[Clinical features and genetic analysis of three patients with Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome due to variants of FOXP3 gene].

OBJECTIVE: To analyze the clinical characteristics of three patients with Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. METHODS: Three patients with IPEX syndrome diagnosed at the Children's Hospital of Fudan University from January 24, 2013 to July 29, 2019 were selected as the study subjects. Their clinical features, laboratory investigations and results of genetic testing were summarized. Treatment and prognosis were also explored. RESULTS: All of the three children had developed the disorder during infancy. One child had initial features including diabetes and diabetic ketoacidosis, whilst the other two had initiated by diarrhea. All patients had gastrointestinal involvement, and one was diagnosed as very early onset inflammatory bowel disease by colonoscopy and biopsy. Two children also had endocrine glands involvement. One child had manifested type 1 diabetes and positivity for thyroglobulin and thyroid peroxidase antibodies, though his thyroid function had remained normal. Another one had hypothyroidism and was treated by levothyroxine. Genetic testing revealed that all children had harbored missense variants of the FOXP3 gene, including c.1222G>A (p.V408M), c.767T>C (p.M256T) and c.1021A>G (p.T341A). The clinical symptoms of one patient were alleviated following allogeneic hematopoietic stem cell transplantation. One patient was stable after treatment with infliximab plus insulin, and one child had died of refractory septic shock and multiple organ dysfunction syndrome at 3 months old. CONCLUSION: FOXP3 gene variant-associated IPEX syndrome may have very early onset and diverse clinical manifestations. For male patients with infantile onset chronic diarrhea, multiple endocrine or multiple system involvement, genetic testing is recommended, which may facilitate early diagnosis, treatment and genetic counseling.

[Analysis of a child with Very early onset inflammatory bowel disease due to compound heterozygous variants of IL10RA and DUOX2 genes].

OBJECTIVE: To explore the genetic basis of a child with Very early onset inflammatory bowel disease (VEOIBD). METHODS: A female child who had presented at the Children's Hospital of Fudan University on May 23, 2018 due to occurrence of diarrhea and fever 6 days after birth was selected as the study subject. Clinical data of the child was collected. Family-based whole-exome sequencing (WES) was carried out. Candidate variant was verified by Sanger sequencing and PCR of the patient and her parents. RESULTS: The child had developed the symptoms 6 days after birth, with main manifestations including diarrhea, fever, failure to thrive, rectovestibular fistula and hypothyroidism. An enterostomy was performed at the age of 3.5 months due to severe intestinal adhesion and obstruction. Based on her clinical manifestations, colonoscopic finding, and results of biopsies, she was diagnosed with VEOIBD in conjunct with congenital hypothyroidism. Replacement treatment of levothyroxine was given since one month of age. Family-based WES revealed that the child has harbored compound heterozygous variants of the DUOX2 gene, namely c.2654G>T (p.R885L) and c.505C>T (p.R169W), in addition with a heterozygous c.301C>T (p.R101W) variant of the IL10RA gene. Re-analysis of the WES data revealed that the patient also had a 333 bp deletion spanning exon 1 of the IL10RA gene (Chr11: 117857034_117857366). CONCLUSION: For patients with VEOIBD, genetic testing is recommended. Presence of additional DUOX2 gene variants might have exacerbated the clinical symptoms in this patient. Above finding has facilitated genetic counseling and prenatal diagnosis for this family, and raised clinicians' awareness of this rare disease.

Clinical outcome of infantile-onset inflammatory bowel disease in 102 patients with interleukin-10 signalling deficiency.

BACKGROUND: Infantile-onset inflammatory bowel disease can be caused by defects in interleukin-10 signalling. The natural history and clinical outcomes of allogeneic haematopoietic stem cell transplantation, medical treatment and surgery have not been thoroughly described. AIMS: This study evaluates disease progression and clinical outcome in patients with interleukin-10 signalling deficiency. METHODS: One hundred and nine patients with interleukin-10 signalling deficiency were retrospectively reviewed from a single tertiary centre. The Kaplan-Meier method was applied to calculate probabilities of survival and interval between transplant and stoma closure. RESULTS: One hundred and nine patients were reviewed, and 102 patients were included in the survival analysis. One hundred and eight patients were identified with IL10RA mutations, and one patient harboured IL10RB mutation. Seventy-three patients received haematopoietic stem cell transplantation. The overall survival after transplantation was 64.2% (95% confidence interval, 52.8 to 75.6), and without transplantation, it was 47.5% (95% confidence interval, 14.8 to 80.2, P = 0.47). The median timeframe between transplant and stoma closure was 19.6 months. The probability of survival was significantly lower in patients with perforation (P < 0.001), ileus (P = 0.038) and without thalidomide treatment (P < 0.001) among patients who did not receive haematopoietic stem cell transplantation. The survival probability was not associated with timeframe between transplant and onset, graft source and genotypes. CONCLUSIONS: The survival probability was not significantly different between patients with transplantation and the non-transplanted patients.

Long-term changes in the gut microbiota after triple therapy, sequential therapy, bismuth quadruple therapy and concomitant therapy for Helicobacter pylori eradication in Chinese children.

BACKGROUND: We previously reported that the administration of 14-day standard triple therapy (TT), sequential therapy (ST), bismuth-based quadruple therapy (BT), and concomitant therapy (CT) as the first-line therapy for Helicobacter pylori infection in Chinese children achieved eradication rates of 74.1%, 69.5%, 89.8%, and 84.6%, respectively. In this follow-up study, we further evaluated the short- and long-term effects of the four regimens on the gut microbiota in these children. METHODS: We prospectively recruited treatment-naïve children with H. pylori infection. Fecal samples were collected at week 0, 2, 6, and 52, and alterations in the gut microbiota were analyzed by 16S rRNA gene sequencing. RESULTS: Sixty-three patients participated in this study (16 with TT, 15 with ST, 16 with BT and 16 with CT). At week 2, the alpha diversity (Shannon and Chao 1 index) was significantly reduced in the TT (p = 0.008, p < 0.001), ST (p < 0.001, p < 0.001), BT (p < 0.001, p < 0.001) and CT groups (p < 0.001, p < 0.001). Some changes persisted in the ST, BT, and CT groups at week 6, and all were restored (expect p = 0.02 with Chao 1 index in the CT group) at week 52. The beta diversity was significantly changed in the BT (p = 0.001) and CT groups (p = 0.001) 2 weeks post-eradication and restored 1 year after therapy. Immediately after therapy, the relative abundance of Proteobacteria was strikingly increased in the ST (p = 0.005), BT (p < 0.001) and CT groups (p < 0.001), and the genus-level analysis showed that the abundances of 23.1%, 43.3%, 78.6%, and 78% of the bacterial genera in the TT, ST, BT, and CT groups were significantly changed. All these changes returned to almost the pre-eradication level 1 year post-eradication. CONCLUSION: Eradication of H. pylori infection can lead to transient dysbiosis of gut microbiota, and these changes almost recovered 1 year post-eradication, which indicates the long-term safety of H. pylori therapy.

Effect of Exclusive Enteral Nutrition on the Disease Process, Nutrition Status, and Gastrointestinal Microbiota for Chinese Children With Crohn's Disease.

BACKGROUND: The aim of this study was to prospectively study the effect of exclusive enteral nutrition (EEN) treatment on Chinese pediatric Crohn's disease (CD) patients. METHODS: Thirty-one newly diagnosed CD patients were enrolled in this study and treated with EEN for 8 weeks. Twelve healthy controls (HCs) donated their fecal samples. Statistical methods were used to compare the differences. RESULTS: According to the Simple Endoscopic Score for CD (SES-CD) at the end of the EEN treatment, 21 patients with SES-CD ≤4 were classified into the remission group (CD-RE), and 10 patients with SES-CD >4 were classified into the nonremission group (CD-NRE). After EEN therapy, there was a significant decrease in the SES-CD, the weighted Pediatric Crohn's Disease Activity Index (wPCDAI), and fecal calprotectin (FCP) in the CD-RE group (all P < .001). The wPCDAI and FCP also decreased in the CD-NRE group (both P < .05). In terms of nutrition improvement, the CD-RE group patients showed more improvement in weight gain, hemoglobin, and serum albumin level than the CD-NRE group patients (all P < .05). For the microbiota, the CD patients had a lower bacterial diversity and different bacterial community compared with HCs. EEN increased overall diversity and was able to shift the dysbiosis in CD patients toward a healthier state. Absence of improvement in wPCDAI and Shannon index at 2 weeks predicts poor response at the end of EEN. CONCLUSION: EEN can be used in most Chinese pediatric CD patients to induce remission and improve nutrition.

Thalidomide Inhibits Angiogenesis via Downregulation of VEGF and Angiopoietin-2 in Crohn's Disease.

Immune-mediated angiogenesis is important in the pathogenesis of inflammatory bowel disease and targeted treatment could alleviate the disease. Thalidomide is an effective drug in inflammatory bowel disease, which might be related to its multiple role in anti-inflammatory, immunoregulatory, and anti-angiogenesis. This study is to investigate the effect of thalidomide on angiogenesis in tissues from patients and in vitro cells. Angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), VEGF, and CD31 expressions in intestinal mucosa from pediatric CD patients before and after thalidomide treatment were measured by immunohistochemistry. Western blotting and polymerase chain reaction were performed to characterize the change of angiogenic factors before and after treatment in remission. Human umbilical vein endothelial cells (HUVECs) treated by thalidomide were used to examine its effect on endothelial cell proliferation and migration and capillary-like structures. Results showed that VEGF and Ang-2 levels were significantly greater in CD patients over controls. Thalidomide produced a significant reduction in protein expression of Ang-2 and VEGF, along with a decrease in mRNA expression of Ang-2. While, Ang-1 level did not show a statistically significant change. Thalidomide significantly inhibited cell proliferation in a dose-dependent manner. It also suppressed VEGF- and Ang-2-induced cell migration and capillary-like tube formation in HUVECs. Therefore, our study suggests that VEGF and Ang-2 levels are up-regulated in pediatric CD patients. It also indicated that thalidomide can be able to deactivate endothelium by the downregulation effect on angiogenic factors by targeting VEGF and Ang-2.

Fecal Microbial Signatures Are Associated With Engraftment Failure Following Umbilical Cord Blood Transplantation in Pediatric Crohn's Disease Patients With IL10RA Deficiency.

OBJECTIVES: Umbilical cord blood transplantation (UCBT) is associated with a relatively high rate of engraftment failure. This study aimed at exploring whether any fecal microbiota could be associated with engraftment failure following UCBT in Crohn's disease patients with IL10RA deficiency. METHODS: Thirteen patients were recruited and their 230 fecal samples were collected longitudinally from immediately before conditioning chemotherapy to 8 weeks post the UCBT. The V3-V4 regions of the bacterial 16S rRNA gene were amplified by PCR and sequenced, followed by bioinformatics analyses. RESULTS: Following the UCBT, 7 out of 13 patients achieved neutrophil and platelet engraftment with a median of 21 and 28 days, respectively (S group), while 6 patients failed to achieve engraftment (F group). In comparison with that in the S group, significantly lower Shannon diversity values on the UCBT day (P = 0.0176) and less abundance of Bifidobacterium longum, Bifidobacterium pseudolongum, Enterobacteriaceae_538000, and one taxon of Lachnospiraceae family was detected in the F group, accompanied by significantly higher abundances of four taxa including Lautropia, Pseudomonas, and species Microvirgula aerodenitrificans during the chemotherapy period as well as UCBT. The abundances of thirty OTUs were correlated significantly with clinical indices. CONCLUSIONS: Microbial indicators of reduced diversity of microbiota and signatures of specific bacterial abundances, such as a lower abundance of Bifidobacterium longum, for engraftment failure would require validation. These indicators may help for the risk stratification in patients with IL10RA deficiency undergoing UCBT.

Intestinal dysbiosis in pediatric Crohn's disease patients with IL10RA mutations.

BACKGROUND: Several studies have employed animal models to explore the association between microbiota and interleukin (IL) 10 signaling; however, limited information is available about the human microbiome. AIM: To characterize the microbiome in patients with IL10RA mutations and to explore the association between gut dysbiosis and disease severity. METHODS: Fecal samples were collected from patients who were diagnosed with loss-of-function mutations in the IL10RA gene between January 2017 and July 2018 at the Children's Hospital of Fudan University. Age-matched volunteer children were recruited as healthy controls. Patients with Crohn's disease (CD) were used as disease controls to standardize the antibiotic exposure. Microbial DNA was extracted from the fecal samples. All analyses were based on the 16S rRNA gene sequencing data. RESULTS: Seventeen patients with IL10RA mutations (IL10RA group), 17 patients with pediatric CD, and 26 healthy children were included. Both patients with IL10RA mutations and those with CD exhibited a reduced diversity of gut microbiome with increased variability. The relative abundance of Firmicutes was substantially increased in the IL10RA group (P = 0.02). On further comparison of the relative abundance of taxa between patients with IL10RA mutations and healthy children, 13 taxa showed significant differences. The IL10RA-specific dysbiosis indices exhibited a significant positive correlation with weighted pediatric CD activity index and simple endoscopic score for CD. CONCLUSION: In patients with IL10RA mutations and early onset inflammatory bowel disease, gut dysbiosis shows a moderate association with disease severity.

Cytomegalovirus enteritis with intractable diarrhea in infants from a tertiary care center in China.

Background: Cytomegalovirus (CMV) is rarely thought to be the cause of significant gastrointestinal infection in immunocompetent children. CMV colitis is seldom observed in young infants. This study aims to examine the clinical features of CMV colitis in Chinese children.Methods: Patients with infantile onset CMV colitis diagnosed in intestinal tissue at Children's Hospital of Fudan University from 1st January 2017, to 31st January 2019 were enrolled. Clinical data were retrieved from medical records, and the literature on infant CMV colitis was also reviewed.Results: Ten patients were included with a median age of 2.5 months [interquartile range 2.0, 6.3 months]. All 10 patients had diarrhea, 10 patients had anemia, seven patients reported hematochezia, five patients had hypoalbuminemia, five patients had retinitis, two patients had hearing impairment, and one patient had perianal abscess and anal fistula. The patients had punched-out ulcerations, longitudinal ulcerations or irregular ulcerations on the rectum and/or colon. Typical histologic evaluation showed crypt distortion and inflammatory infiltration. CMV inclusion bodies were noted in four patients. Immunohistochemistry on intestinal tissue was performed to diagnose CMV, with all patients positive. After follow-up, all patients are clinically recovered or in remission; six patients received antiviral therapy, and five patients had healed ulcers on endoscopic examination.Conclusions: CMV colitis might be a rare cause of intractable diarrhea in immunocompetent children. Clinicians should be aware of the possibility of CMV colitis in patients with intractable diarrhea.

Long-term outcomes of thalidomide in pediatric Crohn's disease.

BACKGROUND AND AIM: In this largest pediatric cohort to date in Asian population, we aimed to report our long-term real-life experience with thalidomide treatment in pediatric Crohn's disease (CD). METHODS: A retrospective single-center analysis of pediatric CD patients treated by thalidomide was conducted. The clinical characteristics and outcomes were extracted. Primary outcomes were clinical response and remission rate at different time points, especially comparing the difference between monogenic and non-monogenic mutation patients. We also evaluated the long-term safety of thalidomide. RESULTS: A total of 62 patients met the inclusion criteria. The median follow-up period was 30.5 months. Among all, 19 patients (30.6%) were diagnosed with monogenic mutation during treatment. Clinical remission rate was 53.2% (33/62) at 6 months, 54.8% (34/62) at 12 months, and 33.9% (21/62) at the end of follow-up. Clinical remission rates between non-monogenic and monogenic groups at the end were statistically different (44.2% [19/43] vs 10.5% [2/19], P < 0.05). At 12 months, 66.7% (30/45) were with normalized C-reactive protein level. Most patients (95.4%, 21/22) discontinued steroids with a median time of 4.4 months. Twelve patients relapsed, but no risk factor was identified to be significantly associated with relapse. A total of 45.2% (28/62) patients experienced an adverse event, in which 22 patients stopped thalidomide due to safety concern. Cumulative dose was not associated with abnormal electromyography but with the occurrence of adverse events. CONCLUSIONS: Thalidomide was clinically efficacious and safe among pediatric CD. Our results suggest that it is an alternative therapy in monogenic mutation patients.

Correction: Clinical and genetic spectrum of children with congenital diarrhea and enteropathy in China.

There is an error in the figure legend of Figure 1(a). The correct figure legend for this figure should be "Fig.1 (a) Hematoxylin and eosin (H&E) analysis of the descending duodenum shows the loss of goblet cells and Paneth cells and the presence of apoptotic cells in patient 48."

Clinical and genetic spectrum of children with congenital diarrhea and enteropathy in China.

PURPOSE: Genetic sequencing for children with congenital diarrhea and enteropathy (CODE) has important implications for the diagnosis, prognosis, and implementation of precision medicine. METHODS: We performed exome sequencing or targeted panel sequencing on 137 children with CODE. Endoscopic, imaging, histological, and immunological assessments were also applied. Patients were divided into three subgroups: watery, fatty, and bloody diarrhea. RESULTS: The median age of onset among patients was 28.0 (interquartile range: 7.5-120.0) days. Genetic diagnosis was achieved in 88/137 (64.2%) of patients. The diagnostic rate was significantly higher in the neonatal group than in the group of patients who had disease onset within 2 years of age (p = 0.033). The diagnostic rates were 71.9% (46/64) for targeted gene panel sequencing and 57.5% (42/73) for exome sequencing (p = 0.081). We identified pathogenic variants in 17 genes. Based on genetic sequencing, 59.9% of patients were diagnosed with medically actionable disorders. Precision medicine was carried out by means of hematopoietic stem cell transplantation for patients with IL10RA, CYBB, or FOXP3 deficiency; pancreatic enzyme replacement for patients with SBDS or UBR1 deficiency; and a special diet for patients with SLC5A1 deficiency. The overall mortality rate was 14.6%. CONCLUSION: Single-gene disorders are common among CODE patients. Genetic diagnosis can improve therapy by enabling precision medicine.

Phenotypic Characterization of Very Early-Onset Inflammatory Bowel Disease with Interleukin-10 Signaling Deficiency: Based on a Large Cohort Study.

BACKGROUND: Interleukin-10 (IL10)/interleukin-10 receptor (IL10R) deficiency is a rare disease with life-threatening infantile-onset colitis. We sought to accurately phenotype this disorder based on a large cohort of patients with a proven defect of IL10 signaling and to clarify the effects of allogeneic hematopoietic stem cell transplantation (HSCT). METHODS: We analyzed the phenotypes of our 61 patients and reviewed 78 other previously reported cases with identified mutations in the genes encoding IL10 or IL10R. We also compared the clinical features of patients with interleukin-10 receptor B (IL10RB), interleukin-10 receptor A (IL10RA), and IL10 mutations. The therapeutic effects of allogeneic HSCT were evaluated. RESULTS: We found that the disease onset time was extremely early: 70.3% within 30 days postnatal and 94.9% within the first 6 months of life. In addition, 94.2% of patients typically presented with perianal lesions. Oral ulcers and skin rash were common extra-intestinal manifestations (33.8% and 51.8%, respectively). There was no statistically significant difference in disease onset time, perianal lesion involvement, or mortality rate among patients with IL10RB, IL10RA, or IL10 deficiency. However, the surgery rate was higher in patients with IL10RB mutations than in those with IL10 or IL10RA mutations (P < 0.05). Compared with those with IL10RA deficiency, a higher percentage (32%, 9 of 28) of patients with IL10RB mutations developed B-cell lymphoma (P < 0.01). Compared with other regions, a higher percentage (98.7%) of IL10RA mutations was detected among patients in East Asia countries (P < 0.01), with hot-spot mutation sites of c.C301T and c.G537A. Allogeneic HSCT is efficacious but has a high mortality rate (17.5%, 7 of 40). CONCLUSIONS: Our study expands the current knowledge on the genotype-correlated phenotypes with a defect of IL10 signaling. B-cell lymphoma was more frequent than would be expected in patients with IL10RB mutations. There may be a unique genetic architecture among Eastern Asia compared with other populations. Although allogeneic HSCT represents a causal therapeutic approach for IL10-and IL10R-deficient patients, a word of caution is warranted.

Infantile Onset Intractable Inflammatory Bowel Disease Due to Novel Heterozygous Mutations in TNFAIP3 (A20).

Background: Mutations in tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a key player in the negative feedback regulation of nuclear factor-κB signaling, have recently been recognized as leading to early onset autoinflammatory and autoimmune syndrome. Here, we have reported the phenotypes of 3 infantile onset intractable inflammatory bowel disease (IBD) patients with TNFAIP3 mutations and reviewed previously reported cases to establish phenotypic features associated with TNFAIP3 monogenicity. Methods: From January 1, 2015, to December 31, 2017, we recruited 58 infantile-onset IBD patients. Targeted sequencing and whole-exome sequencing were performed. Sanger sequencing confirmed the variants and determined the parental origin. We followed all the patients with TNFAIP3 mutations in our cohort and analyzed their clinical data. Results: Genetic screening in all 58 patients with infantile-onset IBD revealed 44 (75.9%) cases of monogenic disorders, and 3 de novo TNFAIP3 mutations were identified, including 1 nonsense and 2 frame shift mutations. All the mutations resulted in premature stop codon. All 3 patients had multiple systemic involvements, with predominant gastrointestinal diseases. Conclusions: Most infantile-onset IBD was associated with monogenetic mutation, and in addition to the 50 reported genes, other rare genetic variants need to be determined. TNFAIP3 may be an important candidate gene. The treatment of TNFAIP3-associated infantile-onset-IBD was challenging. 10.1093/ibd/izy165_video1izy165.video15789607089001.

Mutations in Interleukin-10 Receptor and Clinical Phenotypes in Patients with Very Early Onset Inflammatory Bowel Disease: A Chinese VEO-IBD Collaboration Group Survey.

BACKGROUND: Interleukin-10 (IL10) signaling plays an important role in the pathogenesis of very early onset inflammatory bowel disease (VEO-IBD) in children. However, little is known about the role of the IL10 axis in children with VEO-IBD in China. METHODS: The Chinese VEO-IBD Collaboration Group was created to collect clinical and genetic data from patients deficient in IL10 and the IL10 receptor. High-throughput sequencing was performed to identify mutations in these genes. RESULTS: We identified 32 compound heterozygous mutations and 9 homozygous mutations in IL10 receptor subunit alpha and 1 homozygous mutation in IL10 receptor subunit beta. Among these mutations, 10 novel mutations were identified, and 6 pathogenic mutations had been previously described. In patients with IL10 receptor subunit alpha mutations, c.301C>T (p.R101RW) and c.537 G>A (p.T179T) were the most common mutations. For 88.1% of the patients, the initial symptom was diarrhea, with a time of onset of 10.4 ± 8.0 days. Oral ulcers were the first symptom in 23.8% of the patients, with a time of onset of 9.7 ± 2.8 days. Extraintestinal manifestations included perianal abscesses (22/42), perianal fistulas (23/42), oral ulcers (20/42), and recurrent eczema (15/42). Twelve patients underwent enterostomy. These patients also had lower average Z scores in height-for-age and weight-for-age. Various treatment strategies were used, including fecal microbiota transplantation; however, only hematopoietic stem cell transplantation was efficacious. CONCLUSIONS: This study identified genotypes and phenotypes of Chinese VEO-IBD infants with IL10 receptor mutations. Our study expands the current knowledge on the involvement of the IL10 axis in patients with VEO-IBD.

A20 inhibits lipopolysaccharide-induced inflammation in enterocytes.

AIM: To examine the role of A20 in the regulation of intestinal epithelial cells (IECs) inflammation. METHODS: Using gene transfection, both stable overexpression and knockdown A20-expressed HT-29 cell lines were established. Accordingly, the cells were divided into the following groups: The control group, the A20 overexpression group, the A20 knockdown group and the respective controls. A20 was stimulated with lipopolysaccharide (LPS) in a dose- and time-dependent manner and was detected using western blotting and real-time polymerase chain reaction (PCR) analyses. Immunofluorescence and western blotting analyses were performed to investigate the role of A20 in the regulation of nuclear factor (NF)-κB activation and translocation into the nucleus. ELISA and real-time PCR were performed to examine A20 in regulating the release of the following inflammatory cytokines: Tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and IL-8. RESULTS: The expression of A20 in IECs was inducible. When intestinal epithelial cells were subjected to the stimulation of LPS, the expression of A20 was increased, and the expression of A20 was induced in a dose- and time-dependent manner. The expression of A20 was very low in HT-29 cells without LPS stimulation but rapidly increased and was maintained at a high level 2-4 h after stimulation with LPS. These levels gradually declined with a change in time-course, and the expression of A20 increased with increasing LPS stimulation. Western blotting and immunofluorescence revealed that overexpression of A20 can inhibit NF-κB activation and its translocation to the nucleus. The overexpression of A20 can reduce the levels of proinflammatory cytokines involved in the pathophysiology of inflammatory bowel disease. There was no significant difference in the expression of IL-8 mRNA in the control group, A20 overexpression group or A20 knockdown group without LPS stimulation (P > 0.05); however, while after 2 h, 4 h and 8 h stimulation with LPS, the expression of IL-8 in the A20 overexpression group was lower than the control group and the A20 knockdown group (P < 0.05 or P < 0.01). The expression of TNF-α was different at different time points after 8 h of LPS stimulation (F = 31.33, DF = 5, P < 0.001), and the expression of TNF-α increased as the LPS stimulation time increased. Upon LPS stimulation, lower levels of TNF-α were detected in the A20 overexpression cell lines (P < 0.05). There were no significant differences in the induction of IL-6 and IL-1ß among the control group, A20 overexpression group and A20 knockdown group (P > 0.05). CONCLUSION: A20 plays an important role in limiting inflammation by inhibiting LPS-induced NF-κB responses in the gut luminal. A20 may be a potential therapeutic tool for inflammatory diseases.

Smart hyaluronidase-actived theranostic micelles for dual-modal imaging guided photodynamic therapy.

We here report smart hyaluronidase-actived theranostic nanoparticles based on hyaluronic acid (HA) coupled with chlorin e6 (Ce6) via adipic dihydrazide (ADH) forming HA-ADH-Ce6 conjugates and self-assembling into HACE NPs. The resulting nanoparticles showed stable nano-structure in aqueous condition with uniform size distribution and can be actively disassembled in the presence of hyaluronidase (over-expressed in tumor cells), exhibiting hyaluronidase-responsive "OFF/ON" behavior of fluorescence signal. The HACE NPs were rapidly taken up to human lung cancer cells A549 via CD44 (the HA receptor on the surface of tumor cells) receptor mediated endocytosis. Upon laser irradiation, the HACE NPs realized good near-infrared fluorescence imaging and photoacoustic imaging in the tumor bearing mice, which showed 5-fold higher fluorescence intensity and 3-fold higher photoacoustic (PA) intensity than free Ce6, respectively. In addition, under low dose of laser power, the HACE NPs presented more effective photodynamic therapy to suppression of tumor growth than free Ce6 in vitro and in vivo. Overall, these results suggest that the well-defined HACE NPs is a biocompatible theranostic nanoplatform for in vivo dual-modal tumor imaging and phototherapy simultaneously.

Self-Monitoring Artificial Red Cells with Sufficient Oxygen Supply for Enhanced Photodynamic Therapy.

Photodynamic therapy has been increasingly applied in clinical cancer treatments. However, native hypoxic tumoural microenvironment and lacking oxygen supply are the major barriers hindering photodynamic reactions. To solve this problem, we have developed biomimetic artificial red cells by loading complexes of oxygen-carrier (hemoglobin) and photosensitizer (indocyanine green) for boosted photodynamic strategy. Such nanosystem provides a coupling structure with stable self-oxygen supply and acting as an ideal fluorescent/photoacoustic imaging probe, dynamically monitoring the nanoparticle biodistribution and the treatment of PDT. Upon exposure to near-infrared laser, the remote-triggered photosensitizer generates massive cytotoxic reactive oxygen species (ROS) with sufficient oxygen supply. Importantly, hemoglobin is simultaneously oxidized into the more active and resident ferryl-hemoglobin leading to persistent cytotoxicity. ROS and ferryl-hemoglobin synergistically trigger the oxidative damage of xenograft tumour resulting in complete suppression. The artificial red cells with self-monitoring and boosted photodynamic efficacy could serve as a versatile theranostic platform.

Lipid-Polymer Nanoparticles for Folate-Receptor Targeting Delivery of Doxorubicin.

A biocompatible PLGA-lipid hybrid nanoparticles (NPs) was developed for targeted delivery of anticancer drugs with doxorubicin (DOX). The hydrodynamic diameter and zeta potential of DOX-loaded PLGA-lipid NPs (DNPs) were affected by the mass ratio of Lipid/PLGA or DSPE-PEG-COOH/Lecithin. At the 1:20 drug/polymer mass ratio, the mean hydrodynamic diameter of DNPs was the lowest (99.2 1.83 nm) and the NPs presented the encapsulation efficiency of DOX with 42.69 1.30%. Due to the folate-receptor mediated endocytosis, the PLGA-lipid NPs with folic acid (FA) targeting ligand showed significant higher uptake by folate-receptor-positive MCF-7 cells as compared to PLGA-lipid NPs without folate. Confocal microscopic observation and flow cytometry analysis also supported the enhanced cellular uptake of the FA-targeted NPs. The results indicated that the FA-targeted DNPs exhibited higher cytotoxicity in MCF-7 cells compared with non-targeted NPs. The lipid-polymer nanoparticles provide a solution of biocompatible nanocarrier for cancer targeting therapy.

NIR-driven Smart Theranostic Nanomedicine for On-demand Drug Release and Synergistic Antitumour Therapy.

Smart nanoparticles (NPs) that respond to external and internal stimulations have been developing to achieve optimal drug release in tumour. However, applying these smart NPs to attain high antitumour performance is hampered by limited drug carriers and inefficient spatiotemporal control. Here we report a noninvasive NIR-driven, temperature-sensitive DI-TSL (DOX/ICG-loaded temperature sensitive liposomes) co-encapsulating doxorubicin (DOX) and indocyanine green (ICG). This theranostic system applies thermo-responsive lipid to controllably release drug, utilizes the fluorescence (FL) of DOX/ICG to real-time trace the distribution of NPs, and employs DOX/ICG to treat cancer by chemo/photothermal therapy. DI-TSL exhibits uniform size distribution, excellent FL/size stability, enhanced response to NIR-laser, and 3 times increased drug release through laser irradiation. After endocytosis by MCF-7 breast adenocarcinoma cells, DI-TSL in cellular endosomes can cause hyperthermia through laser irradiation, then endosomes are disrupted and DI-TSL 'opens' to release DOX simultaneously for increased cytotoxicity. Furthermore, DI-TSL shows laser-controlled release of DOX in tumour, enhanced ICG and DOX retention by 7 times and 4 times compared with free drugs. Thermo-sensitive DI-TSL manifests high efficiency to promote cell apoptosis, and completely eradicate tumour without side-effect. DI-TSL may provide a smart strategy to release drugs on demand for combinatorial cancer therapy.