Transcriptome Sequencing of Agave amaniensis Reveals Shoot-Related Expression Patterns of Expansin A Genes in Agave.

Agave species are widely planted for fiber production. However, the molecular basis of agave fiber development has not been well understood. In this study, we performed a transcriptomic analysis in A. amaniensi, a well-known variety with high-quality fiber production. Approximately 43.87 million clean reads were obtained using Illumina sequencing. The de novo assembly produced 66,746 unigrams, 54% of which were annotated in a public database. In the Nr database, 21,490 unigenes of A. amaniensis were shown to be most closely related to Asparagus officinalis. Nine expansin A orthologs with full coding regions were obtained, which were named EXP1a, EXP1b, EXP2, EXP3, EXP4a, EXP4b, EXP11, EXP12, and EXP13. The maximum likelihood phylogenetic tree revealed the species-specific expansion of expansin genes in Arabidopsis, rice and agave. The expression analysis suggested the negative correlation between the expression of expansin genes and the leaf growth rate, except AhEXP11. Moreover, expansin genes were differentially affected by abiotic and biotic stresses. Notably, AhEXP2 expression level was highly upgraded after the infection of Phytophthora nicotiana. Nutrient deficiency also influent expansin genes expression. Together, our research will benefit future studies related to fiber development, disease resistance and nutrient usage in agave.

Regulation of Shh/Bmp4 Signaling Pathway by DNA Methylation in Rectal Nervous System Development of Fetal Rats with Anorectal Malformation.

OBJECTIVE: To study the influence of gene methylation in the Shh/Bmp4 signaling pathway on the enteric nervous system in the rectum of rat embryos with anorectal malformations (ARMs). METHODS: Pregnant Sprague Dawley rats were divided into three groups; two groups treated with either ethylene thiourea (ETU induce ARM) or ETU+5-azacitidine (5-azaC inhibit DNA methylation) and a normal control group. The levels of DNA methyltransferases (DNMT1, DNMT3a, DNMT3b), the methylation status of the Shh gene promoter region and the expression of the key components were detected by PCR, immunohistochemistry and western blotting. RESULTS: The expression of DNMTs in the rectal tissue of the ETU and ETU+5-azaC groups was higher than that of the control. The expression of DNMT1, DNMT3a and methylation level of the Shh gene promoter in the ETU group was higher than in the ETU+5-azaC group (P < 0.01). The methylation level of the Shh gene promoter was higher in the ETU+5-azaC group than in the control. The Shh and Bmp4 expression in the ETU and ETU+5-azaC groups were lower than in the control, and their expression in the ETU group was also lower than in the ETU+5-azaC group. CONCLUSION: The methylation status of genes in the rectum of the ARM rat model may be changed by intervention. The low methylation level of the Shh gene may promote the expression of key Shh/Bmp4 signaling pathway components.

Clinical Characteristics and Management of Colorectal Vascular Malformation in Children: A Retrospective Study of 23 Cases.

INTRODUCTION: The objective of this study is to summarize the clinical characteristics and management of rare diseases of colorectal vascular malformation (CRVM) in children. METHODS: We retrospectively analyzed the clinical data of CRVM patients admitted to the Children's Hospital of Fudan University from 2004 to 2019. RESULTS: A total of 23 cases (16 males, 7 females) were enrolled. The median age of symptom onset was 1.4 years. Hematochezia and anemia were cardinal symptoms. Fourteen patients (60.9%) were misdiagnosed as anal fissures (n = 4), internal hemorrhoids (n = 3), rectal polyps (n = 2), inflammatory bowel disease (n = 2), portal hypertension (n = 2), and Meckel's diverticulum (n = 1), respectively. The average time from symptom onset to diagnosis was 4.5 ± 4.4 years. Other vascular malformations were detected in eight patients (34.8%). All patients showed a positive anomalous vascular image on contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI). The sensitivity of colonoscopy in the diagnosis of CRVM was 82.6% (19/23). A total of 21 patients underwent a modified Soave procedure. The lesions were mostly restricted to the colorectum and showed transmural diffuse distribution, with an average length of 20 ± 5.4 cm. Two patients (9.5%) experienced surgical complications. Bloody stools reappeared in two patients (9.5%), and colonoscopy showed abnormal angiogenesis at the anastomotic site, which were cured by sclerotherapy and/or electrocautery. The median follow-up time was 78 months. Bloody stools were absent at the last time of follow-up, and hemoglobin was in the normal range for all patients. CONCLUSION: The identification of CRVM in children often is delayed. Colonoscopy, CT, and MRI are essential in making the correct diagnosis. The modified Soave procedure is safe and feasible to treat CRVM in children. Endoscopic sclerotherapy and/or electrocautery can be used for residual lesions.

VEGFA rs3025039 and biliary atresia susceptibility in Chinese population: a systematic review and meta-analysis.

Background: Previous studies have suggested an association between vascular endothelial growth factor A (VEGFA) rs3025039 polymorphism and biliary atresia (BA). However, this conclusion is controversial and there is no published pooled evidence of this association. Methods: This study was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. The protocol was registered with PROSPERO (International Prospective Register of Systematic Reviews). A thorough search was performed on databases including PubMed, Embase, and Chinese Biomedical Database up to August 2020. This study included 846 cases of BA and 2821 controls concerning VEGFA rs3025039 polymorphism. We selected relevant studies based on the following inclusion criteria: (1) the study design was case-control and cohort and (2) the patients carried standard clinical diagnoses of BA, etc. The exclusion criteria were as follows: (1) patients with other related diseases, (2) lack of requisite information and (3) duplicate data. The OR (odd ratio) and the corresponding 95% CI (confidence interval) were calculated to estimate the association. Results: This study on VEGFA rs3025039 polymorphism in the Chinese population included 846 cases and 2821 controls. The results showed that there was no significant association between rs3025039 and susceptibility to BA under four genetic models. The results of the subgroup analysis were similar to the overall results. Conclusions: This meta-analysis shows that rs3025039 was not associated with susceptibility to BA in the Chinese population. Further validation may entail additional research. PROSPERO registration number: CRD42020203812.

Aberrant high expression of the TET1 gene in Hirschsprung's disease.

BACKGROUND: The pathogenesis of Hirschsprung's disease (HSCR) remains unclear but might involve genes participating in neural crest development. Gene methylation controls the expression of many genes and is involved in the development and migration of neural crest cells, but the involvement of demethylation in HSCR is unknown. This study aimed to investigate the expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) (a demethylation protein) in patients with HSCR. METHODS: This is a retrospective study of surgical specimens from paediatric patients with and without HSCR (e.g., intussusception and incarcerated hernia) obtained from 07/2015 to 08/2017. TET1 expression was determined by qRT-PCR, western blotting, and immunohistochemistry. The levels of 5-hydroxymethylcytosine were determined by the dot blot assay. RESULTS: The specimens of 35 patients with HSCR and 25 controls were collected. The median TET1 mRNA expression values were 1.028 [HSCR-stenotic (S)], 0.908 [HSCR-dilated (D)], and 0.467 (control) (HSCR-S vs. control: P = 0.002; HSCR-D vs. control: P = 0.008; HSCR-S vs. HSCR-D: P = 0.44). TET1 protein levels followed a similar pattern. The intensity of immunostaining identified higher expression of TET1 in HSCR colon tissues compared with control tissues. The 5-hmC levels in HSCR stenotic segment samples were significantly higher than those in controls. CONCLUSION: The expression of TET1 is higher in paediatric patients with HSCR than in controls. DNA demethylation initiated by TET1 may be related to HSCR, which demonstrates that TET1 may play a role in the development of HSCR.

Characteristics of the Gut Microbiome and IL-13/TGF-ß1 Mediated Fibrosis in Post-Kasai Cholangitis of Biliary Atresia.

Aims: Cholangitis in biliary atresia (BA), which accelerates liver fibrosis progression, is among the most common serious complications after Kasai surgery; however, its etiology remains elusive. Gut microbiome migration may contribute to post-Kasai cholangitis. Further, there is no appropriate model of BA post-Kasai cholangitis for use in investigation of its pathogenesis. Methods: We explored the characteristics of gut microbiome in patients with BA before and after Kasai procedure based on 16S rDNA sequencing. We isolated the dominant strain from patient stool samples and established an in vitro model by infecting patient-derived liver organoids. Bulk RNA-seq was performed, and we conducted qPCR, ELISA, and western blot to explore the mechanism of fibrosis. Results: Gut microbiome diversity was lower in patients after, relative to before, Kasai procedure, while the relative abundance of Klebsiella was higher. Patients who developed cholangitis within 1 month after discharge tended to have simpler gut microbiome composition, dominated by Klebsiella. Klebsiella pneumoniae (KPN) was isolated and used for modeling. RNA-seq showed that BA liver organoids expressed markers of hepatic progenitor cells (KRT19, KRT7, EPCAM, etc.) and that organoids were more stable and less heterogeneous among individuals than liver tissues. After infection with KPN, gene expression patterns in BA liver organoids were enriched in pathways related to infection, apoptosis, and fibrosis. Preliminary experiments indicated the presence of IL-13/TGF-ß1-mediated fibrosis in post-Kasai cholangitis. Conclusions: Our findings using a newly-developed model, demonstrate a key role for Klebsiella, and a potential mechanism underlying fibrosis in post-Kasai cholangitis, mediated by the IL-13/TGF-ß1 pathway.

Skip segment Hirschsprung's disease: report of two rare cases and management.

PURPOSE: Skip segment Hirschsprung's disease (SS-HSCR) is defined as the occurrence of a segment of ganglionated intestine surrounded proximally and distally by aganglionosis. The presence of the skip intestinal segment often leads to clinical misdiagnosis, missed diagnosis or inadequate resection of the lesions. The purpose was to describe two new cases of SS-HSCR with the aim of proposing questions regarding the diagnosis and treatment of this rare disease. METHODS: We reported two cases of infants with SS-HSCR that were admitted to our institution within the last 3 years. RESULTS: One patient had a skip segment of ganglionated intestine in the ascending colon. In the other patient, there were no ganglionic cells in the rectum and appendix, but ganglionic cells were visible in the proximal ascending colon. The entire colons in the both cases were finally resected, and a pull-through operation was performed. CONCLUSION: Multipoint biopsy should be performed when the biopsy results are inconsistent with clinical manifestations. Intraoperative laparoscopic identification of the transition zone may be necessary when TCA is suspected. Multisegment biopsy is needed to as a preventative measure for SS-HSCR if TCA is indicated during surgery. Further study is required to determine the optimal length and method of retention of segments.

Cost-Effectiveness Analysis of Olaparib Maintenance Treatment for Germline BRCA-Mutated Metastatic Pancreatic Cancer.

Background: The PARP inhibitor olaparib has been shown to have clinical efficacy in patients with a germline BRCA mutation and ovarian or breast cancer. However, the high treatment cost associated with this drug limits its viability as a clinical treatment option. This work aims to evaluate the cost-effectiveness of olaparib as a maintenance treatment for metastatic pancreatic cancer from the perspective of the United States and China healthcare systems and provides valuable suggestions for clinical decision making. Method: A three-state Markov model (progression-free, progressed disease, death) was constructed using TreeAge Pro 2020 software to evaluate the economic value of olaparib vs. placebo maintenance treatment for metastatic pancreatic cancer based on the clinical data derived from phase III randomized controlled trial (POLO, ClinicalTrials.gov number, NCT02184195). Total costs, quality-adjusted life years and incremental cost-effectiveness ratio were used as economic indicators for this analysis. A 5-years horizon and 5%/year discount rates were used. One-way sensitivity analysis and probabilistic sensitivity analysis (PSA) were performed to assess the model uncertainty. Results: The incremental cost-effectiveness ratios (ICERs) of the use of olaparib vs. placebo in China and the United States were $6,694/QALY and $13327/QALY, respectively. All ICERs were far below the thresholds of $30829 in China and $50000 in the United States. Sensitivity analysis confirmed a stable economic advantage in the use of olaparib vs. placebo as maintenance therapy in China and the United States. Conclusion: Olaparib was estimated to be more cost effective than placebo for the maintenance therapy of patients with a germline BRCA mutation and pancreatic cancer in China and the United States at thresholds of $30829 and $50000 per QALY, respectively.

Preventive effect of prophylactic intravenous antibiotics against cholangitis in biliary atresia: a randomized controlled trial.

OBJECTIVE: Biliary atresia (BA) is a neonatal liver disease and requires Kasai portoenterostomy. Many patients develop postoperative cholangitis, resulting in a poor prognosis. The preventive strategy of antibiotics is empirical and lacks a standard regimen. We aimed to analyze the effect of different durations of prophylactic intravenous antibiotics against post-Kasai cholangitis. STUDY DESIGN: A single-center, open-labeled, randomized clinical trial was performed from June 2016 to August 2017. One hundred and eighty BA patients were recruited and randomized into a short-term (n = 90) and a long-term (n = 90) treatment group, and prophylactic intravenous antibiotics were used for 7 versus 14 days, respectively. The primary outcome was the overall cholangitis incidence within 6-months post-Kasai portoenterostomy. The secondary outcomes included cholangitis incidence within 1 and 3 months post-Kasai portoenterostomy, the onset and average episodes of cholangitis, jaundice clearance rate, native liver survival rate, and adverse events within 6-months post-Kasai portoenterostomy. RESULTS: The cholangitis incidence within 6-months post-Kasai in the short-term group was similar to the long-term group (62% vs. 70%, p = 0.27) with intention-to-treat and pre-protocol analysis. There was no significant difference in jaundice clearance rate or native liver survival rate between the two groups. However, the percentage of early onset (61% vs. 38%, p = 0.02) and average episodes (2.4 ± 0.2 vs. 1.8 ± 0.1 episodes, p = 0.01) of cholangitis were lower in the long-term group. CONCLUSION: Long-term intravenous antibiotics can be replaced by the short-term regimen in the general protection against post-Kasai cholangitis.

Serum MMP-7 in the Diagnosis of Biliary Atresia.

BACKGROUND: The overlapping features of biliary atresia (BA) and other neonatal cholestasis with alternative causes (non-BA) have posed challenges for diagnosis. Matrix metalloproteinase-7 (MMP-7) has been reported to be promising in diagnosing BA. We aimed to validate the diagnostic accuracy of MMP-7 for BA in a large population sample. METHODS: We enrolled 288 patients with neonatal obstructive jaundice from March 2017 to October 2018. Serum MMP-7 levels were measured by using an enzyme-linked immunosorbent assay. Receiver operating characteristic curves were constructed, and decision curve analysis was done. A Pearson correlation coefficient test was conducted to assess the correlation between MMP-7 levels and other characteristics. RESULTS: The median serum MMP-7 levels were 38.89 ng/mL (interquartile range: 22.96-56.46) for the BA group and 4.4 ng/mL (interquartile range: 2.73-6.56) for the non-BA group (P < .001). The area under the receiver operating characteristic curve value was 0.9829 for MMP-7, and the sensitivity, specificity, positive predictive value, and negative predictive value were 95.19%, 93.07%, 97.27%, and 91.43%, respectively, at a cutoff value of 10.37 ng/mL. When MMP-7 was combined with γ glutamyl transferase, the diagnostic accuracy was slightly improved without significance when compared with MMP-7 alone and had an area under the curve of 0.9880 (P = .08). Decision curve analysis also showed potential for MMP-7 to be used for clinical applications. A significant correlation was found with fibrosis stage from liver biopsy (R = 0.47; P < .001). CONCLUSIONS: MMP-7 demonstrated good accuracy in diagnosing BA and holds promise for future clinical application. Furthermore, its correlation with liver fibrosis indicated its potential use as a therapeutic target or prognostic biomarker.

Intrahepatic cystic lesions in children with biliary atresia after Kasai procedure.

BACKGROUND: Intrahepatic cystic lesion (ICL) is a common complication for biliary atresia post-Kasai portoenterostomy. The purpose of this study was to review the cases in our hospital and assess the correlation between characteristics of ICL and clinical outcomes. METHODS: We retrospectively analyzed 787 cases of biliary atresia from 2012 to 2016. Demographics, clinical details, and postoperative outcomes were reviewed. RESULTS: A total of 76 patients were diagnosed with ICLs using ultrasound post-Kasai procedure, and the incidence was 9.7%. Preoperative characteristics showed no significant differences between ICL (+) and ICL (-) groups. Nearly 70% (53/76) of the patients with ICLs had a history of cholangitis. The 2-year native liver survival rate was 60.4% for those with a history of cholangitis and 87% for those without (P = 0.017). Further analysis showed that the 2-year native liver survival rate was 42.9% for those diagnosed within 3 months post-Kasai procedure, 54.2% for those diagnosed between 3 and 6 months, and 80.0% for those diagnosed beyond 6 months (P = 0.002), while no significance was observed for type (P = 0.094) or site (P = 0.406) of ICL. CONCLUSION: Patients with ICLs had a high incidence of cholangitis. The prognosis was closely related with the history of cholangitis and the onset time of ICLs. LEVEL OF EVIDENCE: Level II.

MicroRNA-29b/142-5p contribute to the pathogenesis of biliary atresia by regulating the IFN-γ gene.

Biliary atresia is one of the most common liver disease in infancy. The cause and pathogenesis remain largely unknown. This study aimed to investigate the potential regulatory effect of miR-29b/142-5p on IFN-γ gene methylation. miRNAs microarray was performed on four pairs of liver and blood specimens from biliary atresia and choledochal cysts. We found the overexpression of miR-142-5p and mRNA level of DNA methyltransferase (DNMT) 1, and miR-29b and DNMT3a/DNMT3b were significantly negatively correlated in biliary atresia livers. Meanwhile, the methylation of the LINE-1, ALU and SAT2 repetitive sequences and the IFN-γ promoter was lower, but the expression of IFN-γ was upregulated. After transfected with DNMTs siRNAs, downregulation of DNMTs exerted a significant hypomethylating effect on the repetitive sequences, which led to upregulation of IFN-γ in Jurkat cells. The direct interactions between miR-29b and DNMT3a/3b, and miR-142-5p and DNMT1 were identified using luciferase reporter assays. By transfecting mimics of miR-29b/142-5p into Jurkat cells, we found overexpression of miR-29b/142-5p markedly suppressed expression of DNMTs. Furthermore, the methylation of repetitive sequences and the IFN-γ promoter region were remarkably downregulated, and with elevated IFN-γ expression. After transfecting the miRNA inhibitors, the levels of DNMTs and the methylation of the IFN-γ gene promoter region was upregulated, while levels of IFN-γ were markedly suppressed. Our study suggested that miRNA-29b/142-5p overexpression and targeted inhibition of DNMTs expression resulted in decreased overall gene methylation and overexpression of the methylation-sensitive IFN-γ gene.

Forkhead box A3 attenuated the progression of fibrosis in a rat model of biliary atresia.

Biliary atresia is a rare, devastating disease of infants where a fibroinflammatory process destroys the bile ducts, leading to fibrosis and biliary cirrhosis, and death if untreated. The cause and pathogenesis remain largely unknown. We tried to investigate factors involved in biliary atresia, especially forkhead box A3 (Foxa3), which might exert a role in the treatment of liver disease. We used RNA sequencing to sequence the whole transcriptomes of livers from six biliary atresia and six choledochal cysts patients. Then, we employed a rat disease model by bile duct ligation (BDL) and adenovirus transduction to address the function of Foxa3 in biliary atresia. We found that tight junction, adherence junction, cell cycle, apoptosis, chemokine singling, VEGF and MAPK signaling pathways were enriched in biliary atresia livers. We showed that Foxa3 expression was notably decreased in liver samples from biliary atresia patients. More importantly, we found that its lower expression predicted a poorer overall survival of biliary atresia patients. Rats that received BDL surgery and Foxa3 expression adenovirus resulted in a significant decrease in the deposition of collagen, and expression of profibrotic cytokines (transforming growth factor-ß and connective tissue growth factor) and fibrosis markers (α-smooth muscle actin, collagen I and collagen III), as compared with rats that received BDL surgery and control adenovirus. Our data suggested a protection role for Foxa3 during the progression of liver fibrosis in biliary atresia, and thereby supported increasing Foxa3 as a targeted treatment strategy.

Outcomes of thoracoscopy versus thoracotomy for esophageal atresia with tracheoesophageal fistula repair: A PRISMA-compliant systematic review and meta-analysis.

BACKGROUND: A thoracoscopic approach for repair of esophageal atresia (EA) with tracheoesophageal fistula (TEF) has become a standard procedure in many pediatric surgical centers. However, whether thoracotomy or thoracoscopy offer advantages in terms of surgical outcomes is not known. METHODS: To evaluate the efficacy and safety of thoracoscopic repair (TR) versus conventional open repair (COR) for EA with TEF.PubMed, Cochrane Library, and EMBASE were searched to identify relevant literature until 2016.Studies comparing surgical outcomes of patients undergoing TR versus COR for EA with TEF were reviewed.The quality of each included study was assessed using the Newcastle-Ottawa scale score. A fixed or random-effect model was applied depending on heterogeneity tests. RESULTS: Eight observational clinical studies involving 452 patients were included in this meta-analysis. The meta-analysis of 2 major postoperative complications (leaks and strictures) did not show significant differences between TR and COR. Overall estimates of the odds ratio (OR) of TR versus COR for leaks and strictures were: 1.57 (95% confidence interval [CI], 0.77-3.20; P = 0.22) and 0.90 (95% CI, 0.27-2.97; P = 0.86), respectively. However, meta-analysis of operation time (OR = 19.59, 95% CI = 0.77-38.40, P = 0.04), timing of extubation (OR = -2.50, 95% CI = -3.39 to -1.62, P < 0.001), time to 1st oral feeding (OR = -2.58, 95% CI = -3.79 to -1.36, P < 0.001), and duration of hospital stay (OR = -10.76, 95% CI = -16.39 to -5.12, P < 0.001) showed significant differences.No randomized controlled trial was included, and most studies had small sample sizes and were based on retrospective analysis. CONCLUSION: TR and COR show a similar complication rates of leaks and strictures for EA/TEF repair. Although associated with a longer operative time, TR has the advantages of an earlier time to extubation and 1st oral feeding, and shorter hospital stay.

Hypermethylation of SHH in the pathogenesis of congenital anorectal malformations.

OBJECTIVE: This study sought to examine promoter methylation and expression of the identified sonic hedgehog (SHH) gene in terminal rectal tissues of children with congenital anorectal malformations (ARMs). METHODS: Tissue samples from the terminal rectum of pediatric patients with ARMs (five cases each of high and intermediate malformation - two cases of rectovesical fistula, two cases of rectourethral prostatic fistula, one case of cloaca with >3cm common channel, four cases of rectourethral bulbar fistula and one case of imperforate anus without fistula, respectively, and ten cases of low malformation - five cases of perineal fistula and five cases of vestibular fistula, respectively), and patients with non-gastrointestinal tract malformation (six cases, anal fistula) were collected and divided into three groups: high-intermediate ARM (ARMhi-int), low ARM (ARMlo), and control (Cont.). Real-time RT-PCR was used to detect mRNA expression levels of the verified differentially methylated gene SHH, and bisulfite genomic sequencing was performed to evaluate DNA methylation in the SHH promoter region. RESULTS: The average methylation levels of the SHH promoter were significantly higher in ARMhi-int (0.850±0.030, P=0.0036) and ARMlo (0.540±0.053, P=0.0087) groups than in Cont. group (0.280±0.032). SHH mRNA expression levels were lower in ARMhi-int (0.340±0.015, P=0.0065) and ARMlo (0.530±0.042, P=0.0156) groups than in Cont. group (0.870±0.046). The average methylation levels of the SHH promoter were higher in ARMhi-int group than in ARMlo group (0.850±0.030 vs. 0.540±0.053, P=0.0095), while SHH expression was significantly reduced in ARMhi-int group compared to ARMlo group (0.340±0.15 vs. 0.530±0.042, P=0.0252). The methylation levels of the SHH promoter in ARMhi-int group were negatively correlated with SHH gene expression (r=-0.89, P<0.01). CONCLUSIONS: The SHH gene, which plays a major role in the development of the anorectum and enteric nervous system, is hypermethylated at its promoter, and this is correlated with low levels of SHH gene expression. This epigenetic modification may therefore be responsible for the observed changes in SHH expression, which could in turn underlie the pathogenesis of congenital ARMs.

Etiologic and clinical analysis of chronic complex anal and rectal inflammation in children less than 3 years old.

OBJECTIVE: To analyze the etiology and clinical diagnostic method for chronic complex anal and rectal inflammation in children less than 3 years old. METHOD: Seven children (5 males and 2 females; 1 year 8 months to 3 years of age at the time of physician evaluation) with chronic complex anal and rectal inflammation were enrolled between May 2008 and May 2013 at our hospital. Clinical history, results of auxiliary examinations, and empirical treatment of the children were analyzed retrospectively combined with the etiologic diagnosis. RESULTS: Four patients were confirmed to have Crohn's disease and one patient was confirmed to have intestinal tuberculosis; two patients were suspected to have Crohn's disease. Anemia and low pre-albumin level were common (seven patients); serologic testing revealed four patients with elevated IgG levels and seven patients with elevated IgA levels; there were no patients with positive tuberculosis antibody titers and two patients were weakly positive for C-ANCA (one patient with Crohn's disease and one patient intestinal tuberculosis). Colonoscopies revealed that the entire colon was affected in one patient, the left hemicolon was affected in four patients, and the sigmoid colon and rectum were affected in two patients. Two patients with Crohn's disease and one patient with intestinal tuberculosis were diagnosed by colonoscopies in combination with histopathologic examinations. Two patients with Crohn's disease were confirmed after empirical drug treatment, and two other patients were not definitely diagnosed. CONCLUSION: The possibility of Crohn's disease or intestinal tuberculosis should be considered in the clinical diagnosis of complex chronic anal and rectal inflammation in younger children. Local surgery is sometimes unnecessary. Empirical drug treatment should be used if necessary.

Identification of HSP90 as potential biomarker of biliary atresia using two-dimensional electrophoresis and mass spectrometry.

Biliary atresia (BA) is a devastating cholestatic liver disease targeting infants. Current diagnosis depends on surgical exploration of the biliary tree. The aim of the present study was to identify potential biomarkers for the diagnosis of biliary atresia (BA). Two-dimensional electrophoresis was utilized for the identification of proteins that were differentially expressed in liver biopsies of 20 BA patients and 12 infants with non-BA neonatal cholestasis (NC) as controls. Using mass spectrometry, we identified 15 proteins with expressions significantly altered. Out of the 15 proteins identified, heat shock protein (HSP) 90 was the most significantly altered and was down-regulated in BA samples compared to NC samples using immunoblotting analysis. Our findings suggest that HSP90 might be a potential biomarker for the diagnosis of BA and may be used for monitoring further development and therapy for BA. This study demonstrated that a comprehensive strategy of proteomic identification combined with further validation should be adopted in biomarker discovery.

Effect of exogenous glial cell-derived neurotrophic factor on development of the enteric nervous system in the rectal end of fetal rats with anorectal malformations.

OBJECTIVE: The objective of this study is to analyze the effect of exogenous glial cell-derived neurotrophic factor (GDNF) in the development of the enteric nervous system (ENS) in the rectal end of fetal rats. MATERIALS AND METHODS: Thirty pregnant Sprague-Dawley rats were categorized randomly into three groups: ethylene thiourea (ETU), ETU + GDNF, and control. On day 10 of gestation, ETU was injected via a gastric tube in the ETU group and ETU + GDNF group. On day 11 of gestation, GDNF was administered through the tail vein in the ETU + GDNF group. On day 20 of gestation, fetal rats were harvested by cesarean section. The prevalence of anorectal malformations (ARMs) in the fetal rats was observed. GDNF expression in the rectal end of fetal rats was detected by immunohistochemical and Western blotting analyses. RESULTS: The prevalence of ARMs in the ETU group and ETU + GDNF group was 51.4 and 52.5 %, respectively, but the difference between the two groups was not significant (P > 0.05). In the rectal end of fetal rats with an anus, GDNF expressions in the three groups were not significantly different (P > 0.05). In the rectal end of fetal rats without an anus: GDNF expression in the ETU + GDNF group was significantly higher than that in the ETU group (P = 0.036); GDNF expression in the rectal end of fetal rats without an anus from the ETU group and ETU + GDNF group was significantly lower than that of fetal rats with an anus (ETU group P = 0.001; ETU + GDNF group P = 0.028). There was a significant difference in the gray level ratio of GDNF and actin between the ETU group and ETU + GDNF group (P < 0.0001), and the expression in the ETU + GDNF group was significantly up-regulated. CONCLUSION: GDNF could not totally prevent the occurrence of ETU-induced ARMs, but it up-regulated expression of the GDNF gene in the wall of the rectal end, thereby promoting the growth of a hypogenetic ENS.

The effect of vitamin A deficiency during pregnancy on anorectal malformations.

OBJECTIVE: The aim of this study was to study the effect of vitamin A deficiency (VAD) on the embryological development of anorectal malformations (ARMs) and the enteric nervous system. MATERIALS AND METHODS: Female Sprague-Dawley rats were divided into 3 groups: VAD group, normal group (negative control), and ethylene thiourea (ETU) group (positive control) with a normal diet. On day 20 of pregnancy, cesarean section was performed on all rats. The incidence of ARMs in the fetal rats and Protein gene product 9.5 (PGP9.5) and S-100 protein expression by immunohistochemistry were determined. RESULTS: The incidence of ARMs in VAD and ETU groups was 64.8% (59/91) and 45.9% (61/133), respectively (P > .05). Anorectal malformations were not found in the normal group. Protein gene product 9.5 and S-100 protein expression in the non-ARM rectums of the VAD group was lower than the ETU (P = .0156 vs P = .0105) and normal groups (P = .0091 vs P = .0024). There was no significant difference in PGP9.5 and S-100 protein expression between ETU and normal groups. In the ARM rectums, PGP9.5 and S-100 protein expression in the VAD group was lower than the ETU group (P < .0001). Protein gene product 9.5 and S-100 protein expression was also lower in ARM than non-ARM rectums in the VAD and ETU groups (P < .0001, P = .0203, and P = .0122, respectively). CONCLUSION: Vitamin A deficiency during pregnancy may result in the embryological development of ARMs. Enteric nervous system development may be related to ARMs.

Preliminary evaluation of anorectal manometry in diagnosing Hirschsprung's disease in neonates.

PURPOSE: The aim of this paper was to assess the clinical value of anorectal manometry (ARMM) in the diagnosing of Hirschsprung's disease (HD) in neonates. METHODS: From January 2003 to June 2005, 75 patients in whom HD was clinically suspected were analyzed. ARMM was performed using a desk, high rate gastrointestinal dynamic detection system and the results were compared with barium enema and rectal suction biopsy. RESULTS: Based on rectal suction biopsies in 52 of 75 patients, the positive, false positive, negative, and false negative rates of ARMM in the diagnosis of HD in neonates were found to be 92.3, 1.9, 1.9, and 3.8%, respectively. Forty-three of 75 patients were diagnosed with HD by both ARMM and barium enema and the diagnoses were validated by pathologic results. The diagnosis of HD was excluded in 18 patients in whom HD was clinically suspected, but in whom the results of ARMM and barium enema were normal. Twelve patients who had ARMM results consistent with HD and a negative barium enema, had serial ARMM performed; a rectoanal inhibitory reflex (RAIR) was elicited in four patients, thereby excluding HD and the remaining eight patients were diagnosed with HD by review of barium enema and pathologic results. One of two patients with a positive barium enema for HD, but an ARMM showing the presence of RAIR was excluded by pathologic results and the other patient was lost to follow-up. The diagnostic accuracies of ARMM and barium enema for HD in neonates were 93.3 and 86.7%, respectively. There was no difference in rectal resting pressure and anal rhythmic wave frequency between neonates with HD and healthy neonates, but neonates with HD had higher anal sphincter pressures than healthy neonates (P=0.0074). CONCLUSIONS: ARMM is a simple, safe, and non-invasive method with high specificity for the diagnosis of HD in neonates.