Polona Juntes

Periodontal Disease Burden and Pathological Changes in Organs of Dogs

Bacterial plaque associated periodontal disease is the most common chronic infection in man and dogs. In man, there is an association between periodontal disease and myocardial infarction and stroke, while in dogs it has also been associated with changes in internal organs. Inflamed periodontal tissues present a ‘periodontal disease burden’ to the host and the extent of this inflammatory disease burden is likely to affect the degree of associated pathological change in distant organs. This hypothesis was investigated in dogs with naturally occurring periodontal disease. Post-mortem investigations including periodontal assessment, standard necropsy, and organ histology were performed on 44 mature toy and miniature Poodles (related, periodontitis predisposed breeds) that died naturally or were euthanized based on clinical disease. Animals with gross primary organ pathology were excluded. The periodontal disease burden was estimated from the total surface area of periodontal pocket epithelium using six measurements of probing depth for each tooth and the tooth circumferences. Ordinal logistic regression (OR) analysis established that for each square centimeter of periodontal disease burden there was a 1.4-times higher likelihood of greater changes being present in the left atrio-ventricular valves (OR = 1.43), plus 1.2 and 1.4 times higher likelihood for greater liver and kidney pathology (OR = 1.21; OR = 1.42), respectively. The results show that there is a link between the estimated ‘periodontal disease burden’ resulting from plaque-bacteria associated periodontal disease and the level of internal pathology in this population, implying that periodontitis might contribute to the development of systemic pathology in dogs.

In vivo toxic and lethal cardiovascular effects of a synthetic polymeric 1,3-dodecylpyridinium salt in rodents

APS12-2 is one in a series of synthetic analogs of the polymeric alkylpyridinium salts isolated from the marine sponge Reniera sarai. As it is a potential candidate for treating non small cell lung cancer (NSCLC), we have studied its possible toxic and lethal effects in vivo. The median lethal dose (LD(50)) of APS12-2 in mice was determined to be 11.5mg/kg. Electrocardiograms, arterial blood pressure and respiratory activity were recorded under general anesthesia in untreated, pharmacologically vagotomized and artificially ventilated rats injected with APS12-2. In one group, the in vivo effects of APS12-2 were studied on nerve-evoked muscle contraction. Administration of APS12-2 at a dose of 8mg/kg caused a progressive reduction of arterial blood pressure to a mid-circulatory value, accompanied by bradycardia, myocardial ischemia, ventricular extrasystoles, and second degree atrio-ventricular block. Similar electrocardiogram and arterial blood pressure changes caused by APS12-2 (8mg/kg) were observed in animals pretreated with atropine and in artificially ventilated animals, indicating that hypoxia and cholinergic effects do not play a crucial role in the toxicity of APS12-2. Application of APS12-2 at sublethal doses (4 and 5.5mg/kg) caused a decrease of arterial blood pressure, followed by an increase slightly above control values. We found that APS12-2 causes lysis of rat erythrocytes in vitro, therefore it is reasonable to expect the same effect in vivo. Indeed, hyperkalemia was observed in the blood of experimental animals. Hyperkalemia probably plays an important role in APS12-2 cardiotoxicity since no evident changes in histopathology of the heart were found. However, acute lesions were observed in the pulmonary vessels of rats after application of 8mg/kg APS12-2. Predominant effects were dilation of interalveolar blood vessels and lysis of aggregated erythrocytes within their lumina.

Ostreolysin induces sustained contraction of porcine coronary arteries and endothelial dysfunction in middle- and large-sized vessels

Ostreolysin (Oly), a cytolytic and cardiotoxic protein from the oyster mushroom (Pleurotus ostreatus), is lethal for mice with an LD(50) of 1170 microg/kg following intravenous application. Its cardiotoxicity is associated with hyperkalemia, which is probably a consequence of potassium released from the lysed cells. Moreover, sub-micromolar concentrations of Oly induce a concentration-dependent increase in rat aortic ring tension, suggesting that ischaemia, and consequent hypoxic injury of cardiomyocytes, could also derive from vasospasm induced by this toxic protein. The purpose of the present study was to demonstrate histopathological lesions caused by Oly after parenteral application to rats, and to define the mechanisms of Oly-induced vasoconstriction using inhibitors verapamil, lanthanum chloride, and selective endothelin receptor antagonist TBC3214, which have different molecular targets, in vitro on porcine coronary artery rings. We found that Oly causes endothelial injury with perivascular oedema in the heart and lungs, as well as myocardial haemorrhages in rats. Treatment of porcine coronary artery rings with Oly causes concentration-dependent vasoconstriction and prevents endothelium-mediated relaxation. Using TBC3214 as a selective blocker of the endothelin A receptor, we showed that vasoconstriction induced by Oly was independent of endothelin release and its effects. Verapamil (1 microM) greatly reduced Oly-evoked contractions of porcine coronary artery rings, while lanthanum abolished them completely. These results provide evidence that the contraction of coronary arteries by Oly is due mainly to the increased influx of Ca(2+) from the extracellular space through voltage-dependent L-type Ca(2+) channels and cation non-selective channels. Experiments suggest that Oly damages endothelial cells both in vitro and in vivo, and probably exhibits direct contractile effects on coronary smooth muscle cells.

Inclusion body hepatitis (IBH) outbreak associated with fowl adenovirus type 8b in broilers

The causative agent of inclusion body hepatitis (IBH) was identified as fowl adenovirus (FAdV) type 8b, a member of the Fowl adenovirus E species, based on PCR results of adenoviral polymerase and the hexon gene in an outbreak of acute mortality that affected a broiler flock of 12,000 animals. In two waves of elevated mortality rate, a total of 264 chickens were found dead. Affected birds showed ruffled feathers, depression, watery droppings and limping. The most common pathological lesions seen on necropsy were pale, swollen and friable livers. On histological examination, acute hepatitis characterized by necrosis of hepatocytes, with large basophilic intranuclear inclusion bodies, were observed. In addition, infectious bursal disease virus and infectious bronchitis virus were detected in the same flock.

Subchronic liver injuries caused by microcystins.

Abstract The subchronic effects of cyanobacterial lyophilizate (CL) containing microcystins on liver were investigated in female New Zealand rabbits. Sterilised CL containing microcystins was injected i.p. Liver toxicity was assessed by histological examination of liver samples. Non-invasive magnetic resonance imaging (MRI) of liver was also performed in order to assess changes in the homogeneity of liver tissue. Subchronical intoxication with microcystins caused morphological changes of liver tissue that were also detected by use of MRI. Histological analysis showed that changes seen on MRI represent liver injury characterised with fatty infiltration and periportal fibrosis. This demonstrates that subchronic exposure to microcystins can lead to liver degeneration, which can easily be detected in vivo by use of MRI.

Enzymatic Degradation of PrPSc by a Protease Secreted from Aeropyrum pernix K1

Background An R30 fraction from the growth medium of Aeropyrum pernix was analyzed for the protease that can digest the pathological prion protein isoform (PrPSc) from different species (human, bovine, deer and mouse). Methodology/Principal Findings Degradation of the PrPSc isoform by the R30 fraction and the purified protease was evaluated using the 6H4 anti-PrP monoclonal antibody. Fragments from the N-terminal and C-terminal of PrPSc were also monitored by Western blotting using the EB8 anti-PrP monoclonal antibody, and by dot blotting using the C7/5 anti-PrP monoclonal antibody, respectively. For detection of smaller peptides from incomplete digestion of PrPSc, the EB8 monoclonal antibody was used after precipitation with sodium phosphotungstate. Characterization of the purified active protease from the R30 fraction was achieved, through purification by fast protein liquid chromatography, and identification by tandem mass spectrometry the serine metalloprotease pernisine. SDS-PAGE and zymography show the purified pernisine plus its proregion with a molecular weight of ca. 45 kDa, and the mature purified pernisine as ca. 23 kDa. The purified pernisine was active between 58°C and 99°C, and between pH 3.5 and 8.0. The temperature and pH optima of the enzymatic activity of the purified pernisine in the presence of 1 mM CaCl2 were 105°C ±0.5°C and pH 6.5±0.2, respectively. Conclusions/Significance Our study has identified and characterized pernisine as a thermostable serine metalloprotease that is secreted from A. pernix and that can digest the pathological prion protein PrPSc.

Electrochemotherapy is highly effective for the treatment of canine perianal hepatoid adenoma and epithelioma

Perianal tumors are common in older male dogs. The usefulness of electrochemotherapy in veterinary oncology has already been demonstrated by clinical studies on different malignancies in companion animals. In a prospective non-randomized study, we evaluated the effectiveness of electrochemotherapy in 5 male dogs with 26 perianal adenocarcinomas and 16 male dogs with 40 benign tumors. After premedication and under general anesthesia, the dogs were treated with intratumoral administration of a chemotherapeutic drug (cisplatin or bleomycin) and exposure of tumors to electric pulses, delivered by two different electroporation protocols (Protocol 1 or Protocol 2). At the end of the observation time (median 14 months), an objective response (OR) was obtained in 62/66 tumors (94%) with 87.9% complete responses (CR). No statistically significant difference in OR rate was observed based on histological type (p = 0.110), previous castration (p = 0.088), chemotherapeutic drug used (p = 0.657), and electroporation protocol (p = 0.337). Tumor size at the beginning of the treatment was the only parameter that influenced the treatment outcome (p = 0.04). No major local or general side-effects were noted. We can conclude that electrochemotherapy is an easy, highly effective, safe and cost-effective local approach for the treatment of primary perianal tumors of dogs, especially hepatoid adenoma and epithelioma.

Influence of conjugated linoleic acid on the porcine immune response and morbidity: a randomized controlled trial

BackgroundConjugated linoleic acid (CLA) has diverse influences on the immune response in different experimental models. In the present study we investigated the effect of CLA feeding on inflammatory and immune responses in a piglet model. We studied the duration of this effect and possible detrimental effects of CLA feeding. After 12 weeks of CLA and control supplementation and washout, animals were sacrificed and parenchymal organs were histologically examined.ResultsIn activated peripheral mononuclear cells interferon-γ was significantly (p = 0.008) lower in the CLA group by the end of the feeding period. This effect disappeared as soon as supplementation was stopped. No differences were found in the tumour necrosis factor-α, interleukin-10 production, serum immunoglobulin-G levels and fat infiltration of the liver, except that fat storage cell infiltration was significantly (p < 0.04) higher in the CLA-fed group. The effect of time for interferon-γ, interleukin-10 and immunoglobulin-G levels was statistically significant.ConclusionAt the end of the feeding period the interferon-γ response was depressed. However, the maturation of the piglet immune system in our young pig model probably outweighs the impact of CLA feeding on the immune response, even though liver fat storage cell infiltration, which plays an important role in liver regeneration, increased during CLA feeding of the piglets.

APS8 Delays Tumor Growth in Mice by Inducing Apoptosis of Lung Adenocarcinoma Cells Expressing High Number of α7 Nicotinic Receptors

The alkylpyridinium polymer APS8, a potent antagonist of α7 nicotinic acetylcholine receptors (nAChRs), selectively induces apoptosis in non-small cell lung cancer cells but not in normal lung fibroblasts. To explore the potential therapeutic value of APS8 for at least certain types of lung cancer, we determined its systemic and organ-specific toxicity in mice, evaluated its antitumor activity against adenocarcinoma xenograft models, and examined the in-vitro mechanisms of APS8 in terms of apoptosis, cytotoxicity, and viability. We also measured Ca2+ influx into cells, and evaluated the effects of APS8 on Ca2+ uptake while siRNA silencing of the gene for α7 nAChRs, CHRNA7. APS8 was not toxic to mice up to 5 mg/kg i.v., and no significant histological changes were observed in mice that survived APS8 treatment. Repetitive intratumoral injections of APS8 (4 mg/kg) significantly delayed growth of A549 cell tumors, and generally prevented regrowth of tumors, but were less effective in reducing growth of HT29 cell tumors. APS8 impaired the viability of A549 cells in a dose-dependent manner and induced apoptosis at micro molar concentrations. Nano molar APS8 caused minor cytotoxic effects, while cell lysis occurred at APS8 >3 µM. Furthermore, Ca2+ uptake was significantly reduced in APS8-treated A549 cells. Observed differences in response to APS8 can be attributed to the number of α7 nAChRs expressed in these cells, with those with more AChRs (i.e., A549 cells) being more sensitive to nAChR antagonists like APS8. We conclude that α7 nAChR antagonists like APS8 have potential to be used as therapeutics for tumors expressing large numbers of α7 nAChRs.

Prions and animal transmissible spongiform encephalopathies

Background. Transmissible spongiform encephalopathies (TSEs) or prion diseases are a unique group of neurodegenerative diseases of animals and humans, which always have a fatal outcome and are transmissible among animals of the same or different species. Scope and Approach. The aim of this work is to review some recent data about animal TSEs, with the emphasis on their causative agents and zoonotic potential, and to discuss why the surveillance and control measures over animal TSEs should remain in force. Key Findings and Conclusions. We still have incomplete knowledge of prions and prion diseases. Scrapie has been present for a very long time and controlled with varied success. Bovine spongiform encephalopathy (BSE) emerged unnoticed, and spread within a few years to epidemic proportions, entailing enormous economic consequences and public concerns. Currently, the classical BSE epidemic is under control, but atypical cases do, and probably will, persist in bovine populations. The Chronic Wasting Disease (CWD) of the cervids has been spreading in North America and has recently been detected in Europe. Preventive measures for the control of classical BSE remain in force, including the feed ban and removal of specified risk materials. However, active BSE surveillance has considerably decreased. In the absence of such preventive and control measures, atypical BSE cases in healthy slaughtered bovines might persist in the human food chain, and BSE prions might resurface. Moreover, other prion strains might emerge and spread undetected if the appropriate preventive and surveillance measures were to cease, leaving behind inestimable consequences.