The relationship between genetic polymorphism of CYP2C19 and the efficacy of Helicobacter pylori eradication therapy in children / 中华儿科杂志

Objective: To investigate the relationship between genetic polymorphisms of cytochrome P450 2C19 (CYP2C19) and the efficacy of Helicobacter pylori (Hp) eradication therapy in children. Methods: The retrospective cohort study was conducted on 125 children with gastroscopy and positive rapid urease test (RUT) from September 2016 to December 2018 who presented to the Children's Hospital of Zhejiang University School of Medicine due to gastrointestinal symptoms including nausea, vomiting, abdominal pain, bloating, acid reflux, heartburn, chest pain, vomiting blood and melena. Hp culture and drug susceptibility test were carried out with gastric antrum mucosa before treatment. All the patients completed 2 weeks of standardized Hp eradication therapy and had 13C urea breath test 1 month after that, which was used to evaluate the curative effect. The DNA of gastric mucosa after RUT was analyzed and CYP2C19 gene polymorphism was detected. Children were grouped according to metabolic type. Combined with the results of Hp culture and drug susceptibility, the relationship between CYP2C19 gene polymorphism and the efficacy of Hp eradicative treatment was analyzed in children. Chi square test was used for row and column variables, and Fisher exact test was used for comparison between groups. Results: One hundred and twenty five children were enrolled in the study, of whom 76 were males and 49 females. The genetic polymorphism of CYP2C19 in these children found poor metabolizer (PM) of 30.4% (38/125), intermediate metabolizer (IM) of 20.8% (26/125), normal metabolizer (NM) of 47.2% (59/125), rapid metabolizer (RM) of 1.6% (2/125), and ultrarapid metabolizer (UM) of 0. There were statistically significant in positive rate of Hp culture among these groups (χ2=124.00, P<0.001). In addition, the successful rates of Hp eradication in PM, IM, NM and RM genotypes were 84.2% (32/38), 53.8% (14/26), 67.8% (40/59), and 0, respectively, with significant differences (χ2=11.35, P=0.010); those in IM genotype was significantly lower than that in PM genotype (P=0.011). With the same standard triple Hp eradicative regimen, the successful rate of Hp eradication for IM type was 8/19, which was lower than that of PM (80.0%, 24/30) and NM type (77.3%, 34/44) (P=0.007 and 0.007, respectively). There was a significant difference in the efficacy of Hp eradication treatment among different genotypes (χ2=9.72, P=0.008). According to the clarithromycin susceptibility result, the successful rate of Hp eradication treatment for IM genotype was 4/15 in the sensitive group and 4/4 in the drug-resistant group (χ2=6.97, P=0.018). Conclusions: The genetic polymorphism of CYP2C19 in children is closely related to the efficacy of Hp eradication treatment. PM has a higher successful rate of eradication treatment than the other genotypes.

Age distribution characteristics of intestinal segmented filamentous bacteria and their relationship with intestinal mucosal immunity in children / 中国当代儿科杂志

OBJECTIVE@#To investigate the age distribution characteristics of intestinal segmented filamentous bacteria (SFB) in children and their relationship with intestinal mucosal immunity.@*METHODS@#The fresh feces of 177 children and the ileocecal fluid of 47 children during colonoscopy were collected. The SFB was determined by real-time PCR. The concentration of secretory immunoglobulin A (sIgA) was determined by enzyme-linked immunosorbent assay. The numbers of interleukin 17A (IL-17A) cells and intraepithelial lymphocytes in the terminal ileum mucosa and the expression of transcription factors associated with the differentiation of T helper (Th) cells, T-box transcription factor (T-bet), forkhead box P3 (FOXP3), and retinoid-related orphan receptor gamma t (ROR-γt), were determined by immunohistochemistry.@*RESULTS@#The positive rate of intestinal SFB in these children was 19.2% (34/177). Trend analysis showed that the positive rate of SFB was correlated with age: the rates for children aged 0-, 1-, 2-, 3-, 4-, 5-, 6-, and 7-15 years were 40%, 47%, 32%, 15%, 12%, 13%, 15% and 4% respectively (P<0.001). The concentration of sIgA in intestinal fluid was significantly higher in SFB-positive children (n=24) than in SFB-negative children (n=23) (P<0.01). The number of intraepithelial lymphocytes in the terminal ileum mucosa and the expression of T-bet, FOXP3, and ROR-γt were not significantly different between the SFB-positive group (n=12) and the SFB-negative group (n=11), but the number of IL-17A cells in the terminal ileum mucosa was significantly lower in the SFB-positive group than in the SFB-negative group (P<0.05).@*CONCLUSIONS@#Intestinal SFB colonization in children is age-related, and the colonization rate is relatively high in children under 3 years old. In SFB-positive children, the secretion of intestinal sIgA is increased, while the number of IL-17A cells in the terminal ileum is reduced.

Association between vitamin D receptor gene polymorphisms and pediatric Crohn's disease in China: a study based on gene sequencing / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the association between TaqI, BsmI, and ApaI polymorphisms of vitamin D receptor (VDR) gene and pediatric Crohn's disease (CD) in China.</p><p><b>METHODS</b>Nineteen children with CD were selected as a case group, and 122 healthy children who underwent physical examination were selected as a control group. Serum 25-hydroxyvitamin D3 [25(OH)D3] levels were measured using ELISA. The TaqI, BsmI, and ApaI polymorphisms of VDR gene were determined by gene sequencing, and the two groups were compared in terms of genotype and allele frequencies.</p><p><b>RESULTS</b>The case group had significantly lower serum 25(OH)D3 levels than the control group (17.3±2.4 ng/mL vs 26.9±2.1 ng/mL; P<0.05). There were no significant differences in the frequencies of genotypes and alleles of TaqI, BsmI, and ApaI polymorphisms between the case and control groups (P>0.05).</p><p><b>CONCLUSIONS</b>Children with CD have low serum 25(OH)D3 levels. TaqI, BsmI, and ApaI polymorphisms of VDR gene may not be associated with susceptibility to CD among the Chinese population.</p>

Changes in tight junction protein expression and permeability of colon mucosa in rats with dextran sulfate sodium-induced inflammatory bowel disease / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To develop an experimental rat model of inflammatory bowel disease (IBD) by administration of dextran sulfate sodium (DSS), and to observe changes in the tight junction protein expression and permeability of colon mucosa.</p><p><b>METHODS</b>Male Sprague-Dawley (SD) rats were randomly divided into control (n=27) and IBD model groups (n=27). In the IBD model group, IBD was induced by 6-day administration of 3% DSS in water followed by 14-day administration of water only. The control group was fed with water only. Pathological changes in colon mucosae were observed on days 7, 14 and 21 after DSS administration. Colon tissue specimens were collected on day 21 for measuring myeloperoxidase (MPO) activity. The transepithelial electric resistance (TEER), transepithelial potential difference (TEPD) and short circuit current (Isc) of the specimens were measured by Ussing chamber. Real-time PCR and Western blot were used to measure the mRNA and protein expression of tight junction proteins in colon epithelia.</p><p><b>RESULTS</b>In the IBD model group, diarrhea, hemafecia and weight loss were seen. Inflammation occurred mainly in the distal colon and was characterized by crypt abscess and inflammatory cell infiltration. The IBD model group showed significantly increased MPO activity (P<0.01), significantly decreased TEER (P<0.01) and TEPD (P<0.01), and significantly increased Isc (P<0.01) compared with the control group. No claudin 2 expression of mRNA and protein was detected in the control group, and they were expressed in the IBD model group. The expression levels of claudin 3, occludin and ZO-1 in the IBD model group were significantly decreased compared with in the control group (P<0.01).</p><p><b>CONCLUSIONS</b>IBD rats show colonic barrier dysfunction and changes in the expression of tight junction proteins. The changes in the expression of tight junction proteins may contribute to colonic barrier dysfunction in cases of IBD in the chronic recovery stage.</p>

Value of protein array in the diagnosis of Helicobactor pylori infection in children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the value of multiple Helicobacter pylori (H.pylori) antibody detection by protein array in the diagnosis of H.pylori infection in children.</p><p><b>METHODS</b>Biopsy specimens obtained by gastroscopy from 120 children with digestive system symptoms were detected by rapid urease test (RUT) and modified Giemsa staining. Positivity in both RUT and Giemsa staining was the "gold criterion" of H.pylori infection. Serum samples of these patients were obtained and the antibodies against cytotoxin associated gene A protein (CagA), vacuolating toxin A (VacA), urease, heat shock protein 60 (Hsp60) and RdxA (nitroreductase) were detected by protein array technique.</p><p><b>RESULTS</b>H.pylori infection was identified according to the "gold criterion" in 60 children. Compared with the "gold criterion", the goodness of fit and the coefficient of contingency in the diagnosis of H.pylori infection of the following four groups antibody detection were all statistically significant (P<0.001): anti-Ure antibody alone, anti-Ure antibody combined with anti-CagA antibody, anti-Ure antibody combined with anti-VacA antibody and anti-Ure antibody combined with anti-CagA and anti-VacA antibody. The sensitivity, specificity and accuracy of the detection of anti-Ure antibody combined with anti-CagA antibody for the diagnosis of H.pylori infection were 81.7%, 91.7% and 86.7%, respectively. The antibody detection showed a high positive predictive value (90.7%) and a high negative predictive value (83.3%).</p><p><b>CONCLUSIONS</b>The antibody detection by protein array, especially the detection of anti-Ure antibody combined with anti-CagA antibody, is valuable in the diagnosis of H.pylori infection.</p>

Effect of zinc deficiency on intestinal mucosal morphology and digestive enzyme activity in growing rat / 中华儿科杂志

<p><b>OBJECTIVE</b>In this study, a growing rat model of zinc deficiency was established to investigate the effect of zinc deficiency on intestinal mucosal morphology and digestive enzyme activity as well as to provide a scientific basis for zinc supplementation therapy in patients with diarrhea.</p><p><b>METHOD</b>Three-week-old weaned Sprague-Dawley male rats (n = 30) were randomly divided into 3 groups with 10 in each: rats in the control group (ZA) were fed with a normal diet containing 30 µg/g zinc; rats in the zinc deficient group (ZD) were fed with a zinc-deficient diet containing 0.4 µg/g zinc (refer to AIN-76 formula); and rats in the paired fed group (PF) were fed with a normal diet, but the food intake was limited to intake of rats in ZD group in the previous day. All rats were provided with deionized water for drinking. Their body weight was measured and the food intake during the previous day was recorded early in the morning of the following day. Symptoms of zinc deficiency, such as anorexia, diarrhea, dermatitis, and growth retardation, were observed. Two weeks later, the rats were sacrificed and serum zinc concentration was measured. Jejunal mucosa was taken for biopsy and was stained with hematoxylin and eosin (HE). The height ratio of the jejunal mucosal villi and crypts was measured. In addition, the activity of lactase in the jejunal mucosal brush border, γ-glutamyl peptidase (GGT), and aminopeptidase N (APN) were measured.</p><p><b>RESULT</b>The average weight of the rats in the ZA, ZD, and PF groups at the beginning of the experiment was (67.4 ± 5.3) g, (64.7 ± 4.8) g, and (66.5 ± 4.1) g, respectively, and the average daily food intake was (11.2 ± 1.0) g, (11.6 ± 1.6) g, and (11.2 ± 1.4) g, respectively. The intergroup differences were not significant. On the 7(th) day of experiment, no significant differences in average food intake were observed between the ZD group and the ZA and PF groups, but the average body weight in the ZD group was significantly lower than that in the ZA and PF groups (P < 0.01). At the end of the experiment (2 weeks), the average weight in the ZD group (112.0 ± 11.5) g was significantly lower than that in the ZA (164.0 ± 15.9) g and PF groups (137.5 ± 16.2) g. The average food intake in the ZD group (13.4 ± 5.1) g was significantly lower than that in the ZA group (18.2 ± 2.4) g (P < 0.01). Serum zinc level in the ZD group (733 ± 231) µg/L was significantly lower than that in the ZA (1553 ± 159) µg/L and PF groups (1457 ± 216) µg/L (P < 0.01). The height ratio of jejunal mucosa villus and crypt in the ZA, ZD, and PF groups was 2.98 ± 0.5, 2.77 ± 0.5, and 2.81 ± 0.7, respectively, and lactase activity was (26.1 ± 15.0) U/mg, (27.4 ± 12.8) U/mg, and (40.8 ± 18.5) U/mg, respectively, without significant intergroup differences. The GGT activity in the jejunal mucosa in the ZD group (12.7 ± 6.5) U/g was significantly lower than that in the ZA (19.1 ± 10.4) U/g and PF groups (18.5 ± 7.7) U/g, but the difference was not significant. The activity of APN in the jejunal mucosa in the ZD group (25.5 ± 7.5) U/g was significantly lower than that in the ZA (48.7 ± 16.8) U/g and PF groups (43.9 ± 14.5) U/g (P < 0.01).</p><p><b>CONCLUSION</b>Zinc deficiency can cause loss of appetite, weight loss, and decreased activity of peptidase in the jejunal mucosal brush border. Zinc deficiency has little effect on the height ratio of the villus and crypt and lactase activity, thereby indicating that zinc deficiency may first affect protein digestion and absorption.</p>

Role of oxidative stress in the pathogenesis of esophageal mucosal injury in children with reflux esophagitis / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the role of oxidative stress in the pathogenesis of esophageal mucosa injury in children with reflux esophagitis (RE).</p><p><b>METHODS</b>Esophageal mucosal samples from 36 children with RE (7 months to 16 years of age) were obtained by gastroscopy. The parameters of oxidative stress, including the contents of malondialdehyde (MDA), glutathione (GSH) and nitric oxide (NO) and total superoxide dismutase (T-SOD) activity in the esophageal mucosa as well as the protein content of the esophageal mucosa, were measured. Twenty children (3 to 16 years of age) without esophageal mucosal injury by gastroscopy served as controls.</p><p><b>RESULTS</b>There was no significant difference in the protein content of the esophageal mucosa between the RE and the control groups. The content of MDA in the RE group (15.36+/- 16.67 nmol/mg) was significantly higher than that in the control group (7.51+/- 6.17 nmol/mg) (P<0.01). The activity of T-SOD in the RE group (30.43+/- 35.09 U/mg) was statistically lower than that in the control group (56.34+/- 51.73 U/mg) (P<0.05). No significant differences were observed in GSH and NO contents between the two groups.</p><p><b>CONCLUSIONS</b>The MDA content increases and the SOD content decreases in the esophageal mucosa in children with RE. This suggests that oxidative stress seems to be an important mediator in generation of esophageal mucosal injury.</p>

Roles of enteric nervous system neurotransmitters and interstitial cells of Cajal in the colon in slow transit constipation in rats / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To evaluate the roles of enteric nervous system neurotransmitters, nitric oxide (NO), substance P (SP) and vasoactive intestinal polypeptide (VIP), and interstitial cells of Cajal (ICC) in the colon in slow transit constipation in rats.</p><p><b>METHODS</b>Thirty-two healthy Wistar rats were randomly assigned to control and constipated groups. In the constipated group, the rats were daily administered with diphenoxylate (8 mg/kg) to develop slow transit constipation, while the control rats were fed with water. The number and the weight of fecal granule and the body weight of rats were recorded every 5 days for 90 days. Transit functions of intestinal movement were examined by an activated charcoal suspension pushing test one week after stopping the administration of diphenoxylate. The levels of NO and SP in the colonic mucosa were measured by nitrate reductase methods and ELISA respectively. The distribution of VIP and ICC positive cells confirmed with symbolic c-kit+ cells in the colonic wall were observed by immunohistochemical methods.</p><p><b>RESULTS</b>The daily number of fecal granule in the constipated group was significantly less than that in the control group (P<0.01). The mean weight of each fecal granule in the constipated group was significantly higher than that in the control group (P<0.01). The discharge time of the first granule of black faeces in the constipated group (430.2+/- 132.1 min) was significantly longer than that in the control group (337.2+/- 74.7 min; P<0.05). There were no significant differences in NO and SP levels and the density of VIP positive cells in the distal colonic segment between the two groups. The number of c-kit+ cells in the distal colonic wall in the constipated group was significantly reduced compared with that in the control group (P<0.05).</p><p><b>CONCLUSIONS</b>The reduction of ICC number in the distal colon may be contributed to the pathogenesis of slow transit constipation in rats.</p>

Establishment of quantifying and typing analysis of 16S rRNA gene by real-time PCR / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore a new method of rapid and reliable diagnosis of bacterial infectious diseases such as purulent meningitis and septicemia.</p><p><b>METHODS</b>A pair of universal primers and a set of probes (including universal fluorescence probe, Gram-positive probe and Gram-negative probe) were designed based on the bacterial highly conserved region of 16S rRNA gene. By using the FQ-PCR method, 12 standard strains, 23 clinical cultural isolations and the controls such as HBV, Cryptococcus histolyticus, Blastomyces albicans and human DNA were detected with the three kinds of probes. The correlation among the results of the three kinds of probes detection was analyzed.</p><p><b>RESULTS</b>The determination of 16S rRNA gene with FQ-PCR was a highly specific and sensitive method and not cross-reactive with human DNA, virus or fungi. The least amount of 10 copies of 16S rRNA gene which corresponded to 2 bacteria could be detected with FQ-PCR. Twelve standard strains and 23 clinical cultural isolations were detected by FQ-PCR with the three kinds of probes mentioned above. All samples presented positive results using the universal probe. The results of 16S rRNA gene detected by the Gramjpositive probe were positive to the 18 G+ strains. The results of 16S rRNA gene detected by the Gram-probe were positive to the 17 G- strains.</p><p><b>CONCLUSIONS</b>The FQ-PCR technique was established for bacteria quantifying and typing using the universal primer and the double type probes. This method was convenient and rapid in detecting, quantifying and typing bacteria, with a high specificity and sensitivity.</p>