Enteric glial cell network function is required for epithelial barrier restitution following intestinal ischemic injury in the early postnatal period.

Ischemic damage to the intestinal epithelial barrier, such as in necrotizing enterocolitis or small intestinal volvulus, is associated with higher mortality rates in younger patients. We have recently reported a powerful pig model to investigate these age-dependent outcomes in which mucosal barrier restitution is strikingly absent in neonates but can be rescued by direct application of homogenized mucosa from older, juvenile pigs by a yet-undefined mechanism. Within the mucosa, a postnatally developing network of enteric glial cells (EGCs) is gaining recognition as a key regulator of the mucosal barrier. Therefore, we hypothesized that the developing EGC network may play an important role in coordinating intestinal barrier repair in neonates. Neonatal and juvenile jejunal mucosa recovering from surgically induced intestinal ischemia was visualized by scanning electron microscopy and the transcriptomic phenotypes were assessed by bulk RNA sequencing. EGC network density and glial activity were examined by Gene Set Enrichment Analysis, three-dimensional (3-D) volume imaging, and Western blot and its function in regulating epithelial restitution was assessed ex vivo in Ussing chamber using the glia-specific inhibitor fluoroacetate (FA), and in vitro by coculture assay. Here we refine and elaborate our translational model, confirming a neonatal phenotype characterized by a complete lack of coordinated reparative signaling in the mucosal microenvironment. Furthermore, we report important evidence that the subepithelial EGC network changes significantly over the early postnatal period and demonstrate that the proximity of a specific functional population of EGC to wounded intestinal epithelium contributes to intestinal barrier restitution following ischemic injury.NEW & NOTEWORTHY This study refines a powerful translational pig model, defining an age-dependent relationship between enteric glia and the intestinal epithelium during intestinal ischemic injury and confirming an important role for enteric glial cell (EGC) activity in driving mucosal barrier restitution. This study suggests that targeting the enteric glial network could lead to novel interventions to improve recovery from intestinal injury in neonatal patients.

Larazotide acetate: a pharmacological peptide approach to tight junction regulation.

Larazotide acetate (LA) is a single-chain peptide of eight amino acids that acts as a tight junction regulator to restore intestinal barrier function. LA is currently being studied in phase III clinical trials and is orally administered to adult patients with celiac disease as an adjunct therapeutic to enhance intestinal barrier function that has been disrupted by gliadin-induced immune reactivity. Mechanistically, LA is thought to act as a zonulin antagonist to reduce zonulin-induced increases in barrier permeability and has been associated with the redistribution and rearrangement of tight junction proteins and actin filaments to restore intestinal barrier function. More recently, LA has been linked to inhibition of myosin light chain kinase, which likely reduces tension on actin filaments, thereby facilitating tight junction closure. Small (rodent) and large (porcine) animal studies have been conducted that demonstrate the importance of LA as a tight junction regulatory peptide in conditions other than celiac disease, including collagen-induced arthritis in mice and intestinal ischemic injury in pigs.

Probiotics, Prebiotics and Epithelial Tight Junctions: A Promising Approach to Modulate Intestinal Barrier Function.

Disruptions in the intestinal epithelial barrier can result in devastating consequences and a multitude of disease syndromes, particularly among preterm neonates. The association between barrier dysfunction and intestinal dysbiosis suggests that the intestinal barrier function is interactive with specific gut commensals and pathogenic microbes. In vitro and in vivo studies demonstrate that probiotic supplementation promotes significant upregulation and relocalization of interepithelial tight junction proteins, which form the microscopic scaffolds of the intestinal barrier. Probiotics facilitate some of these effects through the ligand-mediated stimulation of several toll-like receptors that are expressed by the intestinal epithelium. In particular, bacterial-mediated stimulation of toll-like receptor-2 modulates the expression and localization of specific protein constituents of intestinal tight junctions. Given that ingested prebiotics are robust modulators of the intestinal microbiota, prebiotic supplementation has been similarly investigated as a potential, indirect mechanism of barrier preservation. Emerging evidence suggests that prebiotics may additionally exert a direct effect on intestinal barrier function through mechanisms independent of the gut microbiota. In this review, we summarize current views on the effects of pro- and prebiotics on the intestinal epithelial barrier as well as on non-epithelial cell barrier constituents, such as the enteric glial cell network. Through continued investigation of these bioactive compounds, we can maximize their therapeutic potential for preventing and treating gastrointestinal diseases associated with impaired intestinal barrier function and dysbiosis.

Lubiprostone protects esophageal mucosa from acid injury in porcine esophagus.

Esophageal injury from acid exposure related to gastroesophageal reflux disease is a common problem and a risk factor for development of Barrett's esophagus and esophageal adenocarcinoma. Our previous work highlights the benefits of using porcine esophagus to study human esophageal disease because of the similarities between porcine and human esophagus. In particular, esophageal submucosal glands (ESMGs) are present in human esophagus and proximal porcine esophagus but not in rodent esophagus. Although CFTR is expressed in the ducts of ESMGs, very little is known about CFTR and alternate anion channels, including ClC-2, in the setting of acid-related esophageal injury. After finding evidence of CFTR and ClC-2 in the basal layers of the squamous epithelium, and in the ducts of the ESMGs, we developed an ex vivo porcine model of esophageal acid injury. In this model, esophageal tissue was placed in Ussing chambers to determine the effect of pretreatment with the ClC-2 agonist lubiprostone on tissue damage related to acid exposure. Pretreatment with lubiprostone significantly reduced the level of acid injury and significantly augmented the recovery of the injured tissue (P < 0.05). Evaluation of the interepithelial tight junctions showed well-defined membrane localization of occludin in lubiprostone-treated injured tissues. Pretreatment of tissues with the Na+-K+-2Cl- cotransporter inhibitor bumetanide blocked lubiprostone-induced increases in short-circuit current and inhibited the reparative effect of lubiprostone. Furthermore, inhibition of ClC-2 with ZnCl2 blocked the effects of lubiprostone. We conclude that ClC-2 contributes to esophageal protection from acid exposure, potentially offering a new therapeutic target.NEW & NOTEWORTHY This research is the first to describe the presence of anion channels ClC-2 and CFTR localized to the basal epithelia of porcine esophageal mucosa and the esophageal submucosal glands. In the setting of ex vivo acid exposure, the ClC-2 agonist lubiprostone reduced acid-related injury and enhanced recovery of the epithelial barrier. This work may ultimately provide an alternate mechanism for treating gastroesophageal reflux disease.

Oesophageal eosinophilia accompanies food allergy to hen egg white protein in young pigs.

BACKGROUND: Esophagitis with eosinophilia, inflammation, and fibrosis represent a chronic condition in humans with food allergies. OBJECTIVE: In this investigation, we asked whether esophagitis with an eosinophilic component is observed in young pigs rendered allergic to hen egg white protein (HEWP). METHODS: Food allergy was induced in young pigs using two protocols. In one protocol, sensitized pigs were challenged by gavage with a single dose of HEWP. Clinical signs were monitored for 24 hours, and then, gastrointestinal (GI) tissues were collected for histological examination. The phenotype of circulating, ovalbumin (OVA)-specific T cells also was examined in HEWP challenged animals. In the second protocol, sensitized animals were fed HEWP for 28 days. Animals were then examined by endoscopy and gastrointestinal tissues collected for histological examination. RESULTS: In pigs challenged by gavage with HEWP, clinical signs were noted in 5/6 pigs including diarrhoea, emesis, and skin rash. Clinical signs were not seen in any control group. Histological analysis revealed significant levels of oesophageal eosinophilic infiltration (P < .05) in 4/6 of these animals, with two also displaying eosinophilic infiltration in the stomach. Eosinophils were not increased in ileum or colon samples. Increased numbers of circulating, OVA-specific CD4+ T cells also were observed in pigs that received HEWP by gavage. In the group of animals fed HEWP, endoscopy revealed clinical signs of esophagitis including oedema, granularity, white spots, and furrowing, while histology revealed oedema, immune cell infiltration, and basal zone hyperplasia. CONCLUSIONS AND CLINICAL RELEVANCE: Food allergy in the pig can be associated with esophagitis based on histological and endoscopic findings, including eosinophilic infiltration. The young pig may, therefore, be a useful large animal model for the study of eosinophilic esophagitis in humans.

The Regulation of Intestinal Mucosal Barrier by Myosin Light Chain Kinase/Rho Kinases.

The intestinal epithelial apical junctional complex, which includes tight and adherens junctions, contributes to the intestinal barrier function via their role in regulating paracellular permeability. Myosin light chain II (MLC-2), has been shown to be a critical regulatory protein in altering paracellular permeability during gastrointestinal disorders. Previous studies have demonstrated that phosphorylation of MLC-2 is a biochemical marker for perijunctional actomyosin ring contraction, which increases paracellular permeability by regulating the apical junctional complex. The phosphorylation of MLC-2 is dominantly regulated by myosin light chain kinase- (MLCK-) and Rho-associated coiled-coil containing protein kinase- (ROCK-) mediated pathways. In this review, we aim to summarize the current state of knowledge regarding the role of MLCK- and ROCK-mediated pathways in the regulation of the intestinal barrier during normal homeostasis and digestive diseases. Additionally, we will also suggest potential therapeutic targeting of MLCK- and ROCK-associated pathways in gastrointestinal disorders that compromise the intestinal barrier.

The Integral Role of Tight Junction Proteins in the Repair of Injured Intestinal Epithelium.

The intestinal epithelial monolayer forms a transcellular and paracellular barrier that separates luminal contents from the interstitium. The paracellular barrier consists of a highly organized complex of intercellular junctions that is primarily regulated by apical tight junction proteins and tight junction-associated proteins. This homeostatic barrier can be lost through a multitude of injurious events that cause the disruption of the tight junction complex. Acute repair after injury leading to the reestablishment of the tight junction barrier is crucial for the return of both barrier function as well as other cellular functions, including water regulation and nutrient absorption. This review provides an overview of the tight junction complex components and how they link to other plasmalemmal proteins, such as ion channels and transporters, to induce tight junction closure during repair of acute injury. Understanding the components of interepithelial tight junctions and the mechanisms of tight junction regulation after injury is crucial for developing future therapeutic targets for patients experiencing dysregulated intestinal permeability.

Preservation of reserve intestinal epithelial stem cells following severe ischemic injury.

Intestinal ischemia is an abdominal emergency with a mortality rate >50%, leading to epithelial barrier loss and subsequent sepsis. Epithelial renewal and repair after injury depend on intestinal epithelial stem cells (ISC) that reside within the crypts of Lieberkühn. Two ISC populations critical to epithelial repair have been described: 1) active ISC (aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 positive, sex determining region Y-box 9 positive) and 2) reserve ISC [rISC; less proliferative; homeodomain only protein X (Hopx)+]. Yorkshire crossbred pigs (8-10 wk old) were subjected to 1-4 h of ischemia and 1 h of reperfusion or recovery by reversible mesenteric vascular occlusion. This study was designed to evaluate whether ISC-expressing biomarkers of aISCs or rISCs show differential resistance to ischemic injury and different contributions to the subsequent repair and regenerative responses. Our data demonstrate that, following 3-4 h ischemic injury, aISC undergo apoptosis, whereas rISC are preserved. Furthermore, these rISC are retained ex vivo in spheroids in which cell populations are enriched in the rISC biomarker Hopx. These cells appear to go on to provide a proliferative pool of cells during the recovery period. Taken together, these data indicate that Hopx+ cells are resistant to injury and are the likely source of epithelial renewal following prolonged ischemic injury. It is therefore possible that targeting reserve stem cells will lead to new therapies for patients with severe intestinal injury. NEW & NOTEWORTHY The population of reserve less-proliferative intestinal epithelial stem cells appears resistant to injury despite severe epithelial cell loss, including that of the active stem cell population, which results from prolonged mesenteric ischemia. These cells can change to an activated state and are likely indispensable to regenerative processes. Reserve stem cell targeted therapies may improve treatment and outcome of patients with ischemic disease.

Knockout of ClC-2 reveals critical functions of adherens junctions in colonic homeostasis and tumorigenicity.

Adherens junctions (AJs), together with tight junctions (TJs), form an apical junctional complex that regulates intestinal epithelial cell-to-cell adherence and barrier homeostasis. Within the AJ, membrane-bound E-cadherin binds ß-catenin, which functions as an essential intracellular signaling molecule. We have previously identified a novel protein in the region of the apical junction complex, chloride channel protein-2 (ClC-2), that we have used to study TJ regulation. In this study, we investigated the possible effects of ClC-2 on the regulation of AJs in intestinal mucosal epithelial homeostasis and tumorigenicity. Mucosal homeostasis and junctional proteins were examined in wild-type (WT) and ClC-2 knockout (KO) mice as well as associated colonoids. Tumorigenicity and AJ-associated signaling were evaluated in a murine colitis-associated tumor model and in a colorectal cancer cell line (HT-29). Colonic tissues from ClC-2 KO mice had altered ultrastructural morphology of intercellular junctions with reduced colonocyte differentiation, whereas jejunal tissues had minimal changes. Colonic crypts from ClC-2 KO mice had significantly higher numbers of less-differentiated forms of colonoids compared with WT. Furthermore, the absence of ClC-2 resulted in redistribution of AJ proteins and increased ß-catenin activity. Downregulation of ClC-2 in colorectal cells resulted in significant increases in proliferation associated with disruption of AJs. Colitis-associated tumors in ClC-2 KO mice were significantly increased, associated with ß-catenin transcription factor activation. The absence of ClC-2 results in less differentiated colonic crypts and increased tumorigenicity associated with colitis via dysregulation of AJ proteins and activation of ß-catenin-associated signaling. NEW & NOTEWORTHY Disruption of adherens junctions in the absence of chloride channel protein-2 revealed critical functions of these junctional structures, including maintenance of colonic homeostasis and differentiation as well as driving tumorigenicity by regulating ß-catenin signaling.

IDENTIFICATION OF A NASOCONCHAL PARANASAL SINUS IN THE WHITE RHINOCEROS ( CERATOTHERIUM SIMUM).

African rhinoceros are poached for their horns using indiscriminate and aggressive methods. Rhinoceros that survive these attacks often have severe facial trauma, and treatment is limited by a lack of understanding and published information of the normal anatomy. This study was performed to investigate and describe the anatomy of the most commonly injured area of the head of the white rhinoceros ( Ceratotherium simum). Two white rhinoceros cadaver heads were imaged by computed tomography and grossly dissected. A combined dorsal conchal sinus and nasal sinus (named the nasoconchal sinus) was identified and confirmed to be readily exposed by horn removal. The nasoconchal sinus communicates via a relatively large opening with the middle nasal meatus of the nasal cavity. Awareness of the combined sinus space and its single communicating pathway will assist with accurate assessment and treatment of trauma to the dorsal facial region of the white rhinoceros.

Ductular and proliferative response of esophageal submucosal glands in a porcine model of esophageal injury and repair.

Esophageal injury is a risk factor for diseases such as Barrett's esophagus (BE) and esophageal adenocarcinoma. To improve understanding of signaling pathways associated with both normal and abnormal repair, animal models are needed. Traditional rodent models of esophageal repair are limited by the absence of esophageal submucosal glands (ESMGs), which are present in the human esophagus. Previously, we identified acinar ductal metaplasia in human ESMGs in association with both esophageal injury and cancer. In addition, the SOX9 transcription factor has been associated with generation of columnar epithelium and the pathogenesis of BE and is present in ESMGs. To test our hypothesis that ESMGs activate after esophageal injury with an increase in proliferation, generation of a ductal phenotype, and expression of SOX9, we developed a porcine model of esophageal injury and repair using radiofrequency ablation (RFA). The porcine esophagus contains ESMGs, and RFA produces a consistent and reproducible mucosal injury in the esophagus. Here we present a temporal assessment of this model of esophageal repair. Porcine esophagus was evaluated at 0, 6, 18, 24, 48, and 72 h and 5 and 7 days following RFA and compared with control uninjured esophagus. Following RFA, ESMGs demonstrated an increase in ductal phenotype, echoing our prior studies in humans. Proliferation increased in both squamous epithelium and ESMGs postinjury with a prominent population of SOX9-positive cells in ESMGs postinjury. This model promises to be useful in future experiments evaluating mechanisms of esophageal repair.NEW & NOTEWORTHY A novel porcine model of injury and repair using radiofrequency ablation has been developed, allowing for reproducible injury to the esophagus to study repair in an animal model with esophageal submucosal glands, a key anatomical feature and missing in rodent models but possibly harboring progenitor cells. There is a strong translational component to this porcine model given the anatomical and physiological similarities between pigs and humans.

Myosin light chain kinase mediates intestinal barrier dysfunction via occludin endocytosis during anoxia/reoxygenation injury.

Intestinal anoxia/reoxygenation (A/R) injury induces loss of barrier function followed by epithelial repair. Myosin light chain kinase (MLCK) has been shown to alter barrier function via regulation of interepithelial tight junctions, but has not been studied in intestinal A/R injury. We hypothesized that A/R injury would disrupt tight junction barrier function via MLCK activation and myosin light chain (MLC) phosphorylation. Caco-2BBe1 monolayers were subjected to anoxia for 2 h followed by reoxygenation in 21% O2, after which barrier function was determined by measuring transepithelial electrical resistance (TER) and FITC-dextran flux. Tight junction proteins and MLCK signaling were assessed by Western blotting, real-time PCR, or immunofluorescence microscopy. The role of MLCK was further investigated with select inhibitors (ML-7 and peptide 18) by using in vitro and ex vivo models. Following A/R injury, there was a significant increase in paracellular permeability compared with control cells, as determined by TER and dextran fluxes (P < 0.05). The tight junction protein occludin was internalized during A/R injury and relocalized to the region of the tight junction after 4 h of recovery. MLC phosphorylation was significantly increased by A/R injury (P < 0.05), and treatment with the MLCK inhibitor peptide 18 attenuated the increased epithelial monolayer permeability and occludin endocytosis caused by A/R injury. Application of MLCK inhibitors to ischemia-injured porcine ileal mucosa induced significant increases in TER and reduced mucosal-to-serosal fluxes of 3H-labeled mannitol. These data suggest that MLCK-induced occludin endocytosis mediates intestinal epithelial barrier dysfunction during A/R injury. Our results also indicate that MLCK-dependent occludin regulation may be a target for the therapeutic treatment of ischemia/reperfusion injury.

Animal models of ischemia-reperfusion-induced intestinal injury: progress and promise for translational research.

Research in the field of ischemia-reperfusion injury continues to be plagued by the inability to translate research findings to clinically useful therapies. This may in part relate to the complexity of disease processes that result in intestinal ischemia but may also result from inappropriate research model selection. Research animal models have been integral to the study of ischemia-reperfusion-induced intestinal injury. However, the clinical conditions that compromise intestinal blood flow in clinical patients ranges widely from primary intestinal disease to processes secondary to distant organ failure and generalized systemic disease. Thus models that closely resemble human pathology in clinical conditions as disparate as volvulus, shock, and necrotizing enterocolitis are likely to give the greatest opportunity to understand mechanisms of ischemia that may ultimately translate to patient care. Furthermore, conditions that result in varying levels of ischemia may be further complicated by the reperfusion of blood to tissues that, in some cases, further exacerbates injury. This review assesses animal models of ischemia-reperfusion injury as well as the knowledge that has been derived from each to aid selection of appropriate research models. In addition, a discussion of the future of intestinal ischemia-reperfusion research is provided to place some context on the areas likely to provide the greatest benefit from continued research of ischemia-reperfusion injury.

Protocol to culture enteric glial cells from the submucosal and myenteric plexi of neonatal and juvenile pig colons.

Here, we present our protocol to culture enteric glial cells from the submucosal and myenteric plexus of neonatal and juvenile pig colons. We describe steps for colon isolation, microdissection, and enzymatic and mechanical dissociation. We include procedures for passaging and analyzing cell yield, freeze/thaw efficiency, and purity. This protocol allows for the generation of primary cultures of enteric glial cells from single-cell suspensions of microdissected layers of the colon wall and can be used to culture enteric glia from human colon specimens. For complete details on the use and execution of this protocol, please refer to Ziegler et al.1.

Prebiotic galactooligosaccharide improves piglet growth performance and intestinal health associated with alterations of the hindgut microbiota during the peri-weaning period.

BACKGROUND: Weaning stress reduces growth performance and health of young pigs due in part to an abrupt change in diets from highly digestible milk to fibrous plant-based feedstuffs. This study investigated whether dietary galactooligosaccharide (GOS), supplemented both pre- and post-weaning, could improve growth performance and intestinal health via alterations in the hindgut microbial community. METHODS: Using a 3 × 2 factorial design, during farrowing 288 piglets from 24 litters received either no creep feed (FC), creep without GOS (FG-) or creep with 5% GOS (FG+) followed by a phase 1 nursery diet without (NG-) or with 3.8% GOS (NG+). Pigs were sampled pre- (D22) and post-weaning (D31) to assess intestinal measures. RESULTS: Creep fed pigs grew 19% faster than controls (P < 0.01) prior to weaning, and by the end of the nursery phase (D58), pigs fed GOS pre-farrowing (FG+) were 1.85 kg heavier than controls (P < 0.05). Furthermore, pigs fed GOS in phase 1 of the nursery grew 34% faster (P < 0.04), with greater feed intake and efficiency. Cecal microbial communities clustered distinctly in pre- vs. post-weaned pigs, based on principal coordinate analysis (P < 0.01). No effects of GOS were detected pre-weaning, but gruel creep feeding increased Chao1 α-diversity and altered several genera in the cecal microbiota (P < 0.05). Post-weaning, GOS supplementation increased some genera such as Fusicatenibacter and Collinsella, whereas others decreased such as Campylobacter and Frisingicoccus (P < 0.05). Changes were accompanied by higher molar proportions of butyrate in the cecum of GOS-fed pigs (P < 0.05). CONCLUSIONS: Gruel creep feeding effectively improves suckling pig growth regardless of GOS treatment. When supplemented post-weaning, prebiotic GOS improves piglet growth performance associated with changes in hindgut microbial composition.

A novel case of tracheal injury secondary to gunshot trauma in a white rhinoceros (Ceratotherium simum).

OBJECTIVE: To describe a case of tracheal injury secondary to gunshot trauma in a rhinoceros. ANIMALS: 5-year-old female white rhinoceros (Ceratotherium simum). CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES: The rhinoceros was found alive with an apparent bullet entry wound cranial to the left shoulder. The rhinoceros was agitated and had bilateral epistaxis and increased respiratory noise. Immobilization of the animal facilitated closer examination and initiation of medical therapy. Radiographs obtained of the neck region at this first examination were nondiagnostic. Subsequent immobilization events allowed for further diagnostics and treatment. TREATMENT AND OUTCOME: Initial treatment included a broad-spectrum antibiotic and a corticosteroid. Five days following the injury, the rhinoceros was considered stable, and the animal was immobilized to investigate the cause of the epistaxis and respiratory signs. Tracheoscopy revealed a full-thickness penetrating wound in the mid to caudal region of the trachea, and the surface of a metallic projectile was viewed within the wound. Medical treatment was continued and the rhinoceros was managed conservatively. At 14 days, radiographs of the neck made with a more powerful unit revealed tissue emphysema dorsal to the trachea. A subsequent tracheoscopy 54 days after injury revealed a granulated wound. Follow-up at 4 years after injury determined that the rhinoceros was reported to be behaving normally and had successfully calved. CLINICAL RELEVANCE: Gunshot wounds associated with poaching are a prevalent problem in rhinoceros in Africa. Although more aggressive therapy including surgery may likely be considered in zoo or domestic animals, limited conservative treatment was successful in this wild-managed rhinoceros.

Mechanisms and modeling of wound repair in the intestinal epithelium.

The intestinal epithelial barrier is susceptible to injury from insults, such as ischemia or infectious disease. The epithelium's ability to repair wounded regions is critical to maintaining barrier integrity. Mechanisms of intestinal epithelial repair can be studied with models that recapitulate the in vivo environment. This review focuses on in vitro injury models and intestinal cell lines utilized in such systems. The formation of artificial wounds in a controlled environment allows for the exploration of reparative physiology in cell lines modeling diverse aspects of intestinal physiology. Specifically, the use of intestinal cell lines, IPEC-J2, Caco-2, T-84, HT-29, and IEC-6, to model intestinal epithelium is discussed. Understanding the unique systems available for creating intestinal injury and the differences in monolayers used for in vitro work is essential for designing studies that properly capture relevant physiology for the study of intestinal wound repair.

Risk factors associated with an outbreak of equine coronavirus at a large farm in North Carolina.

Background: Equine coronavirus (ECoV) leads to outbreaks with variable morbidity and mortality. Few previous reports of risk factors for infection are available in the literature. Objectives: To describe unique clinical findings and risk factors for infection and development of clinical disease. Animals: 135 horses on a farm affected by ECoV outbreak. Methods: Retrospective cohort study. Data obtained included age, breed, gender, activity level, housing, and feed at the onset of the outbreak. Factors were evaluated for assessment of risk of infection using simple logistic regression or Fisher's exact test. Significance was set at p ≤ 0.05. Results and findings: Forty-three of 54 (79.6%) horses tested on the farm were positive on fecal PCR for ECoV, and 17 horses (12.6%) developed clinical signs consistent with ECoV. Out of 17 horses in which the presence or absence of signs of colic was noted, 6 of 17 (35.3%) showed signs of colic. Three of these horses had small colon impactions, 2 of which required surgical intervention. Significant risk factors for having positive PCR results included being primarily stalled (OR 167.1, 95% CI 26.4-1719), housing next to a positive horse (OR 7.5, 95% CI 3.1-19.0), being in work (OR 26.9, 95% CI 4.6-281.9), being fed rationed hay vs. ad libitum (OR 1,558, 95% CI 130.8-15,593), and being fed alfalfa hay (OR 1,558, 95% CI 130.8-15,593). Conclusions and clinical importance: This report describes risk factors for ECoV infection many of which were associated with intensive management of show horses. Clinicians should be aware that clinical signs vary and can include severe colic.

A new microphysiological system shows hypoxia primes human ISCs for interleukin-dependent rescue of stem cell activity.

Background & Aims: Hypoxia in the intestinal epithelium can be caused by acute ischemic events or conditions like Inflammatory Bowel Disease (IBD) where immune cell infiltration produces 'inflammatory hypoxia', a chronic condition that starves the mucosa of oxygen. Epithelial regeneration after ischemia and IBD suggests intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of acute and chronic hypoxia on human ISC (hISC) properties have not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs isolated from healthy human tissues. We then test the hypothesis that some inflammation-associated interleukins protect hISCs during prolonged hypoxia. Methods: hISCs were exposed to <1.0% oxygen in the MPS for 6-, 24-, 48- & 72hrs. Viability, HIF1α response, transcriptomics, cell cycle dynamics, and hISC response to cytokines were evaluated. Results: The novel MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs remain viable until 72hrs and exhibit peak HIF1α at 24hrs. hISCs lose stem cell activity at 24hrs that recovers at 48hrs of hypoxia. Hypoxia increases the proportion of hISCs in G1 and regulates hISC capacity to respond to multiple inflammatory signals. Hypoxia induces hISCs to upregulate many interleukin receptors and hISCs demonstrate hypoxia-dependent cell cycle regulation and increased organoid forming efficiency when treated with specific interleukins. Conclusions: Hypoxia primes hISCs to respond differently to interleukins than hISCs in normoxia through a transcriptional response. hISCs slow cell cycle progression and increase hISC activity when treated with hypoxia and specific interleukins. These findings have important implications for epithelial regeneration in the gut during inflammatory events.

Hypoxia Primes Human ISCs for Interleukin-Dependent Rescue of Stem Cell Activity.

BACKGROUND AND AIMS: Hypoxia in the intestinal epithelium can be caused by acute ischemic events or chronic inflammation in which immune cell infiltration produces inflammatory hypoxia starving the mucosa of oxygen. The epithelium has the capacity to regenerate after some ischemic and inflammatory conditions suggesting that intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of hypoxia on human ISC (hISC) function has not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs from healthy donors and test the hypothesis that prolonged hypoxia modulates how hISCs respond to inflammation-associated interleukins (ILs). METHODS: hISCs were exposed to <1.0% oxygen in the MPS for 6, 24, 48, and 72 hours. Viability, hypoxia-inducible factor 1a (HIF1a) response, transcriptomics, cell cycle dynamics, and response to cytokines were evaluated in hISCs under hypoxia. HIF stabilizers and inhibitors were screened to evaluate HIF-dependent responses. RESULTS: The MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs maintain viability until 72 hours and exhibit peak HIF1a at 24 hours. hISC activity was reduced at 24 hours but recovered at 48 hours. Hypoxia induced increases in the proportion of hISCs in G1 and expression changes in 16 IL receptors. Prolyl hydroxylase inhibition failed to reproduce hypoxia-dependent IL-receptor expression patterns. hISC activity increased when treated IL1ß, IL2, IL4, IL6, IL10, IL13, and IL25 and rescued hISC activity caused by 24 hours of hypoxia. CONCLUSIONS: Hypoxia pushes hISCs into a dormant but reversible proliferative state and primes hISCs to respond to a subset of ILs that preserves hISC activity. These findings have important implications for understanding intestinal epithelial regeneration mechanisms caused by inflammatory hypoxia.