Intestinal Crypt Research Articles

Refined Baohe formula protects against 5-fluorouracil-induced intestinal mucositis by modulating AKT pathway in CT-26 tumor-bearing mice

Background5-Fluorouracil (5-FU) is one of the most commonly prescribed anticancer agents. However, intestinal mucositis is a well-known adverse event, which limits its therapeutic use. Refined Baohe formula (RBF) is derived from Baohe Pills with a potent gastrointestinal protective effect. However, the effects of RBF on 5-FU-induced intestinal mucositis remain unknown. Thus, this study investigated the effects and mechanism of RBF on 5-FU-induced intestinal mucositis in a CT-26 xenograft mice colorectal cancer (CRC) model. MethodsThe composition of the RBF preparation was analyzed by high-pressure liquid chromatography. CT-26 cells bearing mice were intraperitoneally administered 150 mg/kg of 5-FU on day 1 to construct a 5-FU-induced intestinal injury model. Mice in the 5-FU + RBF group were intragastrically administrated with RBF (12.4 g/kg) for 4 days. Tumor volume and weight were determined, body weight and diarrhea scores were monitored, leukocytes were calculated, and hematoxylin-eosin staining, immunohistochemistry, and terminal deoxynucleotidyl transferase dUTP nick end labeling were performed to investigate the therapeutic efficiency and underlying mechanisms of RBF on 5-FU-induced intestinal mucositis mice. ResultsRBF didn`t affect the effect of tumor suppression, while attenuated diarrhea associated with 5-FU-induced intestinal mucositis and significantly reduced leucocyte toxicity. Additionally, RBF significantly promoted cell proliferation and inhibited cell apoptosis, and up-regulated CDK4, c-Myc, and B-cell lymphoma-2 expression, while down-regulated Bax expression, as well as enhanced both p-AKT and AKT expression in intestinal crypts of 5-FU-treated mice. ConclusionsRBF attenuated 5-FU-induced intestinal mucositis by inhibiting apoptosis and promoting cell proliferation by modulating the AKT pathway and its downstream effectors in a CT-26 xenograft mouse CRC model.

Mucosal Atrophy Predicts Poorer Outcomes in Pediatric Ulcerative Colitis-A National Inception Cohort Study.

Outcomes in pediatric ulcerative colitis (UC) are heterogeneous and predictors of disease course eagerly sought. Mucosal atrophy (MA) is characterized by histological abnormalities of colonic intestinal glands. To determine the prevalence of MA in a national inception cohort of pediatric UC and its impact on outcomes. Irish children < 16 years old with UC are diagnosed at a single referral center. At diagnosis, patients underwent phenotyping by Paris classification and activity assessment by Pediatric Ulcerative Colitis Activity Index. Biopsies from all colonic segments were evaluated for MA. Patients were followed prospectively. The primary outcome was corticosteroid-free remission at 1 year. Secondary outcomes included relapse, treatment escalation, and colectomy by 2 years. Of 251 pediatric patients with UC (mean age 11.8 years, 55% male), 38 (15%) had MA on diagnostic biopsy. Baseline characteristics were similar between groups with/without MA and there was no difference in steroid-free remission or rates of moderate-severe UC at 1 year. Patients with MA had higher use of steroids (29% vs 15%, P = 0.04) and immunomodulators (40% vs 21%, P = 0.04) at 6 months, higher biologic use at 1 year (34% vs 16%, P = 0.03), earlier first relapse (mean ± SD 29.4 ± 26.1 vs 46.7 ± 43.4 weeks after diagnosis, P = 0.02), and higher colectomy rates by 2 years (21% vs 8%, P = 0.01). Children with MA at diagnosis had higher colectomy rates despite earlier treatment escalation and similar baseline severity scores. We identify MA as a promising new prognostic marker in children with newly diagnosed UC.

Nucleotide Supplements Alter Proliferation and Differentiation of Cultured Human (Caco-2) and Rat (IEC-6) Intestinal Epithelial Cells

The effect of exogenous nucleotides on the proliferation and differentiation of enterocytes was comparatively studied using a human colon tumor cell line (Caco-2) and a normal rat small intestinal crypt cell line (IEC-6). Caco-2 cells exhibited more active endogenous nucleic acid metabolism than did IEC-6 cells, as evidenced by greater cellular pools of nucleotides and their metabolites. To determine the supplemental nutritional effect of nucleotides, a mixture containing equal amounts (10 mg/L) of AMP, CMP, IMP, GMP and UMP was added to the culture medium. The results showed that a nucleotide supplement under normal culture conditions did not affect proliferation and differentiation of Caco-2 cells. In contrast, nucleotide supplements under normal culture conditions promoted proliferation of IEC-6 cells. The addition of nucleotides to the culture medium also enhanced differentiation of IEC-6 cells when grown on an extracellular matrix (Matrigel). Furthermore, when glutamine levels were less than optimal (nutritional stress conditions), nucleotide supplements enhanced growth and maturation of both cell lines. We conclude that the de novo biosynthesis of nucleotides is sufficient to support proliferation of tumor Caco-2 cells but not of the normal crypt IEC-6 cells. Thus, nucleotide supplements may enhance normal enterocyte growth and maturation as well as spare the need for exogenous glutamine in cell maintenance and development.

Effects of Dietary Sodium and Chloride on Slaughter Performance, Digestive Tract Development and Tibia Mineralization of Geese

This study evaluated the slaughter performance, digestive tract development and tibia mineralization effects of sodium (Na) and chloride (Cl) on geese. Four hundred and thirty-four male geese at 29 days were randomly assigned into nine groups with six replicates (eight in each). The experiment employed a 3 × 3 factorial design, with two instances each of three Na levels (0.10%, 0.15%, and 0.20%) and three Cl levels (0.15%, 0.20%, and 0.25%). All experimental birds were husbanded for 42 days. Dietary Na and Cl levels and their interactions (Na ×Cl) had no significant effect on the slaughter, breast, thigh, abdominal fat yield, and digestive tract index of geese (p > 0.05). However, dietary Na and Cl level significantly affected the crypt depth of the jejunum and tibial development. Variations in Na and Cl levels had a significant interaction on the crypt depth of jejunal (p < 0.05), 0.20% Na × 0.25% Cl had a minor crypt depth. Dietary variations in Na and Cl significantly affected the tibial strength, and there was a significant interaction between them (p < 0.05). When Na and Cl were at their maximum (0.20% Na and 0.25% Cl), the strength of the tibia was the lowest. In addition, a single factor (Na or Cl) had no effect (p > 0.05), but its interaction significantly affected the calcium (Ca) content of bone (p < 0.05). When the Na and Cl levels were 0.15% and 0.15%, respectively, the Ca content in bone was the highest. These results suggest that dietary Na and Cl had interactive effects on geese, especially in the development of the tibia. High dietary Na and Cl levels adversely influenced the tibia and intestinal crypt morphology. Therefore, we do not advocate supplementing too much Na or Cl in the diet. Combined with our previous results, for 29-70-day-old geese, it is recommended that dietary Na and Cl levels should be 0.10% and 0.15%, respectively.

Chlorogenic acid improves intestinal morphology by enhancing intestinal stem-cell activity.

Chlorogenic acid (CGA), as one of the most abundant naturally occurring phenolic acids, has been documented to be beneficial for intestinal health. However, the underlying mechanism is still not fully understood. The adult intestinal stem cell is the critical driver of epithelial homeostasis and regeneration. This study hypothesized that CGA exerted intestinal health effects by modulating intestinal stem-cell functions. Lgr5-EGFP mice were treated for 14 days, and intestinal organoids derived from these mice were treated for 3 days, using CGA solution. In comparison with the control group, CGA treatment increased intestinal villous height and crypt depth in mice and augmented the area expansion and the number of budding intestinal organoids. Quantitative polymerase chain reaction (qPCR) analysis revealed that CGA treatment significantly increased the expression of genes coding intestinal stem-cell markers in intestinal tissue and organoids, and upregulated the expression of genes coding secretory cell lineages and enterocytes, although not statistically significantly. Fluorescence-activated cell-sorting analysis further confirmed that CGA augmented the number of stem cells. 5-Ethynyl-2'-deoxyuridine (EdU) incorporation and Ki67 immunostaining results also demonstrated that CGA treatment enhanced intestinal stem-cell proliferation. Altogether, our findings indicate that CGA could activate intestinal stem-cell and epithelial regeneration, which could contribute to the improvement of intestinal morphology or organoid growth of mice. This highlights a promising mechanism for CGA as an excellent candidate for the formulation of dietary supplements and functional foods for intestinal protection. © 2023 Society of Chemical Industry.

Understanding disruption of the gut barrier during inflammation: Should we abandon traditional epithelial cell lines and switch to intestinal organoids?

Disruption of the intestinal epithelial barrier is a hallmark of mucosal inflammation. It increases exposure of the immune system to luminal microbes, triggering a perpetuating inflammatory response. For several decades, the inflammatory stimuli-induced breakdown of the human gut barrier was studied in vitro by using colon cancer derived epithelial cell lines. While providing a wealth of important data, these cell lines do not completely mimic the morphology and function of normal human intestinal epithelial cells (IEC) due to cancer-related chromosomal abnormalities and oncogenic mutations. The development of human intestinal organoids provided a physiologically-relevant experimental platform to study homeostatic regulation and disease-dependent dysfunctions of the intestinal epithelial barrier. There is need to align and integrate the emerging data obtained with intestinal organoids and classical studies that utilized colon cancer cell lines. This review discusses the utilization of human intestinal organoids to dissect the roles and mechanisms of gut barrier disruption during mucosal inflammation. We summarize available data generated with two major types of organoids derived from either intestinal crypts or induced pluripotent stem cells and compare them to the results of earlier studies with conventional cell lines. We identify research areas where the complementary use of colon cancer-derived cell lines and organoids advance our understanding of epithelial barrier dysfunctions in the inflamed gut and identify unique questions that could be addressed only by using the intestinal organoid platforms.

ATG16L1 protects from interferon-γ-induced cell death in the small intestinal crypt

The breakdown of the intestinal mucosal barrier is thought to underlie the progression to Crohn disease (CD), whereby numerous risk factors contribute. For example, a genetic polymorphism of the autophagy gene ATG16L1, associated with an increased risk of developing CD, contributes to the perturbation of the intestinal epithelium. We examined the role of Atg16l1 in protecting the murine small intestinal epithelium from T-cell-mediated damage using the anti-CD3 model of enteropathy. Our work showed that mice specifically deleted for Atg16l1 in intestinal epithelial cells (IECs) (Atg16l1ΔIEC) had exacerbated intestinal damage, characterized by crypt epithelial cell death, heightened inflammation, and decreased survival. Moreover, Atg16l1 deficiency delayed the recovery of the intestinal epithelium, and Atg16l1-deficient IECs were impaired in their proliferative response. Pathology was largely driven by interferon (IFN)-γ signaling in Atg16l1ΔIEC mice. Mechanistically, although survival was rescued by blocking tumor necrosis factor or IFN-γ independently, only anti-IFN-γ treatment abrogated IEC death in Atg16l1ΔIEC mice, thereby decoupling IEC death and survival. In summary, our findings suggest differential roles for IFN-γ and tumor necrosis factor in acute enteropathy and IEC death in the context of autophagy deficiency and suggest that IFN-γ-targeted therapy may be appropriate for patients with CD with variants in ATG16L1.

Bacillus pumilus TS1 alleviates Salmonella Enteritidis-induced intestinal injury in broilers

BackgroundIn the current context of reduced and limited antibiotic use, several pathogens and stressors cause intestinal oxidative stress in poultry, which leads to a reduced feed intake, slow or stagnant growth and development, and even death, resulting in huge economic losses to the poultry breeding industry. Oxidative stress in animals is a non-specific injury for which no targeted drug therapy is available; however, the health of poultry can be improved by adding appropriate feed additives. Bacillus pumilus, as a feed additive, promotes growth and development and reduces intestinal oxidative stress damage in poultry. Heat shock protein 70 (HSP70) senses oxidative damage and repairs unfolded and misfolded proteins; its protective effect has been widely investigated. Mitogen-activated protein kinase/protein kinase C (MAPK/PKC) and hypoxia inducible factor-1 alpha (HIF-1α) are also common proteins associated with inflammatory response induced by several stressors, but there is limited research on these proteins in the context of poultry intestinal Salmonella Enteritidis (SE) infections. In the present study, we isolated a novel strain of Bacillus pumilus with excellent performance from the feces of healthy yaks, named TS1. To investigate the effect of TS1 on SE-induced enteritis in broilers, 120 6-day-old white-feathered broilers were randomly divided into four groups (con, TS1, SE, TS1 + SE). TS1 and TS1 + SE group chickens were fed with 1.4 × 107 colony-forming units per mL of TS1 for 15 days and intraperitoneally injected with SE to establish the oxidative stress model. Then, we investigated whether TS1 protects the intestine of SE-treated broiler chickens using inflammatory cytokine gene expression analysis, stress protein quantification, antioxidant quantification, and histopathological analysis.ResultsThe TS1 + SE group showed lower MDA and higher GSH-Px, SOD, and T-AOC than the SE group. TS1 alleviated the effects of SE on intestinal villus length and crypt depth. Our results suggest that SE exposure increased the expression of inflammatory factors (IL-1β, IL-6, TNF-α, IL-4, and MCP-1), p38 MAPK, and PKCβ and decreased the expression of HSP60, HSP70, and HIF-1α, whereas TS1 alleviated these effects.ConclusionsBacillus pumilus TS1 alleviated oxidative stress damage caused by SE and attenuated the inflammatory response in broilers through MAPK/PKC regulation of HSPs/HIF-1α.

Aryl Hydrocarbon Receptor (AhR) Signaling in Colonic Cells and Tumors.

The aryl hydrocarbon receptor (AhR) is overexpressed in many tumor types and exhibits tumor-specific tumor promoter and tumor suppressor-like activity. In colon cancer, most but not all studies suggest that the AhR exhibits tumor suppressor activity which is enhanced by AhR ligands acting as agonists. Our studies investigated the role of the AhR in colon tumorigenesis using wild-type and AhR-knockout mice, the inflammation model of colon tumorigenesis using mice treated with azoxymethane (AOM)/dextran sodium sulfate (DSS) and APCS580/+; KrasG12D/+ mice all of which form intestinal tumors. The effects of tissue-specific AhR loss in the intestine of the tumor-forming mice on colonic stem cells, organoid-initiating capacity, colon tumor formation and mechanisms of AhR-mediated effects were investigated. Loss of AhR enhanced stem cell and tumor growth and in the AOM/DSS model AhR-dependent suppression of FOXM1 and downstream genes was important for AhR-dependent anticancer activity. Furthermore, the effectiveness of interleukin-22 (IL22) in colonic epithelial cells was also dependent on AhR expression. IL22 induced phosphorylation of STAT3, inhibited colonic organoid growth, promoted colonic cell proliferation in vivo and enhanced DNA repair in AOM/DSS-induced tumors. In this mouse model, the AhR suppressed SOCS3 expression and enhanced IL22-mediated activation of STAT3, whereas the loss of the AhR increased levels of SOCS3 which in turn inhibited IL22-induced STAT3 activation. In the APCS580/+; KrasG12D/+ mouse model, the loss of the AhR enhanced Wnt signaling and colon carcinogenesis. Results in both mouse models of colon carcinogenesis were complemented by single cell transcriptomics on colonic intestinal crypts which also showed that AhR deletion promoted expression of FOXM1-regulated genes in multiple colonic cell subtypes. These results support the role of the AhR as a tumor suppressor-like gene in the colon.

PTBP1 controls intestinal epithelial regeneration through post-transcriptional regulation of gene expression.

The intestinal epithelial regeneration is driven by intestinal stem cells under homeostatic conditions. Differentiated intestinal epithelial cells, such as Paneth cells, are capable of acquiring multipotency and contributing to regeneration upon theloss of intestinal stem cells. Paneth cells also support intestinal stem cell survival and regeneration. We report here that depletion of an RNA-binding protein named polypyrimidine tract binding protein 1 (PTBP1) in mouse intestinal epithelial cells causes intestinal stem cell death and epithelial regeneration failure. Mechanistically, we show that PTBP1 inhibits neuronal-like splicing programs in intestinal crypt cells, which is critical for maintaining intestinal stem cell stemness. This function is achieved at least in part through promoting the non-productive splicing of its paralog PTBP2. Moreover, PTBP1 inhibits the expression of an AKT inhibitor PHLDA3 in Paneth cells and permits AKT activation, which presumably maintains Paneth cell plasticity and function in supporting intestinal stem cell niche. We show that PTBP1 directly binds to a CU-rich region in the 3' UTR of Phlda3, which we demonstrate to be critical for downregulating the mRNA and protein levels of Phlda3. Our results thus reveal the multifaceted in vivo regulation of intestinal epithelial regeneration by PTBP1 at the post-transcriptional level.

LAT1 expression influences Paneth cell number and tumor development in ApcMin/+ mice

BackgroundAmino acid transporters play an important role in supplying nutrition to cells and are associated with cell proliferation. L-type amino acid transporter 1 (LAT1) is highly expressed in many types of cancers and promotes tumor growth; however, how LAT1 affects tumor development is not fully understood.MethodsTo investigate the role of LAT1 in intestinal tumorigenesis, mice carrying LAT1 floxed alleles that also expressed Cre recombinase from the promoter of gene encoding Villin were crossed to an ApcMin/+ background (LAT1fl/fl; vil-cre; ApcMin/+), which were subject to analysis; organoids derived from those mice were also analyzed.ResultsThis study showed that LAT1 was constitutively expressed in normal crypt base cells, and its conditional deletion in the intestinal epithelium resulted in fewer Paneth cells. LAT1 deletion reduced tumor size and number in the small intestine of ApcMin/+ mice. Organoids derived from LAT1-deleted ApcMin/+ intestinal crypts displayed fewer spherical organoids with reduced Wnt/β-catenin target gene expression, suggesting a low tumor-initiation capacity. Wnt3 expression was decreased in the absence of LAT1 in the intestinal epithelium, suggesting that loss of Paneth cells due to LAT1 deficiency reduced the risk of tumor initiation by decreasing Wnt3 production.ConclusionsLAT1 affects intestinal tumor development in a cell-extrinsic manner through reduced Wnt3 expression in Paneth cells. Our findings may partly explain how nutrient availability can affect the risk of tumor development in the intestines.

Quercetin 3-O rutinoside prevents gastrointestinal injury through regulation of apoptosis in 7.5 Gy total body irradiated mice

BackgroundDespite the heightened threat to unforeseen nuclear/radiological exposures worldwide, no countermeasures are currently approved to prevent gastrointestinal (GI) toxicity induced by radiation in humans. PurposeIn this study, we aim to establish the gastroprotective role of flavonoid, Quercetin-3-O-rutinoside (Q-3-R) against 7.5 Gy total body gamma radiation dose that contributes to the hematopoietic syndrome. MethodsQ-3-R (10 mg/kg body weight) was administered intramuscularly to C57BL/6 male mice before exposure to 7.5 Gy and monitored for morbidity and mortality. The GI protection against radiation was ascertained by histopathological and xylose absorption studies. Intestinal apoptosis, crypt proliferation and apoptotic signaling were also investigated in different treatment groups. ResultsWe found that Q-3-R prevented the radiation-induced loss of mitochondrial membrane potential, maintained ATP levels, regulated the apoptotic pathway, and activated crypt cell proliferation in the intestine. Radiation-induced villi and crypt damage as well as mal-absorption were significantly minimized in the Q-3-R treated group. We observed 100% survival post Q-3-R administration against 33.3% lethality in 7.5 Gy (LD33.3/30) exposed C57BL/6 mice. The Q-3-R pre-treated mice that survived the 7.5 Gy dose revealed no pathological changes related to the development of fibrosis in the intestine and thickened mucosal wall till 4 months post irradiation. Complete hematopoietic recovery was observed in these surviving mice when compared to age matched control. ConclusionThe findings revealed that Q-3-R regulated the apoptotic process to achieve GI protection against LD33.3/30 dose (7.5 Gy) that primarily caused death due to hematopoietic failure. The recovery observed in mice survivors suggested that this molecule may also have the potential to minimize side effects on normal tissues during radiotherapy.

Antimicrobial Resistance and Pathogenicity of Aliarcobacter butzleri Isolated from Poultry Meat

Aliarcobacter butzleri (A. butzleri) is an emergent zoonotic food-related pathogen that can be transmitted through the consumption of poultry meat. Data regarding the pathogenicity and resistance of A. butzleri are still scarce, and the presence of virulent MDR strains of this zoonotic pathogen in poultry meat is an issue of particular concern to public health. This study aimed to characterize the pathogenicity and antimicrobial resistance profiles of A. butzleri strains isolated from poultry meat sold at retail markets in São Paulo, Brazil. The minimum inhibitory concentrations of 27 strains were determined using the broth microdilution method. The results showed that 77.7% of the isolates were resistant to clindamycin, 62.9% to florfenicol, 59.2% to nalidixic acid, 11.1% to azithromycin, 7.4% to ciprofloxacin and telithromycin, and 3.7% to erythromycin and tetracycline, although all were susceptible to gentamicin. Moreover, 55.5% of the virulent isolates were also multidrug-resistant (MDR). Three strains were selected for pathogenicity tests in vitro and in vivo. The tested strains expressed weak/moderate biofilm production and showed a diffuse adhesion pattern (3 h) in HeLa cells and toxicity in Vero cells (24 h). Experimental inoculation in 11-week-old chicks induced a transitory inflammatory enteritis. Intestinal hemorrhage and destruction of the intestinal crypts were observed in the rabbit ileal loop test. Considering the fact that Brazil is a major exporter of poultry meat, the data from this study point to the need of improvement of the diagnostic tools, as well as of the adoption of surveillance guidelines and more specific control strategies to ensure food safety, reducing the presence of pathogenic MDR strains in broilers.

Establishment and Evaluation of Intestinal Injury Model of Mouse Acute Graft Versus Host Disease Based on An Organoid Technology

To establish an intestinal organoid model that mimic acute graft versus host disease (aGVHD) caused intestinal injuries by using aGVHD murine model serum and organoid culture system, and explore the changes of aGVHD intestine in vitro by advantage of organoid technology. 20-22 g female C57BL/6 mice and 20-22 g female BALB/c mice were used as donors and recipients for bone marrow transplantation, respectively. Within 4-6 h after receiving a lethal dose (8.0 Gy) of γ ray total body irradiation, a total of 0.25 ml of murine derived bone marrow cells (1×107/mice, n=20) and spleen nucleated cells (5×106/mice, n=20) was infused to establish a mouse model of aGVHD (n=20). The aGVHD mice were anesthetized at the 7th day after transplantation, and the veinal blood was harvested by removing the eyeballs, and the serum was collected by centrifugation. The small intestinal crypts of healthy C57BL/6 mice were harvested and cultivated in 3D culture system that maintaining the growth and proliferation of intestinal stem cells in vitro. In our experiment, 5%, 10%, 20% proportions of aGVHD serum were respectively added into the organoid culture system for 3 days. The formation of small intestinal organoids were observed under an inverted microscope and the morphological characteristics of intestinal organoids in each groups were analyzed. For further evaluation, the aGVHD intestinal organoids were harvested and their pathological changes were observed. Combined with HE staining, intestinal organ morphology evaluation was performed. Combined with Alcian Blue staining, the secretion function of aGVHD intestinal organoids was observed. The distribution and changes of Lgr5+ and Clu+ intestinal stem cells in intestinal organoids were analyzed under the conditions of 5%, 10% and 20% serum concentrations by immunohistochemical stainings. The results of HE staining showed that the integrity of intestinal organoids in the 5% concentration serum group was better than that in the 10% and 20% groups. The 5% concentration serum group showed the highest number of organoids, the highest germination rate and the lowest pathological score among experimental groups, while the 20% group exhibited severe morphological destruction and almost no germination was observed, and the pathological score was the highest among all groups(t=3.668, 4.334,5.309,P<0.05). The results of Alican blue staining showed that the secretion function of intestinal organoids in serum culture of aGVHD in the 20% group was weaker than that of the 5% group and 10% of the organoids, and there was almost no goblet cells, and mucus was stainned in the 20% aGVHD serum group. The immunohistochemical results showed that the number of Lgr5+ cells of intestinal organoids in the 5% group was more than that of the intestinal organoids in the 10% aGVHD serum group and 20% aGVHD serum group. Almost no Clu+ cells were observed in the 5% group. The Lgr5+ cells in the 20% group were seriously injuried and can not be observed. The proportion of Clu+ cells in the 20% group significantly increased. The concentration of aGVHD serum in the culture system can affect the number and secretion function of intestinal organoids as well as the number of intestinal stem cells in organoids. The higher the serum concentration, the greater the risk of organoid injury, which reveal the characteristics of the formation and functional change of aGVHD intestinal organoids, and provide a novel tool for the study of intestinal injury in aGVHD.

Johannes Nathanael Lieberkühn (1711-1756): luminary eighteenth century anatomist and his illuminating discovery of intestinal glands.

Johannes Nathanael Lieberkühn was a prodigious anatomist whose meticulous experiments and precise detailing helped in comprehending the microscopic anatomy of digestive system during early part of eighteenth century. Notably, his inventions in the field of microscopy aptly complemented his quest for anatomical knowledge at microscopic level. He designed a reflector (Lieberkühn reflector) which enhanced the amount of focussed light leading to bright illumination of tissue specimen. He invented the solar microscope which provided excellent resolution of minute anatomical details. Lieberkühn discovered the digestive juice secreting tubular glands (glands of Lieberkühn) present at the base of intestinal villi producing epithelial invaginations (crypts of Lieberkühn). He also described the intricate juxtaposition of blood vessels in relation to a single intestinal villi. Moreover, through empirically designed experimental set up, Lieberkühn was able to demonstrate the flow of lymph from intestinal villi to collecting lymphatic vessels. Also, his grandiose collection of laboratory specimens involving vascular anatomy are a testimony of his untiring efforts in academia. His contributions were seminal in comprehending the anatomy of digestive system and paved the way for future revelations. His work unveiled the enormous scope of microanatomy in medical science and catalysed the advent of histological staining methods a century later.

P060 Restoration of occludin and claudin-1 expression in patients with Crohn’s disease receiving anti-TNF treatment.

Abstract Background Occludin and claudin-1 are integral components of tight junctions (TJs) that contribute to intestinal barrier integrity. The aim of this study was to prospectively evaluate the expression of occludin and claudin-1 in the intestinal mucosa of patients with Crohn’s disease (CD) undergoing treatment with tumor necrosis factor (TNF) inhibitors. Methods Clinical assessment, laboratory testing, and colonoscopy were performed on all participants prior to receiving anti-TNF therapy. Colonic or ileal biopsies were obtained from the affected intestinal areas. A follow-up exam and colonoscopy were performed at least 6 months after the start of anti-TNF therapy, and paired biopsies were obtained from the initially affected segments. The expression of the TJ proteins was evaluated by immunohistochemistry (IHC). Results In this prospective observational study, 21 consecutive patients with active CD [median age 32.5 years (IQR: 18.5-51), 66.7% male] and 10 healthy controls [36 years (IQR: 29-58), 60% male] were enrolled. Patients with CD received treatment with TNF inhibitors (infliximab, adalimumab, and certolizumab: 61.9%, 33.3%, and 4.8%, respectively), according to current guidelines, and a follow-up examination was conducted after a median period of 25 months (IQR: 9.5-66). Before treatment administration, patients with active CD exhibited an increased expression of both occludin [2 (IQR: 1.75-2.75) vs. 1.5 (IQR: 0-1.75); p = 0.032] and claudin-1 [2.5 (IQR: 2-3) vs. 1 (IQR: 0.75-2); p = 0.01] in comparison to healthy controls. In control mucosa, IHC staining for occludin and claudin-1 was weak to moderate, cytoplasmic, and membranous, confined to the surface epithelium, while staining in intestinal crypts was negative. On the other hand, an enhanced expression of both proteins in the enteric crypts and surface epithelium was demonstrated in CD patients upon initial examination. Treatment with TNF inhibitors resulted in a significant reduction of claudin-1 expression (p = 0.018) as well as a notable decline of occludin expression (p = 0.08). Occludin and claudin-1 immunostaining was restricted to the surface epithelium in post-treatment biopsies. In addition, the activity of the disease, as estimated by the Crohn’s Disease Activity Index (r = 0.476; p = 0.014) and C-reactive protein (r = 0.411; p = 0.039), was positively correlated with the expression of claudin-1 in the mucosa. Conclusion Active CD is plagued with a disruption of the expression and distribution of claudin-1 and occludin throughout the affected intestinal mucosa. Successful treatment with biological agents induces the restoration of intestinal barrier function, which is associated with an improvement in the patients' clinical condition.

P098 Novel drug candidate for the treatment of Inflammatory Bowel Disease with unique mechanisms of action

Abstract Background Inflammatory Bowel Disease (IBD) is characterized by chronic inflammation and severe dysfunction of the gastrointestinal tract. Despite significant improvements in therapeutic agents targeting IBD, many subjects with IBD fail to achieve recovery or do not respond to current medication. Moreover, current therapy has been limited to the management of inflammation, while almost no progress has been made in the development of anti-fibrotic therapies for IBD. NEXEL’s novel protein NP-011, targeting αVβ3 and αVβ5 integrins and phosphatidylserine, has shown anti-inflammatory and anti-fibrotic effects in various in vivo models with fibrosis. This study was designed to evaluate the therapeutic potential of NP-011 for the treatment of IBD using mice models. Methods Acute ulcerative colitis was induced by exposure to 2% dextran sodium sulfate (DSS)-treated drinking water from Day 1 to Day 7; Normal control animals did not receive DSS. Animals were then dosed with vehicle (NP-011 storage buffer, n=10), comparative protein, MFG-E8 (n=10), or NP-011 (n=10) daily for five days (IV) from Day 7 to Day 11. All animals were sacrificed on Day 14. The chronic colitis model was induced by exposure to 2% DSS-treated drinking water (initial three consecutive days of each week) and recovered by providing normal water (four days each from Day 3 and Day 10). Animals were dosed with the vehicle, three commercial drugs (n=10, 1.03 mg/kg, on Day 3), and NP-011 (n=10, 320, and 640 ug/kg, on Day 3 and Day 10) by intravenous injection. All animals were sacrificed on Day 18. Colon length and histological changes (inflammation and crypt damage) were analyzed. Results The induction of colitis was characterized by weight loss. Treatment with comparative protein or NP-011 improved body weight gain during recovery. Both proteins suppressed the shortening of the colon and reduced histological score, including intestinal inflammation and crypt damage. Notably, the administration of NP-011 showed higher efficiency than the same dose of the comparative protein in improving these pathologic parameters. NP-011 was also effective in the chronic model of colitis. Colon shortening, inflammation reactions, and crypt damage were markedly attenuated in NP-011-administered mice with comparable efficacy to commercial drugs. Conclusion Treatment with NP-011 demonstrated substantial improvement in histological characteristics of bowel pathology and suppression of colon shortening in the IBD mice model. Taken together, this novel protein, NP-011, could be a promising drug candidate for the treatment of IBD with unique mechanisms of action, including anti-inflammation and anti-fibrosis *JL, GK, and JCK have equally contributed to this study.

P043 ALI culture derived from human organoids recapitulates cell composition during intestinal crypt formation

Abstract Background The ALI (air liquid-interface) culture derived from human primary intestinal epithelium is generating considerable interest for closely reproducing the mature cell features of the differentiated epithelium in a 2D configuration. However, the phenotypic changes occurring in the human ALI culture during cell differentiation have thus far not been explored in depth. Here, we aimed to characterize the cell composition of human ALI culture over time. Methods 3D epithelial organoids (n=3) were expanded from human colonic samples and organoid-derived single cells were seeded on a Transwell. After reaching confluence, the submerged 2D was switched to ALI and maintained for 7 days. The ALI culture was analysed every 24h by qPCR, membrane staining (H&amp;E and immunofluorescence) and trans-epithelial resistance (TER) (Figure 1). Results The confluent pre-ALI (TER&amp;gt;200 Ω*cm2) showed high mRNA expression of stem cell markers (i.e., LGR5, AXIN2), while several differentiation markers (i.e., ZG16, CLCA1, ANPEP, AQP8, BEST4, CHGA) were almost undetectable, thus resembling the stem cell niche compartment. Air exposure of the 2D culture induced an early and transient (limited to ALI-day 1) transcriptional up-regulation of proliferation markers (i.e., MKI67, CDCA7) and OLFM4, followed by a concomitant marked downregulation of stem cell markers, thus mimicking the Transit Amplifying compartment. OLFM4 protein, as observed by immunofluorescence, showed an early cytoplasmic accumulation followed by secretion at later time points (from ALI-day 5). Differentiation towards absorptive and secretory lineages increased gradually from ALI-day 5 and ALI-day 7 onward, respectively, at both the transcriptional and protein level. Expression changes were accompanied by the progressive transition of the ALI monolayer from a squamous to a columnar morphology. In parallel, TER promptly increased upon ALI switch, thus indicating the early establishment of cell-cell junctions. Conclusion By recapitulating the cell composition identified along the crypt, the ALI culture holds promise to be a versatile in vitro model for studying how the different epithelial cell compartments respond to specific environmental stimuli, like those present during an inflammatory flare.

Effects of feeding methods on growth and slaughter performance, blood biochemical indices, and intestinal morphology in Minxinan black rabbits (Oryctolagus cuniculus)

Feed restriction after weaning is a common strategy used in commercial rabbit farms to improve feed efficiency, promote health, and reduce mortality. However, few studies have investigated the feed restrictions of Minxinan black rabbits (Oryctolagus cuniculus). Thus, the effects of feed restriction on growth and slaughter performance, intestinal morphology, and blood biochemical indices of Minxinan black rabbits were evaluated in this study. Rabbits in group A (control group) had ad libitum intake, while those in feed restriction groups (groups B, C, and D) were restricted to 80% of the average daily feed intake (ADFI) of group A the day before. The rabbits in group B were fed once per day at 8:00 am. Rabbits in groups C and D were fed twice per day at 8:00 am (50%) and 4:00 pm (50%) and 8:00 am (30%) and 4:00 pm (70%), respectively. The experimental period lasted for 8 weeks. Compared to that in group A, the diarrhea rate of group C was significantly decreased (P < 0.05), and the ADFI, feed conversion ratio, abdominal fat weight, abdominal fat rate, total protein, albumin, globulin, alanine aminotransferase (ALT), low-density lipoprotein, and intestinal crypt depth of all feed restriction groups were significantly reduced (P < 0.01). Feed conversion ratio in group D was significantly better than that in groups B and C (P < 0.05). The efficiency index (EI) of groups C and D was higher than that of groups A and B (P < 0.01). Triglyceride levels in groups C and D were significantly lower than those in group A. The villus length to crypt depth of the duodenum and jejunum in group D was significantly higher than that in group A (P < 0.01). In conclusion, the following parameters can be improved by feed restriction: feed conversion ratio, diarrhea rate, abdominal fat rate, serum ALT, lipid indices and intestinal health of Minxinan black rabbits, and the EI of the farm. Feeding twice per day, 30% at 8:00 am and 70% at 4:00 pm, had the best comprehensive effects.

EXCESS DIETARY SUGAR ALTERS COLONOCYTE METABOLISM AND IMPAIRS THE EARLY PROLIFERATIVE RESPONSE TO DAMAGE

Abstract The colonic epithelium requires continuous renewal by crypt resident intestinal stem cells (ISCs) and transit amplifying (TA) cells to maintain barrier integrity, especially after inflammatory damage. An important regulator of ISC and TA cell function is dietary metabolites which can affect their proliferative capacity. Over the last 150 years, the diet of high-income countries contains increasing amounts of simple sugars, such as sucrose, but whether excess sugar affects the function of ISCs and TA cells directly is unknown. We used a combination of 3-dimensional colonoids and a mouse model of colon damage/repair (DSS colitis) to demonstrate the direct effect of sugar on the transcriptional, metabolic, and regenerative functions of crypt ISCs and TA cells. We demonstrate that high sugar conditions directly limit murine and human colonoid development, which is associated with a reduction in the expression of proliferative and key stem cell gene signatures. Further, high-glucose conditions led to the accumulation of glycolytic metabolite pyruvate in colonoids, with a concomitant decrease in ATP, suggesting impaired glycolytic fuel metabolism. Treatment of colonoids with DCA, which forces pyruvate into the TCA cycle, restored their growth, ATP levels, and the expression of proliferation and stem cell gene signatures. Similarly, DSS treatment of mice fed a high sugar diet led to massive irreparable damage that was independent of the colonic microbiota and its metabolites. Metabolic analyses of crypt cells from high-sucrose-fed mice revealed increased glycolytic potential without a commensurate increase in aerobic respiration. Rather, epithelium from high-sucrose-fed mice has increased mitochondrial content, without increased levels of ATP, further demonstrating impaired oxidative phosphorylation and fuel metabolism. Finally, we demonstrated impaired proliferative potential of ISCs and reduced number of TA direct daughter cells in high-sucrose-fed mice with DSS damage. Taken together, our results indicate that short-term, excess dietary sucrose can directly modulate intestinal crypt cell metabolism and inhibit ISC/TA cell regenerative proliferation. This knowledge may inform diets that better support the treatment of acute intestinal injury, as is seen in patients with an acute flare of Ulcerative Colitis.