Crypt Regeneration Research Articles

Evidence for a Crucial Role of Paneth Cells in Mediating the Intestinal Response to Injury

The identification of the intestinal stem cell (ISC) markers Lgr5 and Bmi-1 has furthered our understanding of how they accomplish homeostasis in this rapidly self-renewing tissue. Recent work indicates that these markers identify a cycling Lgr5+ ISC which can be replaced by a quiescent Bmi-1+ ISC. Currently, there is little data on how these cells interact to control intestinal crypt homeostasis and regeneration. This interaction likely involves other differentiated cells within the niche as it has previously been demonstrated that the “stemness” of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. To investigate this, we used two conditional mouse models to delete the transcription factor β-catenin within the intestinal crypt. Critically these differ in their ability to drive recombination within Paneth cells and therefore allow us to compare the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. After gene deletion, the intestines in the model in which Paneth cells were retained showed a rapid recovery and repopulation of the crypt-villus axis presumably from either a spared ISC or the hypothetical quiescent ISCs. However, in the absence of Paneth cells the recovery ability was compromised resulting in complete loss of intestinal epithelial integrity. This data indicates that the Paneth cells play a crucial role within the in vivo ISC niche in aiding recovery following substantial insult. Stem Cells2013;31:776–785

Effects of a water-soluble extract of Ganoderma lucidum mycelia on aberrant crypt foci induced by azoxymethane and small-intestinal injury by 5-FU in F344 rats

The present study investigated whether a water-soluble extract from the culture medium of Ganoderma lucidum mycelia (Japanese: Reishi or Mannentake) (designated as MAK) exerted a protective effect against induction of aberrant crypt foci (ACF) by azoxymethane (AOM) and small-intestinal damage induced by the anticancer drug 5-FU. Six-week-old male F344 rats were fed a basic diet (MF), either alone or containing 2.5 % MAK, beginning 1 week before treatment with AOM. The rats were then given subcutaneous injections of AOM (15 mg/kg body weight) once in a week for 3 weeks. Next, beginning 1 day after the final AOM treatment, 25 or 80 mg/kg 5-FU was injected intraperitoneally three times at 5-day intervals. Finally, the rats were killed 3.5 days after the last injection of 5-FU. The large and small intestines were removed, and tissue specimens were examined for both ACF in the large intestine and regeneration of small-intestinal crypts. The number of ACF was significantly decreased by treatment with 25 mg 5-FU and further decreased by 25 mg 5-FU + MAK in comparison with 5-FU alone. Moreover, there was a greater degree of recovery from small-intestinal damage in the 5-FU + MAK groups than in rats that had received 5-FU alone. The present results indicate that MAK ameliorates the colon precancerous lesions induced by AOM and the small-intestinal injury caused by 5-FU, suggesting that MAK could have potential as a preventive agent against colonic precancer, which is a common adverse effect of chemotherapy.

Protective Effects of the Fermented Milk Kefir on X-Ray Irradiation-Induced Intestinal Damage in B6C3F1 Mice

Gastrointestinal damage associated with radiation therapy is currently an inevitable outcome. The protective effect of Kefir was assessed for its usefulness against radiation-induced gastrointestinal damage. A Kefir supernatant was diluted by 2- or 10-fold and administered for 1 week prior to 8 Gray (Gy) X-ray irradiation at a dose rate of 2 Gy/min, with an additional 15 d of administration post-irradiation. The survival rate of control mice with normal drinking water dropped to 70% on days 4 through 9 post-irradiation. On the other hand, 100% of mice in the 10- and 2-fold-diluted Kefir groups survived up to day 9 post-irradiation (p<0.05 and p<0.01, respectively). Examinations for crypt regeneration against 8, 10 and 12 Gy irradiation at a dose rate of 4 Gy/min revealed that the crypt number was significantly increased in the mice administered both diluted Kefir solutions (p<0.01 for each). Histological and immunohistochemical examinations revealed that the diluted Kefir solutions protected the crypts from radiation, and promoted crypt regeneration. In addition, lyophilized Kefir powder was found to significantly recover the testis weights (p<0.05), but had no effects on the body and spleen weights, after 8 Gy irradiation. These findings suggest that Kefir could be a promising candidate as a radiation-protective agent.

Evidence of functional cross talk between the Notch and NF-κB pathways in nonneoplastic hyperproliferating colonic epithelium.

The Notch and NF-κB signaling pathways regulate stem cell function and inflammation in the gut, respectively. We investigate whether a functional cross talk exists between the two pathways during transmissible murine colonic hyperplasia (TMCH) caused by Citrobacter rodentium (CR). During TMCH, NF-κB activity and subunit phosphorylation in colonic crypts of NIH Swiss mice at days 6 and 12 were associated with increases in downstream target CXC chemokine ligand (CXCL)-1/keratinocyte-derived chemokine (KC) expression. Blocking Notch signaling acutely for 5 days with the Notch blocker dibenzazepine (DBZ) failed to inhibit crypt NF-κB activity or CXCL-1/KC expression. Chronic DBZ administration for 10 days, however, blocked Notch and NF-κB signaling in the crypts and abrogated hyperplasia. Intriguingly, chronic Notch inhibition was associated with significant increases in IL-1α, granulocyte colony-stimulating factor, monocyte chemoattractant protein 1, macrophage inflammatory protein 2, and KC in the crypt-denuded lamina propria or whole distal colon, with concomitant increases in myeloperoxidase activity. In core-3(-/-) mice, which are defective in intestinal mucin, DBZ administration replicated the results of NIH Swiss mice; in Apc(Min/+) mice, which are associated with CR-induced elevation of NF-κB-p65(276) expression, DBZ reversed the increase in NF-κB-p65(276), which may have blocked rapid proliferation of the mutated crypts. DBZ further blocked reporter activities involving the NF-κB-luciferase reporter plasmid or the Toll-like receptor 4/NF-κB/SEAPorter HEK-293 reporter cell line, while ectopic expression of Notch-N(ICD) reversed the inhibitory effect. Dietary bael (Aegle marmelos) extract (4%) and curcumin (4%) restored Notch and NF-κB cross talk in NIH Swiss mice, inhibited CR/DBZ-induced apoptosis in the crypts, and promoted crypt regeneration. Thus functional cross talk between the Notch and NF-κB pathways during TMCH regulates hyperplasia and/or inflammation in response to CR infection.

Phloroglucinol Protects Small Intestines of Mice from Ionizing Radiation by Regulating Apoptosis-Related Molecules

Phloroglucinol (PG) is a phenolic compound isolated from Ecklonia cava, a brown algae abundant on Jeju island, Korea. Previous reports have suggested that PG exerts antioxidative and cytoprotective effects against oxidative stress. In this study, we confirmed that PG protected against small intestinal damage caused by ionizing radiation, and we investigated its protective mechanism in detail. Regeneration of intestinal crypts in the PG-treated irradiated group was significantly promoted compared with that in irradiated controls. The expression level of proapoptotic molecules such as p53, Bax, and Bak in the small intestine was downregulated and that of antiapoptotic molecules such as Bcl-2 and Bcl-X(S/L) was augmented in the PG-treated group. On histological observation of the small intestine, PG inhibited the immunoreactivity of p53, Bax, and Bak and increased that of Bcl-2 and Bcl-X(S/L). These results demonstrate the protective mechanisms of PG in mice against intestinal damage from ionizing radiation, providing the benefit of raising the apoptosis threshold of jejunal crypt cells.

Probability of Radiographic Radiation Pneumonitis (RP) and Fibrosis (RF) After Stereotactic Ablative Radiation Therapy (SABR) for Lung Cancer Using NTCP Model

Materials/Methods: Male C57/Bl6 mice eight weeks of age were pretreated with saline or 8 mg of the prolyl hydroxylase inhibitor DMOG 16 hrs and 4 hrs prior to being irradiated with a single fraction of 20 Gy to the abdomen using a modified TLI jig that shields the upper body of the mouse to reduce hematopoietic toxicity. After XRT, the mice got daily doses of saline or DMOG for 5 days. Results: Mice treated with DMOG had a 1.5-fold enrichment of crypt regeneration in the colon as determined by the classical microcolony assay (13.3 3.4 vs. 21.2 1.7 crypts/section, saline vs. DMOG, nZ 10/group, p Z 0.005), which strongly suggests that prolyl hydroxylase inhibition radioprotects the colon. Death from GI radiotoxicity can result from compromised epithelial integrity. Death from GI radiotoxicity can result from compromised epithelial integrity through reduced barrier function and water absorption leading to sepsis, dehydration and electrolyte imbalance. We tested epithelial and barrier function by gavaging irradiated mice with FITC-dextran, which should be undetectable in the blood unless the GI epithelia are compromised. Treatment with DMOG decreased FITC-dextran uptake by 4-fold over controls (52.6 14.7 vs. 12.4 3.4 mcg/mL, n Z 6/group, p Z 0.02). Using metabolic cage analysis, we find that mice treated with DMOG had more formed stools, increased urine output and less weight loss than saline treated mice. Mice treated with only saline had higher sodium and Cr levels, further suggesting that GI function and water balance is improved with DMOG treatment. These physiologic improvements translated to improved overall survival with DMOG treatment after being exposed to 20 Gy of abdominal radiation (p Z 0.01). In addition, mice treated with DMOG within 4 hours after radiation also showed improved survival, although with more mortality than if dosed before receiving radiation. Conclusions: Maintaining epithelial integrity should be a therapeutic goal in treating GI radiotoxicity. Physiologic measurements are an important adjunct to cellular and histologic methods of assessing radioprotection in the gut. Author Disclosure: C.M. Taniguchi: None. T. Atwood: None. A.J. Giaccia: None.

Wnt5a Potentiates TGF-β Signaling to Promote Colonic Crypt Regeneration After Tissue Injury

Reestablishing homeostasis after tissue damage depends on the proper organization of stem cells and their progeny, though the repair mechanisms are unclear. The mammalian intestinal epithelium is well suited to approach this problem, as it is composed of well-delineated units called crypts of Lieberkühn. We found that Wnt5a, a noncanonical Wnt ligand, was required for crypt regeneration after injury in mice. Unlike controls, Wnt5a-deficient mice maintained an expanded population of proliferative epithelial cells in the wound. We used an in vitro system to enrich for intestinal epithelial stem cells to discover that Wnt5a inhibited proliferation of these cells. Surprisingly, the effects of Wnt5a were mediated by activation of transforming growth factor-β (TGF-β) signaling. These findings suggest a Wnt5a-dependent mechanism for forming new crypt units to reestablish homeostasis.

Highlights from the Literature

Highlights from the Literature

A reaction–diffusion mechanism influences cell lineage progression as a basis for formation, regeneration, and stability of intestinal crypts

BackgroundColon crypts, a single sheet of epithelia cells, consist of a periodic pattern of stem cells, transit-amplifying cells, and terminally differentiated cells that constantly renew and turnover. Experimental evidence suggests that Wnt signaling promotes and regulates stem cell division, differentiation, and possible cell migrations while intestinal BMP signaling inhibits stem cell self-renewal and repression in crypt formation. As more molecular details on Wnt and BMP in crypts are being discovered, little is still known about how complex interactions among Wnt, BMP, and different types of cells, and surrounding environments may lead to de novo formation of multiple crypts or how such interactions affect regeneration and stability of crypts.ResultsWe present a mathematical model that contains Wnt and BMP, a cell lineage, and their feedback regulations to study formation, regeneration, and stability of multiple crypts. The computational explorations and linear stability analysis of the model suggest a reaction–diffusion mechanism, which exhibits a short-range activation of Wnt plus a long-range inhibition with modulation of BMP signals in a growing tissue of cell lineage, can account for spontaneous formation of multiple crypts with the spatial and temporal pattern observed in experiments. Through this mechanism, the model can recapitulate some distinctive and important experimental findings such as crypt regeneration and crypt multiplication. BMP is important in maintaining stability of crypts and loss of BMP usually leads to crypt multiplication with a fingering pattern.ConclusionsThe study provides a mechanism for de novo formation of multiple intestinal crypts and demonstrates a synergetic role of Wnt and BMP in regeneration and stability of intestinal crypts. The proposed model presents a robust framework for studying spatial and temporal dynamics of cell lineages in growing tissues driven by multiple signaling molecules.

Crypt Base Columnar Stem Cells in Small Intestines of Mice Are Radioresistant

Adult stem cells have been proposed to be quiescent and radiation resistant, repairing DNA double-strand breaks by nonhomologous end joining. However, the population of putative small intestinal stem cells (ISCs) at position +4 from the crypt base contradicts this model, in that they are highly radiosensitive. Cycling crypt base columnar cells (CBCs) at crypt positions +1-3 recently were defined as an alternative population of ISCs. Little is known about the sensitivity of this stem cell population to radiation. Radiation-induced lethality of CBCs was quantified kinetically in Lgr5-lacZ transgenic mice. γ-H2AX, BRCA1, RAD51, and DNA-PKcs foci were used as DNA repair surrogates to investigate the inherent ability of CBCs to recognize and repair double-strand breaks. 5-ethynyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine incorporation assays were used to study patterns of CBC growth arrest and re-initiation of cell cycling. Apoptosis was evaluated by caspase-3 staining. CBCs are relatively radioresistant, repairing DNA by homologous recombination significantly more efficiently than transit amplifying progenitors or villus cells. CBCs undergo apoptosis less than 24 hours after irradiation (32% ± 2% of total lethality) or mitotic death at 24-48 hours. Survival of CBCs at 2 days predicts crypt regeneration at 3.5 days and lethality from gastrointestinal syndrome. Crypt repopulation originates from CBCs that survive irradiation. Adult ISCs in mice can cycle rapidly yet still be radioresistant. Importantly, homologous recombination can protect adult stem cell populations from genotoxic stress. These findings broaden and refine concepts of the phenotype of adult stem cells.

Murine P-glycoprotein Deficiency Alters Intestinal Injury Repair and Blunts Lipopolysaccharide-Induced Radioprotection

P-glycoprotein (P-gp) has been reported to increase stem cell proliferation and regulate apoptosis. Absence of P-gp results in decreased repair of intestinal epithelial cells after chemical injury. To further explore the mechanisms involved in the effects of P-gp on intestinal injury and repair, we used the well-characterized radiation injury model. In this model, injury repair is mediated by production of prostaglandins (PGE(2)) and lipopolysaccharide (LPS) has been shown to confer radioprotection. B6.mdr1a(-/-) mice and wild-type controls were subjected to 12 Gy total body X-ray irradiation and surviving crypts in the proximal jejunum and distal colon were evaluated 3.5 days after irradiation. B6.mdr1a(-/-) mice exhibited normal baseline stem cell proliferation and COX dependent crypt regeneration after irradiation. However, radiation induced apoptosis was increased and LPS-induced radioprotection was blunted in the C57BL6.mdr1a(-/-) distal colon, compared to B6 wild-type controls. The LPS treatment induced gene expression of the radioprotective cytokine IL-1α, in B6 wild-type controls but not in B6.mdr1a(-/-) animals. Lipopolysaccharid-induced radioprotection was absent in IL-1R1(-/-) animals, indicating a role for IL-1α in radioprotection, and demonstrating that P-gp deficiency interferes with IL-1α gene expression in response to systemic exposure to LPS.

Critical Roles of Notch and Wnt/β-Catenin Pathways in the Regulation of Hyperplasia and/or Colitis in Response to Bacterial Infection

Notch and Wnt/β-catenin signals play essential roles in intestinal development and homeostasis. Citrobacter rodentium induces transmissible murine colonic hyperplasia (TMCH) and various degrees of inflammation, depending upon the genetic background. We aimed at delineating the role of the Notch and Wnt/β-catenin pathways in the regulation of colonic crypt hyperplasia and/or colitis following C. rodentium infection. During TMCH, relative levels of the Notch intracellular domain (NICD) increased significantly, along with increases in Jagged-1 and Hes-1 coinciding with the progression and regression phases of hyperplasia. Blocking of Notch signaling with dibenzazepine (DBZ) for 5 days before the onset of hyperplasia also blocked Wnt/β-catenin signaling. Targeting the Notch pathway for 5 days after the onset of hyperplasia failed to inhibit Wnt/β-catenin-regulated crypt hyperplasia. Chronic DBZ administration for 10 days blocked both Notch and Wnt signaling, disrupted the intestinal barrier, and induced colitis. Core-3(-/-) mice, which are defective in mucin secretion and are susceptible to experimental triggers of colitis, also exhibited significant colitis in response to C. rodentium plus DBZ. Chronic DBZ administration in these mice did not result in depletion of the putative stem cell marker doublecortin-like kinase-1 (DCLK1) in the crypts. Dietary bael (Aegle marmelos) extract (4%) and curcumin (4%) restored signaling via the Notch and Wnt/β-catenin pathways, thereby promoting crypt regeneration, and also replenished the mucus layer, leading to amelioration of C. rodentium- and DBZ-induced colitis in NIH:Swiss mice. Thus, the balancing act between cell proliferation and mucus production to restore barrier integrity seems to depend upon the interplay between the Wnt/β-catenin and Notch pathways in the TMCH model.

Intestinal HIF-2 to protect against radiation-induced gastrointestinal syndrome.

10629 Background: Gastrointestinal (GI) toxicity accounts for significant morbidity during systemic chemotherapy or abdominopelvic radiation therapy. Unfortunately, there are no effective preventative treatments. Hypoxic signaling through hypoxia-inducible factor-1 (HIF-1) and -2 (HIF-2) is critical for intestinal homeostasis during inflammatory challenges and was posited to play a role in radioprotection. Methods: We stabilized both HIF-1 and HIF-2 pharmacologically with the prolyl hydroxylase inhibitor DMOG and with transgenic mice. Male C57/Bl6 mice eight weeks of age were pretreated with saline or 8mg DMOG 16hrs and 4hrs prior to being irradiated with a single fraction of 20Gy to the abdomen using a modified TLI jig that shields the upper body of the mouse to reduce hematopoietic toxicity. After XRT, the mice got daily doses of saline or DMOG for 5 days. Results: Mice treated with DMOG had a 1.5-fold enrichment of crypt regeneration in the colon as determined by microcolony assay (13.3±3.4 vs 21.2±1.7 crypts/section, saline vs DMOG, n=10/group, p=0.005). Death from GI radiotoxicity can result from compromised epithelial integrity. We tested epithelial and barrier function by gavaging irradiated mice with FITC-dextran, which undetectable in the blood unless the GI epithelia is compromised. Treatment with DMOG decreased FITC-dextran uptake by 4-fold over controls (52.6±14.7 vs 12.4±3.4 mcg/ml, n=6/group, p=0.02). These physiologic improvements translated to improved overall survival with DMOG treatment after being exposed to 20Gy of abdominal radiation (p=0.03). To determine if the radioprotective effect of DMOG was specific to HIF-1 or HIF-2, we overexpressed stabilized HIF1 or HIF2 in the intestinal epithelia using a lox-stop-lox (LSL) system and Villin-Cre. The overexpression of HIF1 had no effect on survival (p=0.22), while mice with intestinal-specific HIF2-overexpression had significantly improved survival after receiving 20Gy of abdominal radiation (p=0.001). Conclusions: Activation of HIF-2 is sufficient to protect mice from lethal doses of abdominal radiation may provide a novel class of treatments to protect the GI epithelium from the deleterious side effects of chemotherapy and radiation.

Abstract 1404: Bacterial infection-induced epithelial-to-mesenchymal transition (EMT) of colonic crypt cells with acquired characteristics of stem cells promotes spheroid/organoid formation in vitro and tumorigenesis in vivo

Abstract DCLK-1 (doublecortin-like kinase-1) and LGR5 (leucine-rich-repeat-containing G-protein-coupled receptor) have recently been identified as markers of quiescent and cycling stem cells in the small intestinal crypts, respectively. The epithelial-to-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We aimed at delineating if bacterial infection affects colonic stemness and transdifferentiation and whether these changes facilitate mucosal priming for neoplasia. Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model associated with hyperactive Wnt/β-catenin, Notch and NF-κB pathways in NIH-Swiss mice, we observed dramatic loss of DCLK-1 expression in the distal colonic crypts with significant shift towards stromal staining at peak (12-days post-infection) hyperplasia while staining for both Lgr-5 and Msi-1 increased several fold. When hyperplasia was regressing (at day 34), we observed a dramatic expansion of DCLK-1+ve cells compared to that seen in uninfected control. Colonic crypt cells when isolated from CR-infected mice at 12 days’ post-CR infection and cultured in vitro, formed monolayers, converted to mesenchymal cells, stained positive for vimentin and fibronectin but negative for E-cadherin and cytokeratin suggesting a process of EMT, and also stained positive for DCLK-1 thereby mimicking stromal staining observed in vivo. Trypsinization of these cells followed by growth in soft-agar led to formation of organoids which developed into crypt-like structures (cryptoids) that stained positive for markers of epithelial lineages. Day 34 crypt cells when deposited into the soft-agar also formed spheroids with subsequent transition into organoids/cryptoids in vitro. These changes were observed in culture conditions that did not include intestinotrophic mitogens Noggin or R-spondin. Mice exhibiting 12 or 34 days of TMCH received azoxymethane once for 8h (Gp1) or twice for two weeks (Gp2) and subjected to crypt isolation. Crypt cells from Gp1 animals formed monolayers as well as spheroids in soft-agar but failed to form tumors in athymic/nude mice. Crypt cells, isolated from Gp2 animals failed to form the monolayers but developed into huge spheroids in soft-agar and formed tumors in athymic/nude mice. These studies suggest that both hyperplasia and increased stemness promote cellular transformation in response to a second hit. Thus, the investigation of a model of bacterial infection-induced EMT and/or spheroid/organoid/cryptoid formation with acquired characteristics of stem cells represents an excellent system to study transdifferentiation, crypt regeneration or colon carcinogenesis in response to bacterial infection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1404. doi:1538-7445.AM2012-1404

Activation of two distinct Sox9-EGFP-expressing intestinal stem cell populations during crypt regeneration after irradiation

Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.

Distinct Compartmentalization of NF-κB Activity in Crypt and Crypt-Denuded Lamina Propria Precedes and Accompanies Hyperplasia and/or Colitis following Bacterial Infection

Citrobacter rodentium induces transmissible murine colonic hyperplasia (TMCH) and variable degrees of inflammation and necrosis depending upon the genetic background. Utilizing C. rodentium-induced TMCH in C3H/HeNHsd inbred mice, we observed significant crypt hyperplasia on days 3 and 7 preceding active colitis. NF-κB activity in the crypt-denuded lamina propria (CLP) increased within 24 h postinfection, followed by its activation in the crypts at day 3, which peaked by day 7. Increases in interleukin-α1 (IL-1α), IL-12(p40), and macrophage inflammatory protein 1α (MIP-1α) paralleled NF-κB activation, while increases in IL-1α/β, IL-6/IL-12(p40)/granulocyte colony-stimulating factor (G-CSF)/keratinocyte-derived chemokine (KC)/monocyte chemotactic protein 1 (MCP-1), and MIP-1α followed NF-κB activation leading to significant recruitment of neutrophils to the colonic mucosa and increased colonic myeloperoxidase (MPO) activity. Phosphorylation of the crypt cellular and nuclear p65 subunit at serines 276 and 536 led to functional NF-κB activation that facilitated expression of its downstream target, CXCL-1/KC, during TMCH. Distinct compartmentalization of phosphorylated extracellular signal-regulated kinase 1 and 2 ([ERK1/2] Thr(180)/Tyr(182)) and p38 (Thr(202)/Tyr(204)) in the CLP preceded increases in the crypts. Inhibition of ERK1/2 and p38 suppressed NF-κB activity in both crypts and the CLP. Dietary administration of 6% pectin or 4% curcumin in C. rodentium-infected mice also inhibited NF-κB activity and blocked CD3, F4/80, IL-1α/β, G-CSF/MCP-1/KC, and MPO activity in the CLP while not affecting NF-κB activity in the crypts. Thus, distinct compartmentalization of NF-κB activity in the crypts and the CLP regulates crypt hyperplasia and/or colitis, and dietary intervention may be a novel strategy to modulate NF-κB-dependent protective immunity to facilitate crypt regeneration following C. rodentium-induced pathogenesis.

α-Tocopherol Succinate Protects Mice against Radiation-Induced Gastrointestinal Injury

The purpose of this study was to elucidate the role of α-tocopherol succinate (α-TS) in protecting mice from gastrointestinal syndrome induced by total-body irradiation. CD2F1 mice were injected subcutaneously with 400 mg/kg of α-TS and exposed to different doses of (60)Co γ radiation, and 30-day survival was monitored. Jejunum sections were analyzed for crypts and villi, PUMA (p53 upregulated modulator of apoptosis), and apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling - TUNEL). The crypt regeneration in irradiated mice was evaluated by 5-bromo-2-deoxyuridine (BrdU). Bacterial translocation from gut to heart, spleen and liver in α-TS-treated and irradiated mice was evaluated by bacterial culture on sheep blood agar, colistin-nalidixic acid, and xylose-lysine-desoxycholate medium. Our results demonstrate that α-TS enhanced survival in a significant number of mice irradiated with 9.5, 10, 11 and 11.5 Gy (60)Co γ radiation when administered 24 h before radiation exposure. α-TS also protected the intestinal tissue of irradiated mice in terms of crypt and villus number, villus length and mitotic figures. TS treatment decreased the number of TUNEL- and PUMA-positive cells and increased the number of BrdU-positive cells in jejunum compared to vehicle-treated mice. Further, α-TS inhibited gut bacterial translocation to the heart, spleen and liver in irradiated mice. Our data suggest that α-TS protects mice from radiation-induced gastrointestinal damage by inhibiting apoptosis, promoting regeneration of crypt cells, and inhibiting translocation of gut bacteria.

Bone Marrow Stromal Cell Transplantation Mitigates Radiation-Induced Gastrointestinal Syndrome in Mice

BackgroundNuclear accidents and terrorism presents a serious threat for mass casualty. While bone-marrow transplantation might mitigate hematopoietic syndrome, currently there are no approved medical countermeasures to alleviate radiation-induced gastrointestinal syndrome (RIGS), resulting from direct cytocidal effects on intestinal stem cells (ISC) and crypt stromal cells. We examined whether bone marrow-derived adherent stromal cell transplantation (BMSCT) could restitute irradiated intestinal stem cells niche and mitigate radiation-induced gastrointestinal syndrome.Methodology/Principal FindingsAutologous bone marrow was cultured in mesenchymal basal medium and adherent cells were harvested for transplantation to C57Bl6 mice, 24 and 72 hours after lethal whole body irradiation (10.4 Gy) or abdominal irradiation (16–20 Gy) in a single fraction. Mesenchymal, endothelial and myeloid population were characterized by flow cytometry. Intestinal crypt regeneration and absorptive function was assessed by histopathology and xylose absorption assay, respectively. In contrast to 100% mortality in irradiated controls, BMSCT mitigated RIGS and rescued mice from radiation lethality after 18 Gy of abdominal irradiation or 10.4 Gy whole body irradiation with 100% survival (p<0.0007 and p<0.0009 respectively) beyond 25 days. Transplantation of enriched myeloid and non-myeloid fractions failed to improve survival. BMASCT induced ISC regeneration, restitution of the ISC niche and xylose absorption. Serum levels of intestinal radioprotective factors, such as, R-Spondin1, KGF, PDGF and FGF2, and anti-inflammatory cytokines were elevated, while inflammatory cytokines were down regulated.Conclusion/SignificanceMitigation of lethal intestinal injury, following high doses of irradiation, can be achieved by intravenous transplantation of marrow-derived stromal cells, including mesenchymal, endothelial and macrophage cell population. BMASCT increases blood levels of intestinal growth factors and induces regeneration of the irradiated host ISC niche, thus providing a platform to discover potential radiation mitigators and protectors for acute radiation syndromes and chemo-radiation therapy of abdominal malignancies.

Cellular- and micro-dosimetry of heterogeneously distributed tritium

Purpose: The assessment of radiotoxicity for heterogeneously distributed tritium should be based on the subcellular dose and relative biological effectiveness (RBE) for cell nucleus. In the present work, geometry-dependent absorbed dose and RBE were calculated using Monte Carlo codes for tritium in the cell, cell surface, cytoplasm, or cell nucleus.Materials and methods: Penelope (PENetration and Energy LOss of Positrins and Electrons) code was used to calculate the geometry-dependent absorbed dose, lineal energy, and electron fluence spectrum. RBE for the intestinal crypt regeneration was calculated using a lineal energy-dependent biological weighting function. RBE for the induction of DNA double strand breaks was estimated using a nucleotide-level map for clustered DNA lesions of the Monte Carlo damage simulation (MCDS) code.Results: For a typical cell of 10 μm radius and 5 μm nuclear radius, tritium in the cell nucleus resulted in much higher RBE-weighted absorbed dose than tritium distributed uniformly. Conversely, tritium distributed on the cell surface led to trivial RBE-weighted absorbed dose due to irradiation geometry and great attenuation of beta particles in the cytoplasm. For tritium uniformly distributed in the cell, the RBE-weighted absorbed dose was larger compared to tritium uniformly distributed in the tissue.Conclusions: Cellular- and micro-dosimetry models were developed for the assessment of heterogeneously distributed tritium.

Uncoupling p53 functions in radiation-induced intestinal damage via PUMA and p21.

The role of p53 in tissue protection is not well understood. Loss of p53 blocks apoptosis in the intestinal crypts following irradiation but paradoxically accelerates gastrointestinal (GI) damage and death. PUMA and p21 are the major mediators of p53-dependent apoptosis and cell-cycle checkpoints, respectively. To better understand these two arms of p53 response in radiation-induced GI damage, we compared animal survival, as well as apoptosis, proliferation, cell-cycle progression, DNA damage, and regeneration in the crypts of WT, p53 knockout (KO), PUMA KO, p21 KO, and p21/PUMA double KO (DKO) mice in a whole body irradiation model. Deficiency in p53 or p21 led to shortened survival but accelerated crypt regeneration associated with massive nonapoptotic cell death. Nonapoptotic cell death is characterized by aberrant cell-cycle progression, persistent DNA damage, rampant replication stress, and genome instability. PUMA deficiency alone enhanced survival and crypt regeneration by blocking apoptosis but failed to rescue delayed nonapoptotic crypt death or shortened survival in p21 KO mice. These studies help to better understand p53 functions in tissue injury and regeneration and to potentially improve strategies to protect or mitigate intestinal damage induced by radiation.