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Caspases are cysteine proteases that regulate embryonic development, cell differentiation, tissue homoeostasis, and removal of damaged and harmful cells from the intestine and other parts of the body. Caspase activity is mainly regulated at the posttranslational level, which allows their rapid activation and response to cellular stress and pathogenic stimuli. In most cell types, caspases are initially expressed as inactive proenzymes, which undergo proteolytic cleavage to become functional enzymes. Caspase dysfunction has been associated with intestinal diseases, including inflammatory bowel disease (IBD) and colorectal cancer. Although the roles of caspases have been studied extensively in regulation of apoptosis, recent discoveries have highlighted cell death-independent functions of this protein family. In particular, caspase-1, caspase-4, caspase-5, and caspase-12 are activated during innate immune responses and participate in the formation of the inflammasome. Caspase-8 controls necroptosis of Paneth cells and potentially the death of intestinal epithelial cells in patients with Crohn's disease and appears to be involved in mucosal inflammation. Regulators of caspase-8 might therefore be used to prevent cell death in patients with IBD. Improving our understanding of the regulation and function of caspases in the intestine might lead to new therapeutics for chronic intestinal inflammation and inflammation-associated cancer., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Tumor necrosis factor (TNF) increases intestinal epithelial cell shedding and apoptosis, potentially challenging the barrier between the gastrointestinal lumen and internal tissues. We investigated the mechanism of tight junction remodeling and barrier maintenance as well as the roles of cytoskeletal regulatory molecules during TNF-induced shedding., Methods: We studied wild-type and transgenic mice that express the fluorescent-tagged proteins enhanced green fluorescent protein-occludin or monomeric red fluorescent protein 1-ZO-1. After injection of high doses of TNF (7.5 μg intraperitoneally), laparotomies were performed and segments of small intestine were opened to visualize the mucosa by video confocal microscopy. Pharmacologic inhibitors and knockout mice were used to determine the roles of caspase activation, actomyosin, and microtubule remodeling and membrane trafficking in epithelial shedding., Results: Changes detected included redistribution of the tight junction proteins ZO-1 and occludin to lateral membranes of shedding cells. These proteins ultimately formed a funnel around the shedding cell that defined the site of barrier preservation. Claudins, E-cadherin, F-actin, myosin II, Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK) were also recruited to lateral membranes. Caspase activity, myosin motor activity, and microtubules were required to initiate shedding, whereas completion of the process required microfilament remodeling and ROCK, MLCK, and dynamin II activities., Conclusions: Maintenance of the epithelial barrier during TNF-induced cell shedding is a complex process that involves integration of microtubules, microfilaments, and membrane traffic to remove apoptotic cells. This process is accompanied by redistribution of apical junctional complex proteins to form intercellular barriers between lateral membranes and maintain mucosal function., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Confocal endomicroscopy is an emerging technology that poses the endoscopist with challenges for identifying epithelial structures in the human intestine. We have shown previously that the murine intestinal epithelium is punctuated by gaps caused by cell shedding. The goals of this study were to determine if confocal endomicroscopy could resolve the presence of human epithelial gaps and whether a proinflammatory cytokine could increase cell shedding., Methods: Intestinal mucosa was imaged after staining with acriflavine. Confocal endomicroscopy of 17 patients yielded 6277 images from the human terminal ileum and rectum. Results were validated by parallel studies of anesthetized mice (wild-type and Math1(DeltaIntestine)) using rigid confocal probe microscopy, 2-photon/confocal microscopy, and scanning electron microscopy., Results: Human terminal ileal and rectal epithelium revealed unstained areas with the diameter of an individual epithelial cell, with 2 distinct morphologies. One had a "target" appearance, shown by mouse studies to be goblet cells. The other morphology had no nucleus and was observed by rigid confocal probe microscopy and scanning electron microscopy in the villi of Math1(DeltaIntestine) mice, which lack goblet cells. In the mouse, tumor necrosis factor alpha (0.33 microg/g intraperitoneally) increases cell shedding by 27-fold and caused loss of barrier function across 20% of resultant gaps., Conclusions: Confocal endomicroscopy can distinguish between epithelial discontinuities (gaps) and goblet cells in human intestine. Results suggest that the sealing of epithelial gaps must be considered as a component of the intestinal barrier and has potential implications for intestinal barrier dysfunction in human disease.

Here we give an overview of colorectal cancer screening strategies with an emphasis on the diagnosis and management of rectal cancer. We review the published studies on screening in the high-risk population, including patients with a history of colorectal cancer, inflammatory bowel disease and inherited conditions. In the average-risk population, the evidence base for a number of screening strategies is evaluated, including endoscopy, contrast studies and faecal occult blood testing. Screening guidelines in the high-risk population are predominantly based on case-control studies comparing the incidence of colorectal cancer in screened and control groups. Screening the average-risk population for colorectal cancer reduces cancer-specific mortality by 15% after biennial guaiac faecal occult blood testing and 50-80% after flexible sigmoidoscopy. All of the screening strategies outlined have a greater sensitivity for distal lesions than proximal lesions.

Background & Aims: A number of growth factors affect the regeneration of intestinal epithelia following injury, but the effects of amidated gastrin have not previously been assessed. We therefore investigated the effects of gastrin on intestinal regeneration following a range of stimuli., Methods: Intestinal crypt regeneration was assessed in transgenic mice overexpressing amidated gastrin (INS-GAS) and mice in which hypergastrinemia was induced using omeprazole, following gamma-radiation, 5-fluorouracil, and dextran sulphate sodium (DSS). Abundance of the CCK-2 receptor was assessed in intestinal epithelia and IEC-6 intestinal epithelial cells following gamma-radiation., Results: Four days following 14 Gy gamma-radiation, or 2 injections of 400 mg/kg 5-fluorouracil, INS-GAS mice exhibited significantly increased small intestinal and colonic crypt survival compared with their wild-type counterparts (FVB/N). INS-GAS mice treated with 3% DSS for 5 days showed less weight loss and increased colonic crypt regeneration at 8 days compared with FVB/N. Increased small intestinal and colonic crypt survival was also demonstrated following gamma-radiation in FVB/N mice rendered hypergastrinemic using omeprazole. The increased crypt survival in INS-GAS mice following 14 Gy gamma-radiation was inhibited by administration of a CCK-2 receptor antagonist (YF476). Increased abundance of the CCK-2 receptor was demonstrated in intestinal epithelia following 14 Gy gamma-radiation by Western blotting and immunohistochemistry. Similarly, increased CCK-2 receptor mRNA abundance and increased 125I-gastrin binding was demonstrated in IEC-6 cells following 4 Gy gamma-radiation., Conclusions: Hypergastrinemia increases regeneration of intestinal epithelia following diverse forms of injury. Induction of the CCK-2 receptor in damaged epithelium confers potential for protection against injury by administration of gastrin.

Background & Aims: Epithelial cells of the small intestine migrate to the tip of the villus at which they are shed. It is not understood how the intestinal barrier is maintained during this high cell turnover. The aim of this study was to use high-resolution in vivo light microscopy to investigate the mechanism of epithelial shedding and the site of the permeability barrier during cell shedding., Methods: A laparotomy was performed on anesthetized mice, and a segment of small intestine was opened. The exposed epithelial surface of the intestine was imaged by multiphoton microscopy. Nuclei, cytosol, and cell membranes were imaged using the dyes Hoescht 33258, BCECF, a transgenically expressed fluorescent protein, and the membrane dye DiI. The fluorescent caspase substrate PhiPhiLux was used to detect apoptosis., Results: In the epithelial monolayer, gaps were observed that lacked nuclei or cytosol but appeared to be filled with an impermeable substance. Studies with membrane impermeant fluorophores (Lucifer Yellow and Alexa-dextran) showed that the impermeable substance completely fills the void left by the absent cell. Only a fraction of gaps have either ZO-1 staining or cytoplasmic extensions from neighboring cells at the basal pole. Time-lapse studies reveal that cell shedding results in genesis of a gap and that shedding usually occurs prior to detectable cellular activation of caspase 3 or nuclear condensation., Conclusions: Results suggest that epithelial barrier function is sustained at the apical pole of the epithelial layer, despite discontinuities in the cellular layer.

Before implantation into the uterine wall, the mammalian embryo undergoes a period of cell division, cell shape change, and cell differentiation leading to the formation of an outer epithelium, the trophectoderm. The trophectoderm is the part of the embryo that initiates uterine contact and, after transformation to become the trophoblast, uterine invasion. Similar to the kidney nephron, the trophectoderm is a transporting epithelium with distinct apical and basolateral membrane domains; its function is to facilitate transepithelial Na+ and fluid transport for blastocoel formation. That transport is driven by Na,K-adenosine triphosphatase (ATPase) localized in basolateral membranes of the trophectoderm. Preimplantation embryos express multiple alpha and beta subunit isoforms of Na,K-ATPase, potentially constituting multiple isozymes, but the basolaterally located alpha1beta1 isozyme appears to function uniquely to drive fluid transport. Embryos unable to express alpha1 subunits because of targeted deletion of the gene are able to form a blastocoel, but they fail to maintain their integrity and expire during the peri-implantation period. Preimplantation embryos also express the gamma subunit, a modulator of Na,K-ATPase activity, but targeted deletion of that gene did not reveal an essential developmental role. The preimplantation embryo offers a unique model for understanding the roles of Na,K-ATPase subunit isoforms in epithelial development and transepithelial transport.

Background & Aims: Transgenic mice that overexpress progastrin are more susceptible than either wild-type mice or mice that overexpress amidated gastrin to chemical carcinogen-induced colonic adenomas. We have investigated whether alterations in the regulation of apoptosis or mitosis after DNA damage contribute to the effects of progastrin on murine colonic epithelium., Methods: Apoptosis and mitosis were assessed on a cell positional basis in murine intestinal epithelium after gamma-irradiation. Mice analyzed were progastrin overexpressing, gastrin overexpressing, gastrin knockout, and their wild-type counterparts. The expression of cell cycle regulators was analyzed by gene array and Western blotting., Results: Apoptosis was induced to similar levels in the small intestinal and colonic crypts of all mice 4.5 hours after 8 Gy gamma-radiation. Colonic mitosis was inhibited to almost undetectable levels by 8Gy gamma-radiation in wild-type, gastrin-knockout, and gastrin-overexpressing mice. However, significant colonic mitosis persisted in progastrin-overexpressing mice up to 24 hours after 8Gy gamma-radiation. Increased levels of cdk4 and cyclin D1 proteins were found in the colonic epithelium of progastrin-overexpressing mice relative to wild-type animals after gamma-radiation., Conclusions: After DNA damage by gamma-radiation, mice with elevated progastrin exhibit significantly higher levels of colonic mitosis than wild-type or gastrin-overexpressing mice. Persistently elevated cdk4 and cyclin D1 in progastrin overexpressing mice accounts for the capacity of colon cells to continue with the cell cycle after DNA damage.

Background & Aims: The thiol-containing antioxidant pyrrolidinedithiocarbamate (PDTC) enhances the cytotoxic efficacy of 5-fluorouracil (5-FU) against human colorectal cancer cell lines in vitro and in vivo. This process appears to be mediated by a sustained increase in p21 expression, independent of p53 function, resulting in growth arrest and apoptosis. We determined whether PDTC augmented 5-FU intestinal toxicity in non-tumor-bearing mice., Methods: Apoptotic and mitotic indices were measured in the small and large intestine on a cell positional basis at intervals throughout the 72-hour period after administration of 5-FU (40 mg/kg) and PDTC (250 mg/kg). The proportion of crypts regenerating after 5-FU (600-1200 mg/kg) and PDTC (500 mg/kg) was also measured., Results: 5-FU therapy induces substantial apoptotic cell death with simultaneous inhibition of mitotic activity within the small and large intestinal epithelium. PDTC reduces 5-FU-induced apoptotic events in the colon by 49%, predominantly among clonogenic stem and transit cells while promoting the early recovery of mitotic activity. As a consequence, PDTC increased the proportion of regenerating colonic crypts after 5-FU therapy. PDTC did not, however, significantly modulate 5-FU toxicity in the small intestine., Conclusions: PDTC does not augment the intestinal toxicity of 5-FU and actually protects the colonic mucosa. These results support further investigation of PDTC and related compounds as treatments for colorectal cancer.

Background & Aims: H2O2 causes DNA damage, which activates poly(adenosine diphosphate ribose) polymerase (PARP), a nuclear enzyme that uses nicotinamide adenine dinucleotide (NAD) as a substrate. When DNA strand breaks are extensive, consumption of NAD by PARP can cause adenosine triphosphate depletion. The aim was to study the effect of PARP inhibition on H2O2-induced cell injury in the intestinal epithelial cell line HT-29-18-C1., Methods: Cell injury was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test and flow cytometric analysis., Results: The PARP inhibitors 3-aminobenzamide and nicotinamide both prevented cell death immediately after exposure to 1 mmol/L H2O2 and loss of cellular NAD and adenosine triphosphate. The inactive structural analogues 3-aminobenzoic acid and nicotinic acid had no such protective effect. H2O2 also caused HT-29 cells to detach from the monolayer up to 24 hours after exposure and die by apoptosis in the incubating medium. Flow cytometric analysis showed that 3-aminobenzamide had no effect on this delayed detachment process., Conclusions: H2O2 induces two distinct death pathways in HT-29 cells: one that is immediate and may represent necrosis and another that is delayed, causing cell detachment leading to apoptosis. PARP inhibition prevents necrosis but has no effect on delayed cell detachment leading to apoptosis.

The Medicare payment policy for recombinant human erythropoietin (rHuEPO) treatment for dialysis patients changed in January 1991 from a relatively fixed payment per treatment (allowed charge of $40 per < or = 10,000 units injected) to a more variable payment based on the amount of rHuEPO administered with each treatment (allowed charge of $11 per 1,000 units injected). This change provided an opportunity to examine how payment policy can effect the use, cost, and health outcome of a biotechnology product used in the dialysis population. In cross-sectional (n = 71,880 Medicare-entitled dialysis patients) and longitudinal (n = 29,088 Medicare-entitled dialysis patients) study designs, we used Medicare end-stage renal disease program and claims data in bivariate and multivariate analyses to examine the effect of the change in payment policy for rHuEPO on access to the biotechnology, dosing, costs, and hematocrit, including the prescribing patterns at for-profit versus not-for-profit providers. The observation period included several months before (July 1989 to December 1990) and 6 months after (January to June 1991) the change in Medicare payment policy. The mean dose per treatment during the initial and fourth month of therapy was low (2,742 [95% confidence interval, 2,703 to 2,781] units and 2,632 [95% confidence interval, 2,598 to 2,667] units, respectively, in June 1990) and increased 3.4% and 5.0%, respectively, in the next 6 months prior to the change in Medicare payment policy compared with 14.6% and 14.8%, respectively, in the 6 months following the change in payment policy. The average monthly allowed charge for rHuEPO per dialysis patient receiving rHuEPO decreased from $455 before the policy change to $349 immediately following the policy change, because the allowed charge per unit of rHuEPO was lower when payment became more dependent on the amount of rHuEPO administered with each treatment than when the payment was fixed at $40 per treatment. The average monthly allowed charge for rHuEPO increased to $375 in the sixth month following the change in payment policy as a result of the increase in dose and the new variable payment. The unadjusted and adjusted changes in mean hematocrit 6 months after the payment change were positive but clinically very small (0.3 and 0.2 percentage points, respectively).(ABSTRACT TRUNCATED AT 400 WORDS)

The traditional options available for the correction of hemodialysis-related anemia are blood transfusions and androgen therapy to stimulate erythropoiesis. A new therapeutic option, recombinant human erythropoietin (r-HuEPO; EPOGEN, AMGEN Inc, Thousand Oaks, CA), is currently undergoing clinical trials. Each treatment alternative has certain attendant adverse effects. The adverse effects of transfusion include transmission of infections such as hepatitis or acquired immunodeficiency syndrome, iron overload, and sensitization to histocompatibility antigens. Androgen therapy can cause masculinization of women and children and, in some forms, is associated with a high incidence of abnormal liver function. Treatment with r-HuEPO has some potential adverse effects, including hypertension, thrombosis of arteriovenous fistulae, prolonged duration of dialysis, hyperkalemia, and iron deficiency. Gradual and careful introduction of r-HuEPO should prevent hypertension from becoming problematic.

Water, electrolyte, glucose, and glycine absorption were studied in vivo in successful rat small intestinal transplants. Isolated bowel loops were transplanted from F1 hybrids into parental strain Lewis rats. A 7-day course of cyclosporin A was given for immunosuppression. Absorption was studied using a steady-state perfusion technique at either 9 or 21 days after transplantation. Histologic examination showed there was villus shortening with time but no evidence of rejection. When perfused with isotonic saline, both allografts and controls secreted water. However, allografts and denervated controls secreted chloride, whereas innervated controls absorbed chloride (p less than 0.05). There was a marked reduction in water and sodium absorption from 30 mM glucose-saline in transplanted loops and denervated controls, whereas glucose absorption was relatively preserved in these groups at 9 days (p less than 0.01). These changes could not be accounted for by rejection or ischemia. These studies demonstrate that denervation may be a major limiting factor in intestinal transplantation.

It has recently been postulated that thromboxane A2 may participate in the pathogenesis of acute myohemoglobinuric experimental acute renal failure. To investigate this further, the effect of selective inhibition of thromboxane synthesis on the course of glycerol-induced acute renal failure was determined. Despite significant inhibition of thromboxane synthesis by 4-imidazole-yl-acetophenone, the functional and morphologic disturbance induced by glycerol was unaltered. Moreover, pretreatment with 4-imidazole-yl-acetophenone failed to prevent the fall in renal blood flow seen following glycerol administration. These results argue against a major role for thromboxane A2 in the pathogenesis of this form of experimental acute renal failure.