Langerhans cell histiocytosis in children with refractory diarrhoea and hypoalbuminaemia as the initial presentation: two case reports and a literature review.

Langerhans cell histiocytosis (LCH) involving the gastrointestinal tract is a rare condition for which clinical experience is limited. We describe the cases of two patients who initially presented with chronic diarrhoea, hypoproteinaemia, and intermittent fever. These findings suggest that in cases of refractory diarrhoea accompanied by recurrent hypoalbuminaemia, especially with abdominal rash, LCH should be considered. Gastrointestinal endoscopy, biopsy, and imaging studies are essential for obtaining a definitive diagnosis. This approach might be helpful for the early recognition of gastrointestinal tract involvement in LCH.

Catheter-related bloodstream infections in children with intestinal failure: a 6-year review from an intestinal rehabilitation center in China.

BACKGROUND: Children with intestinal failure (IF) have frequent catheter-related bloodstream infections (CRBSIs). This study aimed to analyze the clinical presentation and laboratory parameters of CRBSIs in children with IF. METHODS: This 6-year retrospective study was conducted among IF children with CRBSIs at an intestinal rehabilitation center in China. Clinical data were collected, including data of temperature and gastrointestinal symptoms. Blood/catheter culture, fecal tests, and calculation of inflammatory index were performed, which were obtained within 1 week since CRBSI onset. RESULTS: Fifty children with 87 CRBSIs were identified, of which there were 17 suspected and 70 confirmed cases. Seventy-two pathogens were cultured from 70 positive blood cultures: 63% were Gram-positive organisms, 23% were Gram-negative organisms, and 11% were fungal organisms. Overall, 48.6% were enteric organisms; 47.2% of bacterial pathogens were consistent between fecal and blood cultures. Moreover, 46.3% fecal routines showed abnormalities including increased white blood cells, occult blood positive and the presence of fat droplets. The consistent symptom at onset of CRBSIs was fever and gastrointestinal symptoms including increased stool output, abdominal distension, or both. C-reactive protein (CRP) and procalcitonin (PCT) were elevated, i.e., 16.5 mg/L [interquartile range (IQR) 8.7-44.7] and 0.48 ng/mL (IQR 0.2-1.76), respectively. CONCLUSIONS: IF children had a high rate of CRBSIs, of which larger proportions were due to Gram-positive and enteric organisms. Fever and/or gastrointestinal symptoms, combined with elevated CRP and PCT, is conducive to the early diagnosis of CRBSIs in IF patients.

Butyrate promotes the adaptation of intestinal smooth muscle cells through the yes-associated protein (YAP) pathway in a rat model of short bowel syndrome.

Our previous study demonstrated that the proliferation of human intestinal smooth muscle (ISM) cells was stimulated by butyrate through the yes-associated protein (YAP) pathway in vitro, suggesting a valuable approach for intestinal adaption of short bowel syndrome (SBS). This study was conducted to confirm these findings in vivo. Three-week-old Sprague-Dawley rats were randomly divided into the following groups: Sham group (bowel transection and reanastomosis), SB W group (80% small bowel resection/water ad libitum), and SB Bu group (80% small bowel resection/50 mM sodium butyrate ad libitum). Morphological changes were determined by hematoxylin and eosin staining; the proliferation rate of ISM cells was examined by Ki67 staining, and apoptosis was determined in the TUNEL assay. Changes in the expression of YAP and its downstream genes were evaluated by quantitative-polymerase chain reaction and western blotting. Fourteen days post-operation, a significant increase in ISM thickness was observed in the SB Bu group compared to the SB W group, accompanied by enhanced proliferation of ISM cells and suppression of apoptosis. Notably, YAP expression was also significantly increased in the SB Bu group, with a 6.5-fold increase in the proportion of YAP-positive ISM cells, 2.2-fold increase in YAP mRNA expression, and 3.4-fold increase in protein expression. In conclusion, our results suggest that butyrate promotes ISM adaption through YAP in vivo, which may be a potential therapeutic approach for SBS patients.

CELF1/p53 axis: a sustained antiproliferative signal leading to villus atrophy under total parenteral nutrition.

Intestinal villus atrophy is a major complication of total parenteral nutrition (TPN). Our previous study revealed that TPN-induced villus atrophy is accompanied by elevated expression of CUGBP, Elav-like family member 1 (CELF1); however, its mechanism of action has not been fully understood. Herein, we report a pivotal role of CELF1/p53 axis, which induces a sustained antiproliferative signal, leading to suppressed proliferation of intestinal epithelial cells (IECs). By using a rat model of TPN, we found synchronous upregulation of CELF1 and p53 in jejunum mucosa, accompanied by a 51% decrease in crypt cell proliferation rate. By using HCT-116 cells as an IEC model in vitro, we found that the expression of CELF1 altered dynamically in parallel to proliferation rate, suggesting a self-adaptive expression pattern in IECs in vitro. Furthermore, ectopic overexpression of CELF1 elicited a significant antiproliferative effect in HCT-116, Caco-2, and IEC-6 cells, whereas knockdown of CELF1 elicited a significant proproliferative effect. Moreover, cell-cycle assay revealed that ectopic overexpression of CELF1 induced sustained G2 arrest and G1 arrest in HCT-116 and IEC-6 cells, respectively, which could be abolished by p53 silencing. Mechanistically, polysomal profiling and nascent protein analysis revealed that regulation of p53 by CELF1 was mediated through accelerating its protein translation in polysomes. Taken together, our findings revealed a sustained suppression of IEC proliferation evoked by CELF1/p53 axis, which may be a potential therapeutic target for the treatment of TPN-induced villus atrophy.-Yan, J.-K., Zhang, T., Dai, L.-N., Gu, B.-L., Zhu, J., Yan, W.-H., Cai, W., Wang, Y. CELF1/p53 axis: a sustained antiproliferative signal leading to villus atrophy under total parenteral nutrition.

Fish oil-derived lipid emulsion induces RIP1-dependent and caspase 8-licensed necroptosis in IEC-6 cells through overproduction of reactive oxygen species.

BACKGROUND: Excessive cell death of enterocytes has been demonstrated to be partially associated with the intravenously-administrated lipid emulsions (LEs) during parenteral nutrition (PN) support. However, as a new generation of LE, the effect of fish oil-derived lipid emulsion (FOLE) on the death of enterocytes remains elusive. METHODS: Intestinal epithelial cells (IEC-6 cell line) were treated with FOLE (0.25-1%) for 24 h. Cell survival was measured by CCK-8 assay, and morphological changes were monitored by time-lapse live cell imaging. The expression of receptor-interacting protein 1/3 (RIP1/3) and caspase 8 was assessed by westernblot, and the formation of necrosome (characterized by the assembly of RIP1/3 complex along with the dissociation of caspase 8) was examined by immunoprecipitation. Additionally, the production of intracellular reactive oxygen species (ROS) was detected by using a ROS detection kit with an oxidation-sensitive probe (DCFH-DA). RESULTS: FOLE dose-dependently induced non-apoptotic, but programmed necroctic cell death (necroptosis) within 4-8 h after treatment. The assembly of RIP1/3 complex along with the dissociation of caspase 8 from RIP1 was observed in FOLE-treated cells. Moreover, FOLE-induced cell death was significantly alleviated by inhibiting RIP1, and was further aggravated by inhibiting caspase 8. In addition, prior to cell death the accumulation of intracellular ROS was significantly increased in FOLE-treated cells (increased by approximately 5-fold versus control, p < 0.001), which could be attenuated by inhibiting RIP1 (decreased by approximately 35% versus FOLE, p < 0.05). CONCLUSIONS: FOLE induces RIP1-dependent and caspase 8-licensed necroptosis through overproduction of ROS in vitro. Our findings may provide novel insights into the clinical applications of FOLE during PN support.

P38 MAPK Pharmacological Inhibitor SB203580 Alleviates Total Parenteral Nutrition-Induced Loss of Intestinal Barrier Function but Promotes Hepatocyte Lipoapoptosis.

BACKGROUND & AIMS: Our previous studies have provided evidence that p38 mitogen-activated protein kinase (MAPK) is involved in total parenteral nutrition (TPN)-associated complications, but its exact effects and mechanisms have not been fully understood. This study aimed to evaluate the roles of p38 MAPK inhibitor SB203580 in the TPN-induced loss of intestinal barrier function and liver disease. METHODS: A rodent model of TPN was used to analyze the roles of SB203580 in TPN-associated complications.Intestinal barrier function was evaluated by transepithelial electrical resistance (TER) and paracellular permeability in Caco-2 cells. The palmitic acid (PA) was used to induce hepatic lipoapoptosis in vitro. The lipoapoptosis was detected using Caspase-3/7 and lipid staining. RESULTS: In the present study, we showed that SB203580 treatment significantly suppressed TPN-mediated intestinal permeability in rats. SB203580 treatment significantly inhibited IL-1ß-induced an increase in tight junction permeability of Caco-2 cells via repressing the p38/ATF-2 signaling. Unexpectedly, SB203580 treatment enhanced hepatic lipoapoptosis in the model of TPN. Palmitic acid (PA)-induced hepatic lipoapoptosis in human liver cells was significantly augmented by the SB203580 treatment. CONCLUSIONS: We demonstrate that the p38 MAPK inhibitor SB203508 ameliorates intestinal barrier function but promotes hepatic lipoapoptosis in model of TPN.

Soybean Oil-Based Lipid Emulsion Increases Intestinal Permeability of Lipopolysaccharide in Caco-2 Cells by Downregulation of P-Glycoprotein via ERK-FOXO 3a Pathway.

BACKGROUND AND AIMS: Elevated intestinal permeability of lipopolysaccharide (LPS) is a major complication for patients with parenteral nutrition (PN), but the pathogenesis is poorly understood. Intestinal P-glycoprotein (P-gp) is one of the efflux transporters that contribute to restricting the permeability of lipopolysaccharide via transcellular route. P-gp expression may be regulated by PN ingredients, and thus this study sought to investigate the effect of PN on the expression of P-gp and to elucidate the underlying mechanism in vitro. METHODS: Caco-2 cells were treated with PN ingredients. Changes in P-gp expression and function were determined and the role of ERK-FOXO 3a pathway was studied. Transport studies of FITC-lipopolysaccharide (FITC-LPS) across Caco-2 cell monolayers were also performed. RESULTS: Among PN ingredients, soybean oil-based lipid emulsion (SOLE) exhibited significant inhibitory effect on P-gp expression and function. This regulation was mediated via activation of ERK pathway with subsequent nuclear exclusion of FOXO 3a. Importantly, P-gp participated in antagonizing the permeation of FITC-LPS (apical to basolateral) across Caco-2 cell monolayers. SOLE significantly increased the permeability of FITC-LPS (apical to basolateral), which was associated with impaired P-gp function. CONCLUSIONS: The expression and function of intestinal P-gp is suppressed by SOLE in vitro.

Hereditary pancreatitis of 3 Chinese children: Case report and literature review.

BACKGROUND: Hereditary pancreatitis (HP) is quite rare and is distinguished by incomplete penetrance presentation as early-onset relapsing pancreatitis, usually beginning in childhood. HP is now known to be commonly relevant to mutations in the PRSS1 (gene-encoding cationic trypsinogen), SPINK1 (serine protease inhibitor, Kazal type 1), CFTR (cystic fibrosis), carboxypeptidase A1 (CPA1), and chymotrypsin C (CTRC) genes as reported in some Caucasian studies. HP has a variable spectrum of severity and may develop complications. METHODS & RESULTS: We describe the clinical course of 3 preschool children, hospitalized with postprandial abdominal pain, whose laboratory tests showed high serum amylase. Similar episodes of abdominal pain led to readmission, and the patients recovered quickly after using symptomatic therapy. The condition of the first boy, who developed a pancreatic tail pseudocyst and splenic infarction, was especially complicated. The boy underwent 2 endoscopic retrograde cholangiopancreatographies and stenting, along with a surgical procedure that completely relieved his symptoms for 3 months. The 3 patients and their parents were given genetic testing. All of the patients carried 1 or more gene mutations inherited from their mothers, fathers, or both parents; however, none of the parents were affected. CONCLUSION: For children with repeated pancreatitis, clinicians should consider HP in the differential diagnosis. It is reliable to perform gene sequencing on suspicious patients and their parents. Multidisciplinary and comprehensive treatment should be recommended to manage HP and its complications. Cholangiopancreatography and stenting is a relatively minimally invasive approach when compared with surgery and can be tried as an early intervention. Surgical procedures should be reserved for patients with complications.

Neutralization of IL-6 and TNF-α ameliorates intestinal permeability in DSS-induced colitis.

The cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) have been implicated as important mediators of the inflammatory reaction in patients with intestinal inflammation. The present study was designed to investigate the roles of these cytokines on mucosal barrier function in a mouse model of acute colitis with using anti-cytokine strategies. Mice received 3% dextran sulfate sodium (DSS) in their drinking water for 7days showed morphological alteration of mucosa and increase of intestinal permeability. Administration of IL-6 monoclonal antibody (mAb) or TNF-α mAb significantly attenuated intestinal permeability. IL-6 mAb and TNF-α mAb treatment also effectively suppressed the expression of claudin-2 and myosin light chain kinase (MLCK). Taken together, we indicated that anti-IL-6 and anti-TNF-α therapy prevent intestinal permeability induced by intestinal inflammation.

CUGBP1 and HuR regulate E-cadherin translation by altering recruitment of E-cadherin mRNA to processing bodies and modulate epithelial barrier function.

The effectiveness and stability of epithelial barrier depend on apical junctional complexes, which consist of tight junctions (TJs) and adherens junctions (AJs). E-cadherin is the primary component of AJs, and it is essential for maintenance of cell-to-cell interactions and regulates the epithelial barrier. However, the exact mechanism underlying E-cadherin expression, particularly at the posttranscriptional level, remains largely unknown. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HU antigen R (HuR) are highly expressed in the intestinal epithelial tissues and modulate the stability and translation of target mRNAs. Here, we present evidence that CUGBP1 and HuR interact directly with the 3'-untranslated region of E-cadherin mRNA and regulate E-cadherin translation. CUGBP1 overexpression in Caco-2 cells inhibited E-cadherin translation by increasing the recruitment of E-cadherin mRNA to processing bodies (PBs), thus resulting in an increase in paracellular permeability. Overexpression of HuR exhibited an opposite effect on E-cadherin expression by preventing the translocation of E-cadherin mRNA to PBs and therefore prevented CUGBP1-induced repression of E-cadherin expression. Elevation of HuR also abolished the CUGBP1-induced epithelial barrier dysfunction. These findings indicate that CUGBP1 and HuR negate each other's effects in regulating E-cadherin translation by altering the recruitment of E-cadherin mRNA to PBs and play an important role in the regulation of intestinal barrier integrity under various pathophysiological conditions.

[Rapid detection of clarithromycin resistant Helicobacter pylori from children's gastric biopsy specimens by polymerase chain reaction-restriction fragment length polymorphism].

OBJECTIVE: Infection with clarithromycin-resistant Helicobacter pylori (Hp) is often predictive of treatment failure. Susceptibility testing for Hp could guide therapy of Hp infections. However, agar dilution approved by the Clinical and Laboratory Standards Institute (CLSI) to test for antimicrobial susceptibility of Hp is time consuming (results are often not available in a week or more). So a more expeditious method is necessary. The purpose of this study was to evaluate polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) test performed directly on gastric biopsy specimen from children to detect 23S rRNA mutations (A2143G and A2144G) indicating clarithromycin resistance. METHODS: All biopsy specimens were derived from patients presenting with upper gastrointestinal symptoms, submitted to endoscopy in the Affiliated Children's Hospital, Zhejiang University School of Medicine from September 2006 to February 2007. No patients had undergone eradication therapy. Thirty-nine samples randomly selected from positive specimens by rapid urease test, were homogenized in 500 microl brucella broth with 30% glycerol. The 200 microl homogenized fluid was used to purify genomic DNA with the kit according to the instructions provided by manufacturer, and the rest was used to isolate Hp strains by culturing. All the Hp isolates were tested for clarithromycin susceptibility with the agar dilution and classified as resistant if the minimum inhibitory concentrations (MIC) exceeded 1 microg/ml. Simultaneously, PCR-RFLP analysis was performed in order to identify 23S rRNA mutations (A2143G and A2144G). Finally, the two methods were compared by statistics. The agar dilution was used as a standard to determine the sensitivity and specificity of the PCR-RFLP assay. RESULTS: Of the 39 samples, agar dilution and PCR-RFLP method respectively detected 13 (33.3%) and 14 (35.9%) clarithromycin-resistant gastric specimens. The sensitivity and specificity of PCR-RFLP for the detection of Hp in biopsy specimens were both 92%. The positive and negative predictive value was 85.7% and 96% respectively. No statistically significant difference was found between the two methods (chi2=0.06, P>0.05). The rate of Hp resistance to clarithromycin significantly increased compared with a previous report from the authors' hospital in 2004 (chi2=6.20, P<0.05). CONCLUSIONS: Rising clarithromycin resistance rates were observed in children who visited the authors' hospital. PCR-RFLP test is reliable and rapid for detection of clarithromycin resistance directly on gastric biopsy specimen from children and may help choose appropriate antibiotic in Hp eradication therapy.

[Preliminary study on in-vitro induction of antibiotic resistance in Helicobacter pylori strains isolated from children].

OBJECTIVE: Many clinical studies indicated that Helicobacter pylori (Hp) strains rarely acquired resistance to amoxicillin but easily to clarithromycin and metronidazole. However, it was unclear whether the antibiotic resistance of Hp strains was induced or passively selected during long-term or frequent treatment with metronidazole, clarithromycin and amoxicillin. To compare the propensity of acquired resistance to antibiotics, Hp strains were exposed to amoxicillin, clarithromycin and metronidazole in vitro in this study. METHODS: All Hp strains were clinical isolates, derived from biopsy specimens of patients taken during endoscopy in the Affiliated Children's Hospital, Zhejiang University School of Medicine from December 2004 to July 2005. To seek susceptible strains, the minimum inhibitory concentrations (MICs) of the three antibiotics were determined by using Epsilometer test (E-test) method. In vitro induction was carried out on serially doubling concentrations of antibiotics incorporated into agar. Isolates were also transferred at least three times on antimicrobial agent-free medium, followed by a redetermination of the final MICs to assess the stability of the selected resistance. RESULTS: 7 strains were exposed to antibiotics in vitro. After 6 - 17 passages on antibiotic plates, 7 and 3 strains respectively acquired resistance to metronidazole and clarithromycin, while none of the strains were resistant to amoxicillin. The inductive folds were different among three groups: 8 - 128 folds in metronidazole group; 1 - 256 folds in clarithromycin group; 2 - 16 folds in amoxicillin group. After three transfers on antimicrobial agent-free medium, the MICs decreased significantly in amoxicillin group (P < 0.05) but had no change in metronidazole group and clarithromycin group (P > 0.05). CONCLUSIONS: The metronidazole resistance in Hp was easily selected. Strains resistant to clarithromycin could be selected, but the amoxicillin resistance could not be selected after in vitro induction for Hp isolated from children. The correlation between in vitro and in vivo outcomes suggests that acquired resistance was the main cause for the resistance in Hp strains. The laboratory results of in vitro antibiotic induction could help predict the actual rate of resistance and select appropriate antibiotics for treatment.

[Prevalence of resistance to metronidazole in Helicobacter pylori from children and induction of resistance in vitro].

OBJECTIVE: To investigate the prevalence of resistance of Helicobacter pylori (H. pylori) to metronidazole (MTZ) and the distribution change of minimal inhibitory concentrations (MICs) in H. pylori from local children, evaluate the applicability of E-test for MIC determination, and display the propensity of acquired-resistance to MTZ after induction of resistance in vitro. METHODS: One group of 44 H. pylori isolates obtained from Oct. 2002 to Nov. 2003 and another 83 H. pylori isolates obtained from Dec. 2004 to Jul. 2005 from the local children who underwent gastroscopy in the Children's Hospital affiliated to Zhejiang University Medical School and were diagnosed as H. pylori-associated gastritis or peptic ulcer were studied. Susceptibility was tested by agar dilution method or E-test method. In 11 randomly selected metronidazole-sensitive isolates (MTZ(S)), resistance was induced in vitro with MTZ. RESULTS: The resistance rate was 31.8% (14/44) in the 44-islates obtained from Oct. 2002 to Nov. 2003 and 51.8% (43/83) (chi(2) = 4.64, P < 0.05) in 83-isolates obtained from Dec. 2004 to Jul. 2005, respectively. The distribution of MICs were < 0.125 - 128 mg/L and 0.25- > 256 mg/L, in which, the MIC(50) was 0.5 mg/L and 16 mg/L, the MIC(90) was 128 mg/L, respectively. Comparing to agar dilution method which is recommended by National Committee for Clinical Laboratory Standards (NCCLS) for MIC, E-test was significantly associated with agar dilution method (chi(2) = 32.38, P < 0.001). The sensitivity, specificity, agreement rate of E-test were 73.08%, 100%, 87.27%, respectively, while there were factors of 2(2) to 2(6) difference in MICs between the results obtained by E-test and agar dilution. For all the 11 MTZ(S) isolates inducted resistance in vitro with MTZ, 16 MICs were achieved through 7 - 9 (7.2 +/- 0.6) passages of induction in vitro, and 100% acquired-resistance to MTZ through 8 - 10 generations; as a result, 10 of 11 MTZ(S) isolates achieved stable high-level resistance (256 mg/L for 2 and > 256 mg/L for 8) and 1 stable 64 mg/L resistance to MTZ. CONCLUSIONS: The prevalence of resistance to MTZ seems to be increasing in H. pylori from local children. To avoid missed diagnosis of H. pylori resistant to MTZ (MTZ(R)), agar dilution method was needed when detecting susceptibility of H. pylori to MTZ. Resistance to MTZ of H. pylori from children is readily induced in vitro.