Interleukin-10 secreted by B-1 cells modulates the phagocytic activity of murine macrophages in vitro

As demonstrated previously in our laboratory, B-1 cells migrate from the peritoneal cavity of mice and home to a distant site of inflammation to become macrophage-like cells. However, the influence that these cells might have on the kinetics and fate of the inflammatory process is not known. Considering that macrophages are pivotal in the inflammatory reaction, we decided to investigate the possible influence B-1 cells could have on macrophage activities in vitro. Our results show that peritoneal macrophages from Xid mice, a mouse strain deprived of B-1 cells, have higher phagocytic indexes for zymozan particles when compared with macrophages from wild-type mice. Moreover, macrophages from wild-type mice have a lower ability to release nitric oxide and hydrogen peroxide when compared with macrophages from Xid mice. Experiments using cocultures of B-1 cells and macrophages from Xid mice in transwell plates demonstrated that B-1 cells down-regulate macrophage activities. These observations also indicate that this phenomenon is not due to a physical interaction between these two cell populations. As B-1 cells are one of the main sources of interleukin (IL)-10, we demonstrate in this study that adherent peritoneal cells from Xid mice produce significantly less amounts of this cytokine in culture when compared with IL-10 production by cells from wild-type mice. When B-1 cells from IL-10 knock-out mice and macrophages from wild-type mice were cocultured in transwell plates, the phagocytic index of macrophages was not altered demonstrating that B-1 cells can influence the effector functions of macrophages in vitro via IL-10 secretion.

Physiological responses of pressed baker's yeast cells pre-treated with citric, malic and succinic acids

The dough-leavening power of baker's yeast, Saccharomyces cerevisiae, is strongly influenced by conditions under which the pressed yeast is maintained prior to bread dough preparation. In this study, the influence of the yeast cell's pre-treatment with organic acids (malic, succinic, and citric acids) was investigated at a wide range of pH values when the pressed yeast samples were exposed to 30 degrees C. Increased fermentative activity was observed immediately after pre-treatment of the cells with organic acids. When the pH of the pressed yeast containing added citric acid was raised from 3.5 to 7.5, increases in both fermentative and maltase activities were obtained. Improvements in viability and levels of total protein were also observed during storage in the presence of citric acid, notably at pH 7.5. Glycerol-3-phosphate dehydrogenase activity and levels of internal glycerol also increased in the presence of citrate. on the other hand, pressed yeast samples containing succinic acid at pH 7.5 showed decreased viability during storage despite the maintenance of high levels of fermentative activity, similar to pressed yeast containing malic acid at pH 4.5 and 7.5. Decreases in intracellular levels of trehalose were observed during storage in all cases. Overall, the results of this study revealed the potential benefits of adding organic acids to pressed yeast preparations for baking purposes.

Male gonadal denervation by guanethidine at pre-puberty: Different doses, different results. The intervention of the pineal deafferentation

Since gonadal denervation and pineal deafferentation by cervical superior ganglionectomy affect sexual development, this study was performed to evaluate testicular steroidogenesis, spermatogenesis and the cervical superior ganglion (CSG) histology in rats treated with guanethidine (GD). The treatment was performed by GD s.c. injections for 3 weeks, from the 21st day of age to the 41st day of age (pre-puberty), when the animals were sacrificed. Different doses were used: group A=10 mg/kg/day, group B=50 mg/kg/day and saline (control group). Testicular denervation was confirmed by HPLC for catecholamines in testicular tissue. Testicular concentrations (TC) of progesterone (P4) and testosterone (T) were measured by RIA. Significantly higher TC of P4 and lower TC of T were observed only in group A in comparison with group B and the control group. No alteration of sperm production was observed in either treated group. Histological analysis of CSG showed only few neuronal alterations in group A rats, while in group B the nervous cells were practically destroyed. This suggests that 10 mg/kg/day GD treatment probably produces a specific blockade of 17 alpha-hydroxylase/17,20 desmolase at pre-puberty leading to a decrease of the androgen production. However, in the 50 mg/kg/day group no differences were observed concerning the steroid profiles, this result being attributed to the extensive damage to the CSG observed only in group B. The CSG destruction causes deafferentation of the pineal gland producing abolishment of the inhibition of the 17 alpha-hydroxylase/17,20 desmolase promoted by melatonin or by an out of phase production of androgen.

Shiitake (Lentinula edodes (Berkeley) Pegler) extracts as a modulator of micronuclei induced in HEp-2 cells

Shiitake (Lentinula edodes (Berkeley) Pegler) is one of the most consumed mushrooms, for both therapeutic purposes and as food, therefore, the study of its biological properties is of great interest for producers and consumers. Aqueous extracts of the shiitake mushroom (L. edodes (Berkeley) Pegler) were evaluated by the micronucleus test (MN) in HEp-2 cells in vitro, to analyze their possible mutagenic and antimutagenic activities. None of the three extract concentrations tested (0.5, 1.0 and 1.5 mg/mL) presented mutagenicity at any of the preparation temperatures (4 degrees C, 22 +/- 2 degrees C and 60 degrees C). In the antimutagenicity evaluation, all extract concentrations at all preparation temperatures presented a strong protective activity for the HEp-2 cells in response to the alkylating agent methyl methanesulfonate (MMS) in the different treatment protocols: pre-treatment, simultaneous treatment and post-treatment. The extracts prepared at 22 +/- 2 degrees C presented the lowest frequencies of MN in the evaluations of mutagenicity and antimutagenicity, indicating these as the best option for potential therapeutic use. (c) 2006 Elsevier Ltd. All rights reserved.

Increased Levels of NOTCH1, NF-kappa B, and Other Interconnected Transcription Factors Characterize Primitive Sets of Hematopoietic Stem Cells

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

Anti-genotoxic effect of aqueous extracts of sun mushroom (Agaricus blazei Murill lineage 99/26) in mammalian cells in vitro

The sun mushroom is the popular name for the Agaricus blazei Murill fungus, a mushroom native to south-eastern Brazil, which has been frequently used in popular medicine mainly in the form of tea to treat various ailments (stress, diabetes, etc.). In the present study, the genotoxic and/or anti-genotoxic effects ofA. blazei on mammalian cells in culture was assessed by checking the increase or reduction of micronucleus (MN) frequency and comets. The sun mushroom (lineage 99/26) was used as aqueous extracts prepared (2.5%) at three different temperatures (60, 25 and 4°C). The in vitro micronucleus (MN) test in binucleated cells and comet assay were used in V79 cells cultivated in HAM-F10+DMEM medium (1:1), supplemented with 10% of fetal bovine serum. The experiments were divided into four treatment types: 1. Negative control; 2. Positive control with MMS; 3. Treatments with the three forms of extracts (60, 25 and 4°C); and 4. Treatments with the extracts in different associations (simultaneous, pre-treatment, post-treatment and simultaneous after pre-incubation for 1 h) with MMS. None of the A. blazei extracts show genotoxic activity. In the comet assay no protecting effect was found. The results obtained in the MN test showed that the three forms of extracts used had protective activity, suggesting that the compound or active ingredients of A. blazei are always present in these extracts. The greater protective efficiency of the simultaneous treatment and simultaneous treatment with pre-incubation mixture with MMS suggests that the extracts have an antimutagenic action of the desmutagenic type. © 2002 Elsevier Science Ltd. All rights reserved.

Non-mutagenic or genotoxic effects of medicinal aqueous extracts from the Agaricus blazei mushroom in V79 cells

Agaricus blazei Murill is a mushroom largely consumed due to its medicinal properties. Effects of aqueous extract from its lineage AB97/11 in 2 fruiting body development stages (closed and opened pileus) were evaluated on chinese hamster V79 cells using cytokinesis blocking micronucleus (CBMN) and comet assays. The cells were treated at 0.15% concentration of aqueous extract prepared at different temperatures: ice-cold (4°C), room temperature (21°C) and warm (60°C). The extracts were applied in mutagenicity and antimutagenicity protocols (simultaneous, pre-incubation and continuous). The results showed that the aqueous extracts of Agaricus blazei lineage AB97/11 obtained at the 3 temperatures and both development stages did not present mutagenic or antimutagenic effect in V79 cells either in CBMN or comet assay.

Genotoxic and antigenotoxic effects of organic extracts of mushroom Agaricus blazei Murrill on V79 cells

Agaricus blazei Murrill, popularly known as the sun mushroom, is a native mushroom in SP, Brazil, that has been widely used in the treatment of cancer and many other pathologies in different parts of the world. A water-soluble protein-polysaccharide complex (1 → 6)β-D-glucan has been isolated from its fruiting body that showed immune-modulation activity. From organic extracts, linoleic acid has been isolated and determined to be the main substance with antimutagenic activity. Using both the micronucleus (MN) and comet (single cell microgel electrophoresis) assays, this study determined the genotoxic and antigenotoxic potential of A. blazei (AB) obtained from commercial sources or the following strains: a) strains AB 97/29 (young and sporulated phases); b) a mixture taken from AB 96/07, AB 96/09 and AB 97/ 11 strains; and c) commercial mushrooms from Londrina, PR and Piedade, SP, designated as AB PR and AB SP, respectively. The extracts from these mushrooms were isolated in chloroform:methanol (3:1) and used in vitro at three different concentrations. V79 cells (Chinese hamster lung cells) were exposed to the extracts under pre-, simultaneous and post-treatment conditions, combined with methyl methanesulfonate (MMS). Under the circumstances of this study, these organic extracts did not show any genotoxic or mutagenic effects, but did protect cells against the induction of micronuclei by MMS. Copyright by the Brazilian Society of Genetics.

Anticlastogenic effect of β-glucan, extracted from Saccharomyces cerevisiae, on cultured cells exposed to ultraviolet radiation

β-glucan is an important polysaccharide due to its medicinal properties of stimulating the immune system and preventing chronic diseases such as cancer. The aim of the present study was to determine the anticlastogenic effect of β-glucan in cells exposed to ultraviolet radiation (UV). Chromosome aberration assay was performed in drug-metabolizing cells (HTC) and non drug-metabolizing cells (CHO-K1 and repair-deficient CHO-xrs5), using different treatment protocols. Continuous treatment (UV + β-glucan) was not effective in reducing the DNA damage only in CHO-xrs5 cells. However, the pre-treatment protocol (β-glucan before UV exposition) was effective in reducing DNA damage only in CHO-K1 cells. In post-treatment (β-glucan after UV exposition) did not show significative anticlastogenic effects, although there was a tendency toward prevention. The data suggest that β-glucan has more than one action mechanism, being capable of exerting desmutagenic as well as bio-antimutagenic action. The findings also suggest that the presence of the xenobiotic metabolizing system can reduce the chemopreventive capacity of β-glucan. Therefore, these results indicate that β-glucan from Saccharomyces cerevisiae can be used in the prevention and/or reduction of DNA damage. © 2012 Springer Science+Business Media B.V.

Assessment of the chemopreventive effect of casearin B, a clerodane diterpene extracted from Casearia sylvestris (Salicaceae)

Studies have shown that Casearia sylvestris compounds protect DNA from damage both in vitro and in vivo. Complementarily, the aim of the present study was to assess the chemopreventive effect of casearin B (CASB) against DNA damage using the Ames test, the comet assay and the DCFDA antioxidant assay. The genotoxicity was assessed by the comet assay in HepG2 cells. CASB was genotoxic at concentrations higher than 0.30μM when incubated with the FPG (formamidopyrimidine-DNA glycosylase) enzyme. For the antigenotoxicity comet assay, CASB protected the DNA from damage caused by H2O2 in the HepG2 cell line in concentrations above 0.04μM with post-treatment, and above 0.08μM with pre-treatment. CASB was not mutagenic (Ames test) in TA 98 and TA 102. In the antimutagenicity assays, the compound was a strong inhibitor against aflatoxin B1 (AFB) in TA 98 (>88.8%), whereas it was moderate (42.7-59.4%) inhibitor against mytomicin C (MMC) in TA 102. Additionally, in the antioxidant assay using DCFDA, CASB reduced reactive oxygen species (ROS) generated by H2O2. In conclusion, CASB was genotoxic to HepG2 cells at high concentrations; was protective of DNA at low concentrations, as shown by the Ames test and comet assay; and was also antioxidant. © 2012 Elsevier Ltd.

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)

Atividade in vivo de parasporina extraída de B. thuringiensis sobre o câncer colorretal murino (CT‐26)

Bacillus thuringiensis (Bt), is an environmental Gram-positive spore-forming bacterium that produces crystalline parasporal protein (Cry) during sporulation. The inclusions often exhibit strong and specific insecticidal activity, making Bt an agent for agricultural controlling insects pest, mites, protozoa and nematodes. Recent studies reported that some of these Crys do not show cytotoxicity against insects but they are capable to kill some human and animal cancer cells. These proteins were denominated parasporins (PS). However, antitumor activity of Bt parasporin on the development of murine colorectal cancer (CT-26), are not well studies and these are no reports on the in vivo effect of these proteins. Thus, the present study evaluated the in vitro and in vivo anti-tumoral activity of Bt parasporin against the murine colorectal cancer line CT-26. Therefore, Balb/c mice were s.c. inoculated with CT-26 cells and weekly treated with parasporin (i.p.) pre-activated by enzymatic digestion with trypsin or proteinase K. Our results have shown, for the first time, that despite the anti-tumor activity in vitro, parasporin crystals couldn’t combat tumor growth in vivo. Instead, this protein was highly toxic, affecting the liver and spleen, with possible effect on other organs, decreasing the survival of treated animals. The results indicate the need for studies to better detoxification or manipulation of parasporin for therapeutic use and new studies for analysis of toxicological effects of repetitive exposure of farmers to this toxin

Non-hematopoietic PAR-2 is essential for matriptase-driven pre-malignant progression and potentiation of ras-mediated squamous cell carcinogenesis

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

Gene expression profiles displayed by peripheral blood mononuclear cells from patients with type 2 diabetes mellitus focusing on biological processes implicated on the pathogenesis of the disease

Patients with type 2 diabetes mellitus (T2DM) exhibit insulin resistance associated with obesity and inflammatory response, besides an increased level of oxidative DNA damage as a consequence of the hyperglycemic condition and the generation of reactive oxygen species (ROS). In order to provide information on the mechanisms involved in the pathophysiology of T2DM, we analyzed the transcriptional expression patterns exhibited by peripheral blood mononuclear cells (PBMCs) from patients with T2DM compared to non-diabetic subjects, by investigating several biological processes: inflammatory and immune responses, responses to oxidative stress and hypoxia, fatty acid processing, and DNA repair. PBMCs were obtained from 20 T2DM patients and eight non-diabetic subjects. Total RNA was hybridized to Agilent whole human genome 4x44K one-color oligo-microarray. Microarray data were analyzed using the GeneSpring GX 11.0 software (Agilent). We used BRB-ArrayTools software (gene set analysis - GSA) to investigate significant gene sets and the Genomica tool to study a possible influence of clinical features on gene expression profiles. We showed that PBMCs from T2DM patients presented significant changes in gene expression, exhibiting 1320 differentially expressed genes compared to the control group. A great number of genes were involved in biological processes implicated in the pathogenesis of T2DM. Among the genes with high fold-change values, the up-regulated ones were associated with fatty acid metabolism and protection against lipid-induced oxidative stress, while the down-regulated ones were implicated in the suppression of pro-inflammatory cytokines production and DNA repair. Moreover, we identified two significant signaling pathways: adipocytokine, related to insulin resistance; and ceramide, related to oxidative stress and induction of apoptosis. In addition, expression profiles were not influenced by patient features, such as age, gender, obesity, pre/post-menopause age, neuropathy, glycemia, and HbA(1c) percentage. Hence, by studying expression profiles of PBMCs, we provided quantitative and qualitative differences and similarities between T2DM patients and non-diabetic individuals, contributing with new perspectives for a better understanding of the disease. (C) 2012 Elsevier B.V. All rights reserved.