Experimental infection of commercial layers using a Salmonella enterica serovar Gallinarum strain: Leukogram and serum acute-phase protein concentrations

The aim of the present study was to evaluate white blood cell counts and serum protein profiles of commercial layers experimentally infected with Salmonella Gallinarum (SG) in order to better understand the pathophysiology of the disease caused by this bacterium. 180 five-day-old commercial layers were divided into 3 groups (G); G1 and G2 received 0.2 mL of inoculate containing 3.3x10 8 CFU or 3.3×10 5 CFU SG resistant to nalidix acid (Nal r)/mL, respectively, directly into their crops. G3 group did not receive the inoculum. Birds were sacrificed 24 hours before (T1) and 24 hours after the infection (T2), and three (T3), five (T4), seven (T5), and ten (T6) days after the administration of the inoculum. White blood cell counts were carried out in a Neubauer hemocytometer and in blood smears. Serum protein concentrations, including acute-phase proteins, were determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Data were submitted to analysis of variance, and means were compared by Tukey's test (P <0.05). G1 and G2 groups presented higher leukocyte counts on T4 and T5, respectively, due to the increase of circulating lymphocytes and heterophils, with a significant difference relative to G3. In electrophoresis, an increase in the serum levels of ceruloplasmin, haptoglobin, and hemopexin and a decrease in transferrin, which are acute-phase proteins, was verified. IgA serum levels did not change; however, IgG concentration increased during the infection. In conclusion, the results provide information for the better understanding of the pathophysiology of fowl typhoid.

Cellular localization of androgen synthesis in equine granulosa-theca cell tumors: Immunohistochemical expression of 17α-hydroxylase/17,20-lyase cytochrome P450

Elevated blood testosterone concentrations, often accompanied by male-typical behaviors, is a common signalment of mares with granulosa-theca cell tumors (GCTCs), but no definitive information exists regarding the cellular differentiation of tumors associated with androgen secretion. This study was conducted to localize and thereby define the cellular expression of 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17), the enzyme most directly responsible for androgen synthesis, in 30 GTCTs and control tissues (gonads and adrenal glands) using immuno-histochemistry (IHC). Immuno-reactivity for P450c17 was evident in approximately half of 30 specimens examined, was most consistent in the interstitial cells surrounding existing or developing cysts, and was less intense in cells within cysts in the smaller proportion of specimens where this was observed. In control tissues, the expression of P450c17 was localized primarily in theca interna of normal ovarian follicles, in theca-lutein cells of some corpora lutea, but not in granulosa-lutein cells. Testicular interstitial cells and islands of adreno-cortical cells located in the adrenal medulla of the adrenal cortex further established the specificity of the antisera used. These data provided the first substantive evidence that polyhedral cells identified previously in GTCTs by histopathology have the potential to synthesize and secrete androgens, similar to theca interna and theca lutein cells in normal equine ovaries. © 2010 Elsevier Inc.

Evaluation of the effects of nicorandil and its molecular precursor (without radical NO) on proliferation and apoptosis of 786-cell

Nicorandil is a nitric oxide (NO) donor used in the treatment of angina symptoms. It has also been reported to protect cells and affect the proliferation and death of cells in some tissues. The molecules that interfere with these processes can cause dysfunction in healthy tissues but can also assist in the therapy of some disorders. In this study we examined the effect of nicorandil and of the molecular precursor that does not have the NO radical (N-(beta-hydroxyethyl) nicotinamide) on the cell proliferation and death of human renal carcinoma cells (786-O) under normal oxygenation conditions. The molecular precursor was used in order to analyze the effects independents of NO. In the cytotoxicity test, nicorandil was shown to be cytotoxic at very high concentrations and it was more cytotoxic than its precursor (cytotoxic at concentrations of 2,000 and 3,000 μg/mL, respectively). We propose that the lower cytotoxicity of the precursor is due to the absence of the NO radical. In this study, the cells exposed to nicorandil showed neither statistically significant changes in cell proliferation nor increases in apoptosis or genotoxicity. The precursor generated similar results to those of nicorandil. We conclude that nicorandil causes no changes in the proliferation or apoptosis of the cell 786-O in normal oxygenation conditions. Moreover, the lack of NO radical in the precursor molecule did not show a different result, except in the cell cytotoxicity. © 2013 Springer Science+Business Media Dordrecht.

High concentrations of glucose reduce the oxidative metabolism of dog neutrophils in vitro

Background: Dogs are commonly affected by hyperglycemic conditions. Hyperglycemia compromises the immune response and favors bacterial infections; however, reports on the effects of glucose on neutrophil oxidative metabolism and apoptosis are conflicting in humans and rare in dogs. Considering the many complex factors that affect neutrophil oxidative metabolism in vivo, we investigated in vitro the specific effect of high concentrations of glucose on superoxide production and apoptosis rate in neutrophils from healthy dogs.Results: The capacity of the neutrophils to reduce tetrazolium nitroblue decreased significantly in the higher concentration of glucose (15.13 ± 9.73% (8 mmol/L) versus 8.93 ± 5.71% (16 mmol/L)). However, there were no changes in tetrazolium nitroblue reduction at different glucose concentrations when the neutrophils were first activated with phorbol myristate acetate. High concentrations of glucose did not affect the viability and apoptosis rate of canine neutrophils either with or without prior camptothecin stimulation. This study provides the first evidence that high concentrations of glucose inhibit the oxidative metabolism of canine neutrophils in vitro in a manner similar to that which occurs in humans, and that the decrease in superoxide production did not increase the apoptosis rate.Conclusions: A high concentration of glucose reduces the oxidative metabolism of canine neutrophils in vitro. It is likely that glucose at high concentrations rapidly affects membrane receptors responsible for the activation of NADPH oxidase in neutrophils; therefore, the nonspecific immune response can be compromised in dogs with acute and chronic hyperglycemic conditions. © 2013 Bosco et al.; licensee BioMed Central Ltd.

Effects of (-)-cubebin (Piper cubeba) on cytotoxicity, mutagenicity and expression of p38 MAP kinase and GSTa2 in a hepatoma cell line

(-)-Cubebin is a lignan extracted from the seeds of the pepper Piper cubeba, a commonly eaten spice with beneficial properties, including trypanocidal, anti-inflammatory, analgesic, anti-proliferative and leishmanicidal activities. Because of its therapeutic potential, we investigated the effects of (-)-cubebin on the cytotoxicity, cell proliferation kinetics, mutagenicity and expression of p38 MAP kinase and glutathione S-transferase a2 (GSTa2) using real-time RT-PCR in Rattus norvegicus hepatoma cells. We found that 280 μM (-)-cubebin was cytotoxic after 24, 48 and 72. h of exposure, but not mutagenic at 0.28 μM, 2.8 μM and 28 μM after 26. h. Similarly, exposure to 0.28 μM, 2.8 μM and 28 μM (-)-cubebin for 24, 48, 72 and 96. h did not alter the cell proliferation kinetics. Cells exposed to 28 μM (-)-cubebin for 24. h did not exhibit changes in p38 MAP kinase and GSTa2 expression, indicating that cellular changes were not induced by extracellular stimuli and that (-)-cubebin is likely not metabolized via this pathway. Our results suggest that high levels of (-)-cubebin should be consumed with caution due to the cytotoxic effect observed at the highest concentration. However, at lower concentrations, no cytotoxic, mutagenic or proliferative effects were observed, providing further evidence of the safety of consuming (-)-cubebin. © 2013 Elsevier Inc.

Mutagenicity of hydroxyurea in lymphocytes from patients with sickle cell disease

Hydroxyurea is commonly used in the treatment of myeloproliferative diseases and in patients with sickle cell disease (SCD). The use of this antineoplastic agent in patients with SCD is justified because of the drug's ability to increase fetal hemoglobin levels, thereby decreasing the severity of SCD. However, high doses or prolonged treatment with hydroxyurea can be cytotoxic or genotoxic for these patients, with an increased risk of developing acute leukemia. This danger can be avoided by monitoring the lymphocytes of patients treated with hydroxyurea. Cytogenetic tests are important endpoints for monitoring the physiological effects of physical and chemical agents, including drugs. In this work, we assessed the genotoxicity of hydroxyurea in short-term cultures of lymphocytes from SCD patients. Hydroxyurea was not cytotoxic or genotoxic at the concentrations tested in the G2 phase of the cell cycle. These results support the use of hydroxyurea in the treatment of SCD, although further work is necessary to understand the effects of this drug in vivo

Therapeutic concentration of morphine reduces oxidative stress in glioma cell line

Morphine is a potent analgesic opioid used extensively for pain treatment. During the last decade, global consumption grew more than 4-fold. However, molecular mechanisms elicited by morphine are not totally understood. Thus, a growing literature indicates that there are additional actions to the analgesic effect. Previous studies about morphine and oxidative stress are controversial and used concentrations outside the range of clinical practice. Therefore, in this study, we hypothesized that a therapeutic concentration of morphine (1 μM) would show a protective effect in a traditional model of oxidative stress. We exposed the C6 glioma cell line to hydrogen peroxide (H₂O₂) and/or morphine for 24 h and evaluated cell viability, lipid peroxidation, and levels of sulfhydryl groups (an indicator of the redox state of the cell). Morphine did not prevent the decrease in cell viability provoked by H₂O₂) but partially prevented lipid peroxidation caused by 0.0025% H₂O₂) (a concentration allowing more than 90% cell viability). Interestingly, this opioid did not alter the increased levels of sulfhydryl groups produced by exposure to 0.0025% H₂O₂), opening the possibility that alternative molecular mechanisms (a direct scavenging activity or the inhibition of NAPDH oxidase) may explain the protective effect registered in the lipid peroxidation assay. Our results demonstrate, for the first time, that morphine in usual analgesic doses may contribute to minimizing oxidative stress in cells of glial origin. This study supports the importance of employing concentrations similar to those used in clinical practice for a better approximation between experimental models and the clinical setting.

The effects of genistein and daidzein on cell proliferation kinetics in HT29 colon cancer cells: the expression of CTNNBIP1 (beta-catenin), APC (adenomatous polyposis coli) and BIRC5 (survivin)

Soybean isoflavonoids have received significant attention due to their potential anticarcinogenic and antiproliferative effects and possible role in many signal transduction pathways. However, their mechanisms of action and their molecular targets remain to be further elucidated. In this paper, we demonstrated that two soybean isoflavones (genistein and daidzein) reduced the proliferation of the human colon adenocarcinoma grade II cell line (HT-29) at concentrations of 25 and 50-100 mu M, respectively. We then investigated the effects of genistein and daidzein by RT-PCR on molecules that involved in tumor development and progression by their regulation of cell proliferation. At a concentration of 50 mu M genistein, there was suppressed expression of beta-catenin (CTNNBIP1). Neither genistein nor daidzein affected APC (adenomatous polyposis coli) or survivin (BIRC5) expression when cells were treated with concentrations of 10 or 50 mu M. These data suggest that the down-regulation of beta-catenin by genistein may constitute an important determinant of the suppression of HT-29 cell growth and may be exploited for the prevention and treatment of colon cancer.

The Occurrence of Autophagic Cell Death in the Tegument of Rabbits Pre-Infested with Rhipicephalus sanguineus and Exposed to Selamectin (Active Principle of Acaricide Pfizer Revolution (R))

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cell cycle kinetics, apoptosis rates, DNA damage and TP53 gene expression in bladder cancer cells treated with allyl isothiocyanate (mustard essential oil)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Control of microorganisms of oral health interest with Arctium lappa L. (burdock) extract non-cytotoxic to cell culture of macrophages (RAW 264.7)

Objectives: To evaluate the antimicrobial activity of Arctium lappa L. extract on Staphylococcus aureus, S. epidermidis, Streptococcus mutans, Candida albicans, C. tropicalis and C. glabrata. In addition, the cytotoxicity of this extract was analyzed on macrophages (RAW 264.7).Design: By broth microdilution method, different concentrations of the extract (250-0.4 mg/mL) were used in order to determine the minimum microbicidal concentration (MMC) in planktonic cultures and the most effective concentration was used on biofilms on discs made of acrylic resin. The cytotoxicity A. lappa L. extract MMC was evaluated on RAW 264.7 by MTT assay and the quantification of IL-1 beta and TNF-alpha by ELISA.Results: The most effective concentration was 250 mg/mL and also promoted significant reduction (log(10)) in the biofilms of S. aureus (0.438 +/- 0.269), S. epiderrnidis (0.377 +/- 0.298), S. mutans (0.244 +/- 0.161) and C. albicans (0.746 +/- 0.209). Cell viability was similar to 100%. The production of IL-beta was similar to the control group (p > 0.05) and there was inhibition of TNF-alpha (p < 0.01).Conclusions: A. lappa L. extract was microbicidal for all the evaluated strains in planktonic cultures, microbiostatic for biofilms and not cytotoxic to the macrophages. (C) 2014 Elsevier Ltd. All rights reserved.

Effect of follicular fluid supplementation during in vitro maturation on total cell number in bovine blastocysts produced in vitro

This study evaluated the influence of follicular fluid (FF) added to the maturation medium on the quality of bovine embryos produced in vitro. In the first experiment, oocytes were matured in media containing five different FF concentrations with different maturation times and classified according to meiotic progression and migration of cortical granules. In the second experiment, oocytes matured in the same media were fertilized at three different maturation times; thereafter, cleavage and blastocyst rates were evaluated. In the third experiment, oocytes were matured in media containing three different FF concentrations at two different maturation times, and embryo quality, inferred by the ratio of inner cell mass and trophectoderm cells compared with total cell number, was evaluated. Higher FF concentration (75 - 100% FF) slowed meiotic progression and CG migration (control - 78.13% vs. treated - 52.58% and control - 52.7% vs. treated - 11.59%, respectively, at 24 h of maturation). Also, FF at concentration of 75% or 100% had a negative influence on cleavage and blastocyst rates (control - 90.13% vs. treated - 82.64% and control - 35.73% vs. treated - 11.57%, respectively, at 24 h of maturation). The 50% FF resulted in embryos with increased inner cell mass numbers (control - 29.91 vs. treated - 35.49, at 24 h of maturation) and total cell numbers (control - 109.53 vs. treated - 120.67, at 26 h of maturation). Even though higher concentration of FF added to the maturation medium reduced embryonic development rates, in lower concentrations, FF slowed the meiotic progression and migration of CG and contributed to increases in inner cell mass number. Thus, FF added to the maturation medium enhances the number of cells in bovine embryos produced in vitro, especially for inner cell mass.

Cell cultures of Maytenus ilicifolia Mart. are richer sources of quinone-methide triterpenoids than plant roots in natura

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transport of amino acids from milk whey by Caco-2 cell monolayer after hydrolytic action of gastrointestinal enzymes

The bioavailability of amino adds from milk whey protein hydrolysates was evaluated using diffusion of the substances through semi-permeable membranes (dialyzability) and transport by Caco-2 cell cultures. The hydrolysates with low degree of hydrolysis (LDH) and high degree of hydrolysis (HDH) were obtained after 120 min of reaction time at 50 degrees C after the initial addition of pepsin, followed by the addition of trypsin, chymotrypsin and carboxypeptidase-A. The proteins and hydrolysates were further subjected to in vitro digestion with pepsin plus pancreatin. HPLC was used to determine the concentrations of dialyzable amino adds (48.4% of the non-hydrolyzed proteins, 63.2% of the LDH sample and 58.3% of the HDH sample), demonstrating the greater dialyzability of the hydrolysates. The LDH and HDH whey protein hydrolysates prepared with pepsin, trypsin, chymotrypsin and carboxypeptidase-A showed only 14.7% and 20.8% of dialyzable small peptides and amino acids, respectively. The efficiency of absorption was demonstrated by the preferential transport of Ile, Lou and Arg through a layer of cells. In the LDH hydrolysate, Tyr was also transported. Prior high- and low-degree hydrolysis of the whey provided transport by 5.7% and 6.6%, respectively, in comparison with 23% for non-hydrolyzed proteins, considering the total amount of these amino adds that was applied to the cells. (C) 2014 Elsevier Ltd. All rights reserved.

Biotransformed soybean extract induces cell death of estrogen-dependent breast cancer cells by modulation of apoptotic proteins

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)