A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium.

Epithelial damage due to gastrointestinal disorders frequently causes severe disease in horses. To study the underlying pathophysiological processes, we aimed to establish equine jejunum and colon enteroids (eqJE, eqCE) mimicking the in vivo epithelium. Therefore, enteroids were cultivated in four different media for differentiation and subsequently characterized histomorphologically, on mRNA and on protein level in comparison to the native epithelium of the same donor horses to identify ideal culture conditions for an in vitro model system. With increasing enterocyte differentiation, the enteroids showed a reduced growth rate as well as a predominantly spherical morphology and less budding compared to enteroids in proliferation medium. Combined or individual withdrawal of stem cell niche pathway components resulted in lower mRNA expression levels of stem cell markers and concomitant differentiation of enterocytes, goblet cells and enteroendocrine cells. For eqCE, withdrawal of Wnt alone was sufficient for the generation of differentiated enterocytes with a close resemblance to the in vivo epithelium. Combined removal of Wnt, R-spondin and Noggin and the addition of DAPT stimulated differentiation of eqJE at a similar level as the in vivo epithelium, particularly with regard to enterocytes. In summary, we successfully defined a medium composition that promotes the formation of eqJE and eqCE consisting of multiple cell types and resembling the in vivo epithelium. Our findings emphasize the importance of adapting culture conditions to the respective species and the intestinal segment. This in vitro model will be used to investigate the pathological mechanisms underlying equine gastrointestinal disorders in future studies.

Short-term glycemic variability in non-diabetic, non-obese dogs assessed by common glycemic variability indices.

Glycemic variability (GV) refers to swings in blood glucose levels and is an emerging measure of glycemic control in clinical practice. It is associated with micro- and macrovascular complications and poor clinical outcomes in diabetic humans. Although an integral part of patient assessment in human patients, it is to a large extent neglected in insulin-treated diabetic dogs. This prospective pilot study was performed to describe canine within-day GV in non-diabetic dogs with the aim to provide a basis for the interpretation of daily glucose profiles, and to promote GV as an accessible tool for future studies in veterinary medicine. Interstitial glucose concentrations of ten non-diabetic, non-obese beagles were continuously measured over a 48-h period using a flash glucose monitoring system. GV was assessed using the common indices MAGE (mean amplitude of glycemic excursion), GVP (Glycemic variability percentage) and CV (coefficient of variation). A total of 2260 sensor measurements were obtained, ranging from 3.7 mmol/L (67 mg/dL) to 8.5 mmol/L (153 mg/dL). Glucose profiles suggested a meal-dependent circadian rhythmicity with small but significant surges during the feeding periods. No differences in GV indices were observed between day and night periods (p > 0.05). The MAGE (mmol/L), GVP (%) and CV (%) were 0.86 (± 0.19), 7.37 (± 1.65), 6.72 (± 0.89) on day one, and 0.83 (± 0.18), 6.95 (± 1.52), 6.72 (± 1.53) on day two, respectively. The results of this study suggest that GV is low in non-diabetic dogs and that glucose concentrations are kept within narrow ranges.

Polarity reversal of canine intestinal organoids reduces proliferation and increases cell death.

Apical-out intestinal organoids are a relatively simple method of gaining access to the apical cell surface and have faced increasing scientific interest over the last few years. Apical-out organoids can thus be used for disease modelling to compare differing effects on the basolateral versus the apical cell surface. However, these 'inside-out' organoids die relatively quickly and cannot be propagated as long as their basal-out counterparts. Here, we show that apical-out organoids have drastically reduced proliferative potential, as evidenced by immunohistochemical staining and the incorporation of the thymidine analogue EdU. At the same time, cell death levels are increased. Nevertheless, these phenomena cannot be explained by an induction of differentiation, as the gene expression of key marker genes for various cell types does not change over time.

Neutralising Effects of Different Antibodies on Clostridioides difficile Toxins TcdA and TcdB in a Translational Approach.

Given the high prevalence of intestinal disease in humans and animals, there is a strong need for clinically relevant models recapitulating gastrointestinal systems, ideally replacing in vivo models in accordance with the principles of the 3R. We established a canine organoid system and analysed the neutralising effects of recombinant versus natural antibodies on Clostridioides difficile toxins A and B in this in vitro system. Sulforhodamine B cytotoxicity assays in 2D and FITC-dextran barrier integrity assays on basal-out and apical-out organoids revealed that recombinant, but not natural antibodies, effectively neutralised C. difficile toxins. Our findings emphasise that canine intestinal organoids can be used to test different components and suggest that they can be further refined to also mirror complex interactions between the intestinal epithelium and other cells.

The World of Organoids: Gastrointestinal Disease Modelling in the Age of 3R and One Health with Specific Relevance to Dogs and Cats.

One Health describes the importance of considering humans, animals, and the environment in health research. One Health and the 3R concept, i.e., the replacement, reduction, and refinement of animal experimentation, shape today's research more and more. The development of organoids from many different organs and animals led to the development of highly sophisticated model systems trying to replace animal experiments. Organoids may be used for disease modelling in various ways elucidating the manifold host-pathogen interactions. This review provides an overview of disease modelling approaches using organoids of different kinds with a special focus on animal organoids and gastrointestinal diseases. We also provide an outlook on how the research field of organoids might develop in the coming years and what opportunities organoids hold for in-depth disease modelling and therapeutic interventions.

Comparison of the effects of open-tube and evacuated tube-assisted sampling methods on thromboelastography variables for blood samples from healthy dogs.

OBJECTIVE: To compare the effects of open-tube blood sampling with previously investigated blood sampling methods via evacuated tube on thromboelastography variables for blood samples from dogs. ANIMALS: 10 healthy Beagles from the research colony owned by the Clinic of Small Animal Internal Medicine, University Veterinary of Medicine, Vienna, were used. PROCEDURES: In this prospective study, blood was sampled from each dog serially into citrate solution-containing tubes via 20-gauge needle. One evacuated tube was filled from a jugular vein via the evacuated tube port, and the second tube was opened and filled by catching blood flowing through the needle from a lateral saphenous vein. Venipuncture quality was scored with a previously described method. Thromboelastography was performed for each sample. RESULTS: Inferential statistics used with the Wilcoxon signed rank test showed significant differences in reaction time (R) of 3.43 ± 0.84 minutes versus 4.53 ± 0.62 minutes (mean ± SD) between evacuated tube assisted and open-tube sampling, respectively. No other significant differences were identified. CLINICAL RELEVANCE: The sampling methods compared have a small but significant effect on R in thromboelastographic analysis for blood samples from healthy dogs. Shear stress by vacuum sampling seems to accelerate coagulation in jugular blood samples harvested by evacuated tube, resulting in a shortened R. Results suggested that the open-tube method avoids shear stress induced activation of coagulation and is an appropriate sampling method for thromboelastography when used within a standardized protocol.

Wolves, dogs and humans in regular contact can mutually impact each other's skin microbiota.

In contrast to humans and dogs, the skin microbiota of wolves is yet to be described. Here, we investigated the skin microbiota of dogs and wolves kept in outdoor packs at the Wolf Science Center (WSC) via 16S rRNA gene amplicon sequencing. Skin swab samples were also collected from human care takers and their pet dogs. When comparing the three canine groups, representing different degrees of human contact to the care takers and each other, the pet dogs showed the highest level of diversity. Additionally, while human skin was dominated by a few abundant phylotypes, the skin microbiota of the care takers who had particularly close contact with the WSC animals was more similar to the microbiota of dogs and wolves compared to the humans who had less contact with these animals. Our results suggest that domestication may have an impact on the diversity of the skin microbiota, and that the canine skin microbiota can be shared with humans, depending on the level of interaction.

Successful Insulin Glargine Treatment in Two Pet Guinea Pigs with Suspected Type 1 Diabetes Mellitus.

Scientific information on spontaneous type I diabetes mellitus (DM) and treatment modalities in guinea pigs is scarce. As most diabetic guinea pigs are overweight and respond to dietary changes, a disorder resembling type II-DM in humans seems to be most prevalent in this species. In the present report, a nine-month-old female intact guinea pig (GP1) was presented because of a cataract and polyphagia. The physical examinations in GP1 and its littermate, GP2, were unremarkable. Laboratory tests revealed hyperglycemia, hyperlipidemia, elevated fructosamine concentrations, and glucosuria in GP1 and GP2. Not responding to dietary changes, an insulin-dependent diabetes mellitus was suspected in both animals. Treatment with 0.5 IU of glargine insulin (Lantus®) per guinea pig subcutaneously (s.c.) once daily was initiated in both animals. Monitoring included repeated clinical evaluations and the measurement of plasma glucose and fructosamine concentrations. Capillary glucose concentration was measured using a glucometer, and glucosuria was monitored by dipstick. Blood glucose concentrations decreased quickly in both GPs, and glucosuria resolved. Including several dose adjustments, DM remained controlled for over 1.5 years. Bilateral cataracts and lens-induced uveitis in GP1 were medically managed with only slight progression. This is the first report of guinea pigs with insulin-dependent diabetes mellitus that were successfully treated with long-acting basal insulin glargine.

Diagnosis of feline pancreatitis with SNAP fPL and Spec fPL.

OBJECTIVES: Pancreatitis is a frequent disease in cats for which the ante-mortem diagnosis remains challenging. Feline pancreatic lipase immunoreactivity (fPLI) has been reported to have a high sensitivity for the diagnosis of pancreatitis. The aim of this study was to compare the rapid in-house test SNAP fPL with the standard test Spec fPL and to evaluate the use of SNAP fPL to diagnose pancreatitis in an emergency setting. METHODS: fPLI of 111 cats with a clinical suspicion of pancreatitis was measured with both SNAP fPL and Spec fPL. Furthermore, clinical signs, haematological and biochemical changes, and abdominal ultrasound findings were recorded. RESULTS: Seventy-eight of 111 cats (70.3%) were tested below the cut-off level for pancreatitis with SNAP, as well as Spec fPL, whereas 21/111 (18.9%) were tested with values above the cut-off level with both tests. In 12/111 (10.8%) cats the results were discordant. The comparison of both tests revealed an agreement of 78/80 (97.5%) when Spec fPL was ⩽3.5 µg/l (negative) and 18/20 (90%) when Spec fPL was ⩾5.4 µg/l (positive). The most common clinical signs in cats with suspected pancreatitis (n = 21) were lethargy (95.2%), reduced appetite and vomiting (90.5% each), dehydration (81.0%), diarrhoea (57.1%), abdominal pain and weight loss (47.6% each). Hyperglycaemia and hyperbilirubinaemia (85.7% each), increased aspartate transaminase (76.2%) and alanine transaminase (47.6%), leucocytosis (61.9%), lymphopenia (57.1%), decreased sodium and chloride (57.1% each), and increased urea (52.4%) were the most common abnormalities in blood work. CONCLUSIONS AND RELEVANCE: Clinical signs, as well as routine blood-work changes, were non-specific and thus proved to be insufficient to diagnose pancreatitis. The combination of SNAP fPL and subsequent Spec fPL, if indicated, provided the opportunity to rule out or to diagnose pancreatitis with a higher certainty than previously known test methods. This study proved SNAP fPL to be a valuable tool to exclude or include pancreatitis in an emergency setting.

Hemocompatibility testing according to ISO 10993-4: discrimination between pyrogen- and device-induced hemostatic activation.

Next to good hemocompatibility performance of new medical devices, which has to be tested according to the ISO 10993-4, the detection of pyrogen-contaminated devices plays a pivotal role for safe device application. During blood contact with pyrogen-contaminated devices, intense inflammatory and hemostatic reactions are feared. The aim of our study was to investigate the influence of pyrogenic contaminations on stents according to the ISO 10993-4. The pyrogens of different origins like lipopolysaccharides (LPS), purified lipoteichoic acid (LTA) or zymosan were used. These pyrogens were dried on stents or dissolved and circulated in a Chandler-loop model for 90 min at 37°C with human blood. Before and after circulation, parameters of the hemostatic system including coagulation, platelets, complement and leukocyte activation were investigated. The complement system was activated by LPS isolated from Klebsiella pneumoniae and Pseudomonas aeruginosa and by LTA. Leukocyte activation was triggered by LPS isolated from K. pneumoniae, LTA and zymosan, whereas coagulation and platelet activation were only slightly influenced. Our data indicate that pyrogen-contaminated devices lead to an alteration in the hemostatic response when compared to depyrogenized devices. Therefore, pyrogenicity testing should be performed prior to hemocompatibility tests according to ISO 10993-4 in order to exclude hemostatic activation induced by pyrogen contaminations.

Highly sensitive pyrogen detection on medical devices by the monocyte activation test.

Pyrogens are components of microorganisms, like bacteria, viruses or fungi, which can induce a complex inflammatory response in the human body. Pyrogen contamination on medical devices prior operation is still critical and associated with severe complications for the patients. The aim of our study was to develop a reliable test, which allows detection of pyrogen contamination on the surface of medical devices. After in vitro pyrogen contamination of different medical devices and incubation in a rotation model, the human whole blood monocyte activation test (MAT), which is based on an IL-1ß-specific ELISA, was employed. Our results show that when combining a modified MAT protocol and a dynamic incubation system, even smallest amounts of pyrogens can be directly detected on the surface of medical devices. Therefore, screening of medical devices prior clinical application using our novel assay, has the potential to significantly reduce complications associated with pyrogen-contaminated medical devices.