Monitoring response to aminobisphosphonate therapy in canine osteosarcoma using dual energy x‐ray absorptiometry (dexa)

Introduction: Palliative therapy is essential to improve the quality of life of dogs with osteosarcoma (OSA), when definitive therapy is not considered a valid option. Bisphosphonates, a novel class of antiosteoclastic drugs, are widely used in humans for several painful osteolytic conditions. Dual energy x‐ray absorptiometry (DEXA) is recognized as a reliable tool to measure bone mineral density (BMD), and to monitor treatment response to bisphosphonates in humans. A prospective evaluation of pamidronate, an injectable aminobisphosphonate, is ongoing in dogs with appendicular OSA. The potential value of DEXA for objective evaluation of BMD variations with palliative therapies is concurrently being assessed. Materials and Methods: Dogs with naturally occurring appendicular OSA treated with pamidronate constitute the patient population. A DEXA scan (QDR‐4500 W, Hologic, Bedford, MA) is performed on day 0 (baseline) and on every treatment day with pamidronate thereafter (every 28 days). For each dog, a whole body scan is performed, followed by a scan of the tumor, and contralateral normal bone. Three regions of interest are subsequently analyzed for BMD changes in tumor and normal bone. Statistical analysis was performed using Student t‐test and paired t‐tests, with significance being set at p < 0.05. Results: Nineteen dogs have been enrolled to date. Seven responders and 6 non‐responders have suitable data for analysis. A significant difference is observed (p = 0.04) between tumor BMD variations of responders and non‐responders at day 28 (mean variations +18.0% and ?4.6% respectively). The changes at day 28 are significant only in the responders (p = 0.038 vs p = 0.05 in non‐responders). When BMD of tumor and normal bone at day 84 is compared to day 0 in six responders, only tumor had a significant increase (p = 0.017 vs p = 0.279, respectively). Conclusions: Objective measurements of response to therapy are essential in pain palliation studies. Increased tumor bone BMD, as obtained by DEXA analysis, may correlate with subjective clinical improvement in pamidronate‐treated dogs with appendicular OSA.     

RNA‐transfected CD40‐activated B cells as a vaccine strategy in canine malignancies

Introduction: Cell‐based vaccine strategies using dendritic cells as cellular adjuvant have entered phase III trials in humans and have been found to be safe, feasible, and potentially efficacious. Canine patients are generally smaller than adult human patients, which makes production of canine dendritic cell (DC) vaccines problematic, given patient size and the small number of available DC precursors. Here we describe feasibility studies of a novel cell‐based vaccine strategy which uses CD40‐activated B‐cells (CD40‐B) loaded with RNA. This strategy is based on our observations that RNA‐transfected human CD40‐B can drive anti‐tumor T cell responses. One advantage of using CD40‐B cells is the ability to expand this cell population ex vivo, allowing for the numbers of cells required for therapeutic vaccines. Methods: Twenty milliliters of blood were drawn from 6 normal dogs and 5 canine lymphoma patients. Peripheral blood mononuclear cells were separated by Ficoll centrifugation. Culture conditions for B cell activation were optimized using CD40‐ligand, canine IL‐4, and Toll‐like receptor stimulus with CpGoligodinucleotides (ODN). Cyclosporine was added to eliminate peripheral T lymphocytes. Proliferation and activation of CD40‐B cells were demonstrated by CFSE dilution of B cells quantified by flow cytometry. Gene transfer was achieved by mRNA electroporation. Results: Marked in vitro stimulation and proliferation of canine peripheral B cells were achieved with soluble trimeric CD40L, canine IL‐4, and ODN. CD40‐B cells showed dramatic upregulation of MHC class II molecules and CD21 (B‐cell activation marker). After two weeks in culture, cells were negative for CD3 and CD4. Canine CD40‐B cells were efficiently transfected with mRNA, with >60% of CD40‐B expressing green fluorescent protein after GFP mRNA electroporation. Conclusion: RNA‐transfected CD40‐B cells can be efficiently generated from normal and tumor‐bearing dogs. These results provide rationale to test tumor RNA‐transfected CD40‐B as a novel therapeutic approach to treating canine malignancies. Clinical trials in canine lymphoma have been proposed.     

Cardiac troponin I in canine patients with lymphoma and osteosarcoma receiving doxorubicin: comparison with clinical heart disease in a retrospective analysis

The cumulative cardiotoxicity that occurs as a result of doxorubicin chemotherapy is irreversible and can affect both quality and quantity of life for the cancer patient. Cardiac troponin I (cTnI) is a sensitive and specific marker of cardiomyocyte death. The purpose of this retrospective study was to evaluate serum concentrations of cTnI in dogs with lymphoma or osteosarcoma given doxorubicin chemotherapy, and with known cardiac outcome, based on a minimum assessment by physical examination and thoracic radiography. Serum samples were also available for cTnI measurement from seven healthy dogs given intracoronary doxorubicin. Serial serum samples obtained before, during and after doxorubicin chemotherapy showed increased cTnI concentrations in some clinical patients following chemotherapy (P = 0.0083 compared to baseline), but this did not correlate with clinical signs of cardiomyopathy. In dogs that subsequently developed cardiomyopathy however, serum cTnI concentrations were elevated before clinical signs became evident (confirmed with echocardiography).     

Treatment of canine haemangiosarcoma with suberoylanilide hydroxamic acid, a histone deacetylase inhibitor

A case report is presented by describing the treatment of a 12‐year‐old dog – diagnosed with haemangiosarcoma (HSA) – with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor. The drug was administered orally, on a daily basis, approximately 2 weeks post‐splenectomy at a dose of 3 mg kg^?1. HSA is a lethal malignancy of the endothelium, which is usually disseminated by the time it is diagnosed. Median survival time, usually, is no longer than 80 days. Following treatment with SAHA, no sign of malignant growth could be discerned by means of diagnostic abdominal ultrasound, chest X‐ray or with the help of clinical symptoms, over a period of >1000 days. The precise mechanism by which HDAC inhibitors exert their anti‐cancer effects is uncertain, but evidence suggests that exposure to SAHA generates hyperacetylated chromosomal histones, which, in turn, facilitates the expression of tumour suppressor genes turned off by epigenetic mechanisms during neoplastic transformation of the endothelium.     

Determination of plasma lysosomal enzyme activity and its relationship to severity of injury following blunt vehicular trauma in dogs

Objective: To determine plasma β‐d ‐glucuronidase (βG) activity in the first 4 hours following injury in dogs struck by a motor vehicle, and to evaluate whether the degree of enzyme activity is correlated with the severity of injury. Design: A prospective clinical study. Setting: Veterinary Medical Teaching Hospital. Animals: Thirteen client‐owned dogs that were presented to the Veterinary Hospital of the University of Pennsylvania between June and August 1999 for blunt vehicular trauma. Ten healthy student and staff‐owned dogs served as controls. Interventions: None. Measurements: Plasma was analyzed for βG enzyme activity at the time of presentation (n=13), and 1 and 4 hours (n=7) following presentation to the Emergency Service for blunt vehicular trauma. The results were compared with enzyme activity from healthy controls evaluated serially over 4 hours. Fluorometric analysis using 96‐well microtiter plates was used to perform the enzyme assays. The relationships between presentation (n=13) and 4 hours (n=7) of enzyme activity and 3 indices of metabolic and physical disturbance (serum pH, serum lactate and Animal Trauma Triage (ATT) score) at the time of presentation were also investigated. Main results: Of the 13 dogs, 7 fulfilled the inclusion criteria for comparison of enzyme activity of the trauma over time. A statistically significant difference in βG activity was found in the trauma group (mean 75.6±10.4 U) at 4 hours following presentation compared with controls (mean 48.0±6.4 U). This difference was suggested by 1 hour following presentation (trauma group, mean 70.4±10.9 U; control group, mean 49.8±5.5 U), although it did not reach statistical significance. Thirteen dogs fulfilled the inclusion criteria for comparison of only presentation enzyme activity with trauma severity score, serum lactate, and serum pH. No statistically significant relationship was found between the βd ‐glucuronidase activity and the presenting ATT score, serum lactate concentration, or serum pH at either presentation or 4 hours, although the power of these analyses was low. Conclusions: These results demonstrate that the activity of βG, a lysosomal enzyme, increases significantly in the systemic circulation in dogs 4 hours following blunt trauma. Additional research to include more severely injured dogs, a larger number of dogs, and to follow the course of injury for a longer period of time would be beneficial to further characterize βG activity following blunt trauma.     

Effect of recombinant canine IFNγ on canine atopic dermatitis evaluated by clinical signs,histopathology and expression of Th1/Th2 cytokine mRNA

Atopic dermatitis (AD) is thought to be caused by immunologic abnormalities expressed as a Th1/Th2 cytokine imbalance in both humans and dogs. Several studies have focused on the therapeutic effects of IFNγ in human AD with successful results; however, the mechanism of action of IFNγ is not fully understood. We investigated the effect of recombinant canine interferon gamma (rCaIFNγ) on 10 dogs with AD and evaluated the ratio of IL‐4 mRNA to IFNγ mRNA in peripheral blood mononuclear cells, serum total IgE levels, and histological changes in skin. After six injections of rCaIFNγ over a span of 2 weeks, seven of the 10 dogs showed improvement, and six of these seven dogs exhibited decreased IL‐4:IFNγ mRNA ratios. Two of the three cases that did not improve had increased IL‐4:IFNγ mRNA ratios. Total serum IgE levels were significantly decreased in nine of 10 cases. The number of IgE‐positive cells detected by immunostaining and the number of mast cells in skin biopsy samples were decreased. A reduction of epidermal cell layers was demonstrated by histopathology after treatment. These results demonstrated that rCaIFNγ may be a novel safe and effective therapeutic option for the treatment of canine AD, and the mechanism of action of rCaIFNγ may be related to the modulation of Th2 cytokines to Th1 cytokines with the reduction of serum IgE production. Funding: Self‐funded.     

Patch test reactions to house dust mites in dogs with atopic dermatitis

The purpose of this study was to determine the percentage of dogs with spontaneous atopic dermatitis that show a positive patch test reaction to a commercially available 20% house dust mites mixture containing equal parts of Dermatophagoides farinae and Dermatophagoides pteronyssinus in white petrolatum. In addition, we evaluated whether skin reactions induced after the epicutaneous application of house dust mites were clinically and histologically similar to naturally developed skin lesions of dogs with atopic dermatitis. Furthermore, we investigated if the reactions induced by house dust mites were true allergic reactions by comparing them to atopic lesional skin and to patch test reactions induced by an irritant substance (sodium lauryl sulphate). White petrolatum alone and nonlesional skin sites were used as negative controls. Macroscopic and microscopic evaluations of the patch test and control sites were performed in a blinded fashion at 48 and 72 h after patch test application. Microscopic results were evaluated in a qualitative and quantitative manner. A chi‐square test for homogenicity was used for the quantitative analysis to compare the proportion of each dermal inflammatory cell type among positive histopathological tested sites. P values ≤ 0.05 were considered significant. The study included 12 healthy nonatopic dogs and 13 dogs with nonseasonal atopic dermatitis. None of the nonatopic dogs reacted to house dust mites and white petrolatum. Ten (77%) of the 13 atopic dogs reacted macroscopically and histopathologically to house dust mites. Macroscopic reactions induced by house dust mites were characterized by erythema, oedema and papules. The macroscopic reactions induced by house dust mites were identical to lesional skin in 20% of the dogs and identical to reactions induced by sodium lauryl sulphate in 40% of the dogs. Qualitative histopathological findings showed that the reactions induced by house dust mites were similar to atopic lesional skin in 80% of the dogs and were similar to sodium lauryl sulphate in 20% of the dogs. Quantitative analyses showed that the proportion of neutrophils in reactions induced by sodium lauryl sulphate was significantly higher (P < 0.05) compared to house dust mites reactions, which could be a differentiator factor between an allergic and an irritant reaction. These results showed that the epicutaneous application of house dust mites in dogs with atopic dermatitis induced histopathological lesions similar to spontaneous atopic lesions in dogs. Therefore, this study demonstrated that house dust mites penetrated the skin of dogs with atopic dermatitis and induced an inflammatory response that resembled a true allergic reaction. Funding: Small Companion Animal Grant, University of Minnesota.     

P‐36 Juvenile idiopathic nasal scaling in three Bengal cats

Affected cats were three Bengals, one male and two females, whose age at onset of lesions ranged from 13‐ to 20‐weeks old. Nasal planum scaling progressed to thick crusting, consequent exfoliation and exposure of underlying erosions. No signs of pruritus or pain were present but mild respiratory signs were noticed. In all cats, haematology and biochemistry were normal, they were FIV and FeLV negative, PCR for herpesvirus, calicivirus and Chlamydia were negative, and viral isolation for calicivirus and herpesvirus was negative. Wood's lamp examination was negative, as were bacterial and fungal cultures. Cytology showed exfoliating keratinocytes. A skin biopsy taken from one case showed no significant changes. Biopsies from the nasal planum of four dead cats with nondermatological conditions (controls) were collected for comparative studies. Morphometry to record the percentage of the granular layer (GL) and stratum corneum (SC) on the total thickness of the epidermis of the nasal planum was performed. The GL and SC accounted for 10.2 and 21.7% of the epidermal thickness in the affected cat, compared to 18.3 and 20.2%, respectively, in the controls. A significant reduction (P < 0.02) of the SC thickness was detected in the affected cat compared to controls. No treatment was instituted as all cats underwent complete (two cats) or nearly complete (one cat) resolution. The reported cases share the same breed, age at onset, type of lesions and a similar outcome. A reduction of the SC thickness in one of the affected cats was recorded. Therefore, an underlying congenital condition is suspected that manifests with high epidermal cell turnover and normal keratinization. Funding: Self‐funded.     

P‐81 A retrospective study of equine sarcoidosis

The purpose of this retrospective study was to evaluate six cases of equine sarcoidosis for initial presenting symptoms, response to therapy and actual outcome. Dermatologists and dermatopathologists from Europe, the United States, Australia and Canada were contacted to obtain these six cases, as this is a rare disease. Signalment, clinical signs, histological findings, clinical management and outcome were determined via a questionnaire and compared to former reports. There was no age or breed predilection, and four of six horses were geldings. Age of onset ranged from 3 months to 17 years. Onset of the disease was insidious or rapid. Interestingly, in five of six cases, scaling began on the trunk (girth and shoulder). Scaling, crusting and alopecia were seen in all six horses. In one horse, clinical signs of systemic disease were reported and included intermittent fever, prescapular lymphadenopathy, depression, poor body condition and nasal discharge. Treatment included phenylbutazone, deworming agents, antibiotics, short‐term low‐dose corticosteroids, and 1–1.5 mg/kg of prednisolone. One horse showed a partial response to trimethoprim and sulfonamide, and five of six went into clinical remission with corticosteroid treatment. Five of six horses were still alive 1 year after diagnosis; one horse was diagnosed <12 months ago. Two horses are in complete remission 4 and 8 years after diagnosis. In both horses, clinical signs recurred after cessation of therapy and went into remission again with reintroduction of treatment. Both of these horses have been in remission for several years without therapy. Funding: Self‐funded.     

Antiviral Effect of Saccharomyces cerevisiaeβ‐glucan to Swine Influenza Virus by Increased Production of Interferon‐γ and Nitric Oxide

The aim of these experiments was to investigate the potential antiviral effect of Saccharomyces cerevisiaeβ‐glucan on the pneumonia induced by swine influenza virus (SIV). Forty colostrum‐deprived 5‐day‐old piglets were randomly divided into four groups of 10. The 20 pigs in groups 1 and 2 were administered Saccharomyces cerevisiaeβ‐glucan orally (50 mg/day/pig; En‐Bio Technology Co., Ltd) for 3 days before SIV infection and those in groups 3 and 4 were given culture medium/diluent alone. Groups 1 and 3 were inoculated intranasally with 3 ml of tissue culture fluid containing 2 × 10⁶ tissue culture infective doses 50% (TCID₅₀)/ml of SIV and those in groups 2 and 4 were exposed in the same manner to uninfected cell culture supernatant. The microscopic lung lesions induced by SIV infection (group 1 pigs) were significantly more severe than those induced by infection in animals pre‐administered β‐glucan (group 3) (P < 0.05). Significantly more SIV nucleic acid was detected in the lungs of pigs experimentally infected with SIV only (group 1) at 5, 7 and 10 days post‐inoculation (dpi) compared with lungs from pigs pre‐administered β‐glucan and infected with SIV (group 3) (P < 0.05). The concentrations of interferon‐γ (IFN‐γ) and nitric oxide (NO) in bronchoalveolar lavage fluid from pigs pre‐administered β‐glucan and infected with SIV (group 3) were significantly higher than for any other group at 7 and 10 dpi for IFN‐γ, and at 5, 7 and 10 dpi for NO (P < 0.05). Saccharomyces cerevisiaeβ‐glucan reduced the pulmonary lesion score and viral replication rate in SIV‐infected pigs. These findings support the potential application of β‐glucan as prophylactic/treatment agent in influenza virus infection.