Perfluorooctanoic Acid (PFOA) Induces Redox Status Disruption in Swine Granulosa Cells

Perfluoroctanoid acid (PFOA) is employed in the production and processing of several plastic materials, mainly during the production of waterproof fabrics or non-stick cookware. PFOA is identified as a "Substance of Very High Concern", as it is classified as a "PBT" (Persistent, Bioac-cumulative and Toxic substance) because of its persistence in the environment and its potential accumulation in organisms. Thus, safe levels of exposure cannot be established and PFOA emis-sions should be minimized. PFOA has been recently linked to several health concerns in humans. In particular, a disruptive effect on redox status homeostasis has been documented, with a po-tential impairment of normal reproductive function which requires an adequate oxidative balance. Therefore, the aim of the present study was to evaluate the effects of PFOA (2, 20 and 200 ng/mL) on ovarian granulosa cells, a model of reproductive cells. The obtained results revealed that PFOA stimulated cell viability (P <0.05). Regarding the effects on free radical production, both O2-, NO and H2O2 were significantly inhibited (P <0.05) while the non-enzymatic antioxidant power was not significantly modified. Collectively, present results deserve attention since free radical mol-ecules play a crucial role in ovarian follicle development leading to a successful ovulatio

The Phytoestrogen Quercetin Impairs Steroidogenesis and Angiogenesis in Swine Granulosa Cells In Vitro

Experimental evidence documents that nutritional phytoestrogens may interact with reproductive functions but the exact mechanism of action is still controversial. Since quercetin is one of the main flavonoids in livestock nutrition, we evaluated its possible effects on cultured swine granulosa cell proliferation, steroidogenesis, and redox status. Moreover, since angiogenesis is essential for follicle development, the effect of the flavonoid on Vascular Endothelial Growth Factor output by granulosa cells was also taken into account. Our data evidence that quercetin does not affect granulosa cell growth while it inhibits progesterone production and modifies estradiol 17β production in a dose-related manner. Additionally, the flavonoid interferes with the angiogenic process by inhibiting VEGF production as well as by altering redox status. Since steroidogenesis and angiogenesis are strictly involved in follicular development, these findings appear particularly relevant, pointing out a possible negative influence of quercetin on ovarian physiology. Therefore, the possible reproductive impact of the flavonoid should be carefully considered in animal nutrition

Potassium bicarbonate and D-ribose effects on A72 canine and HTB-126 human cancer cell line proliferation in vitro

<p>Abstract</p> <p>Background</p> <p>The synergic action of KHCO₃and D-ribose is tested on A72 and HTB-126 cell lines proliferation using K:D-Rib solution. Altered Na⁺/K⁺ATPase expression and activity were shown in patients with cancer. Studies in human epithelial-derived malignancies indicate that K⁺depletion also occurs, contributing to the increased intracellular Na⁺/K⁺ratio <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>. D-ribose transformed to piruvate, enters into the Krebs's cycle and has a key role on energetic metabolism. The up-regulation of glycolysis in tumor cells is already well known and it is the rationale of F¹⁸-FDG PET diagnostic technique. D-ribose is synthesized by the non-oxidative transketolase PPP reaction.</p> <p>Results</p> <p>Results with different K:D-Rib concentrations show that MTT salt interferes with K:D-Rib solution and therefore this method is not reliable. The UV/VIS measurements show that K:D-Rib solutions reduce MTT salt to formazan in absence of cells. Cell proliferation has then been evaluated analysing the digital photos of the Giemsa stained cells with MCID™ software. At 5 mM K:D-Rib concentration, the cell growth arrests between 48 h and 72 h; in fact the cell number after 48 h is around the same with respect to the control after 72 h. In case of HTB-126 human cancer cells, the growth rate was valuated counting the splitting times during 48 days: control cells were split sixteen times while 5 mM treated cells eleven times. Most relevant, the clonogenic assay shows that nine colonies are formed in the control cells while only one is formed in the 5 mM and none in 10 mM treated cells.</p> <p>Conclusions</p> <p>The K:D-Rib solution has an antioxidant behaviour also at low concentrations. Incubation with 5 mM K:D-Rib solution on A72 cells shows a cytostatic effect at 5 mM, but it needs more than 24 h of incubation time to evidence this effect on cell proliferation. At the same concentration on human HTB-126 cells, K:D-Rib solution shows a clear replication slowing but the cytostatic effect at 10 mM K:D-Rib solution only. Results on A72 cells indicate the K⁺uptake could be determinant either to arrest or to slow down cell growth.</p

Platelet-activating factor mediates CD40-dependent angiogenesis and endothelial-smooth muscle cell interaction.

The aim of the present study was to investigate whether stimulation of CD40 expressed by endothelial or smooth muscle cells triggers the synthesis of platelet-activating factor (PAF), an inflammatory mediator with angiogenic properties, and whether PAF contributes to CD40-induced neoangiogenesis. The results obtained indicate that the interaction of CD40 with soluble CD154 or with CD154 expressed on the membrane of leukocytes (CD154-transfected J558 cells) or of activated platelets, stimulated the synthesis of PAF by endothelial cells but not by smooth cells. The synthesis of PAF triggered by activated platelets was inhibited by a soluble CD40-murine Ig fusion protein that prevents the interaction between membrane CD40 and CD154. Studies with specific inhibitors and evaluation of protein phosphorylation indicated the involvement in PAF synthesis of two intracellular signaling pathways leading to cytosolic phospholipase A 2 activation: a phospholipase Cγ-protein kinase C-Raf-p42/p44-mitogen-activated protein kinase (MAPK) and a MAPK kinase-3/6-dependent activation of p38 MAPK. PAF synthesized by endothelial cells after CD40 stimulation was instrumental in the in vitro migration and vessel-like organization of endothelial cells, and in the interaction between endothelial cells and smooth muscle cells, as inferred by the inhibitory effect of two different PAF receptor antagonists, WEB2170 and CV3988. In vivo, blockade of PAF receptors prevented the angiogenic effect triggered by CD40 stimulation in a murine model of s.c. Matrigel implantation. In conclusion, these observations indicate that PAF synthesis induced by stimulation of endothelial CD40 contributes to the formation and organization of new vessels. This may be relevant in the vascular remodeling associated with tumor and inflammatory neoangiogenesis

Evaluation of triclosan effects on cultured swine luteal cells

Triclosan is a chlorinated phenolic, used in many personal and home care products for its powerful antimicrobial effect. Several studies have shown triclosan toxicity and the American Food and Drug Administration (FDA) in 2016 has limited its use. It has been recently included in endocrine-disrupting chemicals (EDCs), a list of chemicals known for their ability to interfere with hormonal signaling with particular critical effects on reproduction both in animals and humans. In order to deepen the knowledge in this specific field, the present study was undertaken to explore the effect of different concentrations of triclosan (1, 10, and 50 µM) on cultured luteal cells, isolated from swine ovaries, evaluating effects on growth Bromodeoxyuridine (BrDU) incorporation and Adenosine TriPhosphate (ATP) production, steroidogenesis (progesterone secretion) and redox status (superoxide and nitric oxide production, enzymatic and non-enzymatic scavenging activity). A biphasic effect was exerted by triclosan on P4 production. In fact, the highest concentration inhibited, while the others stimulated P4 production (p &lt; 0.05). Triclosan significantly inhibited cell proliferation, metabolic activity, and enzymatic scavenger activity (p &lt; 0.05). On the contrary, nitric oxide production was significantly increased by triclosan (p &lt; 0.01), while superoxide anion generation and non-enzymatic scavenging activity were unaffected

CD40-dependent activation of phosphatidylinositol 3-kinase/Akt pathway mediates endothelial cell survival and in vitro angiogenesis.

CD40 has been involved in tumor and inflammatory neoangiogenesis. In this study we determined that stimulation of endothelial CD40 with sCD154 induced resistance to apoptosis and in vitro vessel-like formation by human microvascular endothelial cells (HMEC). These effects were determined to be mediated by CD40-dependent signaling because they were inhibited by a soluble CD40-muIg fusion protein. Moreover, apoptosis of HMEC was associated with an impairment of Akt phosphorylation, which was restored by stimulation with sCD154. The anti-apoptotic effect as well as in vitro vessel-like formation and Akt phosphorylation were inhibited by treatment of HMEC with two unrelated pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002. CD40 stimulation induced a rapid increase in Akt enzymatic activity that was not prevented by cycloheximide, an inhibitor of protein synthesis. The enhanced Akt activity induced by stimulation of endothelial CD40 was temporarily correlated with the association of CD40 with TRAF6, c-Cbl, and the p85 subunit of PI3K. Expression of negative-dominant Akt inhibited the activation of endogenous Akt through CD40 stimulation, despite the observation that association of CD40 with TRAF6, c-Cbl, and PI3K was intact. The defective activation of Akt abrogated not only the anti-apoptotic effect of CD40 stimulation but also the proliferative response, the enhanced motility, and the in vitro formation of vessel-like tubular structures by CD40-stimulated HMEC. In conclusion, these results suggest that endothelial CD40, through activation of the PI3K/Akt signaling pathway, regulates cell survival, proliferation, migration, and vessel-like structure formation, all steps considered critical for angiogenesis

Redox Status in Canine Leishmaniasis

World Health Organization defined Leishmaniasis as ‘one of the priority attention diseases’. Aiming to clarify some aspects of its pathogenetic mechanisms, our study has been focused on the assessment of redox status in dogs, the main reservoir for Leishmania infantum. Forty-five dogs from an endemic area in southern Italy were divided into four different groups (from mild disease with negative to low positive antibody levels to very severe disease with medium to high positive antibody levels) accordingly to the LeishVet group guidelines. Their plasma and/or sera were tested for Reactive Oxygen Species (ROS), namely superoxide anion (O2-), Reactive Nitrogen Species (RNS), such as nitric oxide (NO) and hydroperoxides (ROOH), as well as the activity of the detoxifying enzyme superoxide dismutase (SOD), and the total non-enzymatic antioxidant capacity, as determined by ferric reducing-antioxidant power (FRAP) assay, O2- generation was significantly (p &lt; 0.05) reduced in leishmaniasis affected dogs independently of the clinical stage, while NO production was stimulated (p &lt; 0.05) only in II and III stage patients. No difference could be found for the levels of hydroperoxides and SOD activity between healthy and pathological subjects. FRAP values, were lower in affected dogs but only in stage II. Taken together, although we demonstrate that several redox status parameters are altered in the plasma of dog affected by Leishmaniasis, the oxidative stress changes that are observed in this disease, are possibly mainly due to the cellular blood components i.e. neutrophils, responsible of the elimination of the parasite. Further studies are required to assess the clinical values of the collected data

Effects of orexin B on swine granulosa and endothelial cells

In addition to the well-known central modulatory role of orexins, we recently demonstrated a peripheral involvement in swine granulosa cells for orexin A and in adipose tissue for orexin B (OXB). The aim of present research was to verify immunolocalization of OXB and its potential role in modulating the main features of swine granulosa cells. In particular, we explored the effects on granulosa cell proliferation (through the incorporation of bromodeoxyuridine), cell metabolic activity (as indirect evaluation by the assessment of ATP), steroidogenic activity (by immunoenzymatic examination) and redox status (evaluating the production of superoxide anion by means of the WST test, production of nitric oxide through the use of the Griess test and the non-enzymatic reducing power by FRAP test). Our data point out that OXB does not modify granulosa cell growth, steroidogenesis and superoxide anion generation. On the contrary, the peptide stimulates (p &lt; 0.05) nitric oxide output and non-enzymatic reducing power. Since new vessel growth is crucial for ovarian follicle development, a further aim of this study was to explore the expression of prepro-orexin and the effects of OXB on swine aortic endothelial cells. We found that the peptide is ineffective in modulating cell growth, while it inhibits redox status parameters. In addition, we demonstrated a stimulatory effect on angiogenesis evaluated in fibrin gel angiogenesis assay. Taken together, OXB appears to be potentially involved in the modulation of redox status in granulosa and endothelial cells and we could argue an involvement of the peptide in the follicular angiogenic event

Evaluation of Oxidative Stress Parameters in Healthy Saddle Horses in Relation to Housing Conditions, Presence of Stereotypies, Age, Sex and Breed

Oxidative stress plays an important role in the development of many horse diseases and it has been shown that housing has important implications for the psychophysical well-being of horses. The aim of this study is to determine if there are any dierences between the redox status in horses in relation to housing conditions. The four housing conditions analyzed were: single box, without external access and without contact (Cat A), single box with external access and possibility of partial contact (Cat B), group housing with box and large paddock (Cat C), pasture with more than 7 horses and the possibility of green forage for the whole year (Cat D). A group of 117 healthy horses were selected in several private stables in Northern Italy. All subjects treated with any type of drug were excluded. At the end of the enrollment, the 117 selected horses were divided into the four housing categories. Stereotypies were highest in the group of horses in single box, without external access and without contact (Cat A). Oxidative stress was evaluated by testing plasma or serum samples for the following parameters: superoxide anion (WST), nitric oxide (NO), reactive oxygen species (d-ROMs), ferric reducing ability of plasma (FRAP), and the activity of superoxide dismutase (SOD). Simultaneously with the blood sampling, the owners completed a questionnaire with all the management aspects of the horse (signaling, feeding, equestrian activity, vaccinations, foot management etc.). The statistical evaluation was carried out based on the categories previously described, on the presence and absence of stereotypies and on some signaling data obtained from the questionnaire. There were no significant dierences in the parameters analyzed between the categories. No significant redox status dierences were detected based on the presence or absence of stereotypies. Interestingly, when the age was introduced as selection (&lt;14 and &gt;14 years old) parameter inside the categories, statistical significance was observed for some of the stress markers considered. Finally, independently of the housing conditions, the horses of the most two represented breeds exhibited dierent values of FRAP. All these aspects are commented in the discussion

CyTest – An Innovative Open-source Platform for Training and Testing in Cythopathology

Abstract This paper describes an e-learning platform developed in the context of the European Project CyTest (2014-1-IT01-KA202-002607), dedicated to Cytological Training at European Standard through Telepathology. The main, and novel, feature of our system is the deep integration between virtual microscopy and the training system: images are not simply there to be seen but they are active parts of testing, supporting quantitative measurement of image comprehension, for instance by evaluating the identification of relevant cellular structures by the position of markers put by the student on the image. The solution we developed offers a complete tool for easy creation and interactive access to questions related to images and fully integrates the components of virtual microscopy and teaching, based on state-of-the-art instruments for digital pathology images management, as OMERO, and for training course distribution, as Moodle. The system can be easily extended to support histopathological diagnosis. The software is distributed as Open Source and available on GitHub