Interaction between attaching and effacing Escherichia coli serotypes O157:H7 and O26:K60 in cell culture

Ruminants harbour both O157:H7 and non-O157 Attaching Effacing Escherichia coli (AEEC) strains but to date only nonO157 AEEC have been shown to induce attaching effacing lesions in naturally infected animals. However, O157 may induce lesions in deliberate oral inoculation studies and persistence is considered dependent upon the bacterially encoded locus for enterocyte effacement. In concurrent infections in ruminants it is unclear whether non-O157 AEEC contribute either positively or negatively to the persistence of E. coli O157:H7. To investigate this, and prior to animal studies, E. coli O157:H7 NCTC 12900, a non-toxigenic strain that persists in conventionally reared sheep, and non-toxigenic AEEC O26:K60 isolates of sheep origin were tested for adherence to Hep-2 tissue culture alone and in competition one with another. Applied together, both strains adhered in similar numbers but lower than when either was applied separately. Pre-incubation of tissue culture with either one strain reduced significantly (P < 0.05) the extent of adherence of the strain that was applied second. It was particularly noticeable that AEEC O26 when applied first reduced adherence and inhibited microcolony formation, as demonstrated by confocal microscopy, of E. coli 01 57:H7. The possibility that prior colonisation of a ruminant by non-O157 AEEC such as O26 may antagonise O157 colonisation and persistence in ruminants is discussed. (C) 2004 Elsevier B.V. All rights reserved.

Self-assembled arginine-capped peptide bolaamphiphile nanosheets for cell culture and controlled wettability surfaces

The spontaneous assembly of a peptide bolaamphiphile in water, namely, RFL4FR (R, arginine; F, phenylalanine; L, leucine) is investigated, along with its novel properties in surface modification and usage as substrates for cell culture. RFL4FR self-assembles into nanosheets through lateral association of the peptide backbone. The L4 sequence is located within the core of the nanosheets, whereas the R moieties are exposed to the water at the surface of the nanosheets. Kinetic assays indicate that the self-assembly is driven by a remarkable two-step process, where a nucleation phase is followed by fast growth of nanosheets with an autocatalysis process. The internal structure of the nanosheets is formed from ultrathin bolaamphiphile monolayers with a crystalline orthorhombic symmetry with cross-β organization. We show that human corneal stromal fibroblast (hCSF) cells can grow on polystyrene films coated with films dried from RFL4FR solutions. For the first time, this type of amphiphilic peptide is used as a substrate to modulate the wettability of solid surfaces for cell culture applications.

Nitric oxide detection in cell culture exposed to LPS after Er:YAG laser irradiation

P>Aim To evaluate in vitro the effect of calcium hydroxide [Ca(OH)(2)] and Er:YAG laser on bacterial endotoxin [also known as lipopolysaccharide (LPS)] as determined by nitric oxide (NO) detection in J774 murine macrophage cell line culture. Methodology Samples of LPS solution (50 mu gmL-1), Ca(OH)(2) suspension (25 mg mL-1) and LPS suspension with Ca(OH)(2) were prepared. The studied groups were: I - LPS (control); II - LPS + Ca(OH)(2); III - LPS + Er:YAG laser (15 Hz 140 mJ); IV - LPS + Er:YAG laser (15 Hz 200 mJ); V - LPS + Er:YAG laser (15 Hz 250 mJ), VI - Pyrogen-free water; VII - Ca(OH)(2). Murine macrophage J774 cells were plated and 10 mu L of the samples were added to each well. The supernatants were collected for NO detection by the Griess reaction. Data were analysed statistically by one-way anova and Tukey`s test at 5% significance level. Results The mean and SE (in mu mol L-1) values of NO release were: I - 10.48 +/- 0.58, II - 6.41 +/- 0.90, III - 10.2 +/- 0.60, IV - 8.35 +/- 0.40, V - 10.40 +/- 0.53, VI - 3.75 +/- 0.70, VII - 6.44 +/- 0.60; and the values for the same experiment repeated after 1 week were: I - 21.20 +/- 1.50, II - 9.10 +/- 0.60, III - 19.50 +/- 1.00, IV - 18.50 +/- 0.60, V - 21.30 +/- 0.90, VI - 2.00 +/- 0.20, VII - 6.80 +/- 1.70. There was no significant difference (P > 0.05) between the control and the laser-treated groups (III, IV and V), or comparing groups II, VI and VII to each other (P > 0.05). Group I had significantly higher NO release than group II (P < 0.05). Groups II and VI had similar NO release (P > 0.05). Conclusions Calcium hydroxide inactivated the bacterial endotoxin (LPS) whereas none of the Er:YAG laser parameter settings had the same effectiveness.

Adenosine modulates alpha2-adrenergic receptors through a phospholipase C pathway in brainstem cell culture of rats

Adenosine acts in the nucleus tractus solitarii (NTS), one of the main brain sites related to cardiovascular control. In the present study we show that A(1) adenosine receptor (A(1R)) activation promotes an increase on alpha(2)-adrenoceptor (Alpha(2R)) binding in brainstem cell culture from newborn rats. We investigated the intracellular cascade involved in such modulatory process using different intracellular signaling molecule inhibitors as well as calcium chelators. Phospholipase C, protein kinase Ca(2+)-dependent, IP(3) receptor and intracellular calcium were shown to participate in A(1R)/Alpha(2R) interaction. In conclusion, this result might be important to understand the role of adenosine within the NTS regarding autonomic cardiovascular control. (C) 2009 Elsevier B.V. All rights reserved.

Photodynamic therapy associating Photogem (R) and blue LED on L929 and MDPC-23 cell culture

To evaluate the cytotoxicity of PDT (photodynamic therapy) with Photogem (R) associated to blue LED (light-emitting diode) on L929 and MDPC-23 cell cultures, 30000 cells/cm(2) were seeded in 24-well plates for 48 h, incubated with Photogem (R) (10, 25 or 50 mg/l) and irradiated with an LED source (460 +/- 3 nm; 22 mW/cm(2)) at two energy densities (25.5 or 37.5 J/cm(2)). Cell metabolism was evaluated by the MTT (methyltetrazolium) assay (Dunnet`s post hoc tests) and cell morphology by SEM (scanning electron microscopy). Flow cytometry analysed the type of PDT-induced cell death as well and estimated intracellular production of ROS (reactive oxygen species). There was a statistically significant decrease of mitochondrial activity (90% to 97%) for all Photogem (R) concentrations associated to blue LED, regardless of irradiation time. It was also demonstrated that the mitochondrial activity was not recovered after 12 or 24 h, characterizing irreversible cell damage. PDT-treated cells presented an altered morphology with ill-defined limits. In both cell lines, there was a predominance of necrotic cell death and the presence of Photogem (R) or irradiation increased the intracellular levels of ROS. PDT caused severe toxic effects in normal cell culture, characterized by the reduction of the mitochondrial activity, morphological alterations and induction of necrotic cell death.

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MWr of about 120 kDa and specific PNPP activity of 1200 U/tng. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/ mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 Wing), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and (3glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function. (c) 2006 Elsevier B.V. All rights reserved.

Human breast tumor slices as an alternative approach to cell lines to individualize research for each patient

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

Borderline leprosy: in situ and cytokine profile in supernatant of mononuclear of cell culture

In order to contribute to a better understanding of cytokine participation in borderline leprosy, in the present study we determined - by in vitro and in situ examinations - the production of these cytokine mediation in non-treated borderline tuberculoid (BT) patients and borderline lepromatous (BL) patients. Seven non-treated BT patients, 12 non-treated BL patients, besides 19 healthy individuals (control group), were evaluated. Peripheral blood mononuclear cells (PBMC) were stimulated or not with specific-M. leprae stimulus (whole and sonicated M. leprae antigens) and a non-specific stimulus. After 48 hours, supernatant was collected for TNF-alpha, IFN-gamma, IL-10 and TGF-beta1 cytokine determination by ELISA. Biopsies from cutaneous lesions were submitted to histological analysis and hematoxylin-eosin and Fite-Faraco stainings; the sections then underwent iNOS, IL-10 and TGF-beta1 in situ detection by immunohistochemistry. Cytokine quantification in PBMC supernatants from patients showed that BT patients produced higher levels of IFN-gamma. Compared to healthy individuals, both borderline patient groups produced lower levels of TGF-beta1 while BL patients generated lower IL-10 levels. The in situ iNOS expression was higher in BT patients compared to BL individuals. on the order hand, TGF-beta1 cytokine revealed a higher proportion of immunostained cells in BL patients. There was no significant difference in IL-10 level between BT and BL patients. Regarding cutaneous lesions, in BL patients there was a negative correlation between TGF-beta1 tissue expression and IL-10. Independently of the clinical form, we observed a positive correlation between TGF-beta1 and bacterial index as well as a negative correlation between the TGF-beta1 tissue expression and iNOS. The results even showed a positive correlation between iNOS tissue expression and production of IFN-gamma by PBMC stimulated with M. leprae antigens. Taken together, the histopathological and immunological observations reinforce the notion of immunological instability in borderline leprosy patients and indicating the participation of mixed cytokines profiles in these individuals, specifically a Th1 profile in BT patients and Th2 profile in BL patients, with a possible participation of T-regulatory lymphocytes.

Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culture

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mineral Trioxide Aggregate-based Endodontic Sealer Stimulates Hydroxyapatite Nucleation in Human Osteoblast-like Cell Culture

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Photodynamic therapy associating Photogem (R) and blue LED on L929 and MDPC-23 cell culture

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

Callus cell culture of Pothomorphe umbellata (L.) under stress condition leads to high content of peroxidase enzyme

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

Effect of salt stress on polyamine metabolism in bean cell culture

The adaptive capacity of bean (Phaseolus vulgaris L.) calluses (cultivars IAC-carioca, JALO EEP-558, BAT-93 and IAPAR-14) to salt stress (0-80 mM) was verified to determine the existence of biochemical markers such as organic and inorganic compounds, and metabolism of polyamines. The results obtained demonstrate that salt (NaCl) interfered with all the parameters analyzed and its intensity ranged due to the salt concentration and the cultivars used.