Unity Jeffery

Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia.

Neutrophil extracellular traps (NETs) are webs of DNA and protein with both anti-microbial and pro-thrombotic properties which have not been previously reported in dogs. To confirm dog neutrophils can form NETs, neutrophils were isolated from healthy dogs, and stimulated in vitro with 2μM, 8μM, 31μM, and 125μM platelet activating factor (PAF) or 0.03μM, 0.1μM, 0.4μM, 1.6μM and 6.4μM phorbol-12-myristate-13-acetate (PMA). Extracellular DNA was measured using the cell impermeable dye Sytox Green every hour for 4h. At 4h, extracellular DNA was significantly greater than non-stimulated cells at concentrations ≥31μM and ≥0.1μM for PAF and PMA, respectively. Cells stimulated with 31.25μM PAF reached maximal fluorescence by 1h, whereas maximal fluorescence was not achieved until 2h for cells stimulated with 0.1μM PMA. Immunofluorescent imaging using DAPI and anti-elastase antibody confirmed that extracellular DNA is released as NETs. As NETs have been implicated in thrombosis, nucleosomes, a marker correlated with NET formation, were measured in the serum of dogs with the thrombotic disorder primary immune-mediated hemolytic anemia (IMHA) (n=7) and healthy controls (n=20) using a commercially available ELISA. NETs were significantly higher in IMHA cases than controls (median 0.12 and 0.90, respectively, p=0.01), but there were large positive interferences associated with hemolysis and icterus. In summary, the study is the first to describe NET generation by canine neutrophils and provides preliminary evidence that a marker associated with NETs is elevated in IMHA. However, this apparent elevation must be interpreted with caution due to the effect of interference, emphasizing the need for a more specific and robust assay for NETs in clinical samples.

Positive predictive value of albumin: globulin ratio for feline infectious peritonitis in a mid-western referral hospital population

Low albumin to globulin ratio has been found previously to have a high positive predictive value for feline infectious peritonitis (FIP) in cats with clinical signs highly suggestive of the disease. However, FIP can have a more vague clinical presentation. This retrospective study found that the positive predictive value of an albumin:globulin (A:G) ratio of <0.8 and <0.6 was only 12.5% and 25%, respectively, in a group of 100 cats with one or more clinical signs consistent with FIP. The negative predictive value was 100% and 99% for an A:G ratio of <0.8 and A:G<0.6%, respectively. Therefore, when the prevalence of FIP is low, the A:G ratio is useful to rule out FIP but is not helpful in making a positive diagnosis of FIP.

Canine Neutrophil Extracellular Traps Enhance Clot Formation and Delay Lysis

Autoimmune diseases increase the risk of thrombosis. Neutrophil extracellular traps (NETs) are webs of DNA and protein that may mediate thrombosis in autoimmune diseases. Human and murine studies show NET-releasing neutrophils within a thrombus promote its growth, but it is unclear to what extent NET fragments released into circulation during inflammation are prothrombotic. This study hypothesized that canine NETs promote clot formation and impair lysis even in the absence of neutrophils. NETs were prepared from PMA-stimulated neutrophils and added to fibrinogen and thrombin or to recalcified pooled canine platelet-poor plasma, tissue factor, and tissue plasminogen activator. Clot formation and lysis were measured spectrophotometrically. NETs did not alter fibrin clot formation, but NETs increased maximum clot formation velocity (P = .001) and delayed lysis (P = .009) of plasma clots compared with supernatants from nonstimulated neutrophils. DNase digestion of NETs reduced their effect on clot lysis but not maximum clot formation velocity. This suggested impaired lysis was principally mediated by DNA within NETs but that NET proteins were principally responsible for increased speed of clot formation. Previous reports suggested elastase or histones might be responsible for the effect of NETs on clot formation. Elastase activity was greatly reduced by plasma, and addition of histones to plasma did not increase formation velocity, suggesting these proteins were not responsible for increasing maximum formation velocity. This study showed that NETs enhanced clot formation and impaired clot lysis in canine platelet-poor plasma. These in vitro findings suggest both NET proteins and DNA may contribute to thrombosis in inflammatory disease.

Cell-Free DNA and DNase Activity in Dogs with Immune-Mediated Hemolytic Anemia

Background Immune‐mediated hemolytic anemia (IMHA) in dogs has a high risk of thrombosis and is associated with marked neutrophilia and necrosis. Cell death and release of neutrophil extracellular traps contribute to increased serum concentrations of cell‐free DNA, and in human autoimmune disease reduced DNase activity further increases cell‐free DNA. Free DNA in blood has prothrombotic properties and could contribute to hypercoagulability in IMHA. Hypothesis Cell‐free DNA is elevated and DNase activity reduced in dogs with IMHA compared to healthy dogs. Animals Dogs presenting to two referral hospitals with IMHA (n = 28) and healthy controls (n = 20). Methods Prospective observational study. Blood was collected and death and thrombotic events occurring in the first 14 days after hospitalization recorded. DNA was extracted from plasma with a commercial kit and quantified by PicoGreen fluorescence. DNase activity of serum was measured by radial diffusion assay. Results Cell‐free DNA was significantly higher in cases (median: 45 ng/mL, range: 10–2334 ng/mL) than controls (26 ng/mL, range 1–151 ng/mL, P = 0.0084). DNase activity was not different between cases and controls (P = 0.36). Four cases died and there were five suspected or confirmed thrombotic events. Cell‐free DNA concentration was associated with death (odds ratio for upper quartile versus lower 3 quartiles: 15; 95% confidence interval 1.62–201; P = 0.03) but not thrombosis (P = 0.57). Conclusions and Clinical Importance Cell‐free DNA is elevated in dogs with IMHA and likely reflects increased release rather than impaired degradation of DNA. Cell‐free DNA concentration is potentially associated with death and might be a prognostic indicator, but this requires confirmation in a larger population.

Using the laboratory to predict thrombosis in dogs: An achievable goal?

Thrombosis is a major cause of mortality and morbidity in humans and dogs; however, anti-thrombotic drugs carry a risk of bleeding and increase the cost of patient care. The ability to identify individuals at high risk of thrombosis would allow targeting of anti-coagulant therapy at those most likely to derive a net benefit. Significant advances have been made towards predicting thrombotic risk in humans using laboratory tests individually and as part of risk prediction models. Assays that have shown potential in humans include D-dimers, activated partial thromboplastin time and viscoelastic testing, all of which are available to veterinarians. This review discusses the extent to which these assays are likely to predict thrombosis in dogs, and introduces new research techniques which may have future clinical value.

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release.

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Assessment of novel digital and smartphone goniometers for measurement of canine stifle joint angles

OBJECTIVE To evaluate accuracy and reliability of 3 novel goniometers for measurement of canine stifle joint angles and compare the results with those obtained with a universal goniometer (UG). SAMPLE 8 pelvic limbs from 4 canine cadavers. PROCEDURES Each limb was secured to a wooden platform at 3 arbitrarily selected fixed stifle joint angles. Goniometry was performed with 2 smartphone-based applications (novel goniometers A and B), a digital goniometer (novel goniometer C), and a UG; 3 evaluators performed measurements in triplicate for each angle with each device. Results were compared with stifle joint angle measurements on radiographs (used as a gold standard). Accuracy was determined by calculation of bias and total error, coefficients of variation were calculated to estimate reliability, and strength of linear association between radiographic and goniometer measurements was assessed by calculation of correlation coefficients. RESULTS Mean coefficient of variation was lowest for the UG (4.88%), followed by novel goniometers B (7.37%), A (7.57%), and C (12.71%). Correlation with radiographic measurements was highest for the UG (r = 0.97), followed by novel goniometers B (0.93), A (0.90), and C (0.78). Constant bias was present for all devices except novel goniometer B. The UG and novel goniometer A had positive constant bias; novel goniometer C had negative constant bias. Total error at 50° and 100° angles was > 5% for all devices. CONCLUSIONS AND CLINICAL RELEVANCE None of the devices accurately represented radiographically measured stifle joint angles. Additional veterinary studies are indicated prior to the use of novel goniometers in dogs.

When neuroscience met clinical pathology: partitioning experimental variation to aid data interpretation in neuroscience

In animal experiments, neuroscientists typically assess the effectiveness of interventions by comparing the average response of groups of treated and untreated animals. While providing useful insights, focusing only on group effects risks overemphasis of small, statistically significant but physiologically unimportant, differences. Such differences can be created by analytical variability or physiological within‐individual variation, especially if the number of animals in each group is small enough that one or two outlier values can have considerable impact on the summary measures for the group. Physicians face a similar dilemma when comparing two results from the same patient. To determine whether the change between two values reflects disease progression or known analytical and physiological variation, the magnitude of the difference between two results is compared to the reference change value. These values are generated by quantifying analytical and within‐individual variation, and differences between two results from the same patient are considered clinically meaningful only if they exceed the combined effect of these two sources of ‘noise’. In this article, we describe how the reference change interval can be applied within neuroscience. This form of analysis provides a measure of outcome at an individual level that complements traditional group‐level comparisons, and therefore, introduction of this technique into neuroscience can enrich interpretation of experimental data. It can also safeguard against some of the possible misinterpretations that may occur during analysis of the small experimental groups that are common in neuroscience and, by illuminating analytical error, may aid in design of more efficient experimental methods.

Development of a fibrinolysis assay for canine plasma

Unbalanced coagulation and fibrinolysis leads to hemorrhage or thrombosis. Thromboelastography has been used to characterize hypo- and hyper-fibrinolysis in dogs, however the technique requires specialized instrumentation and proprietary reagents that limit its availability. The aim of this study was to develop a simple microplate method for assessment of fibrinolysis in canine plasma. Plasma from healthy dogs was mixed in a microwell plate with tissue factor, calcium, phospholipid and tissue plasminogen activator. Light absorbance was measured at regular intervals until return to baseline. Peak optical density (milli-absorption units, mAU), formation velocity (mAU/s), lysis velocity (mAU/s) and area under the curve (mAU.s) were calculated. The influence of potential interferents, variation in fibrinogen and ex vivo addition of heparin and aminocaproic acid on assay performance was determined. Inter-day coefficients of variation were ≤15% for all variables. Bilirubin≤1.88mg/dL and hemoglobin≤0.09mg/dL did not interfere with assay variables. Aminocaproic acid (40μg/mL) and heparin (0.125U/mL) caused almost complete inhibition of fibrinolysis and coagulation, respectively. All variables except lysis velocity (R2=0.08) were associated with fibrinogen concentration (R2>0.8). This assay showed acceptable performance characteristics for measurement of fibrinolysis in normal canine plasma. The assay utilizes small volume citrate plasma samples and readily available instrumentation and reagents, is not influenced by mild to moderate hemolysis or icterus and detects the presence of fibrinolysis inhibitors.

Urban environment: a risk factor for canine immune‐mediated disease?

OBJECTIVES To investigate whether dogs living in urban areas are more likely to develop immune-mediated disease than those in rural areas. MATERIALS AND METHODS A case-control study comparing the prevalence of urban home location between dogs with immune-mediated disease and matched controls. Dogs diagnosed with immune-mediated haemolytic anaemia, immune-mediated thrombocytopenia, immune-mediated polyarthritis or meningoencephalomyelitis of unknown origin were identified by case record searches. Breed-matched dogs presenting to the same hospital during the same year as cases were randomly selected as controls. Home locations were classified as rural or urban using the population density of the relevant census tract and conditional logistic regression was used to examine association between home location and immune-mediated disease. RESULTS In the 137 cases and 137 breed-matched controls, the odds ratio for any immune-mediated disease for dogs living in urban (versus rural) areas was 0·94 (95% confidence interval 0·58 to 1·55, P=0·80). Odds ratios for development of immune-mediated haematological diseases, immune-mediated polyarthritis or meningoencephalomyelitis of unknown origin were also not significantly different from the null value. Multivariable analysis including age, gender and season of presentation did not suggest confounding of effect of home location by these additional variables. CLINICAL SIGNIFICANCE This study does not support an association between urban environment and immune-mediated disease in dogs.