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Background Prophylactic replacement therapy in hemophilia A (HA) patients does not adequately prevent bleeds and arthropathic complications. A more refined understanding of the relationship between coagulation factor VIII (FVIII) levels and bleeding risk during protein prophylaxis, or with gene therapy, is needed to improve patient care. Objectives Investigate this relationship in the HA rat, a model exhibiting spontaneous bleeds and development of arthropathy similar to HA patients. Methods Human B domain-deleted FVIII was delivered to HA rats via adeno-associated virus (AAV)-mediated gene transfer or multiple intravenous protein injections. Results and conclusions After 12 weeks of observation, both approaches significantly reduced bleeds per animal and increased the proportion of bleed-free animals compared with controls (43% vs 0%, respectively [AAV]; 75% vs 8%, respectively [injection]). Both approaches resulted in an anti-FVIII inhibitory response in 20% to 37% of treated animals, similar to HA patients. Inhibitory antibodies were refractory to clinical improvement (reduction of bleeds) only in the AAV-based prophylaxis. An integrated model-based analysis of data on FVIII exposure and bleeding events was performed. This predicted the bleeding risk at any given circulating FVIII activity. Specifically, 4.8 or 10 IU/dL FVIII (0.048 and 0.1 IU/mL, respectively) were predicted to reduce bleeding risk by 90% or 95%, respectively, compared with untreated controls. Our data establish the utility of the HA rat model in FVIII prophylaxis studies and describe how FVIII activity affects bleeding risk in this setting. These enable further studies on FVIII prophylaxis focusing on disease complications for an optimized treatment of HA patients.

Background: Hemophilic arthropathy is a debilitating morbidity of hemophilia caused by recurrent joint bleeds. We investigated if the joint bleed volume, before initiation of treatment, was linked to the subsequent degree of histopathological changes and the development of bone pathology in a mouse model of hemophilic arthropathy.Methods: FVIII knock-out (F8-KO) mice were dosed with a micro-CT blood pool agent prior to induction of hemarthrosis. Eight hours after induction, the bleed volume was quantified with micro computed tomography (micro-CT) and recombinant FVIII treatment initiated. On Day 8, inflammation in the knees was characterized by fluorescence molecular tomography. On Day 14, knee pathology was characterized by micro-CT and histopathology. In a second study, contrast agent was injected into the knee of wild-type (WT) mice, followed by histopathological evaluation on Day 14.Results: The average joint bleed volume before treatment was 3.9 mm3. The inflammation-related fluorescent intensities in the injured knees were significantly increased on Day 8. The injured knees had significantly increased synovitis scores, vessel counts, and areas of hemosiderin compared to un-injured knees. However, no cartilage- or bone pathology was observed. The bleed volume before initiation of treatment correlated with the degree of synovitis and was associated with high fluorescent intensity on Day 8. In F8-KO and WT mice, persistence of contrast agent in the joint elicited morphological changes.Conclusion: When applying a delayed on-demand treatment regimen to hemophilic mice subjected to an induced knee hemarthrosis, the degree of histopathological changes on Day 14 reflected the bleed volume prior to initiation of treatment.

Introduction Monitoring of clinical effectiveness of bypassing agents in haemophilia patients is hampered by the lack of validated laboratory assays. Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) have been evaluated for predicting clinical effectiveness of bypassing agents, however, with limited success. Aim Application of a longitudinal model-based approach may allow for a quantitative characterization of the link between ROTEM parameters and the probability of bleeding events. Methods We analyse longitudinal data from haemophilia A rats receiving gene-based FVIIa prophylaxis in terms of total circulatory levels of FVII/FVIIa, clotting time (CT) measured using ROTEM and the probability of bleeding events. Results Using population pharmacokinetic-pharmacodynamic (PKPD) modelling, a PK-CT-repeated time-to-event (RTTE) model was developed composed of three submodels (a) a FVII/FVIIa PK model, (b) a PK-CT model describing the relationship between predicted FVIIa expression and CT and (c) a RTTE model describing the probability of bleeding events as a function of CT. The developed PK-CT-RTTE model accurately described the vector dose-dependent plasma concentration-time profile of total FVII/FVIIa and the exposure-response relationship between AAV-derived FVIIa expression and CT. Importantly, the developed model accurately described the occurrence of bleeding events over time in a quantitative manner, revealing a linear relationship between predicted change from baseline CT and the probability of bleeding events. Conclusion Using PK-CT-RTTE modelling, we demonstrated that ROTEM parameters can accurately predict the probability of bleeding events in a translational animal model of haemophilia A.

Background The anti‐tissue factor plasma inhibitor monoclonal antibody concizumab is under clinical investigation for subcutaneous prophylaxis of hemophilia A/B (HA/HB) with or without inhibitors. Breakthrough bleeds while on concizumab prophylaxis may be treated with bypassing agents (recombinant activated factor VIIa [rFVIIa] and activated prothrombin complex concentrate [APCC]), or with factor VIII (FVIII) or factor IX (FIX). Objectives To evaluate the effect of combining concizumab with rFVIIa, APCC, rFVIII, and rFIX on thrombin generation (TG) potential. Methods Pooled HA plasma was spiked in vitro with concizumab alone or together with rFVIIa, APCC, or rFVIII. rFVIIa, APCC, and rFVIII were added ex vivo to plasma from HA patients receiving concizumab prophylaxis. Pooled HB plasma was spiked with concizumab alone or together with rFIX. TG potential was measured after initiation with tissue factor. Results Concizumab increased thrombin peak in a concentration‐dependent manner. Adding rFVIIa, APCC, rFVIII, or rFIX caused a further increase in thrombin peak. The effects of concizumab and rFVIIa, APCC, rFVIII, or rFIX were mainly additive, with no or up to maximally ~25% extra effect caused by drug‐‐drug interaction. No strong synergistic effects were observed upon combining concizumab with rFVIIa, APCC, rFVIII, or rFIX. The thrombin peak obtained with 0.5 IU/ml rFVIII or rFIX in the presence of concizumab was on occasion slightly higher, but mostly comparable to the thrombin peak with 1 IU/ml rFVIII or rFIX in the absence of concizumab. Conclusion rFVIIa, APCC, rFVIII, and rFIX enhanced plasma TG potential in the presence of concizumab. Dose levels of concomitant use should be adjusted accordingly to balance potential safety concerns while maintaining the necessary hemostatic effect. Please see the video in the Supplementary Material for an animated summary of the data presented.

The aim of this study was to objectively evaluate the biological variation of healthy Asian elephant (Elephas maximus) hematology and biochemistry parameters, therefore enabling evidence-based clinical decision-making to improve patient management. Ten clinically healthy elephants had blood samples collected weekly for 5 wk under standardized conditions. The analytical, between- and within-individual variation, index of individuality, and reference change values were calculated using previously reported methods. Large between-individual variation and small within-individual variation for almost all parameters indicated that individual normal values should be used for interpreting blood results from Asian elephants.

Population-based reference intervals (RIs) are vital tools used to characterize health and disease based on laboratory values. The science and statistical basis for RI generation have evolved over the past 50 yr. Current veterinary-specific guidelines by the American Society of Veterinary Clinical Pathology exist for establishing RIs from nondomestic and wild animals. A list of 35 items that should be included during generation and publication of reference data was distilled from the currently available RI guidelines. The archives of five peer-reviewed journals were searched and 106 articles presenting laboratory reference data from nondomestic or wildlife species were identified and each reviewed by two authors to determine compliance with the list of 35 items. A compliance score was calculated as the number of articles that fulfilled the item out of the number where it would have been appropriate to fulfill the item. Most articles reported the number of reference individuals (compliance score 0.98), their partitioning demographics (compliance score 0.95), and sample collection and handling practices (compliance scores 0.97 and 0.96, respectively). Common deficiencies included omitting discussion of the validation status of the analytical methods for the species being evaluated (compliance score 0.12), documentation of use of exclusion criteria (compliance score 0.51), outlier detection (compliance score 0.43), appropriate statistical methods for the reference population (compliance score 0.34), and calculation and presentation of confidence intervals around the reference limits (compliance score 0.35). Compliance scores were not statistically different when stratified on the number of individuals in the largest and smallest evaluated group or the format of the article (full vs short format). Articles that cited RI generation guidelines fulfilled more of the required steps and provided a more complete description of their data (compliance score 0.74) than those that did not cite guidelines (compliance score 0.58). Additional attention to the science of and recommendations for RI generation is recommended to strengthen the utility of published data.

Background Haemophilic arthropathy is a chronic and debilitating joint disease caused by recurrent spontaneous joint bleeds in patients with haemophilia. Understanding how characteristics of individual joint bleeds relate to the subsequent development of arthropathy could improve management and prevention of this joint disease. Here, we aimed to explore relations between joint bleed characteristics and development of bone pathology in a mouse model of haemophilic arthropathy by using novel in vivo imaging methodology. Methods We characterised induced knee bleeds in a murine model of haemophilic arthropathy by quantitative in vivo fluorescence molecular tomography (FMT) and by measurements of changes in the diameter of the injured knee. Wild-type mice and non-injured haemophilic mice acted as controls. Development of arthropathy was characterised by post mortem evaluation of bone pathology by micro-CT 14 days after bleed-induction. In an in vitro study, we assessed the effect of blood on the quantification of fluorescent signal with FMT. Results In most injured haemophilic mice, we observed significant loss of trabecular bone, and half of the mice developed pathological bone remodelling. Development of pathological bone remodelling was associated with significantly increased fluorescent signal and diameter of the injured knee just 1 day after induction of the bleed. Further, a correlation between the fluorescent signal 1 day after induction of the bleed and loss of trabecular bone reached borderline significance. In the in vitro study, we found that high concentrations of blood significantly decreased the fluorescent signal. Conclusion Our results add novel insights on the pathogenesis of haemophilic arthropathy and underline the importance of the acute phase of joint bleeds for the subsequent development of arthropathy.

Introduction: Haemophilic animal models are used to study blood-induced cartilage damage, but quantitative and sensitive outcome measures are needed. Aim: To develop a novel quantitative method for detecting early cartilage degeneration in a haemophilic rat model of blood-induced joint damage. Methods: The 35Sulphate incorporation (35SO4 2− assay) was applied to tibial and patellar cartilage of wild-type rats to quantify baseline proteoglycan synthesis and to evaluate the effect of 4-day blood exposure in vitro. Next, haemarthrosis was induced in 39 FVIII-deficient rats and characterized by changes in knee joint diameter and development of bone pathology (using micro-CT). Four- and 16-day posthaemarthrosis proteoglycan synthesis rate (PSR) was assessed using the 35SO4 2− assay, with the contralateral knee as control. Results: In vitro, a decrease in PSR in tibial and patellar cartilage was demonstrated following blood exposure. In vivo, joint diameter and development of bone pathology confirmed successful induction of haemarthrosis. In the blood-exposed knee, tibial and patellar PSR was inhibited 4 and 16 days after induced haemarthrosis. Interestingly, at day 16 the proteoglycan synthesis in the contralateral knee was also inhibited to an extent correlating with that of the blood-exposed knee. Conclusion: For the first time, early changes in cartilage matrix synthesis upon blood exposure were quantified with the 35SO4 2− assay in a haemophilic rat model, establishing this assay as a novel method to study blood-induced cartilage damage.

Hemorrhagic disease associated with elephant endotheliotropic herpesvirus infection is the most-frequent cause of mortality in captive Asian elephants ( Elephas maximus). Survival relies on intensive monitoring of hemostatic status. Thromboelastography (TEG) utilizes whole blood samples containing all the blood components of hemostasis and is therefore a sensitive indicator of the clinical status in the patient. This study was performed to assess the practicability of TEG in Asian elephants in a zoo environment. Citrated stabilized whole blood samples were obtained from 44 healthy Asian elephants. Kaolin-activated TEG was performed on whole blood at 60 min and 24 hr postsampling (to replicate shipment to an external laboratory) as well as on freeze-thawed plasma samples, 12-14 mo postsampling. Reference intervals were calculated for fresh whole blood and freeze-thawed plasma samples. In the 24-hr analysis, storage artifacts, likely due to cellular degeneration, resulted in a hypercoagulable thromboelastogram and thus reduced sensitivity for detecting coagulopathies. Therefore, delayed analysis of whole blood samples is not recommended.

Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.

TransCon CNP is a C-type natriuretic peptide (CNP-38) conjugated via a cleavable linker to a polyethylene glycol carrier molecule, designed to provide sustained systemic CNP levels upon weekly subcutaneous administration. TransCon CNP is in clinical development for the treatment of comorbidities associated with achondroplasia. In both mice and cynomolgus monkeys, sustained exposure to CNP via TransCon CNP was more efficacious in stimulating bone growth than intermittent CNP exposure. TransCon CNP was well tolerated with no adverse cardiovascular effects observed at exposure levels exceeding the expected clinical therapeutic exposure. At equivalent dose levels, reductions in blood pressure and/or an increase in heart rate were seen following single subcutaneous injections of the unconjugated CNP-38 molecule or a daily CNP-39 molecule (same amino acid sequence as Vosoritide, USAN:INN). The half-life of the daily CNP-39 molecule in cynomolgus monkey was estimated to be 20 minutes, compared with 90 hours for CNP-38, released from TransCon CNP. Cmax for the CNP-39 molecule (20 µg/kg) was approximately 100-fold higher, compared with the peak CNP level associated with administration of 100 µg/kg CNP as TransCon CNP. Furthermore, CNP exposure for the daily CNP-39 molecule was only evident for up to 2 hours postdose (lower limit of quantification 37 pmol/l), whereas TransCon CNP gave rise to systemic exposure to CNP-38 for at least 7 days postdose. The prolonged CNP exposure and associated hemodynamically safe peak serum concentrations associated with TransCon CNP administration are suggested to improve efficacy, compared with short-lived CNP molecules, due to better therapeutic drug coverage and decreased risk of hypotension. SIGNIFICANCE STATEMENT The hormone C-type natriuretic peptide (CNP) is in clinical development for the treatment of comorbidities associated with achondroplasia, the most common form of human dwarfism. The TransCon Technology was used to design TransCon CNP, a prodrug that slowly releases active CNP in the body over several days. Preclinical data show great promise for TransCon CNP to be an effective and well-tolerated drug that provides sustained levels of CNP in a convenient once-weekly dose, while avoiding high systemic CNP bolus concentrations that can induce cardiovascular side effects.

Background Recombinant factor VIIa (rFVIIa) enhances thrombin generation in a platelet-dependent manner; however, rFVIIa binds activated platelets with relatively low affinity. Triggering receptor expressed on myeloid cells (TREM)-like transcript (TLT)-1 is expressed exclusively on activated platelets. Objective To enhance the potency of rFVIIa via binding TLT-1. Methods Recombinant FVIIa was conjugated to a TLT-1 binding Fab. In vitro potency of this platelet-targeted rFVIIa (PT-rFVIIa) was evaluated using factor X activation assays and by measuring viscoelastic changes in whole blood. In vivo potency was evaluated using a tail vein transection model in F8-/- mice expressing human TLT-1. Results PT-rFVIIa and rFVIIa had similar dissociation constant values for tissue factor binding and similar tissue factor-dependent factor X activation. However, PT-rFVIIa had increased catalytic efficiency on TLT-1-loaded vesicles and activated platelets. The in vitro potency in normal human blood with antibody-induced hemophilia A was dependent on assay conditions used; with maximally activated platelets, the half maximal effective concentration for clot time for PT-rFVIIa was 49-fold lower compared with rFVIIa. In the murine bleeding model, a 53-fold lower half maximal effective concentration was observed for blood loss for PT-rFVIIa, supporting the relevance of the assay conditions with maximally activated platelets. In vitro analysis of blood from subjects with hemophilia A confirmed the data obtained with normal blood. Conclusions Increasing the affinity of rFVIIa to activated platelets resulted in approximately 50-fold increased potency both in vitro and in the mouse model. The correlation of in vivo with in vitro data using maximally activated platelets supports that these assay conditions are relevant when evaluating platelet-targeted hemostatic concepts.

Background Hemophilic arthropathy is a severe complication of hemophilia. It is caused by recurrent bleeding into joint cavities, which leads to synovial inflammation, fibrosis, cartilage degradation, and bone remodeling. Extracellular matrix remodeling of affected tissues is a hallmark of these pathological processes. Objectives The aim of this study was to use serological biomarkers of collagen turnover to evaluate extracellular matrix remodeling in a factor VIII-deficient rat model of hemophilic arthropathy. Methods F8-/- rats and wild-type littermate controls were subjected to repeated knee bleeds induced by needle puncture on days 0 and 14. Development of arthropathy was confirmed by histology upon termination on day 28. Serum samples were collected at baseline and throughout the study, and analyzed for biomarkers of collagen turnover including collagens of the basement membrane (type IV collagen), the interstitial matrix (collagen types III, V, and VI), and cartilage (type II collagen). Results In F8-/- rats, induced knee bleeding and subsequent development of arthropathy caused significant alterations in collagen turnover from baseline to termination, measured as changes in serological biomarkers of basement membrane turnover, interstitial matrix turnover, and cartilage degradation. Biomarkers of type II collagen degradation correlated significantly to cartilage degradation and degree of arthropathy. Hemophilic rats had a 50% higher turnover of the basement membrane than wild-type littermates at baseline. Conclusions Joint bleeding and hemophilic arthropathy cause changes in turnover of extracellular matrix collagens in hemophilic rats. Biomarkers of collagen turnover may be used to monitor joint bleeding and development of blood-induced joint disease in hemophilia. This article is protected by copyright. All rights reserved.

Introduction A major complication of haemophilia is haemophilic arthropathy (HA), a debilitating disorder with an incompletely defined pathobiology. High-resolution imaging may provide new knowledge about onset and progression of HA, and thereby support identification of new treatment opportunities. Recently, a F8−/− rat model of HA was developed. The size of the rat allows for convenient and high resolution imaging of the joints, which could enable in vivo studies of HA development. Aim To determine whether HA in the F8−/− rat can be visualized using ultrasonography (US) and micro-computed tomography (μCT). Methods Sixty F8−/− and 20 wild-type rats were subjected to a single or two induced knee bleeds. F8−/− rats were treated with either recombinant human FVIII (rhFVIII) or vehicle before the induction of knee bleeds. Haemophilic arthropathy was visualized using in vivo US and ex vivo μCT, and the observations correlated with histological evaluation. Results US and μCT detected pathologies in the knee related to HA. There was a strong correlation between disease severity determined by μCT and histopathology. rhFVIII treatment reduced the pathology identified with both imaging techniques. Conclusion US and μCT are suitable imaging techniques for detection of blood-induced joint disease in F8−/− rats and may be used for longitudinal studies of disease progression.

A Danish Government PhD grant and by the University of Pretoria from the Department of Companion Animal Clinical Studies Research Fund and the Faculty of Veterinary Science Research Fund. Additional funding was provided by the Danish funds, The Danish Horse Levy Fund, and The Kustos Foundation of 1881.

UNLABELLED Essentials Validating the F8 rat as a new intermediate-size animal model of hemophilic arthropathy. Factor VIII (FVIII) treated F8(-/-) rats suffered induced hemarthrosis analyzed by histopathology. F8 (-/-) animals develop hemophilic arthropathy upon hemarthrosis, preventable by FVIII treatment. The F8 (-/-) rat presents as a new pharmacologic model of hemophilic arthropathy. SUMMARY Background Translational animal models of hemophilia are valuable for determining the pathobiology of the disease and its co-morbidities (e.g. hemophilic arthropathy, HA). The biologic mechanisms behind the development of HA, a painful and debilitating condition, are not completely understood. We recently characterized a F8(-/-) rat, which could be a new preclinical model of HA. Objectives To establish the F8(-/-) rat as a model of HA by determining if the F8(-/-) rat develops HA resembling human HA after an induced joint bleed and whether a second joint bleed causes further disease progression. Methods Wild-type and F8(-/-) rats were treated with vehicle or recombinant human factor VIII (rhFVIII) prior to a needle-induced joint bleed. Joint swelling was measured prior to injury, the following 7 days and upon euthanasia. Histologic sections of the joint were stained, and athropathic changes identified and scored with regard to synovitis, bone remodelling, cartilage degradation and hemosiderin deposition. Results Vehicle-treated F8(-/-) rats experienced marked joint swelling and developed chronic degenerative joint changes (i.e. fibrosis of the subsynovial membrane, chondrocyte loss and excessive bone remodeling). Treatment with rhFVIII reduced or prevented swelling and degenerative joint changes, returning the F8(-/-) animals to a wild-type phenotype. Conclusion The hemophilic phenotype of the F8(-/-) rat resulted in a persistent hemarthrosis following an induced joint bleed. This caused development of HA resembling human HA, which was prevented by rhFVIII treatment, confirming the potential of the F8(-/-) rat as a model of HA.

A sizable proportion of hemophilia inhibitor patients fails immune tolerance induction and requires bypass agents for long-term bleed management. Recombinant human-activated coagulation Factor VII (rhFVIIa) is an on-demand bypass hemostatic agent for bleeds in hemophilia inhibitor patients. Prophylactic use of rhFVIIa may enable sustained hemostatic management of inhibitor patients, but the critical relationship of rhFVIIa circulating levels and clinical outcome in that setting remains unclear. To address this in vivo, we used the rat hemophilia A (HA) model that exhibits spontaneous bleeds and allows longitudinal studies with sufficient statistical power. We simulated activated Factor VII (FVIIa) prophylaxis by adeno-associated virus (AAV) gene transfer of a rat FVIIa transgene. Compared with naive HA animals, rat FVIIa continuous expression affected the overall observed bleeds, which were resolved with on-demand administration of recombinant rat FVIIa. Specifically, although 91% of naive animals exhibited bleeds, this was reduced to 83% and 33% in animals expressing less than 708 ng/mL (25 nM). Rat FVIIa expression of at least 708 ng/mL was also sufficient to normalize the blood loss after a tail vein injury. Continuous, AAV-mediated rat FVIIa transgene expression had no apparent adverse effects in the hemostatic system of HA rats. This work establishes for the first time a dose dependency and threshold of circulating FVIIa antigen levels for reduction or complete elimination of bleeds in a setting of FVIIa-based HA prophylaxis.

Background Haemophilic arthropathy is the main morbidity of haemophilia. The individual pathological response to the same number of clinically evident joint bleeds is highly variable; thus, it remains unknown if certain joint bleeding characteristics are critical for the development of arthropathy. Aim To study the relation between bleed volume and subsequent development of arthropathy, we aimed to develop quantitative in vivo imaging of active joint bleeds in a mouse model of haemophilia. Methods Haemophilia A (F8-KO) and wild-type (WT) mice were IV-dosed with a micro-CT blood pool contrast agent prior to an induced knee haemarthrosis or sham procedure. The mice were micro-CT scanned five times the following 2 days to characterise and quantify the induced haemarthrosis in vivo. On Day 14, the mice were euthanized and pathological changes evaluated by histology and micro-CT. Additionally, bleeding characteristics in vehicle-treated F8-KO mice were compared with those of recombinant FVIII (rFVIII)-treated F8-KO mice. Results F8-KO mice had a significantly larger bleed volume than WT mice at all scan time points. The bleed volume 12 hours after induction of haemarthrosis correlated with the subsequent degree of arthropathy. Presence of µCT-detectable bone pathology was associated with a significantly increased bleed volume among F8-KO mice. rFVIII treatment significantly reduced bleed volume in F8-KO mice. Conclusion Quantitative in vivo contrast-enhanced micro-CT imaging can be used to characterize and quantify joint bleeds in a mouse model of haemophilic arthropathy. The bleed volume correlates with the subsequent degree of arthropathy.

BACKGROUND: Cranial cruciate ligament rupture is a prevalent injury in dogs, and tibial plateau leveling osteotomy (TPLO) is one of the preferred surgical techniques. Surgical site infection is a possible complication following TPLO and measurement of serum acute phase proteins is suggested to be a way to early recognize and distinguish postoperative infectious complications from normal postoperative inflammatory conditions. In this study we investigate the changes in concentrations of the systemic inflammatory markers C-reactive protein (CRP) and serum amyloid A (SAA) following tibial plateau leveling osteotomy (TPLO) in dogs and evaluate if deviations from the changes expectedly induced by the surgical procedure are useful in early detection of post-surgical infections. Dogs with cranial cruciate ligament injuries treated by TPLO at the Region Animal Hospital of Helsingborg during 2012 were included. Dogs with concurrent diseases, other orthopedic problems, or noninfectious post-surgical complications were excluded. Serial measurements of CRP and SAA concentrations were made. Changes in concentrations were visualized graphically and the discriminative capacity to detect infectious post-surgical complications was tested at different time points.RESULTS: A characteristic pattern of changes in concentrations of CRP and SAA were observed following TPLO with a significant increase 24 h post-surgery in all dogs and baseline-concentrations re-established at day 12. In dogs that developed post-surgical infections, a deviation in form of significantly higher concentrations of CRP and SAA were observed at day 6, compared to un-complicated cases. High-discriminative clinical decision limits of CRP (43.9 mg/L) and SAA (63.8 mg/L) could be established for differentiation of dogs with and without clinical signs of infectious complications at day 6 post-operatively, applicable to reliably rule out presence of infectious complications due to very high sensitivity (no false negatives).CONCLUSIONS: The CRP and SAA levels in dogs with clinical signs of post-surgical infectious complication deviated from the typical levels expected at day 6 after surgery, and clinical decision limits to reliably rule out presence of infectious complications was suggested.

Measurement of low concentrations of C-reactive protein (CRP) in dogs has previously been performed with nonautomated assays. The aim of this study was to validate an automated high-sensitivity CRP (hsCRP) assay, developed by modifying a routinely used canine-specific immunoturbidimetric CRP test (cCRP). Imprecision, linearity under dilution, limit of blank (LOB), limit of detection (LOD), and limit of quantification (LOQ) were determined for the hsCRP test, as well as the presence of prozone effect and interferences. The imprecision, measured as intra-assay variation, was ≤2.7%. The assay was acceptably linear under dilution. An analytically relevant prozone effect was present for samples with CRP concentration >150 mg/L, and there were mild interferences from hemolysis and lipemia. The LOB, LOD, and LOQ were 0.10 mg/L, 0.22 mg/L, and 0.50 mg/L, respectively. A method comparison study with a canine-specific enzyme-linked immunosorbent assay (ELISA) was performed, showing poor agreement between the hsCRP test and the ELISA. An additional aim of the study was to apply the hsCRP test to clinical research samples. Serum samples from 7 dogs undergoing ovariohysterectomy were collected pre- and postoperatively, and CRP was measured with both the cCRP and hsCRP assay. The expected postoperative increase in CRP was detected earlier with the hsCRP test, compared with the cCRP test. The hsCRP assay was further applied on samples from 6 lean and 9 overweight dogs. There was no significant difference in CRP concentration between the groups ( P = 0.06). In conclusion, the hsCRP test had acceptable analytical performance, and the assay was successfully applied to clinical research samples.

Introduction Progressive arthropathy caused by recurrent joint bleeds is a severe complication in haemophilia. Aim We investigated whether biomarkers of cartilage and bone degradation, and inflammation were altered in haemophilia patients and whether these biomarkers could identify haemophilia patients with arthropathy. Methods Serum from 35 haemophilia patients with varying degrees of arthropathy and 43 age- and gender-matched control subjects were analysed. Biomarkers of cartilage degradation (C2M, COMP, CTX-II, ADAMTS5), cartilage formation (PRO-C2), bone formation (PINP), bone resorption (CTX-I) and inflammation (hsCRP, CRPM) were measured by ELISA. Arthropathy was assessed by radiological evaluation (Pettersson score) and physical examination (Gilbert score). Results In patients with haemophilia, cartilage degradation, measured by C2M, CTX-II and COMP, was increased by 25% (P < 0.05) compared with control subjects. Levels of the cartilage degradation enzyme, ADAMTS5, were 10% lower in haemophilia patients (P < 0.05). Bone formation (PINP) was reduced by 25% (P < 0.05) in haemophilia patients, whereas bone resorption (CTX-I) was increased by 30% (P < 0.001). Acute inflammation (hsCRP) was increased by 50% (P < 0.01), whereas chronic inflammation (CRPM) was decreased by 25% (P < 0.0001). The hsCRP/CRPM ratio was 60% higher (P < 0.001) in haemophilia patients relative to control subjects. A biomarker panel combining C2M, CRPM, and ADAMTS5 could distinguish haemophilia patients from control subjects with 85.3% accuracy (P < 0.0001). We found no strong correlation between biomarkers and radiological and physical examination of the joint. Conclusion Biomarkers detect increased cartilage and bone degradation, and altered inflammatory activity in haemophilia patients with arthropathy. These biomarkers could potentially be used to identify patients with progressing joint disease.

We investigated the effects of parasite removal on various blood clinical–chemical variables (BCCVs). BCCVs are indicators of health, reflecting, e.g., homeostasis of liver, kidney function, and bone metabolism. The study was conducted in Norway on chicks of two predatory birds: White-tailed Eagle (Haliaeetus albicilla (L., 1758)) and Northern Goshawk (Accipiter gentilis (L., 1758)). Chicks were treated against both endoparasites (internal parasites) and ectoparasites (external parasites). We treated against ectoparasites by spraying nests with pyrethrins. Within nests, chicks were randomly treated with either an antihelminthic medication (fenbendazole) or sterile water (controls). Treatment against either ectoparasites or endoparasites led to higher levels of the bone and liver enzyme alkaline phosphatase. Bilirubin levels were lower when treated against ectoparasites, whereas bile acids were higher. Anti-endoparasite treatment led to higher creatinine levels. In Northern Goshawks, treating against endoparasites led to higher urea levels and lower potassium levels. Treatment against ectoparasites increased uric acid and urea levels and reduced bilirubin levels and protein:creatinine ratios. In conclusion, anti-parasite treatments led to changes in several BCCVs, suggesting differences in nutrient absorption and physiological state of chicks that are possibly related to the costs of parasitism, but maybe also to the parasite treatment itself. cost of parasitism, BCCVs, blood clinical–chemical variables, raptor, Northern Goshawk, White-tailed Eagle. Résumé : Nous avons étudié les effets du retrait des parasites sur différentes variables chimiocliniques du sang (VCCS). Les VCCS sont des indicateurs de santé, reflétant par exemple l’homéostasie du foie, la fonction rénale et le métabolisme osseux. L’étude a été menée en Norvège sur des oisillons de deux espèces d’oiseau prédateur, le pygargue a` queue blanche (Haliaeetus albicilla (L., 1758)) et l’autour des palombes (Accipiter gentilis (L., 1758)). Les oisillons ont été traités contre les endoparasites (parasites internes) et les ectoparasites (parasites externes). Le traitement contre les ectoparasites consistait a` vaporiser des pyréthrines sur les nids. Dans ces derniers, des oisillons ont été traités aléatoirement avec un médicament helminthicide (fenbendazole) ou de l’eau stérile (témoins). Les traitements contre les ectoparasites et les endoparasites ont tous deux mené a` des teneurs plus élevées en phosphatase alcaline, une enzyme des os et du foie. Les concentrations de bilirubine étaient plus faibles chez les individus traités contre les ectoparasites, alors que les concentrations d’acides biliaires étaient plus élevées chez ces individus. Le traitement contre les endoparasites a mené a` des concentrations de créatinine plus élevées. Chez les autours des palombes, le traitement contre les endoparasites a mené a` des concentrations d’urée plus élevées et de potassium plus faibles. Le traitement contre les ectoparasites s’est traduit par une augmentation des concentrations d’acide urique et d’urée et une diminution des concentrations de bilirubine et des rapports protéines:créatinine. En conclusion, les traitements antiparasitaires ont produit des modifications de plusieurs VCCS, qui indiqueraient des différences sur le plan de l’absorption de nutriments et de l’état physiologique des oisillons possiblement reliées aux coûts du parasitisme, mais aussi, peut-être, au traitement antiparasitaire en soi. [Traduit par la Rédaction] Mots-clés : coût du parasitisme, VCCS, variables chimiocliniques du sang, rapace, autour des palombes, pygargue a` queue blanche. As of 2009, copyright of all articles in NRC Research Press journals remains with the authors. Creative Commons Attribution 4.0 International License (CC BY 4.0)

We used a mouse model of pathogenic (Staphylococcus aureus) and non-pathogenic (teat sealing) mammary inflammation to investigate mRNA expression of several inflammatory cytokines and acute phase proteins (APP) in mammary tissue and liver, and the appearance of some of these factors in plasma and milk. The expression levels of IL1β and TNFα were markedly up-regulated inStaph. aureus-inoculated mammary tissue at 72 h, whilst IL6 was up-regulated to a lesser extent in a way which was not confined to the inoculated glands. APP expression was up-regulated at 48 and 72 h in bothStaph. aureus-inoculated and teat-sealedmammaryglands. These differences between cytokine and APP expression provide additional support for the contention that APPs are produced within the mammary tissue itself during inflammation, rather than in associated immune cells. We propose that measurement of cytokines and APP in combination might provide a tool for diagnostic discrimination between mastitis caused by pathogenic invasion and milk accumulation, and hence allow for better targeting of antibiotic therapy. In comparison with mammary expression, expression of cytokines in liver tissue was up-regulated to a similar or lesser extent, whilst expression of APP was up-regulated to a much greater extent. The first appearance of increased cytokine and APP concentrations in plasma and of milk amyloid A (MAA) in milk occurred in advance of the measurable up-regulation of expression, hence their origin cannot be stated with certainty.

Background Myocardial injury, detected by cardiac troponin I and T (cTnI and cTnT), has been associated with long-term death in the noncardiac human intensive care unit (ICU). Hypothesis Presence of myocardial injury predicts 1-year case fatality in critically ill dogs with systemic inflammation. Animals Thirty-eight dogs with evidence of systemic inflammation and no primary cardiac disease. Methods Prospective cohort study. In dogs admitted to the ICU with evidence of systemic inflammation, blood samples were obtained at ICU admission for measurement of cTnI and cTnT, and cTnI was measured once daily during ICU hospitalization. Receiver operating characteristic (ROC) curves were used to examine prognostic capacity of admission cTnI, admission cTnT, and peak cTnI concentrations. Results One-year case fatality rate was 47% (18/38 dogs). Admission cTnI concentrations were (median [range]) 0.48 [0.004–141.50] ng/mL, and peak cTnI concentrations were 1.21 [0.021–141.50] ng/mL. Admission cTnT concentrations were 15 [

Background Myocardial injury detected by cardiac troponin I and T (cTnI and cTnT) in cardiac disease is associated with increased risk of death in humans and dogs. Hypothesis Presence of myocardial injury predicts long-term death in cats with hypertrophic cardiomyopathy (HCM), and ongoing myocardial injury reflects change in left ventricular wall thickness over time. Animals Thirty-six cats with primary HCM. Methods Prospective cohort study. Cats with HCM were included consecutively and examined every 6 months. Echocardiography, ECG, blood pressure, and serum cTnI and cTnT were evaluated at each visit. Cox proportional hazards regression analysis was performed to evaluate prognostic potential of serum troponin concentrations at admission and subsequent examinations. Correlations were used to examine associations between troponin concentrations and cardiac hypertrophy. Results Troponin concentrations at admission were median [range] 0.14 [0.004–1.02] ng/mL for cTnI, and 13 [13–79.5] ng/L for cTnT. Both were prognostic for death (P = .032 and .026) as were the last available concentrations of each (P = .016 and .003). The final cTnT concentration was a significant predictor of death even when adjusting for the admission concentration (P = .043). In a model containing both markers, only cTnT remained significant (P = .043). Left ventricular free wall thickness at end-diastole (LVFWd) at admission was correlated with cTnI at admission (r = 0.35, P = .035), however no significant correlations (r = 0.2–0.31, P = .074–.26) were found between changes in troponin concentrations and left ventricular thickness over time. Conclusions and Clinical Importance Myocardial injury is part of the pathophysiology leading to disease progression and death. Low sensitivities and specificities prevent outcome prediction in individual cats.

Five acute-phase reactants-serum amyloid A (SAA), C-reactive protein (CRP), haptoglobin, albumin, and iron-were measured using commercially available assays in 110 healthy rhesus macaques (Macaca mulatta), and reference intervals were established for future use in health monitoring of this species. Reference intervals established were as follows: SAA, 29.5-87.7 mg/L; CRP, 0-17.5 mg/L; haptoglobin, 354.3-2,414.7 mg/ L; albumin, 36.1-53.0 g/L; and iron, 13.3-40.2 micromol/L. Furthermore, changes in the acute-phase reactants were studied in two additional groups of animals: eight rhesus macaques suffering from acute traumatic injuries and nine rhesus macaques experimentally infected with Mycobacterium tuberculosis reflecting a chronic active inflammation. In animals with inflammation, SAA and haptoglobin concentrations were moderately increased, while CRP increased more than 200-fold. In addition, marked decreases in albumin and iron concentrations were observed. These results show that SAA, CRP, and haptoglobin are positive acute-phase proteins, whereas albumin and iron are negative acute-phase reactants in rhesus macaques.

Introduction: Concizumab is a humanized monoclonal antibody that inhibits tissue factor pathway inhibitor (TFPI). Concizumab is currently in clinical development as a subcutaneous prophylactic therapy for hemophilia A and B patients with and without inhibitors. Breakthrough bleeding episodes experienced by inhibitor patients while on concizumab prophylaxis may be treated with the bypassing agents recombinant activated factor VII (rFVIIa; NovoSeven®) or activated prothrombin complex concentrate (aPCC; FEIBA®). Aim: To investigate the in vitro effect of rFVIIa and aPCC on hemophilia A plasma containing concizumab using a thrombin generation assay and pooled plasma spiked with concizumab or samples from patients treated prophylactically with concizumab. Methods: Pooled hemophilia A plasma was spiked with concizumab at 1, 3 and 10 nM and patient plasma samples from explorer4 (n=16; hemophilia with inhibitors; NCT03196284) and explorer5 (n=30; hemophilia A; NCT03196297) before and during concizumab prophylaxis at steady state exposure levels were assessed. Samples were spiked with rFVIIa (25 or 75 nM) or aPCC (0.25, 0.5 or 1 U/mL), and analyzed using a thrombin generation assay initiated with tissue factor (PPP-Low, Thrombinoscope). The effects of rFVIIa or aPCC in the absence or presence of concizumab were compared using ANOVA methodology. Results: Addition of rFVIIa or aPCC to hemophilia A plasma with or without inhibitors increased peak thrombin generation both in the absence and presence of concizumab. A significant additional effect of rFVIIa and aPCC was observed for all concizumab concentrations spiked to the plasma pool. Overall, the effects of the combination of concizumab and rFVIIa or aPCC were mainly additive; however, a small but statistically significant drug-drug interaction was observed for rFVIIa (25 nM or 75 nM) and aPCC (0.5 U/ml or 1 U/mL) in the presence of 10 nM concizumab. At this concizumab concentration, the additive effect of aPCC corresponded to 68% of the total observed effect and the additive effect of rFVIIa to 85% of the total observed effect. At lower concizumab concentrations (1 and 3 nM), statistically significant drug-drug effects were only observed in combination with aPCC. No excessive thrombin generation above the level obtained with 1 IU/mL recombinant factor VIII (rFVIII) was observed at 1 nM concizumab combined with either rFVIIa (25 and 75 nM) or aPCC 0.5 U/mL. However, addition of 1 U/mL aPCC to 1 nM concizumab resulted in a thrombin peak modestly above the upper 95% confidence interval of the rFVIII range. In the experiments using plasma from patients treated with concizumab, the increase in thrombin peak upon addition of rFVIIa was within or below the range observed by spiking with 1 IU/mL rFVIII. The increase in thrombin peak upon addition of aPCC was within or above the rFVIII range. The effects of concizumab and rFVIIa or aPCC were mainly additive; however, a small, statistically significant contribution caused by drug-drug interaction was observed for concizumab and rFVIIa (75 nM) in both plasma from patients with and without inhibitors, and for 1 U/mL aPCC in plasma from patients with inhibitors. The additive effects of concizumab and rFVIIa corresponded to between 60% (25 nM rFVIIa, plasma without inhibitors) and 75% (75 nM rFVIIa, inhibitor plasma), and the additive effects of concizumab and 1 U/mL aPCC corresponded to 77% of the total observed effects. Conclusions: Addition of rFVIIa or aPCC to hemophilia A plasma with or without inhibitors increased peak thrombin generation as expected both in the absence and presence of concizumab. Thus, the bypassing agents function as expected in plasma containing concizumab. The effects of concizumab and rFVIIa or aPCC were mainly additive. A small but statistically significant contribution was synergistic in accordance with the concizumab mechanism of action (Hilden I et al, Blood, 2012). These in vitro results support the concomitant use of bypassing agents to treat breakthrough bleeding episodes in hemophilia with inhibitor patients on concizumab prophylactic treatment. Disclosures Kjalke: Novo Nordisk A/S: Employment, Honoraria. Kjelgaard-Hansen:Novo Nordisk A/S: Employment, Equity Ownership. Andersen:Novo Nordisk A/S: Employment, Equity Ownership, Honoraria. Hilden:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties.

Objective To investigate the presence of myocardial injury in dogs hospitalized for snake envenomation and to examine its relationship with systemic inflammation. Design Prospective case-control study. Setting University teaching hospital and small animal referral hospital. Animals Dogs naturally envenomed by the European viper (Vipera berus; n = 24), African puff adder (Bitis arietans; n = 5), or snouted cobra (Naja annulifera; n = 9). Interventions Blood was collected from dogs envenomed by V. berus at admission, 12–24 hours postadmission, and 5–10 days postadmission. Blood was collected from dogs envenomed by B. arietans or N. annulifera at admission, and 12, 24, and 36 hours postadmission. Measurements and Main Results Concentrations of cardiac troponin I (cTnI), a marker of myocardial injury, and C-reactive protein (CRP), a marker of systemic inflammation, were measured in each blood sample. Evidence of myocardial injury was found in 58% of dogs envenomed by V. berus at one or more time points. A significant correlation between cTnI and CRP concentrations was found at all time points. Evidence of myocardial injury was found in 80% of dogs envenomed by B. arietans at one or more time points; however, no correlation was found between cTnI and CRP concentrations. Evidence of myocardial injury was found in 67% of dogs envenomed by N. annulifera at one or more time points. A significant correlation between cTnI and CRP concentrations was found at admission, but not at other time points. Conclusions Myocardial injury frequently occurred in dogs with snake envenomation. While the degree of systemic inflammation was significantly correlated with degree of myocardial injury in V. berus envenomation at all time points, this was not the case in dogs envenomed by N. annulifera or B. arietans. This could be due to differences in the toxic substances of the snake venoms or to differences in the cytokines induced by the venom toxins.

Background There is a paucity of information about the analytical performance of thromboelastography (TEG) in horses, specifically concerning components of variation among different analytical methods. Such data may be obtained by nested analysis of repetitive standardized sampling of healthy individuals. Objectives The objectives were (1) to assess the relative susceptibility to sources of preanalytical variation in a highly standardized setting, (2) to directly compare and evaluate the observed analytical variation, and (3) to assess the applicability of population-based reference intervals. Methods Blood was collected once from 20 healthy adult horses and weekly for 5 consecutive weeks from 8 healthy adult horses. TEG analyses were performed on citrated whole blood using nonactivated TEG and 2 TEG assays with human recombinant tissue factor (TF) or kaolin as activators. Results There were significant differences between nonactivated and activated assays for all measured parameters (P

Objective—To evaluate associations of serum C-reactive protein (CRP) concentration with duration of hospitalization and with outcome in puppies with canine parvoviral enteritis. Design—Prospective observational study. Animals—79 client-owned puppies with naturally acquired canine parvovirus infection. Procedures—All puppies received supportive care. Serum CRP concentration was measured at the time of admission, approximately every 10 to 12 hours for the first 48 hours, and then every 24 hours until discharge from the hospital or death. Associations between outcome and CRP concentration at various time points or changes in CRP concentration over time were assessed via multiple logistic regression. Associations of CRP concentration with survival time and duration of hospitalization among survivors were estimated with Cox proportional hazards regression. Use of CRP concentration to predict outcome was evaluated by means of receiver operating characteristic curve analysis. Results—Serum CRP concentrations at admission and 12 and 24 hours later were positively associated with odds of death, and CRP concentrations at 12 and 24 hours after admission were negatively associated with survival time for puppies. Among survivors, duration of hospitalization was positively associated with CRP concentrations at 12, 24, and 36 hours after admission. Sensitivity and specificity of CRP concentration to differentiate between survivors and nonsurvivors at 24 hours after admission were 86.7% and 78.7%, respectively (considered moderately accurate). Conclusions and Clinical Relevance—Although serum CRP concentration was associated with outcome in puppies with canine parvovirus enteritis, it did not prove to be a good predictor of outcome when used alone.

Background Peritoneal fluid (PF) analysis is a valuable diagnostic tool in equine medicine. Markers such as serum amyloid A (SAA) and haptoglobin (Hp) could facilitate the diagnosis of inflammatory abdominal conditions. Objectives The objectives were to (1) establish reference intervals (RI) for SAA and Hp in serum and PF in healthy horses, (2) compare SAA and Hp concentrations between healthy horses and horses with colic, and (3) to assess the correlation between serum and PF concentrations. Methods Serum amyloid A and Hp concentrations were determined by automated assays in prospectively enrolled healthy reference horses and horses with colic. RIs were calculated, group concentrations were compared by Student's t-test, and Pearson′s correlation for serum and PF concentrations were determined. Results In healthy horses (n = 62) the measurements for SAA were below the detection limit (0.5 mg/L) in 94% of serum samples and 98% of PF samples. Horses with colic (n = 61) had statistically significantly increased SAA concentrations in serum (P

Canine serum amyloid A (SAA) is a useful diagnostic marker of systemic inflammation. A latex agglutination turbidimetric immunoassay (LAT) was validated for automated measurements. The aim of the study was to evaluate the clinical applicability of SAA measured by the LAT. SAA was measured in 7 groups of dogs with and without systemic inflammation (n=247). Overlap performance was investigated. Diagnostic performance was compared to body temperature and leukocyte markers. Clinical decision limits for SAA were estimated. In dogs with neurological, neoplastic or gastrointestinal disorders (n=143), it was investigated whether a higher proportion of SAA positive dogs could be detected in cases of complications with risk of systemic inflammation. Significantly higher concentrations of SAA were measured in dogs with (range [48.75; 5,032 mg/l]), compared to dogs without systemic inflammation [0; 56.4 mg/l]. SAA was a more sensitive and specific marker of systemic inflammation (area under the receiver-operating characteristic curve (AUC) 1.00), compared to body temperature (0.6) and segmented neutrophils (best performing leukocyte marker, 0.84). A clinical decision limit of 56.4 mg/l was established giving close to perfect discrimination between dogs with and without systemic inflammation. Higher proportions of SAA-positive dogs were observed in dogs with neurological, neoplastic and gastrointestinal disorders with complications known to increase risk of systemic inflammation, compared to uncomplicated cases. The automated LAT makes SAA applicable as a relevant diagnostic marker of systemic inflammation in dogs for routine random-access real-time use in a general clinical setting.

Background: Dioctophyme renale is a nematode parasite of dogs, usually found in the right kidney, causing severe damage to the renal parenchyma. Objectives: The objective was to evaluate the acute phase response in dogs naturally infected with this Giant Kidney Worm and the possible effects of nephrectomy on circulating concentrations of select acute phase proteins (APP) such as serum amyloid A (SAA), C-reactive protein (CRP), and haptoglobin (HP). Methods: Nephrectomy was performed in infected dogs and the worms were collected for identification. Blood samples were taken 24 hours before surgery, and 4, 8, and 12 hours postoperatively on the following 10 consecutive days, and 28 days after surgery. Acute phase protein concentrations were determined at all time points. Cortisol concentrations were determined 24 hours before surgery and at recovery (28 days after surgery). One-way ANOVA and Friedman test were used for multiple comparisons; the Wilcoxon-signed rank test was used to compare variables, and Spearman's rho rank test was used to assess the correlation between the number of parasites recovered from the dogs and the APP concentration. Results: Forty-five parasites were recovered from the 12 dogs evaluated in this study. Dogs showed significantly increased HP concentrations (P < .05) but lower CRP and SAA concentrations before surgery, and cortisol concentrations were significantly higher at admission when compared to recovery. No significant correlations were found between the number of parasites and APP concentrations. Conclusion: There is a particular acute phase response profile in dogs with kidney worm infection. Nephrectomy induced a short-term inflammatory process.

HYPERTROPHIC cardiomyopathy (HCM) is the most common cardiac disease in cats, associated with severe myocardial structural and ultrastructural changes (Abbott 2010, Christiansen and others 2015). In human beings, HCM is frequently inherited as an autosomal dominant trait caused by mutations in genes encoding sarcomeric proteins, including the myosin-binding protein C (MYBPC3) and the cardiac troponins (Gomes and Potter 2004, Cambronero and others 2009). In cats, familial mutations have been described in the MYBPC3 protein in Maine Coons (A31P) and ragdolls (R820W) (Meurs and others 2005, 2007). Cats homozygous for these mutations have a high risk of developing a fatal, progressive HCM (Meurs and others 2005, Borgeat and others 2014b). While additional causative mutations associated with feline HCM have yet to be discovered, several other breeds are considered predisposed. Cardiac troponin I (cTnI) is a sensitive and specific marker of cardiomyocyte injury. Serum cTnI is generally increased in HCM patients and is associated with increased risk of mortality (Borgeat and others 2014a, Langhorn and others 2014). The cause of myocardial injury is believed to be mild chronic ischaemia due to a range of neurohormonal, inflammatory and vascular factors (Langhorn and others 2014). Additionally, effects of specific mutations and sex on myocardial injury in HCM have been suspected in human studies (Cambronero and others 2009, Kubo and others 2010), and one study on ragdolls similarly reported a difference in cTnI concentration between mutation-positive and mutation-negative cats (Borgeat and others 2015). The aim of this study was …

Babesia rossi infection causes a severe inflammatory response in the dog, which is the result of the balance between pro- and anti-inflammatory cytokine secretion. The aim of this study was to determine whether changes in cytokine concentrations were present in dogs with babesiosis and whether it was associated with disease outcome. Ninety-seven dogs naturally infected with B. rossi were studied and fifteen healthy dogs were included as controls. Diagnosis of babesiosis was confirmed by polymerase chain reaction and reverse line blot. Blood samples were collected from the jugular vein at admission, prior to any treatment. Cytokine concentrations were assessed using a canine-specific multiplex assay on an automated analyser. Serum concentrations of interleukin (IL)-2, IL-6, IL-8, IL-10, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF) and monocyte chemotactic protein-1 (MCP-1) were measured. Twelve of the Babesia-infected dogs died (12%) and 85 survived (88%). Babesia-infected dogs were also divided into those that presented within 48 hours from displaying clinical signs, and those that presented more than 48 hours after displaying clinical signs. Cytokine concentrations were compared between the different groups using the Mann-Whitney U test. IL-10 and MCP-1 concentrations were significantly elevated for the Babesia-infected dogs compared to the healthy controls. In contrast, the IL-8 concentration was significantly decreased in the Babesia-infected dogs compared to the controls. Concentrations of IL-6 and MCP-1 were significantly increased in the non-survivors compared to the survivors. Concentrations for IL-2, IL-6, IL-18 and GM-CSF were significantly higher in those cases that presented during the more acute stage of the disease. These findings suggest that a mixed cytokine response is present in dogs with babesiosis caused by B. rossi, and that an excessive pro-inflammatory response may result in a poor outcome.

Background The role of antiphospholipid antibodies in the prolonged activated partial thromboplastin time (aPTT) previously identified in healthy Bernese Mountain Dogs remains unknown. In people, an isolated prolonged aPTT without evidence of bleeding might be because of a thrombophilic condition caused by antiphospholipid antibodies. Objective To examine if prolonged aPTT in healthy Bernese Mountain Dogs is because of antiphospholipid antibodies. Animals Twenty-two healthy Bernese Mountain Dogs and 10 healthy adult dogs of various breeds. Methods Prospective case control study. Healthy Bernese Moutain Dogs were examined twice over 6 months. Dogs were investigated for the presence of lupus anticoagulants and anticardiolipin (aCL) antibodies by the use of multiple aPTT tests with low and high lupus anticoagulant sensitivities, a mixing study, and an ELISA test for aCL antibody optical density to detect solid phase antiphospholipid antibodies. Results In all, 15 of 22 healthy Bernese Mountain Dogs were positive for lupus anticoagulants. The Bernese Mountain Dogs had markedly higher levels of aCL antibodies compared with the control dogs (P = .006). In all, 7 of 21 of the Bernese Mountain Dogs were positive for both lupus anticoagulants and aCL antibodies, whereas 4 of 21 Bernese Mountain Dogs were negative for both. Conclusions and Clinical Importance Lupus anticoagulants and aCL antibodies could be the cause of prolonged aPTT in healthy Bernese Mountain Dogs. The importance of the antiphospholipid antibodies in the dogs remains unknown.

Coagulation tests are often performed in dogs suspected of haemostatic dysfunction and are interpreted according to validated laboratory reference intervals (RIs). Breed specific RIs for haematological and biochemical analytes have previously been identified in Bernese Mountain dogs, but it remains to be determined if breed specific RIs are necessary for haemostasis tests. Activated prothromboplastin time (aPTT), prothrombin time (PT), selected coagulation factors, D-dimers, fibrinogen, von Willebrand factor and thromboelastography (TEG) were analyzed in healthy Bernese Mountain dogs using the CLSI model. Three analytes (aPTT, TEG [MA] and TEG [G]) were different according to the CLSI model. For aPTT the new RI was markedly different (0-100s). Whereas the new intervals for TEG (MA) and TEG (G) may be due to breed related biological variation, the cause of the prolonged RI for aPTT is at present uncertain.

Introduction Turoctocog alfa pegol (N8-GP) is a glycoPEGylated recombinant factor VIII (rFVIII) under development for treatment of hemophilia A (HA). Patients with severe HA receiving intravenous (IV) replacement therapy have 20-40% risk of developing inhibitory FVIII antibodies. It is unclear whether the absence of inhibitors in some patients is due to lack of response or due to peripheral tolerance. Induction of peripheral tolerance may be dependent on regulatory T-cells (Tregs) since they are suggested to be involved in regulation of the anti-FVIII antibody response. In this study, tolerance induction following IV rFVIII administration was assessed in humanized HA mice, described as a suitable model for tolerance induction as only a fraction produce anti-FVIII antibodies after IV FVIII exposure (Steinitz KN, et al. Blood 2012;119(17):4073-4082). Aims To explore whether IV exposure to rFVIII induces peripheral tolerance to subcutaneous N8-GP (SC N8-GP) exposure in humanized HA mice, and whether Tregs play a role in tolerance induction. Methods HLA-DR transgenic FVIII-deficient mice lacking murine MHC class II were treated with rFVIII IV once per week for 8 weeks, then challenged with SC N8-GP once a week for 8 weeks. The role of CD25+ Tregs was studied by treating one group of mice with depleting anti-CD25 antibodies during the IV exposure period. The presence of anti-FVIII antibodies was measured prior to IV and SC dosing, and at study end. Mice with anti-FVIII antibodies after IV rFVIII exposure were excluded from the analysis. Results No anti-FVIII antibodies were detected after challenge with SC N8-GP in mice pretreated with IV rFVIII. Depletion of CD25+ Tregs (confirmed by flow cytometry) during the IV exposure did not significantly affect the anti-FVIII antibody response. Conclusion It was demonstrated that peripheral tolerance to SC N8-GP was induced in humanized HA mice not responding to IV rFVIII exposure. Depletion of CD25+ Tregs during IV exposure was insufficient to abolish the induction of tolerance. Disclosures Reedtz-Runge: Novo Nordisk A/S: Employment. Weldingh:Novo Nordisk A/S: Employment. Schiøler Schultz:Novo Nordisk A/S: Employment. Holm Millner:Novo Nordisk A/S: Employment. Rode:Novo Nordisk A/S: Employment. Wiinberg:Novo Nordisk A/S: Employment. Kjelgaard-Hansen:Novo Nordisk A/S: Employment.

Background: The cytokine response in immune-mediated hemolytic anemia (IMHA) is poorly characterized and correlation with outcome is unknown. Hypothesis/Objectives: To determine if cytokine activity is correlated with outcome in dogs with IMHA. Animals: Twenty dogs with primary IMHA and 6 control dogs. Methods: Prospective study on dogs with IMHA with blood sampling at admission. Serum activity of interleukin-2 (IL-2), IL-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-inducible protein-10, interferon-gamma, and keratinocyte chemoattractant (KC) was assessed. Results: Thirty-day case fatality rate was 25% (5/20 dogs). Increased concentrations (median [range]) of IL-2 (45.5 ng/L [0;830] versus 0 ng/L [0;46.8]), IL-10 (8.2 ng/L [0;60.6] versus 0 ng/L [0;88.2]), KC (1.7 μg/L [0.3;4.7] versus 0.5 μg/L [0.2;1.1]), and MCP-1 (162 ng/L [97.6;438] versus 124 ng/L [90.2;168]) were observed in dogs with IMHA compared with controls. The cytokine profile was indicative of a mixture of pro- and anti-inflammatory cytokines of various cellular origins. Cytokines/chemokines strongly associated with macrophage/monocyte activation and recruitment were significantly increased in nonsurvivors compared with survivors; IL-15 (179 ng/L [48.0;570] versus 21.3 ng/L [0;193]), IL-18 (199 ng/L [58.7;915] versus 37.4 ng/L [0;128]), GM-CSF (134 ng/L [70.0;863] versus 57.6 ng/L [0;164]), and MCP-1 (219 ng/L [135;438] versus 159 ng/L [97.6;274]), respectively. Logistic regression suggested increased IL-18 and MCP-1 concentrations were independently associated with mortality in this population (P

Background: In people, increased thrombin-activatable fibrinolysis inhibitor (TAFI) antigen has been associated with increased risk of thrombosis, and decreased TAFI may contribute to bleeding diathesis. TAFI activity in dogs has been described in experimental models, but not in dogs with spontaneous disease. Objective: The aim of this study was to compare TAFI activity in healthy dogs with TAFI activity in dogs with spontaneous disease. Methods: Plasma samples from 20 clinically healthy Beagles and from 35 dogs with various diseases were analyzed using a commercial chromogenic assay that measured TAFI activity relative to activity in standardized pooled human plasma. Results: Median TAFI activity for the 20 Beagles was 46.1% (range 32.2–70.8%) compared with 62.6% (29.1–250%) for the 35 diseased dogs, and 14/35 (40%) had TAFI activities >the upper limit for controls. The highest individual activities (>225%) were in 3 dogs with malignant neoplasms and 1 dog with thrombocytopenia. For data grouped by diagnosis, median TAFI activity was 61.7% for benign neoplasia (n=5), 64.9% for malignant neoplasia (n=8), 75.5% for Angiostrongylus vasorum infection (n=4), 68.8% for bacterial sepsis (n=7), and 58.7% for miscellaneous diseases (n=11). Compared with TAFI activity in control dogs, median TAFI activity was significantly increased only in the group of dogs with bacterial sepsis. Conclusion: Bacterial sepsis was associated with significantly increased TAFI activity, and individual dogs with increased TAFI activities were found in all disease groups. The role of TAFI in the pathogenesis of hemostatic disorders in dogs and its value as a prognostic indicator deserve further investigation.

Background: Hematology and serum biochemistry reference intervals in dogs may be affected by internal factors, such as breed and age, and external factors, such as the environment, diet, and lifestyle. In humans, it is well established that geographic origin and age may have an impact on reference intervals and, therefore, more specific reference intervals are sought for subpopulations. Objective: The objective of this study was to validate and transfer standard laboratory reference intervals for healthy Bernese Mountain dogs and to create new intervals for analytes where the established laboratory reference intervals were rejected. Methods: The procedure was performed using the human Clinical and Laboratory Standards Institute-approved model modified for veterinary use. Thirty-two dogs were included in the study using a direct a priori method, as recommended. Results: While 23 of the standard laboratory reference intervals were readily validated, 7 of the analytes (eosinophils, MCHC, alkaline phosphatase [ALP], γ-glutamyltransferase, total bilirubin, amylase, and cholesterol) required new reference intervals according to the standard. These were calculated using the robust method. In particular, the new reference range for ALP was wide compared with the established laboratory reference interval. No clinical causes were found for differences in the results of these analytes. Conclusion: We found significant differences in 7 hematologic and serum biochemical analytes for which a breed-specific variation appears to be the most plausible explanation. Breed-specific reference intervals for Bernese Mountain dogs will help avoid misinterpretation of laboratory results in the diagnostic process.