*Clinical studies have demonstrated a link between the eosinophil-selective chemokines, eotaxins (eotaxin-1/CCL11 and eotaxin-2/CCL24), eosinophils, and the inflammatory bowel diseases, Crohn's disease and ulcerative colitis (UC). However, the cellular source and individual contribution of the eotaxins to colonic eosinophilic accumulation in inflammatory bowel diseases remain unclear. In this study we demonstrate, by gene array and quantitative PCR, elevated levels of eotaxin-1 mRNA in the rectosigmoid colon of pediatric UC patients. We show that elevated levels of eotaxin-1 mRNA positively correlated with rectosigmoid eosinophil numbers. Further, colonic eosinophils appeared to be degranulating, and the levels positively correlated with disease severity. Using the dextran sodium sulfate (DSS)-induced intestinal epithelial injury model, we show that DSS treatment of mice strongly induced colonic eotaxin-1 and eotaxin-2 expression and eosinophil levels. Analysis of eosinophil-deficient mice defined an effector role for eosinophils in disease pathology. DSS treatment of eotaxin-2 ؊/؊ and eotaxin-1/2 ؊/؊ mice demonstrated that eosinophil recruitment was dependent on eotaxin-1. In situ and immunofluorescence analysis-identified eotaxin-1 expression was restricted to intestinal F4/80 ؉ CD11b ؉ macrophages in DSS-induced epithelial injury and to CD68 ؉ intestinal macrophages and the basolateral compartment of intestinal epithelial cells in pediatric UC. These data demonstrate that intestinal macrophage and epithelial cell-derived eotaxin-1 plays a critical role in the regulation of eosinophil recruitment in colonic eosinophilic disease such as pediatric UC and provides a basis for targeting the eosinophil/eotaxin-1 axis in UC.

Background: While activation of the IL-6-dependent transcription factor signal transducer and activator of transcription 3 (STAT3) has been implicated in the pathogenesis of inflammatory bowel disease (IBD), a direct effect on mucosal gene expression and inflammation has not been shown. We hypothesized that a proinflammatory IL-6:STAT3-dependent biological network would be up regulated in pediatric-onset IBD patients, and would be associated with the severity of mucosal inflammation.

Generation of distinct cell types and numbers in developing cerebral cortex is subject to regulation by extracellular factors that positively or negatively control precursor proliferation. Although signals stimulating proliferation are well described, factors halting cell cycle progression are less well defined. At the molecular level, production and association of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) regulate cycle progression. We now report that the endogenous peptide, pituitary adenylate cyclase activating polypeptide (PACAP), negatively regulates the cell cycle by inhibiting p57Kip2-dependent CDK2 activity in embryonic cortex. Protein levels of CDK2 and members of the CIP/KIP family of CKIs (p27Kip1, p57Kip2) were detected in developing rat cortex from embryonic day 13.5 through postnatal day 2. With advancing development, CDK2 protein levels decreased, whereas CKI expression increased, suggesting that stimulatory and inhibitory cycle proteins control cell cycle exit. Using a well defined, nonsynchronized, 8 hr precursor culture, PACAP decreased the fraction of cells crossing the G1/S boundary, inhibiting DNA synthesis by 35%. CDK2 kinase activity was inhibited 75% by PACAP, whereas kinase protein and its regulatory cyclin E subunit were unaffected. Moreover, decreased kinase activity was accompanied by a twofold increase in levels of p57Kip2 protein, but not p21Cip1 or p27Kip1, suggesting that p57Kip2 mediates PACAP anti-mitogenic effects. Indeed, immunoprecipitation of CDK2 complex revealed increased p57Kip2 association with the kinase and concomitant reduction in free inhibitor after PACAP exposure, suggesting that p57Kip2 interactions directly regulate CDK2 activity. These observations establish a mechanism whereby anti-mitogenic signals actively induce cell cycle withdrawal in developing cortex.

Background Female patients receiving immunosuppressive therapy may be at increased risk for human papillomavirus (HPV) infection and cervical neoplasia. Methods We administered the 3-dose HPV vaccine Gardasil® to 37 females aged 9-26 years with inflammatory bowel disease (IBD) prescribed immunosuppressive therapy (prospective cohort). Geometric mean titers (GMT) in milli-Merck (mMu/mL) units were determined before dose 1 and one month after dose 3 by competitive Luminex immunoassay (cLIA) and qualitatively compared to healthy females of similar age from Merck’s database. Side effects and adverse events were evaluated. Concurrently, in 15 similar IBD patients previously vaccinated by their primary care provider we assessed antibody titers by cLIA and total IgG LIA after dose 3 of vaccine (range 0.5 to 27 months). Results The mean age of prospective patients was 15 years with 51% on anti-TNF therapy and 49% on immunomodulators: 33 of 37 completed all three doses. Seropositivity after dose 3 was 100% for types 6, 11 and 16 and 96% for type 18. GMT for HPV 6, 11, 16 and 18 was 1080, 1682, 3975 and 858, respectively, and did not qualitatively differ from healthy females. No serious adverse events were attributable to the vaccine. In the previously vaccinated cohort, seropositivity was 100% for types 6, 11, and 16, and 40% for type 18 by cLIA (93% for HPV18 by IgG LIA). Titers decreased with time since dose 3. Conclusions In this small study of IBD patients prescribed immunosuppressive therapy, Gardasil® was immunogenic and there were no clinically significant vaccine-associated adverse events.

To evaluate hyponatremia as an independent predictor of mortality in pediatric patients with end-stage liver disease listed for transplantation. We performed a single-center retrospective study of children listed for liver transplantation. We defined hyponatremia as a serum sodium concentration <130 mEq/L that persisted for at least seven days. The primary outcome was death on the waiting list. Ninety-four patients were eligible for the study. The prevalence of hyponatremia was 26%. Kaplan-Meier survival analysis demonstrated that patients with hyponatremia had decreased pretransplant survival compared with patients who maintained a serum sodium >130 mEq/L (p < 0.001). Univariable association analyses demonstrated death on the waiting list was also associated with higher median PELD scores at listing (p = 0.01), non-white race (p = 0.02), and age <1 yr (p = 0.001). Logistic regression analysis identified hyponatremia and non-white race as independently associated with pretransplant mortality [OR = 8.0 (95% CI: 1.4-45.7), p = 0.02 and OR = 6.3 (95% CI: 1.25-33.3), p = 0.03]. When hyponatremia was added to the PELD score, it was significantly better in predicting mortality than the PELD score alone (c-statistic = 0.79, p = 0.03). Hyponatremia identifies a subset of pediatric patients with increased risk of pretransplant mortality and improves the predictive ability of the current PELD score.

Objectives Growth hormone (GH) may reduce symptoms and improve growth in Crohn's Disease (CD). The effect upon mucosal inflammation is not known. We hypothesized that GH would improve both clinical and mucosal disease activity, and stimulate linear growth, in pediatric CD. Methods Twenty patients aged 7-18 receiving corticosteroids (CTX) for active CD were randomized to begin GH, 0.075 mg/kg/day (group A), or continue CTX alone (group B). Clinical and endoscopic disease activity were assessed after 12 weeks. Group B began GH at 12 weeks and clinical disease activity was assessed at 24 weeks. Subjects who experienced a clinical response after 12 weeks of GH therapy continued treatment for an additional 52 weeks, and linear growth was assessed. Results 65% of patients receiving GH achieved clinical remission, compared to 20% treated with CTX alone (p=0.03). While endoscopic disease activity trended towards an improvement at week 12 in group A, this did not differ between the groups. 61% of week 12 GH responders maintained their clinical response through week 64. Mean (95th CI) height Z score on GH increased from -1.1 (-1.6,-0.6) to -0.4 (-1,0.2), p=0.004 during this 52 week extension phase. GH was well tolerated with no unexpected safety signals. Conclusions The addition of GH to CTX therapy did not induce a reduction in mucosal inflammation, relative to CTX alone. However, GH was safe and effective as an adjunct to CTX for treatment of clinical disease activity and growth failure in pediatric CD.

Objectives Genotypic variation in STAT3 increases risk for IBD, and STAT3 dependent inflammatory networks are induced in the colon in these patients. We hypothesized that STAT3 “A” risk allele carriage would be associated with increased cellular STAT3 activation and colon leukocyte recruitment. Methods Colonic expression of genes regulating STAT3 signaling and leukocyte recruitment and function was measured in pediatric CD patients stratified by STAT3 genotype. The frequency of colonic pSTAT3+ and CXCR2+ neutrophils was determined using immunohistochemistry. STAT3 tyrosine phosphorylation (pSTAT3) was measured in circulating leukocytes by flow cytometry, and mechanisms regulating STAT3 activation were tested in IBD EBV-transformed lymphocytes (EBL). Results Colonic expression of IL-6, the STAT3 target gene SOCS3, the neutrophil chemo-attractants IL-8, CXCL1, and CXCL3, and the neutrophil products S100A8, S100A9 and S100A12 were increased in patients carrying the STAT3 “A” risk allele. The frequency of neutrophils expressing the cognate receptor for IL-8, CXCR2, was increased in colonic biopsies from patients carrying the risk allele, and the frequency of pSTAT3+ or CXCR2+ neutrophils correlated with histologic severity. The frequency of CD4+ lymphocytes and granulocytes expressing pSTAT3 was increased in patients carrying the STAT3”A” risk allele. EBL's from patients carrying the STAT3”A” risk allele exhibited increased basal and IL-6 stimulated STAT3 tyrosine phosphorylation, increased transcription of STAT3 and SOCS3 after IL-6 stimulation, and increased membrane localization of the IL-6 receptor, GP130, and JAK2. Conclusions The STAT3 “A” risk allele is associated with increased cellular STAT3 activation and up-regulation of pathways which promote recruitment of CXCR2+ neutrophils to the gut.

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