiate human IBD.

PPARgamma inhibitors reduce tubulin protein levels by a PPARgamma, PPARdelta and proteasome-independent mechanism, resulting in cell cycle arrest, apoptosis and reduced metastasis of colorectal carcinoma cells.

The nuclear transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma) has been identified as an important therapeutic target in murine models of colorectal cancer (CRC). To examine whether PPARgamma inhibition has therapeutic effects in late-stage CRC, the effects of PPARgamma inhibitors on CRC cell survival were examined in CRC cell lines and a murine CRC model. Low doses (0.1-1 microM) of PPARgamma inhibitors (T0070907, GW9662 and BADGE) did not affect cell survival, while higher doses (10-100 microM) of all 3 PPARgamma inhibitors caused caspase-dependent apoptosis in HT-29, Caco-2 and LoVo CRC cell lines. Apoptosis was preceded by altered cell morphology, and this alteration was not prevented by caspase inhibition. PPARgamma inhibitors also caused dual G and M cell cycle arrest, which was not required for apoptosis or for morphologic alterations. Furthermore, PPARgamma inhibitors triggered loss of the microtubule network. Notably, unlike other standard antimicrotubule agents, PPARgamma inhibitors caused microtubule loss by regulating tubulin post-transcriptionally rather than by altering microtubule polymerization or dynamics. Proteasome inhibition by epoxomicin was unable to prevent tubulin loss. siRNA-mediated reduction of PPARgamma and PPARdelta proteins did not replicate the effects of PPARgamma inhibitors or interfere with the inhibitors' effects on apoptosis, cell cycle or tubulin. PPARgamma inhibitors also reduced CRC cell migration and invasion in assays in vitro and reduced both the number and size of metastases in a HT-29/SCID xenograft metastatic model of CRC. These results suggest that PPARgamma inhibitors are a novel potential antimicrotubule therapy for CRC that acts by directly reducing microtubule precursors.

Anti-Saccharomyces cerevisiae antibodies are associated with the development of postoperative fistulas following ileal pouch-anal anastomosis.

Although serologic testing for perinuclear antineutrophil cytoplasmic antibodies (pANCA) and anti-Saccharomyces cerevisiae antibodies (ASCA) is reportedly useful in distinguishing ulcerative colitis (UC) from Crohn's disease (CD), there are few and conflicting reports assessing their utility in predicting postoperative complications after ileal pouch-anal anastomosis (IPAA). We examined the associations between postoperative complications such as pouchitis or fistulas and pANCA and ASCA antibodies in a group of patients who underwent IPAA for UC. We conducted a retrospective chart review of 34 patients initially diagnosed with UC (four of these patients had a diagnosis of indeterminate colitis) who underwent IPAA by a single surgeon, and who had pANCA and ASCA antibody levels measured during their clinical course. Study patients were assigned to four groups based on the pattern of antibody reactivity: pANCA+/ASCA- (16 patients), pANCA-/ASCA+ (nine patients), pANCA+/ASCA+ (five patients), and pANCA-/ASCA- (four patients). The median length of follow-up was 16 months (3-144 months). None of the patients (0 of 16) who were pANCA+/ASCA- had their preoperative diagnosis of UC changed after a median follow-up of 14 months (3-118 months). Of the nine patients with a preoperative diagnosis of UC who were pANCA-/ASCA+, four patients (44%) had their diagnosis changed postoperatively to CD based on clinical findings, with a median follow-up: 15 months (5-98 months). Of 16 patients who underwent IPAA and who were pANCA+/ASCA-, 15 of 16 (93.75%), were free of fistulas postoperatively, with a median follow-up of 14 months (3-118 months). Of nine patients with a preoperative diagnosis of UC who underwent IPAA and who were pANCA-/ASCA+, four of nine (44%; p = 0.04) developed fistulas postoperatively, with a median length of follow-up of 55 months (15-67 months). No relationship between serologic profiles or antibody titer levels and the development of pouchitis was identified. In a cohort of patients undergoing IPAA for UC, serologic profiles may be useful in identifying patients at risk of postoperative fistula formation. Patients who were pANCA-/ASCA+ were at increased risk for the development of fistulas postoperatively compared to patients who were pANCA+/ASCA-, and were also more likely to have their diagnosis changed postoperatively to CD. A larger study is needed to validate these observations.

Inhibition of peroxisome proliferator-activated receptor gamma activity in esophageal carcinoma cells results in a drastic decrease of invasive properties.

Esophageal cancer is difficult to treat because of its rapid progression, and more effective therapeutic approaches are needed. The PPARgamma is a nuclear receptor superfamily member that is expressed in many cancers. PPARgamma expression is a feature of esophageal cancer cell lines, and in the present investigation, the PPARgamma antagonists T0070907 and GW9662 could induce loss of invasion but could not induce growth reduction or apoptosis at low concentrations (< 10 mM). A high concentration of antagonists (50 microM) inhibited cell growth and induced apoptosis, but these effects did not explain our result at the low concentration. Morphological change, decreased expression of the cell signaling pathway and inhibition of cancer cell invasion were observed in the low concentration. This suggested that PPARgamma antagonists inhibited esophageal cancer cell invasion as well as cell adherence, most likely due to alteration in the FAK-MAPK pathway, and this was independent of apoptosis. These results suggested that PPARgamma plays an important role in cancer cell invasion and that it might be a novel target for therapy of esophageal cancer.

Peroxisome proliferator-activated receptor gamma inhibition prevents adhesion to the extracellular matrix and induces anoikis in hepatocellular carcinoma cells.

Activation of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits growth and survival of hepatocellular carcinoma (HCC) cell lines. To further investigate the function of PPARgamma in HCC, PPARgamma expression patterns in primary tumors were examined, and the responses of two HCC cell lines to PPARgamma activation and inhibition were compared. PPARgamma expression was increased in HCC and benign-appearing peritumoral hepatocytes compared with remote benign hepatocytes. Both compound PPARgamma inhibitors and PPARgamma small interfering RNAs prevented HCC cell lines from adhering to the extracellular matrix. Loss of adhesion was followed by caspase-dependent apoptosis (anoikis). PPARgamma inhibitors had no effect on initial beta1 integrin-mediated adhesion, or on total focal adhesion kinase levels but did reduce focal adhesion kinase phosphorylation. The PPARgamma inhibitor T0070907 was significantly more efficient at causing cancer cell death than the activators troglitazone and rosiglitazone. T0070907 caused cell death by reducing adhesion and inducing anoikis, whereas the activators had no direct effect on adhesion and caused cell death at much higher concentrations. In conclusion, PPARgamma overexpression is present in HCC. Inhibition of PPARgamma function causes HCC cell death by preventing adhesion and inducing anoikis-mediated apoptosis. PPARgamma inhibitors represent a potential novel treatment approach to HCC.

Intestinal antiinflammatory effects of thiazolidenedione peroxisome proliferator-activated receptor-gamma ligands on T helper type 1 chemokine regulation include nontranscriptional control mechanisms.

Crohn's disease is associated with an excessive T helper (TH) type 1 inflammatory immune response. Reducing the influx of disease-associated CD4+ TH1 cells into the inflamed intestine is likely to be beneficial in preventing a disease flare-up and even possibly in reducing the effect of acute disease. Thiazolidenedione (TZD) ligands, which activate peroxisome proliferator-activated receptor-gamma (PPARgamma), have been shown to reduce TH1 inflammation in murine models of colitis, primarily in a preventative fashion. To determine whether PPARgamma ligands reduce this inflammation in part by reducing TH1 chemoattractant levels in vivo, the TZD pioglitazone was tested for its effects on a TH1 chemokine (CXCL10) in 2 models of colitis (i.e., dextran sodium sulfate and 2,4,6-dinitrobenzene sulfonic acid-mediated colitis). In both models, CXCL10 levels were significantly reduced by pioglitazone. Because TZDs can affect gene expression either directly, by regulating the binding of PPARgamma to consensus promoter elements, or indirectly, by modulating other signaling pathways that can affect gene transcription, the regulation of CXCL10 by TZDs was investigated in vitro in both HT-29 colon epithelial cells and THP-1 monocyte/macrophage cells. TZDs significantly reduced CXCL10 protein levels from activated HT-29 cells and THP-1-derived macrophages in a dose-dependent manner at nanomolar concentrations. However, TZDs did not affect messenger RNA levels or nuclear factor-kappaB activation at these concentrations in these cells. These findings imply the existence of a novel posttranscriptional regulatory antiinflammatory mechanism by TZDs that is not associated with reductions in nuclear factor-kappaB activation.

Protective effect of endogenous PPARgamma against acute gastric mucosal lesions associated with ischemia-reperfusion.

Acute gastric mucosal lesions (AGMLs) are an important cause of gastrointestinal bleeding. Herein, we demonstrate that peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of a nuclear receptor family, functions as an endogenous anti-inflammatory pathway in a murine model of AGML induced by ischemia-reperfusion (I/R). Treatment with specific PPARgamma ligands such as BRL-49653, pioglitazone, or troglitazone was examined in a model of AGML induced by I/R. PPARgamma-deficient and wild-type mice were also examined for their response to I/R in stomach. Specific PPARgamma ligands exhibited dramatic and rapid protection against AGML formation associated with I/R in mice in a dose-dependent manner. In contrast, the AGML induced by I/R in PPARgamma-deficient mice was more severe than that observed in wild-type mice. Administration of the PPARgamma ligand significantly inhibited the upregulation of TNF-alpha, ICAM-1, inducible nitric oxide synthase, apoptosis, and nitrotyrosine formation induced by I/R in the stomach. These data indicate that an endogenous pathway associated with PPARgamma plays an important role in the pathogenesis of I/R-associated injury in the stomach.

Peroxisome proliferator-activated receptor gamma agonist ligands stimulate a Th2 cytokine response and prevent acute colitis.

Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of a nuclear transcription factor family, has been previously demonstrated to have antiinflammatory activity. The effects of PPARgamma activation in the development of an immune response are less well characterized. Through evaluation of PPARgamma heterozygote mice (PPARgamma(+/-) and specific PPARgamma agonist ligand binding, we evaluated the immunologic effects of PPARgamma activation in a well-described model of colitis. Increased susceptibility to dextran sodium sulfate (DSS)-induced colitis as defined by body weights, histologic injury, and survival was observed in the PPARgamma(+/-) mice in comparison to wild-type mice. Three different PPARgamma ligands (troglitazone, pioglitazone, and rosiglitazone) demonstrated beneficial dose-related treatment effects when administered prior to the onset of colitis. However, no protection was observed when PPARgamma ligand activation occurred after the onset of colitis. The reduction in DSS-induced inflammation noted with PPARgamma ligand treatment was associated with decreased interferon-gamma and tumor necrosis factor-alpha and increased interleukin (IL)-4 and IL- 10 levels as assessed by quantitative reverse transcriptase-polymerase chain reaction. Consistent with this shift towards a T helper (Th2) cytokine dominance, PPARgamma ligand treatment stimulated increased GATA-3 expression. These results indicate that the protective effects exhibited by PPARgamma ligands in intestinal inflammation may be due to immune deviation away from Th1 and towards Th2 cytokine production.