Cryopreservation, early seedling development, and genetic stability of Oncidium flexuosum Sims

An efficient cryopreservation protocol was developed for mature seeds of Oncidium flexuosum Sims. Seed morphology, protocorm formation, and early seedling development were also assessed. The effects of phloroglucinol and Supercool X-1000® as cryoprotectant additives in the vitrification solution were investigated. Dehydration using the plant vitrification solution 2 (PVS2) for 60 and 120 min prior to immersion in liquid nitrogen promoted the highest frequency of in vitro seed germination 6 weeks following culture on half-strength Murashige and Skoog (1/2 MS) medium. Mature seeds submitted to vitrification for 120 min in PVS2 and 1 % phloroglucinol at 0 °C enhanced germination by 68 %, whereas in PVS2 and 1 % Supercool X-1000® germination was just moderately enhanced (26 %). In vitro-germinating seedlings developed healthy shoots and roots without the use of plant growth regulators. After 6 months of growth, there were no differences between in vitro- and ex vitro-grown seedlings for various phenotypic characteristics, including shoot length, number of leaves, number and length of roots, and fresh and dry weight. Seedlings were transferred to greenhouse conditions and successfully acclimatized, further developing into normal plants with over 90 % survival. Comparative analysis of seedlings from control and vitrified seeds using flow cytometry indicated that no change in ploidy levels occurred as a result of cryopreservation, therefore maintaining seedlings genetic stability. In this study, vitrification with PVS2 for 120 min with the addition of 1 % phloroglucinol offers a simple, safe, and feasible protocol for cryopreservation of O. flexuosum mature seeds. © 2013 Springer Science+Business Media Dordrecht.

Avaliação leucocitária de cães com linfoma submetidos ao protocolo de Madison-Wisconsin pela técnica convencional e citometria de fluxo

Lymphoma is the most common hematopoietic tumor in dogs and one of the malignant tumors with higher occurrence in this species. It is a great experimental model due to its resemblance with the non-Hodgkin lymphoma in humans. Considering the importance of the overall changes that result from to this kind of neoplastic tumor and those due to polichemotherapy this study aimed to evaluate the absolute leukon count and the total count of CD45+ cells in the blood of 25 dogs with lymphoma. Findings were crosschecked since diagnose and then once weekly during the first eight sessions of the Madison-Wisconsin chemotherapic protocol. Total granulocyte, lymphocyte and monocyte counts obtained from a conventional automatic counter and by flow cytometry were compared. Results did not reveal statistically significant changes between the two techniques.

Antioxidant effects of quercetin and naringenin are associated with impaired neutrophil microbicidal activity

Naringenin and quercetin are considered antioxidant compounds with promising activity against oxidative damage in human cells. However, no reports have described their effects on reactive oxygen species (ROS) production by phagocytes during microbicidal activity. Thus, the present study evaluated the effects of naringenin and quercetin on ROS production, specifically hypochlorous acid (HOCl), and their involvement in the microbicidal activity of neutrophils. Naringenin and quercetin inhibited HOCl production through different systems, but this inhibition was more pronounced for quercetin, even in the cell-free systems. With regard to the microbicidal activity of neutrophils, both naringenin and quercetin completely inhibited the killing of Staphylococcus aureus. Altogether, these data indicate that the decrease in the oxidant activity of neutrophils induced by these compounds directly impaired the microbicidal activity of neutrophils. Naringenin and quercetin exerted their effects by controlling the effector mechanisms of ROS production, with both positive and negative effects of these antioxidant agents in oxidative stress conditions and on ROS in the microbicidal activity of phagocytes. The present results challenge the traditional view of antioxidants as improvers of pathological conditions. © 2013 Francielli de Cássia Yukari Nishimura et al.

Improvement in clinical signs and cellular immunity of dogs with visceral leishmaniasis using the immunomodulator P-MAPA

This study investigated the immunotherapeutic potential of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride immuno-modulator (P-MAPA) on canine visceral leishmaniasis. Twenty mongrel dogs presenting clinical symptoms compatible with leishmaniasis and diagnosis confirmed by the detection of anti-leishmania antibodies were studied. Ten dogs received 15 doses of the immunomodulator (2.0mg/kg) intramuscularly, and 10 received saline as a placebo. Skin and peripheral blood samples were collected following administration of the immunomodulator. The groups were followed to observe for clinical signals of remission; parasite load in the skin biopsies using real-time PCR, the cytokines IL-2, IL-10 and IFN-γ in the supernatant of peripheral blood mononuclear cells stimulated in vitro with either total promastigote antigen or phytohemagglutinin measured by capture ELISA, and changes in CD4+ and CD8+ T cell subpopulations evaluated by flow cytometry. Comparison between the groups showed that treatment with the immunomodulator promoted improvement in clinical signs and a significant reduction in parasite load in the skin. In peripheral blood mononuclear cell cultures, supernatants showed a decrease in IL-10 levels and an increase in IL-2 and IFN-γ. An increase in CD8+ T cells was observed in peripheral blood. In addition, the in vitro leishmanicidal action of P-MAPA was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and no leishmanicidal activity was detected. These findings suggest that P-MAPA has potential as an immunotherapeutic drug in canine visceral leishmaniasis, since it assists in reestablishing partial immunocompetence of infected dogs. © 2013 Elsevier B.V.

Transdentinal cytotoxicity of experimental adhesive systems of different hydrophilicity applied to ethanol-saturated dentin

The aim of this study was to evaluate the transdentinal cytotoxicity of experimental adhesive systems (EASs) with different hydrophilicity and dentin saturation solutions on odontoblast-like cells. One hundred 0.4-mm-thick dentin discs were mounted in in vitro pulp chambers and assigned to 10 groups. MDPC-23 cells were seeded onto the pulpal side of the discs, incubated for 48 h. The EASs with increasing hydrophilicity (R1, R2, R3 and R4) were applied to the occlusal side after etching and saturation of etched dentin with water or ethanol. R0 (no adhesive) served as controls. R1 is a non-solvated hydrophobic blend, R2 is similar to a simplified etch-and-rinse adhesive system and R3 and R4 are similar to self-etching adhesives. After 24 h, cell metabolism was evaluated by MTT assay (n = 8 discs) and cell morphology was examined by SEM (n = 2 discs). Type of cell death was identified by flow cytometry and the degree of monomer conversion (%DC) was determined by infrared spectroscopy (FTIR) after 10 s or 20 s of photoactivation. Data were analyzed by the Kruskal-Wallis and Mann-Whitney tests (α = 0.05). Dentin saturation with ethanol resulted in higher necrotic cell death ratios for R2, R3 and R4 compared with water saturation, although R2 and R3 induced higher SDH production. Photoactivation for 20 s significantly improved the %DC of all EASs compared with 10 s. A significant positive correlation was observed between the degree of hydrophilicity and %DC. In conclusion, except for R1, dentin saturation with ethanol increased the cytotoxicity of EASs, as expressed by the induction of necrotic cell death. © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Toxic effects of daily applications of 10% carbamide peroxide on odontoblast-like MDPC-23 cells

Background. Tooth bleaching has been widely studied, mainly due to the possible undesirable effects that can be caused by this esthetic procedure. The cytotoxicity of the bleaching agents and its components to pulp cells has been demonstrated in several researches. The aim of this study was to evaluate the toxic effects of successive applications of 10% carbamide peroxide (CP) gel on odontoblast-like cells. Materials and methods. Enamel-dentin discs obtained from bovine incisors were adapted to artificial pulp chambers (APCs). The groups were formed as follows: G1: Without treatment (control group); G2: 10% carbamide peroxide, CP (five applications/one per day); G3: 10% CP (one unique application); and G4: 35% hydrogen peroxide, HP (three applications of 15 min each). After treatment, cell metabolism (MTT), alkaline phosphatase (ALP) activity and plasma membrane damage (flow cytometry) were analyzed. Results. Reductions in cell metabolism and alkaline phosphatase activity along with severe damage of the cytoplasmic membrane were noted in G2. In G3, no damage was observed, compared to the control group. Intermediary values of toxicity were obtained after 35% HP application. Conclusion. It can be concluded that one application of 10% CP did not cause toxic effects in odontoblast-like cells, but the successive application of this product promoted severe cytotoxic effects. The daily application of the bleaching agents, such as used in the at-home bleaching technique, can increase the damages caused by this treatment to the dental pulp cells. © 2013 Informa Healthcare.

Leishmaniasis causes oxidative stress and alteration of oxidative metabolism and viability of neutrophils in dogs

The aim of the present study was to test the hypothesis that oxidative stress and alteration of oxidative metabolism and apoptosis of neutrophils in dogs vary with the stage of leishmaniasis and to determine the contribution of uremia to such alterations. Dogs with leishmaniasis were classified into two stages: moderate (Leish II, n = 20) or very severe (i.e. with concurrent uremia; Leish IV, n = 20) according to the LeishVet Consensus. The two leishmaniasis groups were compared with uremic dogs without leishmaniasis (Uremic, n = 10) and to healthy dogs (Control, n = 30). To determine oxidative stress, total antioxidant/oxidant capacity, lipid peroxidation, total glutathione and the plasma antioxidants albumin, uric acid and bilirubin were quantified. Superoxide production was determined using the hydroethidine probe and viability and apoptosis were measured using annexin V-PE by capillary flow cytometry. Oxidative stress was present in both uremia and leishmaniasis with reduced total antioxidant capacity and was associated with increased induced production of superoxide and apoptosis. The greatest amount of oxidants was observed in animals with moderate disease only. Neutrophils from uremic dogs with and without leishmaniasis had decreased viability and an increased apoptosis rate in addition to increased lipid peroxidation. In conclusion, oxidative stress occurs in both stages of leishmaniasis with differences in intensity and levels of plasma markers; however, uremia does contribute to the decreased spontaneous viability of neutrophils in dogs in the final stage of the disease. © 2013 Elsevier Ltd. All rights reserved.

Comparison of inflammatory cytokine profiles in plasma of patients undergoing otorhinological surgery with propofol or isoflurane anesthesia

Objective and design: The effects of anesthetics on cytokine release in patients without comorbidities who undergo minor surgery are not well defined. We compared inflammatory cytokine profiles in adult patients undergoing minimally invasive surgery who received isoflurane or propofol anesthesia. Methods: Thirty-four patients without comorbidities undergoing minor surgery were randomly assigned to receive an inhaled anesthetic (isoflurane; n = 16) or an intravenous anesthetic (propofol; n = 18). Blood samples were drawn before premedication and anesthesia (T1), 120 min after anesthesia induction (T2), and on the first post-operative day (T3). Plasma concentrations of interleukins (IL-) 1β, 6, 8, 10 and 12 and tumor necrosis factor (TNF)-α were measured using flow cytometry. Results: The pro-inflammatory cytokine IL-6 was increased in the isoflurane group at T2 and T3 compared to T1 (P < 0.01). In the propofol group, IL-6 and IL-8 were significantly increased at T3 compared to T1. However, there were no significant differences in cytokine concentrations between the isoflurane and propofol groups. Conclusion: An inflammatory response occurred earlier in patients who received an inhaled agent compared with an intravenous anesthetic, but no differences in plasma cytokine profiles were evident between isoflurane and propofol anesthesia in patients without comorbidities undergoing minimally invasive surgeries. © 2013 Springer Basel.

Compartmentalization of pro-inflammatory cytokine levels in renal-transplanted pregnant women

Objective: We evaluated whether chronic exposure to immunosuppression in transplant recipients modulate the placental inflammatory cytokine levels associated to gestational tolerance mechanisms. Methods: Serum samples were collected from 12 renal transplanted pregnant under immunosuppressive regimen treatment and 10 healthy women in second/third trimester of gestation. Term placental tissues (decidua and chorionic villi) were also obtained after elective caesarean. Serum IL-1β, IL-6, IL-8, IL-12p70 and TNF-α were measured, as also in placental homogenates, by Cytometric Bead Array (CBA) combined with flow cytometry and, TGF-β and IL-18 were measured by ELISA. Results: Serum levels of IL-6 (p=0.0001) and TNF-α (0.0112) were higher in the 2nd and 3rd trimesters and in decidua the spectrum of increased pro inflammatory cytokines was wider: IL-1β (p=0.0001), IL-6 (p=0.0001), IL-8 (p=0.0001), IL-12p70 (p=0.0001), TGF-β (p=0.0089) and TNF-α (p=0.0002). TGF-β1 was particularly increased in decidual compartment (p=0.001). In the chorionic villous, pro inflammatory profile also were maintained. High IL-1β (p=0.0001), IL-6 (p=0.0001), IL-8 (p=0.0001) and TNF-α (p=0.0001) levels establish a similar pattern to that seem in decidua. Conclusion: Immunosuppressors may impair the immune response, but when associated with pregnancy the cytokine levels seems to shift a proinflammatory profile in placental compartments, which might also impact on the gestational outcomes in transplanted mothers. © 2013 Informa UK Ltd. All rights reserved: reproduction in whole or part not permitted.

CD95 (FAS) and CD178 (FASL) induce the apoptosis of CD4+ and CD8+ cells isolated from the peripheral blood and spleen of dogs naturally infected with Leishmania spp

Infected dogs are urban reservoirs of Leishmania chagasi, which is a causative agent of visceral leishmaniasis (VL). Dogs exhibit immune suppression during the course of this disease, and lymphocyte apoptosis is involved in this process. To investigate apoptosis and the expression levels of FAS-FAS-associated death domain protein (CD95 or APO-1), FASL-FAS ligand protein (CD178), and TRAIL-TNF-related apoptosis-inducing ligand (CD253) receptors in peripheral blood mononuclear cells and spleen leukocytes from 38 symptomatic dogs with moderate VL and 25 healthy dogs were evaluated by flow cytometry. The apoptosis rate of blood and splenic CD4+ and CD8+ cells was higher in infected dogs than in healthy dogs. The expression levels of FAS and FASL in blood and splenic CD4+ cells were lower in infected dogs than in healthy dogs. FAS expression in CD8+ cells was higher in infected dogs than in healthy dogs; in contrast, FASL expression was lower in infected dogs. The expression of the TRAIL receptor increased only in splenic CD8+ cells from infected dogs. The FAS and FAS-L blocking antibodies confirmed the importance of these receptors in apoptosis. Our results enhance the current understanding of the immune response in dogs infected with L. chagasi, facilitating the future development of therapeutic interventions to reduce lymphocyte depletion. © 2013 Elsevier B.V.

Alteration in cellular viability, pro-inflammatory cytokines and nitric oxide production in nephrotoxicity generation by Amphotericin B: Involvement of PKA pathway signaling

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cryoprotective effect of different glycerol concentrations on domestic cat spermatozoa

Cryopreservation of spermatozoa is a pivotal tool in assisted reproduction, and studies aiming to establish optimal freezing/thawing protocols are essential to enhance sperm survival. The objectives of the present study were to (1) compare the cryoprotective efficiency of three different glycerol concentrations (3%, 5%, and 7%) on the basis of post-thaw sperm quality and (2) investigate whether the incidence of morphologically abnormal sperm in fresh samples is related to cryodamage sensitivity. Semen was collected from six tomcats using an artificial vagina (total 18 ejaculates). Each ejaculate was diluted using Tris-egg yolk-based extender (TEY), evaluated, equally divided into three aliquots, and rediluted using TEY with and without glycerol to achieve final concentrations of 3%, 5%, and 7%. Samples were loaded into 0.25 mL straws, equilibrated for 60 minutes at 5 °C, frozen, and then thawed at 46 °C for 12 seconds. Fresh and frozen-thawed samples were evaluated for sperm motion parameters (computer-assisted sperm analysis), plasma membrane integrity (PMI; propidium iodide and carboxyfluorescein diacetate), and DNA integrity (acridine orange). Plasma and acrosomal membrane integrity were assessed by flow cytometry (propidium iodide and fluorescein isothiocyanate-conjugated pea (Pisum sativum) agglutinin) immediately after thawing. Sperm motion parameters were also evaluated at 30 and 60 minutes of postincubation. For all treatment groups, cryopreservation significantly impaired the PMI and sperm motion parameters, except for straightness and amplitude of lateral head displacement. DNA integrity showed a slight reduction (P < 0.05) when 3% glycerol was used. The percentage of total motility, progressive motility, and rapid spermatozoa were significantly lower immediately after thawing and up to 60 minutes of incubation for the 3% glycerol group when compared with 5% and 7%. No difference (P > 0.05) was found for PMI, acrosome integrity, and DNA integrity among post-thaw groups. However, higher (P < 0.05) incidence of viable cells with reacted acrosome and dead cells with intact acrosome were observed with 7% and 3% glycerol, respectively. Percentage of morphologically abnormal spermatozoa in fresh sample was positively correlated with PMI only in the 3% glycerol group and negatively correlated with sperm motility in the 5% and 7% groups. In conclusion, the final concentration of 5% glycerol offered better cryoprotective effect for ejaculated cat sperm, and the relationship found between prefreezing sperm morphology and post-thaw sperm quality showed to be dependent on final glycerol concentration. © 2013 Elsevier Inc.

Avaliação do perfil de expressão gênica de células CD34+ e células CD CD133+ isoladas de medula óssea e de sangue de cordão umbilical

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Metabolismo de glicogênio e relógio biológico em Neurospora crassa: fatores e cofatores de transcrição envolvidos nos processos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação da atividade tripanocida do ácido gálico e seus ésteres contra formas epimastigotas de Trypanosoma cruzi: Rogério Andréo. -

Pós-graduação em Biotecnologia - IQ