Intestinal dysbiosis in pediatric Crohn's disease patients with IL10RA mutations.

BACKGROUND: Several studies have employed animal models to explore the association between microbiota and interleukin (IL) 10 signaling; however, limited information is available about the human microbiome. AIM: To characterize the microbiome in patients with IL10RA mutations and to explore the association between gut dysbiosis and disease severity. METHODS: Fecal samples were collected from patients who were diagnosed with loss-of-function mutations in the IL10RA gene between January 2017 and July 2018 at the Children's Hospital of Fudan University. Age-matched volunteer children were recruited as healthy controls. Patients with Crohn's disease (CD) were used as disease controls to standardize the antibiotic exposure. Microbial DNA was extracted from the fecal samples. All analyses were based on the 16S rRNA gene sequencing data. RESULTS: Seventeen patients with IL10RA mutations (IL10RA group), 17 patients with pediatric CD, and 26 healthy children were included. Both patients with IL10RA mutations and those with CD exhibited a reduced diversity of gut microbiome with increased variability. The relative abundance of Firmicutes was substantially increased in the IL10RA group (P = 0.02). On further comparison of the relative abundance of taxa between patients with IL10RA mutations and healthy children, 13 taxa showed significant differences. The IL10RA-specific dysbiosis indices exhibited a significant positive correlation with weighted pediatric CD activity index and simple endoscopic score for CD. CONCLUSION: In patients with IL10RA mutations and early onset inflammatory bowel disease, gut dysbiosis shows a moderate association with disease severity.

Special clinical characteristics and outcomes in Chinese pediatric patients with early-onset Crohn's disease.

OBJECTIVE: To study the clinical and nutritional characteristics of early-onset Crohn's disease (EO-CD) in China. METHODS: Patients were defined as having EO-CD (age at diagnosis <10 y) or late-onset Crohn's disease (LO-CD; age at diagnosis of 10-17 y). Their characteristics, clinical, and nutritional data were collected at baseline and at each follow-up visit. Statistical analyses were used to compare differences in both groups. RESULTS: From July 1993 to February 2017, of the 137 children enrolled, 68 (49.6%) had EO-CD and 69 (50.4%) had LO-CD. More patients with EO-CD than those with LO-CD presented with diarrhea, hematochezia, growth delay, anemia and skin disease, and had higher pediatric Crohn's disease activity index scores at diagnosis (all P < 0.05). Fewer patients with EO-CD achieved their first remission (42.6% vs 76.8%, P < 0.0001) during follow-up. Patients with EO-CD required a longer treatment time to reach remission (P = 0.0049) and had a higher mortality rate (P = 0.0133), as well as lower height and weight percentiles (P = 0.0200 and 0.0288, respectively), hemoglobin (P = 0.0185) and albumin levels (P = 0.0002), zinc (P = 0.0024) and iron (P = 0.0110) concentrations in blood at diagnosis. CONCLUSION: The EO-CD group had worse clinical outcomes and nutritional status than the LO-CD group.

A20 inhibits lipopolysaccharide-induced inflammation in enterocytes.

AIM: To examine the role of A20 in the regulation of intestinal epithelial cells (IECs) inflammation. METHODS: Using gene transfection, both stable overexpression and knockdown A20-expressed HT-29 cell lines were established. Accordingly, the cells were divided into the following groups: The control group, the A20 overexpression group, the A20 knockdown group and the respective controls. A20 was stimulated with lipopolysaccharide (LPS) in a dose- and time-dependent manner and was detected using western blotting and real-time polymerase chain reaction (PCR) analyses. Immunofluorescence and western blotting analyses were performed to investigate the role of A20 in the regulation of nuclear factor (NF)-κB activation and translocation into the nucleus. ELISA and real-time PCR were performed to examine A20 in regulating the release of the following inflammatory cytokines: Tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and IL-8. RESULTS: The expression of A20 in IECs was inducible. When intestinal epithelial cells were subjected to the stimulation of LPS, the expression of A20 was increased, and the expression of A20 was induced in a dose- and time-dependent manner. The expression of A20 was very low in HT-29 cells without LPS stimulation but rapidly increased and was maintained at a high level 2-4 h after stimulation with LPS. These levels gradually declined with a change in time-course, and the expression of A20 increased with increasing LPS stimulation. Western blotting and immunofluorescence revealed that overexpression of A20 can inhibit NF-κB activation and its translocation to the nucleus. The overexpression of A20 can reduce the levels of proinflammatory cytokines involved in the pathophysiology of inflammatory bowel disease. There was no significant difference in the expression of IL-8 mRNA in the control group, A20 overexpression group or A20 knockdown group without LPS stimulation (P > 0.05); however, while after 2 h, 4 h and 8 h stimulation with LPS, the expression of IL-8 in the A20 overexpression group was lower than the control group and the A20 knockdown group (P < 0.05 or P < 0.01). The expression of TNF-α was different at different time points after 8 h of LPS stimulation (F = 31.33, DF = 5, P < 0.001), and the expression of TNF-α increased as the LPS stimulation time increased. Upon LPS stimulation, lower levels of TNF-α were detected in the A20 overexpression cell lines (P < 0.05). There were no significant differences in the induction of IL-6 and IL-1ß among the control group, A20 overexpression group and A20 knockdown group (P > 0.05). CONCLUSION: A20 plays an important role in limiting inflammation by inhibiting LPS-induced NF-κB responses in the gut luminal. A20 may be a potential therapeutic tool for inflammatory diseases.

Coordination and nursing care of pediatric patients undergoing double balloon enteroscopy.

AIM: To review safety, efficacy, and proper nursing care of double-balloon enteroscopy (DBE) in pediatric patients with small intestinal disease. METHODS: Our study included 37 patients with abdominal pain, diarrhea, passage of blood in the stools, and other symptoms, who underwent DBE from December 2006 to July 2010. DBE was retrograde in 36 procedures, antegrade in six, and from both ends in five. The diagnostic significance and salient points in nursing care are discussed in this article. RESULTS: At least one lesion was discovered in 28 out of 37 patients, which yielded a positive diagnosis in 75.7% of cases. Good bowel preparation and skilled nursing care not only shortened the procedure time, but could also alleviate patient discomfort and enhance the quality of examination. No serious procedure-related complications were observed in any cases. CONCLUSION: DBE is a new modality of endoscopic procedure that improves the standard of diagnosis and treatment of small bowel diseases in children. Good nursing care is essential to the successful execution of the procedure.

[Expression of zinc finger protein A20 in pediatric inflammatory bowel disease].

OBJECTIVE: It is demonstrated that excessive activation of NF-κB is central to the pathogenesis of inflammatory bowel disease (IBD). Zinc finger protein A20 (A20) is a key player in the negative feedback regulation of NF-κB signaling in response to multiple stimuli and has been described as central gatekeeper in inflammation and immunity. Mice genetically deficient in A20 develop severe intestinal inflammation and have increased susceptibility to dextran sodium sulfate (DSS)-induced colitis. Few studies have been done to explore the role of A20 in the pathogenesis of IBD. To clarify the relationship between intestinal inflammation and the expression level of A20 in IBD patients, the expression level of A20 and a series of inflammatory cytokines, such as NF-κB, IL-6, and IL-8, in children with IBD and controls were examined. METHOD: Terminal ileal mucosal samples were obtained via endoscopy. Fifty-seven mucosal samples were divided into 4 groups: normal control group (n = 16), IBD remission group (n = 12), IBD active group (n = 13) and non-IBD enteritis group (n = 16). According to disease activity index scores, the IBD patients were divided into IBD remission group and IBD active group. Normal control group was consisted of patients with functional bowel disorders or intestinal polyps. Non-IBD enteritis was defined as changes in which endoscopy and histological examination showed inflammatory changes but could not be diagnosed as IBD. Real-time PCR was adopted for detecting the mRNA levels of A20, IL-6 and IL-8. Meanwhile immunohistochemistry was performed to measure the expression of A20 and NF-κB. RESULT: (1) The expression of A20 and NF-κB were very low in normal control group, but significantly up-regulated in IBD active group and non-IBD enteritis group (P < 0.01 for both); (2) Compared with normal control group, expression of NF-κB [(9.35 ± 4.84)% vs. (0.57 ± 0.44)%, P < 0.01], IL-6 (t' = 1.34, P > 0.05), IL-8 (t = 1.38, P > 0.05) increased in IBD remission group, while the expression of A20 in both mRNA (t = 1.03, P > 0.05) and protein levels [(0.36 ± 0.18)% vs. (0.87 ± 0.29)%, P < 0.01] decreased; (3) Compared with non-IBD enteritis group, although the expression of NF-κB [(24.17 ± 11.27)% vs. (55.29 ± 21.84)%, P < 0.01], IL-6 (t = 2.22, P < 0.05), IL-8 (t = 2.97, P < 0.01) were highly increased in IBD active group, the expression of A20 in both mRNA(t = 2.26, P < 0.05) and protein levels [(29.23 ± 11.70)% vs. (16.81 ± 5.90)%, P < 0.01]significantly decreased; (4) The expression of IL-6, IL-8 were similar in IBD remission group and non-IBD enteritis group (both P > 0.05), but the expression of A20 was much lower in both mRNA (t = 4.42, P < 0.01) and protein levels [(29.23 ± 11.70)% vs. (0.47 ± 0.25)%, P < 0.01] in IBD remission group. CONCLUSION: The results demonstrate that there is an excessive inflammatory response but insufficient up-regulation of A20 expression in IBD patients. Low levels expression of A20 may play an important role in the pathogenesis of IBD.

Anti-cancer effects of p21WAF1/CIP1 transcriptional activation induced by dsRNAs in human hepatocellular carcinoma cell lines.

AIM: To investigate the anti-cancer effects of p21WAF1/CIP1 transcriptional activation induced by dsRNAs in hepatocellular carcinoma (HCC) cell lines. METHODS: HCC cell lines BEL7402, SMMC-7721, MHCC97L, MHCC97H, and MHCCLM3 were used. HCC cells were treated with dsP21-322 (50 nmol/L), dsControl (50 nmol/L), siP21 (50 nmol/L), or mock transfection. The expression of p21 was detected using quantitative PCR and Western blot. The effects of RNA activation on HCC cells were determined using cell viability assays, apoptosis analyses and clonogenic survival assays. Western blot was also conducted to detect the expression of Bcl-xL, survivin, cleaved caspase-3, cleaved caspase-9 and cleaved PARP. RESULTS: At 72 to 120 h following the transfection, dsP21-322 markedly inhibited the viability of HCC cells and clone formation. At the same times, dsP21-322 caused a significant increase in HCC cell apoptosis, as demonstrated with cytometric analysis. The phenomena were correlated with decreased expression levels of the anti-apoptotic proteins Bcl-xL, surviving, and increased expression of cleaved caspase-3, cleaved caspase-9 and cleaved PARP. CONCLUSION: RNA-induced activation of p21 gene expression may have significant therapeutic potential for the treatment of hepatocellular carcinoma and other cancers.

Treatment of pediatric refractory Crohn's disease with thalidomide.

AIM: To assess the efficacy and tolerability of thalidomide in pediatric Crohn's disease (CD). METHODS: Six patients with refractory CD received thalidomide at an initial dose of 2 mg/kg per day for one month, then increased to 3 mg/kg per day or decreased to 1 mg/kg per day, and again further reduced to 0.5 mg/kg per day, according to the individual patient's response to the drug. RESULTS: Remission was achieved within three months. Dramatic clinical improvement was demonstrated after thalidomide treatment. Endoscopic and pathological improvements were also observed after thalidomide treatment, which was well tolerated by all patients. CONCLUSION: Thalidomide is a useful drug for pediatric refractory CD.