Small Intestinal Crypt Cells Research Articles

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

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

Human Defensin-5 (HD-5) and HD-6 mRNA Produced by Intestinal Crypt Paneth Cells are Decreased in Fecal Samples of Patients Positive for SARS-CoV2

Abstract Alpha-defensins (AD) are antimicrobial peptides (AMP) that are found on mucosal surfaces, providing innate immune defense against pathogens. Human defensin-5 (HD-5) and HD-6 are AD produced only by Paneth cells of small intestinal crypts. SARS-CoV2 virus has been shown to infect the small intestinal cells via the ACE2 receptor and suppress inflammation in the gut. HD-5 and HD-6 bind ACE2 to prevent entry to intestinal cells. Based on the observation that human beta defensin-1 (hBD-1) is downregulated by influenza virus in airway epithelial cells, we hypothesized that HD-5 and HD-6 could be similarly downregulated by SARS-CoV2. To examine this in situ, we sampled patient cohorts with similar co-morbidities, with and without SARS-CoV2 positivity, for HD-5 and HD-6 gene expression via qRT-PCR. Stool samples were collected from all patients, diluted in Qiagen RNA Later and frozen at −80°C prior to total RNA extraction. Results revealed a significant decrease in both HD-5 (p=0.000006) and HD-6 (p=0.002) of SARS-CoV2 positive patients compared with non-Covid19 patients negative for SARS-CoV2. The results were consistent in fecal swabs or fecal streams. Gene expression in other significant markers of inflammation were also blunted: sPLA-2 (p=0.006), alpha-1-anti-trypsin (p=0.0001) and trypsin 2 (p=0.005). Other gut inflammatory markers PSTI, HIP/PAP, trypsin 3 and IL-8 were unchanged. There was a trend in IL-8 upregulation in Covid19 patients. This is the first demonstration of defensin mRNA detection in stool samples. The results suggest that decreasing AMPs may be a mechanism by which the virus increases its chance for survival. Supported by R21DE028378

Olfactomedin 4 Is Not a Precise Marker for Human Intestinal Stem Cells, But Is Involved in Intestinal Carcinogenesis

Olfactomedin 4 Is Not a Precise Marker for Human Intestinal Stem Cells, But Is Involved in Intestinal Carcinogenesis

NF-κB determines Paneth versus goblet cell fate decision in the small intestine.

ABSTRACTAlthough the role of the transcription factor NF-κB in intestinal inflammation and tumor formation has been investigated extensively, a physiological function of NF-κB in sustaining intestinal epithelial homeostasis beyond inflammation has not been demonstrated. Using NF-κB reporter mice, we detected strong NF-κB activity in Paneth cells, in ‘+4/+5’ secretory progenitors and in scattered Lgr5+ crypt base columnar stem cells of small intestinal (SI) crypts. To examine NF–κB functions in SI epithelial self-renewal, mice or SI crypt organoids (‘mini-guts’) with ubiquitously suppressed NF-κB activity were used. We show that NF-κB activity is dispensable for maintaining SI epithelial proliferation, but is essential for ex vivo organoid growth. Furthermore, we demonstrate a dramatic reduction of Paneth cells in the absence of NF-κB activity, concomitant with a significant increase in goblet cells and immature intermediate cells. This indicates that NF-κB is required for proper Paneth versus goblet cell differentiation and for SI epithelial homeostasis, which occurs via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The current study thus presents evidence for an important role for NF-κB in intestinal epithelial self-renewal.

Coronavirus Disease 2019 and Gastrointestinal Involvement: A Systematic Review

<p><strong>Introduction: </strong>The World Health Organization (WHO) announced the Coronavirus 2019 (COVID-19) as a Public Health Emergency of International Concern (PHEIC) toward the end of January 2020. There is still limited evidence to explain the gastrointestinal involvement in COVID-19. In this study, we aimed to further investigate current evidence describing the gastrointestinal involvement in COVID-19 patients.</p><p><strong>Methods: </strong>This systematic review has been registered in PROSPERO (CRD42020181584). A systematic search of literature for observational and randomized controlled trial was conducted in PubMed, PubMed central, and Google Scholar through April 16, 2020. Two reviewers independently searched and selected. The risk of bias was evaluated using the Newcastle-Ottawa Quality assessment tool.<strong> </strong></p><p><strong>Results: </strong>A total of 1,480 articles were screened from which 12 articles with 5584 subjects were selected. SARS-CoV-2 can invade human body by binding to angiotensin converting enzyme 2 (ACE-2) receptor which also located to small intestinal epithelial cells, crypt cells and colon. The virus itself may cause disorders of the intestinal flora. The diagnosis should be based on a set of symptoms diarrhoea, nausea, vomiting, abdominal discomfort or pain, combined with positivity of faecal PCR test. Treatment of COVID-19 mainly is supportive care. The probiotic may modulate the gut microbiota to alter the gastrointestinal symptoms and reduced enteritis, ventilator associated pneumonia, and reverse certain side effect of antibiotics<strong>.</strong></p><p><strong>Conclusion: </strong>Our synthesis of literature showed that there was no good evidence yet in overall area of gastrointestinal manifestations in COVID-19. Future research is needed to explore all areas, especially in mechanism and treatments</p>

The vanillin derivative VND3207 protects intestine against radiation injury by modulating p53/NOXA signaling pathway and restoring the balance of gut microbiota

The intestine is a highly radiosensitive tissue that is susceptible to structural and functional damage due to systemic as well as localized radiation exposure. Unfortunately, no effective prophylactic or therapeutic agents are available at present to manage radiation-induced intestinal injuries. We observed that the vanillin derivative VND3207 improved the survival of lethally irradiated mice by promoting intestinal regeneration and increasing the number of surviving crypts. Pre-treatment with VND3207 significantly increased the number of Lgr5+ intestinal stem cells (ISCs) and their daughter cells, the transient Ki67+ proliferating cells. Mechanistically, VND3207 decreased oxidative DNA damage and lipid peroxidation and maintained endogenous antioxidant status by increasing the level of superoxide dismutase and total antioxidant capacity. In addition, VND3207 maintained appropriate levels of activated p53 that triggered cell cycle arrest but were not sufficient to induce NOXA-mediated apoptosis, thus ensuring DNA damage repair in the irradiated small intestinal crypt cells. Furthermore, VND3207 treatment restores the intestinal bacterial flora structures altered by TBI exposure. In conclusion, VND3207 promoted intestinal repair following radiation injury by reducing reactive oxygen species-induced DNA damage and modulating appropriate levels of activated p53 in intestinal epithelial cells.

Effects of chito-oligosaccharide supplementation with low or medium molecular weight and high degree of deacetylation on growth performance, nutrient digestibility and small intestinal morphology in weaned pigs

Chito-oligosaccharide (COS) supplementation in nursery diets has been found to improve piglets’ performance. However, little is known on which COS characteristics are responsible for its effect. The present study was designed to further study how COS characteristics: molecular weight (MW) and degree of deacetylation (DD); affect the growth performance, nutrient digestibility, and small intestinal modifications in weaning pigs. For this purpose, 48 weanling pigs were divided into 4 groups (12 animals per group) and received either a basal diet or a basal diet supplemented with 150 mg/kg of COS which differed in MW and DD: 8 kDa and ~90% DD (8 kDa~90%), 65 kDa and ~80% DD (65 kDa~80%), and 65 kDa and ~90% DD (65 kDa~90%). The experiment lasted 56 days. There were treatment effects on pigs’ body weight gain (BWG), average daily gain (ADG), average daily feed intake (ADFI), blood urea nitrogen (BUN), digestibility of crude protein, fat and calcium, small intestinal morphology and crypt cell proliferation (P < .05, respectively). Throughout the study, pigs supplemented with 8 kDa~90% COS showed significantly enhanced BWG, ADG, and ADFI when compared to those supplemented with 65 kDa~80% COS (P < .05, respectively). The enhanced growth performances in pigs supplemented with 8 kDa~90% COS was supported by an increase in ADFI (P < .05), and improved ileal digestibility of crude protein and crude fat (P < .01, respectively). Pigs supplemented with 8 kDa~90% COS also had increased absorption capacity as indicated by elongated villus height and increased villus height to crypt depth ratio of the small intestinal segments (P < .05, respectively), and more active cell division as indicated by increased Ki-67 marker at jejunal crypt cells (P < .05) on day 28 and 56 of the experiment. These data suggest that 8 kDa~90% might be the most effective form of COS to improve feed intake, and promote nutrient digestibility and absorption when supplemented at 150 mg/kg in the diet.

Unique regulation of Na-glutamine cotransporter SN2/SNAT5 in rabbit intestinal crypt cells during chronic enteritis.

The only Na‐nutrient cotransporter described in mammalian small intestinal crypt cells is SN2/SNAT5, which facilitates glutamine uptake. In a rabbit model of chronic intestinal inflammation, SN2 stimulation is secondary to an increase in affinity of the cotransporter for glutamine. However, the immune regulation of SN2 in the crypt cells during chronic intestinal inflammation is unknown. We sought to determine the mechanism of regulation of Na‐nutrient cotransporter SN2 by arachidonic acid metabolites in crypt cells. The small intestines of New Zealand white male rabbits were inflamed via inoculation with Eimeria magna oocytes. After 2‐week incubation, control and inflamed rabbits were subjected to intramuscular injections of arachidonyl trifluoromethyl ketone (ATK), piroxicam and MK886 for 48 hrs. After injections, the rabbits were euthanized and crypt cells from small intestines were harvested and used. Results: Treatment of rabbits with ATK prevented the release of AA and reversed stimulation of SN2. Inhibition of cyclooxygenase (COX) with piroxicam did not affect stimulation of SN2. However, inhibition of lipoxygenase (LOX) with MK886, thus reducing leukotriene formation during chronic enteritis, reversed the stimulation of SN2. Kinetic studies showed that the mechanism of restoration of SN2 by ATK or MK886 was secondary to the restoration of the affinity of the cotransporter for glutamine. For all treatment conditions, Western blot analysis revealed no change in SN2 protein levels. COX inhibition proved ineffective at reversing the stimulation of SN2. Thus, this study provides evidence that SN2 stimulation in crypt cells is mediated by the leukotriene pathway during chronic intestinal inflammation.

Oral Administration of Polaprezinc Attenuates Fluorouracil-induced Intestinal Mucositis in a Mouse Model.

5-Fluorouracil (5-FU) has broadly been applied to treat colorectal cancer as one of the most effective chemotherapeutic agents. However, it frequently causes intestinal mucosal injury and related side effects, such as abdominal pain and diarrhoea, which limit the use of 5-FU in a clinic setting. Polaprezinc has gradually become known as a mucosal protective agent for the management of gastric ulcer. This study aimed to investigate the prophylactic efficacy of Polaprezinc administered orally against intestinal mucositis induced by 5-FU in mice on the condition that the antitumour effect could not be compromised. We induced intestinal mucositis in SPF-grade ICR mice with 5-FU, and evaluated intestinal damage in the absence or presence of Polaprezinc. We examined the score of diarrhoea and the loss of weight after the 5-FU treatment and assessed the integrity of villus and the proliferation of small intestine crypt cells by haematoxylin and eosin staining and PCNA immunohistochemical detection. The antitumour effect of 5-FU on colorectal cancer was assessed with or without Polaprezinc in a xenograft model. The result showed that Polaprezinc significantly reduced the elevated diarrhoea score and the body-weight loss caused by 5-FU abolished histological abnormality and crypt cell hypoproliferation in a dose-dependent manner, without affecting 5-FU efficacy on colon xenograft tumour in mice. We conclude that Polaprezinc could inhibit 5-FU-induced diarrhoea and alleviate the weight loss during 5-FU chemotherapy, as a possible candidate for treatment and prevention of intestinal mucositis, through protecting intestinal mucosa and improving the quality of life after chemotherapy.

Abstract 5022: Keratin15 (Krt15) + are radio resistant and tumor-initiating cells in the mouse small intestine

Abstract Background: Intestinal stem cells regulate the emergence of different cell lineages across the crypt-villus axis and orchestrate cellular renewal and replenishment following injury (e.g. radiation). Two major ISC have been identified to date: a rapidly cycling crypt base columnar (CBC) stem cell and a more quiescent reserve stem cell located at the +4 position above the crypt base. CBCs marked by the Wnt target gene Lgr5 are dispensable for homeostasis and are quantitatively ablated by radiation injury. In contrast, quiescent ISCs are indispensable for homeostasis, regenerate CBCs, and are radioresistant. Keratins (Krt) are intermediate filaments required for architectural and mechanical support, but also contribute to cell migration and intracellular signaling. Interestingly, Krt15 has been characterized as a stem cell marker in the hair follicle. Here we described a radio-resistant Krt15+ stem cell population in the small intestine and demonstrate that cells within this population are capable of tumor initiation. Methods: Krt15-CrePR1 mice were crossed with Rosa26 reporter mice to perform lineage-tracing experiments in the mouse small intestine. High-dose radiation was used to investigate the role of Krt15 in tissue regeneration. Finally, Krt15-CrePR1 mice were crossed with Apcfl/fl mice to characterize the tumor-initiating properties of Krt15+ cells. Results: We observed that Krt15 marks a subpopulation of long-lived small intestinal crypt cells that are able to give rise to all differentiated lineages supporting their self-renewing and multipotent capacities. Interestingly, we observed that Krt15+ cells are able to form 3D organoids when seeded in Matrigel as a single cell suspension. Irradiation of Krt15-CrePR1;R26mTomato/mGFP mice with 12 Gy revealed that Krt15+ cells are radio-resistant and participate in tissue regeneration. Interestingly, Krt15 deficiency impairs tissue regeneration following high-dose radiation. Indeed, irradiated Krt15-/- mice display reduced crypt regeneration, crypt length, microcolonies and BrdU incorporation when compared to irradiated Krt15+/+ mice. Finally, we induced Apc loss in Krt15+ cells and observed formation of adenomas as well as adenocarcinomas in all Krt15-CrePR1;Apcfl/fl mice. Metastases were detected in some Krt15-CrePR1;Apcfl/fl mice. Conclusion: We have identified a long-lived Krt15+ subpopulation of intestinal crypt cells displaying stem cell-like properties that are radio-resistant and tumor initiating. This new stem cell population may open new avenues for therapeutic targeting in colon cancer. Citation Format: Veronique Giroux, Julien Stephan, Kathryn E. Hamilton, Priya Chatterjee, Sarah Andres, Andres J. Klein-Szanto, Christopher J. Lengner, Anil K. Rustgi. Keratin15 (Krt15) + are radio resistant and tumor-initiating cells in the mouse small intestine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5022. doi:10.1158/1538-7445.AM2017-5022

Effects of dietary levels of chito-oligosaccharide on ileal digestibility of nutrients, small intestinal morphology and crypt cell proliferation in weaned pigs

There is continued need for novel agents to improve intestinal function in weaned pigs. Some evidence suggests that chito-oligosaccharide (COS) supplements may enhance pigs’ intestinal function after weaning. The present study was designed to examine the effect of COS supplementation on growth performance, nutrient digestibility and small intestinal functions in weaned pigs as an effective alternative to antibiotic addition in post-weaning diets. For the experiment, weanling pigs were divided into 5 groups (13–14 animals per group) and received either a basal diet, a supplemented diet with 75, 150 or 225mg/kg COS, or a supplemented diet with 110mg/kg lincomycin for 56 days. Growth, feed efficiency, hematological and biochemical profiles, nutrient's ileal digestibility, small intestinal morphology and crypt cell proliferation were measured at 28 and 56 days of the experiment. Pigs supplemented with 150mg/kg COS or lincomycin showed: (i) consistently more digestible ileal contents (e.g. crude protein, crude fat, ash, calcium, and phosphorus), (ii) increased absorption capacity (e.g. increased villus height and the villus height/crypt depth ratio for three intestinal segments) on day 28 of the experiment and (iii) more active cell division (as indicated by Ki-67 marker of duodenal and jejunal crypt cells) on day 56 of the experiment (P<0.05, respectively). These data suggest that 150mg/kg COS might be a useful dietary supplement to promote nutrient absorption and digestibility efficiency.

MTOR disruption causes intestinal epithelial cell defects and intestinal atrophy postinjury in mice.

Intestinal stem cells (ISCs) drive small intestinal epithelial homeostasis and regeneration. Mechanistic target of rapamycin (mTOR) regulates stem and progenitor cell metabolism and is frequently dysregulated in human disease, but its physiologic functions in the mammalian small intestinal epithelium remain poorly defined. We disrupted the genes mTOR, Rptor, Rictor, or both Rptor and Rictor in mouse ISCs, progenitors, and differentiated intestinal epithelial cells (IECs) using Villin-Cre. Mutant tissues and wild-type or heterozygous littermate controls were analyzed by histologic immunostaining, immunoblots, and proliferation assays. A total of 10 Gy irradiation was used to injure the intestinal epithelium and induce subsequent crypt regeneration. We report that mTOR supports absorptive enterocytes and secretory Paneth and goblet cell function while negatively regulating chromogranin A-positive enteroendocrine cell number. Through additional Rptor, Rictor, and Rptor/Rictor mutant mouse models, we identify mechanistic target of rapamycin complex 1 as the major IEC regulatory pathway, but mechanistic target of rapamycin complex 2 also contributes to ileal villus maintenance and goblet cell size. Homeostatic adult small intestinal crypt cell proliferation, survival, and canonical wingless-int (WNT) activity are not mTOR dependent, but Olfm4(+) ISC/progenitor population maintenance and crypt regeneration postinjury require mTOR. Overall, we conclude that mTOR regulates multiple IEC lineages and promotes stem and progenitor cell activity during intestinal epithelium repair postinjury.

IGF binding protein-4 is required for the growth effects of glucagon-like peptide-2 in murine intestine.

Glucagon-like peptide-2 (GLP-2) is an enteroendocrine hormone that stimulates the growth of the intestinal epithelium. We have previously demonstrated that GLP-2 exerts its intestinotropic effect through an indirect mechanism that requires both IGF-1 and the intestinal epithelial IGF-1 receptor. However, the biological activity of IGF-1 is modulated by IGF binding proteins (IGFBPs), including IGFBP-4, which is highly expressed in the intestine. To determine the role of IGFBP-4 in the tropic effects of GLP-2, IGFBP-4 knockout (KO) and control mice were treated with degradation-resistant GLP-2 or vehicle for 10 days. Comparable levels of IGFBP-1-3/5-7 mRNAs were observed in the intestinal mucosa of all animals. IGFBP-4 KO mice had greater small intestinal weight and length, and deeper crypts (P < .05) as compared with controls, suggesting that IGFBP-4 has an inhibitory role in basal intestinal growth. However, small intestinal weight, crypt-villus height and crypt cell proliferation increased in response to GLP-2 in control mice (P < .05), and these changes were abrogated with IGFBP-4 KO. In contrast, pregnancy-associated plasma protein-A KO mice, which have increased levels of circulating IGFBP-4, demonstrated a normal intestinotropic response to GLP-2. Finally, GLP-2 treatment of control mice significantly increased IGFBP-4 mRNA expression in the jejunal mucosa (P < .05), a finding that was recapitulated by GLP-2 treatment of fetal rat intestinal cells in culture (10(-8)M for 2 h; P < .05). Collectively, these results indicate that the IGF-I-modulating protein, IGFBP-4, exerts a negative effect on basal intestinal growth but plays a positive regulatory role in the intestinotropic actions of GLP-2.

P.5.f.001 Correlations between pain intensity and cognitive dysfunction in surgery

Intrauterine growth retardation (IUGR) infants have impaired gastrointestinal function with resultant feeding difficulties and predisposition to necrotizing enterocolitis. Supplemented amniotic fluid swallowed by the developing fetus is a potential prenatal treatment for IUGR. Rabbits have naturally occurring IUGR fetuses based on uterine position. To determine intestinal response to epidermal growth factor (EGF) infusion, this rabbit model of IUGR was studied.Eight pregnant rabbits underwent placement of intraamniotic catheters into 2 normal and 2 IUGR fetuses per mother on gestational day 24 of a 31-day gestation. Miniosmotic pumps infused either EGF (about 300 μg/kg/d) or control solution forming 4 study groups (EGF-Favored [Fav] v. Cont-Fav; EGF-IUGR v. Cont-IUGR). On gestational day 31, the fetal gastrointestinal tracts were harvested for analysis. Intestinal epithelial cell proliferation was studied by 5-bromo-2-deoxyuridine (BrdU) incorporation, villus heights were measured, and EGF mRNA was measured by reverse transcriptase polymerase chain reaction (RT-PCR). Statistical analysis was performed using Students’ t test.Fetal survival rate was 87%. EGF-IUGR fetal weights were increased compared with Cont-IUGR fetuses. EGF infusion significantly increased IUGR fetal small intestinal villus height and BrdU-positive small intestinal (SI) crypt cells, all approaching Cont-Fav levels. EGF mRNA was expressed throughout the gastrointestinal tract.Supplemental amniotic EGF normalizes fetal weight and intestinal proliferation in the IUGR fetal rabbit. The inclusion of EGF in supplemental amniotic feeding solutions is supported.

Acid-shock ofCampylobacter jejuniinduces flagellar gene expression and host cell invasion

The bacterial pathogen Campylobacter jejuni is the leading cause of foodborne gastroenteritis in the developed world, with the organism being transmitted by ingestion of contaminated and undercooked poultry. Exposure to acid is an inevitable stressor for C. jejuni during gastric passage, yet the effect of low pH on C. jejuni virulence is still poorly understood. Here, we investigate the effect of acid-shock on C. jejuni viability, gene expression and host-cell invasion. C. jejuni strain NCTC 11168 survived acid exposure at pH 3.5 and above for up to 30 min without a drop in viability, and this exposure induced the expression of flagellar genes transcribed from σ(54)-dependent promoters. Furthermore, acid-shock resulted in increased C. jejuni invasion of m-ICcl2 mouse small intestine crypt cells grown on transwells, but not when the cells were grown on flat-bottomed wells. This suggests that C. jejuni might be invading intestinal epithelial cells at the basolateral side, possibly after paracellular passage. We hypothesize that acid-shock prior to intestinal entry may serve as a signal that primes C. jejuni to express its virulence gene repertoire including flagellar motility genes, but this requires further study in the context of an appropriate colonization or disease model.

Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism

Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter1,2 but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with sub-micron resolution3,4. Here we apply MIMS to diverse organisms, including Drosophila, mice, and humans. We test the “immortal strand hypothesis,” which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labeling mice with 15N-thymidine from gestation through post-natal week 8, we find no 15N label retention by dividing small intestinal crypt cells after 4wk chase. In adult mice administered 15N-thymidine pulse-chase, we find that proliferating crypt cells dilute label consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human hematopoietic system. These studies show that MIMS provides high-resolution quantitation of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research.

EP Receptor Expression in Human Intestinal Epithelium and Localization Relative to the Stem Cell Zone of the Crypts

There is substantial evidence for PGE2 affecting intestinal epithelial proliferation. PGE2 is also reported to be involved in the regulation of growth and differentiation in adult stem cells, both effects mediated by binding to EP-receptors. We have used the Lgr5 as a marker to scrutinize EP-receptor and COX expression in human intestinal epithelial cells with focus on the stem cell area of the crypts. Normal tissue from ileum and colon, but also duodenal biopsies from patients with untreated celiac disease, were investigated by immunohistochemistry and RT-PCR. The combination of fresh flash-frozen tissue and laser microdissection made it possible to isolate RNA from the epithelial cell layer, only. In the small intestine, Lgr5 labels cells are in the +4 position, while in the colon, Lgr5 positive cells are localized to the crypt bottoms. Epithelial crypt cells of normal small intestine expressed neither EP-receptor mRNA nor COX1/2. However, crypt cells in tissue from patients with untreated celiac disease expressed EP2/4 receptor and COX1 mRNA. In the colon, the situation was different. Epithelial crypt cells from normal colon were found to express EP2/4 receptor and COX1/2 transcripts. Thus, there are distinct differences between normal human small intestine and colon with regard to expression of EP2/4 receptors and COX1/2. In normal colon tissue, PGE2-mediated signaling through EP-receptors 2/4 could be involved in regulation of growth and differentiation of the epithelium, while the lack of EP-receptor expression in the small intestinal tissue exclude the possibility of a direct effect of PGE2 on the crypt epithelial cells.

Biological characterization of 2-aminothiazole-derived Cdk4/6 selective inhibitor in vitro and in vivo

Abnormalities in the p16INK4a/ cyclin-dependent kinase (Cdk)4, 6/ Retinoblastoma (Rb) pathway frequently occur in various human cancers. Thus, Cdk4/6 is an attractive target for cancer therapy. Here we report the biological characterization of a 2-aminothiazole-derived Cdk4/6 selective inhibitor, named Compound A in vitro and in vivo. Compound A potently inhibits Cdk4 and Cdk6 with high selectivity (more than 57-fold) against other Cdks and 45 serine/threonine and tyrosine kinases. Compound A inhibits Rb protein (pRb) phosphorylation at Ser780, inhibits E2F-dependent transcription, and induces cell-cycle arrest at G1 in the T98G human glioma cell line. Among 82 human cells derived from various tissues, cell lines derived from hematological cancers (leukemia/lymphoma) tended to be more sensitive to Compound A in cell proliferation assay. Rb-negative cells tended to be insensitive to Compound A, as we had expected. In a nude rat xenograft model, Compound A inhibited pRb phosphorylation and bromodeoxyuridine (BrdU) incorporation in Eol-1 xenograft tumor at plasma concentration of 510 nM. Interestingly Compound A only moderately inhibited those pharmacodynamic and cell cycle parameters of normal crypt cells in small intestine even at 5 times higher plasma concentration. In F344 rats, Compound A did not cause immunosuppression even at 17 times higher plasma conc. These results suggest that Cdk4/6 selective inhibitors only moderately affects on the cell cycle of normal proliferating tissues and has a safer profile than pan-Cdk inhibitor in vivo.

A Quantitative Polymerase Chain Reaction Assay for Detection and Quantification of Lawsonia Intracellularis

Lawsonia intracellularis, a causative agent of porcine proliferative enteropathy, is an obligate intracellular bacterium that is difficult to culture, propagate, and quantify. The intestinal epithelial cell line (IEC-18, derived from rat small intestine crypt cells) has been used to isolate and propagate this pathogen. However, the lack of rapid and simple quantification methods has led to mixed results when using the rat cell line, complicating Lawsonia studies. To overcome these limitations, a SYBR green quantitative polymerase chain reaction (qPCR) assay targeting a unique hypothetical protein was developed for detecting and quantifying L. intracellularis in IEC-18 rat epithelial cells, porcine fecal samples collected from different farms, and experimentally infected pigs. The method was optimized to detect as few as 3 copies per qPCR reaction of the bacterium growing in IEC-18 cells, providing a new and necessary approach to assess the growth of L. intracellularis in these cell lines. Furthermore, the qPCR assay was successful in detecting L. intracellularis in fecal samples collected from pigs with and without a history of Lawsonia infections, as well as in experimentally infected pigs, which further confirmed the suitability of the qPCR assay for studying the epidemiology of this pathogen.

The stem cells of small intestinal crypts: where are they?

Recently, there has been resurgence of interest in the question of small intestinal stem cells, their precise location and numbers in the crypts. In this article, we attempt to re-assess the data, including historical information often omitted in recent studies on the subject. The conclusion we draw is that the evidence supports the concept that active murine small intestinal stem cells in steady state are few in number and are proliferative. There are two evolving, but divergent views on their location (which may be more related to scope of capability and reversibility than to location) several lineage labelling and stem cell self-renewing studies (based on Lgr5 expression) suggest a location intercalated between the Paneth cells (crypt base columnar cells (CBCCs)), or classical cell kinetic, label-retention and radiobiological evidence plus other recent studies, pointing to a location four cell positions luminally from the base of the crypt The latter is supported by recent lineage labelling of Bmi-1-expressing cells and by studies on expression of Wip-1 phosphatase. The situation in the human small intestine remains unclear, but recent mtDNA mutation studies suggest that the stem cells in humans are also located above the Paneth cell zone. There could be a distinct and as yet undiscovered relationship between these observed traits, with stem cell properties both in cells of the crypt base and those at cell position 4.