Serum oxidant-antioxidant status and butyrylcholinesterase activity in dogs with idiopathic epilepsy - A pilot study.

Oxidative stress plays an important role in pathogenesis of idiopathic epilepsy (IE). Although IE is the most common neurological condition, oxidant-antioxidant status in epileptic dogs is still unknown. The aim of this study is to evaluate the serum oxidant-antioxidant status in dogs with newly diagnosed IE. The status in 15 dogs with IE and 15 healthy dogs is estimated through spectrophotometric determination of two oxidant markers: advanced oxidation protein products-albumin index (AOPP) and thiobarbituric acid reactive substances (TBARS); and three antioxidant markers: total thiols (R-SH) level, glutathione (GSH) level, and paraoxonase-1 (PON-1) activity. Also, butyrylcholinesterase (BChE) activity is assessed in both groups of dogs. Higher AOPP is observed in the dogs with newly diagnosed IE, while TBARS level shows no difference when compared to the healthy dogs. In contrast, lower levels of antioxidants (R-SH, GSH, and PON-1) and BChE activity are found in the dogs with IE. No significant differences are observed in the oxidant and antioxidant markers and BChE activity across the investigated IE cases with focal and generalized seizures. Our findings provide evidence that dogs with IE are characterized by an impaired serum oxidant-antioxidant balance and lower BChE activity, which may contribute to a better understanding of IE pathogenesis.

Seasonal differences in the intensity of acute phase response in dogs infected with Babesia canis.

The highest number of acute Babesia canis cases in dogs is recorded over the February-May (Feb-May) period, which also represents the optimal climate conditions for tick activity in Belgrade, Serbia. A possibility that the acute phase response is more intense in dogs developing the disease in the Feb-May period compared with the response in other time periods of the year was tested. A total of 63 client-owned dogs with acute B. canis infection were enrolled and the routine hematology and biochemistry parameters-serum amyloid A (SAA), IgG against B. canis, level of parasitemia, ceruloplasmin (CER), paraoxonase-1 (PON-1), and fibrinogen-were measured. Acute phase indexes (API) were calculated as (SAA×CER)/(Iron×PON-1) and (SAA×CER)/(Albumin×Iron). Statistics included Kruskal-Wallis test and logistic regression analysis. The results showed that in the Feb-May period, the following parameters were lower: creatinine, albumin, iron, and level of parasitemia. Furthermore, increased API values were more probable in the Feb-May than in the other periods. Together, higher acute phase response intensity and presumptive hemodilution in the Feb-May period indicate a more acute course of B. canis infection than in other time periods of the year.

Evidence of acute phase reaction in asymptomatic dogs naturally infected with Babesia canis.

Asymptomatic outdoor dogs can be carriers of Babesia canis, but data describing the development of an acute phase response (APR) are not available. We hypothesised that these dogs have a moderate APR that could be detected by hematological and biochemical changes. Two groups of Babesia-exposed dogs were represented by nine B. canis PCR-positive and twenty B. canis PCR-negative, seroreactive dogs. The control group consisted of ten Babesia-naïve dogs. Serum amyloid A (SAA), paraoxonase-1 (PON-1), complete blood count, and biochemistry parameters were analysed by standard methodologies. Protein and lipoprotein fractions were separated using agarose gel electrophoresis (GE), and the dominant diameters of lipoproteins were assessed on gradient GE. Results were evaluated using non-parametric tests and the Receiver Operating Characteristic curve. SAA (median 39.0 µg/mL, range 2.2-48.8 µg/mL), total protein (median 74.7 g/L, range 57.1-98.3 g/L) and the dominant diameter of α-lipoproteins (median 13.31 nm, range 12.09-14.17 nm) in B. canis PCR-positive dogs were higher relative to dogs in the control group or dogs that were PCR-negative but seroreactive (p < 0.001 for both groups). Mild to moderate anemia (4/29), thrombocytopenia (7/29), and leukocyte counts that were close to the upper limit of the reference range were encountered in both Babesia-exposed groups. When compared to controls, Babesia-exposed dogs displayed decreased a PON-1 activity and protein GE pattern consistent with low-grade chronic inflammation (p < 0.001 for both groups). Dogs with detectable amounts of B. canis DNA in blood contain increased levels of SAA and total protein along with α-lipoproteins that display an increased diameter relative to those dogs with positive Babesia serology but undetectable levels of B. canis DNA in blood.

N-acetyl-L-cysteine protects dental tissue stem cells against oxidative stress in vitro.

OBJECTIVES: The aim of our study was to investigate whether N-acetyl-L-cysteine (NAC) could protect stem cells from exfoliated deciduous teeth (SHED) against oxidative damage, during in vitro cultivation, to preserve regenerative potential of these cells. Accordingly, we examined the potential of cell culture supplementation with NAC in prevention of lipid peroxidation, unfavorable changes of total lipids fatty acid composition, and the effects on the activity of antioxidant enzymes. MATERIAL AND METHODS: We analyzed the extent of oxidative damage in SHED after 48 h treatment with different NAC concentrations. Cellular lipid peroxidation was determined upon reaction with thiobarbituric acid. All enzyme activities were measured spectrophotometrically, based on published methods. Fatty acid methyl esters were analyzed by gas-liquid chromatography. RESULTS: Concentration of 0.1 mM NAC showed the most profound effects on SHED, significantly decreasing levels of lipid peroxidation in comparison to control. This dose also diminished the activities of antioxidant enzymes. Furthermore, NAC treatment significantly changed fatty acid composition of cells, reducing levels of oleic acid and monounsaturated fatty acids and increasing linoleic acid, n-6, and total polyunsaturated fatty acid (PUFA) proportions. CONCLUSION: Low dose of NAC significantly decreased lipid peroxidation and altered fatty acid composition towards increasing PUFA. The reduced oxidative damage of cellular lipids could be strongly related to improved SHED survival in vitro. CLINICAL RELEVANCE: Low doses of antioxidants, applied during stem cells culturing and maintenance, could improve cellular characteristics in vitro. This is prerequisite for successful use of stem cells in various clinical applications.

Acute-phase response in Babesia canis and Dirofilaria immitis co-infections in dogs.

Babesia canis and Dirofilaria immitis are emerging and geographically overlapping vector-borne pathogens in dogs. Infection with B. canis leads to acute-phase response (APR) that can be mild to severe and results in either non-complicated or complicated forms of the disease. The aim of this study was to determine whether acute B. canis infection is more severe in dogs with underlying asymptomatic D. immitis infection. Dogs of both sexes, different ages and breeds, with naturally occurring mono-infections with B. canis (n=13) and D. immitis (n=18) and co-infected dogs (n=7) were enrolled as well as healthy controls (n=15). Routine haematology and biochemistry, agarose gel electrophoresis (agEF) protein fraction separation and enzyme-linked immunosorbent assay (ELISA) for serum amyloid A (SAA) were performed. Based on clinical and laboratory findings, sepsis was diagnosed in the majority of dogs with acute B. canis infection with or without underlying asymptomatic D. immitis infection. Overall, haematology, biochemistry and agEF pattern changes were induced and dictated by acute B. canis infection whether or not the dogs had an asymptomatic D. immitis infection. D. immitis infection slightly influenced the level of anaemia, slightly aggravated the level of dehydration and increased the concentration of γ-globulins in acute-phase B. canis infection. D. immitis infection prevented B. canis-induced leukopenia. SAA equally increased in dogs with acute B. canis infection with or without underlying D. immitis infection. The level of SAA was not changed in dogs with asymptomatic D. immitis when compared to the controls. In conclusion, asymptomatic D. immitis infection does not influence overall APR after acute B. canis infection.

Combined effects of electromagnetic field and low-level laser increase proliferation and alter the morphology of human adipose tissue-derived mesenchymal stem cells.

In recent years, electromagnetic field (EMF) and low-level laser (LLL) have been found to affect various biological processes, the growth and proliferation of cells, and especially that of stem cells. The aim of this study was to investigate the effects of EMF and LLL on proliferation of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and thus to examine the impact of these therapeutic physical modalities on stem cell engraftment. hAT-MSCs were isolated from subcutaneous adipose tissue of six persons ranging in age from 21 to 56 years. EMF was applied for a period of 7 days, once a day for 30 min, via a magnetic cushion surface at a frequency of 50 Hz and an intensity of 3 mT. LLL was applied also for 7 days, once a day for 5 min, at radiation energies of 3 J/cm2, with a wavelength of 808 nm, power output of 200 mW, and power density of 0.2 W/cm2. Nonexposed cells (control) were cultivated under the same culture conditions. Seven days after treatment, the cells were examined for cell viability, proliferation, and morphology. We found that after 7 days, the number of EMF-treated hAT-MSCs was significantly higher than the number of the untreated cells, LLL-treated hAT-MSCs were more numerous than EMF-treated cells, and hAT-MSCs that were treated with the combination of EMF and LLL were the most numerous. EMF and/or LLL treatment did not significantly affect hAT-MSC viability by itself. Changes in cell morphology were also observed, in terms of an increase in cell surface area and fractal dimension in hAT-MSCs treated with EMF and the combination of EMF and LLL. In conclusion, EMF and/or LLL treatment accelerated the proliferation of hAT-MSCs without compromising their viability, and therefore, they may be used in stem cell tissue engineering.

Characterization of deciduous teeth stem cells isolated from crown dental pulp.

BACKGROUND/AIM: The last decade has been profoundly marked by persistent attempts to use ex vivo expanded and manipulated mesenchymal stem cells (MSCs), as a tool in different types of regenerative therapy. In the present study we described immunophenotype and the proliferative and differentiation potential of cells isolated from pulp remnants of exfoliated deciduous teeth in the final phase of root resorption. METHODS: The initial adherent cell population from five donors was obtained by the outgrowth method. Colony forming unit-fibroblast (CFU-F) assay was performed in passage one. Cell expansion was performed until passage three and all tests were done until passage eight. Cells were labeled for early mesenchymal stem cells markers and analysis have been done using flow cytometry. The proliferative potential was assessed by cell counting in defined time points and population doubling time was calculated. Commercial media were used to induce osteoblastic, chondrogenic and adipogenic differentiation. Cytology and histology methods were used for analysis of differentiated cell morphology and extracellular matrix characteristics. RESULTS: According to immunophenotype analyses all undifferentiated cells were positive for the mesenchymal stem cell markers: CD29 and CD73. Some cells expressed CD146 and CD106. The hematopoietic cell marker, CD34, was not detected. In passage one, incidence of CFU-F was 4.7 +/- 0.5/100. Population doubling time did not change significantly during cell subcultivation and was in average 25 h. After induction of differentiation, the multicolony derived cell population had a tri-lineage differentiation potential, since mineralized matrix, cartilage-like tissue and adipocytes were successfully formed after three weeks of incubation. CONCLUSION: Altogether, these data suggest that remnants of deciduous teeth dental pulp contained cell populations with mesenchymal stem cell-like features, with a high proliferation and tri-lineage differentiation potential and that these cultures are suitable for further in vitro evaluation of cell based therapies.