Dana N. LeVine

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.

The use of pooled vs serial urine samples to measure urine protein:creatinine ratios.

BACKGROUND Evaluation of serial urine protein:creatinine (UPC) ratios is important in prognosticating chronic kidney disease and monitoring response to therapeutic interventions. Owing to random biologic variation in dogs with stable glomerular proteinuria, multiple determinations of UPC ratios often are recommended to reliably assess urine protein loss. This can be cost-prohibitive. OBJECTIVE The purpose of this study was to evaluate agreement between the mean of 3 UPC ratios obtained on 3 separate urine samples per dog and a single UPC ratio obtained when aliquots of the separate samples were pooled and analyzed as 1 sample. METHODS Three separate urine samples were collected from each of 25 dogs, both client-owned and members of a research colony. Protein and creatinine concentrations were measured in the supernatant of each sample using a biochemical analyzer, and the mean of the 3 UPC ratios was calculated. A 1.0 mL aliquot of each of the 3 samples from each dog was pooled to create a fourth sample for that dog, and the UPC ratio of the pooled sample was similarly determined. Agreement and correlation between the mean and pooled UPC ratios were assessed using Bland-Altman difference plots and regression analysis, respectively. RESULTS The UPC ratio in the pooled samples was highly correlated (r=.9998, P<.0001) with the mean UPC ratio of the 3 separate samples. Strong agreement between results was demonstrated; a UPC ratio from a pooled sample was at most +/-20% different than the mean UPC ratio obtained from 3 separate samples. CONCLUSIONS Measuring the UPC ratio in a pooled sample containing equal volumes of several different urine specimens from the same dog provides a reliable and cost-effective alternative to assessing multiple UPC ratios on several specimens from the same dog.

Ronidazole pharmacokinetics after intravenous and oral immediate-release capsule administration in healthy cats⋆

Ronidazole (RDZ) is an effective treatment for feline Tritrichomonas foetus infection, but has produced neurotoxicity in some cats. An understanding of the disposition of RDZ in cats is needed in order to make precise dosing recommendations. Single-dose pharmacokinetics of intravenous (IV) RDZ and immediate-release RDZ capsules were evaluated. A single dose of IV RDZ (mean 9.2 mg/kg) and a 95 mg immediate-release RDZ capsule (mean 28.2 mg/kg) were administered to six healthy cats in a randomized crossover design. Plasma samples were collected for 48 h and assayed for RDZ using high pressure liquid chromatography (HPLC). Systemic absorption of oral RDZ was rapid and complete, with detection in the plasma of all cats by 10 min after dosing and a bioavailability of 99.64 (±16.54)%. The clearance of RDZ following IV administration was 0.82 (±0.07) ml/kg/min. The terminal half-life was 9.80 (±0.35) and 10.50 (±0.82) h after IV and oral administration, respectively, with drug detectable in all cats 48 h after both administrations. The high oral bioavailability of RDZ and slow elimination may predispose cats to neurotoxicity with twice-daily administration. Less frequent administration should be considered for further study of effective treatment of T foetus-infected cats.

Development and validation of an endoscopic activity score for canine inflammatory bowel disease

The aim of this study was to develop and prospectively validate a simple endoscopic score of disease activity for dogs with inflammatory bowel disease (IBD). Archived endoscopic still images and video recordings of gastric, duodenal, and colonic endoscopic examinations were displayed to novice and experienced endoscopists for assessment of inflammatory activity using established descriptions. The mucosal appearances evaluated were normal tissue, erosions, friability, increased granularity, lymphangiectasia (duodenum), and mass (colon). Fleiss and Cohens Kappa statistics were used to estimate the inter-observer agreement of the index. For duodenal assessment, there were statistically significant (P <0.05) differences in inter-observer agreement, with experienced endoscopists performing better than novice endoscopists in the accurate identification of mucosal appearance of the duodenum. In contrast, there was no significant difference between novice and experienced endoscopists in their interpretation of gastric (P = 0.10) and colonic (P = 1.0) mucosal appearances. Validation studies using endoscopic video clips to assess the same endoscopic parameters by quantitative (lesion number and severity) and qualitative (presence of mucosal lesions) methods showed moderate-to-substantial agreement between experienced endoscopists. Based on the observations that the quantitative and qualitative scores of mucosal appearances are virtually identical, and that qualitative assessment was performed more quickly and objectively than quantitative assessment, we propose a simple endoscopic activity score based on qualitative criteria alone in dogs with inflammatory bowel disease.

Association of Sphingosine-1-phosphate (S1P)/S1P Receptor-1 Pathway with Cell Proliferation and Survival in Canine Hemangiosarcoma.

Background Sphingosine‐1‐phosphate (S1P) is a key biolipid signaling molecule that regulates cell growth and survival, but it has not been studied in tumors from dogs. Hypothesis/Objectives S1P/S1P1 signaling will contribute to the progression of hemangiosarcoma (HSA). Animals Thirteen spontaneous HSA tissues, 9 HSA cell lines, 8 nonmalignant tissues, including 6 splenic hematomas and 2 livers with vacuolar degeneration, and 1 endothelial cell line derived from a dog with splenic hematoma were used. Methods This was a retrospective case series and in vitro study. Samples were obtained as part of medically necessary diagnostic procedures. Microarray, qRT‐PCR, immunohistochemistry, and immunoblotting were performed to examine S1P1 expression. S1P concentrations were measured by high‐performance liquid chromatography/mass spectrometry. S1P signaling was evaluated by intracellular Ca2+ mobilization; proliferation and survival were evaluated using the MTS assay and Annexin V staining. Results Canine HSA cells expressed higher levels of S1P1 mRNA than nonmalignant endothelial cells. S1P1 protein was present in HSA tissues and cell lines. HSA cells appeared to produce low levels of S1P, but they selectively consumed S1P from the culture media. Exogenous S1P induced an increase in intracellular calcium as well as increased proliferation and viability of HSA cells. Prolonged treatment with FTY720, an inhibitor of S1P1, decreased S1P1 protein expression and induced apoptosis of HSA cells. Conclusions and clinical importance S1P/S1P1 signaling pathway functions to maintain HSA cell viability and proliferation. The data suggest that S1P1 or the S1P pathway in general could be targets for therapeutic intervention for dogs with HSA.

Endoscopic Assessment of the Duodenum in Dogs with Inflammatory Bowel Disease

Background Endoscopy is performed for direct inspection of the mucosa and acquisition of biopsies in dogs with inflammatory bowel disease (IBD). Aim To evaluate the interobserver agreement in the endoscopic assessment of duodenal mucosa in dogs with IBD. Methods Thirty‐five archived endoscopic images of grossly normal (n = 6) and inflamed (n = 29) duodenal mucosa were displayed to 3 expert and 5 trainee endoscopists. Each image was assessed independently by endoscopists for mucosal abnormalities using established indices (of hyperemia, granularity, friability, lymphatic dilatation, and erosions) or interpreted as normal mucosa (trial 1). A repeated trial (trial 2) was performed with the same images presented in random order 1 month later, and accompanied by a visual template. Results There was slight interobserver agreement in initial mucosal assessment for expert and trainee endoscopists in trial 1 (kappa ≤ 0.02, P > .05). Interobserver agreement improved in trial 2 for both expert and trainee endoscopists (kappa = 0.2, P > .05) for experts and (P < .05) for trainees. There was a significant (P < .01) improvement in trainee endoscopy scores of lesions from trial 1 to trial 2. Regression analysis showed a significant (P < .01) difference between expert versus trainee endoscopy scores in trial 1. Repeat lesion assessment aided by use of a visual template (trial 2) improved the overall scores of trainee endoscopists to near that of expert endoscopists (P = .06). Conclusions and Clinical Importance Interobserver agreement of IBD mucosal appearance from endoscopic findings benefitted from operator experience.

Twice-daily dosing of RDZ no longer recommended for treatment of intestinal Tritrichomonas foetus infection

We would like to thank Xenoulis et al1 for their recent retrospective study in which information is provided on the outcome of cats that were treated with ronidazole for Tritrichomonas foetus infection. We agree with the authors’ recommendation of a treatment regimen for infections caused by T foetus of 30 mg/kg ronidazole (RDZ) q24h for 14 days.1 However, they reference a previous study of ours that recommended that cats that relapse should be treated with 30–50 mg/kg RDZ PO q12h.1,2 Based on our recent feline RDZ pharmacokinetic (PK) studies, we no longer recommend twice-daily dosing of RDZ.3 In our PK study, we determined that the half-life of RDZ is long (10.5 h), and that 48 h after a single dose of 30 mg/kg immediaterelease RDZ capsules orally there was still drug remaining in the cats’ plasma.3 Simulations based on our PK studies showed that twice-daily administration of 30 mg/kg RDZ PO would lead to drug accumulation.3 Neurotoxicity, including tremors, ataxia and agitation, is a reported and potentially dangerous side effect of RDZ in cats.4,5 As such, we no longer advocate twice-daily RDZ at this dose. However, it should also be noted that our PK studies did not assess the efficacy of our revised recommendations of once-daily 30 mg/kg RDZ administration for T foetus-infected cats. Such work remains to be done. We wanted to draw readers’ attention to these recent PK studies, so that cats are not inadvertently overdosed and risk neurologic sequelae based on outdated recommendations. References 1 Xenoulis PG, Lopinski DJ, Read SA, et al. Intestinal Tritrichomonas foetus infection in cats: a retrospective study of 104 cases. J Feline Med Surg 2013; 15: 1098–1103. 2 Gookin JL, Copple CN, Papich MG, et al. Efficacy of ronidazole for treatment of feline Tritrichomonas foetus infection. J Vet Intern Med 2006; 20: 536–543. 3 LeVine DN, Papich MG, Gookin JL, et al. Ronidazole pharmacokinetics after intravenous and oral immediaterelease capsule administration in healthy cats. J Feline Med Surg 2011; 13: 244–250. 4 Rosado TW, Specht A and Marks SL. Neurotoxicosis in four cats receiving ronidazole. J Vet Intern Med 2007; 21: 328–331. 5 Pham D. Chronic intermittent diarrhea in a 14-month-old Abyssinian cat. Can Vet J 2009; 50: 85–87.

Ronidazole pharmacokinetics in cats following delivery of a delayed‐release guar gum formulation

Ronidazole (RDZ) is the only known effective treatment for feline diarrhea caused by Tritrichomonas foetus. This study aimed to develop guar gum-coated colon-targeted tablets of RDZ and to determine the pharmacokinetics of this delayed-release formulation in cats. Guar gum-coated tablets were administered orally once to five healthy cats (mean dose 32.3 mg/kg). The tablets were then administered once daily for 5 days to four cats (mean dose 34.5 mg/kg), and absorption studies repeated on day 5. Plasma was collected and analyzed for RDZ concentration, and pharmacokinetic noncompartmental and deconvolution analysis were performed on the data. There was negligible RDZ release until after 6 h, and a delayed peak plasma concentration (mean Cmax 28.9 μg/mL) at approximately 14.5 h, which coincides with colonic arrival in cats. Maximum input rate (mg/kg per hour) occurred between 6 and 16 h. This delayed release of ronidazole from guar gum-coated tablets indicates that release of RDZ may be delayed to deliver the medication to a targeted area of the intestine. Repeated dosing with guar gum tablets to steady-state did not inhibit drug bioavailability or alter the pharmacokinetics. Such targeted RDZ drug delivery may provide improved efficacy and reduce adverse effects in cats.

Force-activatable biosensor enables single platelet force mapping directly by fluorescence imaging

Integrin-transmitted cellular forces are critical for platelet adhesion, activation, aggregation and contraction during hemostasis and thrombosis. Measuring and mapping single platelet forces are desired in both research and clinical applications. Conventional force-to-strain based cell traction force microscopies have low resolution which is not ideal for cellular force mapping in small platelets. To enable platelet force mapping with submicron resolution, we developed a force-activatable biosensor named integrative tension sensor (ITS) which directly converts molecular tensions to fluorescent signals, therefore enabling cellular force mapping directly by fluorescence imaging. With ITS, we mapped cellular forces in single platelets at 0.4µm resolution. We found that platelet force distribution has strong polarization which is sensitive to treatment with the anti-platelet drug tirofiban, suggesting that the ITS force map can report anti-platelet drug efficacy. The ITS also calibrated integrin molecular tensions in platelets and revealed two distinct tension levels: 12-54 piconewton (nominal values) tensions generated during platelet adhesion and tensions above 54 piconewton generated during platelet contraction. Overall, the ITS is a powerful biosensor for the study of platelet mechanobiology, and holds great potential in antithrombotic drug development and assessing platelet activity in health and disease.

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.