Cloning and characterization of a bovine genomic fragment homologous to epidermal growth factor genes

Epidermal growth factor (EGF) is a potent mitogen for a variety of cell types. The 53-amino acid mature EGF protein is encoded by sequences in exons 20 and 21 of a gene spanning over 110 kb. In this study, we report the cloning and characterization of 7.5 kb of bovine genomic sequence homologous to exon 19 through 21 from EGF genes from other mammalian species. The cloned gene fragment had an unusual sequence composition in the form of an in-frame TGA codon in the coding sequence. The sequence was expressed at low levels in kidney tissue and the corresponding cDNA contained the TGA codon. The level of similarity between the bovine exonic sequence and the human, porcine, murine, feline, and canine corresponding sequences varied from 64% to 73%; however, when only sequences encoding the mature EGF protein were compared, the level of similarity between the bovine sequence and the sequence from these species was 59% to 66%. The sequence similarity of the deduced mature protein was lower (34% to 39%) than the sequence similarity of the deduced propeptide. Although the cloned sequences could originate from a bovine EGF pseudogene, the possibility exists that they originate from the functional EGF gene. An as yet unidentified mechanism to by-pass the stop codon would allow the synthesis of a functional EGF protein. Alternatively, the cloned sequence could originate from an EGF-like gene.     

Immunohistochemical Detection of CD4-, CD8- and MHC II-Expressing Immune Cells and Endoglin in the Canine Corpus Luteum at Different Stages of Dioestrus

Specific immunohistochemical methods were applied to detect the presence of CD4-, CD8- and major histocompatibility complex II (MHC II)-expressing immune cells and of endoglin in the canine corpus luteum between days 15 and 75, after ovulation. Corpora lutea were obtained from groups of three clinically healthy beagle bitches, ovariohysterectomized at the respective days. For all four parameters, the effect of time was highly significant. Quantitative evaluation yielded high values on day 15, followed by a decrease on day 30 (CD4, CD8 and endoglin) and day 45 (MHC II). While there were no further changes for cells staining positive for CD4 and endoglin, CD8-positive immune cells increased from day 45 to day 60 to drop again on day 75; MHC II-positive staining increased from day 45 to days 60-75. These data suggest an involvement of the immune system in control of luteal function also in the dog that may have both stimulatory and inhibiting effects.     

Results from the treatment of advanced stage squamous cell carcinoma of the nasal planum in cats, using a combination of intralesional carboplatin and superficial radiotherapy: a pilot study

Six cats with an advanced stage squamous cell carcinoma (SCC) of the nasal planum were treated with a combination of superficial radiotherapy and intralesional carboplatin therapy. This multimodality protocol was well tolerated by the majority of cats and resulted in complete responses in all cats (100%). Median follow‐up for all cats is 268 days, and the median time‐to‐recurrence, time‐to‐progression and overall survival have not yet been reached. Our study, although limited in number of animals and with a relatively short median follow‐up compared to other studies for this disease, suggests that a combination of radiotherapy and intralesional carboplatin is a useful treatment option for an advanced stage SCC of the nasal planum in cats and warrants further application of the multimodality approach presented here.     

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.     

Noninvasive monitoring of doxorubicin cardiotoxicity: a pilot study of two dogs

The cardiotoxicity of anthracycline anticancer agents, most notably doxorubicin, limits their use in treating a wide variety of cancers. Currently available methods for assessment of anthracycline‐induced cardiotoxicity (AIC) often lack specificity and sensitivity. The purpose of this pilot investigation was to determine noninvasive diagnostic methods that are predictive for AIC. Two mongrel dogs were anesthetized for injection of doxorubicin (22.5 mg) directly into the left coronary artery. Doxorubicin injection was repeated 2 weeks later. Dogs were followed for 12 weeks or until heart failure was documented. Dogs were monitored before and at serial time points after doxorubicin using echocardiography and signal‐averaged electrocardiography as well as ambulatory ECG monitoring. Blood was also collected for measurement of cardiac troponin I and T concentrations. Doxorubicin was readministered at 6 and 12 weeks after the second injection, as interim data analysis did not reveal adequate evidence of cardiomyopathy. Dog‐1 appeared to be more sensitive than Dog‐2 to AIC based upon gradual changes in variables of cardiac function and earlier time to failure (113 days). In contrast, Dog‐2 had inconsistent changes in cardiac function and a longer time to failure (139 days). Dog‐1 also had greater peak plasma cardiac troponin I levels (17.96 ng/ml) as compared to peak levels in Dog‐2 (4.08 ng/ml). In addition, Dog‐1 had increased ventricular extrasystolic contractions (VE)(0/day at baseline, 443/day one week prior to death), whereas dog‐2 showed very few VE's throughout. Spectral analysis of ECG data also revealed a significant decrease over time in total spectral power in Dog‐1 from 37,148 at baseline to 603 msec²/Hz one week prior to death, indicating decreased parasympathetic tone. Endomyocardial biopsies revealed minor changes despite significant clinical abnormalities. These data indicate differences in susceptibility to AIC between Dog‐1 and Dog‐2, and suggest a role for these noninvasive measures in monitoring the heart during chemotherapy.     

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.