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Daniele Maggioni,1 Annamaria Cimicata,1 Antonella Praticò,1 Roberta Villa,1 Ferdinando Marco Bianchi,1,2 Silvia Busoli Badiale,1 Claudio Angelinetta1 1Bio Basic Europe Srl, Milan, Italy; 2Dermo-Cosmetic and Medical R&D Center of Bio Basic Europe Srl, Milan, ItalyCorrespondence: Annamaria Cimicata Tel +39 02 4155729Fax +39 0242174243Email annamariacimicata@libero.itIntroduction: Rosacea is a chronic multifactorial skin disorder mainly affecting facial skin with an estimated prevalence of about 5% worldwide. Its main symptoms, occurring early during pathology development, are skin dehydration, redness, erythema, and telangiectasia. Given the lack of a resolutive cure, therapeutic approaches able to relieve the main symptoms are needed.Purpose: The aim of this research article is to evaluate the beneficial effect of a topical product (Serum BK46) on rosacea symptoms.Patients and Methods: A monocentric single-arm, non-blinded study was performed to assess the clinical effect of Serum BK46 in relieving the main symptoms of rosacea: skin dryness, increased trans epidermal water loss (TEWL), redness, and abnormal vascularization. Twenty patients with mild to moderate rosacea were enrolled in the study and asked to apply the product twice per day for 56 days. Skin moisturization, TEWL, and erythema index were instrumentally assessed at baseline and following 24 h and 14, 28 and 56 days of treatment. Clinical parameters, including redness and telangiectasia imperfection visibility, were evaluated on a 5-point scale by a specialized dermatologist at baseline and after 14, 28, and 56 days of treatment. Finally, the visibility of vessel diameter was evaluated at baseline and after 28 and 56 days of treatment.Results: Serum BK46 application restored skin hydration and prevented the loss of water by the skin. Long-term treatment with Serum BK46 significantly reduced skin redness, erythema index, and the visibility of telangiectasia imperfectio

Cancer is a leading cause of death worldwide. Despite therapeutic improvements, some cancers are still untreatable. Recently there has been an increasing interest in the use of natural substances for cancer prevention and treatment. Hibiscus sabdariffa (HS) is a plant, belonging to Malvaceae family, widespread in South Asia and Central Africa. HS extract (HSE) used in folk medicine, gained researchers' interest thanks to its antioxidant, anti-inflammatory, and chemopreventive properties. In the present study, we initially assessed HSE effect on a panel of human tumor cell lines. Then we focused our study on the following that are most sensitive to HSE action cell lines: Multiple Myeloma (MM) cells (RPMI 8226) and Oral Squamous Cell Carcinoma (OSCC) cells (SCC-25). In both RPMI 8226 and SCC-25 cells, HSE impaired cell growth, exerted a reversible cytostatic effect, and reduced cell motility and invasiveness. We evaluated the involvement of MAPKs ERK1/2 and p38 in HSE effects by using specific inhibitors, U0126 and SB203580, respectively. For both SCC-25 and RPMI 8226, HSE cytostatic effect depends on p38 activation, whereas ERK1/2 modulation is crucial for cell motility and invasiveness. Our results suggest that HSE may be a potential therapeutic agent against MM and OSCC.

Neurobasal medium (NBM) is a widely used medium for neuronal cultures, originally formulated to support survival of rat hippocampal neurons, but then optimized for several other neuronal subtypes. In the present study, the toxic effect of NBM on long-term cortical neuron cultures has been reported and investigated. A significant neuronal cell loss was observed 24 h after the total medium change performed at days in vitro 10. The neurotoxic effect was specifically because of NBM-A, a commercially derived modification of classic NBM, as neurons exposed to minimum essential medium for 24 h did not show the same mortality rate. We showed that the toxic effect was mediated by the N-methyl-d-aspartate receptor (NMDAr) as its inactivation partly prevented NBM-induced neuronal loss, and the addition of NMDAr activators, such as l-cysteine or glycine to minimum essential medium, reproduced the same toxicity rate observed in NBM. Besides the toxicity associated with NMDAr activation, the decreased antioxidative defenses also worsen (because of glutathione depletion) neuronal death, thus amplifying the effect of excitotoxic amino acids. Indeed, glutathione supplementation by the addition of its precursor N-acetyl-cysteine resulted in an increase in neuronal survival that partially prevented NBM-A toxicity. These results evidenced, on the one hand, the unsuitability of NBM-A for long-term neuronal culture, and on the other, they highlight the importance of selection of more suitable culture conditions.

Oral cancer, representing all the malignancies arising in the oral cavity, is the eighth most diffused neoplasm worldwide. Despite therapeutic improvements, its survival rate has not changed significantly over the past few decades, with a 5-year survival rate slightly above 50%. In this context, a search for new therapeutic strategies is mandatory. Flavonoids, polyphenolic compounds derived from plants, have a broad spectrum of biological activities, including antioxidant and anticancer. They have been proved to counteract the growth of several types of cancer through multiple mechanisms including the inhibition of cell cycle progression, apoptosis induction, and the modulation of intracellular pathways. Because of their multiple biological activities and their safe toxicological profile, flavonoids have been studied widely in the last decade as potential leads for anticancer therapy. Several studies have reported different flavonoid effects according to cancer cell type. In the present review, therefore, we have evaluated the data available on the effect of flavonoids on oral cancer, with the aim of identifying the molecular mechanisms underlying their potential anticancer properties.

Cholangiocarcinoma (CCA) is the second most frequent primary liver neoplasia. It mainly arises from the malignant transformation of biliary epithelial cells, although it might originate from either hepatic progenitor cells at the Hering canals or transformed hepatocytes. CCA is a highly aggressive tumor with extremely poor prognosis and limited therapeutic approaches. Baicalin (BA) is one of the main bioactive flavonoids identified in the Scutellaria Baicalensis Georgi root dried extract which is extensively used in the Chinese tra-ditional medicine. Together with the anti-inflammatory effect, the anti-neoplastic action is the most relevant BA property demonstrated on cancer cells of different origin. Being aware of the need of new therapeutic weapons for CCA treatment, we in-vestigated whether Baicalin could exert anti-proliferative and anti-migratory effect on EGI-1 cells, a highly metastatic CCA cell line derived from bile duct carcinoma. We first tested different BA concentrations (from 5 to 200µM) in limiting EGI-1 via-bility using MTT assay. After 24h and 48h treatment, 5 and 10µM BA had no effect while rising from 25µM to 200µM (i.e. 25,50,100 and 200µM) BA exerted a significant cell viability reduction already at 24h and increased after 48h BA expo-sure. This reduction well correlated with the adherent absolute cell number de-crease and it cannot be due to BA induced cell cycle impairment after neither 24 nor 48h treatment. We also evaluated the anti-migratory BA potential by a wound healing assay adding different BA concentrations (5, 25, 50,100 and 200µM) to the culture medium immediately after performing a wound on confluent cell cultures. All BA concen-trations but 5µM induced a significant reduction in the EGI-1 migration rate after 24h treatment. Moreover 25, 50 and 10µM BA showed similar migration inhibition extent at 24 and 48h whilst 200µM BA exerted a stronger inhibitory effect already after 24h exposure which increased with time in a significant w

In this study the potential anticancer effect of 2 flavonoids, myiricetin (MYR) and naringenin (NAR) has been evaluated on an oral squamous cell carcinoma (OSCC) cell line, SCC-25, and HaCaT cells. Both the flavonoids inhibited SCC-25 cell growth, although NAR selectively affected cancer cells without impairing HaCaT cell growth. The cell proliferation inhibition by MYR and NAR was not related to apoptosis induction, but on cell cycle impairment, because a G0/G1 and a G2/M blockage was highlighted following 24 h of treatment in SCC-25 and HaCaT cells, respectively. Western blot analysis showed that MYR induced a decrease of Cyclin D1 in SCC-25 and of Cyclin B1b in HaCaT cells, while NAR negatively modulated Cyclin D1 expression in SCC-25 cells. Wound-healing and cell invasion assays demonstrated that both the flavonoids were able to reduce motility on both SCC-25 and HaCaT cells. In conclusion the results of the present study show the anticancer potential of NAR and MYR on OSCC because they exert cytostatic effect by the impairment of cell cycle progression. Moreover both the flavonoids inhibit cell migration, thus highlighting their potential effect as antimetastatic agents. Therefore, MYR and NAR appear as promising candidate as oral cancer chemopreventive agents.

In the present study, we investigated the effect of apigenin, a flavonoid widely present in fruits and vegetables, on a tongue oral cancer-derived cell line (SCC-25) and on a keratinocyte cell line (HaCaT), with the aim of unveiling its antiproliferative mechanisms. The effect of apigenin on cell growth was evaluated by MTT assay, while apoptosis was investigated by phosphatidyl serine membrane translocation and cell cycle distribution by propidium iodide DNA staining through flow cytometry. In addition the expression of cyclins and cyclin-dependent kinases was evaluated by western blotting. A reduction of apigenin-induced cell growth was found in both cell lines, although SCC-25 cells were significantly more sensitive than the immortalized keratinocytes, HaCaT. Moreover, apigenin induced apoptosis and modulated the cell cycle in SCC-25 cells. Apigenin treatment resulted in cell cycle arrest at both G0/G1 and G2/M checkpoints, while western blot analysis revealed the decreased expression of cyclin D1 and E, and inactivation of CDK1 upon apigenin treatment. These results demonstrate the anticancer potential of apigenin in an oral squamous cell carcinoma cell line, suggesting that it may be a very promising chemopreventive agent due to its cancer cell cytotoxic activity and its ability to act as a cell cycle modulating agent at multiple levels.

Epidemiological data consistently demonstrate a reduced cancer risk associated with a polyphenols rich diet. Hibiscus sabdarifa (HS), a polyphenols rich plant widely consumed worldwide as beverage and used in folk medicine, has recently gained interest thanks to its antioxidant, anti-inflammatory and chemopreventive properties. In the present study we investigated the antitumoral potential of HS extract in two different human tumor cell lines: Multiple Myeloma cells (RPMI 8226) and Oral Squamous Cell Carcinoma cells (SCC-25). MTT assays showed that HS extract induced a dose-dependent viability re-duction in both the cells lines. For the subsequent experiments we used HS at the concentration of 5 mg/ml that was the most effective in inducing cell viability reduction after 48h of treatment. Viable cell count using trypan blue staining demonstrated that the HS extract induced decrease in cell growth of both the cell lines and this was due to a reversible cytostatic rather than a cytotoxic effect. Wound-healing and cell invasion assays, respectively performed by a scratch of cell monolayer and Boyden Chamber transwell test, demonstrated that HS ex-tract was able to reduce motility and invasiveness in both RPMI 8226 and SCC-25 cells. The chemical inhibition of ERK1/ERK2 and PI3K, with U0126 and wortmannin re-spectively, reduces proliferation and migration of both SSC-25 and RPMI cells and HB extract treatment played an additive action with the inhibitors. In conclusion, our results suggest that HS extract have antitumoral properties, since it proved to inhibit tumoral cell growth and cell migration and invasiveness. It is interesting to note that HS extract is effective against two very different tumor cell lines. In fact, RPMI 8226 cells are of hematopoietic origin and grow in suspension, whereas SCC-25 cells derive from epithelium and are characterized by adherent cell growth. Therefore, although further studies are needed to clarify the molecular mechanisms involved i

Oral Cancer (OC) is one of the most frequent cancer in Head and Neck district and Oral Squamous Cell Carcinoma (OSCC) constitutes the large majority of the neoplasia arising in oral cavity. OSCC remains a hampering matters for clinics, since the overall disease free survival has not significantly increased during the last decades and invasion to surrounding tissue and to regional lymph nodes is often reported. Therefore new strategies to prevent and inhibit OSCC growth and invasion are highly desirable and new therapeutic approaches are currently tempted also with the use of natural compounds. Myricetin (MYR) and Naringenin (NAR), two naturally occurring flavonoids, widely diffused in plants, fruits and vegetable, have recently gained consideration thanks to their anti oxidant, anti inflammatory and anti tumoral properties. In this study their potential anticancer effect has been evaluated on an OSCC cell line, SCC-25 and on spontaneously immortalized non tumoral keratinocytes, HaCaT cells. MYR and NAR induce a significant cell growth inhibition in SCC-25 cells, in addition NAR selectively affected cancer cells, since it does not impair HaCaT cell growth. Furthermore an additive effect of MYR and NAR has been highlighted. The cell proliferation inhibition is not related to apoptosis induction, as demonstrated by evaluation of phosphatidyl serine membrane translocation and dapi staining. On the contrary MYR and NAR effect depends on the cell cycle progression impairment. Wound-healing and cell invasion assays, respectively performed by cell monolayer scratch and Boyden Chamber transwell test, demonstrate that the two flavonoids are able to reduce motility and invasiveness on both SCC-25 and HaCaT cells. In conclusion the results of the present study show the anticancer potential of NAR and MYR on OSCC, since both flavonoids prevent cancer cell proliferation through a cytostatic effect, by the impairment of cell cycle progression. Moreover both the flavonoids inhibit

Oxygen and glucose deprivation (OGD) due to ischemic events or trauma in the brain result in neuronal loss. The therapeutic approaches available for the treatment of these conditions are not and often the outcome is unfavorable for the patient or at least unpredictable. Stem cells could be useful for the treatment of OGD injured-neurons. Mesenchymal Stem Cells (MSCs), isolated from bone marrow as well as from various tissues, have poor immunogenicity and neuroprotective properties being able to alleviate ischemic brain injuries in animal models. The Endothelial Progenitor Cells (EPCs) are present at low frequencies both in the bone marrow and in the peripheral blood and can be mobilized by the administration of drugs such as statins. They are thought to play a role in the recovery of cerebrovasculature integrity after stroke. In the present study we evaluated the potential neuroprotective effect of human MSC and human EPCs on rat embryonic cortical neurons injured by OGD. OGD was induced by incubating the cortical neurons in a hypoxia chamber in a 95% N2 + 5% CO2 atmosphere at 37°C without glucose for periods ranging from 30 minutes and 6 hours. When the neurons were returned in normoxic atmosphere they were 1) co-cultured with either MSCs or EPCs seeded on a cell culture avoiding neurons and MSCs or EPCs direct contact while sharing the same medium, or 2) cultured in a medium previously conditioned by either MSCs or EPCs. Neuronal survival was evaluated by MTT assay and viable cellular counting. Also neuronal morphology was taken into account to evaluate the potential MSCs and EPCs neuroprotective effect. Both MSCs and EPCs increased neuronal survival after ODG. This effect was observed in absence of a direct contact between MSCs or EPCs and the injured neurons, suggesting that the release of soluble factors may be the main mechanism of action. In conclusion both MSCs and EPCs could represent a potential therapeutic approach for the treatment of brain ischemic inju

Over the last few years the therapeutic approach to demyelinating diseases has radically changed, strategies having been developed aimed at partnering the classic symptomatic treatments with the most advanced regenerative medicine tools. At first, the transplantation of myelinogenic cells, Schwann cells or oligodendrocytes was suggested, but the considerable technical difficulties, (poor availability, difficulties in harvesting and culturing, and the problem of rejection in the event of non-autologous sources), shifted attention towards more versatile cellular types, such as Mesenchymal Stem Cells (MSCs). Recent studies have already demonstrate both in vitro and in vivo that glially-primed MSCs (through exposure to chemical cocktails) have myelogenic abilities. In spite of a large number of papers on glially-differentiated MSCs, little is known about the ability of undifferentiated MSCs to myelinate axons and processes. Here we have demonstrated that also undifferentiated MSCs have the ability to myelinate, since they induce the myelination of rat DRG neuron processes after direct co-culturing. In this process a pivotal role is performed by the p75 receptor. © 2013 Elsevier Inc.

Neurotoxicity is a common dose-limiting side-effect of several drugs (Cavaletti et al., 2008). So far a validated test method to screen drugs neurotoxicity does not exist, therefore in this interdepartment study we have analyzed the effectiveness of a morphometric neurotoxicty assessment model. Drug neurotoxicity evaluation is based on embryonic rat dorsal root ganglia (DRG) organotypic culture. DRG primary sensory neurons are the principal target of drugs neurotoxic action. In fact, primary sensory neurons lie outside the blood-nerve barrier and are supplied by capillaries with fenestrated walls. Moreover, the axons of these cells are among the longest of the entire nervous system and, therefore, are more susceptible to any agent that interferes with the energy metabolism or the structural basis of axonal transport. In particular, in this interdepartment study, the interference of the under study neurotoxic compound with NGF-induced neurite elongation is analysed. The effectiveness and reproducibility of this model, even if commonly used to test drugs, has not yet been demonstrated. In order to assess the validity of this in vitro model, antineoplastic drugs known to be in clinical use and in animal models neurotoxic (paclitaxel and oxaliplatin) or not dangerous (cyclophosphamide and 5-Fluorouracil) have been tested. DRGs explanted from E15 rat embryos have been treated for 24h with drugs concentrations comparable to those achievable in vivo. The length of the longest neurite of each DRG has been measured by ImageJ program. Experiments have been performed by three different blinded researchers in two different laboratories. Mean and standard deviation of each experiment were obtained, subsequently the mean value and standard deviation of the three independent experiments for each researcher were calculated. Data obtained by the three researchers in two different laboratories resulted statistically comparable and no significant differences were detected (One Way Ano

The induction of a peripheral neuropathy is a very common side effect of many chemotherapeutic agents, including platinum compounds, and it often represents the dose limiting factor for drug clinical use. Several strategies have been suggested to reduce drug neurotoxicity without affecting the antineoplastic potential, but up to now results were not encouraging. Recently, it has been demonstrated that Mesenchymal Stem Cells (MSCs) are able to promote the survival and the maturation of untreated sensory neurons of dorsal root ganglia (DRG), which represent also the target of drug neurotoxicity. Aim of this work is to verify the neuroprotective potential of MSCs on rat DRG exposed to cisplatin (CDDP), a chemotherapeutic and neurotoxic agent. DRG post-mitotic explants from E15 rat embryos were exposed for 24 hours to different cisplatin concentrations. After 24 hours, medium was changed and DRG were directly co-cultured with human MSCs (hMSCs) or with hMSCs conditioned medium (hMSC-CM). DRG explants were photographed every day up to 1 month, and the longest neurite of each DRG was measured to evaluate neurotoxicity. DRG survival was estimated by measuring the death area percentage. The survival of CDDP-treated DRG was increased after the co-cultures with hMSCs, and both hMSCs and hMSC-CM were able to improve the neurite outgrowth of untreated and CDDP-treated DRG after 48 hours. This MSC-dependent increase of neurite length was however no longer evident at later times (1 month). This effect on neurite elongation was probably mediated by CSPG, MAG and Nogo, some proteins involved in the modulation of neurite elongation, which resulted expressed and released in the culture medium of hMSCS. Our results demonstrated a neuroprotective effect of hMSCs on CDDP toxicity and evidenced the ability of these cells to modulate neurite elongation. In this way MSCs could represent a possible mean to limit the neurotoxicity on DRG which arises after cisplatin therapy.

Mesenchymal Stem Cells (MSCs) are frequently proposed as potentially suitable for the regenerative therapeutic approach for several neurological diseases both of the central nervous system, such as Multiple Sclerosis, and at the same way also of the peripheral nervous system, thanks to their ability to increase neuronal survival and to release neurotrophic factors. Since there are great differences between neurons of the central and of the peripheral nervous system, in this study we have verified the existence of a different susceptibility of cortical and sensory neurons to the effect of MSCs after different toxic stimuli, in order to mimic the damages observed in some neurological diseases. For this aim we set up direct and indirect co-cultures of MSCs and cortical or sensory neurons previousl exposed to toxic doses of glutamate, as a paradigm of Multiple Sclerosis, or treated with two widely used chemotherapeutic drugs, cisplatin and paclitaxel, which induce peripheral neuropathies. On the same cells we evaluated also the effect of conditioned medium of MSCs, by using morphological and molecular analysis. Neuronal viability was assessed by MTT test and by count of viable cells. Our results demonstrated the protective action of MSC direct and indirect co-cultures only on sensory neurons previously exposed to the toxic agents, while conditioned medium was ineffective to rescue it. On the contrary MSCs failed at all to protect cortical neurons from the drugs used, and their conditioned medium further reduces neuronal viability. We are now investigating the putative interference of MSCs with apoptotic molecules in sensory neurons, while in cortical neurons we are evaluating the possible causes of MSC-medium toxicity by analyzing the factors released. The different effect of MSCs on cortical and sensory neurons protection observed in vitro may be not the same in vivo, where the environments differ, anyway, it suggests to address the use of MSCs against the diseases aff

Este trabalho foi realizado para avaliar a composição química e o perfil de ácidos graxos do músculo Longissimus de bovinos de três grupos genéticos (ZEB – Zebu; LIZ – Limousin vs. Zebu e ANZ – Angus vs. Zebu) alimentados com silagem de sorgo + 1,0% do peso vivo de concentrado ou cana-de-açúcar + 1,2% do peso vivo de concentrado, e abatidos em dois graus de acabamento (3,4 ou 4,8 mm de espessura de gordura de cobertura). Foram utilizados 36 bovinos com idade de 21 meses e peso médio inicial de 330 ± 44 kg terminados em confinamento após período de 115 ou 147 dias de alimentação. Não foram observadas diferenças (P > 0,05) nos teores de umidade (74,09%), cinzas (0,99%), proteína bruta (21,61%), lipídios totais (1,69%) e colesterol total (37,83 mg/100g de músculo) no músculo Longissimus entre os grupos genéticos e as dietas. O músculo Longissimus de bovinos abatidos com 3,4 mm de gordura de cobertura apresentou maior (P < 0,05) teor de umidade (75,40%) e cinzas (1,03%) quando comparado aos abatidos com 4,8 mm (72,78 e 0,96%, respectivamente). Bovinos do grupo genético LIZ apresentaram maiores concentrações (P < 0,05) de ácido eicosapentaenóico (0,45%) e ômega 3 (0,98%) em relação aos ZEB (0,18 e 0,62%, respectivamente) e ANZ (0,25 e 0,70%, respectivamente). A dieta composta por cana-de-açúcar resultou em maior (P < 0,05) deposição do ácido graxo palmitoléico (3,07%) em relação à dieta composta por silagem de sorgo (2,57%). Este resultado foi obtido em função da maior atividade da enzima ?9Dessaturase C16 nos bovinos alimentados com cana de açúcar. A silagem de sorgo proporcionou aumento (P < 0,05) na deposição do ácido 22:4 n-6 (0,14%) em comparação à cana de açúcar (0,10%). O grau de acabamento das carcaças não influenciou (P > 0,05) a composição dos ácidos graxos, com exceção para a razão ômega 6/ômega 3. O abate de bovinos com 3,4 mm de gordura de cobertura proporcionou melhor (P < 0,05) razão ômega 6/ômega 3 (7,33 vs. 8,63). A composição químic, This study was carried out to evaluate the chemical composition and fatty acid profile of Longissimus muscle of cattle from three genetic groups (ZEB - Zebu, LIZ - Limousin vs. Zebu and ANZ - Angus vs. Zebu) fed sorghum silage + 1.0% of body weight of concentrate or sugar cane + 1.2% of body weight of concentrate and were slaughtered at one of two fat thickness levels (3.4 or 4.8 mm fat thickness). Were used 36 bulls with age 21 months and weighing 330 ± 44.0 kg finished in feedlot after 115 or 147 days. There were no differences in moisture content (74.09%), ash (0.99%), protein (21.61%), total lipids (1.69%) and total cholesterol (37.83 mg/100 g of muscle) among the genetic groups and diets. The Longissimus muscle of animals slaughtered at 3.4 mm of fat thickness had higher moisture content (75.40%) and ash (1.03%) when compared to slaughtered 4.8 mm (72.78 and 0.96%, respectively). Genetic groups LIZ showed higher concentrations of eicosapentaenoic acid (0.45%) and omega 3 (0.98%) compared to ZEB (0.18 and 0.62%, respectively) and ANZ (0.25 and 0.70%, respectively). The diet consists of sugar cane resulted in increased deposition of fatty acid palmitoleic (3.07%) in relation to diet composed of sorghum silage (2.57%). This result was obtained due to higher activity of the enzyme ?9desaturase C16 in animals fed with sugar cane. Sorghum silage increased the deposition of acid 22:4 n-6 (0.14%) compared to sugar cane (0.10%). The fat thickness levels of the carcasses had no effect on fatty acid composition, except for the ratio omega 6/omega 3. The slaughter of animals with 3.4 mm of fat thickness provided the best ratio omega 6/omega 3 (7.33 vs. 8.63). The chemical composition and fatty acid profile of Longissimus muscle of animals analyzed in this study show little variation in terms of genetic groups, diets and fat thickness levels.

Este trabalho foi realizado para avaliar a composição química e a composição de ácidos graxos do músculo Longissimus de 33 machos não castrados da raça Nelore com 27 meses de idade e peso médio inicial de 402 ± 7.58 kg terminados em confinamento. Foram usados três tratamentos: Controle – CON, Monensina – MON e Extrato de Própolis – PRO. Os bovinos foram mantidos confinados durante 84 dias e abatidos com peso vivo médio final de 488 ± 24.9 kg. A adição dos aditivos (monensina ou extrato de própolis) não tiveram influência (P > 0,10) na composição química do músculo Longissimus dos bovinos. A composição de ácidos graxos saturados (AGS) foi semelhante entre os tratamentos (P > 0,10). A composição de ácidos graxos monoinsaturados foi diferente entre os tratamentos (P < 0,01). Valor superior foi observado nos bovinos da dieta CON (47,0%). Da mesma forma, houve diferença (P < 0,01) entre tratamentos para os ácidos graxos poliinsaturados (AGPI). Bovinos do tratamento (9,92%) apresentaram maior (P < 0,01) valor para os AGPI. Os bovinos dos tratamentos CON (6,74%) e PRO (6,93%) apresentaram menores valores (P < 0,01) para os AGPI., This work was carried out to evaluate the chemical composition and fatty acid profile of Longissimus muscle of 33 Nellore bulls with 27 months old and initial average weight of 402 ± 7.58 kg finished in feedlot. Three treatments (Control – CON, Monensin – MON and Propolis extract – PRO) were evaluated. The animals were kept in feedlot during 84 days and slaughtered with final average weight with 488 ± 24.9 kg. The addition of additives (monensin or propolis extract) did not influence (P > 0.10) the chemical composition of Longissimus muscle of bulls. Saturated fatty acid (SFA) did not have difference (P > 0.10) among treatments. Monounsaturated fatty acids (MUFA) have difference (P < 0.01) among treatments. The highest value was observed in CON treatment (47.0%). There is also difference (P < 0.01) among treatments for polyunsaturated fatty acids (PUFA). Bulls of MON (9.92%) treatment obtained highest (P < 0.01) value for PUFA. Bulls of CON (6.74%) and PRO (6.93%) have lowest (P < 0.01) values for PUFA.

Multiple sclerosis (MS) is a chronic immuno-mediated inflammatory and demyelinating disease characterised by the presence of both demyelinating lesions and axonal degeneration, which lead to the reduction of nerve conduction velocity and the development of concomitant neurological deficits. Recently, Mesenchymal Stem Cells (MSCs) have been proposed in in vivo studies as promising therapeutic treatment for MS mainly for their capacity to modulate the immune response, moreover, during our previous experiments we found that rat undifferentiated MSC promote Dorsal Root Ganglia neurons survival and maturation. The aim of this study is to verify the potential protective effect of MSCs on an in vitro model of MS represented by rat primary cultures of cortical neurons. Since glutamate excitotoxicity is an important mechanism in neurodegenerative diseases, and it induces neuronal alterations similar to those observed in advanced MS, cortical neurons cultures were treated with different concentrations of glutamate (25, 50, 100, 200 and 500 µM) for 24 hours. After the treatment cortical neurons show a suffering appearance, with damaged axons and cellular degeneration. Neuronal viability, assessed by DAPI staining and by count of viable cells, was glutamate dose-dependent. In order to evaluate the possible positive effect of MSCs on neuronal survival, both direct and indirect co-cultures of MSCs and cortical neurons were set up, and the survival after glutamate treatment analyzed. Cortical neurons treated with glutamate were still suffering after the direct co-cultures with MSCs, while in indirect co-cultures with MSCs neurons were alive, without important signs of axonal degeneration. These preliminary findings suggest that MSCs are able to reduce the cellular death induced by glutamate exposure in cortical neurons and encouraged the further study and characterization of their positive effect

Neurotoxicity is a common dose-limiting side-effect of several drugs. So far a validated test method to screen drugs neurotoxicity does not exist, therefore in this interdepartment study we have analyzed the effectiveness of a neurotoxicty assessment model. Drug neurotoxicity evaluation in this model is based on the ability of NGF exposed embryonic rat dorsal root ganglia (DRG) organotypic culture to grow neurite; in particular the interference of the under study neurotoxic compound with neurite elongation is analysed. The effectiveness and reproducibility of this model , even if commonly used to test drugs , has not yet been demonstrated. In order to assess the validity of this in vitro model, antineoplastic drugs known to be in clinical use and in animal models neurotoxic (paclitaxel and oxaliplatin) or not dangerous (cyclophosphamide and 5-Fluorouracil) have been tested. DRGs explanted from E15 rat embryos have been treated for 24h with drugs concentrations comparable to those achievable in vivo. The length of the longest neurite of each DRG has been measured by ImageJ program. Experiments have been performed by three different blinded researchers in two different laboratories. Mean and standard deviation of each experiment were obtained, subsequently the mean value and standard deviation of the three independent experiments for each researcher were calculated. Data obtained by the three researchers in two different laboratories resulted statistically comparable and no significant differences were detected (One Way Anova analysis of variance and Tukey post test; p<0.05). This interdepartment in vitro study, therefore, indicates that the DRG organotypic culture represents a reliable model to study drug neurotoxicity. The test method permits to make prediction of neurotoxic effects on humans because the concentrations tested are the same to which DRG are exposed during clinical use. This model will allow to reduce animal testing for screening of new drugs and to de

Multiple sclerosis (MS) is a chronic immuno-mediated inflammatory and demyelinating disease characterised by both demyelinating lesions and axonal degeneration, leading to the reduction of nerve conduction velocity and the development of neurological deficits. Recently, some in vivo studies have proposed Mesenchymal Stem Cells (MSCs) as promising therapeutic treatment for MS mainly for their capacity to modulate the immune response, although it is not yet known if other mechanisms, different from immune modulation, are involved in MSCs positive. We investigate the therapeutic potential and the clinical effects of MSCs by using an animal model of multiple sclerosis, represented by Lewis rats affected by acute experimental autoimmune encephalomyelitis (EAE). MSCs were intravenously administered immediately after EAE induction (T0) or one week later (T7), in both the cases before disease onset. The clinical course of acute EAE was ameliorated only in EAE animals in which MSCs were injected one week after disease induction, while EAE rats and EAE rats with MSCs injected at T0 showed similar clinical scores. Moreover, the EAE rats which received MSCs at T7 displayed a cytokines pattern expression comparable to untreated control rats, while both EAE group and EAE+MSCs (T0) group showed an increased expression of pro-inflammatory cytokines. These results evidenced that the intravenous administration of MSCs one week after EAE induction (and before disease onset) induces the amelioration of the clinical scores of EAE-rats, thus supporting the potential role for MSCs in cell therapy in multiple sclerosis. We are now investigating the molecular mechanisms of this positive effect, focusing our attention on the Metalloproteinases pathway, involved in multiple sclerosis and modulated by MSCs.

Twenty-one crossbreeding heifers (½ Nelore vs ½ Aberdeen Angus) with about 307.90 kg of live weight and eighteen months of age were used in a complete randomized design with three treatments and seven repetitions. The treatments were: control heifers, ovariectomized animals and with fifty gunshot spheres in uterine corn. The heifers were fed with 50% of corn silage and 50% concentrated. The feed intake was about 2,5% weight live in dry matter. The heifers gunshot treatments showed was bigger weight gain than the heifers ovariectomized treatments and this more weight gains what control heifers treatments. There was not difference in carcass yield and fat covering amongst treatments. The hematocrito increased in three heifers groups in the first collection (before) for the second collection (after treatments). However, the increase was superior (P < 0,05) in the control heifers group., Foram utilizadas vinte e uma novilhas mestiças ½ Nelore vs ½ Aberdeen Angus, com idade aproximada de dezoito meses e peso médio de 307,90 kg. Os animais foram distribuídos em delineamento inteiramente casualizado, com três tratamentos e sete repetições. Os tratamentos foram compostos por: novilhas vazias, novilhas ovariectomizadas e novilhas com implante de cinqüenta esferas de chumbo esterilizadas no interior do corno uterino. As novilhas foram alimentadas com ração composta de 50% silagem de milho (MS) e 50% concentrado (MS), fornecida na base de 2,5% (MS) do peso vivo dos animais. As novilhas com implante de chumbo apresentaram ganho médio diário superior (P < 0,05) aos das novilhas ovariectomizadas e das vazias. Ainda, as novilhas ovariectomizadas apresentam ganho superior (P < 0,05) aos das novilhas vazias. Todavia, não houve diferença no que se refere ao rendimento de carcaça e cobertura de gordura. O valor do hematócrito aumentou nos três grupos de animais da primeira coleta (antes) para a segunda coleta (depois dos tratamentos). Todavia, este aumento foi maior (P < 0,05) nas novilhas vazias.

Collagen is ubiquitous in the human body and it is the main component of connective tissue. Collagen is also the principal component of endopelvic fascia and it is involved in the physiopathology of female organ prolapse (POP). The role of collagen in the physiopathology of POP has been studied with conflicting results, being difficult to distinguish the cause from the effect and to standardize the samples. The turnover of connective tissue in the vaginal wall is an important process both in healthy women and in women with prolapse. MMP-1, MMP-8 and MMP-13 specifically cleave native triple collagen (I, II, III) helix, yelding two fragments. These fragments are subject to gelatinase (MMP-2 and MMP-9) degradation. MMP-2 and MMP-9 activity, may be regulated at different levels (trascriptional and activation level) influencing the amount of collagen. Aim of this study is to find a correlation between staging of anterior vaginal wall prolapse and morphologic and biochemical (MMP-2 and MMP-9 amount) features of anterior vaginal wall. Anterior vaginal wall full thickness biopsies (eightyfive patients) including endopelvic fascia have been collected during surgical procedures. Recurrence or vault prolapse have been ruled out. POP samples have been classified according to POP-Quantification and they have been divided in three groups: group A (controls), group B (I and II stage prolapse) and group C (III and IV stage prolapse). Each sample has been processed for both microscopic and zymography analysis. Formalin fixed samples have been embedded, cut and stained with Trichromic Masson Goldner (TMG) stains. Microscopic analysis have been performed to evaluate collagen amount, collagen organization, and to assess muscular component. All parameters have been scored semi-quantitatively by two blind observers on vaginal mucosa and underlying fascia. MMP-2 and MMP-9 activity have been evaluated by gelatine zymography. Statistical evaluation have been made by One-Way ANOVA analysis o

Advances in clinical treatment of head and neck squamous cell carcinoma (HNSCC) are hampered by its high infiltrative potential leading to distal metastasis. Since their ability to degrade the basal lamina and extracellular matrix, matrix metalloproteinases (MMP) have a pivotal role in tumor invasion. The overexpression and the aberrant activity of MMPs especially of MMP2 and MMP9, during HNSCC development and progression have been reported. However, up to now little is known about the mechanism of their regulation in HNSCC. It has been demonstrated that MMP2/9 expression is negative regulated by extracellular signal regulated kinase 1 and 2 (ERK1/2) in HNSCC cell lines. ERKs are protein kinases belonging to the mitogen-activated protein kinases family, and they are involved in the regulation of different cellular aspects, from apoptosis to cell proliferation and differentiation. In the present study we evaluated MMP2 and MMP9 activity by gelatine zymography in 16 tissue samples of HNSCC and their paired normal mucosa from patients undergoing surgical treatment. Moreover, ERK1/2 activation was analyzed by immunoblotting. A statistically significant decrease in the levels of activated ERK2 in cancer specimens in comparison with paired normal tissues was observed, whereas a significant increase in the activity of MMP2 was found in cancer specimens. However, the statistical analysis failed to demonstrate a correlation between the increase in MMP2 activity and the reduction of ERK1/2 activation levels. The results obtained, therefore, rule out, for the first time in an ex vivo study, the existence of a negative correlation between ERK1/2 activation and MMP2 activity.

Chemotherapy-induced peripheral neurotoxicity (CIPN) is a side effect limiting cisplatin (CDDP) and docetaxel (DOCE) treatment. Erythropoietin (EPO) is a hematopoietic growth factor also displaying neurotrophic properties. Evidence suggests that EPO's neuroprotective action may rely on PI3K/AKT pathway activation; however, data regarding the EPO neuroprotective mechanism are still limited. This study evaluated the effect of EPO on organotypic cultures of rat dorsal root ganglia (DRG) and in primary cultures of DRG-dissociated sensory neurons exposed to CDDP- and DOCE-induced neurotoxicity, aiming to investigate EPO's neuroprotective mechanism. Subsequently, the levels of AKT expression and activation were analyzed by Western blot in neurons exposed to CDDP or DOCE; AKT's role was further evaluated by using a chemical inhibitor of AKT activation, wortmannin. In these models EPO, was protective against both CDDP- and DOCE-induced cell death and against CDDP-induced neurite elongation reduction. A modulation of AKT activation was observed in CDDP-treated neurons, and the presence of wortmannin prevented EPO's neuroprotective action against CDDP toxicity but did not have any effect on EPO's protection against DOCE-induced toxicity, thus ruling out the PI3K-AKT pathway as the mechanism of EPO's effect in neuronal death prevention after DOCE exposure. Our results confirm in vitro the effectiveness of EPO as a neuroprotectant against both CDDP- and DOCE-induced neurotoxicity. In addition, a role of PI3K/AKT in EPO's protection against CDDP, but not against DOCE, neurotoxicity was shown, suggesting that alternative pathways could be involved in EPO's neuroprotective activity. (

Chemotherapy induced Peripheral Neuropathy (CIPN) is a a well known complication limiting Cisplatin (CDDP) and Docetaxel (DOCE) chemotherapy in patients with cancer. Erythropoietin (EPO) is an heamatopoietic growth factor that has been reported to display neurothrophic properties, in particular it has proved to be neuroprotective in several rodent models of nervous system damage, including CDDP induced peripheral neuropathy. In this study we evaluated the neuroprotective ability of EPO against CDDP and DOCE induced neurotoxicity in primary cultures of sensitive neurons dissociated from Dorsal Root Ganglia (DRG) obtained from 15-day-old rat embryos, aiming to investigate the mechanism underlying EPO neuroprotective action. Briefly, primary cultures of sensitive neurons have been exposed to CDDP (25M), DOCE (100nM) or to the same doses of the chemotherapics but in the presence of EPO (10nM). In this in vitro model EPO turned out to be protective against both CDDP and DOCE induced neurotoxicity, in fact an increased neuronal survival was evident in cultures exposed to the combination treatment with CDDP plus EPO and DOCE plus EPO. Several evidences suggest that AKT pathway may play an important role in EPO neuroprotective action. Therefore we analyzed by western blot the levels of expression and activation (phosphorylation) of AKT in neurons exposed to the CDDP, DOCE or to the cotreatment with EPO. Results obtained indicated that modulation of the levels of phosphorylated AKT (pAKT) occurs following 14 hours treatment with CDDP, when a sharp decrease in the levels of activated AKT was highlighted, on the contrary DOCE induced only a sligth increase in the level of pAKT. Cotreatment with EPO was able to partially prevent CDDP induced decrease in the level of pAKT. Using a specific inhibitor of AKT activation (Wortmannin) we therefore investigate the role of AKT in EPO neuroprotection. The presence of Wortmannin (100nM) did prevent EPO neuroprotective action against CDD

BACKGROUND: Oral squamous cell carcinoma (OSCC) represents the sixth most diffused cancer in developed countries. Mitogen-activated protein kinases (MAPKs) are proteins which transduce a vast array of extracellular signals into intracellular responses. The role of MAPK signalling pathway in cancer is not completely understood. MATERIALS AND METHODS: In this study, we attempted to specifically evaluate the activation state of MAPK in OSCC. MAPK expression and activation were analyzed by immunoblotting in thirty tissue samples of OSCC and their paired nonneoplastic perilesional tissues. On the same tissues, the activation and expression of MAPK JNK/SAPK were also evaluated by ELISA assay. RESULTS: Analyzing the levels of phospho-ERK1/2(p44/p42), a statistically significant reduction was observed in tumors compared to normal tissues. No statistically significant difference between tumor and control tissue was found for p38MAPK or JNK/SAPK. CONCLUSION: These results suggest that a reduction in ERK1/2(p44/p42) phosphorylation is correlated with tumor growth in OSCC., Background: Oral squamous cell carcinoma (OSCC) represents the sixth most diffused cancer in developed countries. Mitogen-activated protein kinases (MAPKs) are proteins which transduce a vast array of extracellular signals into intracellular responses. The role of MAPK signalling pathway in cancer is not completely understood. Materials and Methods: In this study, we attempted to specifically evaluate the activation state of MAPK in OSCC. MAPK expression and activation were analyzed by immunoblotting in thirty tissue samples of OSCC and their paired nonneoplastic perilesional tissues. On the same tissues, the activation and expression of MAPK JNK/SAPK were also evaluated by ELISA assay. Results: Analyzing the levels of phospho-ERK1/2p44/p42, a statistically significant reduction was observed in tumors compared to normal tissues. No statistically significant difference between tumor and control tissue was found for p38MAPK or JNK/SAPK. Conclusion: These results suggest that a reduction in ERK1/2p44/p42 phosphorylation is correlated with tumor growth in OSCC

An unsolved question is how platinum derivatives used for solid cancer therapy cause peripheral neuropathy in patients and apoptosis in "in vitro" models of chemotherapy-induced peripheral neuropathy. DRG neurons from E15 rat embryos were treated with toxic doses of oxaliplatin or cisplatin. Here, the role of MAPKs in neuronal apoptosis was studied. Both oxaliplatin and cisplatin induced a dose-dependent neuronal apoptosis, modulated by the proteins of Bcl-2 family. Regarding MAPKs, platinum derivatives activated p38 while they reduced the active form and the total amount of JNK/Sapk. Both oxaliplatin and cisplatin activated ERKs at early stages, although they behaved differently at later stages. By using specific inhibitors of the various MAPKs it was demonstrated that the platinum-induced neuronal apoptosis is mediated by early p38 and ERK1/2 activation, while JNK/Sapk has a neuroprotective role. These results suggest a role for the different MAPKs in peripheral neuropathies characterized by apoptosis of DRG neurons.

Mitogen-activated protein kinase (MAPK) cascades transmit and amplify signals involved in cell proliferation as well as in cell death. In this study, the potential derangement of MAPK pathways has been evaluated in human squamous cell carcinomas (SCC) of the larynx. The expression and activity of the MAPK p38, ERK1/2p44/p42 and JNK/SAPKp46/p54 have been investigated by immunoblot analysis of tissue homogenates in 27 samples of primary laryngeal cancer and in 27 paired non-neoplastic laryngeal mucosa. On the same tissues, the activation of MAPK JNK/SAPKp46/p54 was also analyzed by an ELISA assay. The results obtained showed that both total and phosphorylated levels of JNK/SAPKp46/p54 and p38 were not different between tumor and normal samples. Conversely, while total protein levels for both ERK1p44 and ERK2p42 were not statistically different between tumor and normal samples, the analysis of the level of the activated forms of ERK1/2 showed a statistically significant decreased phosphorylation of both isoforms in the tumor samples compared to the control tissues. The rate of reduction was similar for both isoforms. Immunohistochemical analysis of all the activated MAPK (p38, JNK/SAPKp46/p54 and ERK1/2p44/p42) in both laryngeal SCC and normal mucosa demonstrated no difference of cellular localization. Activated ERK1/2p44/p42 and activated p38 demonstrated a nucleo-cytoplasmic distribution whereas activated JNK/SAPKp46/p54 were localized into the cytoplasmic membrane. The decreased activity of ERK1/2p44/42 in laryngeal SCC might reflect alterations in tumor suppressing activity or might derive from the interplay among various transduction pathways