Stratification of risk of progression to colectomy in ulcerative colitis via measured and predicted gene expression.

An important goal of clinical genomics is to be able to estimate the risk of adverse disease outcomes. Between 5% and 10% of individuals with ulcerative colitis (UC) require colectomy within 5 years of diagnosis, but polygenic risk scores (PRSs) utilizing findings from genome-wide association studies (GWASs) are unable to provide meaningful prediction of this adverse status. By contrast, in Crohn disease, gene expression profiling of GWAS-significant genes does provide some stratification of risk of progression to complicated disease in the form of a transcriptional risk score (TRS). Here, we demonstrate that a measured TRS based on bulk rectal gene expression in the PROTECT inception cohort study has a positive predictive value approaching 50% for colectomy. Single-cell profiling demonstrates that the genes are active in multiple diverse cell types from both the epithelial and immune compartments. Expression quantitative trait locus (QTL) analysis identifies genes with differential effects at baseline and week 52 follow-up, but for the most part, differential expression associated with colectomy risk is independent of local genetic regulation. Nevertheless, a predicted polygenic transcriptional risk score (PPTRS) derived by summation of transcriptome-wide association study (TWAS) effects identifies UC-affected individuals at 5-fold elevated risk of colectomy with data from the UK Biobank population cohort studies, independently replicated in an NIDDK-IBDGC dataset. Prediction of gene expression from relatively small transcriptome datasets can thus be used in conjunction with TWASs for stratification of risk of disease complications.

Triazine-cored polymeric vectors for antisense oligonucleotide delivery in vitro and in vivo.

BACKGROUND: The polymer-based drug/gene delivery is promising for the treatment of inherent or acquire disease, because of the polymer's structural flexibility, larger capacity for therapeutic agent, low host immunogenicity and less cost. Antisense therapy is an approach to fighting genetic disorders or infections using antisense oligonucleotides (AOs). Unfortunately, the naked AOs showed low therapeutic efficacy in vivo and in clinical trial due to their poor cellular uptake and fast clearance in bloodstream. In this study, a series of triazine-cored amphiphilic polymers (TAPs) were investigated for their potential to enhance delivery of AOs, 2'-O-methyl phosphorothioate RNA (2'-OMePS) and phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. RESULTS: TAPs significantly enhanced AO-induced exon-skipping in a GFP reporter-based myoblast and myotube culture system, and observed cytotoxicity of the TAPs were lower than Endoporter, Lipofectamine-2000 or PEI 25K. Application of optimized formulations of TAPs with AO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in dystrophic mdx mice. The best ones for PMO and 2'-OMePS delivery have reached to 11-, 15-fold compared with the AO only in mdx mice, respectively. CONCLUSION: The study of triazine-cored amphiphilic polymers for AO delivery in vitro and in mdx mice indicated that the carrier's performances are related to the molecular size, compositions and hydrophilic-lipophilic balance (HLB) of the polymers, as well as the AO's structure. Improved exon-skipping efficiency of AOs observed in vitro and in mdx mice accompanied with low cytotoxicity demonstrated TAP polymers are potentials as safe and effective delivery carrier for gene/drug delivery.

Long-Term Treatment of Tamoxifen and Raloxifene Alleviates Dystrophic Phenotype and Enhances Muscle Functions of FKRP Dystroglycanopathy.

The third most common form of limb-girdle muscular dystrophies is caused by mutations of the Fukutin-related protein (FKRP) gene, with no effective therapy available. Selective estrogen receptor modulators, tamoxifen and raloxifene, have been widely used for human conditions for their anti-inflammatory, antifibrosis, prevention of bone loss, and muscle building effects (essential features for muscular dystrophy therapies). We evaluated therapeutic values of tamoxifen and raloxifene in FKRPP448L mutant mouse with severe dystrophic phenotype. The mice were treated with the drugs for 1 year through daily gavage. We demonstrate that tamoxifen and raloxifene significantly ameliorated the disease progression. The improvement includes increase in grip force production, extended running time and distance in treadmill test, and enhancement in cardiac and respiratory functions. Significant reduction in muscle pathology includes diminished fibrosis and fiber degeneration. Tamoxifen and raloxifene also significantly mitigated bone loss. Tamoxifen, but not raloxifene, caused severe adverse effects on male reproductive organs. The results demonstrate that tamoxifen and raloxifene hold significant potential for treating FKRP-related muscular dystrophy and probably other muscular dystrophies. Sex-related differential effects of the drugs call for a careful consideration for the drug and dosage selection in male and female patient populations.

IL-33 promotes growth and liver metastasis of colorectal cancer in mice by remodeling the tumor microenvironment and inducing angiogenesis.

Liver metastasis is the major cause of death from colorectal cancer (CRC). Understanding its mechanisms is necessary for timely diagnosis and development of effective therapies. Interleukin-33 (IL-33) is an IL-1 cytokine family member that uniquely functions as a cytokine and nuclear factor. It is released by necrotic epithelial cells and activated innate immune cells, functioning as an alarmin or an early danger signal. Its role in invoking type 2 immune response has been established; however, it has contrasting roles in tumor development and metastasis. We identified IL-33 as a potently upregulated cytokine in a highly metastatic murine CRC cell line and examined its role in tumor growth and metastasis to the liver. IL-33 was transgenically expressed in murine and human adenocarcinoma and carcinoma cell lines and their growth and spontaneous metastasis to the liver were assessed in orthotopic models of CRC in wild-type C57Bl/6 and Il33 knockout mice. The results showed that increased expression of IL-33 in CRC cells enhanced their tumor take, growth, and liver metastasis. Tumor- rather than host-derived IL-33 induced the enhanced recruitment of CD11b+ GR1+ and CD11b+ F4/80+ myeloid cells to remodel the tumor microenvironment by increased expression of mobilizing cytokines, and tumor angiogenesis by activating endothelial cells. IL-33 expression was elevated in patient tumor tissues, induced early in adenoma development, and activated by pro-inflammatory cytokines derived from the tumor microenvironment. The data suggest that tumor-derived IL-33 modulates the tumor microenvironment to potently promote colon carcinogenesis and liver metastasis, underscoring its potential as a therapeutic target. © 2016 Wiley Periodicals, Inc.

Glucocorticoid Steroid and Alendronate Treatment Alleviates Dystrophic Phenotype with Enhanced Functional Glycosylation of α-Dystroglycan in Mouse Model of Limb-Girdle Muscular Dystrophy with FKRPP448L Mutation.

Fukutin-related protein-muscular dystrophy is characterized by defects in glycosylation of α-dystroglycan with variable clinical phenotypes, most commonly as limb-girdle muscular dystrophy 2I. There is no effective therapy available. Glucocorticoid steroids have become the standard treatment for Duchenne and other muscular dystrophies with serious adverse effects, including excessive weight gain, immune suppression, and bone loss. Bisphosphonates have been used to treat Duchenne muscular dystrophy for prevention of osteoporosis. Herein, we evaluated prednisolone and alendronate for their therapeutic potential in the FKRPP448L-mutant mouse representing moderate limb-girdle muscular dystrophy 2I. Mice were treated with prednisolone, alendronate, and both in combination for up to 6 months. Prednisolone improved muscle pathology with significant reduction in muscle degeneration, but had no effect on serum creatine kinase levels and muscle strength. Alendronate treatment did not ameliorate muscle degeneration, but demonstrated a limited enhancement on muscle function test. Combined treatment of prednisolone and alendronate provided best improvement in muscle pathology with normalized fiber size distribution and significantly reduced serum creatine kinase levels, but had limited effect on muscle force generation. The use of alendronate significantly mitigated the bone loss. Prednisolone alone and in combination with alendronate enhance functionally glycosylated α-dystroglycan. These results, for the first time, demonstrate the efficacy and feasibility of this alliance treatment of the two drugs for fukutin-related protein-muscular dystrophy.

Amniotic fluid inhibits Toll-like receptor 4 signaling in the fetal and neonatal intestinal epithelium.

The fetal intestinal mucosa is characterized by elevated Toll-like receptor 4 (TLR4) expression, which can lead to the development of necrotizing enterocolitis (NEC)--a devastating inflammatory disease of the premature intestine--upon exposure to microbes. To define endogenous strategies that could reduce TLR4 signaling, we hypothesized that amniotic fluid can inhibit TLR4 signaling within the fetal intestine and attenuate experimental NEC, and we sought to determine the mechanisms involved. We show here that microinjection of amniotic fluid into the fetal (embryonic day 18.5) gastrointestinal tract reduced LPS-mediated signaling within the fetal intestinal mucosa. Amniotic fluid is abundant in EGF, which we show is required for its inhibitory effects on TLR4 signaling via peroxisome proliferator-activated receptor, because inhibition of EGF receptor (EGFR) with cetuximab or EGF-depleted amniotic fluid blocked the inhibitory effects of amniotic fluid on TLR4, whereas amniotic fluid did not prevent TLR4 signaling in EGFR- or peroxisome proliferator-activated receptor γ-deficient enterocytes or in mice deficient in intestinal epithelial EGFR, and purified EGF attenuated the exaggerated intestinal mucosal TLR4 signaling in wild-type mice. Moreover, amniotic fluid-mediated TLR4 inhibition reduced the severity of NEC in mice through EGFR activation. Strikingly, NEC development in both mice and humans was associated with reduced EGFR expression that was restored upon the administration of amniotic fluid in mice or recovery from NEC in humans, suggesting that a lack of amniotic fluid-mediated EGFR signaling could predispose to NEC. These findings may explain the unique susceptibility of premature infants to the development of NEC and offer therapeutic approaches to this devastating disease.

Pilot Plant-Based Lifestyle Medicine Program in an Urban Public Healthcare System: Evaluating Demand and Implementation.

Lifestyle interventions that optimize nutrition, physical activity, sleep health, social connections, and stress management, and address substance use, can reduce cardiometabolic risk. Despite substantial evidence that healthful plant-based diets are beneficial for long-term cardiometabolic health and longevity, uncertainty lies in how to implement plant-based lifestyle programs in traditional clinical settings, especially in safety-net contexts with finite resources. In this mixed-methods implementation evaluation of the Plant-Based Lifestyle Medicine Program piloted in a large public healthcare system, we surveyed participants and conducted qualitative interviews and focus groups with stakeholders to assess program demand in the eligible population and feasibility of implementation within the safety-net setting. Program demand was high and exceeded capacity. Participants' main motivations for joining the program included gaining more control over life, reducing medication, and losing weight. The program team, approach, and resources were successful facilitators. However, the program faced administrative and payor-related challenges within the safety-net setting, and participants reported barriers to access. Stakeholders found the program to be valuable, despite challenges in program delivery and access. Findings provide guidance for replication. Future research should focus on randomized controlled trials to assess clinical outcomes as a result of program participation.

Advancing Lifestyle Medicine in New York City's Public Health Care System.

Chronic diseases are the leading cause of death and disability in the United States, and much of this burden can be attributed to lifestyle and behavioral risk factors. Lifestyle medicine is an approach to preventing and treating lifestyle-related chronic disease using evidence-based lifestyle modification as a primary modality. NYC Health + Hospitals, the largest municipal public health care system in the United States, is a national pioneer in incorporating lifestyle medicine systemwide. In 2019, a pilot lifestyle medicine program was launched at NYC Health + Hospitals/Bellevue to improve cardiometabolic health in high-risk patients through intensive support for evidence-based lifestyle changes. Analyses of program data collected from January 29, 2019 to February 26, 2020 demonstrated feasibility, high demand for services, high patient satisfaction, and clinically and statistically significant improvements in cardiometabolic risk factors. This pilot is being expanded to 6 new NYC Health + Hospitals sites spanning all 5 NYC boroughs. As part of the expansion, many changes have been implemented to enhance the original pilot model, scale services effectively, and generate more interest and incentives in lifestyle medicine for staff and patients across the health care system, including a plant-based default meal program for inpatients. This narrative review describes the pilot model and outcomes, the expansion process, and lessons learned to serve as a guide for other health systems.

Long-term rescue of dystrophin expression and improvement in muscle pathology and function in dystrophic mdx mice by peptide-conjugated morpholino.

Exon skipping is capable of correcting frameshift and nonsense mutations in Duchenne muscular dystrophy. Phase 2 clinical trials in the United Kingdom and the Netherlands have reported induction of dystrophin expression in muscle of Duchenne muscular dystrophy patients by systemic administration of both phosphorodiamidate morpholino oligomers (PMO) and 2'-O-methyl phosphorothioate. Peptide-conjugated phosphorodiamidate morpholino offers significantly higher efficiency than phosphorodiamidate morpholino, with the ability to induce near-normal levels of dystrophin, and restores function in both skeletal and cardiac muscle. We examined 1-year systemic efficacy of peptide-conjugated phosphorodiamidate morpholino targeting exon 23 in dystrophic mdx mice. The LD(50) of peptide-conjugated phosphorodiamidate morpholino was determined to be approximately 85 mg/kg. The half-life of dystrophin expression was approximately 2 months in skeletal muscle, but shorter in cardiac muscle. Biweekly injection of 6 mg/kg peptide-conjugated phosphorodiamidate morpholino produced >20% dystrophin expression in all skeletal muscles and ≤5% in cardiac muscle, with improvement in muscle function and pathology and reduction in levels of serum creatine kinase. Monthly injections of 30 mg/kg peptide-conjugated phosphorodiamidate morpholino restored dystrophin to >50% normal levels in skeletal muscle, and 15% in cardiac muscle. This was associated with greatly reduced serum creatine kinase levels, near-normal histology, and functional improvement of skeletal muscle. Our results demonstrate for the first time that regular 1-year administration of peptide-conjugated phosphorodiamidate morpholino can be safely applied to achieve significant therapeutic effects in an animal model.

Optic pathway glioma as part of a constitutional mismatch-repair deficiency syndrome in a patient meeting the criteria for neurofibromatosis type 1.

Patients with constitutional mismatch repair-deficiency (CMMR-D) caused by the biallelic deletions of mismatch repair (MMR) genes have a high likelihood of developing malignancies of the bone marrow, bowel, and brain. Affected individuals often have phenotypic features of neurofibromatosis type 1 (NF-1), including café-au-lait spots. Optic pathway gliomas (OPGs), a common manifestation of NF-1, have not been reported. We report the case of a 3-year-old male with an extensive OPG who met the diagnostic criteria for NF-1. He was subsequently found to have multiple colonic polyps and bi-allelic loss of PMS2. Testing for NF-1 was negative.

Patient-Reported Outcomes from a Pilot Plant-Based Lifestyle Medicine Program in a Safety-Net Setting.

Lifestyle medicine interventions that emphasize healthy behavior changes are growing in popularity in U.S. health systems. Safety-net healthcare settings that serve low-income and uninsured populations most at risk for lifestyle-related disease are ideal venues for lifestyle medicine interventions. Patient-reported outcomes are important indicators of the efficacy of lifestyle medicine interventions. Past research on patient-reported outcomes of lifestyle medicine interventions has occurred outside of traditional healthcare care settings. In this study, we aimed to assess patient-reported outcomes on nutrition knowledge, barriers to adopting a plant-based diet, food and beverage consumption, lifestyle behaviors, self-rated health, and quality-of-life of participants in a pilot plant-based lifestyle medicine program in an urban safety-net healthcare system. We surveyed participants at three time points (baseline, 3 months, 6 months) to measure change over time. After 6 months of participation in the program, nutrition knowledge increased by 7.2 percentage points, participants reported an average of 2.4 fewer barriers to adopting a plant-based diet, the score on a modified healthful plant-based diet index increased by 5.3 points, physical activity increased by 0.7 days per week while hours of media consumption declined by 0.7 h per day, and the percentage of participants who reported that their quality of sleep was "good" or "very good" increased by 12.2 percentage points. Our findings demonstrate that a lifestyle medicine intervention in a safety-net healthcare setting can achieve significant improvements in patient-reported outcomes. Key lessons for other lifestyle medicine interventions include using a multidisciplinary team; addressing all pillars of lifestyle medicine; and the ability for patients to improve knowledge, barriers, skills, and behaviors with adequate support.

Change in cardiometabolic risk factors in a pilot safety-net plant-based lifestyle medicine program.

Introduction: Interventions emphasizing healthful lifestyle behaviors are proliferating in traditional health care settings, yet there is a paucity of published clinical outcomes, outside of pay-out-of-pocket or employee health programs. Methods: We assessed weight, hemoglobin A1c (HbA1c), blood pressure, and cholesterol for 173 patients of the Plant-Based Lifestyle Medicine Program piloted in a New York City safety-net hospital. We used Wilcoxon signed-rank tests to assess changes in means, from baseline to six-months, for the full sample and within baseline diagnoses (i.e., overweight or obesity, type 2 diabetes, prediabetes, hypertension, hyperlipidemia). We calculated the percentage of patients with clinically meaningful changes in outcomes for the full sample and within diagnoses. Findings: The full sample had statistically significant improvements in weight, HbA1c, and diastolic blood pressure. Patients with prediabetes or overweight or obesity experienced significant improvements in weight and those with type 2 diabetes had significant improvements in weight and HbA1c. Patients with hypertension had significant reductions in diastolic blood pressure and weight. Data did not show differences in non-high-density lipoprotein cholesterol (non-HDL-C), but differences in low-density lipoprotein cholesterol (LDL-C) were approaching significance for the full sample and those with hyperlipidemia. The majority of patients achieved clinically meaningful improvements on all outcomes besides systolic blood pressure. Conclusion: Our study demonstrates that a lifestyle medicine intervention within a traditional, safety-net clinical setting improved biomarkers of cardiometabolic disease. Our findings are limited by small sample sizes. Additional large-scale, rigorous studies are needed to further establish the effectiveness of lifestyle medicine interventions in similar settings.

Ribitol dose-dependently enhances matriglycan expression and improves muscle function with prolonged life span in limb girdle muscular dystrophy 2I mouse model.

Limb Girdle Muscular Dystrophy 2I (LGMDR9) is one of the most common LGMD characterized by defects in glycosylation of α-dystroglycan (matriglycan) resulting from mutations of Fukutin-related protein (FKRP). There is no effective therapy currently available. We recently demonstrated that ribitol supplement increases levels of matriglycan in cells in vitro and in FKRP-P448L (P448L) mutant mouse model through drinking water administration. To be clinically relevant, we have now conducted a dose-escalating efficacy study by gavage in P448L mutant mice. Six months of ribitol treatment daily significantly rescued functions of skeletal, respiratory, and cardiac muscles dose-dependently. This was associated with a dose dependent increase in matriglycan and improvement in muscle pathology with reductions in muscle degeneration, inflammatory infiltration and fibrosis. Importantly, ribitol significantly increased life span and muscle functions of the female animals receiving treatment from 10 months of age. The only observed side effect was gastrointestinal tract bloating with loose stool and this effect is also dose dependent. The results validate the mechanism that ribitol as a pre-substrate of glycosyltransferase is able to compensate for the decreased function of mutant FKRP with restoration of matriglycan expression and provide a guidance for future clinical trial design.

Clinical and Host Biological Factors Predict Colectomy Risk in Children Newly Diagnosed With Ulcerative Colitis.

BACKGROUND: Develop a clinical and biological predictive model for colectomy risk in children newly diagnosed with ulcerative colitis (UC). METHODS: This was a multicenter inception cohort study of children (ages 4-17 years) newly diagnosed with UC treated with standardized initial regimens of mesalamine or corticosteroids (CS) depending upon initial disease severity. Therapy escalation to immunomodulators or infliximab was based on predetermined criteria. Patients were phenotyped by clinical activity per the Pediatric Ulcerative Colitis Activity Index (PUCAI), disease extent, endoscopic/histologic severity, and laboratory markers. In addition, RNA sequencing defined pretreatment rectal gene expression and high density DNA genotyping by the Affymetrix UK Biobank Axiom Array. Coprimary outcomes were colectomy over 3 years and time to colectomy. Generalized linear models, Cox proportional hazards multivariate regression modeling, and Kaplan-Meier plots were used. RESULTS: Four hundred twenty-eight patients (mean age 13 years) started initial theapy with mesalamine (n = 136), oral CS (n = 144), or intravenous CS (n = 148). Twenty-five (6%) underwent colectomy at ≤1 year, 33 (9%) at ≤2 years, and 35 (13%) at ≤3 years. Further, 32/35 patients who had colectomy failed infliximab. An initial PUCAI ≥ 65 was highly associated with colectomy (P = 0.0001). A logistic regression model predicting colectomy using the PUCAI, hemoglobin, and erythrocyte sedimentation rate had a receiver operating characteristic area under the curve of 0.78 (95% confidence interval [0.73, 0.84]). Addition of a pretreatment rectal gene expression panel reflecting activation of the innate immune system and response to external stimuli and bacteria to the clinical model improved the receiver operating characteristic area under the curve to 0.87 (95% confidence interval [0.82, 0.91]). CONCLUSIONS: A small group of children newly diagnosed with severe UC still require colectomy despite current therapies. Our gene signature observations suggest additional targets for management of those patients not responding to current medical therapies.

Targeted Assessment of Mucosal Immune Gene Expression Predicts Clinical Outcomes in Children with Ulcerative Colitis.

BACKGROUND AND AIMS: We aimed to determine whether a targeted gene expression panel could predict clinical outcomes in paediatric ulcerative colitis [UC] and investigated putative pathogenic roles of predictive genes. METHODS: In total, 313 rectal RNA samples from a cohort of newly diagnosed paediatric UC patients (PROTECT) were analysed by a real-time PCR microfluidic array for expression of type 1, 2 and 17 inflammation genes. Associations between expression and clinical outcomes were assessed by logistic regression. Identified prognostic markers were further analysed using existing RNA sequencing (RNA-seq) data sets and tissue immunostaining. RESULTS: IL13RA2 was associated with a lower likelihood of corticosteroid-free remission (CSFR) on mesalamine at week 52 (p = .002). A model including IL13RA2 and only baseline clinical parameters was as accurate as an established clinical model, which requires week 4 remission status. RORC was associated with a lower likelihood of colectomy by week 52. A model including RORC and PUCAI predicted colectomy by 52 weeks (area under the receiver operating characteristic curve 0.71). Bulk RNA-seq identified IL13RA2 and RORC as hub genes within UC outcome-associated expression networks related to extracellular matrix and innate immune response, and lipid metabolism and microvillus assembly, respectively. Adult UC single-cell RNA-seq data revealed IL13RA2 and RORC co-expressed genes were localized to inflammatory fibroblasts and undifferentiated epithelial cells, respectively, which was supported by protein immunostaining. CONCLUSION: Targeted assessment of rectal mucosal immune gene expression predicts 52-week CSFR in treatment-naïve paediatric UC patients. Further exploration of IL-13Rɑ2 as a therapeutic target in UC and future studies of the epithelial-specific role of RORC in UC pathogenesis are warranted.

Aminoglycoside Enhances the Delivery of Antisense Morpholino Oligonucleotides In Vitro and in mdx Mice.

Antisense oligonucleotide (AO) therapy has been the specific treatment for Duchenne muscular dystrophy, with ongoing clinical trials. However, therapeutic applications of AOs remain limited, particularly because of the lack of efficient cellular delivery methods imperative for achieving efficacy. In this study, we investigated a few aminoglycosides (AGs) for their potential to improve the delivery of antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. AGs had lower cytotoxicity compared with Endoporter, the currently most effective delivery reagent for PMO in vitro, and improved efficiency in PMO delivery 9- to 15-fold over PMO alone. Significant enhancement in systemic PMO-targeted dystrophin exon 23 skipping was observed in mdx mice, up to a 6-fold increase with AG3 (kanamycin) and AG7 (sisomicin) compared with PMO only. No muscle damage could be detected clearly with the test dosages. These results establish AGs as PMO delivery-enhancing agents for treating muscular dystrophy or other diseases.

Characterization of Stool Virome in Children Newly Diagnosed With Moderate to Severe Ulcerative Colitis.

BACKGROUND: Viral infections have been suggested as possible triggers for the onset of ulcerative colitis (UC). METHODS: We employed VirCapSeq-Vert, a high-throughput sequencing virus capture platform, to examine the stool virome of children with newly diagnosed moderate to severe UC. We surveyed fecal samples collected at presentation, after symptom remission, and from a control group diagnosed with irritable bowel syndrome. RESULTS: Seventy subjects with UC (mean age 13 years, 45 had moderate symptoms, 25 had severe, 69 of 70 had a Mayo endoscopy subscore 2/3) were studied. We detected a wide range of animal viruses that were taxonomically classified into 12 viral families. A virus was present in 50% of fecal samples collected at presentation, 41% of samples collected after remission, and 40% of samples in our control group. The most frequently identified viruses were diet-based gyroviruses. The UC cohort had a significantly higher prevalence of anelloviruses compared with the control cohort. However, we did not identify a single virus that can be implicated in the onset of UC and did not find an association between UC disease severity and viral presence. CONCLUSION: Presence of virus in stool was not associated with the onset of pediatric UC.

Ulcerative colitis mucosal transcriptomes reveal mitochondriopathy and personalized mechanisms underlying disease severity and treatment response.

Molecular mechanisms driving disease course and response to therapy in ulcerative colitis (UC) are not well understood. Here, we use RNAseq to define pre-treatment rectal gene expression, and fecal microbiota profiles, in 206 pediatric UC patients receiving standardised therapy. We validate our key findings in adult and paediatric UC cohorts of 408 participants. We observe a marked suppression of mitochondrial genes and function across cohorts in active UC, and that increasing disease severity is notable for enrichment of adenoma/adenocarcinoma and innate immune genes. A subset of severity genes improves prediction of corticosteroid-induced remission in the discovery cohort; this gene signature is also associated with response to anti-TNFα and anti-α4ß7 integrin in adults. The severity and therapeutic response gene signatures were in turn associated with shifts in microbes previously implicated in mucosal homeostasis. Our data provide insights into UC pathogenesis, and may prioritise future therapies for nonresponders to current approaches.

In Vivo Evaluation of Dystrophin Exon Skipping in mdx Mice.

Dystrophin exon skipping in mdx mice has been the key model for the development of antisense therapy in vivo. Evaluation of exon skipping in this model involves the following two aspects: (1) efficiency and accuracy of exon skipping and levels of dystrophin expression determined by RT-PCR, immunochemistry, and western blotting; (2) therapeutic effects on muscle pathology and functions assessed by histology and functional assays including grip strength measurement, treadmill test, echocardiogram, and hemodynamics for cardiac functions. Here we describe some key considerations and the essential methodologies in detail for exon skipping in mdx mice.

Saponins enhance exon skipping of 2'-O-methyl phosphorothioate oligonucleotide in vitro and in vivo.

BACKGROUND: Antisense oligonucleotide (ASO)-mediated exon skipping has been feasible and promising approach for treating Duchenne muscular dystrophy (DMD) in preclinical and clinical trials, but its therapeutic applications remain challenges due to inefficient delivery. METHODS: We investigated a few Saponins for their potential to improve delivery performance of an antisense 2'-Omethyl phosphorothioate RNA (2'-OMePS) in muscle cells and in dystrophic mdx mice. This study was carried out by evaluating these Saponins' toxicity, cellular uptake, transduction efficiency in vitro, and local delivery in vivo for 2'-OMePS, as well as affinity study between Saponin and 2'-OMePS. RESULTS: The results showed that these Saponins, especially Digitonin and Tomatine, enhance the delivery of 2'-OMePS with comparable efficiency to Lipofectamine 2k (LF-2k) -mediated delivery in vitro. Significant performance was further observed in mdx mice, up to 10-fold with the Digitonin as compared to 2'-OMePS alone. Cytotoxicity of the Digitonin and Glycyrrhizin was much lower than LF-2k in vitro and not clearly detected in vivo under the tested concentrations. CONCLUSION: This study potentiates Saponins as delivery vehicle for 2'-OMePS in vivo for treating DMD or other diseases.