Conduct,Oversight, and Ethical Considerations of Clinical Trials in Companion Animals with Cancer: Report of a Workshop on Best Practice Recommendations

Development of effective and safe treatments for companion animals with cancer requires the collaboration of numerous animal health professionals and the full engagement of animal owners. Establishing ‘Best Practice Recommendations’ for clinical trials in veterinary oncology represents an important step toward meeting the goal of rigorous clinical trial design and conduct that is required to establish valid evidence. Likewise, optimizing patient welfare and owner education and advocacy is crucial to meet the unique ethical obligations to both owners and animals enrolled in these clinical trials and to ensure trust in the team conducting the research. To date, ‘Best Practice Recommendations’ for clinical trial conduct have not been reported for veterinary oncology. This document summarizes the consensus of a workshop held in November, 2014 to identify relevant ethical principles and to ensure responsible conduct of clinical research in companion animals with cancer. It is intended as a working document that will be updated as advances in science and ethical considerations require. To the extent possible, existing guidelines for the conduct and oversight of clinical trials in humans have been adapted for veterinary trials to avoid duplicative effort and to facilitate integration of clinical trials such that translational research with benefits for both companion animals and humans are encouraged.     

Expression of connexin 43 protein in testes, epididymides and prostates of stallions

REASONS FOR PERFORMING STUDY: Connexin 43 (Cx43) is a ubiquitously distributed gap junction protein in testes and other reproductive tissues. Adjacent cells share ions and small metabolites through intercellular channels, which are present in gap junctions. Previously, Cx43 has not been reported in testes, epididymides and prostates either in healthy stallions or cryptorchid horses. OBJECTIVES: To demonstrate the expression pattern of Cx43 in the reproductive tissues of stallions and examine whether naturally occurring bilateral cryptorchidism has any influence on distribution and expression of Cx43. METHODS: The expression and the presence of Cx43 protein were detected by means of immunohistochemistry and Western blot analysis using a polyclonal rabbit anti-Cx43 antibody. RESULTS: In stallions, gap junctions appeared as structures localised to cell-cell contacts between adjacent cells. In testes, Cx43 expression was detected in the interstitial tissue and seminiferous tubules, between Leydig and Sertoli, as well as Sertoli and germ cells. In epididymides, Cx43 was localised between epithelial cells, whereas in prostates, between secretory cells of the glandular epithelium. In the cryptorchid, a clear reduction of Cx43 signal was observed in all reproductive tissues. CONCLUSIONS: Coupling of Leydig cells via gap junctions may suggest that steroidogenic function of the testis is under the influence of these intercellular channels. Within seminiferous tubules, the expression was found to be stage-specific, pointing to its role in coordinating spermatogenesis. Differential distribution of Cx43 protein in the reproductive tract of normal and cryptorchid stallions indicates that expression is clearly dependent on the physiological status of the horse. POTENTIAL RELEVANCE: Detection of Cx43 expression in equine testicular, epididymal, and prostatic cells is important for a better understanding of the role of intercellular membrane channels in direct cell communication within the reproductive tract of stallions.     

Anti-oxidative/oxidative status of rat liver after ovariectomy

The lack of protective action of oestrogens which appears during menopausal period may be the reason of serious metabolic disturbances including oxidative stress. The hypothesis was stated that ovariectomy may induce the variations of antioxidant/oxidant status which can be detected in rat liver. A total of 102 healthy Wistar female rats were included in the experiment and divided into control (CON; n = 6), sham-operated (SHO; n = 48) and ovariectomized (OVX; n = 48) groups. Animals from SHO (n = 6) and OVX (n = 6) groups were killed every week during 8 weeks of experiment to detect dynamic changes in examined parameters. Anti-oxidative enzyme activities [glutathione peroxidase (GSH-Px); superoxide dismutase (SOD)] as well as total antioxidant capacity (TAC) and the intensity of lipid peroxidation, measured by the concentration of N,N,diethyl-p-phenylene diamine (DEPPD) radical, were determined in liver homogenates by the use of spectrophotometric methods. Wave-like patterns of examined parameters within 8 weeks of experiment were detected. GSH-Px activity tended to be higher in OVX animals and was significantly lower at 8th week when compared with 1st week of experiment. SOD activity was higher in SHO animals and showed significant differences between 3rd, 4th, 7th and 1st week. TAC values were significantly higher in OVX when compared with SHO groups in 2nd, 4th, 5th week and significantly lower in 3rd, 6th and 7th week of the experiment. The concentration of DEPPD radicals tended to increase in OVX group. In conclusion, ovariectomy which leads to oestrogen insufficiency is reflected as well in variation of anti-oxidative/oxidative parameters in rat liver homogenates.     

The culturable leaf mycobiome of Viscum album subsp. austriacum

In Poland's pine forests, mistletoe (Viscum album subsp. austriacum) abundance has increased over the past 20 years. This mistletoe infestation has decreased the growth and vigour of Scots pine trees. In this study, we surveyed the culturable fungi from healthy and diseased V. album subsp. austriacum leaves from two stands in Poland. In total, 63 distinct species were identified, 99.8% of which belonged to the phylum Ascomycota. The community compositions of fungi in mistletoe leaves were similar in healthy and diseased leaves as Alternaria alternata, Aureobasidium pullulans, Botryosphaeria visci, Fusarium paeoniae and Microsphaeropsis olivacea were consistently found in leaves of all symptom types. The most frequently isolated fungus from asymptomatic leaves was M. olivacea, followed by A. alternata, A. pullulans and Hypoxylon rubiginosum. In comparison, the most frequently isolated fungi from leaves with rusty-brown necrotic spots were (in decreasing order) M. olivacea, B. visci, F. paeoniae and A. alternata, while the most frequently isolated fungi from leaves with black or dark brown spots were M. olivacea, A. alternata, A. pullulans, Epicoccum layuense and F. paeoniae. This study was the first comprehensive report showing that certain fungal species may be pathogens of V. album subsp. austriacum in Poland. This study was also the first report of F. paeoniae, F. juglandicola, Diaporthe vacuae and Heterotruncatella spartii from V. album, and the first report of D. vacuae and H. spartii in Poland.     

Apoptotic cell death in the porcine endometrium during the oestrous cycle

It has been reported that apoptosis plays an essential role in controlling the physiological cell kinetics in the human and rodent endometrium but this type of death has never been studied in the porcine endometrium. The aim of this study was to investigate the apoptotic cell death in the porcine endometrium during the middle (Days 9-11) and late (Day 13) luteal phase, during the luteolysis (Day 15) and early follicular phase (Days 17-19) of the oestrous cycle. Apoptotic cells were identified by in situ DNA 3'-end labelling method. It was revealed that the greatest number of apoptotic cells in the luminal and glandular epithelium was found on Days 17-19 and on Day 15 of the oestrous cycle, respectively. In the stroma, the greatest number of these cells was found on Days 9-11. Our data have shown that in the porcine endometrium, both epithelial and stromal cells undergo apoptosis and that the number of apoptotic cells varies depending on the phase of the oestrous cycle.     

Erythrocyte osmotic fragility test as the measure of defence against free radicals in rabbits of different age

Peroxidation of the unsaturated bonds of membrane lipids increases fragility and cellular lysis of red blood cells. Erythrocyte susceptibility to the free radicals (peroxyl radicals) generated in vitro by 2,2'-azo-bis(2-amidinopropane) hydrochloride (AAPH) was evaluated and expressed as 50% maximal haemolysis time (HT50) in 3 groups of rabbits of different age. Erythrocytes of 1.5-month-old rabbits were more sensitive to free radicals than those of 3.5- and 6-month-old ones. In the three groups, significant negative correlation (r = -0.8 to -0.98) between the lipid peroxidation rate (thiobarbituric acid reactive substances; TBARS concentration) in blood plasma and the erythrocyte resistance to free radicals was found. This result suggests that the plasma antioxidant defence system is interrelated with that of the red blood cells and that the erythrocytes can be a good model for studies of oxidative stress. The simple haemolysis test reflecting the free radical defence can be useful for evaluating the antioxidant properties of various compounds.     

Cono- and seminiphagous insects of Abies alba Mill. in the Gorce National Park in Poland during 1996?–?1998

A study was conducted on the insects infesting seeds and cones of Abies alba Mill. in the Gorce National Park, in southern Poland during 1996–1998. A total of 856 cones were collected from 66 trees growing at 11 sites located in partially or strictly protected forest reserves. Of this sample, 217 cones with 54,958 seeds were dissected. This study yielded 5,816 specimens of insects, namely: seminiphagous Resseliella piceae Seitn., Megastigmus suspectus Borr. as well as conophagous Earomyia impossibile Morge, Barbara herrichiana Obr. and Dioryctria abietella (Den. et Schiff.). The most abundant species was R. piceae with 4,186 individuals. The index of cone infestation was calculated. It showed great variability between forest reserves and study-years. The numbers of insect individuals, numbers of viable and infertile seeds, and seeds damaged by M. suspectus and R. piceae, in both strict and partial reserves in the consecutive years were compared statistically. The test of significant differences between two frequencies at 0.05 level was used in statistical analysis. In both strict and partial reserves, statistically significant differences were found between studied variables.     

Studies on insects and mites causing galls on the leaves of purple willow Salix purpurea L. in the Ojców National Park in Poland

Analytical studies were conducted on the species composition of galls occurring on the leaves of Salix purpurea L. in the Ojców National Park in 1999–2000. The species composition of galls was identical in two consecutive years. Galls were caused by 5species (a total of 480galls). The most numerous species in both years was Pontania viminalis (L.). Index of occurrence frequency and Agrell's index of species co-existence have been calculated. This index reached the highest values in case of Pontania dolichura (Thoms.) and P. viminalis (L.) in 1999 as well as Phyllocolpa leucaspis (Tischb.) and P. viminalis (L.) in 2000.     

The Effect of Lecithin Supplementation on the Biochemical Profile and Morphological Changes in the Liver of Rats Fed Different Animal Fats

Sixty-four Wistar rats were divided into 8 equal groups and kept for 36 days in individual boxes. Three of the groups were given full synthetic diets containing various animal fats: beef tallow, pork fat or fish oil. A control group was fed the diets without the fat. The other four groups were fed the same diets with lecithin supplementation. At the end of experiment, sections taken from the liver were stained with haematoxylin–eosin and Sudan III to indicate fat infiltration. Liver enzyme levels, total bilirubin, albumin and two products of lipd metabolism were measured in serum from all the animals. The addition of lecithin to the diets did not influence the level of enzymes in the serum except in rats fed the diet containing beef tallow. A relationship between the type of diet, lecithin supplementation and the total cholesterol concentration in serum was observed. The concentration of HDL-cholesterol was only influenced by lecithin supplementation and that of triglycerides by the type of fat in the diet. The addition of lecithin to the diet was associated with proliferation of Kupffer cells, and an increased number of binuclear cells. Fatty degeneration of hepatocytes was less pronounced in all groups following lecithin supplementation. Lecithin supplementation of the diet did not elicit any pathological lesions and may be considered as a hepatocyte protector. This favourable effect of lecithin was most marked in the group of rats fed the fish oil.     

Role of the antioxidants in the protection against oxidative stress in cattle--nonenzymatic mechanisms (Part 2)

Antioxidative status consists of two mechanisms: nonenzymatic and enzymatic mechanisms. Nonenzymatic mechanisms are composed of antioxidants, scavengers of free radicals, transition metal ions, sequester transition metal ions, albumins, ceruloplasmin, and metallothioneins. On the other hand, enzymatic mechanisms are composed of superoxide dismutase (SOD), peroxidase, catalase and reductase. In cattle, characteristics of these mechanisms depend on the nutritional status of anti-oxidant minerals, especially copper, zinc, iron, selenium, silicon, and manganese. The nutritional status of the cattle in different regions of the world and in Poland is often characterised by the lack of these minerals; therefore, there is a great potential for changes in the activity of defence mechanisms against free radicals.