Cloning and variation of ground state intestinal stem cells.

Stem cells of the gastrointestinal tract, pancreas, liver and other columnar epithelia collectively resist cloning in their elemental states. Here we demonstrate the cloning and propagation of highly clonogenic, 'ground state' stem cells of the human intestine and colon. We show that derived stem-cell pedigrees sustain limited copy number and sequence variation despite extensive serial passaging and display exquisitely precise, cell-autonomous commitment to epithelial differentiation consistent with their origins along the intestinal tract. This developmentally patterned and epigenetically maintained commitment of stem cells is likely to enforce the functional specificity of the adult intestinal tract. Using clonally derived colonic epithelia, we show that toxins A or B of the enteric pathogen Clostridium difficile recapitulate the salient features of pseudomembranous colitis. The stability of the epigenetic commitment programs of these stem cells, coupled with their unlimited replicative expansion and maintained clonogenicity, suggests certain advantages for their use in disease modelling and regenerative medicine.

Clinical and biological predictors of response to standardised paediatric colitis therapy (PROTECT): a multicentre inception cohort study.

BACKGROUND: Lack of evidence-based outcomes data leads to uncertainty in developing treatment regimens in children who are newly diagnosed with ulcerative colitis. We hypothesised that pretreatment clinical, transcriptomic, and microbial factors predict disease course. METHODS: In this inception cohort study, we recruited paediatric patients aged 4-17 years with newly diagnosed ulcerative colitis from 29 centres in the USA and Canada. Patients initially received standardised mesalazine or corticosteroids, with pre-established criteria for escalation to immunomodulators (ie, thiopurines) or anti-tumor necrosis factor-α (TNFα) therapy. We used RNA sequencing to define rectal gene expression before treatment, and 16S sequencing to characterise rectal and faecal microbiota. The primary outcome was week 52 corticosteroid-free remission with no therapy beyond mesalazine. We assessed factors associated with the primary outcome using logistic regression models of the per-protocol population. This study is registered with ClinicalTrials.gov, number NCT01536535. FINDINGS: Between July 10, 2012, and April 21, 2015, of 467 patients recruited, 428 started medical therapy, of whom 400 (93%) were evaluable at 52 weeks and 386 (90%) completed the study period with no protocol violations. 150 (38%) of 400 participants achieved week 52 corticosteroid-free remission, of whom 147 (98%) were taking mesalazine and three (2%) were taking no medication. 74 (19%) of 400 were escalated to immunomodulators alone, 123 (31%) anti-TNFα therapy, and 25 (6%) colectomy. Low baseline clinical severity, high baseline haemoglobin, and week 4 clinical remission were associated with achieving week 52 corticosteroid-free remission (n=386, logistic model area under the curve [AUC] 0·70, 95% CI 0·65-0·75; specificity 77%, 95% CI 71-82). Baseline severity and remission by week 4 were validated in an independent cohort of 274 paediatric patients with newly diagnosed ulcerative colitis. After adjusting for clinical predictors, an antimicrobial peptide gene signature (odds ratio [OR] 0·57, 95% CI 0·39-0·81; p=0·002) and abundance of Ruminococcaceae (OR 1·43, 1·02-2·00; p=0·04), and Sutterella (OR 0·81, 0·65-1·00; p=0·05) were independently associated with week 52 corticosteroid-free remission. INTERPRETATION: Our findings support the utility of initial clinical activity and treatment response by 4 weeks to predict week 52 corticosteroid-free remission with mesalazine alone in children who are newly diagnosed with ulcerative colitis. The development of personalised clinical and biological signatures holds the promise of informing ulcerative colitis therapeutic decisions. FUNDING: US National Institutes of Health.

Prediction of complicated disease course for children newly diagnosed with Crohn's disease: a multicentre inception cohort study.

BACKGROUND: Stricturing and penetrating complications account for substantial morbidity and health-care costs in paediatric and adult onset Crohn's disease. Validated models to predict risk for complications are not available, and the effect of treatment on risk is unknown. METHODS: We did a prospective inception cohort study of paediatric patients with newly diagnosed Crohn's disease at 28 sites in the USA and Canada. Genotypes, antimicrobial serologies, ileal gene expression, and ileal, rectal, and faecal microbiota were assessed. A competing-risk model for disease complications was derived and validated in independent groups. Propensity-score matching tested the effect of anti-tumour necrosis factor α (TNFα) therapy exposure within 90 days of diagnosis on complication risk. FINDINGS: Between Nov 1, 2008, and June 30, 2012, we enrolled 913 patients, 78 (9%) of whom experienced Crohn's disease complications. The validated competing-risk model included age, race, disease location, and antimicrobial serologies and provided a sensitivity of 66% (95% CI 51-82) and specificity of 63% (55-71), with a negative predictive value of 95% (94-97). Patients who received early anti-TNFα therapy were less likely to have penetrating complications (hazard ratio [HR] 0·30, 95% CI 0·10-0·89; p=0·0296) but not stricturing complication (1·13, 0·51-2·51; 0·76) than were those who did not receive early anti-TNFα therapy. Ruminococcus was implicated in stricturing complications and Veillonella in penetrating complications. Ileal genes controlling extracellular matrix production were upregulated at diagnosis, and this gene signature was associated with stricturing in the risk model (HR 1·70, 95% CI 1·12-2·57; p=0·0120). When this gene signature was included, the model's specificity improved to 71%. INTERPRETATION: Our findings support the usefulness of risk stratification of paediatric patients with Crohn's disease at diagnosis, and selection of anti-TNFα therapy. FUNDING: Crohn's and Colitis Foundation of America, Cincinnati Children's Hospital Research Foundation Digestive Health Center.

Inflammatory Bowel Disease: Effects on Bone and Mechanisms.

Inflammatory bowel disease (IBD) is associated with decreased bone mass and alterations in bone geometry from the time of diagnosis, before anti-inflammatory therapy is instituted. Deficits in bone mass can persist despite absence of symptoms of active IBD. The effects of IBD on the skeleton are complex. Protein-calorie malnutrition, inactivity, hypogonadism, deficits in calcium intake and vitamin D consumption and synthesis, stunted growth in children, decreased skeletal muscle mass, and inflammation all likely play a role. Preliminary studies suggest that the dysbiotic intestinal microbial flora present in IBD may also affect bone at a distance. Several mechanisms are possible. T cells activated by the gut microbiota may serve as "inflammatory shuttles" between the intestine and bone. Microbe-associated molecular patterns leaked into the circulation in IBD may activate immune responses in the bone marrow by immune cells and by osteocytes, osteoblasts, and osteoclasts that lead to decreased bone formation and increased resorption. Finally, intestinal microbial metabolites such as H2S may also affect bone cell function. Uncovering these mechanisms will enable the design of microbial cocktails to help restore bone mass in patients with IBD.

Distinctive colonic mucosal cytokine signature in new-onset, untreated pediatric Crohn disease.

OBJECTIVE: The aim of the study was to compare the colonic mucosal immune response in children with new, untreated Crohn disease (CD-New), CD in remission (CD-Remission), and unaffected children (CTRL [controls]). METHODS: We performed flow cytometry of mitogen-stimulated colonic lamina propria mononuclear cells isolated from colonic biopsies and 72-hour biopsy explant cultures, and analyzed the supernatant by an unbiased multiplex cytokine array of 45 analytes. RESULTS: Thirty-six children were studied (mean age 14 ± 3 years, 14 girls): 12 CD-New, 11 CD-Remission, and 13 CTRL. We found that stimulation of lamina propria mononuclear cells isolated from colonic biopsies induced comparable intracellular cytokine levels of interferon (IFN-γ), interleukin (IL)-17, and tumor necrosis factor (TNF)-α in T cells from CD-New, CD-Remission, and CTRL, suggesting that mucosal innate inflammation plays a larger role than activated T cells in CD-New. To measure factors released during the ongoing inflammatory response in CD-New, we cultured colonic biopsy explants and uncovered 13/45 factors that were significantly higher in CD-New versus CD-Remission, whereas 10 were increased in CD-New over CTRL. Ingenuity Pathway Analysis software revealed the anticipated interconnectivity of TNF-α, IL-6, and CSF-2 in CD-New of the colon. A novel subnetwork of chemokines was, however, evident, whereas IL-17a appeared as a peripheral factor. Principal component analysis and hierarchal clustering showed that CD-New and CD-Remission separated into distinct subgroups based on the 13 factors. CONCLUSIONS: At diagnosis of inflammatory bowel disease, the colonic cytokine response contains a predominance of innate immune factors, with chemoattractants and vascular adhesion molecules playing a central role.

Bone health in children and adolescents with chronic diseases that may affect the skeleton: the 2013 ISCD Pediatric Official Positions.

The aim of this Task Force was to review the use of dual-energy X-ray absorptiometry (DXA) in children and adolescents with underlying chronic diseases that pose risk factors for compromised bone health, such as inflammation, glucocorticoid therapy, or decreased mobility. The Task Force systematically analyzed more than 270 studies, with an emphasis on those published in the interval since the original 2007 Position Statements. Important developments over this period included prospective cohort studies demonstrating that DXA measures of areal bone mineral density (aBMD) predicted incident fractures and the development of robust reference data and strategies to adjust for bone size in children with growth impairment. In this report, we summarize the current literature on the relationship between DXA-based aBMD and both fracture (vertebral and non-vertebral) outcomes and non-fracture risk factors (e.g., disease characteristics, ambulatory status, and glucocorticoid exposure) in children with chronic illnesses. Most publications described the aBMD profile of children with underlying diseases, as well as the cross-sectional or longitudinal relationship between aBMD and clinically relevant non-fracture outcomes. Studies that addressed the relationship between aBMD and prevalent or incident fractures in children with chronic illnesses are now emerging. In view of these updated data, this report provides guidelines for the use of DXA-based aBMD in this setting. The initial recommendation that DXA is part of a comprehensive skeletal healthy assessment in patients with increased risk of fracture is unchanged. Although the prior guidelines recommended DXA assessment in children with chronic diseases at the time of clinical presentation with ongoing monitoring, this revised Position Statement focuses on the performance of DXA when the patient may benefit from interventions to decrease their elevated risk of a clinically significant fracture and when the DXA results will influence that management.

Health supervision in the management of children and adolescents with IBD: NASPGHAN recommendations.

Ulcerative colitis (UC) and Crohn disease (CD), collectively referred to as inflammatory bowel disease (IBD), are chronic inflammatory disorders that can affect the gastrointestinal tract of children and adults. Like other autoimmune processes, the cause(s) of these disorders remain unknown but likely involves some interplay between genetic vulnerability and environmental factors. Children, in particular with UC or CD, can present to their primary care providers with similar symptoms, including abdominal pain, diarrhea, weight loss, and bloody stool. Although UC and CD are more predominant in adults, epidemiologic studies have demonstrated that a significant percentage of these patients were diagnosed during childhood. The chronic nature of the inflammatory process observed in these children and the waxing and waning nature of their clinical symptoms can be especially disruptive to their physical, social, and academic development. As such, physicians caring for children must consider these diseases when evaluating patients with compatible symptoms. Recent research efforts have made available a variety of more specific and effective pharmacologic agents and improved endoscopic and radiologic assessment tools to assist clinicians in the diagnosis and interval assessment of their patients with IBD; however, as the level of complexity of these interventions has increased, so too has the need for practitioners to become familiar with a wider array of treatments and the risks and benefits of particular diagnostic testing. Nonetheless, in most cases, and especially when frequent visits to subspecialty referral centers are not geographically feasible, primary care providers can be active participants in the management of their pediatric patients with IBD. The goal of this article is to educate and assist pediatricians and adult gastroenterology physicians caring for children with IBD, and in doing so, help to develop more collaborative care plans between primary care and subspecialty providers.

Effect of gluten-free diet on growth and glycemic control in children with type 1 diabetes and asymptomatic celiac disease.

We aimed to evaluate the effects of a gluten-free diet on growth and glycemic control in children with type 1 diabetes mellitus (DM) and asymptomatic, biopsy-proven celiac disease (CD). Each case of CD was compared to two children with DM and no CD. We studied weight, height, and hemoglobin A1c (HgbAlc) up to 12 months pre- and post- CD diagnosis in 29 cases and 58 controls. The change in body mass index (deltaBMI Z-score) over 2 years was significantly higher in CD cases vs. controls (mean +/- SD 0.33 +/- 0.74 vs. +/- 0.08 +/- 0.46; p = 0.023). However, BMI Z-score did not change in CD patients diagnosed with DM for > 1 year. Mean HgbA1c was similar between groups throughout the study. In conclusion, children with asymptomatic CD and DM do not have significant changes in BMI, height Z-score or metabolic control 1 year post-diagnosis.

Cloning of ground-state intestinal stem cells from endoscopic biopsy samples.

'Adult' or 'somatic' stem cells harbor an intrinsic ability to regenerate tissues. Heterogeneity of such stem cells along the gastrointestinal tract yields the known segmental specificity of this organ and may contribute to the pathology of certain enteric conditions. Here we detail technology for the generation of 'libraries' of clonogenic cells from 1-mm-diamter endoscopic biopsy samples from the human gastrointestinal tract. Each of the 150-300 independent clones in a typical stem cell library can be clonally expanded to billions of cells in a few weeks while maintaining genomic stability and the ability to undergo multipotent differentiation to the specific epithelia from which the sample originated. The key to this methodology is the intrinsic immortality of normal intestinal stem cells (ISCs) and culture systems that maintain them as highly immature, ground-state ISCs marked by a single-cell clonogenicity of 70% and a corresponding 250-fold proliferative advantage over spheroid technologies. Clonal approaches such as this enhance the resolution of molecular genetics, make genome editing easier, and may be useful in regenerative medicine, unravelling heterogeneity in disease, and facilitating drug discovery.

A two-year longitudinal study of persistent lean tissue deficits in children with Crohn's disease.

BACKGROUND & AIMS: Deficits in lean body mass have been reported in cross-sectional studies of children with Crohn's disease, but no longitudinal data exist from diagnosis. We observed the effects of Crohn's disease on body composition and bone mineral content (BMC), beginning at diagnosis and followed prospectively for 2 years. METHODS: The study was conducted at 2 tertiary care centers for pediatric inflammatory bowel diseases. At diagnosis we recorded age, weight, height, levels of serum interleukin-6 and insulin-like growth factor-1, sexual maturation stage, disease activity, z scores for body mass index (BMI), fat-free mass (FFM), and bone mineral content (BMC). z Scores were adjusted for height and age, when appropriate. Measurements were made yearly in patients with Crohn's disease (n = 42) but only at the start of the study in controls (n = 81). RESULTS: BMI and FFM z scores were significantly reduced at the time of diagnosis in children with Crohn's disease, compared with controls. During the 2-year study period, the BMI z scores normalized in patients with Crohn's disease, but the FFM z scores did not increase significantly. The BMC z scores increased significantly, but they were still lower than control values after 2 years. Changes in BMC during a period of 2 years were associated with increases in FFM (R(2) = 0.318, P < .01). CONCLUSIONS: The correction of BMI that is associated with clinical improvement in children with Crohn's disease 2 years after diagnosis results from gains in fat mass. Significant FFM deficits might hamper normal acquisition of bone mass in children with Crohn's disease because of a lack of mechanical strain.

Impact of a Quality Improvement Initiative to Optimize the Discharge Process of Pediatric Gastroenterology Patients at an Academic Children's Hospital.

Discharge is an important and complex process that can be optimized to reduce inpatient healthcare inefficiency and waste. This study aimed to increase the percentage of patients discharged before 1 pm by 20% from an academic inpatient pediatric gastroenterology service (IPGS), over 6 months. METHODS: We conducted a preintervention and postintervention study of patients discharged from IPGS. Patients discharged from January to June 2016, and those following our intervention from June to December 2016, were studied. Interventions included (1) implementation of the electronic medical record medical and logistical discharge criteria checklists for the 4 most common IPGS discharge diagnoses, (2) standardization of the rounds process to prioritize discharge, (3) education of nursing staff and families about the role they played in discharge. Process, outcome, and balancing measures were analyzed. RESULTS: Three hundred fifty-five total discharges were studied. Between the preintervention and postintervention groups, there were no significant improvements in discharge order time, physical discharge time, discharge response time, or discharges before 1 pm. The balancing measure of 30-day readmission was unaffected. However, length of stay (LOS) index, calculated as the ratio of actual to expected LOS, improved; when translated into days, LOS declined by 1 day, with potential associated savings of $373,000. CONCLUSIONS: Interventions to improve discharge timeliness on IPGS service demonstrated mixed effectiveness. Only LOS index improved. Further iterative quality improvement interventions are needed to continue optimizing discharge timeliness and change the culture of pediatric discharge on inpatient subspecialty services in academic children's hospitals.

Unlimited expansion of intestinal stem cells from a wide range of ages.

The recent technical advance in cloning and culturing ground-state intestinal stem cells (ISC) provides us an opportunity of accurate assessment of age-related impact on the function of highly proliferative intestinal stem cells. Our ability of indefinitely and robustly expanding single-stem-cell derived pedigrees in vitro allows us to study intestinal stem cells at the clonal level. Interestingly, comparable number of ISC clones was yielded from 1mm endoscopic biopsy of all donors despite the age. They were passaged in vitro as pedigrees and expanded to 1 billion cells in approximately sixty days without changes in stemness demonstrated by clonogenicity and multipotency. Therefore, our study shows that ISCs from a wide range of ages can be cloned and expanded to unlimited number in vitro with similar efficiency and stability. These patient-derived ISCs harbor intrinsic immortality and are ideal for autologous transplantation, supporting the promise of adult-stem-cell based personalized medicine.

Challenges in IBD Research: Precision Medicine.

Precision medicine is part of five focus areas of the Challenges in IBD research document, which also includes preclinical human IBD mechanisms, environmental triggers, novel technologies, and pragmatic clinical research. The Challenges in IBD Research document provides a comprehensive overview of current gaps in inflammatory bowel diseases (IBD) research and delivers actionable approaches to address them. It is the result of a multidisciplinary input from scientists, clinicians, patients, and funders, and represents a valuable resource for patient centric research prioritization. In particular, the precision medicine section is focused on highlighting the main gap areas that must be addressed to get closer to treatments tailored to the biological and clinical characteristics of each patient, which is the aim of precision medicine. The main gaps were identified in: 1) understanding and predicting the natural history of IBD: disease susceptibility, activity, and behavior; 2) predicting disease course and treatment response; and 3) optimizing current and developing new molecular technologies. Suggested approaches to bridge these gaps include prospective longitudinal cohort studies to identify and validate precision biomarkers for prognostication of disease course, and prediction and monitoring of treatment response. To achieve this, harmonization across studies is key as well as development of standardized methods and infrastructure. The implementation of state-of-the-art molecular technologies, systems biology and machine learning approaches for multi-omics and clinical data integration and analysis will be also fundamental. Finally, randomized biomarker-stratified trials will be critical to evaluate the clinical utility of validated signatures and biomarkers in improving patient outcomes and cost-effective care.