Human intestinal enteroids as a model of Clostridioides difficile-induced enteritis.

Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton. C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models is limited. Moreover, no model is available for examining C. difficile-induced enteritis, an understudied health problem. We hypothesized that human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells, which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic C. difficile strains elicited cell rounding in a strain-dependent manner, and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of C. difficile infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium.NEW & NOTEWORTHY In this article, we developed a novel model of Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear C. difficile-conditioned media suitable for live-cell imaging.

Dietary supplementation with olive mill wastewaters induces modifications on chicken jejunum epithelial cell transcriptome and modulates jejunum morphology.

BACKGROUND: The Mediterranean diet is considered one of the healthier food habits and olive oil is one of its key components. Olive oil polyphenols are known to induce beneficial effects in several pathological conditions, such as inflammatory bowel disease, and to contrast the proliferation of cancer cells or hypercholesterolemia. Polyphenols are also present in waste products derived from the olive industry: olive mill wastewaters (OMWW) are rich in polyphenols and there is an increasing interest in using OMWW in animal nutrition. OMWW are attributed with positive effects in promoting chicken performance and the quality of food-derived products. However, a tissue-specific transcriptome target analysis of chickens fed with OMWW has never been attempted. RESULTS: We explored the effect of dietary OMWW on the intestinal function in broilers. A morphological analysis of the jejunum revealed that OMWW reduced crypt depth, whereas no significant modifications were observed for villus height and the villus height/crypt depth ratio. An RNA Sequencing analysis was performed on isolated, intestinal, epithelial cells and 280 differentially expressed genes were found using a count-based approach. An enrichment analysis revealed that the majority of up regulated genes in the OMWW group were over-represented by the regulation of viral genome replication-related GO-Terms, whereas down regulated genes were mainly involved in cholesterol and lipid metabolism. CONCLUSIONS: Our study showed how an industrial waste product can be recycled as a feed additive with a positive relapse. OMWW dietary supplementation can be a nutritional strategy to improve chicken performance and health, prevent intestinal damage, enhance innate immunity and regulate cholesterol metabolism and fat deposition.

Both NHERF3 and NHERF2 are necessary for multiple aspects of acute regulation of NHE3 by elevated Ca2+, cGMP, and lysophosphatidic acid.

The intestinal epithelial brush border Na+/H+ exchanger NHE3 accounts for a large component of intestinal Na absorption. NHE3 is regulated during digestion by signaling complexes on its COOH terminus that include the four multi-PDZ domain-containing NHERF family proteins. All bind to NHE3 and take part in different aspects of NHE3 regulation. Because the roles of each NHERF appear to vary on the basis of the cell model or intestinal segment studied and because of our recent finding that a NHERF3-NHERF2 heterodimer appears important for NHE3 regulation in Caco-2 cells, we examined the role of NHERF3 and NHERF2 in C57BL/6 mouse jejunum using homozygous NHERF2 and NHERF3 knockout mice. NHE3 activity was determined with two-photon microscopy and the dual-emission pH-sensitive dye SNARF-4F. The jejunal apical membrane of NHERF3-null mice appeared similar to wild-type (WT) mice in surface area, microvillus number, and height, which is similar to results previously reported for jejunum of NHERF2-null mice. NHE3 basal activity was not different from WT in either NHERF2- or NHERF3-null jejunum, while d-glucose-stimulated NHE3 activity was reduced in NHERF2, but similar to WT in NHERF3 KO. LPA stimulation and UTP (elevated Ca2+) and cGMP inhibition of NHE3 were markedly reduced in both NHERF2- and NHERF3-null jejunum. Forskolin inhibited NHE3 in NHERF3-null jejunum, but the extent of inhibition was reduced compared with WT. The forskolin inhibition of NHE3 in NHERF2-null mice was too inconsistent to determine whether there was an effect and whether it was altered compared with the WT response. These results demonstrate similar requirement for NHERF2 and NHERF3 in mouse jejunal NHE3 regulation by LPA, Ca2+, and cGMP. The explanation for the similarity is not known but is consistent with involvement of a brush-border NHERF3-NHERF2 heterodimer or sequential NHERF-dependent effects in these aspects of NHE3 regulation. NEW & NOTEWORTHY NHERF2 and NHERF3 are apical membrane multi-PDZ domain-containing proteins that are involved in regulation of intestinal NHE3. This study demonstrates that NHERF2 and NHERF3 have overlapping roles in NHE3 stimulation by LPA and inhibition by elevated Ca2+ and cGMP. These results are consistent with their role being as a NHERF3-NHERF2 heterodimer or via sequential NHERF-dependent signaling steps, and they begin to clarify a role for multiple NHERF proteins in NHE3 regulation.

Dynamic regulation of extracellular ATP in Escherichia coli.

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

A canine model of experimental infection with Cryptosporidium canis.

Cryptosporidium is a genus of protozoal parasites that affects the gastrointestinal epithelium of a variety of hosts. Several models of experimental infection have been described to study the susceptibility, infectivity and pathogenicity among different Cryptosporidium species and isolates. This study aimed to establish an experimental infection of Cryptodporidium canis in canids. Infectivity and pathogenicity have been measured by evaluating the clinical status, pattern of oocyst excretion and histological examination. Results showed that C. canis was not infective for immunocompetent dogs or mice with severe combined immunodeficiency syndrome (SCID). Oocysts were first detected in the feces of immunosuppressed dogs on day 3 post-infection (p.i.), with levels peaking twice on days 10 and 17 p.i. during the patent period. cryptosporidial developmental stages were found in the duodenum and jejunum of dogs in histological sections stained with hematoxylin and eosin (H & E) and using scanning electron microscopy (SEM). Histopathological changes in the intestinal tract of infected dogs were characterized by epithelial metaplasia and dilatation; the integrity of intestinal mucosal epithelial cells was distinctly damaged with whole sheets of cilia sloughed away. Ultrastructural observation data were consistent with histological observations. Based on these findings, the canine model described in this work will be useful to evaluate clinical, parasitological and histological aspects of C. canis infection and will be useful for the further understanding of cryptosporidiosis, drug development, and vaccine development.

Distinct cytoprotective roles of pyruvate and ATP by glucose metabolism on epithelial necroptosis and crypt proliferation in ischaemic gut.

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.

Targeting palmitoyl acyltransferase ZDHHC21 improves gut epithelial barrier dysfunction resulting from burn-induced systemic inflammation.

Clinical studies in burn patients demonstrate a close association between leaky guts and increased incidence or severity of sepsis and other complications. Severe thermal injury triggers intestinal inflammation that contributes to intestinal epithelial hyperpermeability, which exacerbates systemic response leading to multiple organ failure and sepsis. In this study, we identified a significant function of a particular palmitoyl acyltransferase, zinc finger DHHC domain-containing protein-21 (ZDHHC21), in mediating signaling events required for gut hyperpermeability induced by inflammation. Using quantitative PCR, we show that ZDHHC21 mRNA production was enhanced twofold when intestinal epithelial cells were treated with TNF-α-IFN-γ in vitro. In addition, pharmacological targeting of palmitoyl acyltransferases with 2-bromopalmitate (2-BP) showed significant improvement in TNF-α-IFN-γ-mediated epithelial barrier dysfunction by using electric cell-substrate impedance-sensing assays, as well as FITC-labeled dextran permeability assays. Using acyl-biotin exchange assay and click chemistry, we show that TNF-α-IFN-γ treatment of intestinal epithelial cells results in enhanced detection of total palmitoylated proteins and this response is inhibited by 2-BP. Using ZDHHC21-deficient mice or wild-type mice treated with 2-BP, we showed that mice with impaired ZDHHC21 expression or pharmacological inhibition resulted in attenuated intestinal barrier dysfunction caused by thermal injury. Moreover, hematoxylin and eosin staining of the small intestine, as well as transmission electron microscopy, showed that mice with genetic interruption of ZDHHC21 had attenuated villus structure disorganization associated with thermal injury-induced intestinal barrier damage. Taken together, these results suggest an important role of ZDHHC21 in mediating gut hyperpermeability resulting from thermal injury.NEW & NOTEWORTHY Increased mucosal permeability in the gut is one of the major complications following severe burn. Here we report the novel finding that zinc finger DHHC domain-containing protein-21 (ZDHHC21) mediates gut epithelial hyperpermeability resulting from an experimental model of thermal injury. The hyperpermeability response was significantly attenuated with a pharmacological inhibitor of palmitoyl acyltransferases and in mice with genetic ablation of ZDHHC21. These findings suggest that ZDHHC21 may serve as a novel therapeutic target for treating burn-induced intestinal barrier dysfunction.

Interactive effects of temperature and dietary supplementation of arginine or guanidinoacetic acid on nutritional and physiological responses in male broiler chickens.

1. The aim of this experiment was to study the interactive effect of rearing temperature and dietary supplementation of arginine (Arg) or guanidinoacetic acid (GAA) on performance, gut morphology and ascites indices in broiler chickens raised under the same condition in the first 2 weeks and then reared under normal (23-26°C) or subnormal (17°C) ambient temperatures for the next 3 weeks. 2. This experiment was conducted as a split plot with 900 Ross 308 male broiler chicks that were allocated to two houses (as main plots); each consisted of 5 treatments (as sub-plots) with 6 replicates of 15 birds. The 5 diets were (1) control, (2) control + 0.60 g/kg GAA, (3) control + 1.20 g/kg GAA, (4) control + 0.86 g/kg Arg and (5) control + 1.72 g/kg Arg. 3. Feed intake (0-35 d) of birds fed on a diet containing 1.2 g GAA/kg and reared under normal temperature was reduced compared to control fed birds. Birds fed on a diet containing 1.72 g/kg Arg and reared under subnormal temperature had higher weight gain compared to those fed on control or GAA-added diets in overall study period. 4. Supplementation of diets with Arg alleviated the adverse effect of cold stress as reflected by reduction in blood haematocrit (41% vs. 37%), and right ventricle to total ventricle ratio (0.28 vs. 0.25) at 35 d of age. Addition of Arg to the diet of birds reared under cold stress resulted in a higher jejunal villus surface area compared to those fed on control or GAA-added diets. 5. Findings of this study revealed that Arg or GAA supplementation of diets did not affect performance of birds under normal temperatures, but Arg supplementation of the diet significantly alleviated the adverse effect of cold stress on performance, gut development and ascites syndrome. In addition, GAA supplementation at 1.2 g/kg improved jejunal villus surface area in birds raised under subnormal temperature.

Identification of intestinal ion transport defects in microvillus inclusion disease.

Loss of function mutations in the actin motor myosin Vb (Myo5b) lead to microvillus inclusion disease (MVID) and death in newborns and children. MVID results in secretory diarrhea, brush border (BB) defects, villus atrophy, and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl(-)) and sodium (Na(+)) is unknown. The present study used Myo5b loss-of-function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal stimulated emission depletion (gSTED) imaging, immunohistochemical staining, transmission electron microscopy, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression, and function of the major BB ion transporters NHE3 (Na(+)), CFTR (Cl(-)), and SLC26A3 (DRA) (Cl(-)/HCO3 (-)) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory cryptlike enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BB membrane of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation, and a cryptlike phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3, and DRA contribute to Cl(-) and Na(+) stool loss in MVID diarrhea.

Nitric oxide-induced oxidative stress impairs pacemaker function of murine interstitial cells of Cajal during inflammation.

The pacemaker function of interstitial cells of Cajal (ICC) is impaired during intestinal inflammation. The aim of this study is to clarify the pathophysiological mechanisms of ICC dysfunction during inflammatory condition by using intestinal cell clusters. Cell clusters were prepared from smooth muscle layer of murine jejunum and treated with interferon-gamma and lipopolysaccharide (IFN-γ+LPS) for 24h to induce inflammation. Pacemaker function of ICC was monitored by measuring cytosolic Ca(2+) oscillation in the presence of nifedipine. Treatment with IFN-γ+LPS impaired the pacemaker activity of ICC with increasing mRNA level of interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6 in cell clusters; however, treatment with these cytokines individually had little effect on pacemaker activity of ICC. Treatment with IFN-γ+LPS also induced the expression of inducible nitric oxide synthase (iNOS) in smooth muscle cells and resident macrophages, but not in ICC. Pretreatment with NOS inhibitor, L-NAME or iNOS inhibitor, 1400W ameliorated IFN-γ+LPS-induced pacemaker dysfunction of ICC. Pretreatment with guanylate cyclase inhibitor, ODQ did not, but antioxidant, apocynin, to suppress NO-induced oxidative stress, significantly suppressed the impairment of ICC function induced by IFN-γ+LPS. Treatment with IFN-γ+LPS also decreased c-Kit-positive ICC, which was prevented by pretreatment with L-NAME. However, apoptotic ICC were not detected in IFN-γ+LPS-treated clusters, suggesting IFN-γ+LPS stimulation just changed the phenotype of ICC but not induced cell death. Moreover, ultrastructure of ICC was not disturbed by IFN-γ+LPS. In conclusion, ICC dysfunction during inflammation is induced by NO-induced oxidative stress rather than NO/cGMP signaling. NO-induced oxidative stress might be the main factor to induce phenotypic changes of ICC.

[COMPARATIVE ANALYSIS OF TIGHT JUNCTIONS OF EPITHELIUM OF RATS JEJUNUM UNDER THE EFFECT OF LIPOPOLYSACCHARIDE AND CHOLERA TOXIN].

AIM: Comparative study of tight junctions and ultrastructure alterations of enterocytes of mucous membranes of jejunum of rats under the effect of lipopolysaccharides and cholera toxin. MATERIALS AND METHODS: Lipopolysaccharides (Sigma-Aldrich, Germany) and cholera toxin (Sigma-Aldrich, Germany) were used. The study was carried out in Wistar line rats. Effect of lipopolysaccharides and cholera toxin on epitheliocytes was carried out by a method of withdrawal of segments of rat jejunum and their incubation with the specified substances. Comparative analysis of ultrathin sections of enterocytes of jejunum of rats and tight junctions between them was carried out in control and under the effect of lipopolysaccharides and cholera toxin. RESULTS: Effect of lipopolysaccharides on ultrastructure of enterocytes of rat jejunum manifested in the change of cell form as a result of increase of intercellular space without destruction of tight junctions. Disappearance of desmosomes, increase of nuclei and more pronounced ER were noted in some epitheliocytes. Effect of cholerogen on epitheliocytes of mucous membrane of rat jejunum by a number of signs is similar to the effect of lipopolysaccharides, that manifested in an alteration of ultrastructure of cell, the form of those also transformed as a result of an increase of intercellular space, this process was not accompanied by destruction of tight junctions. Disappearance of folding of the lateral region of plasmatic membrane of cells and a reduction of a number of microvilli was observed under the effect of cholera toxin. CONCLUSION: A similar character of effect of lipopolysaccharides and cholera toxins on ultrastructure of cells and region of tight junctions of enterocytes of rat jejunum was detected, both substances caused an increase of intercellular space without the destruction of tight junctions.

Probing endocytosis from the enterocyte brush border using fluorescent lipophilic dyes: lipid sorting at the apical cell surface.

The small intestinal brush border is a specialized cell membrane that needs to withstand the solubilizing effect of bile salts during assimilation of dietary nutrients and to achieve detergent resistance; it is highly enriched in glycolipids organized in lipid raft microdomains. In the present work, the fluorescent lipophilic probes FM 1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide), FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)hexatrienyl)pyridinium dibromide), TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate), and CellMask Orange plasma membrane stain were used to study endocytosis from the enterocyte brush border of organ-cultured porcine mucosal explants. All the dyes readily incorporated into the brush border but were not detectably endocytosed by 5 min, indicating a slow uptake compared with other cell types. At later time points, FM 1-43 clearly appeared in distinct punctae in the terminal web region, previously shown to represent early endosomes (TWEEs). In contrast, the other dyes were relatively "endocytosis resistant" to varying degrees for periods up to 2 h, indicating an active sorting of lipids in the brush border prior to internalization. For some of the dyes, a diphenylhexatriene motif in the lipophilic tail seemed to confer the relative endocytosis resistance. Lipid sorting by selective endocytosis therefore may be a process in the enterocytes aimed to generate and maintain a unique lipid composition in the brush border.

[REARRANGEMENT OF THE LYMPH TISSUE IN THE MICE SPLEEN AND JEJUNUM WALL DURING THE GROUND-BASED REPRODUCTION OF THE CONDITIONS OF ANIMAL MAINTENANCE IN THE BIOSATTELITE BION-M1 MISSION].

Purpose of the investigation was microscopic examination of changes in cyto architectonics of the spleen and jejunum lymph (immune) tissue in 19-20-week C57BL/6N male mice exposed to some conditions their counterparts had lived in during the 30-d Bion-M1 mission (ground experiment). Local deviations in reactions of the morphofunctional zones of these organs were found. In the spleen, reaction in the centers of lymph nodules generation or the B-lymphocytes maturation zone grows strong. Changes in the cell composition of periarterial lymph sheaths that constitute the morphological site of T-lymphocytes accumulation suggest inhibition of its functional activity. Cell composition of the jejunum wall structure implies a decline of the jejunal immune activity. Our investigation of the organs taken from the ground control mice maintained in the flight BIOS-MLZh module evidences that unceasing noise, hypokinesia, isolation, and paste-like feed weaken general immunity of laboratory animals.

Stimulation of intestinal growth and function with DPP4 inhibition in a mouse short bowel syndrome model.

Glucagon-like peptide-2 (GLP-2) has been shown to be effective in patients with short bowel syndrome (SBS), but it is rapidly inactivated by dipeptidyl peptidase IV (DPP4). We used an orally active DPP4 inhibitor (DPP4-I), MK-0626, to determine the efficacy of this approach to promote adaptation after SBS, determined optimal dosing, and identified further functional actions in a mouse model of SBS. Ten-week-old mice underwent a 50% proximal small bowel resection. Dose optimization was determined over a 3-day post-small bowel resection period. The established optimal dose was given for 7, 30, and 90 days and for 7 days followed by a 23-day washout period. Adaptive response was assessed by morphology, intestinal epithelial cell (IEC) proliferation (proliferating cell nuclear antigen), epithelial barrier function (transepithelial resistance), RT-PCR for intestinal transport proteins and GLP-2 receptor, IGF type 1 receptor, and GLP-2 plasma levels. Glucose-stimulated sodium transport was assessed for intestinal absorptive function. Seven days of DPP4-I treatment facilitated an increase in GLP-2 receptor levels, intestinal growth, and IEC proliferation. Treatment led to differential effects over time, with greater absorptive function at early time points and enhanced proliferation at later time points. Interestingly, adaptation continued in the group treated for 7 days followed by a 23-day washout. DPP4-I enhanced IEC proliferative action up to 90 days postresection, but this action seemed to peak by 30 days, as did GLP-2 plasma levels. Thus DPP4-I treatment may prove to be a viable option for accelerating intestinal adaptation with SBS.

Coated zinc oxide improves intestinal immunity function and regulates microbiota composition in weaned piglets.

The present study was conducted to test the hypothesis that low concentrations of coated ZnO, as a substitute for a high concentration of ZnO (2250 mg Zn/kg), could improve intestinal immunity function and regulate microbiota composition, thus alleviating the incidence of diarrhoea in weaned piglets. A total of eighty-four cross-bred piglets, weaned at an age of 28 (SEM 1) d, were allocated randomly, on the basis of average initial body weight (7·72 (SEM 0·65) kg), to seven treatment groups as follows: a 250 mg Zn (ZnO)/kg group (low Zn; LZ) and a 2250 mg Zn (ZnO)/kg group (high Zn; HZ) that were offered diets containing ZnO at 250 and 2250 mg Zn/kg, respectively; and five experimental groups in which coated ZnO was added at 250, 380, 570, 760 and 1140 mg Zn/kg basal diet, respectively. The trial lasted 2 weeks. The results indicated that, compared with LZ treatment, supplementation with coated ZnO at 380 or 570 mg Zn/kg reduced (P< 0·05) diarrhoea index, increased (P< 0·05) duodenal villus height and the ratio of villus height:crypt depth, up-regulated (P< 0·05) the gene expression of insulin-like growth factor 1, zonula occludens protein-1, occludin, IL-10 and transforming growth factor ß1, and elevated (P< 0·05) secretory IgA concentration in the jejunal mucosa. Microbiota richness and the Shannon diversity index were also decreased (P< 0·05). Furthermore, piglets in the group fed coated ZnO at 380 or 570 mg Zn/kg did not differ from those in the HZ-fed group in relation to the aforementioned parameters. Collectively, a low concentration of coated ZnO (380 or 570 mg Zn/kg) can alleviate the incidence of diarrhoea by promoting intestinal development, protecting the intestinal mucosal barrier from damage, stimulating the mucosal immune system and regulating the microbiota composition.

[Histopathological changes in EV71-infected mouse model:a transmission electron microscopic study].

OBJECTIVE: To document ultrastructural changes of brain, spinal cord, skeletal muscle, jejunum and lung of EV71 infection mouse model, and to explore the myotropism and pathogenesis of EV71 in nervous system. METHODS: Ten-day-old suckling mice were infected with EV71 strain via the intraperitoneal route. Mice with paralysis were scarified on day 4 post infection and the brain, spinal cord, skeletal muscle, jejunum and lung were sampled for transmission electron microscopy and light microscopy. RESULTS: Lesions in brain were generally mild with inner chamber swelling in some of mitochondria. Myelin sheaths of medullated fibers were split with vacuolated changes. The Nissl bodies in anterior motor neurons disappeared along with mitochondria swelling, rough endoplasmic reticulum swelling and degranulation. Cytoplasm of anterior motor neurons showed cribriform appearance accompanied by neuronophagia. The bands of skeletal muscle in the infected group disappeared with degeneration and karyopyknosis in myocytes, in addition to mitochondrial swelling. Microvilli of epithelium in jejunum became loosely arranged along with formation of spiral medullary sheath structure and mitochondria swelling. Interstitial pneumonia was observed in lungs with type II pneumocyte proliferation and evacuation of the multilamellar bodies. CONCLUSIONS: EV71 infection causes severe myositis in the mouse model suggesting a strong myotropism of EV71 virus. The presence of lesions of various degrees in central nervous system and changes in anterior motor neurons may be associated with limb paralysis.

Diarrhoea-predominant irritable bowel syndrome: an organic disorder with structural abnormalities in the jejunal epithelial barrier.

OBJECTIVE: Recently, the authors demonstrated altered gene expression in the jejunal mucosa of diarrhoea-predominant irritable bowel syndrome patients (IBS-D); specifically, the authors showed that genes related to mast cells and the intercellular apical junction complex (AJC) were expressed differently than in healthy subjects. The aim of the authors here was to determine whether these alterations are associated with structural abnormalities in AJC and their relationship with mast cell activation and IBS-D clinical manifestations. DESIGN: A clinical assessment and a jejunal biopsy were obtained in IBS-D patients (n=45) and healthy subjects (n=30). Mucosal mast cell number and activation were determined by quantifying CD117(+) cells/hpf and tryptase expression, respectively. Expression and distribution of AJC specific proteins were evaluated by western blot and confocal microscopy. AJC ultrastructure was assessed by transmission electron microscopy. RESULTS: Compared with healthy subjects, IBS-D patients exhibited: (a) increased mast cell counts and activation; (b) increased protein expression of claudin-2, reduced occludin phosphorylation and enhanced redistribution from the membrane to the cytoplasm; and (c) increased myosin kinase expression, reduced myosin phosphatase and, consequently, enhanced phosphorylation of myosin. These molecular alterations were associated with ultrastructural abnormalities at the AJC, specifically, perijunctional cytoskeleton condensation and enlarged apical intercellular distance. Moreover, AJC structural alterations positively correlated both with mast cell activation and clinical symptoms. CONCLUSION: The jejunal mucosa of IBS-D patients displays disrupted apical junctional complex integrity associated with mast cell activation and clinical manifestations. These results provide evidence for the organic nature of IBS-D, a heretofore model disease of functional gastrointestinal disorders.

Intestinal stem cells remain viable after prolonged tissue storage.

Intestinal stem cells (ISCs) are responsible for renewal of the epithelium both during normal homeostasis and following injury. As such, they have significant therapeutic potential. However, whether ISCs can survive tissue storage is unknown. We hypothesize that, although the majority of epithelial cells might die, ISCs would remain viable for at least 24 h at 4 °C. To explore this hypothesis, jejuna of C57Bl6/J or Lgr5-LacZ mice were removed and either processed immediately or placed in phosphate-buffered saline at 4 °C. Delayed isolation of epithelium was performed after 24, 30, or 48 h storage. At the light microscope level, despite extensive apoptosis of villus epithelial cells, small intestinal crypts remained morphologically intact for 30 h and ISCs were identifiable via Lgr5-LacZ positivity. Electron microscopy showed that ISCs retained high integrity for 24 h. When assessed by flow cytometry, ISCs were more resistant to degeneration than the rest of the epithelium, including neighboring Paneth cells, with higher viability across all time points. Cultured isolated crypts showed no loss of capacity to form complex enteroids after 24 h tissue storage, with efficiencies after 7 days of culture remaining above 80 %. By 30 h storage, efficiencies declined but budding capability was retained. We conclude that, with delay in isolation, ISCs remain viable and retain their proliferative capacity. In contrast, the remainder of the epithelium, including the Paneth cells, exhibits degeneration and programmed cell death. If these findings are recapitulated in human tissue, storage at 4 °C might offer a valuable temporal window for the harvesting of crypts or ISCs for therapeutic application.

The role of neuropeptide Y and aquaporin 4 in the pathogenesis of intestinal dysfunction caused by traumatic brain injury.

BACKGROUND: Although the exact incidence is unknown, traumatic brain injury (TBI) can lead to intestinal dysfunction. It has important influence on the early nutrition and prognosis of TBI patients. Experiments were designed to study the roles of neuropeptide Y (NPY) and aquaporin 4 (AQP4) in the pathogenesis of intestinal dysfunction caused by TBI and to find some new solutions for the treatment of intestinal dysfunction after TBI. METHODS: Forty adult male Wistar rats were randomly divided into control, mild trauma, moderate trauma, and severe trauma groups. TBI was induced by Feeney's impact method. Control animals were sham operated but not subjected to the impact test. All rats were killed 24 h after surgery. Blood samples were obtained from the abdominal aorta for enzyme-linked immunosorbent assay measurement of NPY concentrations. Jejunum segments 15 cm distal to the Treitz ligament were taken for analysis of NPY and AQP4 expression by polymerase chain reaction, Western blot, and immunohistochemistry. Pathologic changes in intestinal cell structure and ultrastructure were studied by light microscopy and transmission electron microscopy. RESULTS: The specimens from different groups showed different degrees of structural changes, ranging from swelling and degeneration of villous epithelial cells to extensive denudation and collapse of the villi. The more severe the trauma, the more serious the degree of intestinal mucosal injury. Intestinal smooth muscle also showed varying degrees of edema and structural disorder. Electron microscopy showed that intestinal mitochondria had varying degrees of swelling and the structure of mitochondrial crista was disordered and even fractured. Plasma concentrations of NPY and jejunal gene and protein expressions of NPY and AQP4 increased significantly following TBI (P < 0.05), with greater increases at higher levels of injury. Moreover, there were positive correlations between NPY and AQP4 (P < 0.05). CONCLUSIONS: Increasing grades of TBI caused increasing degrees of intestinal ischemia and edema, and thus caused increasingly severe intestinal dysfunction. AQP4 and NPY may be involved in the pathogenesis of intestinal dysfunction after TBI. Increased NPY levels may be responsible for intestinal ischemia and hypoxia, and AQP4 may play an important role in intestinal edema. Increased NPY levels may be one of the main causes for the increase in AQP4 after TBI.

Octreotide alleviates obesity by reducing intestinal glucose absorption and inhibiting low-grade inflammation.

PURPOSE: To investigate the role of octreotide, a somatostatin (SST) analog with anti-inflammatory effects, on the digestive and absorptive functions of jejunum in rats fed a high-fat diet, as well as its therapeutic prospects for diet-induced obesity. METHODS: Rats were divided into three groups with different diet and treatment for the 176-day experiment: (1) control, 18 rats fed with standard chow, (2) high-fat control, 19 rats fed with high-fat chow, and (3) high-fat octreotide, 21 rats fed with high-fat chow and treated with octreotide for the last 8 days of the experiment. Plasma tumor necrosis factor-α (TNF-α) was measured by ELISA and SST by radioimmunoassay. Disaccharidase activity in the jejunal homogenate was determined. SST and Na⁺-dependent glucose transporter 1 (SGLT-1) in the jejunal mucosa were visualized by immunohistochemistry. SGLT-1 was quantified by reverse transcription polymerase chain reaction and Western blot assays. RESULTS: After 176 days, the fat/body weight ratio, villus height, maltase, SGLT-1, and plasma TNF-α in the high-fat control rats were much higher than those in the control rats (p < 0.01 or p < 0.05) and were significantly lower in the high-fat + octreotide rats (p < 0.01 or p < 0.05). SST levels were dramatically different in the intestinal mucosa of the two high-fat groups (231.12 ± 98.18 pg/mg in the high-fat controls and 480.01 ± 286.65 pg/mg in the octreotide group). CONCLUSIONS: The low-grade inflammation induced by high-fat diet apparently reduced the secretion of intestinal SST, which increased intestinal absorption of energy and nutrients and formation of adipose tissues. Octreotide effectively reversed this process, a finding that has far-reaching significance for the regulation of energy balance.