Normal Intestine Research Articles

Fruit Consumption is Associated with Alterations in Microbial Composition and Lower Rates of Pouchitis.

Patients with ulcerative colitis [UC] who undergo proctocolectomy with an ileal pouch-anal anastomosis commonly develop pouch inflammation [pouchitis]. Pouchitis develops in a previously normal small intestine and may involve environmental factors. We explored whether diet and microbiota alterations contributed to the pathogenesis of pouchitis. Patients were recruited and prospectively followed at a comprehensive pouch clinic. Pouch behaviour was clinically defined as a normal pouch [NP] or pouchitis. Patients completed Food Frequency Questionnaires [FFQs]. Faecal samples were analysed for microbial composition [16S rRNA gene pyrosequencing]. Nutritional evaluation was performed in 172 patients [59% females], and of these, faecal microbial analysis was performed in 75 patients (microbiota cohort: NP [n = 22], pouchitis [n = 53]). Of the entire cohort, a subgroup of 39 [22.6%] patients had NP at recruitment [NP cohort]. Of these, 5 [12.8%] developed pouchitis within a year. Patients at the lowest tertile of fruit consumption [<1.45 servings/day] had higher rates of pouchitis compared with those with higher consumption [30.8% vs 3.8%, log rank, p = 0.03]. Fruit consumption was correlated with microbial diversity [r = 0.35, p = 0.002] and with the abundance of several microbial genera, including Faecalibacterium [r = 0.29, p = 0.01], Lachnospira [r = 0.38, p = 0.001], and a previously uncharacterized genus from the Ruminococcaceae family [r = 0.25, p = 0.05]. Reduction in fruit consumption over time was associated with disease recurrence and with reduced microbial diversity [Δ = -0.8 ± 0.3, p = 0.008]. Fruit consumption is associated with modification of microbial composition, and lower consumption was correlated with the development of pouchitis. Thus, fruit consumption may protect against intestinal inflammation via alteration of microbial composition.

Immunohistochemical Profile of Tumor Suppressor Proteins RASSF1A and LATS1/2 in Relation to p73 and YAP Expression, of Human Inflammatory Bowel Disease and Normal Intestine.

The intestinal neoplastic transformation is a possible risk of chronic inflammatory bowel disease (IBD). Previous evidence in mice IBD provides a role for the RAS-association domain family tumor suppressor protein 1 A (RASSF1A), in the repairing process following mucosa epithelium damage, through cooperation with the HIPPO-signaling molecules p73, and YAP. HIPPO pathway which has been implicated in stem cell activity includes as key components for signal transduction the large tumor suppressor homology Ser/Thr kinases LATS1/2. The aim of this study was to assess immunohistochemically, using specific antibodies, the RASSF1A and LATS1/2 expression patterns in a cohort of patients with IBD including 52 ulcerative colitis (UC), 24 Crohn's disease (CD) and 24 IBD unclassified (IBD-U), compared with normal intestine from non-IBD patients (control group). The relationship between subtypes of IBD and RASSF1A and LATS1/2 expression, both individually and related to p73 and YAP/pYAP(Ser127) proteins was also investigated. Quantitative analyses of the immunohistochemical findings in mucosa cells revealed a significantly decreased expression in UC and IBD-U for RASSF1A expression and a significantly elevated expression in UC, IBD-U, and CD for LATS1/2 expression compared with normal mucosa (P < 0.05). However, ROC curve analysis showed that only LATS1/2 could differentiate IBD from control group. RASSF1A expression was significantly correlated with LATS1/2 in UC with dysplasia (P < 0.0001), and p73 in UC (P < 0.001), and IBD-U (P < 0.02). The expression of all proteins did not differ significantly between subtypes of IBD (P ≥ 0.05). RASSF1A-LATS1/2 co-expression was mainly observed in IBD samples. These findings suggest that tumor suppression proteins RASSF1A and LATS1/2 may be involved in the pathogenesis of human IBD and imply a potential cooperation of RASSF1A, and HIPPO signaling pathways in human bowel inflammation.

The double stranded RNA analog poly-IC elicits both robust IFN-λ production and oncolytic activity in human gastrointestinal cancer cells

PurposeType III IFN (IFN-λ) is the dominant frontline response over type I IFN in human normal intestinal epithelial cells upon viral infection, this response being mimicked by the dsRNA analog poly-IC. Poly-IC also induces cell death in murine intestinal crypts ex vivo. Here we examined whether these innate defense functions of normal intestinal epithelial cells are recapitulated in gastrointestinal carcinoma cells so that they could be harnessed to exert both immunoadjuvant and oncolytic functions, an unknown issue yet.Experimental designFour human gastrointestinal carcinoma cell lines versus the Jurkat lymphoma cell line were used to assess the effects of intracellular poly-IC on i) IFN-λ secretion and cell proliferation and ii) role of NFκB signaling using the NFκB inhibitory peptide SN50 as a screening probe and a siRNA approach.ResultsPoly-IC induced in all cell lines except Jurkat both a robust IFN-λ secretion and a cytoreductive effect on adherent cells, restricted to proliferating cells and associated with cellular shedding and reduced clonogenicity of the shed cells. Collectively these findings demonstrate the oncolytic activity of poly-IC. Inhibiting NFκB in T84 cells using a siRNA approach decreased IFN-λ production without protecting the cells from the poly-IC oncolytic effects. In line with these findings IFN-λ, that upregulated the anti-viral protein MxA, was unable per se to alter T84 cell proliferation.ConclusionOur demonstration that poly-IC-induced concomitant recapitulation of two innate functions of normal intestine, i.e. IFN-λ production and cell death, by human gastrointestinal cancer cells opens new perspectives in gastrointestinal cancer treatment.

Abstract A69: Mutagenicity of urea cycle dysregulation and its implications for cancer immunotherapy

Abstract Immune checkpoint therapy leads to durable clinical responses in many cancer patients, but fails in others. To improve the clinical response to immunotherapy, it is highly important to identify predictive biomarkers. While checkpoint genes’ expression levels, tumor neo-antigen load and microsatellite instability (MSI) have been associated with enhanced response to checkpoint immunotherapies, they yet provide only a modest predictive signal and hence there is a need to identify additional predictive factors. Specifically, while there is growing evidence that metabolic alterations can affect the tumor and modulate the immune response, the potential effects of altered cancer metabolism on tumor mutagenesis and immunotherapy remain unexplored. The urea cycle (UC) converts excess nitrogen derived from the breakdown of nitrogen-containing molecules (e.g., ammonia) to urea, a relatively non-toxic and disposable nitrogenous compound. We and others have shown that silencing of the UC enzyme ASS1 promotes cancer proliferation by diverting its substrate aspartate toward CAD enzyme, which mediates the first three reactions in the pyrimidine synthesis pathway. We now demonstrate, by analysis of the TCGA data, tumor samples and cancer cell line experiments, that UC dysregulation (UCD) is a much wider common metabolic phenomenon that maximizes nitrogen utilization in cancer, favoring pyrimidine synthesis over urea disposal. Of note, while UCD is significantly associated with decreased cancer patient survival, the overall mutational load is not. Remarkably, we find that the UCD changes the 1:1 purine (R)-to-pyrimidine (Y) ratio in favor of pyrimidine in cancer cells. Moreover, in analysis of both TCGA data and UC perturbed cancer cells we find that: (a) UCD is significantly associated with a novel and unique pattern of purine-to-pyrimidine transversion mutational bias across many cancer types at the DNA coding (sense) strand, and (b) this trend becomes stronger and more significant at both the mRNA and protein levels, testifying to its functional implications. Notably, the overall mutational load in cancer is negatively correlated with UCD, testifying to their independence. To test whether the mutational bias is associated with better immunotherapy response, we analyze published data of three large melanoma cohorts. We find that responders of both anti-PD1 and anti-CTLA4 therapy exhibit significantly higher UCD and R-&amp;gt;Y mutational bias than non-responders. We further observe that the peptides carrying transverse R-&amp;gt;Y mutations are preferentially presented as neoantigens in responders independent of mutational load, and this trend becomes significant for more clonal neoantigens, promoting UCD as a potential biomarker for the success of immunotherapy. Finally, as nitrogen metabolites are excreted in the urine, we hypothesize that these changes may be detectable in urine of UCD-cancers. We observe increased levels of pyrimidine derived metabolites in the urine of mice bearing colon tumors associated with UCD in the tumors compared to normal intestine. In an analogous manner, we find significantly higher levels pyrimidine derived metabolites in the urine of human patients with prostate cancer compared to controls. Collectively, these results support our hypothesis that UCD is a prevalent metabolic phenomenon in cancer, generating mutational biased neo-peptides, worsening patients’ prognosis and yet enhancing the response to immune therapy independent of mutational load and MSI. Taken together, our findings point to the important role of UCD in a broad spectrum of cancers, to the potential use of UCD related metabolites as cancer biomarkers, and last but not least, to the role of UCD in predicting response to immune check point therapy. Broadly, our results suggest future therapeutic interventions aiming to increase UCD levels to enhance the coverage and efficiency of cancer immunotherapy. Citation Format: Joo Sang Lee, Narin Carmel, Hiren Karathia, Noam Auslander, Shiran Rabinovich, Rom Keshet, Noa Stettner, Alon Silberman, Lilach Agemy, Daniel Helbling, Raya Eilam, Qin Sun, Alexander Brandis, Hila Weiss, David Dimmock, Noam Stern-Ginossar, Avigdor Scherz, Igor Ulitsky, Sandesh CS Nagamani, Ronit Elhasid, Sridhar Hannenhalli, Eytan Ruppin, Ayelet Erez. Mutagenicity of urea cycle dysregulation and its implications for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A69.

Contrast Radiography in Zarudni’s Spur-Thighed Tortoises (Testudo graeca zarudnyi) by Gastrografin®

Objective- In this study, the general anatomical features of the digestive tube and the transit time of the digestive tube of the Zarudni’s spur-thighed tortoises were examined by contrast radiology.Design - Experimental study.Animals - 4 adult female Zarudni’s spur-thighed tortoises (Testudo graeca zarudnyi).Procedures- At a temperature of 25-27°c a set of dorsoventral radiograph was taken to locate the Gastrografin position.Results- The normal gastric, small intestine and large intestine anatomy were obtained and the mean gastric, small intestine and large intestine transit times were 0.2 hr, 2.1 hr, and 27 hr, respectively. Our results showed some differences in the gastrointestinal transit time with that of other species.Conclusion and Clinical Relevance- The noninvasive diagnostic imaging techniques provide detailed information concerning gastrointestinal tract. Since there have not been any anatomical and radiological studies on this species in Iran, results of this study can use as a reference in this species.

Abstract 1262: Type-2 cGMP-dependent protein kinase has a tumor-suppressive role in the colon epithelium

Abstract The cGMP level plays a central role in regulating homeostasis in the colon epithelium and is emerging as a potential target for colon cancer prevention. The signaling components downstream of cGMP, and the tumor suppressive mechanism remain poorly understood. The present study has examined the expression of cGMP-dependent protein kinase isoforms (PKG1, PKG2) in normal intestinal mucosa and in colon cancer from human specimens. Immunohistochemical analysis detected PKG1 in supportive cells in the lamina propria and in smooth-muscle tissue, but not in the epithelium of normal intestine and colon. In contrast, PKG2 was detected exclusively in the epithelium. In colon cancer, PKG1 was restricted to the stroma where it colocalized with vimentin, whereas PKG2 was only detected in the tumor epithelium where it colocalized with cytokeratin. This pattern of PKG isoform expression was similar in the mouse intestine and colon, where only PKG2 was detected in purified colonic crypts. PKG2 knockout (KO) mice were used interrogate possible anti-carcinogenic roles in the colon epithelium. When subjected to the AOM/DSS model of colon carcinogenesis, the PKG2 KO animals produced significantly more polyps than wild type controls (1.75-fold, p=0.0037). While the polyps from PKG2 KO animals were slightly smaller than those from wild type, the trend was not significant (p=0.13). Wild type and PKG2 KO mice responded equally to DSS treatment, but the PKG2-deficient animals exhibited crypt hyperplasia and increased apoptosis in luminal epithelium relative to wild type animals. Taken together these findings suggest that PKG2 has a tumor suppressive role in the colon epithelium, and that reducing the proliferative compartment may be part of the mechanism. Citation Format: Bianca N. Islam, Sarah K. Sharman, Yali Hou, Justin Ashby, Rui Wang, Subbaramiah Sridhar, Ravindra Kolhe, Darren D. Browning. Type-2 cGMP-dependent protein kinase has a tumor-suppressive role in the colon epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1262.

Abstract 979: EGLN inhibition reduces gastrointestinal radiation toxicity and improves survival in a murine model of locally advanced pancreatic cancer

Abstract Rationale: Pancreatic cancer is almost always fatal since chemotherapy is only modestly effective and surgery is often not possible. Radiation therapy (RT) can be a definitive treatment in localized pancreatic cancer, but gastrointestinal toxicity to the nearby duodenum limits its efficacy. There are no known medications that can prevent or treat this gastrointestinal radiotoxicity, but we find that FG-4592, a small molecule inhibitor of EGLN proteins, reduces radiation damage to the intestines by selectively enhancing HIF expression within normal intestines but not within tumors. Methods: We developed a murine model of spontaneous pancreatic cancer (KrasLSL/+; Trp53FL/+; Ptf1aCre/+) that behaves similarly to locally advanced disease. KPC mice were screened for tumors by weekly ultrasounds and upon diagnosis were sequentially assigned to receive one of four possible treatments: Vehicle only (VEH), FG-4592 (FG), RT concurrent with vehicle (RT+VEH), or RT concurrent with FG-4592 (RT+FG). Mice were enrolled on a rolling basis and the total time from diagnosis to treatment end was approximately three weeks. Radiation treatments were planned to administer a total of 75Gy in 15 daily fractions. Results: A total of 70 KPC mice were evaluated. The administration of high-dose radiation therapy of &amp;gt;65Gy (high-dose) with or without radioprotection improved survival compared to all animals that received ≤65Gy (low-dose) of treatment or no RT (37 days [95% CI 33-41] vs. 15 days [10-21] vs. 15 days [10-20], respectively; p=0.005). KPC animals that received dose-escalated radiation lived significantly longer compared to unirradiated controls that received vehicle or FG-4592 alone. The median overall survival was compared amongst the treatment groups and was highest (p&amp;lt;.001) for mice that received RT with FG-4592 for radioprotection (43 days, [95% CI 39-47]) versus RT with vehicle (36 days, [95% CI 10-62]), FG alone (29 days, [95% CI 21-37]), or vehicle alone (9 days, [95% CI 5-13]). High-dose RT reduced death from local progression as determined by reduced jaundice and obstruction on necropsy which was present in 64% (25/39) of mice who did not receive RT versus 0% (0/29) who received RT with or without radioprotection (p=0.02). The addition of FG-4592 to RT further improved survival compared to RT + vehicle controls by eliminating radiation-induced gastrointestinal toxicity. Approximately 56% (5/9) of animals that received high-dose RT without radioprotection died from fatal GI bleeding, whereas no bleeding was seen in animals who received high dose RT+ FG-4592 (P&amp;lt;.0001). Thus, these experiments demonstrate a proof of concept that selective radioprotection of the intestinal tract by EGLN inhibition enables higher, and potentially curative, doses of cytotoxic therapy without any tumor protection. Citation Format: Cullen M. Taniguchi, Tara N. Fujimoto, Lauren E. Colbert, Jessica M. Moilkentine, Amit Deorukhkar, Laura J. Baseler, Ramesh Tailor, Gabriel O. Sawakuchi. EGLN inhibition reduces gastrointestinal radiation toxicity and improves survival in a murine model of locally advanced pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 979.

PO-049 EGFR blockade induces a paneth cell-like phenotype with rewired signalling dependencies in CRC tumoursat maximal response

IntroductionAnti-EGFR therapies with the monoclonal antibodies cetuximab and panitumumab have improved survival in colorectal cancer (CRC) patients; nevertheless, incomplete mass obliteration and eventual relapse are a common setback, even after a plateau of maximal response. Preclinical data suggest that tumour recurrence may be fueled by a reservoir of so-called ‘drug-tolerant persisters’ that engage non-mutational routes of adaptation to therapy. Yet, the molecular underpinnings that sustain residual disease, as well as the strategies to oppose it, are largely unexplored.Material and methodsThe effects of targeted therapies were evaluated in patient-derived xenografts. The biochemical and biological consequences of drug exposure were gauged by immunohistochemistry and morphometric analyses (in vivo), and by time-lapse imaging, Western Blot, Cell Titer-Glo and Caspase-Glo assays (in vitro). Transcriptional perturbations were assessed by microarray analysis and/or RT-qPCR. The activity of transcriptional modulators was measured by reporter assays in vitro.Results and discussionsResidual tumours surviving cetuximab treatment exhibited a quiescent, Wnt-high, and secretory/Paneth cell-like state as a distinctive trait. This pattern outlines that of EGFR-inhibited quiescent stem cells of the normal intestine, suggesting that developmental trajectories are somehow coopted by cancer cells to face external insults. Such phenotype was reversible with drug suspension, pointing to non-genetic plasticity as a determinant of cancer cell reprogramming. Residual tumours also displayed lower expression of EGFR-activating ligands, congruent with reduced EGFR dependency, and showed rewired reliance on compensatory HER2/HER3 activity, as well as persistent PI3K signalling. Mechanistically, the acquisition of Paneth cell-like features was mediated, at least partly, by inactivation of YAP – a key driver of intestinal cell regeneration. Therapeutically, combined blockade of EGFR and PI3K/AKT lessened residual disease burden, but did not lead to long-term disease control. However, treatment with panHER, a mixture of antibodies concurrently targeting EGFR, HER2, and HER3, reduced tumour volumes and delayed tumour relapse after therapy cessation.ConclusionDrug tolerance in cetuximab-sensitive CRC models involves a switch towards a Paneth-cell like state typified by sustained HER2/HER3 and PI3K signalling. Treatment with panHER effectively exhausted residual tumour burden and impeded/delayed late relapse.

PO-330 Investigating tissue- and age-specific microsatellite instability in mice with different levels of MLH1, a central mismatch repair protein

IntroductionLynch syndrome (LS) is an inherited condition in which individuals harbour germline mutations in one or more of the DNA mismatch repair (MMR) genes, typically MLH1 or MSH2, predisposing them to cancers, in particular non-polyposis colorectal cancer. Tumours in LS are characterised by high microsatellite instability (MSI), which reflects defective MMR. MSI in colorectal cancer results from epigenetic inactivation or loss of heterozygosity (LOH) of the wild-type MMR allele, leading to absence of MMR proteins. However, some evidence suggests haploinsufficiency of MMR proteins, i.e. heterozygosity may lead to loss of genome integrity. For instance, Mlh1 +/-mice display increased tumour incidence and individuals with LS show subtle MSI in their peripheral blood leukocytes.Our aim is to understand how MMR protein levels affect in vivo tandem DNA repeat instability. A key outstanding question is why certain tissues, in particular the intestine and colon in humans, are especially prone to MSI. We hypothesise that normal levels of MMR proteins are particularly critical for genome stability in these tissues.Material and methodsWe are using mouse genetics to find out how the levels of MMR proteins contribute to in vivo tandem DNA repeat instability. We are analysing tissue- and age- specific MSI in mice expressing different levels of MMR proteins. We are using single molecule PCR followed by capillary electrophoresis to investigate microsatellite instability. Data is being analysed using Fragman R package. Our group holds a National Animal Experiment Board license to perform animal experiments.Results and discussionsOur preliminary results in old Mlh1 +/-mice show a very subtle but detectable MSI increase in small intestine compared to Mlh1+/+mice, the observed difference being predominantly due to an increase in deletion-mutant alleles.ConclusionOur result show subtle increase in MSI in macroscopically normal intestine of Mlh1 +/-mice, which indicates inactivation of just one Mlh1 allele is sufficient to induce genome instability.

Role of the Wilms' tumor suppressor gene Wt1 in pancreatic development.

The Wilms tumor suppressor gene (Wt1) encodes a transcription factor involved in the development of a number of organs, but the role played by Wt1 in pancreatic development is unknown. The pancreas contains a population of pancreatic stellate cells (PSC) very important for pancreatic physiology. We described elsewhere that hepatic stellate cells originate from the WT1-expressing liver mesothelium. Thus, we checked if the origin of PSCs was similar. WT1 expression is restricted to the pancreatic mesothelium. Between embryonic day (E) 10.5 and E15.5, this mesothelium gives rise to mesenchymal cells that contribute to a major part of the PSC and other cell types including endothelial cells. Most WT1 systemic mutants show abnormal localization of the dorsal pancreas within the mesentery and intestinal malrotation by E14.0. Embryos with conditional deletion of WT1 between E9.5 and E12.5 showed normal dorsal pancreatic bud and intestine, but the number of acini in the ventral bud was reduced approximately 30% by E16.5. Proliferation of acinar cells was reduced in WT1 systemic mutants, but pancreatic differentiation was not impaired. Thus, mesothelial-derived cells constitute an important subpopulation of pancreatic mesodermal cells. WT1 expression is not essential for pancreas development, although it influences intestinal rotation and correct localization of the dorsal pancreas within the mesogastrium. Developmental Dynamics 247:924-933, 2018. © 2018 Wiley Periodicals, Inc.

EXPRESSION OF MUC2 IN NORMAL GASTRIC MUCOSA, INTESTINAL METAPLASIA AND GASTRIC CARCINOMA BY IMMUNOHISTOCHEMISTRY.

14Mar 2018 EXPRESSION OF MUC2 IN NORMAL GASTRIC MUCOSA, INTESTINAL METAPLASIA AND GASTRIC CARCINOMA BY IMMUNOHISTOCHEMISTRY. Shuaeb Bhat , R. P. Tania , Irfan Ansari , Shaffy Thukral , Saleem Hussain , Shareefa Akhter , Fatima Rahim and B. R. Sahaf. Senior Resident, Department of Hematopathology, Sher-I-Kashmir Institute Of Medical Science, Soura, India. Consultant Pathologist, Department of Pathology, Shusrusha Hospital, Mumbai, India. Associate Professor, Department of Pathology, College of Medicine, Taibah University. Assistant Professor, Department of Pathology, Adesh Medical College, Bathinda, India. Technologist, Department of Hematopathology, Sher-I-Kashmir Institute Of Medical Science, Soura, India. Lecturer, Department of Pathology, SKIMS Medical College, Bemina, J &K, India.

Intestinal absorption differences of major bioactive compounds of Gegenqinlian Decoction between normal and bacterial diarrheal mini-pigs in vitro and in situ

Intestinal condition plays an important role in drug absorption and metabolism, thus the effects of varied gastrointestinal diseases such as infectious diarrhea on the intestinal function are crucial for drug absorption. However, due to the lack of suitable models, the differences of absorption and metabolism of drugs between the diarrheal and normal intestines are rarely reported. Thus, in this study, Escherichia coli diarrhea model was induced in mini-pigs and single-pass intestinal perfusion and intestinal mucosal enzyme metabolism experiments were conducted. A simple and rapid ultrahigh performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to determine the concentrations of 9 major components in Gegen Qinlian decoction (GQD). Samples were pretreated by protein precipitation with methanol and naringin and prednisolone were used as internal standards. The validated method demonstrated adequate sensitivity, selectivity, and process efficiency for the bioanalysis of 9 compounds. Results of intestinal perfusion showed that puerarin, daidzein, daidzin and baicalin and berberine were absorbed faster in diarrheal jejunum than in normal intestines (p < 0.05). However, puerarin, daidzin and liquiritin were metabolized more slowly in diarrheal intestine after incubation compared with the normal group (p < 0.05). The concentrations of daidzein in both perfusion and metabolism and wogonin in metabolism were significantly increased (p < 0.05). In conclusion, absorption and metabolism of GQD were significantly different between the diarrheal and normal intestines, which suggest that bacterial diarrheal mini-pigs model can be used in the intestinal absorption study and is worthy to be applied in the other intestinal absorption study of anti- diarrheal drugs.

Transplantation of placenta-derived multipotent cells in rats with dimethylhydrazine-induced colon cancer decreases survival rate.

Transplantation of placenta-derived multipotent cells (PDMCs) is a promising treatment method for many diseases. However, the impact of PDMCs on colon cancer has not yet been studied. PDMCs were obtained from rat placentas by culturing tissue explants. Colon cancer was experimentally induced in male albino Wistar rats by administering 20 mg/kg dimethylhydrazine (DMH) once a week for 20 consecutive weeks. The administration of the PDMCs was performed at the 20th week after the first DMH injection. The number and size of each tumour lesion were calculated in the 5th week after transplantation. The tumour type was determined by standard histological methods. To study the engraftment of PDMCs in the body of rats, the cells were transduced with enhanced green fluorescent protein. Cell engraftment was determined by assessing the presence of EGFP by PCR and immunohistochemistry. Survival of all rats was monitored daily. Allogeneic transplantation of PDMCs to rats at middle phase of DMH-induced colon carcinogenesis did not significantly influence the number of neoplasms and the parameters of mean and total tumour area, but led to an increase in size of the most invasiveness tumours. Intravenous allogeneic transplantation of PDMCs reduced the survival rate of rats with colon cancer by 17 days. PDMCs from rats engrafted into tissues of the normal intestine, tumours, lungs, liver, and spleen of rats for five weeks after intravenous transplantation. These results suggest that intravenous allogeneic transplantation of PDMCs promotes colon cancer progression and has a negative impact on survival of rats.

Proinflammatory Role of Monocyte-Derived CX3CR1int Macrophages in Helicobacter hepaticus-Induced Colitis.

ABSTRACTCells of the monocyte/macrophage lineage play important roles in the pathogenesis of inflammatory bowel diseases, but they are also present in the normal healthy intestine, where they are critical for maintaining homeostasis. It has been unclear whether the proinflammatory roles of intestinal macrophages reflect altered behavior of the existing resident cells, or whether they involve recruitment of a distinct cell type. Here, we have explored these ideas using the model of colitis induced by Helicobacter hepaticus in the context of neutralization or deletion of interleukin-10 (IL-10). Granulocytes and monocytes made up most of the inflammatory myeloid infiltrates found in the colon of H. hepaticus-infected colitic mice, rising to a peak within 2 weeks of H. hepaticus inoculation but taking several months to resolve completely. The inflammatory response was dependent on the combined presence of H. hepaticus and absence of IL-10 and was accompanied by increased production of inflammatory mediators such as IL-1β, tumor necrosis factor alpha (TNF-α), IL-6, and IL-23p19 by infiltrating myeloid cells, mostly relatively immature cells of the macrophage lineage that express intermediate levels of CX3CR1. In contrast, the population of mature CX3CR1hi macrophages did not expand as markedly during colitis, and these cells made little contribution to inflammatory mediator production. Taking into account their numerical dominance in the myeloid compartment, we conclude that newly recruited monocytes are the main source of proinflammatory mediators in colitis induced in the absence of IL-10 signaling and that altered behavior of mature macrophages is not a major component of this pathology.

Non-Canonical Hedgehog Signaling Is a Positive Regulator of the WNT Pathway and Is Required for the Survival of Colon Cancer Stem Cells

Colon cancer is a heterogeneous tumor driven by asubpopulation of cancer stem cells (CSCs). To study CSCs in colon cancer, we used limiting dilution spheroid and serial xenotransplantation assays to functionally define the frequency of CSCs in a panel of patient-derived cancer organoids. These studies demonstrated cancer organoids to be enriched for CSCs, which varied in frequency between tumors. Whole-transcriptome analysis identified WNT and Hedgehog signaling components to be enhanced in CSC-enriched tumors and in aldehyde dehydrogenase (ALDH)-positive CSCs. Canonical GLI-dependent Hedgehog signaling is a negative regulator of WNT signaling in normal intestine and intestinal tumors. Here, we show that Hedgehog signaling incolon CSCs is autocrine SHH-dependent, non-canonicalPTCH1 dependent, and GLI independent. Inaddition, using small-molecule inhibitors and RNAi against SHH-palmitoylating Hedgehog acyltransferase (HHAT), we demonstrate that non-canonical Hedgehog signaling is a positive regulator of WNT signaling and required for colon CSC survival.

Findings of Retrograde Contrast Study Through Double-balloon Enteroscopy Predict the Risk of Bowel Resections in Patients with Crohn's Disease with Small Bowel Stenosis.

Patients with Crohn's disease (CD) with small bowel strictures are at risk of surgery. Double-balloon enteroscopy (DBE) can evaluate the status of the small intestine, and retrograde contrast through the scope enables the surgeon to obtain information beyond the reach of the scope. This study aimed to examine whether a retrograde contrast study through DBE could be used as a predictor of subsequent surgery in patients with CD with small intestinal strictures. The findings of DBE with retrograde contrast in 48 patients CD with small bowel strictures were examined. Of the 48 patients, 14 (29%) underwent surgery for small intestinal strictures during a median observation period of 2.4 years (interquartile range: 1.4-3.7 yr). According to the results of the multivariate analysis, a maximum length of strictures ≥20 mm and the ratio of the maximum diameter of prestenotic dilations to the diameter of the normal small intestine ≥1.4 were independent risk factors of surgery for small intestinal strictures (risk ratio = 7.6 [95% confidence interval, 1.8-42.0], P = 0.006; and risk ratio = 52.0 [95% confidence interval, 3.5-2485.1], P = 0.002, respectively). The latter predicted subsequent surgery with 92% sensitivity and 88% specificity. Cumulative surgery-free rates were discriminated significantly according to the presence or absence of these 2 risk factors (log-rank test: P < 0.001). Findings of retrograde contrast through DBE are helpful to predict risk of surgery in patients with CD with small intestinal strictures.

The role of intermediate filaments in maintaining integrity and function of intestinal epithelial cells after massive bowel resection in a rat.

Intermediate filaments (IFs) are a part of the cytoskeleton that extend throughout the cytoplasm of all cells and function in the maintenance of cell-shape by bearing tension and serving as structural components of the nuclear lamina. In normal intestine, IFs provide a tissue-specific three-dimensional scaffolding with unique context-dependent organizational features. The purpose of this study was to evaluate the role of IFs during intestinal adaptation in a rat model of short bowel syndrome (SBS). Male rats were divided into two groups: Sham rats underwent bowel transection and SBS rats underwent a 75% bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined 2weeks after operation. Illumina's Digital Gene Expression (DGE) analysis was used to determine the cytoskeleton-related gene expression profiling. IF-related genes and protein expression were determined using real-time PCR, Western blotting and immunohistochemistry. Massive small bowel resection resulted in a significant increase in enterocyte proliferation and concomitant increase in cell apoptosis. From the total number of 20,000 probes, 16 cytoskeleton-related genes were investigated. Between these genes, only myosin and tubulin levels were upregulated in SBS compared to sham animals. Between IF-related genes, desmin, vimentin and lamin levels were down-regulated and keratin and neurofilament remain unchanged. The levels of TGF-β, vimentin and desmin gene and protein were down-regulated in resected rats (vs sham animals). Two weeks following massive bowel resection in rats, the accelerated cell turnover was accompanied by a stimulated microfilaments and microtubules, and by inhibited intermediate filaments. Resistance to cell compression rather that maintenance of cell-shape by bearing tension are responsible for contraction, motility and postmitotic cell separation in a late stage of intestinal adaptation.

Rack1 maintains intestinal homeostasis by protecting the integrity of the epithelial barrier.

Previously, we generated mouse models of Rack1 deficiency to identify key functions for Rack1 in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia. However, other than low body weight, we did not detect an overt phenotype in mice constitutively deleted of Rack1 in intestinal epithelia ( vil-Cre: Rack1fl/fl mice), presumably because Rack1 was deleted in <10% of the total surface area of the epithelia. To assess the effect of Rack1 loss throughout the entire intestinal epithelia, we generated another mouse model of Rack1 deficiency, vil-Cre-ERT2: Rack1fl/fl. Within 5-10 days of the initial tamoxifen treatment, the mice lost over 20% of their body weight, developed severe diarrhea that for some was bloody, became critically ill, and died, if not euthanized. Necropsies revealed mildly distended, fluid-, gas-, and sometimes blood-filled loops of small and large bowel, inguinal lymphadenopathy, and thrombocytosis. Rack1 was deleted in nearly 100% of the epithelia in both the small intestine and colon when assessed by immunofluorescent or immunoblot analyses. Rack1 expression in other tissues and organs was not different than in control mice, indicating tissue specificity of the recombination. Histopathology revealed a patchy, erosive, hemorrhagic, inflammatory enterocolitis with denuded, sloughed off surface epithelium, and crypt hyperplasia. These results suggest a protective function for Rack1 in maintaining the integrity of intestinal epithelia and for survival. NEW & NOTEWORTHY Our findings reveal a novel function for Rack1 in maintaining intestinal homeostasis by protecting the epithelial barrier. Rack1 loss results in a patchy, erosive, hemorrhagic, inflammatory enterocolitis, which resembles that of inflammatory bowel diseases (IBD) in humans. Understanding mechanisms that protect barrier function in normal intestine and how loss of that protection contributes to the pathogenesis of IBD could lead to improved therapies for these and other erosive diseases of the gastrointestinal tract.

Pathogen invasion changes the intestinal microbiota composition and induces innate immune responses in the zebrafish intestine

Numerous bacteria are harbored in the animal digestive tract and are impacted by several factors. Intestinal microbiota homeostasis is critical for maintaining the health of an organism. However, how pathogen invasion affects the microbiota composition has not been fully clarified. The mechanisms for preventing invasion by pathogenic microorganisms are yet to be elucidated. Zebrafish is a useful model for developmental biology, and studies in this organism have gradually become focused on intestinal immunity. In this study, we analyzed the microbiota of normal cultivated and infected zebrafish intestines, the aquarium water and feed samples. We found that the predominant bacteria in the zebrafish intestine belonged to Gammaproteobacteria (67%) and that feed and environment merely influenced intestinal microbiota composition only partially. Intestinal microbiota changed after a pathogenic bacterial challenge. At the genus level, the abundance of some pathogenic intestinal bacteria increased, and these genera included Halomonas (50%), Pelagibacterium (3.6%), Aeromonas (2.6%), Nesterenkonia (1%), Chryseobacterium (3.4‰), Mesorhizobium (1.4‰), Vibrio (1‰), Mycoplasma (0.7‰) and Methylobacterium (0.6‰) in IAh group. However, the abundance of some beneficial intestinal bacteria decreased, and these genera included Nitratireductor (0.8‰), Enterococcus (0.8‰), Brevundimonas (0.7‰), Lactococcus (0.7‰) and Lactobacillus (0.4‰). Additionally, we investigated the innate immune responses after infection. ROS levels in intestine increased in the early stages after a challenge and recovered subsequently. The mRNA levels of antimicrobial peptide genes lectin, hepcidin and defensin1, were upregulated in the intestine after pathogen infection. These results suggested that the invasion of pathogen could change the intestinal microbiota composition and induce intestinal innate immune responses in zebrafish.

The length of the large intestine in children determined by computed tomography scan.

Little information is available on the length of the normal large intestine and its component parts in children. This information would be useful for procedures such as colonoscopy. The aim of this study was to investigate the length of the large intestine and its component parts in New Zealand children. Archival deidentified pediatric supine abdominopelvic computed tomography (CT) scans were retrospectively analyzed. After exclusion criteria, a total of 112 scans (57 males and 55 females) were included in the study and divided into three age groups: 0-2 years (n = 33), 4-6 years (n = 40), and 9-11 years of age (n = 39). The length of the large bowel increased from a mean of 52 cm in children aged <2 years to 73 cm at 4-6 years and 95 cm at 9-11 years. In all age groups, the transverse colon was the longest segment, contributing ∼30% of the total length of the large bowel. In comparison to total large bowel length, the mean proportional length of the rectum (9-12%), sigmoid colon (23-27%), descending colon (19-22%), transverse colon (27-32%), and ascending colon (14-17%) varied little between the three age groups. There were no significant differences between males and females in all age groups. The cecum was located in the right upper quadrant in 27% of children aged 0-2 years but in the right lower quadrant in all 9-11 year olds. These data provide useful information on the length of the large intestine and its component parts in living children, which are particularly relevant to pediatric colonoscopy and surgery. Clin. Anat. 30:887-893, 2017. © 2017 Wiley Periodicals, Inc.