Optimization of the non-invasive 13C-sucrose breath test in a rat model of methotrexate-induced mucositis.

PURPOSE: In order to determine the sensitivity and specificity of the test and to optimize experimental conditions utilizing the SBT in a rat model of chemotherapy-induced small intestinal damage. METHODS: Initially, a 13C-sucrose dose-response study was performed in rats to determine an optimal sucrose concentration for the SBT; then applied to assess chemotherapy-induced intestinal damage. A further study was conducted to establish a SBT time-course of methotrexate-induced small intestinal damage and repair. Animals were killed at 96 or 144 h. RESULTS: A sucrose concentration of 0.25 g/ml was optimal (20% CV) for reproducibility and detection of intestinal damage. Maximal damage occurred at 72 h, small intestinal repair was initiated by 96 h and continued at 144 h post-MTX, as determined by the SBT and confirmed by biochemical analyses. Levels of sensitivity and specificity for the SBT were 98 and 94%, respectively. CONCLUSIONS: The SBT is a reliable non-invasive marker of small intestinal health and damage with a high degree of sensitivity and specificity.

Lactoferrin and desferrioxamine are ineffective in the treatment of Helicobacter pylori infection and may enhance H. pylori growth and gastric inflammation in mice.

AIMS: To evaluate the efficacy of bovine lactoferrin (BLf), recombinant human lactoferrin (rHLf) and desferrioxamine against Helicobacter pylori in vitro and in mice and also to determine whether BLf or rHLf alter gastric inflammation. METHODS AND RESULTS: In vitro: Broth dilution susceptibility tests were performed using different concentrations of desferrioxamine, BLf and rHLf. Murine trials: In the prevention trial, C57BL/6 female mice were treated with BLf or rHLF, and then infected with the SS1 strain of H. pylori. In the treatment trial, mice were gavaged with either BLf, rHLf or desferrioxamine. In addition, gastric myeloperoxidase activity (MPO) was measured to assess gastric inflammation. Desferoxamine was found to have a direct bactericidal effect, while BLf and rHLf only partially suppressed H. pylori growth in vitro. However, in both prevention and treatment trials all three forms of treatment failed to reduce H. pylori load in mice. Gastric MPO activity and H. pylori load were noted to be higher with lactoferrin treatments. CONCLUSIONS: Our study does not support the use of BLf or rHLF in the treatment of human H. pylori infection. Interestingly, H. pylori growth and gastric inflammation appear to be enhanced by lactoferrin treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: The mouse model is ideal for testing novel H. pylori eradicating agents.

The effects of formula feeding on physiological and immunological parameters in the gut of neonatal rats.

A unique model of formula feeding in the neonatal rat was utilized to investigate the effects of an enterally delivered artificial milk formula on clinically relevant immunological and biological characteristics in the gut, compared to naturally reared pups. Hooded Wistar rat pups were randomly allocated to two treatment groups: formula-fed (FF) or naturally suckled (NS). A flexible silastic intra-gastric cannula was surgically implanted into the FF pups, through which an artificial rat milk supplement was continuously delivered from day 4 to day 10 of life. Rat pups were sacrificed at 10 days of age. Body weight, small intestinal weight, mucosal CD8(+) cell numbers, and ileal lactase activity in FF animals were significantly decreased compared to their NS counterparts (P < 0.05). Numbers of eosinophils, mucosal mast cells, CD4(+) T-cells, ileal villus height and gastric emptying times were significantly increased in FF pups (P < 0.05). We have developed a new rat model of artificial feeding which possesses important immunological and biological similarities to the premature human infant.

A method for collecting breath samples from individual chickens for analysis of 13CO(2), H(2), and CH(4).

This study describes experiments using simple helmets to collect breath samples from individual birds for measurement of (13)CO(2), H(2), and CH(4), which form the basis for various diagnostic tests for intestinal dysfunction in humans. Peak enrichment in (13)C in breath CO(2) occurred between 5 and 30 min postingestion by 18-d-old chickens administered a gelatin capsule containing approximately 3.6 mg of (13)C-octanoic acid dissolved in vegetable oil. For 25-d-old chickens given 10 mL of homogenized cooked corn by oral gavage, peak enrichment occurred 60 to 90 min postingestion. In fully fed 25-d-old chickens, H(2) and CH(4) concentrations in breath ranged from 7 to 115 ppm and from 0 to 5.5 ppm, respectively. Following an overnight fast, H(2) and CH(4) concentrations in breath ranged from 0.5 to 7.5 ppm and 0 to 3.0 ppm, respectively, in the same chickens. Ranges in H(2) (1.0 to 56.5 ppm) and CH(4) (0 to 8.0 ppm) concentrations widened considerably 3 h after oral gavage with approximately 130 mg of lactulose (an indigestible disaccharide) dissolved in 5 mL of water. The results from these investigations indicate that collection of re-breathed air samples from chickens is plausible, which opens the way for development of noninvasive methods for evaluating gastrointestinal functions in chickens.

Non-invasive tests in animal models and humans: a new paradigm for assessing efficacy of biologics including prebiotics and probiotics.

Newer biological agents that are designed to have multiple effects on a host require better ways to determine both their safety and toxicity. Indeed ecologically potent factors such as agents that can alter the gut milieu and change host responses are now being realized as a viable alternative to more focused pharmaceuticals. Even in the pharmaceutical arena there is a growing awareness of the preventative and therapeutic potential of alternative agents. Probiotics and prebiotics amongst other agents fall into this category and can have both direct and indirect effects on the pathogenesis and progress of disease. This review details some of the new approaches using non-invasive tests to enable firstly a better definition of a stressed through to a damaged gastrointestinal mucosa. They constitute ways to apply dynamic function testing in animal models and humans to provide reference points to which other measurements can be related e.g. altered circulating cytokines, altered gene expression. As such this phenotypic scaffold, alone and combined with newer molecular parameters, will improve our understanding of the interaction of luminal factors within the alimentary tract and the impact that these have on physiologically challenged mucosa and in disease both at the gastrointestinal level and also in remote organs. Practically, the dynamic function tests, primarily breath tests, can now be used as diagnostic and prognostic indicators of the efficacy of new biologics such as probiotics and prebiotics that in part elicit their effects by altering the ecology of particular regions of the intestine.

Yoghurts containing probiotics reduce disruption of the small intestinal barrier in methotrexate-treated rats.

Small intestinal permeability was employed to assess the efficacy of commercially available yoghurts containing probiotics in a rat model of methotrexate (MTX)-induced mucositis. Male Sprague-Dawley rats were allocated to four groups (n = 8): MTX + water, MTX + cow's milk yoghurt (CY; fermented with Lactobacillus johnsonii), MTX + sheep's milk yoghurt (SY; containing Lactobacillus bulgaricus and Streptococcus thermophilus), and saline. Treatment gavage occurred twice daily for 7 days pre-MTX and 5 days post-MTX. Intestinal permeability was assessed on days -7, -1, 2, and 5 of the trial. Intestinal sections were collected at sacrifice for histological and biochemical analyses. Histology revealed that rats receiving CY and SY did not have a significantly damaged duodenum compared to controls. However, an improved small intestinal barrier function was evident, determined by a decreased lactulose/mannitol ratio. Probiotics containing SY and CY may be useful in preventing disruption to intestinal barrier function in MTX-induced mucositis.

Short-term zinc supplementation attenuates Helicobacter felis-induced gastritis in the mouse.

BACKGROUND: Mucosal damage by H. pylori infection is mainly caused by neutrophils producing large quantities of reactive oxygen species (ROS). Metallothionein (MT) an intracellular, low-molecular, cysteine-rich protein, which is inducible by dietary zinc (Zn), has been implicated in sequestering ROS. This study examines the effects of Zn supplementation on Helicobacter colonisation and associated gastritis and the relationship with gastric MT levels. METHODS: C57Bl/6 mice were inoculated with either 10(8) H. pylori or H. felis and were infected for 4 weeks or 6 and 12 weeks, respectively. Mice infected with H. pylori (4 weeks) or H. felis (6 weeks) were treated with either Zn acetate (ZnA; 1 mg/ml), or Zn sulphate (ZnSO4; 5 mg/ml) for 2 weeks with 0.1 ml oro-gastric gavage twice daily. H. pylori load and H. felis colonisation density were determined by culture and microscopy, respectively. MT levels and H. felis-induced gastritis were also determined. RESULTS: Zn treatment showed no significant difference in Helicobacter load and gastric MT, however, ZnSO4 treatment showed a significant (p<0.05) increased in gastric MT in H. felis infected mice. Both Zn-treated groups showed a significant (p<0.05) difference in gastritis score in the antrum of the stomach within the basal and submucosal compartments compared to H. felis-infected controls. CONCLUSIONS: We found that H. felis-induced gastritis can be attenuated by short-term treatment of Zn. This observation suggests that Zn alone may be effective for the suppression of gastric mucosal inflammation induced by Helicobacter.

First generation of pentazole (HN5, pentazolic acid), the final azole, and a zinc pentazolate salt in solution: A new N-dearylation of 1-(p-methoxyphenyl) pyrazoles, a 2-(p-methoxyphenyl) tetrazole and application of the methodology to 1-(p-methoxyphenyl) pentazole.

Ceric ammonium nitrate (CAN) in methanol-water gave a new N-dearylation of a series of substituted 1-(p-methoxyphenyl) pyrazoles and a 2-(p-methoxyphenyl)tetrazole producing p-benzoquinone and the parent azole in a mole for mole ratio. Application of this reaction to 1-(p-methoxyphenyl) pentazole at -40 degrees C produced p-benzoquinone. 15N NMR spectra suggest that pentazole, HN5, was also produced and held in solution as N5- with Zn2+ ion. The 15N signal from N5- was -10.0 +/- 2.0 ppm in agreement with calculated values.

Improvement in intestinal permeability precedes morphometric recovery of the small intestine in coeliac disease.

It is often difficult to assess small bowel recovery in adults with coeliac disease on a gluten-free diet (GFD). This prospective study compares changes in intestinal permeability with changes in intestinal biopsy at various intervals after commencing a GFD. Intestinal permeability was measured by lactulose/rhamnose absorption from 1 week to 24 months after commencing a GFD. Intestinal morphometry was measured by villus area, crypt length and mitotic count per crypt at diagnosis and after commencing a GFD. Median intestinal permeability values decreased from 0.47 (n = 35) at diagnosis to 0.25 (n = 17) after 1 week and to 0.16 (n = 18) after 2 months of a GFD. Rhamnose absorption improved significantly at an early stage, from 6.6% (untreated) to 15.4% at 3 months of a GFD, whereas the decrease in lactulose permeation took longer: from 3.4% (untreated) to 0.8% after 12 months of a GFD. Mean villus area (n = 29) was reduced to 16% of control values at diagnosis, and improved to a maximum of 48% after 6 months on a GFD, but did not change thereafter. Mean crypt length and mitotic count per crypt were increased by 222% and 356% respectively at diagnosis, and these parameters remained elevated at 172% and 216% above control values after 6 months of a GFD. We conclude that intestinal permeability improves within 2 months after starting a GFD, but that measurable intestinal biopsy improvement requires ingestion of a GFD for at least 3-6 months, and even then remains incomplete.

A new tricyclic ring and a nitrogen-sulfur analogue of the tri-pentagon bowl. Substituted 5,6,7,7a-tetrahydropyrrolo[2,1-b]-1,3,4-thiadiazole- endo-6,7-dicarboxyimides.

The first examples of a bowl-shaped tricyclic nitrogen-sulfur analogue of the tri-pentagon bowl are reported; substituted 5,6,7,7a-tetrahydropyrrolo[2,1-b]-1,3,4-thiadiazole-endo-6,7-dicarboxyimides are synthesised by a relatively simple route.

Probiotics in pediatric gastrointestinal disorders.

Probiotics have been defined most recently as living microorganisms which, upon ingestion in certain numbers, exact health benefits beyond inherent general nutrition. They have been a part of human nutrition for centuries, but in recent years they have been more closely studied for their potential to improve health and treat disease. This review of probiotics is not extensive, highlighting the most recent reviews and well controlled clinical studies in both animals and humans. The safety issues are also discussed as well as potential mechanisms of action. The importance of studying each probiotic bacterium individually in each condition where a health benefit is claimed is highlighted by Lactobacillus GG, the most widely studied probiotic which has proven benefit in reducing the severity and duration of viral diarrhea but no benefit against bacterial diarrhea.

Assessment of gastric emptying in the mouse using the [13C]-octanoic acid breath test.

1. Gastric emptying studies in small laboratory animals are hampered by the deficiency of a technique that is non-invasive and repeatable. The aim of the present study was to adapt the non-invasive [13C]-octanoic acid breath test, which has been validated in humans, to assess both liquid and solid gastric emptying in the mouse. 2. Gastric emptying rates were investigated for a liquid meal (Intralipid; Kabi Pharmacia AB, Stockholm, Sweden; n = 7) and two solid meals (egg yolk and mouse chow; n = 7) incorporating [13C]-octanoic acid. All meals were analysed for natural enrichment of [13C]. Mathematical analysis of the 13CO2 excretion rate allowed the determination of gastric emptying parameters. 3. Gastric emptying of Intralipid was more rapid than egg yolk (P < 0.0001). Gastric emptying of mouse chow could not be assessed due to intragastric separation of [13C]-octanoic acid and natural [13C] enrichment of the pellet. 4. The [13C]-octanoic acid breath test can reproducibly assess both liquid and solid gastric emptying non-invasively in the mouse. This method can now be used to assess gastric emptying in drug studies and disease studies for which there are established mouse models.