Systemic iron reduction via an iron deficient diet decreases the severity of knee cartilage lesions in the Dunkin-Hartley guinea pig model of osteoarthritis.

OBJECTIVE: Iron accumulation is emerging as a player in aging-related disorders due to its propensity for generating reactive oxygen species (ROS). Studies investigating the role of iron in the pathogenesis of primary osteoarthritis (OA) are limited. We designed a proof-of-principle study to determine the effect of systemic iron deficiency, via an iron deficient diet, on knee OA in an animal model. METHODS: Twelve-week-old male Hartley guinea pigs received the standard diet (n = 6) or a diet devoid of iron (n = 6) for 19-weeks. Iron levels were determined in the serum, liver, and articular cartilage. Knees were collected to assess structural changes related to OA (microcomputed tomography, histopathology). Immunohistochemistry was performed to evaluate the presence and distribution of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) and ROS-driven 4-hydroxynonenal (4-HNE)-induced protein adducts. Transcript expression was also assessed. RESULTS: Relative to control animals, an iron deficient diet reduced the concentration of this mineral in serum, liver, and articular cartilage. Iron deficient animals had lower histologic OA scores; decreased subchondral bone mineral density was also noted. This reduction in knee joint pathology was accompanied by a decrease in: ADAMTS4 in synovium; and 4-HNE protein adducts from lipid peroxidation in both the menisci and articular cartilage of iron deficient animals. Expression of iron-related genes in these tissues was also altered in treated animals. CONCLUSIONS: Results from this study suggest that systemic iron levels may play a role in knee OA pathogenesis, with a short-term deficit in dietary iron reducing the severity of knee cartilage lesions.

Multiplate platelet aggregometry in dogs undergoing laparoscopic liver biopsy for diagnosis of chronic hepatopathy.

OBJECTIVES: To assess platelet function via the Multiplate analyser in dogs undergoing laparoscopic liver biopsy for diagnosis of chronic hepatopathy. MATERIALS AND METHODS: Twenty-seven client-owned dogs were prospectively enrolled. Before laparoscopic liver biopsy, whole blood impedance platelet aggregometry via the Multiplate analyser was performed. Buccal mucosal bleeding time was performed in 23 of 27 dogs. Tissue factor-activated thromboelastography was also performed, in addition to plasma-based coagulation testing. Descriptive statistics were calculated and the prevalence of platelet function abnormalities and results of other biochemical and coagulation testing were reported. RESULTS: Seventeen (63%) of 27 dogs had evidence of decreased platelet function as assessed by aggregometry, with all 17 dogs having decreased responsiveness to adenosine diphosphate, and 11 of 17 dogs demonstrating decreased responsiveness to arachidonic acid. Based on maximum amplitude, most dogs were classified as normocoagulable on thromboelastography (15/25; 60%). Other frequent coagulation abnormalities included increased D-dimers (20/27;74%), thrombocytopenia (11/27; 41%), hypofibrinogenemia (4/27; 15%), and decreased antithrombin (4/27; 15%). CLINICAL SIGNIFICANCE: Decreased platelet function as assessed by whole blood impedance aggregometry was common in dogs with chronic liver disease. Further study is necessary to determine whether this finding is repeatable or indicative of increased bleeding risk.

Phase I clinical trial of an antithrombotic drug protocol combining apixaban and clopidogrel in dogs.

INTRODUCTION: Combining an antiplatelet drug, clopidogrel, with the direct oral Factor Xa inhibitor, apixaban, could provide an effective antithrombotic strategy in dogs. Thus, a limited 3 + 3 phase I dose-escalation clinical trial in healthy dogs was conducted to evaluate bleeding (primary end-point) and pharmacodynamic (PD)/pharmacokinetic (PK) parameters (secondary end-point). ANIMALS: Eleven beagle dogs, median body weight 10.2 kg (9.7-10.9 kg), were enrolled. METHODS: Four doses of apixaban (three dogs/dose) administered for eight days. Clopidogrel dose was fixed at 18.75 mg per os (PO) q 24 h with escalation of apixaban dose at 5 mg PO q 12 h, 5 mg PO q 8 h, 10 mg PO q 12 h, and 10 mg PO q 8 h. Laboratory testing included fecal occult blood, coagulation parameters, Factor X activity, apixaban concentration, platelet aggregometry, and thromboelastography on days 1, 3, and 8. RESULTS: Evidence of bleeding was not observed at any dosage. Dose-dependent changes in PD/PK parameters between baseline and 3 h post-medication were observed including a prolongation of prothrombin time, a prolongation of activated partial thromboplastin time, a decrease of Factor X activity level, and increased apixaban concentration. CONCLUSIONS: The combination of apixaban at a dosage range of approximately 0.5 mg/kg PO q 12 h to 1 mg/kg PO q 8 h and clopidogrel at approximately 1.8 mg/kg PO q 24 h did not cause bleeding over a one-week period in healthy dogs. Clinically relevant changes in PD/PK data occur at all dosage levels. This study provides a starting point for longer-term clinical trials to determine safety and efficacy.

Transferrin receptor expression in serum exosomes as a marker of regenerative anaemia in the horse.

REASONS FOR PERFORMING STUDY: Evaluation of erythrocyte regeneration in horses is challenging, as they do not release reticulocytes into the peripheral blood. This study investigated transferrin receptor 1 (TfR1) expression in exosomes as a noninvasive method of characterising the regenerative response in anaemic horses. OBJECTIVES: To quantify TfR1 in ultraprecipitate of serum in horses before and after phlebotomy-induced anaemia, and to identify exosomes as the source of TfR1. The hypothesis was that serum exosomal TfR1 expression would increase during a regenerative response. STUDY DESIGN: Experimental model of anaemia. METHODS: Six horses were phlebotomised to achieve a 25% decrease in packed cell volume. Transferrin receptor 1 quantity in exosomes was determined by western blot and relative densitometry before and after phlebotomy. The size and density of the TfR1-associated particles were confirmed by transmission electron microscopy and density gradient centrifugation, respectively. RESULTS: Regenerative anaemia was confirmed by decreased packed cell volumes and decreased myeloid:erythroid ratios in the bone marrow. In all 6 horses, TfR1 expression increased between Days 7 and 10. Mean TfR1 levels peaked on Day 10 and at 3-fold higher than levels on Day 0. Appropriately sized particles were evident on transmission electron microscopy and sucrose density gradient fractions expected to contain exosomes also contained TfR1. CONCLUSIONS: These data indicate that TfR1 expression in serum exosomes may provide a marker for regeneration in anaemic horses.

Antiphospholipid antibodies in dogs with immune mediated hemolytic anemia, spontaneous thrombosis, and hyperadrenocorticism.

BACKGROUND: The pathophysiology of thrombus formation in canine IMHA and other diseases remains unclear. Antiphospholipid antibodies (aPL) are an important cause of thrombosis in humans and might cause thrombosis in dogs. HYPOTHESIS: Dogs with IMHA, spontaneous thrombosis, and hyperadrenocorticism will have increased levels of aPL and lupus anticoagulants (LA), compared with healthy and sick dogs. ANIMALS: Thre aPL were measured in healthy controls (n = 40-45); sick dogs without thrombosis (n = 86); IMHA (n = 37); spontaneous thrombosis (ST, n = 11); and hyperadrenocorticism (n = 17). Four groups of dogs were also tested for the presence of LA: healthy controls (n = 40); sick dogs without thrombosis (n = 13); IMHA (n = 13); and ST (n = 5). METHODS: Prospective cohort study. Dogs were tested for aPL by an ELISA and for LA by the dilute Russell's Viper venom time (dRVVT). Median values were compared by Kruskal-Wallis (aPL) or ANOVA (LA), and an odds ratio for development of thrombosis in dogs positive for aPL was calculated. RESULTS: aPL are uncommon in healthy dogs. A total of 13/86 sick dogs without thrombosis, 7/37 dogs with IMHA, 1/11 dogs with ST, and 3/17 dogs with HAC were positive for protein binding-dependent aPL. There was no significant difference in the number of dogs positive for aPL for any of the study groups, and there was no increased risk for thrombosis in dogs positive for aPL. No dogs had LA. CONCLUSIONS: Our preliminary research does not support a strong role for aPL for the development of thrombosis in dogs with IMHA and other thrombotic diseases, although future studies are warranted.

Anemia is associated with decreased survival time in dogs with lymphoma.

BACKGROUND: Anemia is a common complication in human patients with neoplasia and has been associated with decreased survival time and a poorer quality of life. HYPOTHESIS: The presence of anemia at diagnosis is negatively associated with survival and remission times in dogs with lymphoma, but not in dogs with osteosarcoma. ANIMALS: Eighty-four dogs with lymphoma and 91 dogs with osteosarcoma that presented for treatment at the Animal Cancer Center, Colorado State University. METHODS: Retrospective, case-control study. Medical records were reviewed to determine the presence or absence of anemia (PCV < 40) at initial presentation. Median survival and remission times were identified by the Kaplan-Meier product limit method and the association between anemia and survival was determined by a multivariable Cox proportional hazard regression analysis. RESULTS: Cancer-related anemia is more frequent in dogs with lymphoma than in control dogs or dogs with osteosarcoma. Dogs with lymphoma and anemia had a significantly decreased survival time compared with dogs without anemia. There was no effect of anemia on remission time in dogs with lymphoma. Anemic dogs with osteosarcoma did not have decreased survival or remission time compared with nonanemic dogs with osteosarcoma. CONCLUSIONS AND CLINICAL IMPORTANCE: Shortened survival time in dogs with lymphoma and anemia at initial presentation has important prognostic significance. Understanding cancer-related anemia in dogs might offer new opportunities to improve quality of life and survival times in these patients.

Defective gamma-glutamyl carboxylase activity and bleeding in Rambouillet sheep.

A flock of Rambouillet sheep was examined because of increased lamb mortality caused by ineffective hemostasis at parturition. Neonatal-affected lambs presented with inadequate hemostasis at the umbilicus, pale mucus membranes, and markedly prolonged activated clotting time. Affected lambs had consistently prolonged 1-stage prothrombin times and activated partial thromboplastin times that supported a defect in the common pathway or defects in both the intrinsic and extrinsic pathway of the coagulation cascade. Decreased activity of vitamin K-dependent procoagulant factors II, VII, IX, and X in male and female lambs suggested either a defect of the hepatic enzyme gamma-glutamyl carboxylase, or vitamin K(1) 2,3 epoxide reductase. Affected lamb hepatic gamma-glutamyl carboxylase activity was markedly decreased compared with that of age- and sex-matched control lambs, while vitamin K(1) 2,3 epoxide reductase and glucose-6-phosphatase activities were similar between an affected and normal lamb. Subcutaneous vitamin K(1) supplementation did not increase vitamin K-dependent procoagulant factor activities in 3 lambs administered vitamin K(1) daily. These data confirm defective gamma-glutamyl carboxylase activity as the cause of impaired coagulation of sheep in this flock. This flock represents the only viable animal model of hereditarily defective gamma-glutamyl carboxylase activity.

Hemochromatosis secondary to repeated blood transfusions in a dog.

Hemochromatosis was presumptively diagnosed using cytologic examination of liver tissue from an aged male Miniature Schnauzer. The dog was presented after receiving whole blood transfusion every 6-8 weeks for 3 years to treat pure red cell aplasia. The cytologic specimen contained clusters of hepatocytes with abundant intracytoplasmic gold-yellow pigment granules and clumps of extracellular, green-black, globular pigment, both interpreted to be hemosiderin. Histologic sections of liver revealed hepatocellular degeneration with bridging portal fibrosis, lobular atrophy, biliary hyperplasia, and diffuse, severe hemosiderin accumulation. Serum iron and ferritin levels, and dry-weight iron concentrations of liver, heart, and kidneys were markedly increased. Hemosiderin accumulation was confirmed in hepatocytes of cytologic and histologic specimens using Perl's Prussian blue staining. This report is the first description of transfusional hemochromatosis in a dog and is the first to describe its cytologic appearance in a veterinary patient.

Infection with Leishmania major stimulates haematopoiesis in susceptible BALB/c mice and suppresses haematopoiesis in resistant CBA mice.

Cytokine responses to Leishmania infection begin very early in infection, and differ between susceptible and resistant mice. Susceptibility to chronic Leishmania infection has been associated with increased haematopoiesis. To analyse the effect that acute infection with L. major has on bone-marrow haematopoiesis in susceptible (BALB/c) and resistant (CBA) mice, we enumerated erythroid progenitors and granulocyte-monocyte progenitors 3 days after infection. We found that haematopoiesis was stimulated in BALB/c mice infected with L. major, while haematopoiesis was inhibited in CBA mice. We found that this effect could be partially explained by cytokine production: interleukin-4 was involved in stimulation of BALB/c haematopoiesis and tumour necrosis factor-alpha was involved in inhibition of CBA haematopoiesis. Our conclusions are that haematopoietic changes occur shortly after L. major infection, and may be related to disease outcome.

Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes.

Although the diagnosis of canine leukemia and lymphoma in advanced stages is usually uncomplicated, some presentations of the disease can be a diagnostic challenge. In certain situations, lymphoma and leukemia can be difficult to distinguish from a benign reactive proliferation of lymphocytes. Because clonality is the hallmark of malignancy, we have developed an assay that uses the polymerase chain reaction to amplify the variable regions of immunoglobulin genes and T-cell receptor genes to detect the presence of a clonal lymphocyte population. The assay detected clonally rearranged antigen receptor genes in 91% of the 77 dogs with lymphoid malignancy. Of the 24 dogs tested, that were either healthy or had clearly defined conditions not related to lymphoid malignancy, a clonally rearranged antigen receptor gene was found in one (a dog with Ehrlichia canis infection). Gene rearrangement was appropriate for the immunophenotype (immunoglobulin gene rearrangement in B-cell leukemias and T-cell receptor gene rearrangement in T-cell leukemias). Dilution analysis showed that the clonal rearrangement could be detected when 0.1-10% of the DNA was derived from neoplastic cells, depending on the source tissue. Potential applications of this assay include the diagnosis of lymphoma or leukemia in biopsy samples, cavity fluids, fine needle aspirates, bone marrow and peripheral blood; the determination of lineage (B or T cell); staging of lymphoma; and detection of residual disease after chemotherapy.

Use of a PCR assay to assess the prevalence and risk factors for Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' in cats in the United Kingdom.

Blood samples from 426 healthy and sick cats in the UK were tested in a PCR assay for 'Candidatus Mycoplasma haemominutum' and Mycoplasma haemofelis (basonym Haemobartonella felis). Seventy-two of the cats (16.9 per cent) were positive for 'Candidatus M. haemominutum' alone, six (1.4 per cent) were positive for M. haemofelis alone and one (0.2 per cent) was positive for both. Logistic regression analysis indicated that older male cats were significantly more likely to be infected with 'Candidatus M. haemominutum', but there was no significant association between it and any of the haematological variables measured. M. haemofelis infection was uncommon in the anaemic cats sampled, and there were too few positive cases for multivariable analysis to be performed for M. haemofelis-positive status.