Feeding FIDO : development, validation and application of a dynamic, in vitro model of the gastrointestinal tract of the dog

To produce a complete and balanced dog diet it is necessary to know the nutritional needs of the dog as well as the availability of nutrients from the diet. Petfood manufacturers are restricted in performing (invasive) studies with animals for ethical reasons. Therefore, it is necessary to search for in vitro alternatives to these studies as explained in Chapter 1. Based on a literature study on the physiology of the GI tract of the healthy, adult dog (Chapter 2), the dynamic in vitro model for human subjects, pigs and calves, as described by Minekus (1995), was modified to mimic the physiological conditions of the dog. The model is named FIDO ( f unctional gastro i ntestinal do g model).The aim of the study was to develop the in vitro model simulating the GI tract of the dog. The model should simulate the physiological conditions in the GI tract of the dog as closely as possible. In that way luminal processes as well as physical and chemical properties of diets could be investigated. This thesis describes the developmental experiments, the validation experiments in comparison to dog studies as well as application studies.Validation of the modelTechnical validationThe study started with an extended literature review on the physiology of the stomach and small intestine of the healthy, adult dog with a special emphasis on those parameters which are relevant for the development of the dog model (Chapter 2). With respect to digestion of food, such parameters as transit times, pH values, concentrations of electrolytes and activities of enzymes are important to mimic physiological conditions as closely as possible. Data found in the literature were translated to a computer program to simulate these parameters in FIDO. The features of the model are described in Chapters 2 to 7. Based on the simulation of the physiology of the dog dry and canned dog foods were used to test the technical possibilities of the model. After some technical modifications to the gastric compartment and the pre-filters connected to the jejunal and ileal compartments, a study was performed to test the effect of particle size of dry dog food on gastric emptying. Particles≤3 mm emptied more slowly than particles≤1 mm. The effect of transit time on the availability for absorption of nitrogen and calcium of canned dog food was also investigated. Like in vivo , in FIDO less nitrogen and calcium were available for absorption with faster transit times (Chapter 3).Validation in vitro versus in vivoValidation of the model in comparison to the in vivo situation was the next step in the development of the dog model (Chapter 4). Ileal protein digestibility and availability for absorption of nitrogen of eight different dry dog foods were tested in the model. The results were compared with data found in vivo with ileally cannulated dogs, performed at the University of Illinois. The experiments proved to be very reproducible and the results found in FIDO are similar to those found in vivo in the dogs.Based on these findings and those of former experiments simulating pigs, calves and human beings, it can be concluded that the model is a suitable tool as an alternative to animal experiments in nutritional research.Application of the modelThe validation study (Chapter 3) showed a low availability for absorption of calcium in the model. Based on these results it was decided to study calcium and phosphorus availability from three commercially available canned dog foods (Chapter 4). Effects of addition of calcium-phosphorus supplements or the enzyme phytase and the effect of a lower pH in the small intestine were also included in this study. A human standard breakfast was used as a control diet, because this diet had a high calcium availability under human conditions in the diet.The results showed that the canned dog foods had a low availability for absorption for calcium (maximum 21%) and phosphorus (maximum 44%). Differences in relative availability of calcium and phosphorus were found among these diets, which can probably be explained by the source of these minerals. Also the low availability of these minerals can be attributable to the source. Another possible explanation of the low availability are the (saturated) fatty acids in the diet, which can form calcium soaps in the model.The advantage of FIDO is that the availability for absorption of calcium and other nutrients can be studied without the influence of the physiological status of the animal. The real amount absorbed by the animal, however, cannot be studied. Absorption by the animal depends on its needs and the absorption is hence dependent on two different mechanisms: passive and active absorption. To get more insight into the absorption of calcium through the intestinal wall of the dog, experiments were performed with Great Danes (8 and 20 weeks of age; in cooperation with the Veterinary Faculty of Utrecht University), FIDO and intestinal segments (Chapter 7). The effect of vitamin D was taken into account in this study by studying two levels in the diet. The three different methods ( in vivo , FIDO and intestinal segments) are complementary and can be used to get a better understanding of the regulation of calcium absorption in the dog.In Chapter 6 experiments are described to investigate the effect of gelling agents on the digestibility and availability for absorption of nutrients ('chunks in gravy' products). Three different (combinations of) gelling agents added to the gravy were used in this study in different concentrations (0.2% and 0.5%): carrageenan plus guar gum, carrageenan plus locust bean meal, and wheat starch. A diet without gelling agent was used as a control diet. Neither the gelling agent nor the concentration had any effect on digestibility of proteins and carbohydrates, availability for absorption of calcium and phosphorus, viscosity or buffering capacity in the intestinal content. From the results it can be concluded that addition of the gelling agents used does not affect the nutritional quality of the diets at the low concentrations tested.ConclusionsThe dynamic in vitro model of the GI tract of dogs simulates the physiological parameters very accurately and reproducibly. Transit time of food has an effect on digestibility and availability for absorption, just like in dogs. Also ileal protein digestibility in the model is similar to the data found in vivo . It is possible to investigate specific questions regarding dog food in this model (e.g. the effect of gelling agents on digestibility). Another important aspect is the fact that the experiments in FIDO can be performed under highly standardized condition, in contrast to in vivo studies in which biological variance among animals plays a role. This comes to expression in the reproducibility and sensitivity of the results from FIDO compared to results of in vivo studies.The dynamic in vitro model of the gastrointestinal tract of the dog is a suitable alternative to in vivo studies with respect to digestibility and availability for absorption of nutrients from different types of dog food, such as canned and dry dog foods.</p

Recombinant human erythropoietin α modulates the effects of radiotherapy on colorectal cancer microvessels

Recent data suggest that recombinant human erythropoietin (rhEPO) modulates tumour growth and therapy response. The purpose of the present study was to examine the modulation of radiotherapy (RT) effects on tumour microvessels by rhEPO in a rat colorectal cancer model. Before and after 5 × 5 Gy of RT, dynamic contrast-enhanced -magnetic resonance imaging was performed and endothelial permeability surface product (PS), plasma flow (F), and blood volume (V) were modelled. Imaging was combined with pO2 measurements, analysis of microvessel density, microvessel diameter, microvessel fractal dimension, and expression of vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 α (HIF-1α), Bax, and Bcl-2. We found that RT significantly reduced PS and V in control rats, but not in rhEPO-treated rats, whereas F was unaffected by RT. Oxygenation was significantly better in rhEPO-treated animals, and RT induced a heterogeneous reoxygenation in both groups. Microvessel diameter was significantly larger in rhEPO animals, whereas VEGF expression was significantly lower in the rhEPO group. No differences were observed in HIF-1α, Bax, or Bcl-2 expression. We conclude that rhEPO results in spatially heterogeneous modulation of RT effects on tumour microvessels. Direct effects of rhEPO on neoplastic endothelium are likely to explain these findings in addition to indirect effects induced by increased oxygenation

Stress Strengthens Memory of First Impressions of Others' Positive Personality Traits

Encounters with strangers bear potential for social conflict and stress, but also allow the formation of alliances. First impressions of other people play a critical role in the formation of alliances, since they provide a learned base to infer the other's future social attitude. Stress can facilitate emotional memories but it is unknown whether stress strengthens our memory for newly acquired impressions of other people's personality traits. To answer this question, we subjected 60 students (37 females, 23 males) to an impression-formation task, viewing portraits together with brief positive vs. negative behavior descriptions, followed by a 3-min cold pressor stress test or a non-stressful control procedure. The next day, novel and old portraits were paired with single trait adjectives, the old portraits with a trait adjective matching the previous day's behavior description. After a filler task, portraits were presented again and subjects were asked to recall the trait adjective. Cued recall was higher for old (previously implied) than the novel portraits' trait adjectives, indicating validity of the applied test procedures. Overall, recall rate of implied trait adjectives did not differ between the stress and the control group. However, while the control group showed a better memory performance for others' implied negative personality traits, the stress group showed enhanced recall for others' implied positive personality traits. This result indicates that post-learning stress affects consolidation of first impressions in a valence-specific manner. We propose that the stress-induced strengthening of memory of others' positive traits forms an important cue for the formation of alliances in stressful conditions

Second asymptomatic carotid surgery trial (ACST-2): a randomised comparison of carotid artery stenting versus carotid endarterectomy

Background: Among asymptomatic patients with severe carotid artery stenosis but no recent stroke or transient cerebral ischaemia, either carotid artery stenting (CAS) or carotid endarterectomy (CEA) can restore patency and reduce long-term stroke risks. However, from recent national registry data, each option causes about 1% procedural risk of disabling stroke or death. Comparison of their long-term protective effects requires large-scale randomised evidence. Methods: ACST-2 is an international multicentre randomised trial of CAS versus CEA among asymptomatic patients with severe stenosis thought to require intervention, interpreted with all other relevant trials. Patients were eligible if they had severe unilateral or bilateral carotid artery stenosis and both doctor and patient agreed that a carotid procedure should be undertaken, but they were substantially uncertain which one to choose. Patients were randomly allocated to CAS or CEA and followed up at 1 month and then annually, for a mean 5 years. Procedural events were those within 30 days of the intervention. Intention-to-treat analyses are provided. Analyses including procedural hazards use tabular methods. Analyses and meta-analyses of non-procedural strokes use Kaplan-Meier and log-rank methods. The trial is registered with the ISRCTN registry, ISRCTN21144362. Findings: Between Jan 15, 2008, and Dec 31, 2020, 3625 patients in 130 centres were randomly allocated, 1811 to CAS and 1814 to CEA, with good compliance, good medical therapy and a mean 5 years of follow-up. Overall, 1% had disabling stroke or death procedurally (15 allocated to CAS and 18 to CEA) and 2% had non-disabling procedural stroke (48 allocated to CAS and 29 to CEA). Kaplan-Meier estimates of 5-year non-procedural stroke were 2·5% in each group for fatal or disabling stroke, and 5·3% with CAS versus 4·5% with CEA for any stroke (rate ratio [RR] 1·16, 95% CI 0·86–1·57; p=0·33). Combining RRs for any non-procedural stroke in all CAS versus CEA trials, the RR was similar in symptomatic and asymptomatic patients (overall RR 1·11, 95% CI 0·91–1·32; p=0·21). Interpretation: Serious complications are similarly uncommon after competent CAS and CEA, and the long-term effects of these two carotid artery procedures on fatal or disabling stroke are comparable. Funding: UK Medical Research Council and Health Technology Assessment Programme

A review of the physiology of the canine digestive tract related to the development of in vitro systems.

Food and nutrition studies in animals and human beings often meet with technical difficulties and sometimes with ethical questions. An alternative to research in living animals is the dynamic multicompartmental in vitro model for the gastrointestinal tract described by Minekus et al. (1995) and Havenaar & Minekus (1996). The dynamic conditions that are simulated in this model are peristaltic movements, transit times, pH responses, secretion of enzymes and electrolytes and absorption of nutrients and water. To obtain data for an in vitro model of the dog gastrointestinal tract, the literature was surveyed for physiological responses to different types of dog food. These included: values of enzyme activities, electrolyte concentrations, gastric emptying and intestinal transit times, pH values, secretion and composition of bile and absorption rates in different parts of the dog gastrointestinal tract. The review focuses on research carried out on healthy, adult dogs of 10-20 kg and on parameters related to the oral cavity, stomach and small intestine. This literature research gives sufficient data on the physiology of the canine digestive tract for the development of an in vitro dynamic model that adequately simulates the functions of the stomach and small intestine of the dog

A novel stepwise analysis procedure of genome-wide expression profiles identifies transcript signatures of thiamine genes as classifiers of mitochondrial mutants

To extract functional information on genes and processes from large expression datasets, analysis methods are required that can computationally deal with these amounts of data, are tunable to specific research questions, and construct classifiers that are not overspecific to the dataset at hand. To satisfy these requirements, a stepwise procedure that combines elements from principal component analysis and discriminant analysis, was developed to specifically retrieve genes involved in processes of interest and classify samples based upon those genes. In a global expression dataset of 300 gene knock-outs in Saccharomyces cerevisiae, the procedure successfully classified samples with similar 'cellular component' Gene Ontology annotations of the knock-out gene by expression signatures of limited numbers of genes. The genes discriminating 'mitochondrion' from the other subgroups were evaluated in more detail. The thiamine pathway turned out to be one of the processes involved and was successfully evaluated in a logistic model to predict whether yeast knock-outs were mitochondrial or not. Further, this pathway is biologically related to the mitochondrial system. Hence, this strongly indicates that our approach is effective and efficient in extracting meaningful information from large microarray experiments and assigning functions to yet uncharacterized genes.status: publishe