Intestinal Healing Research Articles

Differential analysis of sorting nexin 10 and sterol regulatory element-binding protein 2 expression in inflammatory bowel disease.

Sorting nexin 10 (SNX10) expression induces intestinal barrier dysfunction and inflammatory responses; in contrast, its inhibition promotes intestinal mucosal healing through sterol regulatory element-binding protein 2 (SREBP2)-mediated cholesterol synthesis. However, its regulatory mechanism for the pathogenesis of inflammatory bowel disease (IBD) remains unclear. In this study, we examined SNX10 and SREBP2 expression in ulcerative colitis (UC) and Crohn's disease (CD). A total of 30 and 28 patients with UC and CD, respectively, were recruited. The expression of SNX10 and SREBP2 in the colonic mucosa was measured by immunohistochemistry (IHC). We discovered that patients with CD had significantly higher expression levels of SNX10 and SREBP2 than patients with UC and healthy controls. In addition, the expression of SREBP2 in patients with UC was significantly higher than that in healthy controls. In our study, we indicated that SNX10 and SREBP2 may serve as biomarkers for identifying patients with UC and CD, thereby providing a clinical therapeutic strategy for the treatment of IBD by inhibiting SNX10.

Role of the serosa in intestinal anastomotic healing: insights from in-depth histological analysis of human and murine anastomoses.

Anastomotic leakage following colorectal surgery remains a significant complication despite advances in surgical techniques. Recent findings on serosal injury repair in coelomic cavities, such as the peritoneum, challenge the current understanding of the cellular origins and mechanisms underlying intestinal anastomotic healing. Understanding the contribution of each layer of the intestinal wall during anastomotic healing is needed to find new therapeutic strategies to prevent anastomotic leakage. The aim of this experimental study was to investigate the role of the serosal layer of the intestinal wall in anastomotic healing. Comprehensive histologic analysis of human and murine anastomoses was performed to elucidate histologic changes in the different intestinal layers during anastomotic healing. In vivo staining of the extracellular matrix (ECM) in the serosal layer was performed using a fluorophore-conjugated N-hydroxysuccinimide-ester before anastomosis surgery in a murine model. Histological examination of both human and murine anastomoses revealed that closure of the serosal layer occurred first during the healing process. In vivo serosal ECM staining demonstrated that a significant portion of the newly formed ECM within the anastomosis was indeed deposited onto the serosal layer. Furthermore, mesenchymal cells within the anastomotic scar were positive for mesothelial cell markers, podoplanin and Wilms tumour protein. In this experimental study, the results suggest that serosal scar formation is an important mechanism for anastomotic integrity in intestinal anastomoses. Mesothelial cells may significantly contribute to scar formation during anastomotic healing through epithelial-to-mesenchymal transition, potentially suggesting a novel therapeutic target to prevent anastomotic leakage by enhancing physiological healing processes.

The effects of combustible cigarettes and electronic nicotine delivery systems on immune cell-driven inflammation and mucosal healing in ulcerative colitis.

The effects of combustible cigarettes (CCs) and electronic nicotine delivery systems (ENDS) on immune cell-driven colon inflammation and intestinal healing of patients with ulcerative colitis (UC) are still unknown and, therefore, were examined in this study. Intracellular staining and flow cytometry analysis of immune cells isolated from UC patients who used ENDS (UCENDS), CCs (UCCC) and who were non-smokers (UCAIR) were performed to elucidate cellular mechanisms which were responsible for CCs and ENDS-dependent modulation of immune response during UC progression. Additionally, dextran sulfate sodium (DSS)-colitis was induced in ENDS/CC/air-exposed mice (DSSENDS/ DSSCC/DSSAIR groups) to support clinical findings. Significantly increased number of immunosuppressive, IL-10, TGF-β and IL-35-producing, FoxP3-expressing CD3+CD4+T regulatory cells (Tregs) was observed in the blood of UCENDS patients while reduced presence of inflammatory, TNF-α and IFN-γ-producing, Tbx21-expressing CD3+CD4+ Th1, IL-4-producing Gata3-expresing Th2 and IL-17, IL-22-producing, RORγT, IL-23R-expressing Th17 cells were noticed in the blood of UCCC patients. Exposure to either CCs or ENDS was associated with enhanced mucosal healing, ameliorated spontaneous recovery and improved survival of DSS-treated mice. An expansion of immunosuppressive cells (IL-10-producing tolerogenic CD11c+ dendritic cells, alternatively activated CD206, Arginase 1-expressing, IL-10-producing F4/80+macrophages, IL-10-producing FoxP3-expressing Tregs) was noticed in the colons of DSSENDS-treated mice, while reduced number of inflammatory, IL-17- and IL-4-producing T lymphocytes was observed in the colons of DSSCC-compared to DSSAIR-treated mice. Despite different mechanisms of action, both ENDS and CCs attenuated on-going colon inflammation, enhanced healing and ameliorated recovery of injured intestines of DSS-treated mice and UC patients. This is the first study that compared the effects of CCs and ENDS on immune cells of patients suffering from ulcerative colitis, providing new information about molecular and cellular mechanisms which were responsible for ENDS and CCs-dependent modulation of immune cell-driven colon injury and inflammation. Obtained results showed that both ENDS and CCs had capacity to attenuate detrimental immune response, enhanced healing and ameliorated recovery of injured intestines.

Short cycles of remote ischemic preconditioning had no effect on tensile strength in small intestinal anastomoses: an experimental animal study

PurposeThis study aimed to investigate the effect of remote ischemic preconditioning (RIPC) on the healing of small intestinal anastomoses, evaluated by tensile strength and histologic wound healing on postoperative day 5. MethodsA total of 22 female pigs were randomized 1:1 into either an intervention or control group. The intervention group received 5 cycles of 3-minute ischemia followed by 3-minute reperfusion on the right forelimb. Two end-to-end anastomoses, a distal and a proximal, were created in the small intestine 30 and 60 min after RIPC, respectively. On postoperative day 5, the anastomoses were harvested and underwent a maximal anastomotic tensile strength (MATS) test (MATS 1–3) followed by histologic analyses. ResultsMATS 1, when a tear became visible in the serosa, was significantly increased in the proximal anastomoses of the RIPC group compared with the control group (4.91 N vs 3.83 N; P = .005). No other significant differences were found when comparing these 2 groups. ConclusionOur study showed no convincing results of RIPC on intestinal anastomotic healing to recommend its use in a general clinical setting. Further animal studies on RIPC’s effect after relative or absolute intestinal ischemia may be recommended.

Focal Adhesion Kinase and Colony Stimulating Factors: Intestinal Homeostasis and Innate Immunity Crosstalk.

Thousands struggle with acute and chronic intestinal injury due to various causes. Epithelial intestinal healing is dependent on phenotypic transitions to a mobile phenotype. Focal adhesion kinase (FAK) is a ubiquitous protein that is essential for cell mobility. This phenotype change is mediated by FAK activation and proves to be a promising target for pharmaceutical intervention. While FAK is crucial for intestinal healing, new evidence connects FAK with innate immunity and the importance it plays in macrophage/monocyte chemotaxis, as well as other intracellular signaling cascades. These cascades play a part in macrophage/monocyte polarization, maturation, and inflammation that is associated with intestinal injury. Colony stimulating factors (CSFs) such as macrophage colony stimulating factor (M-CSF/CSF-1) and granulocyte macrophage colony stimulating factor (GM-CSF/CSF-2) play a critical role in maintaining homeostasis within intestinal mucosa by crosstalk capabilities between macrophages and epithelial cells. The communication between these cells is imperative in orchestrating healing upon injury. Diving deeper into these connections may allow us a greater insight into the role that our immune system plays in healing, as well as a better comprehension of inflammatory diseases of the gut.

Risks of Cancer Associated with Therapeutic Drugs for Inflammatory Bowel Disease

Crohn's disease and ulcerative colitis are lifelong chronic inflammatory conditions, with many patients requiring ongoing immunomodulatory drug therapy for maintenance treatment. Recent therapeutic goals in inflammatory bowel disease (IBD) are not only aimed at symptomatic remission but also at achieving mucosal healing to improve the natural course of the disease. In this context, therapeutic approaches are being applied in clinical settings that involve early and appropriate use of drugs, such as immunomodulators or biologics, that have the potential to induce healing of the inflamed intestine before irreversible intestinal damage occurs. All drugs that continuously control intestinal inflammation in IBD can heal the mucosa and potentially reduce the incidence of colitis-associated bowel cancer; however, the continuous use of immunosuppressants can potentially increase the risk of malignancies. The safety issues of the drugs used in clinical practice are partly confirmed during their development processes or shortly after initial marketing, but in other cases, they are estimated through post-marketing case reports or epidemiological studies, sometimes decades after drug approval. This review explores the risks associated with malignancies related to the treatment of IBD, focusing on drugs currently approved in Republic of Korea.

Lactoferrin promotes intestinal stem cell‐mediated epithelial regeneration by activating Wnt signaling

AbstractNutritional intervention can greatly benefit people who suffer from side effects of medical treatments by improving intestinal post‐damage recovery. The recovery process is dependent on the regeneration of intestinal epithelial cells, which is driven by Lgr5+ intestinal stem cells (ISCs). Lactoferrin (LF) is a natural milk‐abundant protein with several gut health‐related functions such as antibacterial and immunoregulation activities, thus protecting intestine from damage. However, it is unclear whether LF also plays a role in the repair of the injured intestine, despite limited clues on its ability to modulate intestinal epithelial cell growth in vitro. Here, we show that LF accelerates intestinal epithelial recovery after both dextran sulfate sodium challenge on mice and TNF‐α treatment on intestinal organoids. Furthermore, we find LF gives rise to ISC‐mediated epithelial regeneration by directly activating the Lgr5+ stem cells. Finally, we identify Lrp5/Wnt signaling as the key pathway for LF‐enhancing ISC stemness and function. Overall, our study reveals the potential of LF as a nutrient that can be applied to promote intestinal healing.

The reparative immunologic consequences of stem cell transplantation as a cellular therapy for refractory Crohn's disease.

Treatment strategies for Crohn's disease (CD) suppress diverse inflammatory pathways but many patients remain refractory to treatment. Autologous hematopoietic stem cell transplantation (SCT) has emerged as a therapy for medically refractory CD. SCT was developed to rescue cancer patients from myelosuppressive chemotherapy but its use for CD and other immune diseases necessitates reimagining SCT as a cellular therapy that restores appropriately responsive immune cell populations from hematopoietic progenitors in the stem cell autograft (i.e. immune "reset"). Here we present a paradigm to understand SCT as a cellular therapy for immune diseases and reveal how SCT re-establishes cellular immunity utilizing high-dimensional cellular phenotyping and functional studies of the stem cell grafts. Immunophenotyping using CyTOF, single cell RNA sequencing (scRNA-seq) and T cell receptor (TCR) sequencing was performed on peripheral blood and intestinal tissue samples from refractory CD patients who underwent SCT. The stem cell graft from these patients was analyzed using flow cytometry and functionally interrogated using a murine model for engraftment. Our study revealed a remodeling of intestinal macrophages capable of supporting mucosal healing that was independently validated using multimodal studies of immune reconstitution events including CyTOF and scRNA-seq. Functional interrogation of hematopoietic stem cells (HSCs) using a xenograft model demonstrated that HSCs shape the timing of immune reconstitution, the selected reconstitution of specific cell lineages and potentially the clinical efficacy of SCT. These studies indicate that SCT serves as a myeloid-directed cellular therapy re-establishing homeostatic intestinal macrophages that support intestinal healing and suggest refractory CD evolves from impairment of restorative functions in myeloid cells. Furthermore, we report heterogeneity among HSCs from CD patients which may drive SCT outcomes and suggests an unrecognized impact of CD pathophysiology on HSC in the marrow niche.

Knockout Genes in Bowel Anastomoses: A Systematic Review of Literature Outcomes.

The intestinal wound healing process is a complex event of three overlapping phases: exudative, proliferative, and remodeling. Although some mechanisms have been extensively described, the intestinal healing process is still not fully understood. There are some similarities but also some differences compared to other tissues. The aim of this systematic review was to summarize all studies with knockout (KO) experimental models in bowel anastomoses, underline any recent knowledge, and clarify further the cellular and molecular mechanisms of the intestinal healing process. A systematic review protocol was performed. Medline, EMBASE, and Scopus were comprehensively searched. a total of eight studies were included. The silenced genes included interleukin-10, the four-and-one-half LIM domain-containing protein 2 (FHL2), cyclooxygenase-2 (COX-2), annexin A1 (ANXA-1), thrombin-activatable fibrinolysis inhibitor (TAFI), and heparin-binding epidermal growth factor (HB-EGF) gene. Surgically, an end-to-end bowel anastomosis was performed in the majority of the studies. Increased inflammatory cell infiltration in the anastomotic site was found in IL-10-, annexin-A1-, and TAFI-deficient mice compared to controls. COX-1 deficiency showed decreased angiogenesis at the anastomotic site. Administration of prostaglandin E2 in COX-2-deficient mice partially improved anastomotic leak rates, while treatment of ANXA1 KO mice with Ac2-26 nanoparticles reduced colitis activity and increased weight recovery following surgery. our findings provide new insights into improving intestinal wound healing by amplifying the aforementioned genes using appropriate gene therapies. Further research is required to clarify further the cellular and micromolecular mechanisms of intestinal healing.

Oral administration of CXCL12-expressing Limosilactobacillus reuteri improves colitis by local immunomodulatory actions in preclinical models.

Treatments of colitis, inflammation of the intestine, rely on induction of immune suppression associated with systemic adverse events, including recurrent infections. This treatment strategy is specifically problematic in the increasing population of patients with cancer with immune checkpoint inhibitor (ICI)-induced colitis, as immune suppression also interferes with the ICI-treatment response. Thus, there is a need for local-acting treatments that reduce inflammation and enhance intestinal healing. Here, we investigated the effect and safety of bacterial delivery of short-lived immunomodulating chemokines to the inflamed intestine in mice with colitis. Colitis was induced by dextran sulfate sodium (DSS) alone or in combination with ICI (anti-PD1 and anti-CTLA-4), and Limosilactobacillus reuteri R2LC (L. reuteri R2LC) genetically modified to express the chemokine CXCL12-1α (R2LC_CXCL12, emilimogene sigulactibac) was given perorally. In addition, the pharmacology and safety of the formulated drug candidate, ILP100-Oral, were evaluated in rabbits. Peroral CXCL12-producing L. reuteri R2LC significantly improved colitis symptoms already after 2 days in mice with overt DSS and ICI-induced colitis, which in benchmarking experiments was demonstrated to be superior to treatments with anti-TNF-α, anti-α4β7, and corticosteroids. The mechanism of action involved chemokine delivery to Peyer's patches (PPs), confirmed by local CXCR4 signaling, and increased numbers of colonic, regulatory immune cells expressing IL-10 and TGF-β1. No systemic exposure or engraftment could be detected in mice, and product feasibility, pharmacology, and safety were confirmed in rabbits. In conclusion, peroral CXCL12-producing L. reuteri R2LC efficiently ameliorates colitis, enhances mucosal healing, and has a favorable safety profile.NEW & NOTEWORTHY Colitis symptoms are efficiently reduced by peroral administration of probiotic bacteria genetically modified to deliver CXCL12 locally to the inflamed intestine in several mouse models.

Single Cell Analysis of Human Colonoids Exposed to Uranium-Bearing Dust.

Uranium exposure remains an important environmental legacy and physiological health concern, with hundreds of abandoned uranium mines located in the Southwestern United States largely impacting underserved indigenous communities. The negative effects of heavy metals on barrier permeability and inhibition of intestinal epithelial healing have been described; however, transcriptomic changes within the intestinal epithelial cells and impacts on lineage differentiation are largely unknown. Herein, we sought to determine the molecular and cellular changes that occur in the colon in response to uranium bearing dust (UBD) exposure. Human colonoids from three biologically distinct donors were acutely exposed to UBD then digested for single cell RNA sequencing to define the molecular changes that occur to specific identities of colonic epithelial cells. Validation in colonoids was assessed using morphological and imaging techniques. Human colonoids acutely exposed to UBD exhibited disrupted proliferation and hyperplastic differentiation of the secretory lineage cell, enteroendocrine cells (EEC). Single-cell RNA sequencing also showed more EEC subtypes present in UBD-exposed colonoids. These findings highlight the significance of crypt-based proliferative cells and secretory cell differentiation using human colonoids to model major colonic responses to uranium-bearing particulate dust exposure. https://doi.org/10.1289/EHP13855.

Tannic acid and zinc ion coordination of nanase for the treatment of inflammatory bowel disease by promoting mucosal repair and removing reactive oxygen and nitrogen species

Colon mucosal overexpression of reactive oxygen and nitrogen species (RONS) accelerates the development of inflammatory bowel disease (IBD) and destroys the mucosa and its barrier. IBD can be alleviated by removing RONS from the inflamed colon. The preparation of strong and efficient nanoantioxidants remains a challenge despite the development of numerous nanoantioxidants. In this paper, Zn-TA nanoparticles with fine hollow microstructure (HZn-TA) were successfully prepared and could be effectively used to treat IBD. In the first step, ZIF-8 nanoparticles were synthesized by a one-pot method. On this basis, HZn-TA nanoparticles were etched by TA, and a multifunctional nanase was developed for the treatment of IBD. RONS, including reactive oxygen species (ROS) and nitric oxide (NO), can be eliminated to increase cell survival following Hydrogen peroxide (H2O2) stimulation, including reactive oxygen species (ROS) and nitric oxide (NO with hydrogen peroxide (H2O2). In a model for preventing and delaying acute colitis, clearance of RONS has been shown to reduce intestinal inflammation in mice by reducing colon damage, proinflammatory cytokine levels, the spleen index, and body weight. Intestinal mucosal healing can be promoted by HZn-TA nanoparticles, which can upregulate zonula occludens protein 1 (ZO-1) and claudin-1 expression. Based on the results of this study, HZn-TA nanoparticles were able to effectively treat IBD with minimal adverse effects by being biocompatible, multienzyme active, and capable of scavenging RONS. Therefore, we pioneered the application of HZn-TA nanoparticles for the treatment of IBD, which are capable of clearing RONS without significant adverse effects. Statement of significance➢ HZn-TA nanoparticles were successfully prepared and could be effectively used to treat IBD.➢ Intestinal mucosal healing can be promoted by HZn-TA nanoparticles, which can upregulate ZO-1 and claudin-1 expression.➢ HZn-TA nanoparticles were able to effectively treat IBD with minimal adverse effects by being biocompatible, multienzyme active, and capable of scavenging RONS.

A179 UNDERSTANDING THE NEED FOR A ZINC DIET TO TREAT ZINC DEFICIENCY IN PATIENTS WITH CELIAC DISEASE

Abstract Background Nutritional deficiencies in celiac disease (CeD) are caused by delayed intestinal healing and a nutrient-lacking gluten-free diet (GFD). At our specialized adult celiac clinic, zinc (Zn) was the most common deficiency present in patients and is clinically treated through Zn supplementation. However, Zn supplements in excess may cause inhibition of other minerals, and gastrointestinal side effects. Alternatively, a Zn diet may be a sustainable option to treat zinc deficiency, however patient preference regarding this treatment method is unknown. Aims To understand patient preference of zinc supplementation compared to a zinc diet to treat Zn deficiency in CeD. We also aim to determine the tolerability and effectiveness of Zn supplements to reduce gastrointestinal and extraintestinal symptoms in the CeD population. Methods This observational prospective pragmatic study was conducted from March 2022-September 2023. Eligible participants had bloodwork indicating plasma Zn levels ampersand:003C 9.4 μ/mol . All participants were prescribed 25 mg per day of Zn gluconate supplements throughout the study. Patients were provided a RedCap link where they completed questionnaires at Visit 1 and at their next clinic appointment in 3-6 months (Visit 2). These surveys included; Celiac Symptom Index (CSI) Questionnaire 2) Extraintestinal Symptoms Visual Analogue Scale (ES-VAS) and 3) Patient Perception Survey (PPS). Continuous data are expressed as Mean (SD), and categorical data is reported as frequencies (%). The Wilcoxon test was used to assess changes in symptoms. Results A total of 42 participants completed the Visit 1 survey, of which 17 participants had their follow up clinic appointment (Visit 2). Out of these 17 participants, 15 were female (88.2%) and the mean age was 33.7 years (+/- 10.6). The mean CSI score at Visit 1 was 35.3 (+/- 10.3) and 33.7 (+/- 13.0) at Visit 2, indicating moderate disease symptoms (p=0.552). The most common extraintestinal symptoms was anxiety (ES-VAS: 5.7), foggy mind (ES-VAS: 4.1), fatigue (ES-VAS: 4.1) and depression (ES-VAS: 3.5). There were no significant changes in extraintestinal symptoms between clinic visits. Out of 42 participants, 15 (35.7%) prefer to treat their Zn deficiency through diet, 26 (59.5%) through supplement, and 1 (2.4%) of participants had no preference. Additionally, 35 (85.4%) participants indicated that they were “likely” to comply to a Zn-diet treatment. Conclusions CeD patients did not experience improvement in gastrointestinal or extraintestinal symptoms on Zn supplementation. However, a portion of CeD patients prefer to treat and are likely to comply with a Zn diet to treat their deficiency. Based on this knowledge, further studies should investigate the efficacy of a Zn diet compared to supplements CeD patients. Funding Agencies CIHR

Investigation of Gastrointestinal Toxicities Associated with Concurrent Abdominal Radiation Therapy and the Tyrosine Kinase Inhibitor Sunitinib in a Mouse Model.

Tyrosine kinase inhibitors (TKIs) may be combined with radiation therapy (RT) to enhance tumor control; however, increased incidences of gastrointestinal (GI) toxicity have been reported with this combination. We hypothesize that toxicity is due to compromised intestinal healing caused by inhibition of vascular repair and proliferation pathways. This study explores underlying tissue toxicity associated with abdominal RT and concurrent sunitinib in a mouse model. Four groups of CD-1 mice were treated with 12 Gy abdominal RT, oral sunitinib, abdominal RT + sunitinib, or sham treatment. Mice received oral sunitinib or the vehicle via gavage for 14 days. On day 7, mice were irradiated with 12 Gy abdominal RT or sham treated. Mice were euthanized on day 14 and intestinal tract was harvested for semiquantitative histopathologic evaluation and immunohistochemical quantification of proliferation (Ki67) and vascular density (CD31). Non-irradiated groups had stable weights while abdominal irradiation resulted in weight loss, with mice receiving RT + SUN having greater weight loss than mice receiving RT alone. Semiquantitative analysis showed significant increases in inflammation in irradiated groups. The difference in the density of CD31+ cells was significantly increased in RT alone compared to SUN alone. Ki67+ density was not significant. In summary, we identify a lack of angiogenic response in irradiated GI tissues when abdominal RT is combined with a TKI, which may correlate with clinical toxicities seen in canine and human patients receiving combined treatment.

LEUCINE-RICH ALPHA 2 GLYCOPROTEIN: A USEFUL BIOMARKER TO DISCRIMINATE SMALL INTESTINAL MUCOSAL HEALING IN C-REACTIVE PROTEIN-NEGATIVE CROHN’S DISEASE

Abstract BACKGROUND The treatment goals for inflammatory bowel disease, including Crohn's disease (CD), have shifted from clinical remission to mucosal healing. However, frequent endoscopies burden patients. Leucine-rich alpha 2 glycoprotein (LRG), produced by hepatocytes, neutrophils, and the intestinal epithelium, is more gut-specific than C-reactive protein (CRP). We examined whether LRG, as a biomarker, can detect small intestinal mucosal healing in CD patients, especially in those who are CRP-negative. METHOD 1) This study examined CD patients who visited our department from May 2021 to April 2023, had LRG measured, and underwent image evaluation using capsule endoscopy or balloon-assisted endoscopy. We analyzed their clinical background, looked for a correlation between LRG and CRP, assessed the ROC-AUC of LRG and CRP for mucosal healing, and measured the LRG and CRP cutoff values to determine mucosal healing. 2) Next, in CRP-negative CD patients, we analyzed the ROC-AUC of LRG for mucosal healing and LRG cutoff value to determine mucosal healing. Endoscopic evaluation was done within 3 months before and after LRG measurement. Absent open ulcers indicated mucosal healing. The upper limit of normal CRP value was <3 mg/L. We used the Spearman correlation coefficient to determine the correlation. RESULTS 1) Here, 67 CD patients were analyzed; 43 (64%) were men. The median age (range) of onset was 25.6 (3-57) years. The median disease duration (range) was 7.6 (0-27) years. B1, B2, and B3 of Montreal Classification were established in 31 (46.2%), 21 (31.3%), and 15 (22.3%) patients, respectively. The median (range) of LRG was 21.7 (7.6-67.4) μg/mL. The median (range) of CRP was 10.8 (0.0-86.9) mg/L. LRG and CRP were strongly correlated (r=0.878, p<0.001). The ROC-AUC of LRG for mucosal healing was 0.839 (95% confidence interval [CI] 0.760-0.918). LRG cutoff value for discriminating mucosal healing was 15.5 μg/mL (sensitivity 77%, specificity 75%). The ROC-AUC of CRP for mucosal healing was 0.811 (95% CI 0.721-0.900). The CRP cutoff value to differentiate mucosal healing was 0.9 mg/L (sensitivity 55%, specificity 90%). 2) Forty CRP-negative CD patients were analyzed; 23 (57%) were men. The median age (range) of onset was 21.9 (3-57) years. The median disease duration (range) was 7.0 (0-27) years. B1, B2, and B3 of Montreal Classification were established in 17 (42.5%), 12 (30.0%), and 11 (27.5%) patients, respectively. The median (range) of LRG was 12.3 (7.6-23.5) μg/mL. The ROC-AUC of LRG for mucosal healing was 0.817 (95% CI 0.696-0.938). The LRG cutoff value to differentiate mucosal healing was 14.1 μg/mL (sensitivity 61%, specificity 76%). CONCLUSION LRG may be a better biomarker than CRP for assessing endoscopic remission of the small intestinal mucosa in CD. In CRP-negative CD patients, LRG level of ≥14.1 μg/mL could indicate small intestinal ulcers.

P431 Assessing intestinal barrier healing by fusing ultra-high magnification endoscope and automated spatial multispectral imaging analysis in PSC-colitis patients

Abstract Background PSC colitis is associated with an increased risk of colorectal carcinoma. Endoscopic monitoring is crucial since endoscopic and histological activity can persist in PSC even during clinical remission. Recent evidence suggests that the presence of barrier dysfunction may predict relapse in quiescent patients. This study aims to assess mucosal and barrier healing in quiescent PSC colitis and evaluate the potential to predict outcomes by combining an ultra-high magnification endocytoscope (ECS) with intestinal barrier protein markers. Methods 23 PSC-colitis patients in clinical remission requiring surveillance colonoscopy were prospectively enrolled. Ileal and colonic mucosa were assessed with high definition, virtual chromoendoscopy and ultra-high magnification ECS. Endoscopic remission was defined as MES ≤1, UCEIS≤1, PICaSSO≤3, while histological remission was defined as RHI≤3 without lamina propria or epithelial neutrophils, NHI ≤1 and PHRI=0. A novel ECS score was developed to assess ileal and colonic intestinal barrier (Table 1). The expression of tight junction proteins (Claudin-2, Occludin, and JAM-A) was evaluated through immunohistochemistry and multiplex immunofluorescence, and it was quantified as cell density and mean expression using the automated inForm® Akoya Biosciences digital multiplex platform. The occurrence of significant adverse outcomes (steroid use, flare-ups, hospitalization, colectomy) was evaluated over a 12-month follow-up period. Results Of 18 patients in endoscopic remission, 12 and 13 were in histological remission according to RHI and NHI, respectively. The ECS score correlated moderately with endoscopic score (r=0.57 for MES, r=0.44 for UCEIS and r=0.54 for PICaSSO) and strongly with histological scores (r=0.56 for RHI, r=0.61 for NHI and r=0.62 for PHRI), especially in the right colon. Only 5 patients experienced adverse outcomes. Notably, barrier healing, as indicated by an ECS score<3 in the ileum and <5 in the colon, was significantly associated with better outcomes. The three barrier proteins showed a distinct mucosal localisation in both ileum and colon, with higher cell density of Occludin and Claudin-2 in the epithelium, while higher JAM-A cell density in the lamina propria. The combined assessment of the intestinal barrier by ECS and Claudin-2 cell density significantly predicted outcomes in both colon and ileum (p=0.02 and p=0.04, respectively). Conclusion ECS combined with automated multispectral imaging analysis of intestinal barrier integrity is an innovative tool to assess barrier healing precisely and predicts significant adverse outcomes in PSC colitis patients in clinical, endoscopic, and histological remission.

Restore Intestinal Barrier Integrity: An Approach for Inflammatory Bowel Disease Therapy.

The intestinal barrier maintained by various types of columnar epithelial cells, plays a crucial role in regulating the interactions between the intestinal contents (such as the intestinal microbiota), the immune system, and other components. Dysfunction of the intestinal mucosa is a significant pathophysiological mechanism and clinical manifestation of inflammatory bowel disease (IBD). However, current therapies for IBD primarily focus on suppressing inflammation, and no disease-modifying treatments specifically target the epithelial barrier. Given the side effects associated with chronic immunotherapy, effective alternative therapies that promote mucosal healing are highly attractive. In this review, we examined the function of intestinal epithelial barrier function and the mechanisms of behind its disruption in IBD. We illustrated the complex process of intestinal mucosal healing and proposed therapeutic approaches to promote mucosal healing strategies in IBD. These included the application of stem cell transplantation and organ-like tissue engineering approaches to generate new intestinal tissue. Finally, we discussed potential strategies to restore the function of the intestinal barrier as a treatment for IBD.

Results of the use of human platelet-rich fibrin membrane applied on colorrhaphy in nourished and malnourished rats.

To evaluate the effects of platelet-rich fibrin (PRF) on the healing of intestinal sutures in rats. Forty rats were distributed into four groups. Two groups were treated with a standard diet and considered nourished (I and II). Two other groups were treated with cornmeal and considered malnourished (III and IV). All animals underwent cecotomy and cecorrhaphy. Groups II and IV had sutures overlapped with human PRF membrane. The following parameters were evaluated: animal weight, death, rupture site, rupture pressure, collagen, and reticulin dosage in the suture line. The use of PRF did not influence deaths, rupture pressure or rupture location. For malnourished animals, a significant difference was observed in relation to the rupture site, corresponding to the suture line (p = 0.038) and reticulin dosage (p = 0.040), when PRF was used. There was no difference in relation to burst pressures. The use of PRF did not influence intestinal healing in nourished rats. In the group of malnourished animals, its use favored healing.

Celiac Disease: Promising Biomarkers for Follow-Up.

Celiac disease is a common gastroenterological illness. Current diagnostics of the disease are based on serological markers and histology of duodenal biopsies. Hitherto, a strict gluten-free diet is the only effective treatment and is necessary for good control of the disease. Serological tests in current use have very high specificity and sensitivity for diagnostics, but in follow-up they have some limitations. Their levels do not accurately reflect mucosal healing, and they are unable to detect minimal transgressions in the diet. This problem is significant in patients with IgA deficiency, and there exist no robust follow-up tools for monitoring these patients' adherence to treatment. For their follow-up, we currently use IgG-based tests, and these antibodies persist for a long time even when a patient has stopped consuming gluten. More accurate and specific biomarkers are definitely needed. Adherence to a gluten-free diet is essential not only for intestinal mucosa healing and alleviation of symptoms but also for preventing complications associated with celiac disease. Here, we summarize current evidence regarding noninvasive biomarkers potentially useful for follow-up not only of patients with IgA deficiency but for all patients with celiac disease. We describe several very promising biomarkers with potential to be part of clinical practice in the near future.

CHRONIC IFNΓ-DRIVEN INFLAMMATION IMPAIRS INTESTINAL TOLERANCE AND REGENERATION IN AGING

Abstract The intestine’s remarkable regenerative capacity decreases with aging as the stem cellniche interaction is impaired. Beyond defects in the stem cell-niche interaction, there is a direct correlation between age-related changes in immunity and increased susceptibility to infections. However, how the aging immune system affects the regenerative capacity of the intestine to challenges such as infections is unclear. Here, we infected young and aged mice with Citrobacter rodentium, a model that activates the immune response and causes epithelial damage. Young mice successfully healed, while old mice succumbed to infection. In the aged infected intestines, we identified elevated basal levels of interferon γ (IFNγ) that significantly increased in lymphoid cells, CD4+, and CD8+ T cells. Additionally, aged post-infected intestines exhibited dramatic epithelial barrier disruption, partly due to a decrease in Lgr5+ stem cells and an imbalance of cell differentiation towards goblet cell production, raising the question if the aging intestine is more susceptible to infection due to persistent exposure to IFNγ. To test the response of young and aged intestinal epithelium to continuous IFNγ exposure, we treated young and aged organoids with IFNγ. We found that old organoids undergo massive apoptotic cell death as early as 24 hours post-IFNγ treatment. In contrast, young organoids were unaffected, demonstrating that IFNγ drives the age-related impairment of intestinal healing. Although a short-term boost of IFNγ is necessary for intestinal regeneration in adults, our findings indicate that prolonged IFNγ activation associated with aging can impede intestinal repair.