In vivo fluorescence molecular tomography of induced haemarthrosis in haemophilic mice: link between bleeding characteristics and development of bone pathology.

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.

Serological biomarkers detect active joint destruction and inflammation in patients with haemophilic arthropathy.

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.

Visualization of haemophilic arthropathy in F8-/- rats by ultrasonography and micro-computed tomography.

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.

Altered collagen turnover in factor VIII-deficient rats with hemophilic arthropathy identifies potential novel serological biomarkers in hemophilia.

Essentials Joint bleeding in hemophilia may induce significant remodeling of the extracellular matrix. Biomarkers of collagen turnover were investigated in a F8-/- rat model of hemophilic arthropathy. Biomarkers of cartilage degradation increased significantly during development of arthropathy. Basement membrane and interstitial matrix turnover changed significantly following hemarthrosis. SUMMARY: 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 after 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, measured as changes in serological biomarkers of basement membrane turnover, interstitial matrix turnover and cartilage degradation. Biomarkers of type II collagen degradation correlated significantly with 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.

The F8(-/-) rat as a model of hemophilic arthropathy.

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.

Cardiac troponin I and T as prognostic markers in cats with hypertrophic cardiomyopathy.

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.

Prediction of long-term outcome by measurement of serum concentration of cardiac troponins in critically ill dogs with systemic inflammation.

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 [<13-3744] ng/L. For each marker, non-survivors had significantly higher concentrations than survivors (P = .0082-.038). ROC analyses revealed areas under curves [95% CI] of 0.707 [0.537-0.843] for peak cTnI and 0.739 [0.571-0.867] for admission cTnT, respectively. At the optimal cut-off, concentrations were 1.17 ng/mL (peak cTnI) and 23 ng/L (admission cTnT), sensitivities were 72% and 72%, and specificities were 70% and 80%, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: While peak cTnI and admission cTnT are significantly related to 1-year case fatality in critically ill dogs with systemic inflammation, low sensitivities and specificities prevent their prediction of long-term outcome in individual patients. Troponins might play a role in identification of dogs at long-term risk of death.

Prognostic importance of myocardial injury in critically ill dogs with systemic inflammation.

BACKGROUND: In noncardiac critical disease in humans, myocardial injury as detected by cardiac troponin I and T (cTnI and cTnT) has been linked to high intensive care unit (ICU) death independent of prognostic composite scoring. HYPOTHESIS: Presence of myocardial injury predicts short-term death in critically ill dogs with systemic inflammation and provides additional prognostic information when combined with established canine prognostic composite scores. ANIMALS: Forty-two dogs admitted to the ICU with evidence of systemic inflammation and no primary cardiac disease. METHODS: Prospective cohort study. Blood samples were obtained at ICU admission for the measurement of cTnI and cTnT, C-reactive protein, and several cytokines. The acute patient physiologic and laboratory evaluation (APPLE) score and the survival prediction index were calculated within the first 24 hours of admission. Receiver operating characteristic (ROC) curves were used to examine the prognostic capacity of each biomarker and severity score. Multiple logistic regression analysis was performed to evaluate whether cardiac markers significantly contributed to severity scores. RESULTS: Twenty-eight day case fatality rate was 26% (11/42 dogs). cTnI concentrations were (median [range]) 0.416 [0.004-141.5] ng/mL and cTnT concentrations were 13.5 [<13-3,744] ng/L. cTnI, cTnT, and the APPLE score were all significant prognosticators with areas under the ROC curves [95% CI] of 0.801 [0.649; 0.907], 0.790 [0.637; 0.900], and 0.776 [0.621; 0.889], respectively. cTnI significantly contributed to the APPLE score in providing additional prognostic specificity (P = .025). CONCLUSIONS AND CLINICAL IMPORTANCE: Markers of myocardial injury predict short-term death in dogs with systemic inflammation and cTnI significantly contributes to the APPLE score.

Hemostatic abnormalities in uncomplicated babesiosis (Babesia rossi) in dogs.

BACKGROUND: Babesiosis in dogs is associated with severe thrombocytopenia; yet infected dogs rarely show clinical signs of hemorrhage. HYPOTHESIS: Dogs with uncomplicated babesiosis have normal hemostatic capacity despite severe thrombocytopenia. ANIMALS: Nineteen client-owned dogs with uncomplicated babesiosis; 10 healthy controls. METHODS: A prospective, cross-sectional, observational study. Thromboelastography (TEG), prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, D-dimers, and antithrombin (AT) measured in both groups. Statistical significance set at P < .01. RESULTS: Babesiosis group hematocrit and platelet count significantly lower than controls (0.29 versus 0.50 L/L; P < .001 and 20.0 versus 374.5 × 10(9)/L; P < .001, respectively). Except for K, no significant difference in TEG variables between groups. Hemostatic variables for babesiosis group versus controls (mean ± SD); R: 5.9 ± 1.8 versus 4.6 ± 0.9 min (P = .048); K: 2.8 ± 1.1 versus 1.9 ± 0.6 min (P = .003); angle: 55.5 ± 11.7 versus 62.2 ± 4.1 degrees (P = .036); MA: 48.4 ± 9.7 versus 57.2 ± 5.2 mm (P = 0.013); G: 5.1 ± 1.9 versus 6.9 ± 1.5 dyn/cm(2) (P = .019); LY30 (median, range): 0 (0-5.7) versus 0.6% (0-6.1) (P = .152); and LY60: 0 (0-8.8) versus 3.1% (0-13.1) (P = .012). AT activity significantly lower (105.2 ± 16.5 versus 127.8 ± 15.4%; P = .001). Fibrinogen concentration significantly higher in babesiosis group (5.7 ± 1.3 versus. 3.0 ± 0.7 g/L; P < .001). CONCLUSION AND CLINICAL IMPORTANCE: Despite severe thrombocytopenia, dogs with uncomplicated babesiosis did not have clinical signs of hemorrhage and TEG variables were normal, which could indicate a normocoagulable state.

Plasma IL-8 concentrations are increased in dogs with spirocercosis.

The nematode Spirocerca lupi (S. lupi) induces sarcoma in the dog oesophagus in about 25% of cases. The aim of this study was to compare the differences in the cytokine milieu between dogs with neoplastic (n=29) and non-neoplastic disease (n=49) and age- and gender-matched healthy controls (n=25). We measured IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, GM-CSF and MCP-1 in a specific canine multiplex immunoassay kit. Cytokine concentrations were compared between the different groups using the Kruskal-Wallis test followed by Dunn's test. Only IL-8 and IL-18 showed significant differences in their plasma concentration among the three groups. Kruskal-Wallis test revealed a significant (p=0.001) difference in IL-8 concentration between the neoplastic group (634pg/ml), the non-neoplastic (429 pg/ml) and the control groups (150 pg/ml). Post-test analysis revealed a significance difference between the two S. lupi groups and the control group (p<0.01). The highest IL-18 concentration was found in the non-neoplastic group (53 pg/ml), followed by the control group (46 pg/ml) and finally the neoplastic group (33 pg/ml). IL-18 concentrations were significantly higher in the non-neoplastic group than in the neoplastic group (p=0.05). The increased IL-8 in the spirocercosis groups is consistent with the neutrophilic infiltrate in spirocercosis lesions and in those of other inflammatory-induced neoplasias such as Barret's oesophagus and Helicobacter gastritis. IL-18 showed negative regulatory effect in several worm infections and it is possible that it plays the same role in spirocercosis, allowing the worm to evade the host response and to induce neoplastic transformation.

Evaluation of an automated assay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline, and equine SAA.

Major acute phase proteins (APPs) have proven diagnostically useful in dogs, cats and horses with routine use facilitated by commercially available automated heterologous assays. An automated assay applicable across all three species would highly facilitate further dissemination of routine use, and the aim of this study was to validate an automated latex agglutination turbidimetric immunoassay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline and equine SAA. Serum samples from 60 dogs, 40 cats and 40 horses were included. Intra- and inter-assay imprecision, linearity and detection limit (DL) were determined to assess analytical performance. To assess clinical performance, equine and feline SAA measurements were compared with parallel measurements using a previously validated automated SAA assay in a method comparison setting, and by assessing overlap performance of canine SAA in healthy dogs and diseased dogs with and without systemic inflammation. Intra- and inter-assay CVs ranged between 1.9-4.6% and between 3.0-14.5%, respectively. Acceptable linearity within a clinically relevant range of SAA concentrations was observed for all three species. The DL was 1.06 mg/L. Method comparison revealed acceptable agreement of the two assays measuring feline and equine SAA, and the overlap performance of canine SAA was acceptable. The tested assay measured SAA in canine, feline and equine serum with analytical and overlap performance acceptable for clinical purposes so improving practical aspects of clinical APP application. The monoclonal nature of the antibodies suggests strong, long-term inter-batch performance stability.

The impact of Staphylococcus aureus concentration on the development of pulmonary lesions and cytokine expression after intravenous inoculation of pigs.

Acute respiratory distress syndrome is a common complication in severe sepsis. In pigs, the lungs play an important role in clearing systemic bacterial infections due to pulmonary intravascular macrophages found specifically in pigs. However, this increases the exposure of the porcine lungs to pathogens and potential injury. The authors propose that increasing the concentration of the inoculum without changing the bacterial dose will lead to severe sepsis with pronounced pulmonary lesions. This could potentially create a risk of cytokine spillover to the circulation, leading to an increased systemic response. Eight Danish Landrace pigs, approximately 10 weeks old, were inoculated twice with a low or once with a high concentration of Staphylococcus aureus. Three pigs were sham-inoculated. The animals were grouped based on macro- and microscopic lung lesions. The mRNA expression of local pulmonary inflammatory markers was compared to protein levels of systemic inflammatory markers. The most severe pulmonary lesions were observed in animals receiving the high S. aureus concentration, indicating that severity of lesions is dependent on inoculum concentration rather than total numbers of bacteria. Furthermore, local mRNA expression of inflammatory cytokines appeared to be dependent on the magnitude and severity of tissue destruction, including the ability to confine the lesions. Increasing mRNA levels of serum amyloid A could be a confident marker of severity of pulmonary lesions. Since no correlation was observed between local and systemic levels of inflammatory cytokines, this finding could indicate an ability of the porcine lung to compartmentalize the local inflammatory response and thus restrict systemic contribution.

Less invasive blood sampling in the animal laboratory: clinical chemistry and haematology of blood obtained by the Triatominae bug Dipetalogaster maximus.

Dipetalogaster maximus (Dipmax), a blood-sucking bug belonging to the family Reduviidae, has been used to obtain blood samples, for example for clinical chemistry and haematology, in a variety of zoo animals and wildlife. Using this bug allows stress-free blood sampling as the bug is able to draw blood without the mammal noticing the bug. In laboratory animal science, the need for blood samples from unstressed animals may arise, especially in animal behaviour research. The use of Dipmax bugs may prove a valuable tool for this purpose. To validate the method, we compared an array of standard blood parameters sampled from New Zealand White rabbits, sampled either by the use of bugs or by the conventional method; puncture of vena auricularis caudalis. The overall hypothesis was that there was no significant difference in clinical chemistry and haematological parameters between the bug method and the conventional method. A total of 17 clinical parameters as well as 12 haematological parameters were measured and compared in New Zealand White rabbits. The results showed that for 13 of these 29 analysed parameters, the bug method and the conventional method did not give significantly different results, and the obtained results were thus directly comparable. For the remaining parameters the obtained results were significantly different. However, all parameters were measurable in the bug samples. The influences of the bug metabolism on these parameters are discussed.

Serial assessment of the coagulation status of dogs with immune-mediated haemolytic anaemia using thromboelastography.

This study investigated the coagulation status of dogs with immune-mediated haemolytic anaemia (IMHA) over time. Thirty animals with primary IMHA were blood sampled on three occasions over a 5 day period and assays performed included prothrombin time, activated partial thromboplastin time, D-dimer and fibrinogen concentration, antithrombin activity and recalcified unactivated thromboelastography (TEG). Based on TEG, dogs with IMHA were significantly hypercoagulable vs. controls (P<0.001) and over the 5 day period, 3/4 of the TEG parameters reflected increased clotting kinetics (P ≤ 0.02). The 30 day survival of these patients was 80% and, at hospital admission, the TEG maximum amplitude (MA) was significantly higher in survivors than non-survivors (P=0.015). Each unit increase in MA was associated with an increased odds of 30 day survival of 1.13 (95%; CI 1.02-1.25). Based on TEG, most dogs with IMHA were hypercoagulable on admission and their clotting kinetics increased with time. Relative hypocoagulability identified by TEG at initial assessment was found to be a negative prognostic indicator.

The presence of antiphospholipid antibodies in healthy Bernese Mountain Dogs.

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.

Use of serum concentrations of interleukin-18 and monocyte chemoattractant protein-1 as prognostic indicators in primary immune-mediated hemolytic anemia in dogs.

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<.05, Wald's type 3). CONCLUSIONS AND CLINICAL IMPORTANCE: A mixed cytokine response is present in dogs with IMHA and mediators of macrophage activation and recruitment might serve as prognostic indicators.

C-reactive protein in canine babesiosis caused by Babesia rossi and its association with outcome.

C-reactive protein (CRP) is a positive major acute-phase protein in dogs and can be used as a predictive marker for risk of disease and to monitor the response to treatment. Increased concentrations in certain diseases are associated with poor outcome. This cross-sectional, observational study of 75 dogs naturally infected with Babesia rossi was designed to examine the relationship between outcome and CRP concentration at admission and the magnitude of CRP change 24 hours after admission. Diagnosis was confirmed by polymerase chain reaction (PCR) and reverse line blot. CRP concentrations were determined by an automated human CRP Turbidometric Immunoassay, previously validated for use in dogs. There was no significant difference in mean CRP concentration between survivors (n = 57), 107.5 +/- 49.5 mg/l and non-survivors (n = 11), 122.1 +/- 64.6 mg/l at admission and using the exact logistic regression, adjusting for age and sex, there was no association with outcome (P = 0.53). Multiple regression analysis failed to show a significant relationship between admission CRP concentration and number of days of hospitalisation in the survivors, adjusting for age and sex (P = 0.65). Similarly, no significance was found in the relationship between the magnitude of change in CRP concentration 24 hours after admission, and the number of days of hospitalisation in survivors, (P = 0.34). It is concluded that CRP concentration, as a measure of the acute phase response, is not associated with outcome in canine babesiosis, and inflammation is unlikely to be the only cause of severity of disease.

Evaluation of a commercially available apparatus for measuring the acute phase protein serum amyloid A in horses.

The aim of this study was to investigate the reliability of an immunoturbidometric assay for measuring the acute phase protein serum amyloid A (SAA) in horses in clinical practice. The assay was compared to a previously validated assay, and overlap performance was assessed by measuring the concentration of SAA in clinically healthy horses and horses with inflammatory and non-inflammatory diseases. In pools of serum with low and high SAA concentrations the assay's intra-assay coefficients of variation were 11.7 per cent and 4.6 per cent, and its interassay coefficients of variation were 9.1 per cent and 5.6 per cent, respectively. Slight inaccuracies were observed, but they were negligible in comparison with the range of the SAA response. The assay systematically underestimated the concentrations of SAA in comparison with the results of the validated assay. The assay detected the expected difference in SAA concentrations between the healthy and diseased horses.