Untersuchungen zu biochemischen Markern des Knochen- und Energiestoffwechsels am Osteoporose-induzierten Schafmodell

Die Untersuchungen erfolgten im Rahmen des Sonderforschungsbereichs/ Transregio 79 (2010-2014): „Werkstoffe für die Geweberegeneration im systemisch erkrankten Knochen“ Teilprojekt T1: „Etablierung und Qualitätssicherung osteoporotischer Tiermodelle“. Die Versuche am Tier wurden in der Zentralen Forschungseinrichtung der Johann Wolfgang Goethe-Universität Frankfurt am Main durchgeführt. Der Tierversuchsantrag (GE: F31/36) wurde durch das Regierungs-präsidium Darmstadt am 27.09.2012 genehmigt. Serum und Urinproben, die für diese Arbeit relevant waren, wurden am Klinikum Veterinärmedizin, Klinische Pathophysiologie und Klinische Laboratoriumsdiagnostik der Justus-Liebig-Universität Gießen analysiert. Ziel dieses interdisziplinierten Forschungsprojektes war es, neue Knochenersatzmaterialien und Implantatwerkstoffe für den systemisch erkrankten Knochen des Menschen zu generieren. Für die dafür nötigen präklinischen Studien ist die Etablierung eines Tiermodells, welches die Prozesse am menschlichen Knochen in ähnlicher Weise widerspiegeln kann, unerlässlich. Ziel dieser Arbeit war es, die Interaktion zwischen Knochen- und Energiestoffwechsel im Osteoporose-induzierten Schafmodell zu evaluieren, die beim Schaf bisher wenig bekannt ist. Schlussfolgernd kann festgehalten werden, dass diese Studie durch die Kombinationsinduktion einer Osteoporose mittels Ovarektomie, defizitärer Diät (Calcium und Vitamin D) und Glukokortikoiden eine Basis für weitere Forschungen wie z.B. dem Einsatz neuartiger Biomaterialien zur Frakturversorgung am Tiermodell Schaf darstellt. In diesem Modell waren deutliche Veränderungen im Knochenstoffwechsel sowie eine Interaktion zwischen Knochen- und Energiestoffwechsel im Sinne einer transienten Insulinresistenz induzierbar, wobei jedoch die frühesten und ausgeprägtesten Veränderungen in der OVXDC-Gruppe auftraten, was darauf schließen lässt, dass Glukokortikoide beim Schaf einen schnelleren Effekt auf den Knochen- und Energiestoffwechsel haben als eine Ovarektomie und Diät alleine. Daher ist für präklinische Studien ein Kombinations-Schema inklusive Glukokortikoidgabe empfehlenswert, wobei jedoch die Menge an Methylprednisolon noch anzupassen ist, um starke Nebenwirkungen bei den Schafen zu vermeiden.The investigations were conducted within the scope of the sub-project T1: "Establishment and Quality Assurance in Osteoporotic Animal Models" of the Collaborative Research Centre/Transregio 79 (2010-2014): "Materials for Tissue Regeneration in Systemically Diseased Bones". The animal experiments were conducted in the Central Research Facility of the Goethe University of Frankfurt. The study was ethically approved by the Darmstadt Regional Council on 27/09/2012 (GE: F31/36). Serum and urine samples that were relevant to this study were analysed at the Faculty of Veterinary Medicine, Clinical Pathophysiology and Clinical Laboratory Diagnostics of the Justus Liebig University Gießen, Germany. The goal of this interdisciplinary research project was to develop new synthetic materials as surrogate for bones and materials for implants for systemically diseased human bones. For the required pre-clinical studies, it is essential to establish an animal model capable to reflect the processes in human bones in a similar way. The goal of this study was to evaluate the interaction between bone and energy metabolism in a sheep model with induced osteoporosis for which hitherto only scarce knowledge is existent. Overall, it can be concluded that osteoporosis in the sheep can be induced by a combination of ovariectomy, deficient diet (calcium and vitamin D) and glucocorticoids, so that the study is a useful basis for further research, e.g. use of novel biomaterials for fracture treatment in a sheep animal model. In this model, it was possible to induce significant abnormalities of the bone metabolism and the interaction between bone and energy metabolism consistent with a transient insulin resistance, whereby the earliest and most severe changes occurred in the OVXDC group, which suggests that glucocorticoids have a quicker effect on bone and energy metabolism in sheep than an ovariectomy and diet alone. A combined scheme including glucocorticoids is therefore recommendable for pre-clinical studies, although the dosage of methylprednisolone still needs to be adjusted to avoid strong clinical side effects in the sheep

Evaluation of a novel quantitative canine species-specific point-of-care assay for C-reactive protein

Background: Species-specific point-of-care tests (POCT) permit a rapid analysis of canine C-reactive protein (CRP), enabling veterinarians to include CRP in clinical decisions. Aim of the study was to evaluate a novel POCT for canine CRP (Point Stripâ TM Canine CRP Assay) run on a small in-house-analyzer (Point Reader TM V) using lithium heparin plasma and to compare assay performance to an already established canine CRP assay (Gentian Canine CRP Immunoassay) run on two different bench top analyzers serving as reference methods (ABX Pentra 400, AU 5800). Linearity was assessed by stepwise dilution of plasma samples with high CRP concentrations. Limit of quantification (LoQ) was determined by repeated measurements of samples with low CRP concentrations. Coefficient of variation (CV) at low (10-50 mg/l), moderate (50-100 mg/l), and high (100-200 mg/l) CRP concentrations was investigated as well as possible interferences. Method comparison study was performed using 45 samples of healthy and diseased dogs. Quality criteria were fulfilled if the total observed error (TEobs=2CV%+bias%) was below the minimal total allowable error of 44.4% (TE min). Additionally, a reference range (n =60 healthy dogs) was established. Results: Linearity was present at CRP concentrations of 10-132 mg/l (&#8793; 361 mg/l CRP with reference method) with a LoQ set at 10 mg/l. At moderate to high CRP concentrations, intra- and inter-assay CVs were< =8% and <=11% respectively, while CVs<=22% and <=28% were present at low concentrations. No interferences were observed at concentrations of 4 g/l hemoglobin, 800 mg/l bilirubin and 8 g/l triglycerides. Method comparison study demonstrated an excellent correlation with both reference methods (r =0.98 for ABX Pentra 400; 0.99 for AU 5800), though revealing a proportional bias of 19.7% (ABX Pentra 400) and 10.7% (AU 5800) respectively. TEobs was 26.7-31.9% and 16.7-21.9% and thus < TEmin. Healthy dogs presented with CRP values <=11.9 mg/l. Conclusions The POCT precisely detects canine CRP at clinically relevant moderate and high CRP concentrations. The assay correlates well with both reference methods. Due to the bias, however, follow-up examinations should be performed with the same assay and analyzer

Evaluation of a novel quantitative canine species-specific point-of-care assay for C-reactive protein

Abstract Background Species-specific point-of-care tests (POCT) permit a rapid analysis of canine C-reactive protein (CRP), enabling veterinarians to include CRP in clinical decisions. Aim of the study was to evaluate a novel POCT for canine CRP (Point Strip™ Canine CRP Assay) run on a small in-house-analyzer (Point Reader™ V) using lithium heparin plasma and to compare assay performance to an already established canine CRP assay (Gentian Canine CRP Immunoassay) run on two different bench top analyzers serving as reference methods (ABX Pentra 400, AU 5800). Linearity was assessed by stepwise dilution of plasma samples with high CRP concentrations. Limit of quantification (LoQ) was determined by repeated measurements of samples with low CRP concentrations. Coefficient of variation (CV) at low (10–50 mg/l), moderate (50–100 mg/l), and high (100–200 mg/l) CRP concentrations was investigated as well as possible interferences. Method comparison study was performed using 45 samples of healthy and diseased dogs. Quality criteria were fulfilled if the total observed error (TEobs = 2CV% + bias%) was below the minimal total allowable error of 44.4% (TE min). Additionally, a reference range (n = 60 healthy dogs) was established. Results Linearity was present at CRP concentrations of 10–132 mg/l (≙ 361 mg/l CRP with reference method) with a LoQ set at 10 mg/l. At moderate to high CRP concentrations, intra- and inter-assay CVs were ≤ 8% and ≤ 11% respectively, while CVs ≤ 22% and ≤ 28% were present at low concentrations. No interferences were observed at concentrations of 4 g/l hemoglobin, 800 mg/l bilirubin and 8 g/l triglycerides. Method comparison study demonstrated an excellent correlation with both reference methods (r = 0.98 for ABX Pentra 400; 0.99 for AU 5800), though revealing a proportional bias of 19.7% (ABX Pentra 400) and 10.7% (AU 5800) respectively. TEobs was 26.7–31.9% and 16.7–21.9% and thus < TEmin. Healthy dogs presented with CRP values ≤11.9 mg/l. Conclusions The POCT precisely detects canine CRP at clinically relevant moderate and high CRP concentrations. The assay correlates well with both reference methods. Due to the bias, however, follow-up examinations should be performed with the same assay and analyzer

Method Validation and Establishment of Reference Intervals for an Insulin-like Growth Factor-1 Chemiluminescent Immunoassay in Cats

Previously, radioimmunoassay (RIA) has been the only assay to measure insulin-like growth factor-1 (IGF-1) to diagnose hypersomatotropism (HS). Due to radiation concerns, availability, and the cost of IGF-1 RIA, validation of assays for automated analysers such as a chemiluminescent immunoassay (CLIA) is needed. The aim of this study was to validate a CLIA for measurement of feline IGF-1 (IMMULITE 2000® XPi, Siemens Medical Solutions Diagnostics, Malvern, PA, USA) compared to IGF1 RIA, establish reference interval (RI), and determine a cut-off value for diagnosis of HS in diabetic cats. Validation of assay performance included precision, linearity, and recovery studies. Right-sided RI was determined using surplus serum of 50 healthy adult cats. Surplus serum samples of diabetic cats with known IGF-1 concentration with (n = 32/68) and without HS (n = 36/68) were used for method comparison with RIA. The cut-off for diagnosis of HS was established using receiver operating characteristic (ROC) analysis. The intra-assay coefficient of variation (CV) was ≤4.7%, and the inter-assay CV was ≤5.6% for samples with low, medium, and high IGF-1 concentration. Linearity was excellent (R2 > 0.99). The correlation between CLIA and RIA was very high (rs = 0.97), with a mean negative bias for CLIA of 24.5%. The upper limit of RI was 670 ng/mL. ROC analysis showed an area under the curve of 0.94, with best cut-off for diagnosis of HS at 746 ng/mL (sensitivity, 84.4%; specificity, 97.2%). The performance of CLIA was good, and the RI and cut-off for HS diagnosis established in this study allow for CLIA to be used in routine work-up of diabetic cats

Feline paraneoplastic alopecia associated with metastasising intestinal carcinoma

Case summary A 10-year-old male neutered British Shorthair cat was presented with a 6 month history of lethargy, weight loss and alopecia. Clinical examination revealed widespread alopecia of the ventral abdomen and hindlimbs. The skin in these areas was smooth and shiny and hairs could be easily epilated. Spontaneous pruritus was observed. Cytological examination of superficial impression smears showed a severe Malassezia species dermatitis and pyoderma. Ectoparasites could not be detected and no sign of dermatophytosis was visible in trichograms and Wood’s lamp analysis. Abdominal ultrasound found a focally thickened wall of the large intestine and multiple nodules in the liver. Fine-needle aspirates from lymph nodes, liver and altered colonic wall were consistent with an undifferentiated malignant neoplasia. The cat was euthanased at the owners’ request, owing to potential neoplasia with metastatic spread. At necropsy a metastasising carcinoma of the colonic wall was found, as well as a paraneoplastic alopecia. Relevance and novel information Feline paraneoplastic alopecia has been reported in association with pancreatic carcinoma, bile duct carcinoma and hepatocellular carcinoma, as well as with neuroendocrine pancreatic carcinoma and hepatosplenic plasma cell tumour. This is the first reported case of feline paraneoplastic alopecia associated with a colon carcinoma