Bovine toll-like receptor 9: a comparative analysis of molecular structure, function and expression

Non-methylated CpG motifs, present in viral and bacterial DNA, are one of many pathogen-associated molecular patterns (PAMP) recognized by the mammalian innate immune system. Recognition of this PAMP occurs through a specific interaction with toll-like receptor 9 (TLR9) and this interaction can induce cytokine responses that influence both innate and adaptive immune responses. Previous investigations determined that both the flanking sequences in synthetic CpG oligodeoxynucleotides (CpG ODN) and the cellular pattern of TLR9 expression can influence species-specific responses to CpG ODN. Therefore, the structure, function and cellular distribution of bovine TLR9 were compared with what is known for mice and human. Analysis of the bovine TLR9 gene revealed greater sequence homology between cattle and humans than cattle and mice Similar CpG motifs induced optimal activation of both human and bovine leukocytes and these motifs were distinct from those which activated mouse leukocytes. Functional analyses with CpG ODN stimulated bovine blood leukocytes revealed that class A CpG ODN were more potent inducers of interferon-alpha (IFN-alpha) than class B CpG ODN. Furthermore, magnetic activated cell sorting of bovine blood leukocyte subpopulations implicated dendritic cells but not monocytes in the regulation of CpG ODN-induced IFN secretion. Thus, the cellular pattern of CpG ODN-induced responses in cattle shared many similarities with human leukocytes. Collectively, these analyses revealed substantial conservation of TLR9 structure and TLR9 function in blood leukocytes of humans, cattle and other domestic species.     

Pharmacokinetic and Phase I Evaluation of Carboplatin in Dogs

Thirty dogs with spontaneously occurring malignant neoplasms were treated monthly with carboplatin (CBDCA) given as a 30-minute intravenous infusion in a dose escalation study. Twenty-eight dogs were considered evaluable for toxicity. The maximally tolerated dose of CBDCA was conceptually defined as that dose, determined by logistic regression analyses of toxicity data, resulting in a 50% incidence of moderate toxicity (MOD₅₀) or a 5% incidence of severe toxicity (SEV₅). Each designated maximally tolerated dose was calculated for the first course of treatment only and for the first and second courses of treatment combined to estimate cumulative drug toxicity. The MOD₅₀ and SEV₅ for the first treatment course were 340 and 278 mg/M², respectively. MOD₅₀ and SEV₅ values for the first plus second treatment courses were 327 and 231 mg/M², respectively. The nadir of neutrophil and platelet counts occurred approximately 14 days after treatment. The mean neutrophil and platelet values for all dogs experiencing myelosuppression during the first two treatment courses were 1541/μL and 62,600/μL, respectively. Nonparametric pharmacokinetic analysis of plasma CBDCA values suggested that half-life (T_1/2), area-under-the-curve and total body clearance (CL_b) were not dose dependent. Volume of distribution (VD_ss) significantly increased with dose only between 100 and 150 mg/M², not between 150 and 300 mg/M². Dose-independent serum CBDCA pharmacokinetic disposition indicates that detailed investigation of tissue CBDCA distribution would be warranted and may identify novel dosing strategies that could improve the therapeutic index of CBDCA by minimizing toxicity. (Journal of Veterinary Internal Medicine 1993; 7:235–240. Copyright © 1993 by the American College of Veterinary Internal Medicine.)     

Clinical evaluation of pimecrolimus eye drops for treatment of canine keratoconjunctivitis sicca: a comparison with cyclosporine A

The aim of this study was to evaluate the efficacy of pimecrolimus oil-based eye drops in alleviating the clinical signs of keratoconjunctivitis sicca (KCS) in dogs and to compare the efficacy with that of cyclosporine A (CsA) ointment. An open-label, multicenter study enrolling 44 dogs previously untreated with CsA was conducted. Dogs were randomly assigned to a treatment group and medicated twice daily for 8 weeks. After that time the mean increase (+/-SEM) in the Schirmer tear test was 9.2+/-1.6 mm/min in the pimecrolimus group and 5.8+/-1.1 mm/min in the CsA group (P=0.085). The improvement in clinical signs of inflammation in eyes treated with pimecrolimus was significantly greater than in eyes treated with CsA (P=0.02). The results show that 1% pimecrolimus oily eye drops are as safe as and more effective than CsA ointment in controlling KCS in dogs.     

VH1-44 gene usage defines a subset of canine B-cell lymphomas associated with better patient survival

The use of specific immunoglobulin heavy chain variable region (VH) genes has been associated with increased patient survival in human B-cell lymphomas (hBCL). Given the similarity of human and canine BCL (cBCL) in morphology and clinical treatment, we examined the choice of VH in cBCL and determined whether VH gene selection was a distinct feature associated with survival time in dogs. VH gene selection and mutational status in 52 cBCL, including 29 diffuse large B-cell lymphomas (cDLBCL, the most common subtype of cBCL), were analyzed by comparison with the 80 published canine germline VH gene sequences. We further examined the prognostic impact of the subgroups defined by these features on canine survival. We found that VH1-44 was preferentially expressed in the majority of the 52 cBCLs (60%) as well as in the majority of the cDLBCL subset (59%). VH1-44 gene expression was associated with a statistically better overall survival (p = 0.039) in cBCL patients, as well as in the cDLBCL subset of patients (p = 0.038). These findings suggest that VH gene selection in cBCL is not random and may therefore have functional implications for cBCL lymphomagenesis, in addition to being a useful prognostic biomarker.     

Chemotactic response of bovine neutrophils to Pasteurella haemolytica culture fluid

Bovine neutrophil chemotactic activity was detected in the supernatant fluid of logarithmic phase cultures of P. haemolytica serotype 1. The chemoattractant was produced under culture conditions suitable for P. haemolytica leukotoxin production. An inverse correlation existed between the leukotoxin LC50 and the chemotactic activity in the culture fluid. Elimination of leukotoxin activity by heating, dilution or ultrafiltration, exposed the chemotactic activity in the culture fluid. The chemoattractant was partially resistant to heating (60 degrees C, 30 min), and had an apparent molecular weight greater than 100,000. Detection of chemotactic activity in both the concentrate and filtrate after XM300 filtration suggested that there might be more than one component with chemotactic activity or else that polymerization was occurring. Production of a potent neutrophil chemoattractant by P. haemolytica may explain the rapid infiltration of neutrophils that occurs during the early stages of bovine pneumonic pasteurellosis.     

Ultrasonographic findings in dogs with hyperammonemia: 90 cases (2000-2002)

OBJECTIVE: To determine ultrasonographic abnormalities in dogs with hyperammonemia. DESIGN: Retrospective study. ANIMALS: 90 client-owned dogs with hyperammonemia. PROCEDURE: Ultrasonography of the abdominal vessels and organs was performed in a systematic way. Dogs in which the ultrasonographic diagnosis was a congenital portosystemic shunt were included only if they underwent laparotomy or necropsy. Dogs in which the abdominal vasculature appeared normal and dogs in which the ultrasonographic diagnosis was acquired portosystemic shunts and portal hypertension were included only if liver biopsy specimens were submitted for histologic examination. RESULTS: Ultrasonography excluded portosystemic shunting in 11 dogs. Acquired portosystemic shunts were found in 17 dogs, of which 3 had arterioportal fistulae and 14 had other hepatic abnormalities. Congenital portosystemic shunts were found in 61 dogs, of which 19 had intrahepatic shunts and 42 had extrahepatic shunts. Intrahepatic shunts originated from the left portal branch in 14 dogs and the right portal branch in 5. Extrahepatic shunts originated from the splenic vein, the right gastric vein, or both and entered the caudal vena cava or the thorax. Ultrasonography revealed splenic-caval shunts in 24 dogs, right gastric-caval shunts in 9 dogs, splenic-azygos shunts in 8 dogs, and a right gastric-azygos shunt in 1 dog. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that ultrasonography is a reliable diagnostic method to noninvasively characterize the underlying disease in dogs with hyperammonemia. A dilated left testicular or ovarian vein was a reliable indicator of acquired portosystemic shunts.     

Site of acid-base relevant ion transfer in the gills of rainbow trout (Oncorhynchus mykiss) exposed to environmental hypercapnia

In order to delineate the contribution of primary vs. secondary circulatory circuits in the gill for acid-base and ionic regulation, the flow and composition of the fluids in the central venous sinus (CVS) and the systemic circuit of rainbow trout were studied by application of a previously developed microcannulation technique during normocapnic and hypercapnic conditions. The average haematocrit (Hct) of blood from dorsal aorta (DA) and sinus venosus (SV) ranged from 20.1 to 26.7%, whereas average Hct in the fluid from the branchial vein (BV), representing drainage from the central venous sinus (CVS), was in the range of 4.2 to 7.0%. Under normocapnic conditions, the largest fraction of cardiac output, 92.9%, was directed through the systemic vascular circuit, whereas the CVS circuit was perfused with 7.1 % of cardiac output. Hypercapnia did not significantly affect the blood flow distribution between the two circuits. The pattern of acid-base regulation in dorsal aortic blood reflected the characteristic response of fish exposed to environmental hypercapnia. Upon initiation of environmental hypercapnia (2% CO₂), plasma P_{CO 2} was elevated in all three flow compartments (CVS, DA, SV), inducing an extracellular respiratory acidosis of about 0.4 pH units. pH and [HCO₃ ^-] values in the DA were consistently lower, than those for both CVS and SV sites throughout the hypercapnic period. During the 8h of exposure plasma bicarbonate concentration was elevated by about 12 mM, complemented by a fall in extracellular [Cl^-] of about 10 mM in all three compartments. The amount of HCO₃ ^- gained at the CVS site during 8h of hypercapnia (3.3 mmol · kg^-1) exceeds the amount accumulated in the extracellular space (2.1 mmol·kg^-1), suggesting the CVS as the main site of ionic acid-base regulation in trout.