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The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggered an unprecedented public health crisis known as the coronavirus disease 2019 (COVID-19) pandemic. Gastrointestinal (GI) symptoms develop in patients during acute infection and persist after recovery from airway distress in a chronic form of the disease (long COVID). A high incidence of irritable bowel syndrome (IBS) manifested by severe abdominal pain and defecation pattern changes is reported in COVID patients. Although COVID is primarily considered a respiratory disease, fecal shedding of SARS-CoV-2 antigens positively correlates with bowel symptoms. Active viral infection in the GI tract was identified by human intestinal organoid studies showing SARS-CoV-2 replication in gut epithelial cells. In this review, we highlight the key findings in post-COVID bowel symptoms and explore possible mechanisms underlying the pathophysiology of the illness. These mechanisms include mucosal inflammation, gut barrier dysfunction, and microbiota dysbiosis during viral infection. Viral shedding through the GI route may be the primary factor causing the alteration of the microbiome ecosystem, particularly the virome. Recent evidence in experimental models suggested that microbiome dysbiosis could be further aggravated by epithelial barrier damage and immune activation. Moreover, altered microbiota composition has been associated with dysregulated serotonin pathways, resulting in intestinal nerve hypersensitivity. These mechanisms may explain the development of post-infectious IBS-like symptoms in long COVID. Understanding how coronavirus infection affects gut pathophysiology, including microbiome changes, would benefit the therapeutic advancement for managing post-infectious bowel symptoms., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

Background: Superior mesenteric ischemia/reperfusion (I/R) causes barrier dysfunction and facilitates bacterial translocation (BT) in the small intestine, which can even lead to systemic sepsis. Our previous research showed that luminal administration of glucose and its anaerobic glycolytic metabolites exerted cytoprotective effects on epithelial cells and ameliorated I/R-induced BT in the liver and spleen. Notably, the reduction of BT occurs over the whole intestinal tract, not only restricted in the ligated glucose-containing loop., Objectives: In this study, we hypothesized that local jejunal glucose-contacting might confer on the remote intestinal epithelium regeneration potential, fortify their barrier function and goblet cell secretory activity., Methods: Two 10-cm jejunal segments were isolated in Wistar rats. One segment was ligatured at both ends and infused with Krebs buffer containing 0- or 50-mM glucose (local loop), whereas the adjacent segment was left unaltered and not exposed to glucose (remote loop). The rats then underwent either a sham operation or I/R challenge by occlusion of the superior mesenteric artery for 20 min, followed by reperfusion for 1 h., Results: Enteral addition of glucose in the local jejunum loop alleviated ischemia-induced barrier defects, histopathological scores, cell death, and mucosal inflammation (myeloperoxidase and inflammatory cytokine production) in the remote jejunum. After ischemia, goblet cells in the remote jejunum showed cavitation of mucin granules and low MUC2 expression. Local addition of glucose enhanced MUC2 synthesis and stimulated a jet-like mucus secretion in the remote jejunum, which was accompanied by the restoration of crypt activity., Conclusions: Our results showed local enteral glucose effectively mitigates I/R-induced barrier dysfunction, suggesting that local glucose-stimulated mucus secretion by remote goblet cells may serve to mitigate mucosal inflammation and BT. We provide a more precise barrier protection role of enteral glucose upon I/R challenge, presenting new opportunities for future therapeutic potential., (© 2024 The Author(s).)

Objectives: Aberrant serotonin (5-hydroxytryptamine, 5-HT) metabolism and neurite outgrowth were associated with abdominal pain in irritable bowel syndrome (IBS). We previously demonstrated that 5-HT receptor subtype 7 (5-HT₇) was involved in visceral hypersensitivity of IBS-like mouse models. The aim was to compare the analgesic effects of a novel 5-HT₇ antagonist to reference standards in mouse models and investigate the mechanisms of 5-HT₇-dependent neuroplasticity., Methods: Two mouse models, including Giardia post-infection combined with water avoidance stress (GW) and post-resolution of trinitrobenzene sulfonic acid-induced colitis (PT) were used. Mice were orally administered CYY1005 (CYY, a novel 5-HT₇ antagonist), alosetron (ALN, a 5-HT₃ antagonist), and loperamide (LPM, an opioid receptor agonist) prior to measurement of visceromotor responses (VMR). Levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin receptors (NTRs) were assessed., Results: Peroral CYY was more potent than ALN or LPM in reducing VMR values in GW and PT mice. Increased mucosal 5-HT₇-expressing nerve fibers were associated with elevated Gap43 levels in the mouse colon. We observed higher colonic Ntrk2 and Ngfr expression in GW mice, and increased Bdnf expression in PT mice compared with control mice. Human SH-SY5Y cells stimulated with mouse colonic supernatant or exogenous serotonin exhibited longer nerve fibers, which CYY dose-dependently inhibited. Serotonin increased Ntrk1 and Ngfr expression via 5-HT₇ but not 5-HT₃ or 5-HT₄, while Ntrk2 upregulation was dependent on all three 5-HT receptor subtypes., Conclusions: Stronger analgesic effects by peroral CYY were observed compared with reference standards in two IBS-like mouse models. The 5-HT₇-dependent NTR upregulation and neurite elongation may be involved in intestinal hypernociception., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Background: Factors that contribute to inflammatory bowel disease [IBD] pathogenesis include genetic polymorphisms, barrier loss, and microbial dysbiosis. A major knowledge gap exists in the origins of the colitogenic microbiome and its relationship with barrier impairment. Epithelial myosin light chain kinase [MLCK] is a critical regulator of the paracellular barrier, but the effects of MLCK activation on the intraepithelial bacteria [IEB] and dysbiosis are incompletely understood. We hypothesise that MLCK-dependent bacterial endocytosis promotes pathobiont conversion and shapes a colitogenic microbiome., Methods: To explore this, transgenic [Tg] mice with barrier loss induced by intestinal epithelium-specific expression of a constitutively active MLCK were compared with wild-type [WT] mice., Results: When progeny of homozygous MLCK-Tg mice were separated after weaning by genotype [Tg/Tg, Tg/WT, WT/WT], increased IEB numbers associated with dysbiosis and more severe colitis were present in Tg/Tg and Tg/WT mice, relative to WT/WT mice. Cohousing with MLCK-Tg mice induced dysbiosis, increased IEB abundance, and exacerbated colitis in WT mice. Conversely, MLCK-Tg mice colonised with WT microbiota at birth displayed increased Escherichia abundance and greater colitis severity by 6 weeks of age. Microarray analysis revealed circadian rhythm disruption in WT mice co-housed with MLCK-Tg mice relative to WT mice housed only with WT mice. This circadian disruption required Rac1/STAT3-dependent microbial invasion but not MLCK activity, and resulted in increased proinflammatory cytokines and glucocorticoid downregulation., Conclusions: The data demonstrate that barrier dysfunction induces dysbiosis and expansion of invasive microbes that lead to circadian disruption and mucosal inflammation. These results suggest that barrier-protective or bacterium-targeted precision medicine approaches may be of benefit to IBD patients., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

Irinotecan ( 1 ), a prodrug of SN38 ( 2 ) approved by the US Food and Drug Administration for treating colorectal cancer, lacks specificity and causes many side effects. To increase the selectivity and therapeutic efficacy of this drug, we designed and synthesized conjugates of SN38 and glucose transporter inhibitors (phlorizin ( 5 ) or phloretin ( 6 )), which could be hydrolyzed by glutathione or cathepsin to release SN38 in the tumor microenvironment, as a proof of concept. These conjugates ( 8 , 9 , and 10 ) displayed better antitumor efficacy with lower systemic exposure to SN38 in an orthotopic colorectal cancer mouse model compared with irinotecan at the same dosage. Further, no major adverse effects of the conjugates were observed during treatment. Biodistribution studies showed that conjugate 10 could induce higher concentrations of free SN38 in tumor tissues than irinotecan at the same dosage. Thus, the developed conjugates exhibit potential for treating colorectal cancer.

Irritable bowel syndrome (IBS) is characterized by visceral hypersensitivity (VH) associated with abnormal serotonin/5-hydroxytryptamine (5-HT) metabolism and neurotrophin-dependent mucosal neurite outgrowth. The underlying mechanisms of VH remain poorly understood. We investigated the role of 5-HT 7 receptor in mucosal innervation and intestinal hyperalgesia. A high density of mucosal nerve fibres stained for 5-HT 7 was observed in colonoscopic biopsy specimens from IBS patients compared with those from healthy controls. Staining of 5-HT 3 and 5-HT 4 receptors was observed mainly in colonic epithelia with comparable levels between IBS and controls. Visceromotor responses to colorectal distension were evaluated in two mouse models, one postinfectious with Giardia and subjected to water avoidance stress (GW) and the other postinflammatory with trinitrobenzene sulfonic acid-induced colitis (PT). Increased VH was associated with higher mucosal density of 5-HT 7 -expressing nerve fibres and elevated neurotrophin and neurotrophin receptor levels in the GW and PT mice. The increased VH was inhibited by intraperitoneal injection of SB-269970 (a selective 5-HT 7 antagonist). Peroral multiple doses of CYY1005 (a novel 5-HT 7 ligand) decreased VH and reduced mucosal density of 5-HT 7 -expressing nerve fibres in mouse colon. Human neuroblastoma SH-SY5Y cells incubated with bacteria-free mouse colonic supernatant, 5-HT, nerve growth factor, or brain-derived neurotrophic factor exhibited nerve fibre elongation, which was inhibited by 5-HT 7 antagonists. Gene silencing of HTR7 also reduced the nerve fibre length. Activation of 5-HT 7 upregulated NGF and BDNF gene expression, while stimulation with neurotrophins increased the levels of tryptophan hydroxylase 2 and 5-HT 7 in neurons. A positive-feedback loop was observed between serotonin and neurotrophin pathways via 5-HT 7 activation to aggravate fibre elongation, whereby 5-HT 3 and 5-HT 4 had no roles. In conclusion, 5-HT 7 -dependent mucosal neurite outgrowth contributed to VH. A novel 5-HT 7 antagonist could be used as peroral analgesics for IBS-related pain., (© 2022. The Author(s).)

Powdery mildew was found on Aristolochia debilis ( Aristolochiaceae ) in Jiangsu Province and Shandong Province, China. This fungus is characterized by having long conidiophore foot-cells which are straight or curved at the base, and chasmothecia with numerous appendages. Phylogenetic analysis using internal transcribed spacer sequences showed that five sequences on A. debilis determined in this study and two sequences retrieved from Erysiphe sp. on A. debilis formed an independent cluster within the Erysiphe aquilegiae clade with 58% bootstrap support. This powdery mildew differs from allied species of the E. aquilegiae clade in producing longer conidia and conidiophores with longer foot-cells, which are often curved at the base. Morphological observations and molecular phylogenetic analysis revealed a new powdery mildew species, described as Erysiphe ruyongzhengiana ., (2022, by The Mycological Society of Japan.)

Many cities advocate retrofitting green roofs and green walls (GRGW) to create additional green areas, especially in cramped urban areas. Yet, worldwide, only a handful of studies have evaluated the public views towards the benefits and negative issues and promotion policies of this innovative greening option. To address this gap in the literature, we conducted a survey (N = 500) of residents' opinions towards GRGW in Tokyo, a city with mandatory installation of GRGW for almost two decades. Respondents mostly agreed with the contribution of GRGW to thermal comfort, air quality, and cityscape but weakly endorsed other potential benefits. High costs as well as mosquitoes and plant litter nuisances were the most recognized negative issues. Mandatory installation was the least preferred promotion policy. Instead, respondents expected installation on public buildings and provision of installation guidance. Respondents predominantly held a "moderate" view towards both the benefits and negative issues, showing indifferent attitudes towards GRGW. Income level and housing type shaped the overall perceptions, whereas age, sex, and current living environment influenced perceptions of individual aspects. Our findings signified a need for a bottom-up strategy to heighten public awareness for the advanced development of GRGW to complement and prime the top-down mandatory installation policy., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background & Aims: Microbiota dysbiosis and mucosa-associated bacteria are involved in colorectal cancer progression. We hypothesize that an interaction between virulent pathobionts and epithelial defense promotes tumorigenesis., Methods: Chemical-induced CRC mouse model was treated with antibiotics at various phases. Colonic tissues and fecal samples were collected in a time-serial mode and analyzed by gene microarray and 16S rRNA sequencing. Intraepithelial bacteria were isolated using a gentamicin resistance assay, and challenged in epithelial cultures., Results: Our study showed that antibiotic treatment at midphase but not early or late phase reduced mouse tumor burden, suggesting a time-specific host-microbe interplay. A unique antimicrobial transcriptome profile showing an inverse relationship between autophagy and oxidative stress genes was correlated with a transient surge in microbial diversity and virulence emergence in mouse stool during cancer initiation. Gavage with fimA/fimH/htrA-expressing invasive Escherichia coli isolated from colonocytes increased tumor burden in recipient mice, whereas inoculation of bacteria deleted of htrA or triple genes did not. The invasive E.coli suppressed epithelial autophagy activity through reduction of microtubule-associated protein 1 light-chain 3 transcripts and caused dual oxidase 2-dependent free radical overproduction and tumor cell hyperproliferation. A novel alternating spheroid culture model was developed for sequential bacterial challenge to address the long-term changes in host-microbe interaction for chronic tumor growth. Epithelial cells with single bacterial encounter showed a reduction in transcript levels of autophagy genes while those sequentially challenged with invasive E.coli showed heightened autophagy gene expression to eliminate intracellular microbes, implicating that bacteria-dependent cell hyperproliferation could be terminated at late phases. Finally, the presence of bacterial htrA and altered antimicrobial gene expression were observed in human colorectal cancer specimens., Conclusions: Invasive pathobionts contribute to cancer initiation during a key time frame by counterbalancing autophagy and oxidative stress in the colonic epithelium. Monitoring gut microbiota and antimicrobial patterns may help identify the window of opportunity for intervention with bacterium-targeted precision medicine., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Altered glucose metabolism is associated with chemoresistance in colorectal cancer (CRC). This study aimed to illustrate the molecular mechanisms of glucose-mediated chemoresistance against irinotecan, a topoisomerase I inhibitor, focusing on the distinct roles of metabolites such as pyruvate and ATP in modulating cell death and proliferation., Methods: Four human CRC cell lines, tumorspheres, and mouse xenograft models were treated with various doses of irinotecan in the presence of various concentrations of glucose, pyruvate, or ATP-encapsulated liposomes., Results: In this study, human CRC cell lines treated with irinotecan in high glucose displayed increased cell viability and larger xenograft tumor sizes in mouse models compared to those treated in normal glucose concentrations. Irinotecan induced apoptosis and necroptosis, both mitigated by high glucose. Liposomal ATP prevented irinotecan-induced apoptosis, while it did not affect necroptosis. In contrast, pyruvate attenuated the receptor-interacting protein kinase 1/3-dependent necroptosis via free radical scavenging without modulating apoptotic levels. Regarding the cell cycle, liposomal ATP aggravated the irinotecan-induced G0/G1 shift, whereas pyruvate diminished the G0/G1 shift, showing opposite effects on proliferation. Last, tumorsphere structural damage, an index of solid tumor responsiveness to chemotherapy, was determined. Liposomal ATP increased tumorsphere size while pyruvate prevented the deformation of spheroid mass., Conclusions: Glucose metabolites confer tumor chemoresistance via multiple modes of action. Glycolytic pyruvate attenuated irinotecan-induced necroptosis and potentiated drug insensitivity by shifting cells from a proliferative to a quiescent state. On the other hand, ATP decreased irinotecan-induced apoptosis and promoted active cell proliferation, contributing to tumor recurrence. Our findings challenged the traditional view of ATP as the main factor for irinotecan chemoresistance and provided novel insights of pyruvate acting as an antioxidant responsible for drug insensitivity, which may shed light on the development of new therapies against recalcitrant cancers., (© 2022 S. Karger AG, Basel.)

Myosin light chain kinase (MLCK) regulates actinomyosin contraction. Two splice variants of long MLCK are expressed in epithelial cells and divergently regulate gut barrier functions; reduced MLCK levels in human colorectal cancers (CRC) with unclarified significance have been reported. CRC are solid tumors clonally sustained by stem cells highly expressing CD44 and CD133. The aim was to investigate the role of MLCK splice variants in CRC tumorigenesis. We found lower MLCK1/2 and higher CD44 expression in human CRC, but no change in CD133 or LGR5. Large-scale bioinformatics showed an inverse relationship between MYLK and CD44 in human sample gene datasets. A 3-fold increased tumor burden was observed in MLCK(-/-) mice compared with wild-type (WT) mice in a chemical-induced CRC model. Primary tumorspheres derived from the MLCK(-/-) mice displayed larger sizes and higher CD44 transcript levels than those from the WT mice. Bioinformatics revealed binding of TEAD4 (a transcriptional enhancer factor family member in the Hippo pathway) to CD44 promoter, which was confirmed by luciferase reporter assay. Individually expressing MLCK1 and MLCK2 variants in the MLCK-knockout (KO) Caco-2 cells inhibited the nuclear localization of TEAD4 cofactors, VGLL3 and YAP1, respectively, and both variants reduced the CD44 transcription. Accelerated cell cycle transit was observed in the MLCK-KO cells, whereby expression of MLCK1/2 variants counterbalanced the cell hyperproliferation. In conclusion, MLCK1/2 variants are novel tumor suppressors by downregulating the TEAD4/CD44 axis via reducing nuclear translocation of distinct transcriptional coactivators. The reduction of epithelial MLCKs, especially isoform 2, may drive cancer stemness and tumorigenesis., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Overexpression of glucose transporters (GLUTs) in colorectal cancer cells is associated with 5-fluorouracil ( 1 , 5-FU) resistance and poor clinical outcomes. We designed and synthesized a novel GLUT-targeting drug conjugate, triggered by glutathione in the tumor microenvironment, that releases 5-FU and GLUTs inhibitor (phlorizin ( 2 ) and phloretin ( 3 )). Using an orthotopic colorectal cancer mice model, we showed that the conjugate exhibited better antitumor efficacy than 5-FU, with much lower exposure of 5-FU during treatment and without significant side effects. Our study establishes a GLUT-targeting theranostic incorporating a disulfide linker between the targeting module and cytotoxic payload as a potential antitumor therapy.

Both protein kinase C (PKC) and reactive oxygen species (ROS) are well-known signaling messengers cross-talking with each other to activate mitogen-activated protein kinases (MAPKs) for progression of hepatocellular carcinoma (HCC). However, the underlying mechanisms are not well elucidated. Especially, whether mitochondrial ROS (mtROS) is involved and how it triggers MAPK signaling are intriguing. In this study, we found mtROS generation and phosphorylation of MAPKs were mediated by PKCδ in HCCs treated with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Heat shock protein 60 (HSP60), one of the chaperones in mitochondria was the major protein oxidized in TPA-treated HCCs. Moreover, depletion of HSP60 or expression of HSP60 cysteine mutant prevented TPA-induced phosphorylation of MAPKs. To delineate how HSP60 mediated MAPK activation, the role of Raf kinase inhibitor protein (RKIP), a negative regulator of MAPK, was investigated. TPA dissociated RKIP from HSP60 in both mitochondria and cytosol, concurrently with translocation of HSP60 and MAPK from mitochondria to cytosol, which was associated with robust phosphorylation of MAPKs in the cytosol. Moreover, TPA induced opposite phenotypical changes of HCCs, G1 cell cycle arrest, and cell migration, which were prevented by mtROS scavengers and depletion of PKCδ and HSP60. Consistently, TPA increased the migration-related genes, hydrogen peroxide inducible clone5, matrix metalloproteinase-1/3, lamininγ2, and suppressed the cell cycle regulator cyclin E1 (CCNE1) via PKCδ/mtROS/HSP60/MAPK-axis. Finally, c-jun and c-fos were required for TPA-induced expression of the migration-related genes and a novel microRNA, miR-6134, was responsible for TPA-induced suppression of CCNE1. In conclusion, PKCδ cross-talked with mtROS to trigger HSP60 oxidation for release of RKIP to activate MAPK, regulating gene expression for migration, and G1 cell cycle arrest in HCC. Targeted therapy aiming at key players like PKCδ, RKIP, and HSP60 is promising for preventing HCC progression., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Inflammatory bowel disease (IBD) is characterized by abnormal host-microbe interactions. Proinflammatory cytokine IFNγ and a novel TNF superfamily member, TL1A, have been implicated in epithelial barrier dysfunction. The divergent regulatory mechanisms of transcellular versus paracellular hyperpermeability remain poorly understood. Intestinal epithelia express two splice variants of long myosin light chain kinase (MLCK), of which the full-length MLCK1 differ from the shorter isoform MLCK2 by a Src kinase phosphorylation site. The aim was to investigate the roles of MLCK splice variants in gut barrier defects under proinflammatory stress. Upregulated expression of TL1A, IFNγ, and two MLCK variants was observed in human IBD biopsy specimens. The presence of intraepithelial bacteria preceded tight junction (TJ) damage in dextran sodium sulfate-treated and TL1A-transgenic mouse models. Lack of barrier defects was observed in long MLCK(-/-) mice. TL1A induced MLCK-dependent terminal web (TW) contraction, brush border fanning, and transepithelial bacterial internalization. The bacterial taxa identified in the inflamed colonocytes included Escherichia, Enterococcus, Staphylococcus,and Lactobacillus. Recombinant TL1A and IFNγ at low doses induced PI3K/Akt/MLCK2-dependent bacterial endocytosis, whereas high-dose IFNγ caused TJ opening via the iNOS/Src/MLCK1 axis. Bacterial internalization was recapitulated in MLCK-knockout cells individually expressing MLCK2 but not MLCK1. Immunostaining showed different subcellular sites of phosphorylated MLC localized to the TJ and TW in the MLCK1- and MLCK2-expressing cells, respectively. In conclusion, proinflammatory cytokines induced bacterial influx through transcellular and paracellular routes via divergent pathways orchestrated by distinct MLCK isoforms. Bacterial transcytosis induced by TL1A may be an alternative route causing symptom flares in IBD., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Chronic inflammation is a major risk factor for colitis-associated colorectal carcinoma (CRC). Macrophages play a key role in altering the tumor microenvironment by producing pro-inflammatory and anti-inflammatory cytokines. Our previous studies showed that glucose metabolism conferred death resistance for tumor progression and exerted anti-inflammatory effects in ischemic gut mucosa. However, the effect of glucose and cancer metabolites in modulating macrophage cytokine profiles remains poorly defined. We used an in vitro system to mimic intestinal microenvironment and to investigate the roles of glucose and cancer metabolites in the cross-talk between carcinoma cells and macrophages. Human monocyte-derived THP-1 macrophages were stimulated with bacterial lipopolysaccharide (LPS) in the presence of conditioned media (CM) collected from human CRC Caco-2 cells incubated in either glucose-free or glucose-containing media. Our results demonstrated that glucose modulated the macrophage cytokine production, including decreased LPS-induced pro-inflammatory cytokines (i.e., tumor necrosis factor [TNF]α and interleukin [IL]-6) and increased anti-inflammatory cytokine (i.e., IL-10), at resting state. Moreover, glucose-containing CM reduced the macrophage secretion of TNFα and IL-8 but elevated the IL-12 and IL-23 levels, showing an opposite pattern of distinct pro-inflammatory cytokines modulated by cancer glucose metabolites. In contrast, LPS-induced production of macrophage inflammatory protein-1 (a macrophage-derived chemoattractant for granulocytes) was not altered by glucose or CM, indicating that resident macrophages may play a more dominant role than infiltrating granulocytes for responding to cancer metabolites. In conclusion, glucose metabolites from CRC triggered distinct changes in the cytokine profiles in macrophages. The downregulation of death-inducing TNFα and upregulation of Th1/17-polarizing IL-12/IL-23 axis in macrophages caused by exposure to cancer-derived glucose metabolites may contribute to tumor progression., Competing Interests: None

Reprogrammed glucose metabolism and increased glycolysis have been implicated in tumor chemoresistance. The aim was to investigate the distinct roles of the glucose metabolites pyruvate and ATP in chemoresistance mechanisms, including cell death and proliferation. Our data showed higher glucose transporters in colorectal cancer (CRC) from non-responsive patients than those responsive to chemotherapy. Human CRC cell lines exposed to 5-fluorouracil (5-FU) displayed elevated cell viability and larger tumors in xenograft mouse models if cultured in high-glucose medium. Glucose conferred resistance to 5-FU-induced necroptosis via pyruvate scavenging of mitochondrial free radicals, whereas ATP replenishment had no effect on cell death. Glucose attenuated the 5-FU-induced G0/G1 shift but not the S phase arrest. Opposing effects were observed by glucose metabolites; ATP increased while pyruvate decreased the G0/G1 shift. Lastly, 5-FU-induced tumor spheroid destruction was prevented by glucose and pyruvate, but not by ATP. Our finding argues against ATP as the main effector for glucose-mediated chemoresistance and supports a key role of glycolytic pyruvate as an antioxidant for dual modes of action: necroptosis reduction and a cell cycle shift to a quiescent state., (Copyright © 2019 Huang, Huang, Pai, Lin, Lee, Liang and Yu.)

Commensal microbes are involved in intestinal homeostasis, and the dysregulation of host-microbe interactions may lead to the development of local and systemic disorders. Recent evidence indicated that microbiota dysbiosis plays a key role in the pathogenesis of inflammatory bowel disease and metabolism-related disorders. The circadian clock system originally identified in the brain was later found in the gastrointestinal tract. Although the light-controlled central clock in the brain is responsible for the synchronization of peripheral clocks, the timing of meal consumption serves as another cue for the rhythmic setting of gastrointestinal digestion, absorption, and epithelial renewal and barrier functions. Multiple lines of evidence have indicated that in addition to daylight and food intake, microbiota (as an environmental factor) are involved in the circadian control of gut homeostasis. Recent studies demonstrated that microbial metabolites and innate signaling orchestrate the host circadian rhythm, revealing unforeseen molecular mechanisms underlying the regulatory role of microbiota in intestinal physiology and systemic metabolism. In this review, we discuss the host-microbe interplay that contributes to the regulation of intestinal clock signals and physiological functions and explore how microbiota dysbiosis may cause misalignment of circadian systems leading to the development of chronic inflammatory and metabolic diseases.

Inflammatory bowel disease (IBD) is a multifactorial disease which arises as a result of the interaction of genetic, environmental, barrier and microbial factors leading to chronic inflammation in the intestine. Patients with IBD had a higher risk of developing colorectal carcinoma (CRC), of which the subset was classified as colitis-associated cancers. Genetic polymorphism of innate immune receptors had long been considered a major risk factor for IBD, and the mutations were also recently observed in CRC. Altered microbial composition (termed microbiota dybiosis) and dysfunctional gut barrier manifested by epithelial hyperpermeability and high amount of mucosa-associated bacteria were observed in IBD and CRC patients. The findings suggested that aberrant immune responses to penetrating commensal microbes may play key roles in fueling disease progression. Accumulative evidence demonstrated that mucosa-associated bacteria harbored colitogenic and protumoral properties in experimental models, supporting an active role of bacteria as pathobionts (commensal-derived opportunistic pathogens). Nevertheless, the host factors involved in bacterial dysbiosis and conversion mechanisms from lumen-dwelling commensals to mucosal pathobionts remain unclear. Based on the observation of gut leakiness in patients and the evidence of epithelial hyperpermeability prior to the onset of mucosal histopathology in colitic animals, it was postulated that the epithelial barrier dysfunction associated with mucosal enrichment of specific bacterial strains may predispose the shift to disease-associated microbiota. The speculation of leaky gut as an initiating factor for microbiota dysbiosis that eventually led to pathological consequences was proposed as the "common ground hypothesis", which will be highlighted in this review. Overall, the understanding of the core interplay between gut microbiota and epithelial barriers at early subclinical phases will shed light to novel therapeutic strategies to manage chronic inflammatory disorders and colitis-associated cancers.

Cadherin-17 is an adhesion molecule expressed specifically in intestinal epithelial cells. It is frequently underexpressed in human colorectal cancer. The physiological function of cadherin-17 and its role in tumourigenesis have not yet been determined. We used the transcription activator-like effector nuclease technique to generate a Cdh17 knockout (KO) mouse model. Intestinal tissues were analysed with histological, immunohistochemical and ultrastructural methods. Colitis was induced by oral administration of dextran sulphate sodium (DSS), and, to study effects on intestinal tumourigenesis, mice were given azoxymethane (AOM) and DSS to induce colitis-associated cancer. Cdh17 KO mice were viable and fertile. The histology of their small and large intestines was similar to that of wild-type mice. The junctional architecture of the intestinal epithelium was preserved. The loss of cadherin-17 resulted in increased permeability and susceptibility to DSS-induced colitis. The AOM/DSS model demonstrated that Cdh17 KO enhanced tumour formation and progression in the intestine. Increased nuclear translocation of Yap1, but not of β-catenin, was identified in the tumours of Cdh17 KO mice. In conclusion, cadherin-17 plays a crucial role in intestinal homeostasis by limiting the permeability of the intestinal epithelium. Cadherin-17 is also a tumour suppressor for intestinal epithelia. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Key Points: Balloon-assisted enteroscopy (BAE) is an emerging standard procedure by utilizing distensible balloons to facilitate deep endoscopy in the small and large intestine. Sporadic cases of bacteraemia were found after BAE. Balloon distension by BAE caused gut tissue hypoxia. The impact of balloon distension-induced hypoxia on intestinal barriers remains unclear. Murine models of BAE by colonic balloon distension showed that short- and long-term hypoxia evoked opposite effects on epithelial tight junctions (TJs). Short-term hypoxia fortified TJ integrity, whereas long-term hypoxia caused damage to barrier function. Our data showed for the first time the molecular mechanisms and signalling pathways of epithelial barrier fortification and TJ reorganization by short-term hypoxia for the maintenance of gut homeostasis. The findings suggest avoiding prolonged balloon distension during BAE to reduce the risk of hypoxia-induced gut barrier dysfunction., Abstract: Balloon-assisted enteroscopy (BAE) is an emerging standard procedure that uses distensible balloons to facilitate deep endoscopy. Intestines are known to harbour an abundant microflora. Whether balloon distension causes perturbation of blood flow and gut barrier dysfunction, and elicits risk of bacterial translocation remains unknown. Our aims were to (1) conduct a prospective study to gather microbiological and molecular evidence of bacterial translocation by BAE in patients, (2) establish a murine model of colonic balloon distension to investigate tissue hypoxia and intestinal barrier, and (3) assess the effect of short- and long-term hypoxia on epithelial permeability using cell lines. Thirteen patients were enrolled for BAE procedures, and blood samples were obtained before and after BAE for paired comparison. Four of the 13 patients (30.8%) had positive bacterial DNA in blood after BAE. Post-BAE endotoxaemia was higher than the pre-BAE level. Nevertheless, no clinical symptom of sepsis or fever was reported. To mimic clinical BAE, mice were subjected to colonic balloon distension. Local tissue hypoxia was observed during balloon inflation, and reoxygenation after deflation. A trend of increased gut permeability was seen after long-term distension, whereas a significant reduction of permeability was observed by short-term distension in the proximal colon. Human colonic epithelial Caco-2 cells exposed to hypoxia for 5-20 min exhibited increased tight junctional assembly, while those exposed to longer hypoxia displayed barrier disruption. In conclusion, sporadic cases of bacteraemia were found after BAE, without septic symptoms. Short-term hypoxia by balloon distension yielded a protective effect whereas long-term hypoxia caused damage to the gut barrier., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

A role of gut microbiota in colorectal cancer (CRC) growth was first suggested in germ-free rats almost 50 years ago, and the existence of disease-associated bacteria (termed pathobionts) had becoming increasingly evident from experimental data of fecal transplantation, and microbial gavage or monoassociation. Altered bacterial compositions in fecal and mucosal specimens were observed in CRC patients compared to healthy subjects. Microbial fluctuations were found at various cancer stages; an increase of bacterial diversity was noted in the adenoma specimens, while a reduction of bacterial richness was documented in CRC samples. The bacterial species enriched in the human cancerous tissues included Escherichia coli , Fusobacterium nucleatum , and enterotoxigenic Bacteroides fragilis . The causal relationship of gut bacteria in tumorigenesis was established by introducing particular bacterial strains in in situ mouse CRC models. Detailed experimental protocols of bacterial gavage and the advantages and caveats of different experimental models are summarized in this review. The microbial genotoxins, enterotoxins, and virulence factors implicated in the mechanisms of bacteria-driven tumorigenesis are described. In conclusion, intestinal microbiota is involved in colon tumorigenesis. Bacteria-targeting intervention would be the next challenge for CRC., Competing Interests: CONFLICT OF INTEREST: No potential conflict of interest relevant to this article was reported.

Single-layered intestinal epithelia play key roles in the maintenance of gut homeostasis and barrier integrity. Various types of epithelial cell death, including apoptosis, necrosis, and necroptosis, have been detected in ischemic and hypoxic stress conditions, thus resulting in bacterial translocation and gut-derived septic complications. Cytoprotective strategies, such as enteral glucose uptake, rescue intestinal epithelium from cell death after ischemic and hypoxic injury. Although glucose metabolism and energy production are generally considered to be the key factors in cytoprotection, the precise modes and sites of action have not been clarified. Our recent studies have demonstrated that energy restoration promotes crypt hyperplasia but does not prevent epithelial cell death under ischemic stress. On the other hand, glycolytic pyruvate prevents epithelial cells from undergoing apoptosis and necroptosis by scavenging free radicals in an ATP-independent manner. Distinct gut protective mechanisms involving ATP, pyruvate, glucose metabolic enzymes, and sodium-dependent glucose transporter activation are discussed here. Overall, glucose-mediated cytoprotection may be a universal mechanism that has evolved in epithelial cells for the maintenance of intestinal homeostasis. Enteral glucose supplementation is beneficial as a perioperative supportive therapy for the protection of gut barrier integrity.

Background: Intestinal ischemia/reperfusion (I/R) causes barrier impairment and bacterial influx. Protection against I/R injury in sterile organs by hypoxic preconditioning (HPC) had been attributed to erythropoietic and angiogenic responses. Our previous study showed attenuation of intestinal I/R injury by HPC for 21 days in a neutrophil-dependent manner., Aim: To investigate the underlying mechanisms of neutrophil priming by HPC, and explore whether adoptive transfer of primed neutrophils is sufficient to ameliorate intestinal I/R injury., Methods: Rats raised in normoxia (NM) and HPC for 3 or 7 days were subjected to sham operation or superior mesenteric artery occlusion for I/R challenge. Neutrophils isolated from rats raised in NM or HPC for 21 days were intravenously injected into naïve controls prior to I/R., Results: Similar to the protective effect of HPC-21d, I/R-induced mucosal damage was attenuated by HPC-7d but not by HPC-3d. Naïve rats reconstituted with neutrophils of HPC-21d rats showed increase in intestinal phagocytic infiltration and myeloperoxidase activity, and barrier protection against I/R insult. Elevated free radical production, and higher bactericidal and phagocytic activity were observed in HPC neutrophils compared to NM controls. Moreover, increased serum levels of tumor necrosis factor α (TNFα) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were seen in HPC rats. Naïve neutrophils incubated with HPC serum or recombinant TNFα, but not CINC-1, exhibited heightened respiratory burst and bactericidal activity. Lastly, neutrophil priming effect was abolished by neutralization of TNFα in HPC serum., Conclusions: TNFα-primed neutrophils by HPC act as effectors cells for enhancing barrier integrity under gut ischemia.

Background: Endotoxins contribute to systemic inflammatory response and microcirculatory dysfunctions under conditions of sepsis. Polymyxin B hemoperfusion (PMX-HP) is used to remove circulating endotoxins and improve clinical outcomes. This study aims to investigate the effect of PMX-HP on microcirculation in septic pigs., Materials and Methods: By using a septic pig model, we tested the hypothesis that PMX-HP can correct intestinal microcirculation, tissue oxygenation saturation, and histopathologic alterations. A total of 18 male pigs were divided into three groups: (1) sham; (2) sepsis (fecal peritonitis); and (3) sepsis + PMX-HP groups. A sidestream dark field video microscope was used to record microcirculation throughout the terminal ileal mucosa, colon mucosa, kidney surface, and sublingual area. A superficial tissue oxygenation monitor employing the light reflectance spectroscopy technique was used to measure the tissue oxygen saturation. Hematoxylin and eosin staining was used for histologic examination., Results: The perfused small vessel density and tissue oxygen saturation of the ileal mucosa at 6 h were higher in the sepsis + PMX-HP group than those in the sepsis group. The fluid amount and norepinephrine infusion rate between the sepsis group and sepsis + PMX-HP groups did not differ significantly. The histologic score for the ileal mucosa was lower in the sepsis + PMX-HP group than that in the sepsis group. Finally, the urine output was higher in the sepsis + PMX-HP group than it was in the sepsis group., Conclusions: This study demonstrates that PMX-HP attenuates microcirculatory dysfunction, tissue desaturation, and histopathologic alterations in the ileal mucosa in septic pigs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Key Points: Intestinal ischaemia causes epithelial death and crypt dysfunction, leading to barrier defects and gut bacteria-derived septic complications. Enteral glucose protects against ischaemic injury; however, the roles played by glucose metabolites such as pyruvate and ATP on epithelial death and crypt dysfunction remain elusive. A novel form of necrotic death that involves the assembly and phosphorylation of receptor interacting protein kinase 1/3 complex was found in ischaemic enterocytes. Pyruvate suppressed epithelial cell death in an ATP-independent manner and failed to maintain crypt function. Conversely, replenishment of ATP partly restored crypt proliferation but had no effect on epithelial necroptosis in ischaemic gut. Our data argue against the traditional view of ATP as the main cytoprotective factor by glucose metabolism, and indicate a novel anti-necroptotic role of glycolytic pyruvate under ischaemic stress., Abstract: Mesenteric ischaemia/reperfusion induces epithelial death in both forms of apoptosis and necrosis, leading to villus denudation and gut barrier damage. It remains unclear whether programmed cell necrosis [i.e. receptor-interacting protein kinase (RIP)-dependent necroptosis] is involved in ischaemic injury. Previous studies have demonstrated that enteral glucose uptake by sodium-glucose transporter 1 ameliorated ischaemia/reperfusion-induced epithelial injury, partly via anti-apoptotic signalling and maintenance of crypt proliferation. Glucose metabolism is generally assumed to be cytoprotective; however, the roles played by glucose metabolites (e.g. pyruvate and ATP) on epithelial cell death and crypt dysfunction remain elusive. The present study aimed to investigate the cytoprotective effects exerted by distinct glycolytic metabolites in ischaemic gut. Wistar rats subjected to mesenteric ischaemia were enterally instilled glucose, pyruvate or liposomal ATP. The results showed that intestinal ischaemia caused RIP1-dependent epithelial necroptosis and villus destruction accompanied by a reduction in crypt proliferation. Enteral glucose uptake decreased epithelial cell death and increased crypt proliferation, and ameliorated mucosal histological damage. Instillation of cell-permeable pyruvate suppressed epithelial cell death in an ATP-independent manner and improved the villus morphology but failed to maintain crypt function. Conversely, the administration of liposomal ATP partly restored crypt proliferation but did not reduce epithelial necroptosis and histopathological injury. Lastly, glucose and pyruvate attenuated mucosal-to-serosal macromolecular flux and prevented enteric bacterial translocation upon blood reperfusion. In conclusion, glucose metabolites protect against ischaemic injury through distinct modes and sites, including inhibition of epithelial necroptosis by pyruvate and the promotion of crypt proliferation by ATP., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Background and Aim: Irritable bowel syndrome is characterized by abdominal pain and altered bowel habits and may occur following stressful events or infectious gastroenteritis such as giardiasis. Recent findings revealed a link between cholecystokinin (CCK), neurotrophin synthesis, and intestinal hyperalgesia. The aim was to investigate the role of CCK in visceral hypersensitivity using mouse models challenged with a bout of infection with Giardia lamblia or psychological stress, either alone or in combination., Methods: Abdominal pain was evaluated by visceromoter response to colorectal distension. Nerve fibers in intestinal tissues were stained using immunohistochemistry (PGP9.5). Human neuroblastoma SH-SY5Y cells incubated with bacterial-free mouse gut supernatant or recombinant CCK-8S were assessed for neurite outgrowth and nerve growth factor (NGF) production., Results: Intestinal hypersensitivity was induced by either stress or Giardia infection, and a trend of increased pain was seen following dual stimuli. Increased CCK levels and PGP9.5 immunoreactivity were found in colonic mucosa of mice following stress and/or infection. Inhibitors to the CCK-A receptor (L-364718) or CCK-B receptor (L-365260) blocked visceral hypersensitivity caused by stress, but not when induced by giardiasis. Nerve fiber elongation and NGF synthesis were observed in SH-SY5Y cells after incubation with colonic supernatants from mice given the dual stimuli, or after treatment with CCK-8S. Increased nerve fiber length by colonic supernatant and CCK-8S was attenuated by L-365260 or neutralizing anti-NGF., Conclusions: This new model successfully recapitulates intestinal hypernociception induced by stress or Giardia. Colonic CCK contributes to visceral hypersensitivity caused by stress, but not by Giardia, partly via NGF-dependent neurite outgrowth., (© 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Colorectal carcinogenesis is affected by overexpression of the lipopolysaccharide (LPS) receptors CD14 and TLR4, which antagonize each other by affecting epithelial cell proliferation and apoptosis. Eritoran is an investigational drug for sepsis treatment that resembles the lipid A moiety of LPS and therefore acts as a TLR4 inhibitor. In the present study, we explored the potential therapeutic uses and mechanisms of action of eritoran in reducing colon cancer progression. Eritoran administration via intracolonic, intragastric, or intravenous routes significantly reduced tumor burden in a chemically induced mouse model of colorectal carcinoma. Decreased proliferation and increased apoptosis were observed in mouse tumor cells after eritoran treatment. In vitro cultures of mouse primary tumor spheroids and human cancer cell lines displayed increased cell proliferation and cell-cycle progression following LPS challenge. This effect was inhibited by eritoran and by silencing CD14 or TLR4. In contrast, apoptosis induced by eritoran was eliminated by silencing CD14 or protein kinase Cζ (PKCζ) but not TLR4. Lastly, LPS and eritoran caused hyperphosphorylation of PKCζ in a CD14-dependent and TLR4-independent manner. Blocking PKCζ activation by a Src kinase inhibitor and a PKCζ-pseudosubstrate prevented eritoran-induced apoptosis. In summary, our work offers a preclinical proof of concept for the exploration of eritoran as a clinical treatment, with a mechanistic rationale to reposition this drug to improve the management of colorectal cancer. Cancer Res; 76(16); 4684-95. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: Dexmedetomidine reduces cytokine production in septic patients and reduces inflammation and mortality in experimental models of endotoxemia and sepsis. This study investigated whether dexmedetomidine attenuates endothelial dysfunction, intestinal microcirculatory dysfunction, and intestinal epithelial barrier disruption in endotoxemic rats., Methods: Ninety-two male Wistar rats were randomly assigned to the following four groups: (1) Sham; (2) lipopolysaccharide, received IV lipopolysaccharide 15 and 10 mg/kg at 0 and 120 min; (3) dexmedetomidine, received IV dexmedetomidine for 240 min; and (4) lipopolysaccharide + dexmedetomidine, received both lipopolysaccharide and dexmedetomidine. Sidestream dark-field videomicroscope, tissue oxygen monitor, and full-field laser perfusion image were used to investigate the microcirculation of the terminal ileum. Serum endocan level was measured. The Ussing chamber permeability assay, lumen-to-blood gadodiamide passage by magnetic resonance imaging, and bacterial translocation were conducted to determine epithelial barrier function. Mucosal apoptotic levels and tight junctional integrity were also examined., Results: The density of perfused small vessels in mucosa, serosal muscular layer, and Peyer patch in the lipopolysaccharide + dexmedetomidine group was higher than that of the lipopolysaccharide group. Serum endocan level was lower in the lipopolysaccharide + dexmedetomidine group than in the lipopolysaccharide group. Mucosal ratio of cleaved to full-length occludin and spleen bacterial counts were significantly lower in the lipopolysaccharide + dexmedetomidine group than in the lipopolysaccharide group., Conclusion: The study finding suggests that dexmedetomidine protects against intestinal epithelial barrier disruption in endotoxemic rats by attenuating intestinal microcirculatory dysfunction and reducing mucosal cell death and tight junctional damage. (Anesthesiology 2016; 125:355-67).

Background: Gastrointestinal bypass changes the gut microbiota and decreases systemic endotoxemia in obese subjects. Epithelial barrier integrity is crucial for confining enteric bacteria in the lumen and preventing gut-derived endotoxemia. The effect of bypass surgery on intestinal barrier functions remains poorly understood. This study aimed to evaluate the changes in intestinal permeability and gut barrier between rats receiving Roux-en-Y duodenojejunal bypass (DJB) or sham operation (SO)., Methods: Eighteen Sprague-Dawley rats were assigned to DJB or SO groups. Tissues of the alimentary, biliopancreatic, and common limbs in the small intestine, and the colon, were collected 2 weeks after operation. Mucosa-associated bacteria were quantified by colony forming units. Intestinal permeability was determined by mucosal-to-serosal dextran flux measured in Ussing chambers. Expression of occludin and proliferating cell nuclear antigen (PCNA) in the intestinal mucosa was examined by western blots., Results: Enteric bacterial numbers were increased in the alimentary and common limbs after DJB. Reduced dextran permeability was found in the alimentary limb, common limb, and colon after DJB. Moreover, increased villus height and crypt depth were found to be associated with higher mucosal levels of occludin and PCNA levels in the alimentary and common limbs after DJB., Conclusions: DJB in rats altered gut microbiota and reduced intestinal permeability due to increased epithelial proliferation and tight junctional protein expression. Our results show that bypass surgery led to fortification of the intestinal barrier functions, which may provide an explanation for the decreased risk of systemic endotoxemia in postoperative patients.

Colon cancers develop adaptive mechanisms to survive under extreme conditions and display hallmarks of unlimited proliferation and resistance to cell death. The deregulation of cell death is a key factor that contributes to chemoresistance in tumors. In a physiological context, balance between cell proliferation and death, and protection against cell damage are fundamental processes for maintaining gut epithelial homeostasis. The mechanisms underlying anti-death cytoprotection and tumor resistance often bear common pathways, and although distinguishing them would be a challenge, it would also provide an opportunity to develop advanced anti-cancer therapeutics. This review will outline cell death pathways (i.e., apoptosis, necrosis, and necroptosis), and discuss cytoprotective strategies in normal intestinal epithelium and death resistance mechanisms of colon tumor. In colorectal cancers, the intracellular mechanisms of death resistance include the direct alteration of apoptotic and necroptotic machinery and the upstream events modulating death effectors such as tumor suppressor gene inactivation and pro-survival signaling pathways. The autocrine, paracrine and exogenous factors within a tumor microenvironment can also instigate resistance against apoptotic and necroptotic cell death in colon cancers through changes in receptor signaling or transporter uptake. The roles of cyclooxygenase-2/prostaglandin E2, growth factors, glucose, and bacterial lipopolysaccharides in colorectal cancer will be highlighted. Targeting anti-death pathways in the colon cancer tissue might be a promising approach outside of anti-proliferation and anti-angiogenesis strategies for developing novel drugs to treat refractory tumors.

Gut microbiota and lipopolysaccharide (LPS) signaling have been associated with colon cancer development. Our recent findings demonstrated that LPS receptor subunits expressed on colonocytes have antagonistic roles in cell death and tumorigenesis: epithelial toll-like receptor 4 (TLR4) confers resistance to the apoptosis induced by its co-receptor CD14 and contributes to epithelial transition to cancer.

Co-existing paracellular and transcellular barrier defect in intestinal epithelium was documented in inflammatory bowel disease, celiac disease, and intestinal obstruction. Mechanisms regarding tight junction disruption have been extensively studied; however, limited progress has been made in research on bacterial transcytosis. Densely packed brush border (BB), with cholesterol-based lipid rafts in the intermicrovillous membrane invagination, serves as an ultrastructural barrier to prevent direct contact of luminal microbes with the cellular soma. Evidence in in vitro epithelial cell cultures and in vivo animal models of bowel obstruction and antibiotic-resistant bacterial infection had indicated that nonpathogenic, noninvasive enteric bacteria may hijack the lipid raft-mediated endocytic pathways. Our studies have shown that low dose interferon-gamma (IFNγ) causes long myosin light chain kinase (MLCK)-dependent terminal web (TW) contraction and BB fanning, allowing bacteria to pass through the consequently widened intermicrovillous cleft to be endocytosed via caveolin-associated lipid rafts. Activation of intracellular innate immune receptors by bacteria-containing endosomes may further induce inflammatory and oxidative stress, leading to secondary tight junction damage. The finding of bacterial internalization preceding tight junction damage suggests that abnormal bacterial uptake by epithelial cells may contribute to the initiation or relapse of chronic intestinal inflammation.

Antibiotic usage promotes intestinal colonization of antibiotic-resistant bacteria. However, whether resistant bacteria gain dominance in enteric microflora or disseminate to extraintestinal viscera remains unclear. Our aim was to investigate temporal diversity changes in microbiota and transepithelial routes of bacterial translocation after antibiotic-resistant enterobacterial colonization. Mice drinking water with or without antibiotics were intragastrically gavaged with ampicillin-resistant (Amp-r) nonpathogenic Escherichia coli (E. coli) and given normal water afterward. The composition and spatial distribution of intestinal bacteria were evaluated using 16S rDNA sequencing and fluorescence in situ hybridization. Bacterial endocytosis in epithelial cells was examined using gentamicin resistance assay and transmission electromicroscopy. Paracellular permeability was assessed by tight junctional immunostaining and measured by tissue conductance and luminal-to-serosal dextran fluxes. Our results showed that antibiotic treatment enabled intestinal colonization and transient dominance of orally acquired Amp-r E. coli in mice. The colonized Amp-r E. coli peaked on day 3 postinoculation and was competed out after 1 wk, as evidenced by the recovery of commensals, such as Escherichia, Bacteroides, Lachnospiraceae, Clostridium, and Lactobacillus. Mucosal penetration and extraintestinal dissemination of exogenous and endogenous enterobacteria were correlated with abnormal epithelial transcytosis but uncoupled with paracellular tight junctional damage. In conclusion, antibiotic-induced enteric dysbiosis predisposes to exogenous infection and causes systemic dissemination of both antibiotic-resistant and commensal enterobacteria through transcytotic routes across epithelial layers. These results may help explain the susceptibility to sepsis in antibiotic-resistant enteric bacterial infection., (Copyright © 2014 the American Physiological Society.)

Background: Recent studies of Giardia lamblia outbreaks have indicated that 40-80% of infected patients experience long-lasting functional gastrointestinal disorders after parasitic clearance. Our aim was to assess changes in the intestinal barrier and spatial distribution of commensal bacteria in the post-clearance phase of Giardia infection., Methods: Mice were orogastrically inoculated with G. lamblia trophozoites (strain GS/M) or pair-fed with saline and were sacrificed on post-infective (PI) days 7 (colonization phase) and 35 (post-clearance phase). Gut epithelial barrier function was assessed by Western blotting for occludin cleavage and luminal-to-serosal macromolecular permeability. Gut-associated, superficial adherent, and mucosal endocytosed bacteria were measured by agar culturing and were examined by fluorescence in situ hybridization. Intracellular bacteria cultured from isolated mucosal cells were characterized by 16S rDNA sequencing. Neutrophil-specific esterase staining, a myeloperoxidase activity assay, and enzyme-linked immunosorbent assays for cytokine concentrations were used to verify intestinal tissue inflammation., Results: Tight junctional damage was detected in the intestinal mucosa of Giardia-infected mice on PI days 7 and 35. Although intestinal bacterial overgrowth was evident only during parasite colonization (PI day 7), enhanced mucosal adherence and endocytosis of bacteria were observed on PI days 7 and 35. Multiple bacterial strains, including Bacillus, Lactobacillus, Staphylococcus, and Phenylobacterium, penetrated the gut mucosa in the post-infective phase. The mucosal influx of bacteria coincided with increases in neutrophil infiltration and myeloperoxidase activity on PI days 7 and 35. Elevated intestinal IFNγ, TNFα, and IL-1β levels also were detected on PI day 35., Conclusions: Giardia-infected mice showed persistent tight junctional damage and bacterial penetration, accompanied by mucosal inflammation, after parasite clearance. These novel findings suggest that the host's unresolved immune reactions toward its own microbiota, due to an impaired epithelial barrier, may partly contribute to the development of post-infective gut disorders.

Intestinal ischemia/reperfusion (I/R) induces mucosal barrier dysfunction and bacterial translocation (BT). Neutrophil-derived oxidative free radicals have been incriminated in the pathogenesis of ischemic injury in various organs, but their role in the bacteria-containing intestinal tract is debatable. Primed neutrophils are characterized by a faster and higher respiratory burst activity associated with more robust bactericidal effects on exposure to a second stimulus. Hypoxic preconditioning (HPC) attenuates ischemic injury in brain, heart, lung and kidney; no reports were found in the gut. Our aim is to investigate whether neutrophil priming by HPC protects against intestinal I/R-induced barrier damage and bacterial influx. Rats were raised in normoxia (NM) or kept in a hypobaric hypoxic chamber (380 Torr) 17 h/day for 3 weeks for HPC, followed by sham operation or intestinal I/R. Gut permeability was determined by using an ex vivo macromolecular flux assay and an in vivo magnetic resonance imaging-based method. Liver and spleen homogenates were plated for bacterial culturing. Rats raised in HPC showed diminished levels of BT, and partially improved mucosal histopathology and epithelial barrier function compared with the NM groups after intestinal I/R. Augmented cytokine-induced neutrophil chemoattractant (CINC)-1 and -3 levels and myeloperoxidase activity correlated with enhanced infiltration of neutrophils in intestines of HPC-I/R compared with NM-I/R rats. HPC alone caused blood neutrophil priming, as shown by elevated production of superoxide and hydrogen peroxide on stimulation, increased membrane translocation of cytosolic p47(phox) and p67(phox), as well as augmented bacterial-killing and phagocytotic activities. Neutrophil depletion reversed the mucosal protection by HPC, and aggravated intestinal leakiness and BT following I/R. In conclusion, neutrophil priming by HPC protects against I/R-induced BT via direct antimicrobial activity by oxidative respiratory bursts and through promotion of epithelial barrier integrity for luminal confinement of enteric bacteria.

The gastrointestinal tract is the largest reservoir of commensal bacteria in the human body, providing nutrients and space for the survival of microbes while concurrently operating mucosal barriers to confine the microbial population. The epithelial cells linked by tight junctions not only physically separate the microbiota from the lamina propria, but also secrete proinflammatory cytokines and reactive oxygen species in response to pathogen invasion and metabolic stress and serve as a sentinel to the underlying immune cells. Accumulating evidence indicates that commensal bacteria are involved in various physiological functions in the gut and microbial imbalances (dysbiosis) may cause pathology. Commensal bacteria are involved in the regulation of intestinal epithelial cell turnover, promotion of epithelial restitution and reorganization of tight junctions, all of which are pivotal for fortifying barrier function. Recent studies indicate that aberrant bacterial lipopolysaccharide-mediated signaling in gut mucosa may be involved in the pathogenesis of chronic inflammation and carcinogenesis. Our perception of enteric commensals has now changed from one of opportunistic pathogens to active participants in maintaining intestinal homeostasis. This review attempts to explain the dynamic interaction between the intestinal epithelium and commensal bacteria in disease and health status.

Intestinal epithelial barrier plays a critical role in the maintenance of gut homeostasis by limiting the penetration of luminal bacteria and dietary allergens, yet allowing antigen sampling for the generation of tolerance. Undigested proteins normally do not gain access to the lamina propria due to physical exclusion by tight junctions at the cell-cell contact sites and intracellular degradation by lysosomal enzymes in enterocytes. An intriguing question then arises: how do macromolecular food antigens cross the epithelial barrier? This review discusses the epithelial barrier dysfunction in sensitized intestine with special emphasis on the molecular mechanism of the enhanced transcytotic rates of allergens. The sensitization phase of allergy is characterized by antigen-induced cross-linking of IgE bound to high affinity FcεRI on mast cell surface, leading to anaphylactic responses. Recent studies have demonstrated that prior to mast cell activation, food allergens are transported in large quantity across the epithelium and are protected from lysosomal degradation by binding to cell surface IgE and low-affinity receptor CD23/FcεRII. Improved immunotherapies are currently under study including anti-IgE and anti-CD23 antibodies for the management of atopic disorders.

Objective: Gut barrier dysfunction and bacterial translocation occur in various disorders, including intestinal obstruction. Overexpression of inducible nitric oxide synthase is implicated in the pathogenesis of bacterial translocation, of which the molecular mechanism remains unclear. Epithelial permeability is regulated by tight junction reorganization and myosin light chain phosphorylation. Our aim was to investigate the roles of Rho-associated kinase and protein kinase C ζ in epithelial nitric oxide synthase-mediated barrier damage., Design: Animal study and cell cultures., Setting: Research laboratory., Subjects: BALB/c mice., Interventions: : Mouse distal small intestine was obstructed in vivo by a 10-cm loop ligation in which vehicle, L-Nil (a nitric oxide synthase inhibitor), or Y27632 (a Rho-associated kinase inhibitor) was luminally administered. After obstruction for 24 hrs, intestinal tissues were mounted on Ussing chambers for macromolecular flux. Liver and spleen tissues were assessed for bacterial counts. Caco-2 cells were exposed to 1 mM S-nitroso-N-acetylpenicillamine (a nitric oxide donor) for 24 hrs, and transepithelial resistance and permeability were evaluated., Measurements and Main Results: Mice with intestinal obstruction displayed epithelial barrier dysfunctions, such as permeability rise and bacterial translocation, associated with tight junction disruption and myosin light chain phosphorylation. Increased inducible nitric oxide synthase and phosphorylated protein kinase C ζ were observed in villus epithelium. Enteric instillation of L-Nil and Y27632 attenuated the functional and structural barrier damage caused by intestinal obstruction. L-Nil decreased intestinal obstruction-induced myosin light chain, myosin phosphatase target subunit 1, and protein kinase C ζ phosphorylation, suggesting that inducible nitric oxide synthase is upstream of Rho-associated kinase and protein kinase C ζ signaling. The intestinal phosphorylated myosin light chain level did not increase in inducible nitric oxide synthase(-/-) mice following intestinal obstruction. In vitro studies showed that S-nitroso-N-acetylpenicillamine-induced transepithelial resistance drop and permeability rise was independent of cell apoptosis. Y27632 inhibited S-nitroso-N-acetylpenicillamine-induced myosin light chain phosphorylation and permeability rise. S-nitroso-N-acetylpenicillamine also triggered phosphorylation and membrane translocation of protein kinase C ζ. Inhibitory protein kinase C ζ pseudosubstrate blocked S-nitroso-N-acetylpenicillamine-induced tight junction reorganization, but not myosin light chain phosphorylation., Conclusions: Epithelial inducible nitric oxide synthase activates two distinct signals, protein kinase C ζ and Rho-associated kinase, to disrupt tight junctions leading to bacterial influx.

The rapid rise of allergic disorders in developed countries has been attributed to the hygiene hypothesis, implicating that increased environmental sanitation in early childhood may be associated with higher incidence of hypersensitivity. Intestinal epithelial barriers play a crucial role in the maintenance of gut homeostasis by limiting penetration of luminal bacteria and dietary allergens, yet allowing antigen sampling via the follicle-associated epithelium for generation of tolerance. However, this intricate balance is upset in allergic intestines, whereby luminal proteins with antigenic properties gain access to the subepithelial compartment and stimulate mast cell degranulation. Recent studies demonstrated that food allergens were protected from lysosomal degradation, and were transported in large quantities across the epithelium by binding to cell surface IgE/CD23 (FcepsilonRII) that prevented the antigenic protein from lysosomal degradation in enterocytes. IL-4 (a Th2-type cytokine) not only increased production of IgE from B cells, but also upregulated the expression of CD23 on intestinal epithelial cells. Further studies indicated that CD23 was responsible for the bidirectional transport of IgE across epithelium. The presence of IgE/CD23 opens a gate for intact dietary allergens to transcytose across the epithelial cells, and thus foments the mast cell-dependent anaphylactic responses. The understanding of the molecular mechanism responsible for epithelial barrier defects may be helpful in designing novel therapies to treat food allergy and other allergic diseases.

Objectives: To develop an in vivo intestinal permeability assay applying magnetic resonance imaging (MRI) to monitor real-time gut barrier defects in animal models of acute mesenteric ischemia/reperfusion (I/R) insult., Materials and Methods: Twenty Wistar rats were divided to 2 groups for I/R challenge or sham controls. I/R rats received occlusion of superior mesenteric artery for 20 minutes and reperfusion for 1 hour. Sham-operation controls received laparotomy without manipulation of artery. To assess gut permeability, a 10-cm jejunal sac was created distal to the ligament of Treitz in both groups of rats after laparotomy, and a contrast agent (gadodiamide) was injected into the lumen of the ligated intestinal sac. The signals produced by gadodamide in the liver, kidney, and plasma before and after the start of reperfusion were examined by 1.5 Tesla MRI (GE Signa Excite), and the increment of signal-to-noise ratio (SNR) in these organs that parallels the luminal-to-serosal flux rate of the probe was used as an indicator of gut permeability. At the end of procedures, jejunal tissues and mucosal scrapings were collected for histologic examination and Western blotting for epithelial tight junctional proteins. Moreover, liver and spleen homogenates were cultured on fresh blood agar plates to measure the bacterial colony-forming units per gram of tissue., Results: In I/R rats, disrupted villous structure and decreased epithelial tight junctional expression were seen in the jejunum associated with massive enteric bacterial translocation to the liver and spleen. The SNR in the liver of I/R rats was higher than sham controls (2.65 +/- 0.56 vs. 0.65 +/- 0.26, P < 0.01) at 15 minutes postreperfusion. Elevation of SNR in the kidney was also found in I/R rats compared with sham controls (11.61 +/- 2.07 vs. 3.06 +/- 1.15, P < 0.05). The plasma gadodiamide concentration in I/R rats was significantly increased compared with sham controls (0.220 +/- 0.044 vs. 0.006 +/- 0.004 mM, P < 0.01) at 15 minutes postreperfusion., Conclusions: This novel MRI-based intestinal permeability assay has shown a significant increase in the signal intensity in liver, kidney, and plasma samples that correlated with mucosal barrier defects in experimental models of acute mesenteric I/R.