Ustekinumab in pediatric patients with Crohn's disease: safety, and efficacy results from a multicenter retrospective study in China.

Background: Ustekinumab (UST) is approved as an effective therapy for Crohn's disease (CD) in adults. Off-label use is increasing in the pediatric population, more data on safety and efficacy in pediatric patients with CD is urgently needed. Aims: This study aimed to evaluate the clinical efficacy and safety of UST in children and adolescents with Crohn's disease. Methods: This multicenter retrospective study carried out at three tertiary care centers, and identified children who received their first dose of UST at 18 years old or younger and followed up for a minimum of 24 weeks. Data on demographics, disease behavior, location and activity, treatment history were collected. The primary outcomes were clinical remission at weeks 24-32 and weeks 48-56 of UST therapy. Secondary outcomes were clinical response at the same time points, endoscopic remission, changes in C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin and fecal calprotectin, improvement in growth parameters, and rate of adverse events. Results: Sixteen patients were included, and 11/13 (84.6%) continued to receive UST after 1 year. Our data demonstrate that the clinical remission rates were 41.7% at weeks 24∼32 with the Weighted pediatric CD activity index (wPCDAI) was lower than baseline (43.8, IQR: 31.3-51.9 vs.15, IQR: 5.6-25, p < 0.001) and 75% at weeks 48-56 with wPCDAI was lower than baseline (42.5, IQR: 23.8-50 vs. 7.5, IQR: 0-13.8, p = 0.004). Five of eleven children achieved endoscopic remission. No serious adverse events were recorded during the study period. Conclusions: UST is efficacious and safe in pediatric patients with CD. Pediatric patients could benefit from UST as either a primary or secondary biologic therapy for the induction, or maintenance of remission of CD.

Application of Dynamic Contrast-Enhanced Ultrasound in Evaluation the Activity of Crohn's Disease.

BACKGROUND AND OBJECTIVE: The dynamic assessment of disease activity during the follow-up of patients with Crohn's disease (CD) remains a significant challenge. In this study, we aimed to identify the role of dynamic contrast-enhanced ultrasound (DCE-US) in the evaluation of activity of CD. METHODS: In the retrospective study, patients diagnosed with CD in our hospital were included. All the diagnoses were confirmed by clinical symptoms and ileocolonoscopical results. All patients underwent intestinal ultrasound and contrast-enhanced ultrasound (CEUS) examinations within 1 week of the ileocolonoscopy examinations. Acuson Sequoia (Siemens Healthineers, Mountain View, CA, USA) and Resona R9 Elite (Mindray Medical Systems, China) with curved array and Line array transducers were used. The CEUS examination was performed with SonoVue (Bracco SpA, Milan, Italy). DCE-US analysis was performed by UltraOffice (version: 0.3-2010, Mindray Medical Systems, China) software. Two regions of interest (ROIs) were set in the anterior section of the infected bowel wall and its surrounding normal bowel wall 2 cm distant from the inflamed area. Time-intensity curves (TICs) were generated and quantitative perfusion parameters were obtained after curve fittings. The Simple Endoscopic Score for Crohn's disease (SES-CD) was regarded as the reference standard to evaluate the activity of CD. The receiver operating characteristic curve (ROC) analyses were used to determine the diagnostic efficiency of DCE-US quantitative parameters. RESULTS: From March 2023 to November 2023, 52 CD patients were included. According to SES-CD score, all patients were divided into active group with the SES-CD score > 5 (n = 39) and inactive group SES-CD score < 5 (n = 13). Most of the active CD patients showed bowel wall thickness (BWT) > 4.2 mm (97.4%, 38/39) or mesenteric fat hypertrophy (MFH) on intestinal ultrasound (US) scan (69.2%, 27/39). Color Doppler signal of the bowel wall mostly showed spotty or short striped blood flow signal in active CD patients (56.4%, 22/39). According to CEUS enhancement patterns, most active CD patients showed a complete hyperenhancement of the entire intestinal wall (61.5%, 24/39). The TICs of active CD showed an earlier enhancement, higher peak intensity, and faster decline. Among all CEUS quantitative parameters, amplitude-derived parameters peak enhancement (PE), wash-in area under the curve (WiAUC), wash-in rate (WiR), wash-in perfusion index (WiPI), and wash-out rate (WoR) were significantly higher in active CD than in inactive CD (p < 0.05). The combined AUROC of intestinal ultrasound features and DCE-US quantitative perfusion parameters in the diagnosis of active CD was 0.987, with 97.4% sensitivity, 100% specificity, and 98.1% accuracy. CONCLUSIONS: DCE-US with quantitative perfusion parameters is a potential useful noninvasive imaging method to evaluate the activity of Crohn's disease.

The association of three vaccination doses with reduced gastrointestinal symptoms after severe acute respiratory syndrome coronavirus 2 infections in patients with inflammatory bowel disease.

Background: The protective efficacy of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination against the new-onset gastrointestinal (GI) symptoms following COVID-19 infection is critical among patients with inflammatory bowel disease (IBD); however, the optimal protective vaccine dose remains unknown. Therefore, this study aimed to clarify whether there is a correlation between SARS-CoV-2 vaccinations and GI symptoms following Omicron infection in patients with IBD. Methods: We conducted a multicenter cross-sectional study of IBD patients among three tertiary hospitals in eastern China. Professional physicians collected all data using online questionnaires. The patients were stratified into four groups: patients who were unvaccinated and patients who received one, two, or three vaccination doses. The primary outcome was the presence of any new-onset GI symptoms after SARS-CoV-2 infection before a negative SARS-CoV-2 nucleic acid test or a negative self-testing for antigens. Results: In total, 536 patients with IBD (175 unvaccinated, 31 vaccinated, 166 vaccinated with two doses, and 164 vaccinated with three doses) reported having COVID-19 infection. Compared with the unvaccinated, the three vaccination doses group was associated with reduced GI symptoms after infection (adjusted odds ratio = 0.56, 95% confidence interval 0.34-0.90, P < 0.05). Reduced diarrhea (adjusted odds ratio = 0.54, 95% confidence interval 0.31-0.92, P < 0.05) and nausea or vomiting (adjusted odds ratio = 0.45, 95% confidence interval 0.21-0.92, P < 0.05) were observed in the three vaccination doses group compared with the unvaccinated group. Conclusions: In conclusion, in the 536 patients with IBD who reported COVID-19 infection, we found that the three vaccination doses, but not the one or two doses group, were associated with reduced GI symptoms after infection compared with the unvaccinated group.

AMBRA1 promotes intestinal inflammation by antagonizing PP4R1/PP4c mediated IKK dephosphorylation in an autophagy-independent manner.

IκB kinase (IKK) complex is central regulators of the NF-κB pathway, and dysregulation of IKK phosphorylation leads to hyperactivation of proinflammatory response in various chronic inflammatory diseases, including inflammatory bowel disease (IBD). However, the dynamic modulation of IKK phosphorylation and dephosphorylation in intestinal inflammation remains uncharacterized. Here, we found that autophagy/beclin-1 regulator 1 (AMBRA1) was highly expressed in inflamed colons in a colitis mouse model and in clinical IBD samples. Importantly, AMBRA1 deletion significantly decreased proinflammatory cytokine expression and enhanced the therapeutic effect of infliximab on intestinal inflammation. Mechanistically, the N-term F1 domain of AMBRA1 was required for AMBRA1 to competitively interact with protein phosphatase 4 regulatory subunit 1 (PP4R1) and catalytic protein phosphatase 4 (PP4c) to suppress their interactions with IKK, promote the dissociation of the PP4R1/PP4c complex, and antagonize the dephosphorylation activity of this complex towards the IKK complex. In response to TNF-α stimulation, IKKα phosphorylates AMBRA1 at S1043 to stabilize AMBRA1 expression by impairing its binding to Cullin4A (CUL4A) to decrease its CUL4A-mediated K48-linked ubiquitination. Overall, our study identifies an autophagy-independent function of AMBRA1 as a positive modulator of IKK phosphorylation to promote intestinal inflammation, thus providing a new targeted therapeutic strategy for patients with refractory IBD.

Development of a scoring system for predicting the severity of ulcerative colitis.

BACKGROUND AND STUDY AIMS: Monitoring disease activity in ulcerative colitis (UC) is critical in preventing long-term complications. This study aims to develop a scoring system using non-invasive indicators to predict endoscopic activities for ulcerative colitis (UC) patients. PATIENTS AND METHODS: All enrolled patients with UC admitted to Shanghai Xinhua Hospital between June 2017 and January 2021 were enrolled, and their clinical data were retrospectively collected and a number of serological biomarkers concentrations were analyzed. Patients were categorized into mild and moderate-to-severe disease groups. Univariate and multivariate logistic regression was used to predict moderate-to-severe endoscopic activities, which were then incorporated into a nomogram to establish a prediction scoring model. RESULT: Overall, 231 patients were divided into a mild group (n = 111, 48.0%) and a moderate-to-severe group (n = 120, 52.0%). The following variables were independently associated with the disease severity and were subsequently included into the prediction model: Proteinase 3 antineutrophil cytoplasmic antibody (PR3-ANCA), C-reactive protein (CRP), hemoglobin(Hb), IL-10, stool frequency ≥ 5 times/day and hematochezia. Incorporating these 6 factors, the nomogram showed good discrimination with C-index of 0.819 and reliable calibration. A scoring model was established with the area under the curve 0.818. Moreover, PR3-ANCA and CRP correlated with the duration of hospital stay. CONCLUSION: We developed a predictive model for endoscopic disease activities by using noninvasive factors based on PR3-ANCA, CRP, Hb, IL-10, stool frequency and hematochezia. This prediction model might assist clinicians in managing patients with UC.

Oral colon-targeted responsive alginate/hyaluronic acid-based hydrogel propels the application of infliximab in colitis.

Currently, commercialized infliximab (IFX) has rapidly propelled the clinical treatment of IBD, however, its inherent attributes, such as off-target effects and rapid metabolism, severely limit practical applications. Moreover, high doses injection of IFX can result in IBD treatment failure, which may induce other side effects. In this study, an colon microenvironment-responsive hydrogel (AL/HA hydrogel), consisting of acid-resistant sodium alginate and colon-degraded and targeted hyaluronic acid, was constructed by simple Ca2+/Zn2+ cross-linking. The ion-mediated hydrogel exhibited the protective effect of gastrointestinal tract to avoid early drug leakage, while the inflammation environments showed well-controlled drug release and significant biodegradable behaviors. Additionally, oral hydrogel exhibited long-standing enteritis areas compared with normal mice. Therefore, hydrogel-assisted enteritis treatment has great potential in IBD as an oral agent. After that, IFX was packaged in hydrogel to fabricate a facile oral antibody delivery system to treat IBD. IFX-embedded hydrogel showed remarkable therapeutic effect on IBD compared with free IFX. Surprisingly, oral hydrogel below 7 times IFX achieve the same amount of IFX-infused treatment that will further help alleviate the drawbacks of IFX. Our work elaborated on the efficacy of oral AL/HA@IFX in IBD, providing a guarantee for the future of promoted clinical transformation.

Premorbid Steatohepatitis Increases the Seriousness of Dextran Sulfate Sodium-induced Ulcerative Colitis in Mice.

Background and Aims: The concurrence of nonalcoholic steatohepatitis (NASH) and ulcerative colitis (UC) is increasingly seen in clinical practice, but the underlying mechanisms remain unclear. This study aimed to develop a mouse model of the phenomenon by combining high-fat high-cholesterol diet (HFHCD)-induced NASH and dextran sulfate sodium (DSS)-induced UC, that would support mechanistic studies. Methods: Male C57BL/6 mice were randomly assigned to two groups receiving either a chow diet or HFHCD for 12 weeks of NASH modeling. The mice were the divided into four subgroups for UC modeling: (1) A control group given a chow diet with normal drinking water; (2) A colitis group given chow diet with 2% DSS in drinking water; (3) A steatohepatitis group given HFHCD with normal drinking water; and (4) A steatohepatitis + colitis group given HFHCD with 2% DSS in drinking water. Results: NASH plus UC had high mortality (58.3%). Neither NASH nor UC alone were fatal. Although DSS-induced colitis did not exacerbate histological liver injury in HFHCD-fed mice, premorbid NASH significantly increased UC-related gut injury compared with UC alone. It was characterized by a significantly shorter colon, more colonic congestion, and a higher histopathological score (p<0.05). Inflammatory (tumor necrosis factor-alpha, interleukin 1 beta, C-C motif chemokine ligand 2, and nuclear factor kappa B) and apoptotic (Bcl2, Bad, Bim, and Bax) signaling pathways were significantly altered in distal colon tissues collected from mice with steatohepatitis + colitis compared with the other experimental groups. Conclusions: Premorbid steatohepatitis significantly aggravated DSS-induced colitis and brought about a lethal phenotype. Potential links between NASH and UC pathogeneses can be investigated using this model.

Accelerated Infliximab Induction for Severe Lower Gastrointestinal Bleeding in a Young Patient with Crohn's Disease: A Case Report.

BACKGROUND: Severe lower gastrointestinal bleeding (SLGIB) is a rare complication of Crohn's disease (CD). The treatment of these patients is a clinical challenge. Monoclonal anti-TNFα antibody (IFX) can induce relatively fast mucosal healing. It has been reported for the treatment of SLGIB, but there are few reports on accelerated IFX induction in CD patients with SLGIB. CASE SUMMARY: A 16-year-old boy with a history of recurrent oral ulcers for nearly 1 year presented to the Gastroenterology Department of our hospital complaining of recurrent periumbilical pain for more than 1 mo and having bloody stool 4 times within 2 wk. Colonoscopy showed multiple areas of inflammation of the colon and a sigmoid colon ulcer with active bleeding. Hemostasis was immediately performed under endoscopy. The physical examination of the patient showed scattered small ulcers in the lower lip of the mouth and small cracks in the perianal area. Combined with his medical history, physical examination, laboratory examinations with high C-reactive protein (CRP), platelet count (PLT), erythrocyte sedimentation rate (ESR) and fecal calprotectin levels, imaging examinations and pathology, a diagnosis of CD was taken into consideration. According to the pediatric CD activity index 47.5, methylprednisolone (40 mg QD) was given intravenously. The abdominal pain disappeared, and CRP, PLT, and ESR levels decreased significantly after the treatment. Unfortunately, he had a large amount of bloody stool again after 1 wk of methylprednisolone treatment, and his hemoglobin level decreased quickly. Although infliximab (IFX) (5 mg/kg) was given as a combination therapy regimen, he still had bloody stool with his hemoglobin level decreasing from 112 g/L to 80 g/L in a short time, so-called SLGIB. With informed consent, accelerated IFX (5 mg/kg) induction was given 7 days after initial presentation. The bleeding then stopped. Eight weeks after the treatment, repeat colonoscopy showed mucosal healing; thus far, no recurrent bleeding has occurred, and the patient is symptom-free. CONCLUSION: This case highlights the importance of accelerated IFX induction in SLGIB secondary to CD, especially after steroid hormone treatment.

Artemisinin ameliorates intestinal inflammation by skewing macrophages to the M2 phenotype and inhibiting epithelial-mesenchymal transition.

BACKGROUND: Inflammatory bowel disease (IBD) is a self-destructive intestinal disease whose etiology is unclear but complex and the effective treatment is deficient. Increasing evidences have indicated that immune dysfunction and epithelial-mesenchymal transition (EMT)-related intestinal mucosal barrier impaired hold critical position in the pathogenesis of IBD. Artemisinin (ART) is a sesquiterpenoid compound extracted from Chinese herbal medicine which has good immunomodulatory effects. Studies have shown that artemisinin and its analogues have therapeutic effects on a variety of tumors and immune-related disorders. The purpose of current study was to research the effect and mechanism about artemisinin-induced macrophage polarization to M2 phenotype and inhibiting the process of EMT. METHODS: In vitro, the anti-inflammatory effect of artemisinin is mainly verified by RAW264.7 cells and tissue (colon tissue and PBMC) from CD patients with active intestinal inflammation. RAW264.7 cells stimulated with LPS to induce inflammatory state and ART were used as therapeutic treatment in different concentration. Then the expression levels of pro-inflammatory factors, macrophage polarization and ERK pathway were analyzed. Colon tissue and PBMC from CD patients were treated with ART in different concentrations and macrophage polarization, pro-inflammatory factors expression, EMT-related protein were analyzed. In vivo, DSS-induced colitis mice were treated by ART for seven days. The DAI score was calculated and the colons and spleens were harvested after the animals were sacrificed. The expression of macrophage markers and EMT-related markers in the intestines of mice in each group were monitored by qPCR and western blot. RESULT: ART treatment could decrease the levels of pro-inflammatory coefficient expressed in theRAW264.7 cells and human PBMC. Moreover, ART could ameliorate the intestinal inflammation in vivo through down-regulating the expression of pro-inflammatory factors, promoting macrophage polarization to M2 phenotype and inhibiting the process of EMT. CONCLUSION: Taken together, our findings demonstrated that artemisinin might ameliorate inflammation by inducing macrophage polarization to M2 phenotype and inhibiting the process of EMT, suggesting that ART may be applied to the rehabilitation of IBD in the future.

ABIN3 Negatively Regulates Necroptosis-induced Intestinal Inflammation Through Recruiting A20 and Restricting the Ubiquitination of RIPK3 in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: There is evidence for a disturbed necroptosis function in many inflammatory diseases, but its role in inflammatory bowel diseases [IBD] and the underlying mechanisms are unclear. Here, we studied the functional significance and molecular mechanisms of ABIN3, a ubiquitin-binding protein, in regulating the ubiquitination and activation of necroptosis in IBD. METHODS: The expression of necroptosis hallmarks and ABIN3 were assessed in inflamed samples of IBD patients, dextran sodium sulphate [DSS]-induced colitis models, and azoxymethane [AOM]/DSS models in mice. ABIN3 was overexpressed and silenced to explore its function in regulating necroptosis, inflammation, and intestinal barrier function. Immuoprecipitiation [IP] and co-IP assays were performed to investigate the cross-talk between ABIN3 and deubiquitinating enzyme A20, and the mechanisms of coordinating ubiquitination modification to regulate necroptosis. RESULTS: Excessive necroptosis is an important contributory factor towards the uncontrolled inflammation and intestinal barrier defects in IBD and experimental colitis. Blocking necroptosis by Nec-1s or GSK'872 significantly prevented cell death and alleviated DSS-induced colitis in vivo, whereas in the AOM/DSS model, necroptosis inhibitors aggravated the severity of colitis-associated colon carcinogenesis [CAC]. Mechanistically, ABIN3 is rapidly recruited to the TNF-RSC complex, which interacts and coordinates with deubiquitinating enzyme A20 to control the K63 deubiquitination modification and subsequent activation of the critical necroptosis kinase, RIPK3, to suppress necroptosis. CONCLUSIONS: ABIN3 regulates inflammatory response and intestinal barrier function by interacting with A20 and coordinating the K63 deubiquitination modification of necroptosis in IBD.

Cost-Effective Fabrication of Transparent Strain Sensors via Micro-Scale 3D Printing and Imprinting.

The development of strain sensors with high sensitivity and stretchability is essential for health monitoring, electronic skin, wearable devices, and human-computer interactions. However, sensors that combine high sensitivity and ultra-wide detection generally require complex preparation processes. Here, a novel flexible strain sensor with high sensitivity and transparency was proposed by filling a multiwalled carbon nanotube (MWCNT) solution into polydimethylsiloxane (PDMS) channel films fabricated via an electric field-driven (EFD) 3D printing and molding hybrid process. The fabricated flexible strain sensor with embedded MWCNT networks had superior gauge factors of 90, 285, and 1500 at strains of 6.6%, 14%, and 20%, respectively. In addition, the flexible strain sensors with an optical transparency of 84% offered good stability and durability with no significant change in resistance after 8000 stretch-release cycles. Finally, the fabricated flexible strain sensors with embedded MWCNT networks showed good practical performance and could be attached to the skin to monitor various human movements such as wrist flexion, finger flexion, neck flexion, blinking activity, food swallowing, and facial expression recognition. These are good application strategies for wearable devices and health monitoring.

Boosting mTOR-dependent autophagy via upstream TLR4-MyD88-MAPK signalling and downstream NF-κB pathway quenches intestinal inflammation and oxidative stress injury.

BACKGROUND AND AIMS: Defective autophagy has been proposed as an important event in a growing number of autoimmune and inflammatory diseases such as rheumatoid arthritis and lupus. However, the precise role of mechanistic target of rapamycin (mTOR)-dependent autophagy and its underlying regulatory mechanisms in the intestinal epithelium in response to inflammation and oxidative stress remain poorly understood. METHODS: The levels of p-mTOR, LC3B, p62 and autophagy in mice and LPS-treated cells were examined by immunoblotting, immunohistochemistry, confocal microscopy and transmission electron microscopy (TEM). We evaluated the expression of IL-1ß, IL-8, TNF-α, MDA, SOD and T-AOC by quantitative real time-polymerase chain reaction (qRT-PCR) and commercially available kits after silencing of mTOR and ATG5. In vivo modulation of mTOR and autophagy was achieved by using AZD8055, rapamycin and 3-methyladenine. Finally, to verify the involvement of TLR4 signalling and the NF-κB pathway in cells and active ulcerative colitis (UC) patients, immunofluorescence, qRT-PCR, immunoblotting and TEM were performed to determine TLR4 signalling relevance to autophagy and inflammation. RESULTS: The mTOR-dependent autophagic flux impairment in a murine model of colitis, human intestinal epithelial cells and active UC patients is probably regulated by TLR4-MyD88-MAPK signalling and the NF-κB pathway. Silencing mTOR remarkably attenuated, whereas inhibiting ATG5 aggravated, LPS-induced inflammation and oxidative injury. Pharmacological administration of mTOR inhibitors and autophagy stimulators markedly ameliorated experimental colitis and oxidative stress in vivo. CONCLUSIONS: Our findings not only shed light on the regulatory mechanism of mTOR-dependent autophagy, but also provided potential therapeutic targets for intestinal inflammatory diseases such as refractory inflammatory bowel disease.

miR-155 Regulates IL-10-Producing CD24hiCD27+ B Cells and Impairs Their Function in Patients with Crohn's Disease.

Regulatory interleukin-10 (IL-10)-producing B cells (B10 cells) play a critical role in preventing and curing autoimmune diseases in experimental mouse models. However, the precise cellular and molecular mechanisms of action of B10 cells in humans, especially in patients with Crohn's disease (CD), remain to be determined. miR-155 regulates many physiological and pathological conditions, including inflammation such as that in CD. In this study, we aimed to explore the effect of miRNA-155 on IL-10 production by B cells in healthy controls (HCs) and CD patients. Interestingly, we found that CD24hiCD27+ B cells express high levels of miRNA-155 and IL-10, which are positively correlated. Additionally, CD24hiCD27+ B cells express higher levels of Toll-like receptor 9 than those found in other B cell subsets. Overexpression of miRNA-155 promotes IL-10 production, while inhibition of miRNA-155 decreases IL-10 production. We determined that miR-155 directly inhibits the expression of Jarid2, which reduces H3K27me3 binding to the IL10 promoter and increases IL-10 gene expression. In coculture systems, the CD24hiCD27+ B cells from HCs suppressed the secretion of TNFα and IFNγ by monocytes and T cells, respectively. However, the number and function of CD24hiCD27+ B cells from CD patients were decreased. Moreover, we found that miR-155 induces CD24hiCD27+ B cells to produce higher levels of TNFα instead of IL-10 in CD patients than in the controls and that the increased number of IL-10+TNFα+ B cells reduces the induction of Foxp3 expression and the inhibition of IFNγ production by CD4+CD25- T cells, as well as TNFα production by monocytes. Our study demonstrates the critical role of miRNA-155 in the regulation of IL-10 production by B cells and reveals the novel molecular mechanism underlying the functional impairment of B10 cells in CD patients. Our study has the potential to drive the development of B10 cell-based strategies to ameliorate disease progression in CD patients.

Protein Arginine Methyltransferase 5 Inhibition Upregulates Foxp3+ Regulatory T Cells Frequency and Function during the Ulcerative Colitis.

Ulcerative colitis (UC) pathogenesis is related to imbalance of immune responses, and the equilibrium between inflammatory T cells and Foxp3+ regulatory T cells (Tregs) plays an important role in the intestinal homeostasis. Protein arginine methyltransferases (PRMTs) regulate chromatin remodeling and gene expression. Here, we investigated whether inhibition of PRMTs affects colitis pathogenesis in mice and inflammatory bowel disease patients and further explored the underlying mechanisms. In this study, we found that protein arginine N-methyltransferase inhibitor 1 (AMI-1) treatments increased Tregs frequency, function, and reduced colitis incidence. Adoptive transfer of AMI-1-treated Tregs could reduce the colitis incidence. Colitis was associated with increased local PRMT5 expression, which was inhibited by AMI-1 treatment. Additionally, PRMT5 knockdown T cells produced a better response to TGFß and promoted Tregs differentiation through decreased DNA methyltransferase 1 (DNMT1) expression. PRMT5 also enhanced H3K27me3 and DNMT1 binding to Foxp3 promoter, which restricted Tregs differentiation. Furthermore, PRMT5 knockdown led to decreased Foxp3 promoter methylation during Tregs induction. PRMT5 expression had a negative relationship with Tregs in UC patients, knockdown of PRMT5 expression increased Tregs frequency and decreased TNFα, IL-6, and IL-13 levels. Our study outlines a novel regulation of PRMT5 on Tregs development and function. Strategies to decrease PRMT5 expression might have therapeutic potential to control UC.

Inhibition of Autophagic Degradation Process Contributes to Claudin-2 Expression Increase and Epithelial Tight Junction Dysfunction in TNF-α Treated Cell Monolayers.

Tight junction dysfunction plays a vital role in some chronic inflammatory diseases. Pro-inflammatory cytokines, especially tumor necrosis factor alpha (TNF-α), act as important factors in intestinal epithelial tight junction dysfunction during inflammatory conditions. Autophagy has also been shown to be crucial in tight junction function and claudin-2 expression, but whether autophagy has an effect on the change of claudin-2 expression and tight junction function induced by TNF-α is still unknown. To answer this question, we examined the expression of claudin-2 protein, transepithelial electrical resistance (TER), and permeability of cell monolayers, autophagy flux change, and lysosomal pH after TNF-α with or without PP242 treatment. Our study showed that claudin-2 expression, intestinal permeability, microtubule-associated protein 1 light chain 3B II (LC3B-II) and sequestosome 1 (P62) expression largely increased while TER values decreased in TNF-α treated cell monolayers. Further research using 3-methyladenine (3-MA), bafilomycin A1, and ad-mCherry-GFP-LC3B adenovirus demonstrated that LC3B-II increase induced by TNF-α was attributed to the inhibition of autophagic degradation. Moreover, both qualitative and quantitative method confirmed the increase of lysosomal pH, and mammalian target of rapamycin (mTOR) inhibitor PP242 treatment relieved this elevation. Moreover, PP242 treatment also alleviated the change of autophagy flux, TER, and claudin-2 expression induced by TNF-α. Therefore, we conclude that increase of claudin-2 levels and intestinal epithelial tight junction dysfunction are partly caused by the inhibition of autophagic degradation in TNF-α treated cell monolayers.

Expression and purification of functional HMGB1 A box by fusion with SUMO.

High­mobility­group­box chromosomal protein 1 (HMGB1) is a ubiquitous and abundant nuclear protein in eukaryotic cells. Nuclear HMGB1 serves an important role in maintaining nuclear stability under stress. However, extracellular HMGB1 exerts actions, which are distinctly different compared with these intracellular functions. HMGB1, when released extracellularly, is a potent innate signal, which initiates host defense mechanisms or tissue regeneration. HMGB1 has two DNA­binding domains: HMG A box and B box. The HMGB1 A box exhibits an antagonistic, anti­inflammatory effect, and is a potential therapeutic target, however, the large­scale expression and purification of the HMGB1 A box with high efficiency remains to be reported. In the present study, a SUMO­fusion expression system was used to express and purify high levels of functional HMGB1 A box to meet the requirements of therapeutic protein production.

Integrin antagonists are effective and safe for Crohn's disease: a meta-analysis.

AIM: To evaluate the efficacy and safety of integrin antagonists, including natalizumab and vedolizumab, in Crohn's disease (CD). METHODS: We carried out a literature search in PubMed, MEDLINE, EMBASE and the Cochrane Library to screen for citations from January 1990 to August 2014. Data analysis was performed using Review Manager version 5.2. RESULTS: A total of 1340 patients from five studies were involved in this meta-analysis. During 6-12 wk treatment, integrin antagonists increased the rate of clinical response and remission with OR = 1.69, 95%CI: 1.37-2.09 and 1.84, 95%CI: 1.44-2.34, respectively. No significant difference was found between integrin antagonists and placebo treatments regarding their adverse reactions (OR = 1.07, 95%CI: 0.83-1.38) and serious adverse reactions (OR = 0.81, 95%CI: 0.57-1.15). CONCLUSION: The results prove the efficacy and safety of integrin antagonists for CD treatment, although the treatment strategies varied.

Infliximab preferentially induces clinical remission and mucosal healing in short course Crohn's disease with luminal lesions through balancing abnormal immune response in gut mucosa.

This study was undertaken to evaluate the efficacy of infliximab (IFX) in treatment of Crohn's disease (CD) patients. 106 CD patients were undergoing treatment with IFX from five hospitals in Shanghai, China. Clinical remission to IFX induction therapy was defined as Crohn's disease activity index (CDAI) < 150. Clinical response was assessed by a decrease in CDAI ≥ 70, and the failure as a CDAI was not significantly changed or increased. Ten weeks after therapy, 61 (57.5%) patients achieved clinical remission, 17 (16.0%) had clinical response, and the remaining 28 (26.4%) were failed. In remission group, significant changes were observed in CDAI, the Simple Endoscopic Score for Crohn's Disease (SES-CD), and serum indexes. Patients with short disease duration (22.2 ± 23.2 months) and luminal lesions showed better effects compared to those with long disease duration (71.0 ± 58.2 months) or stricturing and penetrating lesions. IFX markedly downregulated Th1/Th17-mediated immune response but promoted IL-25 production in intestinal mucosa from remission group. No serious adverse events occurred to terminate treatment. Taken together, our studies demonstrated that IFX is efficacious and safe in inducing clinical remission, promoting mucosal healing, and downregulating Th1/Th17-mediated immune response in short course CD patients with luminal lesions.

Depletion of thymosin ß4 promotes the proliferation, migration, and activation of human hepatic stellate cells.

BACKGROUND & AIMS: It has recently been reported that thymosin beta-4 (Tß4) has anti-fibrogenic effects in human hepatic stellate cells (HSCs) in vitro, but the mechanisms underlying these effects remain unclear. The aim of this study was to investigate the roles of Tß4 in the proliferation, migration, and activation of HSCs. METHODS: Enzyme-linked immunosorbent assays (ELISA), immunohistochemistry, and western blot assays were utilized to determine the expression levels of Tß4 in serum, liver tissues, and LX-2 cells. Tß4 was depleted in LX-2 cells using small interfering RNAs (siRNAs). Cell proliferation was analyzed using cell counting kit-8 (CCK-8) viability assays, and cell migration was investigated using wound-healing and transwell migration assays. RESULTS: The expression of Tß4 was significantly reduced during the progression of liver fibrosis. The depletion of Tß4 significantly promoted the proliferation and migration of LX-2 cells via the activation of the PI3K/Akt signaling pathway. The pro-migratory and pro-proliferative effects of Tß4 depletion in LX-2 cells can be counteracted by treatment with the Akt inhibitor MK-2206. In addition, Tß4 depletion was also associated with the activation of HSCs via the enhanced expression of α-smooth muscle actin (α-SMA) and vimentin. CONCLUSIONS: Our results suggest that Tß4 participates in liver fibrosis by inhibiting the migration, proliferation, and activation of HSCs and that Tß4 may be an effective target in the treatment of liver fibrosis.

ß-catenin is overexpressed in hepatic fibrosis and blockage of Wnt/ß-catenin signaling inhibits hepatic stellate cell activation.

ß-catenin, a core component of Wnt/ß-catenin signaling, has been shown to be an important regulator of cellular proliferation and differentiation. Abnormal activation of Wnt/ß-catenin signaling promotes tissue fibrogenesis. In the present study, the role of ß-catenin during liver fibrogenesis was analyzed and the functional effects of ß-catenin gene silencing in hepatic stellate cells (HSCs) using small interfering (si)RNA were investigated. The expression of ß-catenin in human hepatic fibrosis tissues of different grades and normal human hepatic tissues was examined using immunohistochemistry. To inhibit the Wnt/ß-catenin signaling pathway, siRNA for ß-catenin was developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000. ß-catenin expression was evaluated by quantitative polymerase chain reaction (qPCR) and western blot analysis. The expression of collagen types Ⅰ and Ⅲ was evaluated by qPCR and immunofluorescent staining. Cellular proliferation and the cell cycle were analyzed using a methyl thiazolyl tetrazolium assay. Apoptosis was assessed by Annexin V staining. A higher expression level of ß-catenin was identified in the patients with high-grade hepatic fibrosis in comparison with that of the normal controls. Additionally, ß-catenin siRNA molecules were successfully transfected into HSCs and induced inhibition of ß-catenin expression in a time-dependent manner. ß-catenin siRNA treatment also inhibited synthesis of collagen types Ⅰ and Ⅲ in transfected HSCs. Furthermore, compared with those of the control group, siRNA-mediated knockdown of ß-catenin in HSC-T6 cells inhibited cell proliferation and resulted in cell apoptosis. This study suggests a significant functional role for ß-catenin in the development of liver fibrosis and demonstrates that downregulation of the Wnt/ß-catenin signaling pathway inhibits HSC activation. Thus, this study provides a novel strategy for the treatment of hepatic fibrosis.