Association between gut microbiota and Hirschsprung disease: a bidirectional two-sample Mendelian randomization study.

Background: Several studies have pointed to the critical role of gut microbiota (GM) and their metabolites in Hirschsprung disease (HSCR) pathogenesis. However, the detailed causal relationship between GM and HSCR remains unknown. Methods: In this study, we used two-sample Mendelian randomization (MR) analysis to investigate the causal relationship between GM and HSCR, based on the MiBioGen Consortium's genome-wide association study (GWAS) and the GWAS Catalog's HSCR data. Reverse MR analysis was performed subsequently, and the sensitivity analysis, Cochran's Q-test, MR pleiotropy residual sum, outlier (MR-PRESSO), and the MR-Egger intercept were used to analyze heterogeneity or horizontal pleiotropy. 16S rDNA sequencing and targeted mass spectrometry were developed for initial validation. Results: In the forward MR analysis, inverse-variance weighted (IVW) estimates suggested that Eggerthella (OR: 2.66, 95%CI: 1.23-5.74, p = 0.01) was a risk factor for HSCR, while Peptococcus (OR: 0.37, 95%CI: 0.18-0.73, p = 0.004), Ruminococcus2 (OR: 0.32, 95%CI: 0.11-0.91, p = 0.03), Clostridiaceae1 (OR: 0.22, 95%CI: 0.06-0.78, p = 0.02), Mollicutes RF9 (OR: 0.27, 95%CI: 0.09-0.8, p = 0.02), Ruminococcaceae (OR: 0.16, 95%CI: 0.04-0.66, p = 0.01), and Paraprevotella (OR: 0.45, 95%CI: 0.21-0.98, p = 0.04) were protective factors for HSCR, which had no heterogeneity or horizontal pleiotropy. However, reverse MR analysis showed that HSCR (OR: 1.02, 95%CI: 1-1.03, p = 0.049) is the risk factor for Eggerthella. Furthermore, some of the above microbiota and short-chain fatty acids (SCFAs) were altered in HSCR, showing a correlation. Conclusion: Our analysis established the relationship between specific GM and HSCR, identifying specific bacteria as protective or risk factors. Significant microbiota and SCFAs were altered in HSCR, underlining the importance of further study and providing new insights into the pathogenesis and treatment.

Identifying the potential transcriptional regulatory network in Hirschsprung disease by integrated analysis of microarray datasets.

Objective: Hirschsprung disease (HSCR) is one of the common neurocristopathies in children, which is associated with at least 20 genes and involves a complex regulatory mechanism. Transcriptional regulatory network (TRN) has been commonly reported in regulating gene expression and enteric nervous system development but remains to be investigated in HSCR. This study aimed to identify the potential TRN implicated in the pathogenesis and diagnosis of HSCR. Methods: Based on three microarray datasets from the Gene Expression Omnibus database, the multiMiR package was used to investigate the microRNA (miRNA)-target interactions, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Then, we collected transcription factors (TFs) from the TransmiR database to construct the TF-miRNA-mRNA regulatory network and used cytoHubba to identify the key modules. Finally, the receiver operating characteristic (ROC) curve was determined and the integrated diagnostic models were established based on machine learning by the support vector machine method. Results: We identified 58 hub differentially expressed microRNAs (DEMis) and 16 differentially expressed mRNAs (DEMs). The robust target genes of DEMis and DEMs mainly enriched in several GO/KEGG terms, including neurogenesis, cell-substrate adhesion, PI3K-Akt, Ras/mitogen-activated protein kinase and Rho/ROCK signaling. Moreover, 2 TFs (TP53 and TWIST1), 4 miRNAs (has-miR-107, has-miR-10b-5p, has-miR-659-3p, and has-miR-371a-5p), and 4 mRNAs (PIM3, CHUK, F2RL1, and CA1) were identified to construct the TF-miRNA-mRNA regulatory network. ROC analysis revealed a strong diagnostic value of the key TRN regulons (all area under the curve values were more than 0.8). Conclusion: This study suggests a potential role of the TF-miRNA-mRNA network that can help enrich the connotation of HSCR pathogenesis and diagnosis and provide new horizons for treatment.

Fecal microbiota transplantation enhances cell therapy in a rat model of hypoganglionosis by SCFA-induced MEK1/2 signaling pathway.

Hirschsprung disease (HSCR), one of several neurocristopathies in children, is characterized by nerve loss in the large intestine and is mainly treated by surgery, which causes severe complications. Enteric neural crest-derived cell (ENCC) transplantation is a potential therapeutic strategy; however, so far with poor efficacy. Here, we assessed whether and how fecal microbiota transplantation (FMT) could improve ENCC transplantation in a rat model of hypoganglionosis; a condition similar to HSCR, with less intestinal innervation. We found that the hypoganglionosis intestinal microenvironment negatively influenced the ENCC functional phenotype in vitro and in vivo. Combining 16S rDNA sequencing and targeted mass spectrometry revealed microbial dysbiosis and reduced short-chain fatty acid (SCFA) production in the hypoganglionic gut. FMT increased the abundance of Bacteroides and Clostridium, SCFA production, and improved outcomes following ENCC transplantation. SCFAs alone stimulated ENCC proliferation, migration, and supported ENCC transplantation. Transcriptome-wide mRNA sequencing identified MAPK signaling as the top differentially regulated pathway in response to SCFA exposure, and inhibition of MEK1/2 signaling abrogated the SCFA-mediated effects on ENCC. This study demonstrates that FMT improves cell therapy for hypoganglionosis via short-chain fatty acid metabolism-induced MEK1/2 signaling.

miR-378c suppresses Wilms tumor development via negatively regulating CAMKK2.

Wilms tumor is a rare kidney malignancy primarily developed in children. Treatment for Wilms tumor includes surgery, radiotherapy, and chemotherapy. Recent studies have demonstrated that microRNAs (miRNAs) play important roles in regulating Wilms tumor development. In this study, we aimed to elucidate the expression and function of miR-378c in Wilms tumor. Quantitative real-time PCR (qRT-PCR) results showed that miR-378c was downregulated in Wilms tumor tissues and cell lines. Functionally, further CCK-8, would healing, and transwell assays revealed that overexpression of miR-378c impaired Wilms tumor cell growth and metastasis in vitro. In addition, xenograft assay showed that miR-378c overexpression inhibited Wilms tumor development in vivo. Mechanistically, luciferase reporter assay confirmed that miR-378c directly targets CAMKK2 in Wilms tumor. qRT-PCR and western blot assays demonstrated that CAMKK2 was highly expressed in Wilms tumor tissues and cell lines. Rescue experiments were performed to further evaluate the functional relationship between miR-378c and CAMKK2. Overexpression of miR-378c suppressed Wilms tumor cell metastasis via negatively regulating CAMKK2 expression. Consistently, inhibition of miR-378c enhanced Wilms tumor cell malignancy behavior via augmenting CAMKK2 expression, which could be abrogated by CAMKK2 knockdown. In summary, our findings suggest that miR-378c inhibits the development and metastasis of Wilms tumor via negatively regulating CAMKK2 expression, which could be utilized to develop new therapy strategy.

Phenotypic and functional comparison of rat enteric neural crest-derived cells during fetal and early-postnatal stages.

In our previous study, we showed that with increasing time in culture, the growth characteristics of enteric neural crest-derived cells (ENCCs) change, and that the proliferation, migration and neural differentiation potential of these cells in vitro notably diminish. However, there are no studies on the developmental differences in these characteristics between fetal and early-postnatal stages in vitro or in vivo. In this study, we isolated fetal (embryonic day 14.5) and postnatal (postnatal day 2) ENCCs from the intestines of rats. Fetal ENCCs had greater maximum cross-sectional area of the neurospheres, stronger migration ability, and reduced apoptosis, compared with postnatal ENCCs. However, fetal and postnatal ENCCs had a similar differentiation ability. Fetal and postnatal ENCCs both survived after transplant into a rat model of Hirschsprung's disease. In these rats with Hirschsprung's disease, the number of ganglionic cells in the myenteric plexus was higher and the distal intestinal pressure change was greater in animals treated with fetal ENCCs compared with those treated with postnatal ENCCs. These findings suggest that, compared with postnatal ENCCs, fetal ENCCs exhibit higher survival and proliferation and migration abilities, and are therefore a more appropriate seed cell for the treatment of Hirschsprung's disease. This study was approved by the Animal Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University (approval No. 2016086) on March 3, 2016.

Involvement of the lncRNA AFAP1-AS1/microRNA-195/E2F3 axis in proliferation and migration of enteric neural crest stem cells of Hirschsprung's disease.

NEW FINDINGS: What is the central question of this study? Long non-coding RNAs (lncRNAs) are widely involved in the progression of Hirschsprung's disease (HSCR), but the role of actin filament associated protein 1 antisense RNA1 (AFAP1-AS1), an lncRNA, in HSCR has not been explored before. What is the main finding and its importance? Downregulation of AFAP1-AS1 blocks enteric neural crest stem cell proliferation, differentiation, migration and invasion and promotes the occurrence of HSCR via the miR-195/E2F3 axis, indicating thatAFAP1-AS might be a potential biomarker for HSCR patients. ABSTRACT: Long non-coding RNAs (lncRNAs) are involved in several human disorders. Nevertheless, it remains unclear whether they are implicated in the phenotypes of enteric neural crest stem cells (ENCSCs) in Hirschsprung's disease (HSCR). Therefore, we designed this study to explore the pathogenicity of AFAP1-AS1 for HSCR. Microarray analysis and bioinformatic tools were used to screen out the differentially lncRNAs and microRNAs (miRNAs) in patients with HSCR. Small interference RNA transfection was applied to carry out functional experiments in ENCSCs. Cellular activities were detected by cell counting kit-8, 5-ethynyl-2'-deoxyuridine, Transwell assays and flow cytometry. Finally, rescue experiments were performed to examine the cofunction of AFAP1-AS1 and miR-195 and of miR-195 and E2F transcription factor 3 (E2F3). AFAP1-AS1 was reduced in HSCR patients. Meanwhile, knockdown of AFAP1-AS1 reduced the cell migratory and proliferative capacities and facilitated cell apoptosis along with G0/G1 phase arrest. E2F3 was diminished when miR-195 was upregulated, and AFAP1-AS1 inhibition reduced its ability to bind to miR-195. Altogether, AFAP1-AS1 silencing acts as an endogenous RNA by interacting with miR-195 to alter E2F3 expression, thus conferring repressive effects on ENCSC activity and promoting HSCR progression.

NEAT1 Negatively Regulates Cell Proliferation and Migration of Neuroblastoma Cells by miR-183-5p/FOXP1 Via the ERK/AKT Pathway.

Neuroblastoma, a malignant tumor of the sympathetic nervous system, is an aggressive extracranial tumor in childhood. Long noncoding RNAs (lncRNAs) have been discovered to play a key role in the eukaryotic regulatory gene network and be involved in a wide variety of biological processes. We observed that the expression of lncRNA nuclear-enriched abundant transcript-1 (NEAT1) was significantly decreased in human neuroblastoma tissues and cell lines, compared with the normal. We observed cell proliferation, migration, and invasion with Cell Counting Kit-8 assay, colony formation assay, and Transwell assay to investigate the effects of NEAT1, miR-183-5p, or FOXP1 on neuroblastoma cells. And we also used StarBase and luciferase reporter gene assay to predict and confirm the interaction of NEAT1, miR-183-5p, and FOXP1 in neuroblastoma cells. First, overexpression of NEAT1 suppressed cell proliferation and played a key role in cell migration and invasion. In addition, NEAT1 was demonstrated to directly interact with miR-183-5p and exerted its antioncogenic role in neuroblastoma by negatively regulating miR-183-5p expression. miR-183-5p suppressed the expression of FOXP1 and regulated cell proliferation and migration by directly targeting FOXP1 mRNA 3'-untranslated region. Moreover, FOXP1 antagonized the effect of miR-183-5p on the phosphorylation of extracellular-regulated kinase/protein kinase B (ERK/AKT), while FOXP1 siRNA increased the reduced phosphorylation of ERK/AKT caused by miR-183-5p inhibitor in neuroblastoma cells. Taken together, these data showed that NEAT1 negatively regulated cell proliferation and migration of neuroblastoma by the miR-183-5p/FOXP1 axis via suppression of the ERK/AKT pathway. Our findings may provide a new target for the study of pathogenesis and treatment of neuroblastoma.

Lack of associations between LIG3 gene polymorphisms and neuroblastoma susceptibility in Chinese children.

Accumulating evidence suggests that dysregulation of the DNA non-homologous end-joining (NHEJ) repair system is a causative factor in many cancers, including high-risk neuroblastoma. A number of studies have shown that polymorphisms in the DNA ligase III (LIG3) gene, one of the key genes in the error-prone alternative NHEJ (a-NHEJ) pathway for DNA double-strand break (DSB) repair, are associated with a variety of cancers. Nevertheless, whether LIG3 polymorphisms contribute to neuroblastoma risk remains unknown. We investigated the correlation between neuroblastoma susceptibility and two LIG3 polymorphisms (rs1052536 C>T and rs4796030 A>C) among 469 neuroblastoma patients and 998 healthy controls from China. Our results failed to detect any relationship between the analyzed SNPs and neuroblastoma risk in either overall analysis or stratification analysis. These results suggest that rs1052536 C>T and rs4796030 A>C are unrelated to neuroblastoma susceptibility in the Chinese population. Further studies with larger sample sizes and multiple ethnicities are necessary to verify our results.

PARP1 gene polymorphisms and neuroblastoma susceptibility in Chinese children.

Neuroblastoma is a heterogeneous cancer frequently occurring in childhood. Germline mutations of PARP1 oncogene are implicated in several types of cancer. However, whether common single nucleotide polymorphisms (SNPs) in PARP1 gene are associated with neuroblastoma risk has received relatively few attentions. In this multi-center study, we aimed to elucidate the contributing role of PARP1 SNPs in neuroblastoma risk. We successfully genotyped three potentially functional PARP1 SNPs (rs1136410 A>G, rs2666428 T>C, rs8679 A>G) in 469 neuroblastoma cases and 998 controls. We did not detect any significant association between the analyzed SNPs and neuroblastoma risk in single SNP analysis. However, stratified analysis revealed that rs1136410 AG/GG carriers were more likely to develop tumors arising from mediastinum (AG/GG vs. AA: adjusted OR=1.65, 95% CI=1.06-2.56, P=0.028). Moreover, rs2666428 TC/CC carriers were at significantly lower risk to develop tumors from "other sites" (TC/CC vs. TT: adjusted OR=0.44, 95% CI=0.20-0.96, P=0.040). Our findings failed to provide evidence of the conferring role of the PARP1 gene polymorphisms in the risk of neuroblastoma. Further investigations of the association between PARP1 gene SNPs and neuroblastoma risk are warranted.

Correlation of spatio-temporal characteristics of intestinal inflammation with IL-17 in a rat model of hypoganglionosis.

Interleukin 17 expression is increased in children with Hirschsprung disease, which is characterized by intestinal inflammation. This study designed to exploit the characteristics of intestinal inflammation and examine the correlation of interleukin 17 in this process of hypoganglionosis model established by benzalkonium chloride treatment. Colon sections from female rats were treated with benzalkonium chloride to induce hypoganglionosis or with saline alone as a sham control. C-reactive protein and tumor necrosis factor-ɑ were used as markers of inflammation. Expression of C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 was assessed in colon tissue and blood serum on days 7, 14 and 21 after treatment. The correlation between C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 expression was estimated using the Spearman's rank-correlation coefficient. C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 were strongly expressed in submucosa and mucosa layers and serum from treated animals. The expression of C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 maintained the highest level at Day 21. Only C-reactive protein and tumor necrosis factor-ɑ expression was increased in control animals and only on day 7. Spearman's rank correlation coefficient was significant in C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 at Day 7, 14 and 21. Concomitant upregulation of C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 and significant positive correlations between C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 may imply that interleukin 17 is involved in spatio-temporal inflammation induced by benzalkonium chloride.

Sirolimus as initial therapy for kaposiform hemangioendothelioma and tufted angioma.

BACKGROUND: Sirolimus has been used to manage various complex vascular anomalies. Kaposiform hemangioendothelioma and tufted angioma may develop Kasabach-Merritt phenomenon in infancy. METHODS: We retrospectively reviewed the clinical and laboratory data of eight patients with kaposiform hemangioendothelioma and tufted angioma who were initially treated using oral sirolimus in our center, including six with Kasabach-Merritt phenomenon. RESULTS: Five girls and three boys seen between September 2012 and March 2015 were included. Age at initiation of sirolimus ranged from 30 days to 14 weeks (mean±SD 8.6 ± 3.5 weeks). Six of these eight patients had kaposiform hemangioendothelioma, and two had a tufted angioma. Platelet count before start of oral sirolimus ranged from 5 × 109 /L to 189 × 109 /L ((78.8 ± 65.2)×109 /L) and fibrinogen level from 68 to 215 mg/dL (123.1 ± 50.5 mg/dL). All patients received standard doses of sirolimus (0.05 mg/kg orally, twice daily) as initial therapy. All patients with thrombocytopenia or hypofibrinogenemia reached a normal platelet count and a normal fibrinogen level within 3 to 4 weeks after sirolimus treatment. Length of treatment ranged from 12 to 79 weeks (39.9 ± 15.3 weeks). Two patients developed grade 2 oral mucositis during treatment. CONCLUSION: Sirolimus as first-line therapy shows great promise in the treatment of kaposiform hemangioendothelioma and tufted angioma.

Upregulation of MiR-369-3p suppresses cell migration and proliferation by targeting SOX4 in Hirschsprung's disease.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital digestive disease in the new born. miR-369-3p has been reported to be involved in many human diseases. However, the relationship between miR-369-3p and HSCR remains largely unknown. METHODS: In this study, qRT-PCR was used to detect the relative expression of miR-369-3p in 60 HSCR bowel tissue samples and 47 matched controls. Bioinformatic analysis and dual-luciferase reporter assay were performed to evaluate the target for miR-369-3p. Cell Counting Kit-8 (CCK-8) assay, Transwell assay, wound healing assay and flow cytometry were employed to investigate the biological function of miR-369-3p in human SH-SY5Y and 293T cell lines. RESULTS: We found that ganglion cell numbers were remarkably reduced while miR-369-3p was significantly upregulated in HSCR tissues compared to that in adjacent normal tissues (P<0.01). Dual-luciferase reporter assay showed that the 3'-UTR of SOX4 was a direct target to miR-369-3p. Moreover, an increased level of miR-369-3p was inversely correlated with decreased levels of SOX4 mRNA and protein (P<0.05, respectively). Dysregulation of miR-369-3p and SOX4 significantly suppressed cell proliferation and migration in SH-SY5Y and 293T cell lines in vitro (P<0.05, respectively). CONCLUSION: Our study demonstrates that aberrant expression of miR-369-3p might play a crucial role in the development HSCR by regulating SOX4 expression, which may infer that it is an effective diagnostic target in the pathogenesis of HSCR, but investigation is still needed to explore the underlying mechanism.

Identifying key genes associated with Hirschsprung's disease based on bioinformatics analysis of RNA-sequencing data.

BACKGROUND: Hirschsprung's disease (HSCR) is a type of megacolon induced by deficiency or dysfunction of ganglion cells in the distal intestine and is associated with developmental disorders of the enteric nervous system. To explore the mechanisms of HSCR, we analyzed the RNA-sequencing data of the expansion and the narrow segments of colon tissues separated from children with HSCR. METHODS: RNA-sequencing of the expansion segments and the narrow segments of colon tissues isolated from children with HSCR was performed. After differentially expressed genes (DEGs) were identified using the edgeR package in R, functional and pathway enrichment analyses of DEGs were carried out using DAVID software. To further screen the key genes, protein-protein interaction (PPI) network and module analyses were conducted separately using Cytoscape software. RESULTS: A total of 117 DEGs were identified in the expansion segment samples, including 47 up-regulated and 70 down-regulated genes. Functional enrichment analysis suggested that FOS and DUSP1 were implicated in response to endogenous stimulus. In the PPI network analysis, FOS (degree=20), EGR1 (degree=16), ATF3 (degree=9), NOS1 (degree=8), CCL5 (degree=8), DUSP1 (degree=7), CXCL3 (degree=6), VIP (degree=6), FOSB (degree=5), and NOS2 (degree=4) had higher degrees, which could interact with other genes. In addition, two significant modules (module 1 and module 2) were identified from the PPI network. CONCLUSIONS: Several genes (including FOS, EGR1, ATF3, NOS1, CCL5, DUSP1, CXCL3, VIP, FOSB, and NOS2) might be involved in the development of HSCR through their effect on the nervous system.

Combination of exogenous cell transplantation and 5-HT4 receptor agonism induce endogenous enteric neural crest-derived cells in a rat hypoganglionosis model.

Enteric neural crest-derived cells (ENCCs) can migrate into endogenous ganglia and differentiate into progeny cells, and have even partially rescued bowel function; however, poor reliability and limited functional recovery after ENCC transplantation have yet to be addressed. Here, we investigated the induction of endogenous ENCCs by combining exogenous ENCC transplantation with a 5-HT4 receptor agonist mosapride in a rat model of hypoganglionosis, established by benzalkonium chloride treatment. ENCCs, isolated from the gut of newborn rats, were labeled with a lentiviral eGFP reporter. ENCCs and rats were treated with the 5-HT4 receptor agonist/antagonist. The labeled ENCCs were then transplanted into the muscular layer of benzalkonium chloride-treated colons. At given days post-intervention, colonic tissue samples were removed for histological analysis. ENCCs and neurons were detected by eGFP expression and immunoreactivity to p75NTR and peripherin, respectively. eGFP-positive ENCCs and neurons could survive and maintain levels of fluorescence after transplantation. With longer times post-intervention, the number of peripherin-positive cells gradually increased in all groups. Significantly more peripherin-positive cells were found following ENCCs plus mosapride treatment, compared with the other groups. These results show that exogenous ENCCs combined with the 5-HT4 receptor agonist effectively induced endogenous ENCCs proliferation and differentiation in a rat hypoganglionosis model.

Combination of basic fibroblast growth factor and epidermal growth factor enhances proliferation and neuronal/glial differential of postnatal human enteric neurosphere cells in vitro.

Human enteric neural stem cells (hENSCs) proliferate and differentiate into neurons and glial cells in response to a complex network of neurotrophic factors to form the enteric nervous system. The primary aim of this study was to determine the effect of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on in-vitro expansion and differentiation of postnatal hENSCs-containing enteric neurosphere cells. Enteric neurosphere cells were isolated from rectal polyp specimens of 75 children (age, 1-13 years) and conditioned with bFGF, EGF, bFGF+EGF, or plain culture media. Proliferation of enteric neurosphere cells was examined using the methyl thiazolyl tetrazolium colorimetric assay over 7 days of culture. Fetal bovine serum (10%) was added to induce the differentiation of parental enteric neurosphere cells, and differentiated offspring cells were immunophenotyped against p75 neutrophin receptor (neural stem cells), peripherin (neuronal cells), and glial fibrillary acidic protein (glial cells). Combining bFGF and EGF significantly improved the proliferation of enteric neurosphere cells compared with bFGF or EGF alone (both P<0.01) throughout 7 days of culture. The addition of bFGF drove a significantly greater proportion of enteric neurosphere cells to differentiate into neuronal cells than that of EGF (P<0.01), whereas addition of EGF resulted in significantly more glial differentiation compared with addition of bFGF (P<0.01). Combining bFGF and EGF drove enteric neurosphere cells to differentiate into neuronal cells in a proportion similar to glial cells. Our results showed that the combination of bFGF and EGF significantly enhanced the proliferation and differentiation of postnatal hENSCs-containing enteric neurosphere cells in vitro.

Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro.

A growing body of evidence supports the potential use of enteric neural crest-derived cells (ENCCs) as a cell replacement therapy for Hirschsprung's disease. Based on previous observations of robust propagation of primary ENCCs, as opposed to their progeny, it is suggested that their therapeutic potential after in vitro expansion may be restricted. We therefore examined the growth and differentiation activities and phenotypic characteristics of continuous ENCC cultures. ENCCs were isolated from the intestines of postnatal rats and were identified using an immunocytochemical approach. During continuous ENCC culture expansion, proliferation, migration, apoptosis, and differentiation potentials were monitored. The Cell Counting Kit-8 was used for assessment of ENCC vitality, Transwell inserts for cell migration, immunocytochemistry for cell counts and identification, and flow cytometry for apoptosis. Over six continuous generations, ENCC proliferation potency was reduced and with prolonged culture, the ratio of migratory ENCCs was decreased. The percentage of apoptosis showed an upward trend with prolonged intragenerational culture, but showed a downward trend with prolonged culture of combined generations. Furthermore, the percentage of peripherin(+) cells decreased whilst the percentage of GFAP(+) cells increased with age. The results demonstrated that alterations in ENCC growth characteristics occur with increased culture time, which may partially account for the poor results of proposed cell therapies.

Detection of autophagy in Hirschsprung's disease: implication for its role in aganglionosis.

Hirschsprung's disease (HD) is a common congenital gastrointestinal malformation, characterized by the lack of ganglion cells from the distal rectum to the proximal bowel, but the pathogenesis is not well understood. This paper evaluates the effects of autophagy in HD. Using electron microscopy, the autophagosomes were detected in three segments: narrow segment (NS), transitional segment (TS), and dilated segment (DS). Typical autophagosome structures are found in the Auerbach plexus of both NS and TS. Real-time PCR results showed that Beclin1 (NS vs. TS, P<0.01) and LC3 (NS vs. TS, P<0.05) mRNA were the highest in the NS, but p75 (NS vs. TS, P<0.01) was the highest in the DS. Correlation analysis results showed a positive correlation between Beclin1 and LC3 mRNA levels (R=0.736, P=0.000), whereas inverse correlations were found between p75 and Beclin1/LC3 mRNA levels (p75 vs. Beclin1: R=-0.714, P=0.000; p75 vs. LC3: R=-0.619, P=0.000). Immunohistochemistry analyses indicated a consistent result with mRNA levels, by increased Beclin1-positive and LC3-positive neurons, but reduced p75-positive neurons in the Auerbach plexus of TS compared with DS. These findings indicated that autophagy exists in the bowel of patients with HD. On the basis of the detection of the highest expression of the autophagy genes in NS, autophagy may additionally cause the lack of neurons.

Dramatic Response of Topical Doxycycline in Umbilical Granuloma: Report of 84 Cases.

We previously observed that topical doxycycline powder was effective in the treatment of umbilical granuloma. This study aims to evaluate the efficacy of this agent. The patients were randomly assigned into inpatient group and outpatient group. Doxycycline powder was administered topically once daily for 5 days. The protocol was restarted if no response observed. Eighty-four patients were included in this study. With one course of therapy, the overall cure rate was 82.14% (69/84), and the statistical difference in response rate was not significant between the 2 groups (P > .05). With 2 courses of therapy, the overall cure rate was 94.05% (79/84). No complication was observed during treatment. No recurrence was observed during follow-up. The treatment of umbilical granuloma using topical doxycycline is safe and efficacious. The administration of the agent can be performed conveniently by parents at home. This protocol could be considered to be the first treatment option for this disorder in neonates and infants.

Comparison of surgical stress between laparoscopic and open appendectomy in children.

PURPOSE: The present study aimed to evaluate laparoscopic appendectomy (LA) in comparison with conventional open appendectomy (OA) in children, with special emphasis on the extent of surgical trauma after LA and OA, and to assess whether LA had any clear advantages compared with conventional OA. METHODS: A total of 160 patients with a median age of 7.9 years (range 3-15 years) were studied. Sixty-nine of them underwent LA, and the remaining 91 underwent OA. Serum interleukin (IL) 6 and C-reactive protein (CRP) levels which are thought to play a pivotal role in the pathogenesis of surgical trauma and can also be used to monitor the magnitude of surgical trauma were measured using an enzyme-linked immunosorbent assay before surgery and 12 hours after surgery. In addition, we compared operating time, hospital stay, incidence of wound infection, and incidence of intra-abdominal infection. RESULTS: The operative time of normal and suppurative appendix in the laparoscopic group was significantly shorter than that in the open group, respectively, but the operative time of gangrenous appendix was not different between the laparoscopic group and open group. The hospital stay in the laparoscopic group was also significantly shorter than that in the open group. Postoperatively, 1 patient had port-site infection in the laparoscopic group, whereas 10 had wound infection in the open group; this difference was highly significant (chi2 = 4.19, P < .05). Three patients in the open group and 2 patients in the laparoscopic group had intra-abdominal infection, and the difference had no statistically significant difference (chi2 = 0.10, P < .05). Preoperative IL-6 levels were not different between the 2 groups, but the rise (preoperative vs postoperative) of IL-6 in the laparoscopic group was remarkably less than that in the open group. Similar results were obtained for CRP; serum CRP levels in the basal state were not different between the 2 groups, but the rise (preoperative vs postoperative) of CRP in the laparoscopic group was also substantially less compared with that in the open group. CONCLUSIONS: LA for children was as safe and effective as the open procedure and had significant advantages over OA because of less operating time, less postoperative complications, less surgical trauma, and more rapid postoperative recovery.

Role of urokinase plasminogen activator and its receptor in metastasis and invasion of neuroblastoma.

BACKGROUND/PURPOSE: Urokinase plasminogen activator (uPA) is a serine proteinase that has been suggested to play an important role in tumor invasion and metastasis. It binds to a specific membrane receptor, uPA receptor (uPAR), and activates plasminogen to form plasmin, which participates in tissue degradation and proteolysis. Binding of uPA to its receptor accelerates the activation of uPA from pro-uPA, enhancing the activity of the uPA/uPAR cascade. Because of the high metastatic and invasive potential of neuroblastoma (NB) cells, the authors have analyzed in the current study, the concomitant of uPA and its receptor in NB. METHODS: The expression and distribution of uPA and uPAR were analyzed by immunostaining in 52 neuroblastoma tissues; at the same time we use the reverse transcriptase polymerase chain reaction (RT-PCR) for neuroendocrine protein gene products 9.5 (PGP 9.5) mRNA to detect small numbers of NB cells in the peripheral blood and bone marrow (BM) and study the relationship uPA and uPAR to the ability of invasion and metastasis of NB cells. To identify risk factors for disease progression, the authors performed a retrospective analysis of clinical (age, sex, and risk group) and tumor biologic markers (histology, MYCN, DNA ploidy, chromosome 1 p, PGP9.5, uPA, uPAR, and combined uPA and uPAR) in all patients. Survival curves were estimated using the Kaplan-Meier method. Univariate analysis was performed with the log-rank test. Multivariate analysis was performed using the Cox proportional hazards regression model. RESULTS: The results of immunohistochemistry showed that uPA and uPAR were localized mainly in the membrane and cytoplasm of tumor cells. The positive rate of uPA in the high-risk group (23 of 25, 92.0%) was remarkably higher than that in intermediate-risk group (8 of 17, 47.1%) and low-risk group (3 of 10, 30.0%), in UH (26 of 29, 89.7%) was higher than in FH (8 of 23, 34.8%), respectively, and statistical significance was remarkable both P < .01). Similar results were obtained for uPAR. The positive rate of uPAR in the high-risk group (22 of 25, 88.0%) was substantially higher compared with that in intermediate-risk group (6 of 17, 35.3%) and low-risk group (2 of 10, 20.0%; P < .01). The positive rate of uPAR in UH (24 of 29, 82.8%) was higher compared with that in FH (6 of 23, 26.1%), and statistical significance was remarkable (P < .01). PGP9.5 mRNA in peripheral blood and BM was detected in 24 of 52 (45.2%) patients. The positive rate of PGP 9.5 mRNA in peripheral blood and BM in the cases positive for uPA (22 of 34, 64.7%) was markedly higher than that in the cases negative for uPA (11.1%, 2 of 18), and statistical significance was remarkable (P < .01). There was significant difference in the positive rate of PGP9.5 mRNA between the group positive for uPAR (66.7%, 20 of 30) and the group negative for uPAR (18.2%, 4 of 22), and a larger difference was found between the group positive for both uPA and uPAR (73.1%, 19 of 26) and the group negative for uPA or uPAR (19.2%, 5 of 26). The overall survival (OS) and event-free survival (EFS) rates at 5 years for all patients were, respectively, 70% +/- 3% and 63% +/- 3% with a median follow-up of 65 months (range 13 to 20). Among all the biologic and clinical features analyzed, multivariate analysis using Cox proportional hazards regression showed that age, MYCN, and combined uPA and uPAR remained significant predictors for both OS and EFS (P < .01, respectively). Both EFS rate and OS rate were significantly better for patients who positively expressed uPA and uPAR than those who negatively expressed uPA or uPAR. CONCLUSIONS: This study showed that uPA and uPAR were overexpressed in high-risk and UH tumor of NB, and that overexpression of both factors was associated with the ability of invasion, metastasis, and prognosis of NB. The presence of high levels of combined uPA and uPAR may be a new prognostic marker that would allow us to identify patients with poorer prognosis who might benefit from more aggressive surgical and adjuvant treatment.