Application of enzyme-linked immunosorbent assay to detect antimicrobial peptides in human intestinal lumen.

Intestinal transplantation is the definitive treatment for intestinal failure. However, tissue rejection and graft-versus-host disease are relatively common complications, necessitating aggressive immunosuppression that can itself pose further complications. Tracking intraluminal markers in ileal effluent from standard ileostomies may present a noninvasive and sensitive way to detect developing pathology within the intestinal graft. This would be an improvement compared to current assessments, which are limited by poor sensitivity and specificity, contributing to under or over-immunosuppression, respectively, and by the need for invasive biopsies. Herein, we report an approach to reproducibly analyze ileal fluid obtained through stoma sampling for antimicrobial peptide/protein concentrations, reasoning that these molecules may provide an assessment of intestinal homeostasis and levels of intestinal inflammation over time. Concentrations of lysozyme (LYZ), myeloperoxidase (MPO), calprotectin (S100A8/A9) and ß-defensin 2 (DEFB2) were assessed using adaptations of commercially available enzyme-linked immunosorbent assays (ELISAs). The concentration of α-defensin 5 (DEFA5) was assessed using a newly developed sandwich ELISA. Our data support that with proper preparation of ileal effluent specimens, precise and replicable determination of antimicrobial peptide/protein concentrations can be achieved for each of these target molecules via ELISA. This approach may prove to be reliable as a clinically useful assessment of intestinal homeostasis over time for patients with ileostomies.

A battery-less implantable glucose sensor based on electrical impedance spectroscopy.

The ability to perform accurate continuous glucose monitoring without blood sampling has revolutionised the management of diabetes. Newer methods that can allow measurements during longer periods are necessary to substantially improve patients' quality of life. This paper presents an alternative method for glucose monitoring which is based on electrical impedance spectroscopy. A battery-less implantable bioimpedance spectroscope was designed, built, and used in an in vivo study on pigs. After a recovery period of 14 days post surgery, a total of 236 subcutaneous bioimpedance measurements obtained from intravenous glucose tolerance tests, with glucose concentration ranges between 77.4 and 523.8 mg/dL, were analyzed. The results show that glucose concentrations estimated by subcutaneous bioimpedance measurements correlate very well to the blood glucose reference values. The pigs were clinically healthy throughout the study, and the postmortem examinations revealed no signs of adverse effects related to the sensor. The implantation of the sensor requires minor surgery. The implant, being externally powered, could in principle last indefinitely. These encouraging results demonstrate the potential of the bioimpedance method to be used in future continuous glucose monitoring systems.

Age Rather Than Supplementation with Oat ß-Glucan Influences Development of the Intestinal Microbiota and SCFA Concentrations in Suckling Piglets.

The effects of early supplementation with oat ß-glucan during the suckling period on piglet gut microbiota composition, concentrations of short-chain fatty acids, and gut physiological markers were assessed. Fifty piglets from five litters, balanced for sex and birth weight, were divided within litters into two treatment groups: ß-glucan and control. Piglets in the ß-glucan group received the supplement three times/week from day 7 of age until weaning. Rectal swab samples were collected from 10 piglets per treatment group (balanced across litters) from week 1 to week 4, and plasma samples were collected at 1, 3, and 4 weeks of age. Additional samples of intestinal tissues and jugular and portal vein plasma were collected from 10 animals at weaning (one per treatment group and litter). The concentrations of short-chain fatty acids in plasma and the microbiota composition in rectal swabs were mainly influenced by piglet age, rather than the supplement. There were significant differences in microbiota composition between litters and several correlations between concentrations of short-chain fatty acids in plasma and specific microbial taxa in rectal swabs. Overall, ß-glucan supplementation did not have any clear impact on the gut environment in suckling piglets, whereas a clear age-related pattern emerged.

Training Pigs for Oral Glucose Tolerance Test-Six Years' Experience of a Refined Model.

Animal models of human diseases are important in biomedical research. When using animals for scientific purposes, the 3Rs (replace, reduce, refine) should be considered. Refinement of animal models is essential to ensure best use of animals, which is important for ethical reasons and to retrieve reliable research data. The present publication describes improvements to an oral glucose tolerance test (OGTT) model for pigs published in 2016. Historical data from 42 pigs were used to describe improvements in the training technique over six years. Pigs of various breeds and ages can be trained to bottle-feed glucose dissolved in water to undergo OGTT. This publication describes different tips and techniques to apply for successful training and will help researchers to minimize exclusions of pigs due to unsuccessful training. The improvements are an important contribution to the 3Rs.

Exploring the GLP-1-GLP-1R axis in porcine pancreas and gastrointestinal tract in vivo by ex vivo autoradiography.

INTRODUCTION: Glucagon-like peptide-1 (GLP-1) increases insulin secretion from pancreatic beta-cells and GLP-1 receptor (GLP-1R) agonists are widely used as treatment for type 2 diabetes mellitus. Studying occupancy of the GLP-1R in various tissues is challenging due to lack of quantitative, repeatable assessments of GLP-1R density. The present study aimed to describe the quantitative distribution of GLP-1Rs and occupancy by endogenous GLP-1 during oral glucose tolerance test (OGTT) in pigs, a species that is used in biomedical research to model humans. RESEARCH DESIGN AND METHODS: GLP-1R distribution and occupancy were measured in pancreas and gastrointestinal tract by ex vivo autoradiography using the GLP-1R-specific radioligand 177Lu-exendin-4 in two groups of pigs, control or bottle-fed an oral glucose load. Positron emission tomography (PET) data from pigs injected with 68Ga-exendin-4 in a previous study were used to retrieve data on biodistribution of GLP-1R in the gastrointestinal tract. RESULTS: High homogenous uptake of 177Lu-exendin-4 was found in pancreas, and even higher uptake in areas of duodenum. Low uptake of 177Lu-exendin-4 was found in stomach, jejunum, ileum and colon. During OGTT, there was no increase in plasma GLP-1 concentrations and occupancy of GLP-1Rs was low. The ex vivo autoradiography results were highly consistent with to the biodistribution of 68Ga-exendin-4 in pigs scanned by PET. CONCLUSION: We identified areas with similarities as well as important differences regarding GLP-1R distribution and occupancy in pigs compared with humans. First, there was strong ligand binding in the exocrine pancreas in islets. Second, GLP-1 secretion during OGTT is minimal and GLP-1 might not be an important incretin in pigs under physiological conditions. These findings offer new insights on the relevance of porcine diabetes models.

Bioavailability of subcutaneous and intramuscular administrated buprenorphine in New Zealand White rabbits.

BACKGROUND: Buprenorphine is one of the most used analgesics for postoperative pain in rabbits. The recommended dose in rabbits (0.01-0.05 mg/kg) is the same for intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration, despite lack of pharmacokinetic data. Five male and five female New Zealand White rabbits (mean ± SD body weight 3.1 ± 0.3 kg) were administered 0.05 mg/kg buprenorphine by the IV, IM and SC routes and 0.1 mg/kg by the SC route, in a cross-over design with two-week wash-out periods between treatments. Blood was collected before, and up to 8 h post buprenorphine injection, for determination of serum levels by UPHLC-MS/MS. RESULTS: The area under the time concentration curve (AUC0-t) was lower after SC (398 ± 155 ng/mL/min) than IM (696 ± 168 ng/mL/min, p < 0.001) and IV (789 ± 189 ng/mL/min, p < 0.001) administration. The maximum serum concentration was lower after SC (2.2 ± 1.4 ng/mL) than after IM (11 ± 3.2 ng/mL) administration (p < 0.001). The bioavailability was lower after SC (50 ± 19%) than after IM (95 ± 21%) administration (p = 0.006). The elimination half-life was longer after SC (260 ± 120 min) than after IM (148 ± 26 min, p = 0.002) as well as IV (139 ± 33 min) injection (p < 0.001). An increase in the SC dose from 0.05 to 0.1 mg/kg resulted in an increase in the area under the time concentration curve of 50% in female (p = 0.022) and 165% in male rabbits (p < 0.001). The bioavailability did not change in the females (36 ± 14%, p = 0.6), whereas it increased in the males (71 ± 23%, p = 0.008). CONCLUSIONS: The lower bioavailability of 0.05 mg/kg buprenorphine after SC administration could explain the lack of efficacy seen in clinical pain studies in rabbits, using this route. For immediate pain relief, IV or IM administration is therefore be recommended, whereas SC administration may be useful to sustain analgesic serum levels, once efficient pain relief has been achieved. The current data do not support an increase in dose to compensate for the lower SC bioavailability.

The effect of housing environment on bone healing in a critical radius defect in New Zealand White rabbits.

In animal studies on bone healing, the effect of housing space and physical activity are seldom taken into account. Bone formation was evaluated in New Zealand White rabbits (mean ± SEM BW: 3.9 ± 0.11 kg) with a critical bone defect after 12 weeks of rehabilitation in pair-housing in 3 m2 large floor pens (Floor, n = 10) or standard single housing in 0.43 m2 cages (Cage, n = 10). In the randomised full-factorial study, a bone replica of calcium phosphate cement (CPC, n = 10) or autologous bone (AB, n = 10) was implanted in the unilateral 20 mm radius defect. Post-mortem, the oxidative capacity was measured by citrate synthase (CS) activity in M. quadriceps and the defect filling volume and density evaluated by microcomputer tomography (µ-CT). Histology sections were evaluated by subjective scoring and histomorphometry. Fourteen rabbits remained until the end of the study. Group Floor (n = 7; 3 CPC + 4 AB) had a higher CS activity and a larger bone defect filling volume and lower density by µ-CT measurements than group Cage (n = 7; 3 CPC + 4 AB). Three out of four rabbits in AB-Floor presented fusion of the defect with reorganisation of trabecular bone, whereas three of four in AB-Cage showed areas of incomplete healing. Floor rabbits had a higher score of bony fusion between the radius and ulna than Cage rabbits. There were no differences between groups in histomorphometry. The study found that a larger housing space increased physical activity and promoted bone formation.

Nursing and training of pigs used in renal transplantation studies.

The pig is commonly used in renal transplantation studies since the porcine kidney resembles the human kidney. To meet the requirements of intense caretaking and examination without stress, a 2-week socialisation and training programme was developed. Conventional cross-breed pigs (n = 36) with high health status were trained for 15 min/day in a four-step training programme before kidney transplantation. The systematic training resulted in calm animals, which allowed for ultrasound examination, blood sampling and urine sampling without restraint. When a 2-methacryloyloxyethyl phosphorylcholine polymer-coated jugular catheter introduced via the auricular vein was used for post-operative blood sampling, clotting was avoided. To assess renal function, urinary output was observed and creatinine and cystatin C were measured; the latter was not found to be useful in recently transplanted pigs. The results presented contribute to the 3Rs (refine, reduce, replace).

Pancreatic perfusion and its response to glucose as measured by simultaneous PET/MRI.

AIMS: Perfusion of the pancreas and the islets of Langerhans is sensitive to physiological stimuli and is dysregulated in metabolic disease. Pancreatic perfusion can be assessed by both positron emission tomography (PET) and magnetic resonance imaging (MRI), but the methods have not been directly compared or benchmarked against the gold-standard microsphere technique. METHODS: Pigs (n = 4) were examined by [15O]H2O PET and intravoxel incoherent motion (IVIM) MRI technique simultaneously using a hybrid PET/MRI scanner. The pancreatic perfusion was measured both at basal conditions and after intravenous (IV) administration of up to 0.5 g/kg glucose. RESULTS: Pancreatic perfusion increased by 35%, 157%, and 29% after IV 0.5 g/kg glucose compared to during basal conditions, as assessed by [15O]H2O PET, IVIM MRI, and microspheres, respectively. There was a correlation between pancreatic perfusion as assessed by [15O]H2O PET and IVIM MRI (r = 0.81, R2 = 0.65, p < 0.01). The absolute quantification of pancreatic perfusion (ml/min/g) by [15O]H2O PET was within a 15% error of margin of the microsphere technique. CONCLUSION: Pancreatic perfusion by [15O]H2O PET was in agreement with the microsphere technique assessment. The IVIM MRI method has the potential to replace [15O]H2O PET if the pancreatic perfusion is sufficiently large, but not when absolute quantitation is required.

Anaesthesia and changes in parameters that reflect glucose metabolism in pigs - a pilot study.

Pigs are commonly used in diabetes research due to their many physiological similarities to humans. They are especially useful in imaging procedures because of their large size. However, to achieve imaging procedures the pig must lie completely still, and thus needs to be anaesthetized. Most anaesthetic drugs used in laboratory animals affect carbohydrate metabolism by the inhibition of insulin release. The aim of this pilot study was primarily to develop an anaesthetic protocol for pigs that did not have an effect on blood glucose levels throughout the 3 h of anaesthesia; and secondly, to evaluate the most promising protocol in combination with an oral glucose tolerance test (OGTT). Two anaesthetic protocols were used in four growing pigs. Intravenous propofol infusion caused hyperglycaemia in three out of four pigs within 5-10 min after induction and was therefore excluded. Intravenous infusion with tiletamine, zolazepam and butorphanol (TZB) for 3 h did not affect blood glucose levels. The pigs underwent OGTT twice, once without anaesthesia and once with TZB induction after glucose intake. Anaesthesia during OGTT resulted in a lower area under the curve (AUC) of glucose ( P < 0.05), higher AUC of glucagon ( P < 0.05) and an insulin response less than 10% of that during OGTT without anaesthesia. In conclusion, long-term infusion anaesthesia with TZB does not affect glucose homeostasis in pigs. However, the protocol is not effective when combined with OGTT, as glucose, insulin and glucagon levels are affected.

Establishment of a Refined Oral Glucose Tolerance Test in Pigs, and Assessment of Insulin, Glucagon and Glucagon-Like Peptide-1 Responses.

Diabetes mellitus is increasing worldwide and reliable animal models are important for progression of the research field. The pig is a commonly used large animal model in diabetes research and the present study aimed to refine a model for oral glucose tolerance test (OGTT) in young growing pigs, as well as describing intravenous glucose tolerance test (IVGTT) in the same age group. The refined porcine OGTT will reflect that used in children and adolescents. Eighteen pigs were obtained one week after weaning and trained for two weeks to bottle-feed glucose solution, mimicking the human OGTT. The pigs subsequently underwent OGTT (1.75 g/kg BW) and IVGTT (0.5 g/kg BW). Blood samples were collected from indwelling vein catheters for measurements of glucose and the diabetes related hormones insulin, glucagon and active glucagon-like peptide-1. The study confirmed that pigs can be trained to bottle-feed glucose dissolved in water and thereby undergo an OGTT more similar to the human standard OGTT than previously described methods in pigs. With the refined method for OGTT, oral intake only consists of glucose and water, which is an advantage over previously described methods in pigs where glucose is given together with feed which will affect glucose absorption. Patterns of hormonal secretion in response to oral and intravenous glucose were similar to those in humans; however, the pigs were more glucose tolerant with lower insulin levels than humans. In translational medicine, this refined OGTT and IVGTT methods provide important tools in diabetes research when pigs are used as models for children and adolescents in diabetes research.