A coumarin-based fluorescent probe for specific detection of cysteine in the lysosome of living cells.

Cysteine (Cys), the only amino acid in the 20 natural amino acids that contains a reduced sulfhydryl group, plays important roles in the balance of redox homeostasis in biological systems. Lysosome is an important organelle containing a variety of hydrolases and has been proved to be the decomposition center of a variety of exogenous and endogenous macromolecular substances. In this research, a coumarin-based fluorescent probe MCA for the detection of Cys in lysosomes of living cells was developed. Due to the acrylate moiety, this probe exhibited high sensitivity (detection limit = 6.8 nM) and selectivity towards Cys superior to other analytes. Moreover, the probe was proved to be lysosome-targetable and showed good cell imaging ability and low cell toxicity.

Impact of Preoperative Exclusive Enteral Nutrition on Postoperative Complications and Recurrence After Bowel Resection in Patients with Active Crohn's Disease.

BACKGROUND: The impact of preoperative enteral nutrition (EN) on postoperative complications and recurrence in Crohn's disease (CD) has not been investigated to date. The purpose of the present study was to determine the effect of preoperative exclusive EN on postoperative complications and recurrence after bowel resection in patients with active CD. METHODS: Patient data were obtained from a prospectively maintained database. 81 patients who received bowel resection for ileal or ileocolonic CD were studied. Before operation, 42 CD patients received exclusive EN for 4 weeks, and the other patients had no nutritional therapy. All patients were followed up regularly for 2 years after surgery, and ileocolonoscopy was performed every 6 months after bowel resection. RESULTS: Patients receiving exclusive EN had a dramatic improvement of nutritional (BMI, albumin, pre-albumin, and Hb) and inflammatory (CRP and CDAI) status compared with baseline after the EN therapy for 4 weeks (P < 0.05). Furthermore, significantly lower incidence of both infectious and non-infectious complications was observed in patients receiving exclusive EN compared with those received no nutritional therapy (P < 0.05). Exclusive EN therapy for 4 weeks significantly reduced endoscopic recurrence rates after resection for CD 6 months after operation. However, during the 2-year follow-up, incidence of clinical recurrence was similar in both groups (P > 0.05). CONCLUSIONS: Preoperative exclusive EN therapy for 4 weeks reduced postoperative complications, which may be associated with improvement of nutritional and inflammatory status in patients with active CD.

Anti-mouse CD52 monoclonal antibody ameliorates intestinal epithelial barrier function in interleukin-10 knockout mice with spontaneous chronic colitis.

Intestinal inflammation causes tight junction changes and death of epithelial cells, and plays an important role in the development of Crohn's disease (CD). CD52 monoclonal antibody (CD52 mAb) directly targets the cell surface CD52 and is effective in depleting mature lymphocytes by cytolytic effects in vivo, leading to long-lasting changes in adaptive immunity. The aim of this study was to investigate the therapeutic effect of CD52 mAb on epithelial barrier function in animal models of IBD. Interleukin-10 knockout mice (IL-10(-/-) ) of 16 weeks with established colitis were treated with CD52 mAb once a week for 2 weeks. Severity of colitis, CD4(+) lymphocytes and cytokines in the lamina propria, epithelial expression of tight junction proteins, morphology of tight junctions, tumour necrosis factor-α (TNF-α)/TNF receptor 2 (TNFR2) mRNA expression, myosin light chain kinase (MLCK) expression and activity, as well as epithelial apoptosis in proximal colon were measured at the end of the experiment. CD52 mAb treatment effectively attenuated colitis associated with decreased lamina propria CD4(+) lymphocytes and interferon-γ/IL-17 responses in colonic mucosa in IL-10(-/-) mice. After CD52 mAb treatment, attenuation of colonic permeability, increased epithelial expression and correct localization of tight junction proteins (occludin and zona occludens protein-1), as well as ameliorated tight junction morphology were observed in IL-10(-/-) mice. CD52 mAb treatment also effectively suppressed the epithelial apoptosis, mucosa TNF-α mRNA expression, epithelial expression of long MLCK, TNFR2 and phosphorylation of MLC. Our results indicated that anti-CD52 therapy may inhibit TNF-α/TNFR2-mediated epithelial apoptosis and MLCK-dependent tight junction permeability by depleting activated T cells in the gut mucosa.

DHA protects against experimental colitis in IL-10-deficient mice associated with the modulation of intestinal epithelial barrier function.

A defect in the intestinal barrier is one of the characteristics of Crohn's disease (CD). The tight junction (TJ) changes and death of epithelial cells caused by intestinal inflammation play an important role in the development of CD. DHA, a long-chain PUFA, has been shown to be helpful in treating inflammatory bowel disease in experimental models by inhibiting the NF-κB pathway. The present study aimed at investigating the specific effect of DHA on the intestinal barrier function in IL-10-deficient mice. IL-10-deficient mice (IL-10(-/-)) at 16 weeks of age with established colitis were treated with DHA (i.g. 35.5 mg/kg per d) for 2 weeks. The severity of their colitis, levels of pro-inflammatory cytokines, epithelial gene expression, the distributions of TJ proteins (occludin and zona occludens (ZO)-1), and epithelial apoptosis in the proximal colon were measured at the end of the experiment. DHA treatment attenuated the established colitis and was associated with reduced infiltration of inflammatory cells in the colonic mucosa, lower mean histological scores and decreased levels of pro-inflammatory cytokines (IL-17, TNF-α and interferon-γ). Moreover, enhanced barrier function was observed in the DHA-treated mice that resulted from attenuated colonic permeability, rescued expression and corrected distributions of occludin and ZO-1. The results of the present study indicate that DHA therapy may ameliorate experimental colitis in IL-10(-/-) mice by improving the intestinal epithelial barrier function.

Ultrasound as a diagnostic tool in detecting active Crohn's disease: a meta-analysis of prospective studies.

OBJECTIVES: To evaluate the diagnostic accuracy of ultrasound in assessing active Crohn's disease (CD) in adults. METHODS: We systematically searched PubMed, EMBASE, Web of Science and the Cochrane Library for prospective studies in which ultrasound was performed to evaluate active CD in adults from January 1993 to April 2013. Pooled sensitivity, specificity, positive and negative likelihood ratios (LR+ and LR-) and diagnostic odds ratios (DORs) in a per-patient-based analysis were estimated. Additionally, the area under the receiver-operating characteristic (ROC) curve was calculated to measure the diagnostic accuracy of ultrasound in patients with known or suspected CD. RESULTS: Fifteen studies involving 1,558 patients were included in this meta-analysis. Overall, the pooled sensitivity (0.88) and LR- (0.14) were not heterogeneous, whereas the pooled specificity (0.97, I(2) = 72.9 %) and LR + (15.10, I(2) = 71.8 %) were. The DOR of ultrasound for assessing active CD was 121.70, with significant heterogeneity (I(2) = 63.3 %). A symmetrical summary ROC curve was plotted showing that the area under the curve was 0.94, indicating good diagnostic accuracy. Meta-regression and subgroup analysis showed that the disease location may be a major cause of heterogeneity. CONCLUSIONS: This meta-analysis suggests that ultrasound has high diagnostic accuracy in detecting active CD in adults, especially for small bowel CD. KEY POINTS: • Ultrasound can detect active Crohn's disease (CD). • A meta-analysis of ultrasound for detecting active CD was carried out. • This revealed that ultrasound has high diagnostic accuracy for detecting CD.

Anti-mouse CD52 monoclonal antibody ameliorates iron-deficient anaemia in IL-10 knockout mice.

Approximately 50 % of patients with inflammatory bowel disease (IBD) suffer from anaemia, with Fe deficiency being the most common cause. CD52 monoclonal antibody (mAb) targets the cell surface CD52 and is effective in depleting lymphocytes through cytolytic effects in vivo. The aim of the present study was to investigate the therapeutic effect of anti-mouse CD52 mAb on Fe-deficient anaemia in IBD. IL-10 knockout mice (IL-10- / -) of 12 weeks with established colitis were treated with anti-mouse CD52 mAb once per week for 2 weeks. Severity of colitis, blood T lymphocytes, blood Hb, haematocrit, plasma erythropoietin (EPO), serum Fe concentration, transferrin saturation, splenic Fe stores, expression of liver hepcidin mRNA, Western blotting of the phosphorylated form of Smad1/5/8 and total Smad1 were measured at the end of the experiment. IL-10- / - mice treated with CD52 mAb showed a reduction in the percentage of CD4+ and CD4+CD45+ T cells in blood and weight loss typically associated with colonic inflammation, serum levels of EPO, the expression of liver hepcidin mRNA and total Smad1 protein, while they showed an increase in Hb concentrations, haematocrit, levels of serum Fe, transferrin saturation and splenic Fe stores. The present results indicated that anti-CD52 therapy may ameliorate Fe-deficient anaemia by reducing colonic inflammation. These findings may open novel horizons in the treatment of patients with IBD by resetting of immunological homeostasis in the gut by depleting the activated T cells in the gut mucosa.

TUBB3 Promotes Growth and Invasion of Gallbladder Cancer Cells by Akt/mTOR Signal Pathway.

Aberrant expression of ß-tubulin-III (TUBB3) is known that related to aggressive tumor features and poor clinical outcomes. However, there is limited research about TUBB3 expression and its role in the outcomes and the progression of gallbladder cancer. We have measured TUBB3 level in gallbladder cancer samples and cell lines from 2012 to 2016, and tested the effects of TUBB3 in cancer cell growth, apoptosis and cell cycle arrest by using appropriate methods. The results revealed that TUBB3 was significantly over-expressed in gallbladder cancer samples and cell lines, and high TUBB3 level contributed to shorter overall survival in patients. The knockdown of TUBB3 with sh-TUBB3 inhibited the proliferation, migration and invasion of cancer cells. Meanwhile, it promotes apoptosis and changes the cell cycle distribution. Suppression of TUBB3 expression could increase p21 and cyclin B1 expression, and decrease cyclin D1. Xenograft mouse model also showed that low expression of TUBB3 reduced the growth of established gallbladder cancer xenograft in vivo. Furthermore, TUBB3 knockdown significantly decreased phosphorylation of Akt and mTOR in vitro and in vivo. TUBB3 can induce the development of gallbladder cancer by Akt/mTOR signal pathway and we point out a potential therapeutic target for gallbladder cancer treatment.

Docosahexaenoic Acid Attenuated Experimental Chronic Colitis in Interleukin 10-Deficient Mice by Enhancing Autophagy Through Inhibition of the mTOR Pathway.

BACKGROUND: In the battle against Crohn's disease, autophagy stimulation is a promising therapeutic option-one both new and newly rediscovered. In experimental models, docosahexaenoic acid (DHA)-a long-chain polyunsaturated fatty acid-has been demonstrated to be useful in the treatment of inflammatory bowel disease through inhibition of the nuclear factor-κB pathway. However, the impact of DHA on autophagy in the colon remains unclear. METHODS: Mice were divided into 3 groups: wild type (placebo), the interleukin 10 knockout group (IL-10-/-, placebo), and the DHA group (IL-10-/-, DHA). DHA was administered to IL-10-/- mice by gavage at a dosage of 35.5 mg/kg/d for 2 weeks. The severity of colitis, expression of proinflammatory cytokines, expression/distribution of LC3B, and mTOR signaling pathway were evaluated in the proximal colon tissues collected from all mice at the end of the experiment. RESULTS: DHA administration ameliorated experimental colitis in the IL-10-/- mice, as demonstrated by decreased proinflammatory cytokines (TNF-α and IFN-γ), reduced infiltration of inflammatory cells, and lowered histologic scores of the proximal colon mucosa. Moreover, in the DHA-treated mice, enhanced autophagy was observed to be associated with (1) increased expression and restoration of the distribution integrity of LC3B in the colon and (2) inhibition of the mTOR signaling pathway. CONCLUSION: This study showed that DHA therapy could attenuate experimental chronic colitis in IL-10-/- mice by triggering autophagy via inhibition of the mTOR pathway.

Dietary Non-digestible Polysaccharides Ameliorate Intestinal Epithelial Barrier Dysfunction in IL-10 Knockout Mice.

BACKGROUND: Enteral nutrition [EN] was reported to be as effective as steroids in achieving short-term remission in patients with Crohn's disease [CD], and exclusive EN [EEN] is widely used as primary therapy in children with CD. The aim of this study was to investigate the effect of a specific multi-fibre mix [MF], designed to match the fibre content of a healthy diet, on intestinal epithelial barrier function in IL-10 knockout [IL-10(-/-)] mice with spontaneous chronic colitis. METHODS: IL-10(-/-) mice aged 16 weeks, with established colitis, were used for the experiments with multi-fibre mix diet [MF] for 4 weeks. Severity of colitis, levels of short cahin fatty acids [SCFA] in caecum contents, expression of STAT 3 and STAT 4 proteins, CD4(+) CD45(+) lymphocytes, CD4(+)Foxp3(+) regulatory T cells [Tregs] and cytokines in the lamina propria [LP], epithelial expression of tight junction proteins, TNF-α/TNFR2 mRNA expression, and epithelial apoptosis in the proximal colon were measured at the end of the experiment. RESULTS: MF feeding effectively attenuated disease activity index and colitis associated with decreased lamina propria CD4(+) CD45(+) lymphocytes, IFN-γ/IL-17A mRNA expression, and p-STAT 3 and p-STAT 4 expression in colonic mucosa of IL-10(-/-) mice [p < 0.05]. Furthermore, CD4(+)Foxp3(+) Tregs in the LP and concentrations of total SCFA, acetate, propionate, and butyrate in the caecum were markedly increased after MF feeding in IL-10(-/-) mice. After MF feeding, increased epithelial expression and correct localisation of tight junction proteins [occludin and zona occludens protein 1], as well as reduced TNF-α/TNFR2 mRNA expression and epithelial apoptosis, were also observed in IL-10(-/-) mice. CONCLUSIONS: These results indicated that EEN supplemented with the tested fibre mix, known to modulate the intestinal microbiota composition and SCFA production, could possibly improve efficacy in inducing remission in patients with active CD.

Effectiveness of a clinical pathway for inpatients undergoing ileal/ileocecal resection for chronic radiation enteritis with intestinal obstruction.

Surgery is associated with elevated morbidity and mortality in chronic radiation enteritis (CRE). The objective of this study was to evaluate the effect of a fast-track clinical pathway (CP) on postoperative outcomes in patients undergoing ileal/ileocecal resection for CRE with intestinal obstruction. There were 85 patients with CRE (January 2011 to March 2013) with intestinal obstruction admitted to our department for ileal/ileocecal resection. The patients were divided into a prepathway group and a pathway group. The clinical outcomes were then assessed and compared. The postoperative lengths of hospital stay were 8.52 days for the pathway group and 11.32 days for the prepathway group (P = 0.02). The pathway group had a lower stoma rate (21.6 vs 56%, P = 0.033) and fewer postoperative moderate to severe complications (8.1 vs 25%, P = 0.043) compared with the prepathway group. Implementation of the CP may reduce stoma rate, postoperative moderate to severe complications, and postoperative length of hospital stay for patients undergoing ileal/ileocecal resection for the treatment of CRE with intestinal obstruction.

Laquinimod ameliorates spontaneous colitis in interleukin-10-gene-deficient mice with improved barrier function.

BACKGROUND AND AIMS: Crohn's disease is an autoimmune disease associated with imbalanced mucosal immunity, mediated with increased Th1 and Th17 cells. Laquinimod, an immunomodulatory drug, has shown efficacy in regulating the differentiation of T cells. The aim of the study was to investigate the therapeutic effect of laquinimod on spontaneous colitis in interleukin-10-gene-deficient mice, an animal model of Crohn's disease. METHODS: Male Il10(-/-) mice aged 16weeks in the laquinimod group were treated with laquinimod with distilled water at a dose of 25mg/kg by oral gavage, 3 times a week. Il10(-/-) mice in the IL-10-KO group and wild type mice received equal volume of phosphate buffered saline by oral gavage, 3 times a week. After 4weeks, mice were sacrificed for analysis. Severity of colitis, epithelial expression of T-cell-associated cytokines, expression and distribution of tight junction proteins in the lamina propria and NF-κB signaling pathway associated mRNA expression were measured at the end of the experiment. RESULTS: Laquinimod treatment ameliorated spontaneous colitis in Il10(-/-) mice, which was associated with decreased T-cell-associated pro-inflammatory cytokines. Increased expression and correct distribution of tight junction proteins (occludin and ZO-1) were found in Il10(-/-) mice treated with laquinimod. In addition, in mice treated with laquinimod, NF-κB signaling pathway associated mRNA in the colon was also downregulated. CONCLUSIONS: Our results indicated that laquinimod treatment ameliorates colitis in Il10(-/-) mice and improves intestinal barrier function, which may support a new therapeutic approach to Crohn's disease.

Tripterygium wilfordii Hook F as Maintenance Treatment for Crohn's Disease.

BACKGROUND: The traditional Chinese medicine, Tripterygium wilfordii Hook F (TwHF) is widely used to treat Crohn's disease (CD) in China. METHODS: The authors compared different doses of TwHF with mesalazine in 198 patients with CD over a 52-week period. Subjects were randomized to receive mesalazine (3 g/d), low-dose TwHF (1.5 mg·kg·d), or high-dose TwHF (2.0 mg·kg·d). RESULTS: A total of 137 patients completed the study. At week 52, a significant lower proportion of patients in the high-dose TwHF group (7/71) had clinical recurrence compared with patients in the low-dose TwHF (15/68, P = 0.047) or mesalazine group (17/59, P = 0.006), whereas the difference between the low-dose TwHF group and the mesalazine group was not significant (P = 0.503). Patients receiving mesalazine experienced less adverse events than those receiving high-dose TwHF (P = 0.029) and those receiving low-dose TwHF (P = 0.048), but no significant difference was found about drug adverse events resulted withdrawal in the 3 groups (P > 0.05). In addition, compared with low-dose TwHF and mesalazine, the authors also detected significant superiority of high-dose TwHF arm in the decrease of CDAI and SESCD (P < 0.05). CONCLUSION: A 2.0 mg/kg daily TwHF was well tolerated and prolonged remission in patients with CD.

SEW2871 protects from experimental colitis through reduced epithelial cell apoptosis and improved barrier function in interleukin-10 gene-deficient mice.

Loss of intestinal epithelial barrier function including typical tight junction changes and epithelial cell apoptosis plays an important role in Crohn's disease. SEW2871, a selective sphingosine-1-phosphate type-1 receptor agonist, has been proven to be efficient in protecting against colitis in IL-10(-/-) mice in our previous study. Here we performed additional studies to investigate whether treatment with SEW2871 was associated with an improved epithelial barrier function in IL-10(-/-) mice. SEW2871 was administered by gavage at a dose of 20 mg/kg/day for 2 weeks to IL-10(-/-) mice. Severity of colitis, CD4+ T cells in colon lamina propria and proinflammatory cytokine productions were evaluated. Furthermore, intestinal permeability, tight junction (occludin and ZO-1) expressions and distributions, as well as epithelial cell apoptosis, were also assessed. SEW2871 treatment attenuated established colitis associated with decreased CD4+ T cells in colon lamina propria and reduced TNF-α and IFN-γ levels. Moreover, enhanced barrier function, which resulted from ameliorated tight junction (occludin and ZO-1) expressions and suppressed epithelial cell apoptosis, was found to contribute to the therapeutic effects. SEW2871 treatment protects from colitis in IL-10(-/-) mice through reduced epithelial cell apoptosis and improved barrier function. Thus, targeting sphingosine-1-phosphate may represent a new therapeutic approach in Crohn's disease.

Celastrol ameliorates experimental colitis in IL-10 deficient mice via the up-regulation of autophagy.

BACKGROUND: Celastrol had been proved effective in the treatment for IBD, probably with the modulation of oxidative stress, inflammatory cytokines and intestinal homeostasis. This study was aimed to investigate whether celastrol could ameliorate the inflammation of IL-10 deficient mice, a murine model of Crohn's disease (CD) with the induction of autophagy. MATERIAL AND METHODS: The mice included were divided into four groups, ##WT group, IL-10(-/-) group, Cel group and Control group (celastrol+3-Methyladenine). Celastrol (2 mg/kg) treatment by gavage was administered to mice daily over one week. 3-Methyladenine (autophagy inhibitors) was administered at a dose of 30 mg/kg by intraperitoneal injection. The histological evaluation of the colon, tissue myeloperoxidase (MPO), and colon inflammation of mice in the four groups was evaluated and compared. Furthermore, the PI3K/Akt/mTOR pathway and the status of autophagy in intestine affected by celastrol were also assessed. RESULTS: The one-week administration of celastrol ameliorated established colitis in IL-10 deficient mice, associated with a reduction of marked histological inflammation, a decreased colon MPO concentration and suppression of colonic proinflammatory cytokine. Furthermore, the decreased neutrophil infiltration in proximal colon and improvement of inflammation in the Cel group was much more obvious than that in the Control group. The Western blotting analysis of the PI3K/Akt/mTOR pathway and autophagy showed that celastrol treatment up-regulated the autophagy of colon tissue by suppressing the PI3K/Akt/mTOR signaling pathway. CONCLUSIONS: Celastrol ameliorates experimental colitis in IL-10 deficient mice via the up-regulation of autophagy by suppressing the PI3K/Akt/mTOR signaling pathway.

Impact of enteral nutrition on energy metabolism in patients with Crohn's disease.

AIM: To investigate the impact of enteral nutrition (EN) on the body composition and metabolism in patients with Crohn's disease (CD). METHODS: Sixty-one patients diagnosed with CD were enrolled in this study. They were given only EN (enteral nutritional suspension, TPF, non-elemental diet) support for 4 wk, without any treatment with corticosteroids, immunosuppressive drugs, infliximab or by surgical operation. Body composition statistics such as weight, body mass index, skeletal muscle mass (SMM), fat mass, protein mass and inflammation indexes such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and CD activity index (CDAI) were recorded before and after EN support. RESULTS: The 61 patients were divided into three groups according to CDAI before and after EN support: A (active phase into remission via EN, n=21), B (remained in active phase before and after EN, n=19) and C (in remission before and after EN, n=21). Patients in group A had a significant increase in SMM (22.11±4.77 kg vs 23.23±4.49 kg, P=0.044), protein mass (8.01±1.57 kg vs 8.44±1.45 kg, P=0.019) and decrease in resting energy expenditure (REE) per kilogram (27.42±5.01 kcal/kg per day vs 22.62±5.45 kcal/kg per day, P<0.05). There was no significant difference between predicted and measured REE in active CD patients according to the Harris-Benedict equation. There was no linear correlation between the measured REE and CRP, ESR or CDAI in active CD patients. CONCLUSION: EN could decrease the hypermetabolism in active CD patients by reducing the inflammatory response.