Inducible generalized activation of hSTING-N154S expression in mice leads to lethal hypercytokinemia: a model for "cytokine storm".

Excessive levels of circulating proinflammatory mediators, known as "hypercytokinemia," that are generated by overwhelming immune system activation can lead to death due to critical organ failure and thrombotic events. Hypercytokinemia has been frequently associated with a variety of infectious and autoimmune diseases, with severe acute respiratory syndrome coronavirus 2 infection currently being the commonest cause, of what has been termed the cytokine storm. Among its various functions within the host, STING (stimulator of interferon genes) is critical in the defense against certain viruses and other pathogens. STING activation, particularly within cells of the innate immune system, triggers potent type I interferon and proinflammatory cytokine production. We thus hypothesized that generalized expression of a constitutively active STING mutant in mice would lead to hypercytokinemia. To test this, a Cre-loxP-based system was used to cause the inducible expression of a constitutively active hSTING mutant (hSTING-N154S) in any tissue or cell type. Herein, we employed a tamoxifen-inducible ubiquitin C-CreERT2 transgenic to obtain generalized expression of the hSTING-N154S protein, thereby triggering the production of IFN-ß and multiple proinflammatory cytokines. This required euthanizing the mice within 3 to 4 d after tamoxifen administration. This preclinical model will allow for the rapid identification of compounds aimed at either preventing or ameliorating the lethal effects of hypercytokinemia.

PNKP is required for maintaining the integrity of progenitor cell populations in adult mice.

DNA repair proteins are critical to the maintenance of genomic integrity. Specific types of genotoxic factors, including reactive oxygen species generated during normal cellular metabolism or as a result of exposure to exogenous oxidative agents, frequently leads to "ragged" single-strand DNA breaks. The latter exhibits abnormal free DNA ends containing either a 5'-hydroxyl or 3'-phosphate requiring correction by the dual function enzyme, polynucleotide kinase phosphatase (PNKP), before DNA polymerase and ligation reactions can occur to seal the break. Pnkp gene deletion during early murine development leads to lethality; in contrast, the role of PNKP in adult mice is unknown. To investigate the latter, we used an inducible conditional mutagenesis approach to cause global disruption of the Pnkp gene in adult mice. This resulted in a premature aging-like phenotype, characterized by impaired growth of hair follicles, seminiferous tubules, and neural progenitor cell populations. These results point to an important role for PNKP in maintaining the normal growth and survival of these murine progenitor populations.

A synthetically lethal nanomedicine delivering novel inhibitors of polynucleotide kinase 3'-phosphatase (PNKP) for targeted therapy of PTEN-deficient colorectal cancer.

Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a major tumor-suppressor protein that is lost in up to 75% of aggressive colorectal cancers (CRC). The co-depletion of PTEN and a DNA repair protein, polynucleotide kinase 3'-phosphatase (PNKP), has been shown to lead to synthetic lethality in several cancer types including CRC. This finding inspired the development of novel PNKP inhibitors as potential new drugs against PTEN-deficient CRC. Here, we report on the in vitro and in vivo evaluation of a nano-encapsulated potent, but poorly water-soluble lead PNKP inhibitor, A83B4C63, as a new targeted therapeutic for PTEN-deficient CRC. Our data confirmed the binding of A83B4C63, as free or nanoparticle (NP) formulation, to intracellular PNKP using the cellular thermal shift assay (CETSA), in vitro and in vivo. Dose escalating toxicity studies in healthy CD-1 mice, based on measurement of animal weight changes and biochemical blood analysis, revealed the safety of both free and nano-encapsulated A83B4C63, at assessed doses of ≤50 mg/kg. Nano-carriers of A83B4C63 effectively inhibited the growth of HCT116/PTEN-/- xenografts in NIH-III nude mice following intravenous (IV) administration, but not that of wild-type HCT116/PTEN+/+ xenografts. This was in contrast to IV administration of A83B4C63 solubilized with the aid of Cremophor EL: Ethanol (CE), which led to similar tumor growth to that of formulation excipients (NP or CE without drug) or 5% dextrose. This observation was attributed to the higher levels of A83B4C63 delivered to tumor tissue by its NP formulation. Our data provide evidence for the success of NPs of A83B4C63, as novel synthetically lethal nano-therapeutics in the treatment of PTEN-deficient CRC. This research also highlights the potential of successful application of nanomedicine in the drug development process.

Nano-Delivery of a Novel Inhibitor of Polynucleotide Kinase/Phosphatase (PNKP) for Targeted Sensitization of Colorectal Cancer to Radiation-Induced DNA Damage.

Inhibition of the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) increases the sensitivity of cancer cells to DNA damage by ionizing radiation (IR). We have developed a novel inhibitor of PNKP, i.e., A83B4C63, as a potential radio-sensitizer for the treatment of solid tumors. Systemic delivery of A83B4C63, however, may sensitize both cancer and normal cells to DNA damaging therapeutics. Preferential delivery of A83B4C63 to solid tumors by nanoparticles (NP) was proposed to reduce potential side effects of this PNKP inhibitor to normal tissue, particularly when combined with DNA damaging therapies. Here, we investigated the radio-sensitizing activity of A83B4C63 encapsulated in NPs (NP/A83) based on methoxy poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) (mPEO-b-PBCL) or solubilized with the aid of Cremophor EL: Ethanol (CE/A83) in human HCT116 colorectal cancer (CRC) models. Levels of γ-H2AX were measured and the biodistribution of CE/A83 and NP/A83 administered intravenously was determined in subcutaneous HCT116 CRC xenografts. The radio-sensitization effect of A83B4C63 was measured following fractionated tumor irradiation using an image-guided Small Animal Radiation Research Platform (SARRP), with 24 h pre-administration of CE/A83 and NP/A83 to Luc+/HCT116 bearing mice. Therapeutic effects were analyzed by monitoring tumor growth and functional imaging using Positron Emission Tomography (PET) and [18F]-fluoro-3'-deoxy-3'-L:-fluorothymidine ([18F]FLT) as a radiotracer for cell proliferation. The results showed an increased persistence of DNA damage in cells treated with a combination of CE/A83 or NP/A83 and IR compared to those only exposed to IR. Significantly higher tumor growth delay in mice treated with a combination of IR and NP/A83 than those treated with IR plus CE/A83 was observed. [18F]FLT PET displayed significant functional changes for tumor proliferation for the drug-loaded NP. This observation was attributed to the higher A83B4C63 levels in the tumors for NP/A83-treated mice compared to those treated with CE/A83. Overall, the results demonstrated a potential for A83B4C63-loaded NP as a novel radio-sensitizer for the treatment of CRC.

Stimulator of interferon genes (STING) activation exacerbates experimental colitis in mice.

Detection of cytoplasmic DNA by the host's innate immune system is essential for microbial and endogenous pathogen recognition. In mammalian cells, an important sensor is the stimulator of interferon genes (STING) protein, which upon activation by bacterially-derived cyclic dinucleotides (cDNs) or cytosolic dsDNA (dsDNA), triggers type I interferons and pro-inflammatory cytokine production. Given the abundance of bacterially-derived cDNs in the gut, we determined whether STING deletion, or stimulation, acts to modulate the severity of intestinal inflammation in the dextran sodium sulphate (DSS) model of colitis. DSS was administered to Tmem173gt (STING-mutant) mice and to wild-type mice co-treated with DSS and a STING agonist. Colitis severity was markedly reduced in the DSS-treated Tmem173gt mice and greatly exacerbated in wild-type mice co-treated with the STING agonist. STING expression levels were also assessed in colonic tissues, murine bone marrow derived macrophages (BMDMs), and human THP-1 cells. M1 and M2 polarized THP-1 and murine BMDMs were also stimulated with STING agonists and ligands to assess their responses. STING expression was increased in both murine and human M1 polarized macrophages and a STING agonist repolarized M2 macrophages towards an M1-like subtype. Our results suggest that STING is involved in the host's response to acutely-induced colitis.

Expression of a constitutively active human STING mutant in hematopoietic cells produces an Ifnar1-dependent vasculopathy in mice.

STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disorder characterized by blood vessel occlusions, acral necrosis, myositis, rashes, and pulmonary inflammation that are the result of activating mutations in the STimulator of Interferon Genes (STING). We generated a transgenic line that recapitulates many of the phenotypic aspects of SAVI by targeting the expression of the human STING-N154S-mutant protein to the murine hematopoietic compartment. hSTING-N154S mice demonstrated failure to gain weight, lymphopenia, progressive paw swelling accompanied by inflammatory infiltrates, severe myositis, and ear and tail necrosis. However, no significant lung inflammation was observed. X-ray microscopy imaging revealed vasculopathy characterized by arteriole occlusions and venous thromboses. Type I interferons and proinflammatory mediators were elevated in hSTING-N154S sera. Importantly, the phenotype was prevented in hSTING-N154S mice lacking the type I interferon receptor gene (Ifnar1). This model, based on a mutant human STING protein, may shed light on the pathophysiological mechanisms operative in SAVI.

Orally administered indomethacin acutely reduces cellular prion protein in the small intestine and modestly increases survival of mice exposed to infectious prions.

OBJECTIVE: The oral uptake of infectious prions represents a common way to acquire a prion disease; thus, host factors, such as gut inflammation and intestinal "leakiness", have the potential to influence infectivity. For example, the ingestion of nonsteroidal anti-inflammatory drugs (NSAIDs) is known to induce intestinal inflammation and increase intestinal permeability. Previously, we reported that normal cellular prion protein (PrP(C)) expression was increased in experimental colitis, and since the level of PrP(C) expressed is a determinant of prion disease propagation, we hypothesized that NSAID administration prior to the oral inoculation of mice with infectious prions would increase intestinal PrP(C) expression and accelerate the onset of neurological disease. MATERIALS AND METHODS: In the long-term experiments, one group of mice was gavaged with indomethacin, followed by a second gavage with brain homogenate containing mouse-adapted scrapie (ME7). Control mice received ME7 brain homogenate alone. Brain and splenic tissues were harvested at several time points for immunoblotting, including at the onset of clinical signs of disease. In a second series of experiments, mice were gavaged with indomethacin to assess the acute effects of this treatment on intestinal PrP(C) expression. RESULTS: Acutely, NSAID treatment reduced intestinal PrP(C) expression, and chronically, there was a modest delay in the onset of neurological disease. CONCLUSION: In contrast to our hypothesis, brief exposure to an NSAID decreased intestinal PrP(C) expression and led to a modest survival advantage following oral ingestion of infectious prions.

Endogenous prion protein attenuates experimentally induced colitis.

Although the cellular prion protein (PrP(C)) is expressed in the enteric nervous system and lamina propria, its function(s) in the gut is unknown. Because PrP(C) may exert a cytoprotective effect in response to various physiologic stressors, we hypothesized that PrP(C) expression levels might modulate the severity of experimental colitis. We evaluated the course of dextran sodium sulfate (DSS)-induced colitis in hemizygous Tga20 transgenic mice (approximately sevenfold overexpression of PrP(C)), Prnp(-/-) mice, and wild-type mice. On day 7, colon length, disease severity, and histologic activity indices were determined. Unlike DSS-treated wild-type and Prnp(-/-) animals, PrP(C) overexpressing mice were resistant to colitis induction, exhibited much milder histopathologic features, and did not exhibit weight loss or colonic shortening. In keeping with these results, pro-survival molecule expression and/or phosphorylation levels were elevated in DSS-treated Tga20 mice, whereas pro-inflammatory cytokine production and pSTAT3 levels were reduced. In contrast, DSS-treated Prnp(-/-) mice exhibited increased BAD protein expression and a cytokine expression profile predicted to favor inflammation and differentiation. PrP(C) expression from both the endogenous Prnp locus or the Tga20 transgene was increased in the colons of DSS-treated mice. Considered together, these findings demonstrate that PrP(C) has a previously unrecognized cytoprotective and/or anti-inflammatory function within the murine colon.

Prion protein expression level alters regional copper, iron and zinc content in the mouse brain.

The central role of the prion protein (PrP) in a family of fatal neurodegenerate diseases has garnered considerable research interest over the past two decades. Moreover, the role of PrP in neuronal development, as well as its apparent role in metal homeostasis, is increasingly of interest. The host-encoded form of the prion protein (PrP(C)) binds multiple copper atoms via its N-terminal domain and can influence brain copper and iron levels. The importance of PrP(C) to the regulation of brain metal homeostasis and metal distribution, however, is not fully understood. We therefore employed synchrotron-based X-ray fluorescence imaging to map the level and distributions of several key metals in the brains of mice that express different levels of PrP(C). Brain sections from wild-type, prion gene knockout (Prnp(-/-)) and PrP(C) over-expressing mice revealed striking variation in the levels of iron, copper, and even zinc in specific brain regions as a function of PrP(C) expression. Our results indicate that one important function of PrP(C) may be to regulate the amount and distribution of specific metals within the central nervous system. This raises the possibility that PrP(C) levels, or its activity, might regulate the progression of diseases in which altered metal homeostasis is thought to play a pathogenic role such as Alzheimer's, Parkinson's and Wilson's diseases and disorders such as hemochromatosis.

Hydrogen sulphide synthesis in the rat and mouse gastrointestinal tract.

AIMS: Hydrogen sulphide (H2S) exerts several anti-inflammatory effects, accelerates the healing of experimental gastric ulcers, and can stimulate intestinal secretion. Little is known about H2S synthesis in the gastrointestinal tract. The aim of this study was to characterize H2S synthesis throughout the gastrointestinal tract. METHODS: H2S synthesis in various gastrointestinal tissues of rats and mice was determined. The effects and selectivity of inhibitors of two key enzymes for H2S synthesis, cystathionine-gamma-lyase and cystathionine-beta-synthase, were examined. Cystathionine-gamma-lyase and cystathionine-beta-synthase expression was evaluated by Western blotting and immunohistochemistry. Cystathionine-gamma-lyase and cystathionine-beta-synthase expression in biopsies of human colon was also examined. RESULTS: H2S synthesis was variable throughout the gastrointestinal tract in parallel with variations in cystathionine-gamma-lyase and cystathionine-beta-synthase expression. The efficacy of cystathionine-beta-synthase and cystathionine-gamma-lyase inhibitors to reduce H2S synthesis in these tissues was also variable. Cystathionine-beta-synthase is the predominant source of H2S synthesis in the colon of rodents. Cystathionine-gamma-lyase and cystathionine-beta-synthase were also expressed in healthy human colon biopsies. CONCLUSIONS: The capacity for H2S synthesis varies throughout the rodent gastrointestinal tract, as does the distribution and contribution of the two key enzymes. Investigation of additional enzymatic sources of H2S and the development of more selective inhibitors are suggested.

Endogenous and exogenous hydrogen sulfide promotes resolution of colitis in rats.

BACKGROUND & AIMS: Hydrogen sulfide (H(2)S) is an endogenous gaseous mediator of mucosal defense with antiinflammatory effects that promote ulcer healing. The effects of H(2)S during the pathogenesis of colitis have not been established. We analyzed the contribution of H(2)S to inflammation and ulceration of the colon in a rat model of colitis. METHODS: Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. The ability of the colon to synthesize H(2)S was studied over the course of the resolution of the colitis. Expression of 2 enzymes involved in the synthesis of H(2)S and the effects of inhibitors of these enzymes were examined. We also examined the effects of H(2)S donors on the resolution of colitis. RESULTS: The capacity for the colon to produce H(2)S increased markedly over the first days after induction of colitis and then declined toward control levels as the colitis was resolved. Inhibition of colonic H(2)S synthesis markedly exacerbated the colitis, resulting in significant mortality. Inhibition of H(2)S synthesis in healthy rats resulted in inflammation and mucosal injury in the small intestine and colon along with down-regulation of cyclooxygenase-2 messenger RNA expression and prostaglandin synthesis. Intracolonic administration of H(2)S donors significantly reduced the severity of colitis and reduced colonic expression of messenger RNA for the proinflammatory cytokine tumor necrosis factor alpha. CONCLUSIONS: In rats, H(2)S modulates physiological inflammation and contributes to the resolution of colitis.

Annexin-1 modulates repair of gastric mucosal injury.

Annexin-1 is a glucocorticoid-inducible protein that plays an important effector role in the resolution of inflammation and has recently been shown to contribute to the resistance of the stomach to injury. Using an integrated genetic and pharmacological approach, we have tested the hypothesis that annexin-1 contributes to the healing of mucosal injury, given that such injury is accompanied by an inflammatory response, which is often associated with an overexpression of annexin-1 expression. Gastric ulcers were induced in mice through serosal application of acetic acid. Annexin-1 expression during the healing of the ulcers was examined. The effects on gastric ulcer healing of treatment with an annexin-1 mimetic (Ac2-26), an antagonist of the annexin-1 receptor (Boc2), or a glucocorticoid (dexamethasone) were examined. Finally, susceptibility to and healing of indomethacin-induced gastric lesions were compared in wild-type and annexin-1-deficient mice. Expression of annexin-1 was significantly increased in the gastric ulcer margin throughout the healing process. Treatment with an annexin-1 mimetic (Ac2-26) significantly enhanced gastric ulcer healing. In contrast, both dexamethasone and an formyl peptide receptor-like-1 (FPRL-1) antagonist impaired the early phase of ulcer healing. Annexin-1-deficient mice exhibited the same susceptibility as wild-type mice to indomethacin-induced gastric damage, but the healing of that damage was impaired in the former. These data support the hypothesis that annexin-1 contributes significantly to the process of healing of gastric mucosal damage.

Hydrogen sulfide enhances ulcer healing in rats.

Hydrogen sulfide is an endogenous mediator that relaxes vascular smooth muscle, exhibits several antiinflammatory activities, and contributes to gastric mucosal defense. This study was performed to examine the role of hydrogen sulfide in the resolution of injury; specifically, the healing of gastric ulcers. Ulcers were induced in rats by serosal application of acetic acid. This elicited a marked increase in gastric expression of the two key enzymes in hydrogen sulfide synthesis (cystathionine-beta-synthase and cystathionine-gamma-lyase) and in hydrogen sulfide synthesis. Twice-daily treatment for a week with hydrogen sulfide donors significantly increased the extent of healing of gastric ulcers as compared to vehicle-treatment. Similar treatment with L-cysteine, a precursor for hydrogen sulfide, also accelerated healing of the ulcers, and the effect was abolished by cotreatment with an inhibitor of cystathionine-gamma-lyase. The beneficial effects of hydrogen sulfide on ulcer healing were not dependent on nitric oxide synthesis, nor did they appear to occur through activation of ATP-sensitive K+ channels. These results suggest that hydrogen sulfide is produced in the gastric mucosa in response to injury and acts to promote healing. The results further suggest that drugs releasing hydrogen sulfide could be employed to accelerate healing of gastric ulcers, and possibly of other wounds.

Enteric neural pathways mediate the anti-inflammatory actions of glucagon-like peptide 2.

Glucagon-like peptide-2 (GLP-2) is an important regulator of nutritional absorptive capacity with anti-inflammatory actions. We hypothesized that GLP-2 reduces intestinal mucosal inflammation by activation of vasoactive intestinal polypeptide (VIP) neurons of the submucosal plexus. Ileitis or colitis was induced in rats by injection of trinitrobenzene sulfonic acid (TNBS), or colitis was induced by administration of dextran sodium sulfate (DSS) in drinking water. Subsets of animals received (1-33)-GLP-2 (50 mug/kg sc bid) either immediately or 2 days after the establishment of inflammation and were followed for 3-5 days. The involvement of VIP neurons was assessed by concomitant administration of GLP-2 and the VIP antagonist [Lys(1)-Pro(2,5)-Arg(3,4)-Tyr(6)]VIP and by immunohistochemical labeling of GLP-2-activated neurons. In all models, GLP-2 treatment, whether given immediately or delayed until inflammation was established, resulted in significant improvements in animal weights, mucosal inflammation indices (myeloperoxidase levels, histological mucosal scores), and reduced levels of inflammatory cytokines (IFN-gamma, TNF-alpha, IL-1beta) and inducible nitric oxide synthase, with increased levels of IL-10 in TNBS ileitis and DSS colitis. Reduced rates of crypt cell proliferation and of apoptosis within crypts in inflamed tissues were also noted with GLP-2 treatment. These effects were abolished with coadministration of GLP-2 and the VIP antagonist. GLP-2 was shown to activate neurons and to increase the number of cells expressing VIP in the submucosal plexus of the ileum. These findings suggest that GLP-2 acts as an anti-inflammatory agent through activation of enteric VIP neurons, independent of proliferative effects. They support further studies to examine the role of neural signaling in the regulation of intestinal inflammation.

Gastrointestinal inflammation: a central component of mucosal defense and repair.

The mucosal layer of the gastrointestinal (GI) tract is able to resist digestion by the endogenous substances that we secrete to digest foodstuffs. So-called "mucosal defense" is multi-factorial and can be modulated by a wide range of substances, many of which are classically regarded as inflammatory mediators. Damage to the GI mucosa, and its subsequent repair, are also modulated by various inflammatory mediators. In this article, we provide a review of some of the key inflammatory mediators that modulate GI mucosal defense, injury, and repair. Among the mediators discussed are nitric oxide, polyamines, the eicosanoids (prostaglandins and lipoxins), protease-activated receptors, and cytokines. Many of these endogenous factors, or the enzymes involved in their synthesis, are considered potential therapeutic targets for the treatment of diseases of the digestive tract that are characterized by inflammation and ulceration.

Acute denervation alters the epithelial response to adrenoceptor activation through an increase in alpha1-adrenoceptor expression on villus enterocytes.

Loss of sympathetic input due to intestinal denervation results in hypersensitivity and increased intestinal secretion. It is unknown whether denervation-induced alterations in intestinal epithelial physiology are the result of changes in adrenoceptors on enterocytes (ENTs). The purpose of this study was to examine adrenoceptor distribution and pharmacology on small intestinal ENTs following acute intestinal denervation. Lewis rats underwent small bowel transplantation (SBT) or sham operation and proximal small intestinal segments were harvested 1, 2 and 4 weeks postoperatively. Intestinal electrolyte movement was assessed using short-circuit current (Isc) measurements of stripped epithelial sheets following stimulation with phenylephrine (PE), an alpha(1)-adrenoceptor agonist. The presence of adrenoceptor subtypes on separated villus and crypt ENTs was assessed using flow cytometry. Alpha(1)-adrenoceptors were found on approximately 27% of jejunal villus ENTs, but not crypt ENTs, following acute extrinsic denervation. ENTs from the Lewis rat have few beta-adrenoceptors. Alpha(1)-adrenoceptor stimulation of acutely denervated intestinal epithelial sheets decreased Isc by -13.45%. This effect was mediated by a reduction in chloride (Cl(-)) secretion; the absence of Cl(-) reversed the Isc to +13.79%. In conclusion, loss of sympathetic innervation to the gastrointestinal epithelium causes acute upregulation of alpha(1)-adrenoceptors on villus ENTs, leading to inhibition of Cl(-) secretion at the villus tip. The increase in adrenoceptors may reflect a compensatory mechanism to combat the increased secretory state of the bowel due to the loss of the sympathetic innervation and tonic control over intestinal secretion.

A pilot study of the use of epidermal growth factor in pediatric short bowel syndrome.

BACKGROUND: This study examined the effects of enterally administered epidermal growth factor (EGF) on nutrient absorption and tolerance of enteral feeds in pediatric patients with short bowel syndrome (SBS). METHODS: Patients identified with severe SBS (<25% bowel length predicted for age) were prospectively enrolled in treatment using human recombinant EGF (1-53); 100 microg/kg per day given mixed with enteral feeds and patients were treated for 6 weeks. End points followed were patient weight, tolerance of enteral feeds, nutrient absorption, and intestinal permeability as determined using carbohydrate probes and hematologic values for liver function parameters. RESULTS: Five patients were treated with EGF; all showed a significant improvement in carbohydrate absorption (3-0 methylglucose): absorption 24.7% +/- 9.7% pretreatment vs 34.1% +/- 13.8% posttreatment and improved tolerance of enteral feeds (enteral energy as % of total energy, 25% +/- 28% pretreatment vs 36% +/- 24% posttreatment; mean +/- SD; P < .05 by Wilcoxon's signed rank test). Epidermal growth factor treatment was not associated with significant changes in intestinal permeability, the rate of weight gain, or liver function tests. During the treatment phase, no patients developed episodes of sepsis; however, within 2 weeks of discontinuation of EGF treatment, 3 patients developed septic episodes. No adverse effects of EGF administration were noted. CONCLUSIONS: These results suggest that enteral treatment with EGF in pediatric SBS improves nutrient absorption, increases tolerance with enteral feeds, and may improve the infection rate. Further studies exploring treatment strategies including the timing and duration of EGF administration are indicated.

The effects of systemic hypoxia on colon anastomotic healing: an animal model.

PURPOSE: Acute postoperative systemic hypoxia occurs frequently in the clinical setting following intestinal resection, as a result of complications such as pneumonia, pulmonary edema, or the acute respiratory distress syndrome. Although it is well established that oxygen is essential for metabolism in general and intestinal anastomotic healing, the mechanisms by which systemic hypoxia affect this process are not clear. The purpose of this study was to establish an animal model to simulate acute systemic hypoxia and to examine the effects on anastomotic healing. We investigated the hypothesis that systemic hypoxia impairs anastomotic healing in the colon by disrupting revascularization via changes in the expression of two putative angiogenic factors: inducible nitric oxide synthase and vascular endothelial growth factor. METHODS: Phase I: Juvenile male Sprague-Dawley rats underwent carotid artery cannulation. In a controlled environment the FiO2 was incrementally decreased from 21 to 9 percent and the resultant PaO2 measured. Phase II: Animals underwent colonic transection with immediate reanastomosis and were placed in either a normoxic (FiO2 21 percent) or hypoxic (FiO2 11 percent) environment for seven days. Perianastomotic in vivo tissue oxygen saturation was measured before segmental colon resection in each of the animals and at seven days before measurement of anastomotic bursting pressure. Perianastomotic tissue samples were assessed by Western blot assay for the expression of vascular endothelial growth factor and inducible nitric oxide synthase protein. Sections from each tissue sample were taken and evaluated by a pathologist blinded to treatment group for determination of anastomotic healing score. RESULTS: Phase I: Incrementally decreasing the FiO2 resulted in a progressive decrease in PaO2 (r2 = 0.77). Phase II: Animals maintained in a hypoxic environment had a significant decrease in tissue oxygen saturation (73 +/- 9 percent vs. 94 +/- 3 percent; P < 0.0001) and anastomotic bursting pressure (118 +/- 18 mmHg vs. 207 +/- 30 mmHg; P < 0.0001) compared with normoxic controls. Systemic hypoxia induced a significant increase, when compared with normoxic controls, in vascular endothelial growth factor (247.1 +/- 9.5 vs. 142.2 +/- 10.6; P < 0.0001) and inducible nitric oxide synthase (259.6 +/- 21.1 vs. 120.2 +/- 10.9; P < 0.0001) protein expression and led to a significant decrease in the overall wound-healing score. CONCLUSION: This study validates a new animal model to study the effects of acute systemic hypoxia on colonic anastomotic healing. In this model, systemic hypoxia directly translated into local tissue hypoxia, and anastomotic healing was impaired. Contrary to our original hypothesis, hypoxia led to a significant increase in vascular endothelial growth factor and inducible nitric oxide synthase protein expression at the colonic anastomotic site. Impairment in anastomotic integrity despite upregulation of these angiogenic factors could be a result of the inability of wounded tissue to respond to vascular endothelial growth factor and inducible nitric oxide synthase or alternatively, hypoxia may adversely affect collagen synthesis and deposition directly.

Effect of the anticoagulant, pindone, on the breeding performance and survival of merino sheep, Ovis aries.

The effect of the anticoagulant, pindone, on the breeding performance and survival of relatively free-ranging merino sheep was assessed. Pindone (2-pivalyl-1, 3-indandione) was administered orally as a single (10, 3, or 2 mg pindone kg(-1) over three consecutive days) or multiple exposure (dosing regime repeated after a further 8 days). Prothrombin times (PT) increased up to 4-fold in treated sheep, and haemorrhage occurred in some instances, particularly with the double dose treatment. Deaths of sheep also occurred, usually when the sheep were placed under added stress, particularly that associated with shearing. The breeding performance of pregnant ewes dosed with pindone was reduced, mainly due to an increase in stillborn and nonviable lambs (i.e. deaths within 2 days of birth). The motility of sperm in treated rams was also affected. Pindone persisted in the blood (maximum, 13.2 mg L(-1)) for up to 14 days after the last dose, and the half-life (t1/2) was estimated at approximately 5 days depending upon the dosing regime. Other tissue residues ranged from 17 (fat) to 39 (liver) mg kg(-1). The implications of these findings for ongoing responsible use of pindone (anticoagulants) in pest control programs are also discussed.

3-0 methylglucose uptake as a marker of nutrient absorption and bowel length in pediatric patients.

BACKGROUND: Inert carbohydrate probes are commonly used to assess intestinal permeability; we have previously shown that the actively transported moiety 3-0 methylglucose (3-0 MG) is a useful marker of intestinal surface area and nutrient absorption in animal models of short bowel syndrome (SBS). This study examines the correlation of 3-0 MG absorption with nutrient absorption, bowel length, and the tolerance of enteral feeds in pediatric patients. METHODS: Fifteen children (1 month to 15 years in age) were studied after intestinal surgery. All had a stoma, 2 were > 1 year of age, the remainder had surgical intervention as a neonate or within the first month of life. Eight had SBS (50% expected bowel length for age). Bowel length was measured intraoperatively. Nutrient absorption was quantified with a 48-hour bowel study, measuring fat, protein, and carbohydrate output directly. 3-0 MG absorption and intestinal permeability were quantified using a solution containing 30 mg/mL 3-0 MG, 20 mg/mL mannitol and 30 mg/mL lactulose (osmolarity 352, given at 1 mL/kg via feeding tube). Subsequent urine production was collected for 8 hours, and probe recovery measured using HPLC. RESULTS: 3-0 MG absorption was significantly correlated with nutrient absorption. The correlation with protein absorption was r2 = .59, fat r2 = .62 and carbohydrate r2 = .56. The correlation between 3-0 MG absorption and bowel length was r2 = .58. 3-0 MG absorption was significantly lower in SBS patients vs patients with normal bowel length (15.8 +/- 6.7 vs 30.5 +/- 10.2%). 3-0 MG absorption also correlated with the ability to tolerate enteral feeds (r2 = .38; p < .03 for all comparisons). CONCLUSIONS: 3-0 MG may be a useful marker of nutrient absorption and bowel length in pediatric patients with short bowel syndrome. The simplicity and reproducibility of the method make it an attractive option for following patient outcomes. Further studies are suggested to determine the utility of these markers in directing the clinical management of patients.