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The present study examined self-tolerance for T cell receptor (TCR) alpha beta intestinal intraepithelial lymphocytes (iIELs) using the 2C transgenic (Tg) mouse model specific for a peptide antigen (Ag) presented by the class I major histocompatibility complex H-2Ld. Although Tg+ T cells were largely deleted from the periphery of Ag+ mice, equivalent numbers of Tg iIELs were present in Ag+ compared to Ag- mice. Tg iIELs in Ag- mice contained CD8 alpha beta, CD8 alpha alpha, and CD4-CD8- subsets, whereas only CD8 alpha alpha and CD4-CD8- Tg iIEL subsets were detected in Ag+ mice. Analysis of surface markers revealed that Tg iIELs in Ag+ mice expressed decreased levels of Thy-1 and increased CD45R/B220 as compared to Ag- Tg iIELs. In response to activation with exogenous peptide or immobilized anti-TCR mAB, iIELs from Ag- mice proliferated at high levels and produced interleukin (IL)-2 and interferon (IFN)-gamma, while Tg+ iIELs from Ag+ mice proliferated at low levels and failed to produce detectable IL-2 or IFN-gamma. Activation of sorted iIEL subsets from Ag- mice revealed that CD8 alpha alpha and CD4-CD8- subsets produced low levels of IL-2 and IFN-gamma in response to activation with antigen-presenting cells and added peptide or immobilized anti-TCR mAb, while CD8 alpha beta + iIELs responded to endogenous levels of peptide. In response to APC and exogenous peptide, sorted iIEL subsets from Ag+ mice produced IL-2 and IFN-gamma, and proliferated at greatly reduced levels compared to corresponding subsets from Ag- mice. Analysis of cytokine mRNA levels revealed that activation in vitro induced IL-2 mRNA only in Ag-, but not Ag+ iIELs, whereas a high level of IL-4 mRNA induction was detected in Tg+ iIELs from Ag+ mice, and to a lesser degree, from Ag- mice. These data suggest that tolerance for Tg+ iIELs resulted in the deletion of CD8 alpha beta + subsets and the persistence of Tg+ iIEL subsets with decreased sensitivity to endogenous levels of self-peptide. A comparison of the cytokine profiles expressed by Tg+ iIEL subsets in Ag- and Ag+ mice suggested that tolerance induction had involved the functional deviation of cells from TC1 (T helper-1-like) to a less inflammatory TC2 (T helper-2-like) phenotype capable of mediating humoral immune responses in the mucosa.

Mitochondria are dynamic organelles that function in cellular energy metabolism, intracellular and extracellular signalling, cellular fate and stress responses. Mitochondria of the intestinal epithelium, the cellular interface between self and enteric microbiota, have emerged as crucial in intestinal health. Mitochondrial dysfunction occurs in gastrointestinal diseases, including inflammatory bowel diseases and colorectal cancer. In this Review, we provide an overview of the current understanding of intestinal epithelial cell mitochondrial metabolism, function and signalling to affect tissue homeostasis, including gut microbiota composition. We also discuss mitochondrial-targeted therapeutics for inflammatory bowel diseases and colorectal cancer and the evolving concept of mitochondrial impairment as a consequence versus initiator of the disease., (© 2024. Springer Nature Limited.)

Introduction: A significant proportion of patients with acute severe ulcerative colitis (ASUC) require colectomy., Methods: Patients with ASUC treated with upadacitinib and intravenous corticosteroids at 5 hospitals are presented. The primary outcome was 90-day colectomy rate. Secondary outcomes included frequency of steroid-free clinical remission, adverse events, and all-cause readmissions., Results: Of the 25 patients with ASUC treated with upadacitinib, 6 (24%) patients underwent colectomy, 15 (83%) of the 18 patients with available data and who did not undergo colectomy experienced steroid-free clinical remission (1 patient did not have complete data), 1 (4%) patient experienced a venous thromboembolic event, while 5 (20%) patients were readmitted., Discussion: Upadacitinib along with intravenous corticosteroids may be an effective treatment for ASUC., (Copyright © 2024 by The American College of Gastroenterology.)

Background: Rural residence has been associated with a lower incidence of inflammatory bowel disease (IBD) but higher health care utilization and worse outcomes. Socioeconomic status is intrinsically tied to both IBD incidence and outcomes. Inflammatory bowel disease outcomes have not been investigated in Appalachia: a rural, economically distressed region rife with risk factors for both increased incidence and unfavorable outcomes., Methods: Hospital inpatient discharge and outpatient services databases were utilized to assess outcomes in patients diagnosed with either Crohn's disease (CD) or ulcerative colitis (UC) in Kentucky. Encounters were classified by patient residence in Appalachian or non-Appalachian counties. Data were reported as crude and age-adjusted rates of visits per 100,000 population per year collected in 2016 to 2019. National inpatient discharge data from 2019, stratified by rural and urban classification codes, were utilized to compare Kentucky to national trends., Results: Crude and age-adjusted rates of inpatient, emergency department and outpatient encounters were higher in the Appalachian cohort for all 4 years observed. Appalachian inpatient encounters are more frequently associated with a surgical procedure (Appalachian, 676, 24.7% vs non-Appalachian, 1408, 22.2%; P = .0091). In 2019, the Kentucky Appalachian cohort had significantly higher crude and age-adjusted rates of inpatient discharges for all IBD diagnoses compared with national rural and nonrural populations (crude 55.2; 95% CI, 50.9-59.5; age-adjusted 56.7; 95% CI, 52.1-61.3)., Conclusions: There is disproportionately higher IBD health care utilization in Appalachian Kentucky compared with all cohorts, including the national rural population. There is a need for aggressive investigation into root causes of these disparate outcomes and identification of barriers to appropriate IBD care., (© The Author(s) 2023. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Traumatic brain injury (TBI) increases gastrointestinal morbidity and associated mortality. Clinical and preclinical studies implicate gut dysbiosis as a consequence of TBI and an amplifier of brain damage. However, little is known about the association of gut dysbiosis with structural and functional changes of the gastrointestinal tract after an isolated TBI. To assess gastrointestinal dysfunction, mice received a controlled cortical impact or sham brain injury and intestinal permeability was assessed at 4 h, 8 h, 1 d, and 3 d after injury by oral administration of 4 kDa FITC Dextran prior to euthanasia. Quantification of serum fluorescence revealed an acute, short-lived increase in permeability 4 h after TBI. Despite transient intestinal dysfunction, no overt morphological changes were evident in the ileum or colon across timepoints from 4 h to 4 wks post-injury. To elucidate the timeline of microbiome changes after TBI, 16 s gene sequencing was performed on DNA extracted from fecal samples collected prior to and over the first month after TBI. Differential abundance analysis revealed that the phylum Verrucomicrobiota was increased at 1, 2, and 3 d after TBI. The Verrucomicrobiota species was identified by qPCR as Akkermansia muciniphila, an obligate anaerobe that resides in the intestinal mucus bilayer and produces short chain fatty acids (e.g. butyrate) utilized by intestinal epithelial cells. We postulated that TBI promotes intestinal changes favorable for the bloom of A. muciniphila. Consistent with this premise, the relative area of mucus-producing goblet cells in the medial colon was significantly increased at 1 d after injury, while colon hypoxia was significantly increased at 3 d. Our findings reveal acute gastrointestinal functional changes coupled with an increase of beneficial bacteria suggesting a potential compensatory response to systemic stress after TBI., (© 2024. The Author(s).)

The white-bellied pangolin ( Phataginus tricuspis ) is the world's most trafficked mammal and is at risk of extinction. Reducing the illegal wildlife trade requires an understanding of its origins. Using a genomic approach for tracing confiscations and analyzing 111 samples collected from known geographic localities in Africa and 643 seized scales from Asia between 2012 and 2018, we found that poaching pressures shifted over time from West to Central Africa. Recently, Cameroon's southern border has emerged as a site of intense poaching. Using data from seizures representing nearly 1 million African pangolins, we identified Nigeria as one important hub for trafficking, where scales are amassed and transshipped to markets in Asia. This origin-to-destination approach offers new opportunities to disrupt the illegal wildlife trade and to guide anti-trafficking measures.

Background: There are many ways that palliative care can support patients with heart failure, but the role of palliative care in supporting patients who are considering or are already using advanced cardiac therapies is less clear. Objective: To understand referring providers' perspectives about the role of palliative care in the treatment of patients with heart failure considering or using advanced cardiac therapies. Design: Qualitative study using a semistructured interview guide. Setting/Subjects: This study was conducted at an academic medical center in the United States with an integrated cardiac palliative care program. Interviews were conducted with cardiology providers, including cardiologists, cardiac surgeons, and nurse practitioners who care for patients with heart failure and who are considering or receiving advanced cardiac therapies. Measurements: Interview transcripts were analyzed deductively and inductively to reveal themes in providers' perspectives. Results: Five themes were identified about the role of palliative care when advanced therapies were considered or being used: (1) educating patients; (2) supporting goal-concordant care; (3) managing symptoms; (4) addressing psychosocial needs; and (5) managing end-of-life care. Providers suggested palliative care could be a facilitator of advanced therapies, rather than merely something to add to end-of-life care. Conclusions: Cardiology providers recognize the value of integrating palliative care across the heart failure disease trajectory to provide therapy options, support decision-making processes, and provide goal-concordant care for patients considering or receiving advanced therapies. Increasing awareness of opportunities to integrate palliative care throughout the treatment of these patients may help cardiology providers better coordinate with palliative care specialists to improve patient care.

The development of 3D organoids of the small intestine is a tremendous breakthrough in drug development and biological research. However, the development of colonic organoids (i.e., colonoids) is particularly challenging due to a lack of simple, cost-effective protocols for colonoid cultivation. Here, intestinal homogenates are described as a supplement to the culture medium for maintaining and replicating colonic stem cells. Colonoids generated by this cultivation protocol demonstrate substantial proliferation and differentiation (3 months). There is a similarity between cultured colonoids and primary colon tissue regarding structure and functionality. To evaluate the functionality of colonoids, permeability testing is performed with suspensions of 4 and 40 kDa fluorescein isothiocyanate-dextran (FITC-DEX). It is observed that neither can permeate the healthy epithelial barrier. The P-glycoprotein receptor, a vital drug efflux pump mitigating potential drug toxicity, is functionally manipulated, as evidenced by its inhibition function by verapamil and monitoring uptake of Rhodamin 123. In addition, Forskolin treatment which affects chloride transport results in organoid swelling; this confirms the functional expression of the CFTR transporter in the colonoids. This protocol to generate colonoids is promising for high-throughput drug screening, toxicity testing, and oral drug development., (© 2023 The Authors. Advanced Biology published by Wiley-VCH GmbH.)

Background: Palliative care integration into cardiology is growing, allowing primary cardiology care teams increasing opportunities to utilize palliative care to support processes such as advance care planning (ACP). Objective: The aim of the study is to understand perspectives of cardiac care team members about the involvement and impact of palliative care on ACP in heart failure. Design: A qualitative study using a semistructured interview guide was performed. Settings/Subjects: Interviews were conducted with cardiac care team members, including cardiologists, cardiac surgeons, and nurse practitioners, at a large academic medical center in the United States with an integrated cardiac palliative care team. Measurements: Deductive and inductive thematic analysis of interview transcripts enabled characterization of themes around the role of palliative care in ACP. Results: Two themes were identified with regard to providers' perspectives about ACP: (1) different levels of comfort with initiating and conducting ACP conversations and (2) different opinions about the desired role of palliative care in the ACP process. In exploring these themes, we characterized four distinct approaches to ACP with palliative care as a novel framework for planning consultation. Conclusions: The different approaches to ACP and the implications for how cardiac providers interact with the palliative care team present an important opportunity to guide ACP consultation in practice. Adoption of this framework may help cardiac providers enhance the process of care delivery and ACP in important ways that improve care for their patients.

Background: Characteristics of patients undergoing outpatient evaluation for advanced cardiac therapies are largely unknown. Objective: To describe demographics, baseline quality of life, and frailty of patients undergoing evaluation for advanced therapies at the time of presentation for evaluation in an outpatient cardiac palliative care clinic and examine key quality of life differences across patients. Design: Retrospective chart review to report baseline demographics and quality of life. Settings/Subjects: Patients at a large academic medical center in the United States referred for advanced cardiac therapies in 2021. Measurements: Depression and anxiety were measured using the Hospital Anxiety and Depression Scale; quality of life was measured using the Functional Assessment of Chronic Illness Therapy-Palliative care (FACIT-Pal) tool; and frailty was measured using the Fried Frailty Phenotype. Differences in quality of life by disease etiology, demographic characteristics, and frailty were assessed. Results: Fifty-four patients were seen in the outpatient cardiac palliative care clinic for advanced therapy evaluation. Most were Caucasian (80%) and male (74%). Patients traveled an average of 61 minutes to the clinic. All but five lived in a rural, medically underserved, or health professional shortage area. Forty percent scored abnormal or borderline abnormal for anxiety; 22% scored abnormal or borderline abnormal for depression. The FACIT-Pal mean score was 129 (standard deviation 23), with emotional and functional well-being domains contributing most to poor quality of life. Seventy-one percent were frail. Patients with a nonischemic etiology had a 3.32 times higher rate of anxiety than nonischemic patients (95% confidence interval = 1.05-10.54, p  = 0.041). Conclusion: As patients undergoing transplant evaluation have high levels of depression, anxiety, and frailty, interdisciplinary care teams will be needed to properly manage the needs of this complex population. These results can inform efforts to integrate palliative care into advanced cardiac therapy and improve patients' experiences.

Background: Patients with heart failure frequently have significant disease burden and complex psychosocial needs. The integration of palliative care into the management of these patients can decrease symptom burden throughout their course of illness. Therefore, in 2009, we established a cardiac palliative care clinic colocated with heart failure providers in a large academic heart hospital. Objective: To better understand the facilitators and barriers to integrating palliative care into our heart failure management service. Design: Qualitative study using a semistructured interview guide. Setting, Subjects: Between October 2020 and January 2021, we invited all 25 primary cardiac providers at our academic medical center in the midwestern United States to participate in semistructured qualitative interviews to discuss their experiences with the cardiac palliative care clinic. Measurements: Interview transcripts were analyzed using a deductive-dominant thematic analysis approach to reveal emerging themes. Results: Providers noted that the integration of palliative care into the treatment of patients with heart failure was helped and hindered primarily by issues related to operations and communications. Operational themes about clinic proximity and the use of telehealth as well as communication themes around provider-provider communication and the understanding of palliative care were particularly salient. Conclusions: The facilitators and barriers identified have broad applicability that are independent of the etiological nature (e.g., cancer, pulmonary, neurological) of any specialty or palliative care clinic. Moreover, the strategies we used to implement improvements in our clinic may be of benefit to other practice models such as independent and embedded clinics.

Background: The total artificial heart (TAH) is an implanted device approved as a modality to stabilize patients with severe biventricular heart failure or persistent ventricular arrhythmias for evaluation and bridge to transplantation. According to the Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS), about 450 patients received a TAH between 2006 and 2018. Patients being evaluated for a TAH are often critically ill and a TAH offers the best chance at survival. Given the prognostic uncertainty of these patients, there is a crucial need for preparedness planning to help patients and their caregivers plan for living and supporting a loved one with a TAH., Aim: To describe an approach to preparedness planning and highlight the importance of palliative care., Methods: We reviewed the current needs and approaches to preparedness planning for a TAH. We categorized our findings and suggest a guide to maximize conversations with patients and their decision makers., Results: We identified four critical areas to address: the decision maker, minimal acceptable outcome/maximal acceptable burden, living with the device, and dying with the device. We suggest using a framework of mental and physical outcomes and locations of care as a way to identify minimal acceptable outcome and maximal acceptable burden., Conclusion: Decision making for a TAH is complex. There is an urgency and patients do not always have capacity. Identifying legal decision makers and social support is critical. The surrogate decision makers should be included in preparedness planning including discussions about end-of-life care and treatment discontinuation. Having palliative care as members of the interdisciplinary mechanical circulatory support team can assist in these preparedness conversations., Competing Interests: Competing interests: BCL is an unpaid member of the Finance Committee with the International Society of Heart and Lung Transplantation. NAM is the Site Prinicpal Investigator for the EXACT trial with Xylocor. He also receives consulting fees through Medtronic, Abbott, SynCardia, Carmat and Xylocor. He serves on the Clinical Events Committee for Carmat, DSBM-Prevent II for Abott and Medical Advisory Board for Xylocor. He holds the following patents: (USPTO 17/184,781) Prevention and Treatment of Viral Infection and Viral Infection-Induced Organ Failure, filing date 25 February 2021 and (USPTO 63/240,253) Devices and Methods for Left Atrial Appendage Occlusion, filing date 2 September 2022. TAB has a role with the American Academy of Hospice and Palliative Medicine Finance Committee and holds stock options with Perfect Health (ConcertoCare)., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Workplace violence in healthcare institutions is becoming more frequent. The objective of this study was to better understand the nature of threat and physical acts of violence from heart and lung transplant patients and families toward healthcare providers and suggest programmatic mitigation strategies. We administered a brief survey to attendees at the 2022 International Society of Heart and Lung Transplantation Conference in Boston, Massachusetts. A total of 108 participants responded. Threats of physical violence were reported by forty-five participants (42%), were more frequently reported by nurses and advanced practice providers than physicians (67% and 75% vs. 34%; p < 0.001) and were more prevalent in the United States than abroad (49% vs. 21%; p = 0.026). Acts of physical violence were reported by one out of every eight providers. Violence against providers in transplant programs warrants closer review by health systems in order to ensure the safety of team members.

Flies of the family Calliphoridae, commonly called blow flies, are important in the decomposition process. Knowledge on their succession pattern on corpses, species identification and the duration of their life cycle stages can be useful in forensic investigations especially when estimating the post-mortem interval. We performed linear-based morphometrics on the cephalopharyngeal skeleton of four blow fly species found in Jamaica to distinguish species and determine larval development stage. We collected eggs from pigs' heads used as bait in the field and conducted rearing exercises in the laboratory. We used the internal skeletonized structure, the cephalopharyngeal skeleton, to develop a practical and efficient method for species identification. For the first instar, we found species can be differentiated using all the measurements analysed in the study. We found that the mouth hook length may be useful in distinguishing larvae in the second instar. For the larvae in the third instar, the whole length of the skeleton, from mouth hook to length of the dorsal cornue, may be useful for separating species. We provide information on the cephalopharyngeal skeleton of Lucilia lucigerens (James), a blow fly species endemic to Jamaica, for the first time. Our work provides relevant information that could be utilized for species identification and life stage determination if fly evidence is to be incorporated in forensic investigations in Jamaica., (© 2022. Sociedade Entomológica do Brasil.)

Background: The Gemini trial failed to detect a significant difference in response rate for patients with ulcerative colitis (UC) randomized to standard (every 8 week) vedolizumab dosing vs escalated (every 4 week) dosing. Subsequent real-world data imply the Gemini trial design may have obscured a benefit of escalated dosing., Aims: We investigated outcomes after vedolizumab dose escalation for patients with UC. We also explored potential clinical predictors of dose escalation requirement., Methods: In this retrospective study, we included patients with UC who received vedolizumab between 1/2017-1/2019. We compared rates of clinical response (decrease in partial Mayo score by ≥ 2) and remission (partial Mayo < 2) for standard vs escalated dosing., Results: Among the 90 patients reviewed, 52 achieved and maintained remission on standard dosing. The average time to remission with standard dosing was 33.3 ± 6.6 weeks. After an average of 56.3 ± 7.4 weeks standard dosing, 24 patients (22 "partial responders" and 2 "non-responders") were dose-escalated. Of the 22 "partial responders" dose-escalated, 10 (45%) achieved remission, 10 (45%) achieved further improvement. Neither "non-responder" demonstrated further clinical benefit. Prior anti-tumor necrosis factor (anti-TNF) biologic exposure predicted dose escalation requirement (p = 0.008). Patients requiring dose escalation had more severe disease at baseline as measured by both full Mayo (p = 0.009) and partial Mayo scores (p = 0.01)., Conclusions: We show dose escalation benefited patients with UC who exhibit a "partial response" to standard dosing. Early vedolizumab dose escalation should be considered in both patients with severe disease and those with prior anti-TNF experience.

Background: Chronic opioid use is associated with poorer clinical outcomes in inflammatory bowel disease., Aims: To investigate an association between chronic opioid use and persistence with biologic agents in management of inflammatory bowel disease., Methods: A total of 16 624 patients diagnosed with inflammatory bowel disease and receiving a first-time biologic prescription from 2011 to 2016 were identified retrospectively from the Truven MarketScan Database. A cohort of 1768 patients were identified as chronic opioid users utilising outpatient prescription claims. Utilisation patterns of biologic therapies were assessed from inpatient administration and outpatient claims data, including persistence calculations. Information on healthcare utilisation and common comorbidities was also collected. A Cox regression model was constructed to assess the hazard of chronic opioid use on early discontinuation of biologic therapy controlling for disease severity., Results: A mean 1.5 different biologic agents were utilised by inflammatory bowel disease patients with chronic opioid use (vs 1.37 in the comparator group; P < 0.0001). A lower proportion of the chronic opioid use cohort persisted on biologic therapies to the end of the study period (16.2% vs 33.5% P < 0.0001). Inflammatory bowel disease patients with chronic opioid use utilised more healthcare resources and had a higher rate of comorbidities than the reference cohort. Patients with chronic opioid use were 23% more likely (hazard ratio 1.23; 95% CI [1.16-1.31]) to be non-persistent with biologic therapy while accounting for relevant markers of disease acuity., Conclusions: Chronic opioid use is associated with increased hazard of biologic discontinuation in inflammatory bowel disease. Symptoms of opioid withdrawal may mimic IBD flares thereby leading providers to inappropriately switch biologic therapies and compromise disease control., (© 2021 John Wiley & Sons Ltd.)

Background: Clinicians often utilize off-label dose escalation of ustekinumab (UST) in Crohn's disease (CD) patients with disease refractory to standard dosing. Previous studies report mixed results with dose escalation of UST., Methods: A retrospective observational study of 143 adult patients with CD receiving UST over a 33-month time period was conducted. Patients receiving UST at standard dosage for a minimum of 16 weeks were included in the analysis. Primary outcomes collected were clinical response [Physician Global Assessment Score (PGA) by >1] and remission (PGA = 0). Changes in clinical parameters were calculated for dose-escalated patients beginning with the time of dose switch (~42 weeks) and compared with a group of patients who were classified as "failing" standard dosing at 42 weeks who were not dose escalated., Results: Dose escalation improved PGA by 0.47 ± 0.19 compared with patients remaining on every 8 weeks dosing (Q8 week), who worsened by 0.23 ± 0.23 ( p  < 0.05). Dose escalation decreased CRP 0.33 ± 0.19 mg/L and increased serum albumin 0.23 ± 0.06 g/dL ( p  < 0.05). Surprisingly, disease duration and prior CD surgeries inversely correlated with the need for dose escalation., Conclusion: Our results support UST Q4 week dose escalation for selected CD patients who fail to achieve remission on standard Q8 week dosing. Dose escalation improves clinical outcomes, prevents worsening disease severity, and positively impacts CRP and albumin levels. Together these data indicate that clinicians should attempt Q4 week UST dosing in refractory CD patients before switching to an alternative class of biologic therapy., Competing Interests: Conflict of interest statement: TAB has consulted and received honoraria for speaker’s bureau activities for Takeda and AbbVie pharmaceutical companies. None of the other authors have any conflict of interest to disclose., (© The Author(s), 2020.)

Chronic liver injury is a risk factor for cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms that regulate the decision between normal injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a tumor stem cell marker, is induced during cirrhosis and HCC. Here, we demonstrate that DCLK1-overexpressing primary human hepatocytes formed spheroids in suspension cultures. Spheroids derived from DCLK1-overexpressing hepatoma cells showed high level expression of active β-catenin, α-fetoprotein, and SOX9, suggesting that DCLK1 overexpression induces clonogenicity and dedifferentiated phenotypes in hepatoma cells. DCLK1 overexpression in hepatoma cells also increased phosphorylation of GSK-3β at Ser 9 . This was associated with an induction of a 48-kDa active β-catenin with a preserved hypophosphorylated N-terminus that interacted with nuclear TCF-4 resulting in luciferase reporter activity and cyclin D1 expression. DCLK1 downregulation inhibited 48-kDa β-catenin expression. The proteasome inhibitor bortezomib did not block the 48-kDa β-catenin, instead, caused a threefold accumulation, suggesting a proteasome-independent mechanism. Liver tissues from patients with cirrhosis and HCC revealed epithelial co-staining of DCLK1 and active β-catenin, and cleaved E-cadherin. Repopulated DCLK1-overexpressing primary human hepatocytes in humanized FRG mouse livers demonstrated active β-catenin. In conclusion, DCLK1 regulates oncogenic signaling and clonogenicity of hepatocytes by a novel non-canonical/atypical β-catenin-dependent mechanism.

Background and Aims: Intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. Disruption of this homeostasis is associated with disorders such as inflammatory bowel disease (IBD). We investigated the role of Sirtuin 2 (SIRT2), a NAD-dependent protein deacetylase, in intestinal epithelial cell (IEC) proliferation and differentiation and the mechanism by which SIRT2 contributes to maintenance of intestinal cell homeostasis., Methods: IECs were collected from SIRT2-deficient mice and patients with IBD. Expression of SIRT2, differentiation markers (mucin2, intestinal alkaline phosphatase, villin, Na,K-ATPase, and lysozyme) and Wnt target genes (EPHB2, AXIN2, and cyclin D1) was determined by western blot, real-time RT-PCR, or immunohistochemical (IHC) staining. IECs were treated with TNF or transfected with siRNA targeting SIRT2. Proliferation was determined by villus height and crypt depth, and Ki67 and cyclin D1 IHC staining. For studies using organoids, intestinal crypts were isolated., Results: Increased SIRT2 expression was localized to the more differentiated region of the intestine. In contrast, SIRT2 deficiency impaired proliferation and differentiation and altered stemness in the small intestinal epithelium ex vivo and in vivo. SIRT2-deficient mice showed decreased intestinal enterocyte and goblet cell differentiation but increased the Paneth cell lineage and increased proliferation of IECs. Moreover, we found that SIRT2 inhibits Wnt/β-catenin signaling, which critically regulates IEC proliferation and differentiation. Consistent with a distinct role for SIRT2 in maintenance of gut homeostasis, intestinal mucosa from IBD patients exhibited decreased SIRT2 expression., Conclusion: We demonstrate that SIRT2, which is decreased in intestinal tissues from IBD patients, regulates Wnt-β-catenin signaling and is important for maintenance of IEC proliferation and differentiation., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancer cells are known to upregulate aerobic glycolysis to promote growth, proliferation, and survival. However, the role of mitochondrial respiration in tumorigenesis remains elusive. Here we report that inhibition of mitochondrial function by silencing TFAM, a key transcription factor essential for mitochondrial DNA (mtDNA) replication and the transcription of mtDNA-encoded genes, markedly reduced tumor-initiating potential of colon cancer cells. Knockdown of TFAM significantly decreased mitochondrial respiration in colon cancer cells; however, the cellular levels of ATP remained largely unchanged as a result of increased glycolysis. This metabolic alteration rendered cancer cells highly susceptible to glucose deprivation. Interestingly, upregulation of glycolysis was independent of hypoxia-inducible factor-1 (HIF1) as TFAM knockdown cells fail to stabilize HIF1α under hypoxic conditions. Moreover, knockdown of TFAM results in decreased expression of genes-associated cancer stem cells downstream of Wnt/β-catenin signaling. Metabolic analysis reveals that the level of α-ketoglutarate (α-KG) was significantly upregulated in TFAM knockout cells. Silencing of prolyl hydroxylase domain-containing protein 2 (PHD2), a α-KG-dependent dioxyenase, rescued the expression of target genes of both HIF1α and Wnt/β-catenin. Furthermore, intestinal-specific knockout of TFAM prevents tumor formation in Apc-mutant mouse models of colon cancer. Taken together, our findings identify a novel role of mitochondria-mediated retrograde signaling in regulating Wnt signaling and tumor initiation in colon cancer.

Vitamin C (ascorbic acid) and vitamin B 3 (niacin) have been extensively studied since the 20th century. In the area of stem cell biology, vitamin C has shown its direct impact toward homeostasis and epigenetic changes (D'Aniello et al., Stem Cells International, 2017, 1-16). Vitamin B 3 aids in maintaining healthy intestinal homeostasis and reducing gut inflammation by participating in the rapamycin signaling pathway (Kumar et al., The American Journal of Physiology-Gastrointestinal and Liver Physiology, 2013). In this study, vitamin C and vitamin B 3 (600 and 1,200 μg/mL) have been explored as potential new biomaterials to study their effects on four types of intestinal stem cells which are isolated from mice bearing different microbiota. We observed that C3H ASF and 129 ASF IL-10 are more sensitive towardB7 600 μg/mL vitamin B 3 and 1,200 μg/mL vitamin C. The lowest growth rate and viability for all types of organoids was with 1,200 μg/mL vitamin C. From quantitative polymerase chain reaction analysis (qPCR analysis), MUC2 was upregulated for 129 ASF and C3H Conv when exposed to 600 μg/mL and 1,200 μg/mL vitamin C. It suggests that large amounts of glycoprotein may be produced after adding high concentrations of vitamin C. Since inflammatory bowel disease has low level of MUC2, this finding may be helpful in restoring mucosal health by upregulating the MUC2 gene while altering patient's microbiota (Sibila et al., Annals of the American Thoracic Society, 2016). These results are expected to have a positive translational impact because this bottom-up strategy would be instrumental in developing Vitamin C and B3 based orally available therapeutic strategies and formula for advancing the fields of gastrointestinal regenerative medicine., (© 2019 Wiley Periodicals, Inc.)

Necrotizing enterocolitis (NEC) is a devastating disease affecting premature infants with intestinal inflammation and necrosis. The neonatal intestinal inflammatory response is rich in macrophages, and blood monocyte count is low in human NEC. We previously found that NF-κB mediates the intestinal injury in experimental NEC. However, the role of NF-κB in myeloid cells during NEC remains unclear. Herein, inhibitor of kappaB kinase β (IKKβ), a critical kinase mediating NF-κB activation, was deleted in lysozyme M (Lysm)-expressing cells, which were found to be Cd11b + Ly6c + monocytes but not Cd11b + Ly6c - macrophages in the dam-fed neonatal mouse intestine. NEC induced differentiation of monocytes into intestinal macrophages and up-regulation of monocyte recruitment genes (eg, L-selectin) in the macrophage compartment in wild-type mice, but not in pups with IKKβ deletion in Lysm + cells. Thus, NF-κB is required for NEC-induced monocyte activation, recruitment, and differentiation in neonatal intestines. Furthermore, pups with Lysm-IKKβ deletion had improved survival and decreased incidence of severe NEC compared with littermate controls. Decreased NEC severity was not associated with an improved intestinal barrier. In contrast, NEC was unabated in mice with IKKβ deletion in intestinal epithelial cells. Together, these data suggest that recruitment of Ly6c + monocytes into the intestine, NF-κB activation in these cells, and differentiation of Ly6c + monocytes into macrophages are critical cellular and molecular events in NEC development to promote disease., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Ulcerative colitis (UC) patients exhibit elevated histamine, but how histamine exacerbates disease is unclear as targeting histamine 1 receptor (H1R) or H2R is clinically ineffective. We hypothesized that histamine functioned instead through the other colon-expressed histamine receptor, H4R. In humans, UC patient biopsies exhibited increased H4R RNA and protein expression over control tissue, and immunohistochemistry showed that H4R was in proximity to immunopathogenic myeloperoxidase-positive neutrophils. To characterize this association further, we employed both the oxazolone (Ox)- and dextran sulfate sodium (DSS)-induced experimental colitis mouse models and also found upregulated H4R expression. Mast cell (MC)-derived histamine and H4R drove experimental colitis, as H4R -/- mice had lower symptom scores, neutrophil-recruitment mediators (colonic interleukin-6 (IL-6), CXCL1, CXCL2), and mucosal neutrophil infiltration than wild-type (WT) mice, as did MC-deficient Kit W-sh/W-sh mice reconstituted with histidine decarboxylase-deficient (HDC -/- ) bone marrow-derived MCs compared with WT-reconstituted mice; adaptive responses remained intact. Furthermore, Rag2 -/- × H4R -/- mice had reduced survival, exacerbated colitis, and increased bacterial translocation than Rag2 -/- mice, revealing an innate protective antibacterial role for H4R. Taken together, colonic MC-derived histamine initiates granulocyte infiltration into the colonic mucosa through H4R, suggesting alternative therapeutic targets beyond adaptive immunity for UC.

Inflammatory bowel disease (IBD) causes inflammation to the gastrointestinal tract. Local administration of anti-inflammatory drugs such as 5-aminosalicylic acid (5-ASA) can alleviate the symptoms of IBD. The application of nanoparticles for IBD treatment in direct rectal administration showed high drug availability and treatment efficacy. However, relying on size-dependent adsorption of smaller particles is not sufficient for making the formulation capable of targeting. Intestinal organoids can improve the functionality of the nanoparticles due to their ability to adsorb small nanoparticle inside the lumen and attach to the damaged area. In this study, intestinal organoids were used as carriers of 5-ASA-loaded poly(lactic-co-glycolic acid) nanoparticles. The nanoparticle sizes, confirmed by scanning electron microscopy, were 200-300 nm and the zeta potential were negative. The nanoparticles did not have any noticeable pernicious effect on organoid growth and viability. After mixing the nanoparticles with Matrigel and organoids, Rhodamine B loaded inside the nanoparticles was highly detected inside the organoid's lumen after 3 days by confocal fluorescent microscopy and no longer detected in the lumen after day 4. It may be attributed to the ability of the lumen to digest particles. Thus, the organoid Trojan horse system is a possible approach for delivering drugs to inflamed areas. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 876-886, 2018., (© 2017 Wiley Periodicals, Inc.)

The intestinal epithelium of the gastrointestinal (GI) tract constantly renews itself to absorb nutrients and provide protection for the body from the outside world. Since the intestinal epithelium is constantly exposed to various chemicals and dietary components, it is critical to determine which constituents promote or inhibit intestinal epithelium health and growth rate. Intestinal organoids, three-dimensional miniature models of the intestines, represent an ex vivo tool to investigate intestinal physiology and growth patterns. In this study, we measured the growth rates of murine intestinal organoids exposed to various concentrations of different dietary constituents. Results indicate that caffeic acid inhibited organoid growth in a concentration-dependent manner, curcumin exhibited variable effectiveness, and vitamin C had no effect on organoid growth.

Nuclear activation of Wnt/β-catenin signaling is required for cell proliferation in inflammation and cancer. Studies from our group indicate that β-catenin activation in colitis and colorectal cancer (CRC) correlates with increased nuclear levels of β-catenin phosphorylated at serine 552 (pβ-Cat 552 ). Biochemical analysis of nuclear extracts from cancer biopsies revealed the existence of low molecular weight (LMW) pβ-Cat 552 , increased to the exclusion of full size (FS) forms of β-catenin. LMW β-catenin lacks both termini, leaving residues in the armadillo repeat intact. Further experiments showed that TCF4 predominantly binds LMW pβ-Cat 552 in the nucleus of inflamed and cancerous cells. Nuclear chromatin bound localization of LMW pβ-Cat 552 was blocked in cells by inhibition of proteasomal chymotrypsin-like activity but not by other protease inhibitors. K48 polyubiquitinated FS and LMW β-catenin were increased by treatment with bortezomib. Overexpressed in vitro double truncated β-catenin increased transcriptional activity, cell proliferation and growth of tumor xenografts compared to FS β-catenin. Serine 552-> alanin substitution abrogated K48 polyubiquitination,  β-catenin nuclear translocation and tumor xenograft growth. These data suggest that a novel proteasome-dependent posttranslational modification of β-catenin enhances transcriptional activation. Discovery of this pathway may be helpful in the development of diagnostic and therapeutic tools in colitis and cancer.

TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn's disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/β-catenin-mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/β-catenin signaling correlates with inflammation status. TNF-deficient ( Tnf -/- ) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM-derived cells and TNFR expression by radioresistant IECs. Wild-type→ Tnfr1/2 -/- BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/β-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/β-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced β-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/β-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Aim: To assess dietary myo-inositol in reducing stem cell activation in colitis, and validate pβ-catenin S552 as a biomarker of recurrent dysplasia., Methods: We examined the effects of dietary myo-inositol treatment on inflammation, pβ-catenin S552 and pAkt levels by histology and western blot in IL-10 -/- and dextran sodium sulfate-treated colitic mice. Additionally, we assessed nuclear pβ-catenin S552 in patients treated with myo-inositol in a clinical trial, and in patients with and without a history of colitis-induced dysplasia., Results: In mice, pβ-catenin S552 staining faithfully reported the effects of myo-inositol in reducing inflammation and intestinal stem cell activation. In a pilot clinical trial of myo-inositol administration in patients with a history of low grade dysplasia (LGD), two patients had reduced numbers of intestinal stem cell activation compared to the placebo control patient. In humans, pβ-catenin S552 staining discriminated ulcerative colitis patients with a history of LGD from those with benign disease., Conclusion: Enumerating crypts with increased numbers of pβ-catenin S552 - positive cells can be utilized as a biomarker in colitis-associated cancer chemoprevention trials., Competing Interests: Conflict-of-interest statement: The authors have no conflicts-of-interest to report.

Background and Aims: Patients with Crohn's disease (CD) are often prescribed opioids chronically to manage pain associated with their disease. However, little evidence exists to support this practice. Here, we examine newly diagnosed patients with CD with and without chronic opioid use (COU) and sought to identify predictors and consequences of COU., Methods: A nationally representative administrative health care claims that data set identified newly diagnosed patients with CD. Their data were examined during the periods 6 months before and 2 years after diagnosis. Multivariable logistic regression was used to assess predictors of COU at diagnosis., Results: The final study cohort consisted of 47,164 patients with CD. Of them, 3.8% were identified with new COU. Chronic opioid users were more likely women, older, and likely who had more surgeries, endoscopies, admissions, and medication usage compared with other patients. Features detected before CD diagnosis that correlated with COU after diagnosis included previous opioid use (odds ratio [OR] = 6.6), chronic pain (OR = 1.36), arthritis (OR = 1.95), and mental disorders (OR = 1.58). Interestingly, emergency department visits before CD Dx increased the risk of COU (OR = 1.11), whereas endoscopy reduced COU risk (OR = 0.88)., Conclusions: This study presents a nationally representative assessment of COU in newly diagnosed patients with CD. The results may be used to determine the impact of COU in this population and to alert clinicians to those patients with CD at high risk of COU. Chronic opioids are consistently associated with indicators of more severe disease; however, additional research is needed to determine whether COU drives disease severity or vice versa.

Background & Aims: Total parenteral nutrition (TPN), a crucial treatment for patients who cannot receive enteral nutrition, is associated with mucosal atrophy, barrier dysfunction, and infectious complications. Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) improve intestinal epithelial cell (IEC) responses and attenuate mucosal atrophy in several TPN models. However, it remains unclear whether these 2 factors use distinct or overlapping signaling pathways to improve IEC responses. We investigated the interaction of GLP-2 and EGF signaling in a mouse TPN model and in patients deprived of enteral nutrition., Methods: Adult C57BL/6J, IEC-Egfr knock out (KO) and IEC-pik3r1 KO mice receiving TPN or enteral nutrition were treated with EGF or GLP-2 alone or in combination with reciprocal receptor inhibitors, GLP-2(3-33) or gefitinib. Jejunum was collected and mucosal atrophy and IEC responses were assessed by histologic, gene, and protein expression analyses. In patients undergoing planned looped ileostomies, fed and unfed ileum was analyzed., Results: Enteral nutrient deprivation reduced endogenous EGF and GLP-2 signaling in mice and human beings. In the mouse TPN model, exogenous EGF or GLP-2 attenuated mucosal atrophy and restored IEC proliferation. The beneficial effects of EGF and GLP-2 were decreased upon Gefitinib treatment and in TPN-treated IEC-Egfr KO mice, showing epidermal growth factor-receptor dependency for these IEC responses. By contrast, in TPN-treated IEC-pi3kr1 KO mice, the beneficial actions of EGF were lost, although GLP-2 still attenuated mucosal atrophy., Conclusions: Upon enteral nutrient deprivation, exogenous GLP-2 and EGF show strong interdependency for improving IEC responses. Understanding the differential requirements for phosphatidylinositol 3-kinase/phosphoAKT (Ser473) signaling may help improve future therapies to prevent mucosal atrophy.

Intestinal adaptation to small-bowel resection (SBR) after necrotizing enterocolitis expands absorptive surface areas and promotes enteral autonomy. Survivin increases proliferation and blunts apoptosis. The current study examines survivin in intestinal epithelial cells after ileocecal resection. Wild-type and epithelial Pik3r1 (p85α)-deficient mice underwent sham surgery or 30% resection. RNA and protein were isolated from small bowel to determine levels of β-catenin target gene expression, activated caspase-3, survivin, p85α, and Trp53. Healthy and post-resection human infant small-bowel sections were analyzed for survivin, Ki-67, and TP53 by immunohistochemistry. Five days after ileocecal resection, epithelial levels of survivin increased relative to sham-operated on mice, which correlated with reduced cleaved caspase-3, p85α, and Trp53. At baseline, p85α-deficient intestinal epithelial cells had less Trp53 and more survivin, and relative responses to resection were blunted compared with wild-type. In infant small bowel, survivin in transit amplifying cells increased 71% after SBR. Resection increased proliferation and decreased numbers of TP53-positive epithelial cells. Data suggest that ileocecal resection reduces p85α, which lowers TP53 activation and releases survivin promoter repression. The subsequent increase in survivin among transit amplifying cells promotes epithelial cell proliferation and lengthens crypts. These findings suggest that SBR reduces p85α and TP53, which increases survivin and intestinal epithelial cell expansion during therapeutic adaptation in patients with short bowel syndrome., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Wnt/β-catenin signaling is required for crypt structure maintenance. We previously observed nuclear accumulation of Ser-552 phosphorylated β-catenin (pβ-Cat(Ser-552)) in intestinal epithelial cells (IEC) during colitis and colitis-associated cancer. Data here delineate a novel multiprotein cytosolic complex (MCC) involved in β-catenin signaling in the intestine. The MCC contains p85α, the class IA subunit of PI3K, along with β-catenin, 14-3-3ζ, Akt, and p110α. MCC levels in IEC increase in colitis and colitis-associated cancer patients. IEC-specific p85α-deficient (p85(ΔIEC)) mice develop more severe dextran sodium sulfate colitis due to delayed ulcer healing and reduced epithelial β-catenin activation. In colonic IEC, p85α deficiency did not alter PI3K signaling. In vitro shRNA depletion of individual complex members disrupts the MCC and reduces β-catenin signaling. Despite worse colitis, p85(ΔIEC) mice have reduced tumor burden after azoxymethane/dextran sodium sulfate treatment. Together the data indicate that the β-catenin MCC is needed for mucosal repair and carcinogenesis. This novel MCC may be an attractive therapeutic target in preventing cancer in colitis patients., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Near-infrared fluorescence (NIRF) endoscopy has a great potential for efficient early detection of dysplastic lesions in the colon. For preclinical studies, we developed a small animal NIRF endoscope and successfully used this device to identify dysplastic lesions in a murine model of chronic colitis. In this chapter, we present a step-by-step protocol for using NIRF endoscopy to examine the location, the size, and the borders of the dysplastic lesions developed in murine colitis. Our studies suggest that NIRF endoscopy is a specific and sensitive technique that provides a unique opportunity to analyze early stages of tumorigenesis in animal models of colon cancer and to perform surveillance colonoscopy in patients with colitis-associated colon cancer.

A common feature of inflammatory bowel disease (IBD) is the loss of intestinal epithelial barrier function due to excessive apoptosis of intestinal epithelial cells (IECs). However, the molecular mechanism underlying increased IEC apoptosis remains unclear. Here, we investigated the role of PHLPP, a novel family of protein phosphatases, in regulating inflammation-induced IEC apoptosis in mouse models of colitis. Both Phlpp1 and Phlpp2 genes were deleted in mice. Compared with wild-type mice, PHLPP double knockout (DKO) mice were protected from colitis induced by DSS as demonstrated by lower histopathological scores, and this reduced susceptibility to colitis was associated with decreased apoptosis and increased Akt activity in IECs in vivo. In addition, epithelial organoids derived from PHLPP DKO mice were more resistant to inflammation-induced apoptosis while inhibition of Akt activity abolished the protective effect of PHLPP-loss. Furthermore, we found that PHLPP expression was significantly reduced in IECs following the induction of colitis by DSS and in human IBD patient samples. This inflammation-induced downregulation of PHLPP was partially blocked by treating cells with a proteasome inhibitor. Taken together, our results indicated that proteasome-mediated degradation of PHLPP at the onset of inflammation plays an important role in protecting IEC injury by inhibiting apoptosis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Long-term outcomes of intestinal transplantation are limited by infection and rejection. To understand the underlying immune mechanisms, graft infiltrating and peripheral blood cells were analyzed using multiple ex vivo assays in intestinal transplantation recipients., Methods: Infiltrating cells from rejected (graft enterectomy for rejection) and accepted or quiescent (stoma closure in stable transplant recipients) grafts were isolated and phenotypically characterized as to subsets and Toll-like receptor expressions as well as functionally tested for antimicrobial and antidonor immune responses. Multiparameter antidonor immunity was also assessed serially in the peripheral blood., Results: The graft infiltrating lymphocytes were mostly of recipient origin in all patients tested. In rejecting grafts, the predominant populations were TcRαβ(+)CD3(+)CD8(+) T cells, and CD14(+) monocytes that coexpressed Toll-like receptor-2, receptor-3, receptor-4, receptor-5, and receptor-9, suggesting innate immune activation. In quiescent allografts the major cell subsets were CD13(+)CD14(-) monocytes and CD4(+)CD25(+) T cells with possible regulatory functions. Infiltrating cells from rejected but not quiescent grafts proliferated in response to enteric bacterial and donor antigens as well as killed donor targets. Serial follow-up of peripheral blood indicated donor-specific posttransplant unresponsiveness in micro-cell-mediated lympholysis (m-CML) and mixed lymphocyte reaction (MLR) in recipients with quiescent grafts, but not in recipients with multiple rejection episodes. Enzyme-Linked ImmunoSpot assays yielded parallel results: granzyme-B with micro-cell-mediated lympholysis and interferon-γ with MLR tests., Conclusion: These results were consistent with the notion that rejection was associated with innate and acquired antimicrobial and antidonor immune reactivity and that patients with stable grafts were free from these deleterious effects.

Previous studies suggest an interdependent relationship between liver and intestine for cholesterol elimination from the body. We hypothesized that a combination of ursodiol (Urso) and ezetimibe (EZ) could increase biliary secretion and reduce cholesterol reabsorption, respectively, to promote cholesterol excretion. Treatment with Urso increased hepatic ABCG5 ABCG8 (G5G8) protein and both biliary and fecal sterols in a dose-dependent manner. To determine whether the drug combination (Urso-EZ) further increased cholesterol excretion, mice were treated with Urso alone or in combination with two doses of EZ. EZ produced an additive and dose-dependent increase in fecal neutral sterol (FNS) elimination in the presence of Urso. Finally, we sequentially treated wide-type and G5G8-deficient mice with Urso and Urso-EZ to determine the extent to which these effects were G5G8 dependent. Although biliary and FNS were invariably lower in G5G8 KO mice, the relative increase in FNS following treatment with Urso alone or the Urso-EZ combination was not affected by genotype. In conclusion, Urso increases G5G8, biliary cholesterol secretion, and FNS and acts additively with EZ to promote fecal sterol excretion. However, the stimulatory effect of these agents was not G5G8 dependent., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Background: Psychotherapy is not routinely recommended for in ulcerative colitis (UC). Gut-directed hypnotherapy (HYP) has been linked to improved function in the gastrointestinal tract and may operate through immune-mediated pathways in chronic diseases., Aims: To determine the feasibility and acceptability of HYP and estimate the impact of HYP on clinical remission status over a 1-year period in patients with an historical flare rate of 1.3 times per year., Methods: A total of 54 patients were randomised at a single site to seven sessions of gut-directed HYP (n = 26) or attention control (CON; n = 29) and followed for 1 year. The primary outcome was the proportion of participants in each condition that had remained clinically asymptomatic (clinical remission) through 52 weeks post treatment., Results: One-way analysis of variance comparing HYP and CON subjects on number of days to clinical relapse favoured the HYP condition [F = 4.8 (1, 48), P = 0.03] by 78 days. Chi-squared analysis comparing the groups on proportion maintaining remission at 1 year was also significant [χ²(1) = 3.9, P = 0.04], with 68% of HYP and 40% of CON patients maintaining remission for 1 year. There were no significant differences between groups over time in quality of life, medication adherence, perceived stress or psychological factors., Conclusion: This is the first prospective study that has demonstrated a significant effect of a psychological intervention on prolonging clinical remission in patients with quiescent ulcerative colitis (Clinical Trial # NCT00798642)., (© 2013 John Wiley & Sons Ltd.)

Background: Mesalamine, 5-aminosalicylic acid (5-ASA), is a potent antioxidant and is known to enhance peroxisome proliferator-activated receptor γ activity in the intestine. Our previous studies suggested reduced Phosphoinositide 3-Kinase (PI3K)/β-catenin signaling as a mechanism for 5-ASA chemoprevention in chronic ulcerative colitis (CUC). We now hypothesize that 5-ASA mediates changes in intestinal epithelial cell (IEC) reactive oxygen species during colitis to affect phosphatase and tensin homolog (PTEN), PI3K, and β-catenin signaling., Methods: Here, we examined effects of 5-ASA on oxidant-induced cell signaling pathways in HT-29 cells, IECs from mice, and biopsy tissue from control and CUC patients. Samples were selected to control for inflammation between untreated and 5-ASA-treated CUC patients., Results: Direct evaluation of IEC in H2O2-stimulated whole colonic crypts indicated that 5-ASA reduces reactive oxygen species levels in lower crypt IECs where long-lived progenitor cells reside. Analysis of biopsies from patient samples revealed that 5-ASA increases expression of the antioxidant catalase in CUC patients. Also, 5-ASA increased nuclear peroxisome proliferator-activated receptor γ protein and target gene expression. Data showed 5-ASA-induced peroxisome proliferator-activated receptor γ DNA binding to the PTEN promoter (chromatin immunoprecipitation) and reduced both phosphorylated and oxidized (inactive) PTEN protein levels. Analysis of patient samples revealed 5-ASA that also reduced levels of active phosphorylated Akt in inflamed colitis tissue. Reduced PI3K/Akt signaling and expression of β-catenin target genes in 5-ASA-treated CUC patients additionally suggests enhanced PTEN activity as well., Conclusions: Therefore, 5-ASA reduces CUC-induced reactive oxygen species in colonic progenitor cells and enhances PTEN activity, thus attenuating PI3K/Akt signaling. These data suggest that the antioxidant properties of 5-ASA may be the predominant mechanism for 5-ASA chemoprevention.

Background: Surveillance colonoscopy using random biopsies to detect colitis-associated cancer (CAC) suffers from poor sensitivity. Although chromoendoscopy improves detection, acceptance in the community has been slow. Here, we examine the usefulness of near infrared fluorescence (NIRF) endoscopy to image molecular probes for cathepsin activity in colitis-induced dysplasia., Methods: In patient samples, cathepsin activity was correlated with colitis and dysplasia. In mice, cathepsin activity was detected as fluorescent hydrolysis product of substrate-based probes after injection into Il10(-/-) colitic mice. Fluorescence colonoscopy and colonic whole-mount imaging were performed before complete sectioning and pathology review of resected colons., Results: Cathepsin activity was 5-fold and 8-fold higher in dysplasia and CAC, respectively, compared with areas of mild colitis in patient tissue sections. The signal-to-noise ratios for dysplastic lesions seen by endoscopy in Il10(-/-) mice were 5.2 ± 1.3 (P = 0.0001). Lesions with increased NIRF emissions were classified as raised or flat dysplasia, lymphoid tissue, and ulcers. Using images collected by endoscopy, a receiver operating characteristic curve for correctly diagnosing dysplasia was calculated. The area under the curve was 0.927. At a cutoff of 1000 mean fluorescence intensity, the sensitivity and specificity for detecting dysplasia were 100% and 83%, respectively. Analysis revealed that focally enhanced NIRF emissions derived from increased numbers of infiltrating myeloid-derived suppressor cells and macrophages with equivalent cathepsin activity., Conclusions: These studies indicate that cathepsin substrate-based probe imaging correctly identifies dysplastic foci within chronically inflamed colons. Combined white light and NIRF endoscopy presents unique advantages that may increase sensitivity and specificity of surveillance colonoscopy in patients with CAC.