Stable expression of a human-like sialylated recombinant thyrotropin in a chinese hamster ovary cell line expressing 'alfa'2,6-sialyltransferase

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

Optimization of a multielectrode array (MEA)-based approach to study the impact of Aβ on the SH-SY5Y cell line

The human brain stores, integrates, and transmits information recurring to millions of neurons, interconnected by countless synapses. Though neurons communicate through chemical signaling, information is coded and conducted in the form of electrical signals. Neuroelectrophysiology focus on the study of this type of signaling. Both intra and extracellular approaches are used in research, but none holds as much potential in high-throughput screening and drug discovery, as extracellular recordings using multielectrode arrays (MEAs). MEAs measure neuronal activity, both in vitro and in vivo. Their key advantage is the capability to record electrical activity at multiple sites simultaneously. Alzheimer’s disease (AD) is the most common neurodegenerative disease and one of the leading causes of death worldwide. It is characterized by neurofibrillar tangles and aggregates of amyloid-β (Aβ) peptides, which lead to the loss of synapses and ultimately neuronal death. Currently, there is no cure and the drugs available can only delay its progression. In vitro MEA assays enable rapid screening of neuroprotective and neuroharming compounds. Therefore, MEA recordings are of great use in both AD basic and clinical research. The main aim of this thesis was to optimize the formation of SH-SY5Y neuronal networks on MEAs. These can be extremely useful for facilities that do not have access to primary neuronal cultures, but can also save resources and facilitate obtaining faster high-throughput results to those that do. Adhesion-mediating compounds proved to impact cell morphology, viability and exhibition of spontaneous electrical activity. Moreover, SH-SY5Y cells were successfully differentiated and demonstrated acute effects on neuronal function after Aβ addition. This effect on electrical signaling was dependent on Aβ oligomers concentration. The results here presented allow us to conclude that the SH-SY5Y cell line can be successfully differentiated in properly coated MEAs and be used for assessing acute Aβ effects on neuronal signaling.

Cyto- and Genotoxicity assessment of manufactured nano cerium dioxide in the A549 cell line

In the past decades the growing application of nanomaterials (NMs) in diverse consumer products has raised various concerns in the field of toxicology. They have been extensively used in a broad range of applications and cover most of the industrial sectors as well as the medicine and the environmental areas. The most common scenarios for human exposure to NMs are occupational, environmental and as consumers and inhalation is the most frequent route of exposure, especially in occupational settings. Cerium dioxide NMs (nano-CeO2) are widely used in a number of applications such as in cosmetics, outdoor paints, wood care products as well as fuel catalysts. For such reason, nano-CeO2 is one of the selected NMs for priority testing within the sponsorship program of the Working Party of Manufactured Nanomaterials of the OECD. In this context, the aim of this study is to assess the safety of nano-CeO2 (NM-212, Joint Research Center Repository) through the characterization of its cytotoxicity and genotoxicity in a human alveolar epithelial cell line. A dispersion of the NM in water plus 0.05% BSA was prepared and sonicated during 16 minutes, according to a standardized protocol. DLS analysis was used to characterize the quality of the NM dispersion in the culture medium. To evaluate the cytotoxicity of nano-CeO2 in the A549 cell line, the colorimetric MTT assay was performed; the capacity of cells to proliferate when exposed to CeO2 was also assessed with the Clonogenic assay. The genotoxicity of this NM was evaluated by the Comet Assay (3 and 24h of exposure) to quantify DNA breaks and the FPG-modified comet assay to assess oxidative DNA damage. The Cytokinesis-Block Micronucleus (CBMN) assay was used to further detect chromosome breaks or loss. The nano-CeO2 particles are spherical, displaying a diameter of 33 nm and 28 m2/g of surface area. The results of the MTT assay did not show any decreased in cells viability following treatment with a dose-range of nano-CeO2 during 24h. Nevertheless, the highest concentrations of this NM were able to significantly reduce the colony forming ability of A549 cells, suggesting that a prolonged exposure may be cytotoxic to these cells. Data from both genotoxicity assays revealed that nano-CeO2 was neither able to induce DNA breaks nor oxidative DNA damage. Likewise, no significant micronucleus induction was observed. Taken together, the present results indicate that this nano-CeO2 is not genotoxic in this alveolar cell line under the tested conditions, although further studies should be performed, e.g., gene mutation in somatic cells and in vivo chromosome damage (rodent micronucleus assay) to ensure its safety to human health.

MRI indices of functional recovery following intracerebral implantation of a human neural stem cell line in a pre-clinical model of ischaemic stroke

Stem cell therapy for ischaemic stroke is an emerging field in light of an increasing number of patients surviving with permanent disability. Several allogenic and autologous cells types are now in clinical trials with preliminary evidence of safety. Some clinical studies have reported functional improvements in some patients. After initial safety evaluation in a Phase 1 study, the conditionally immortalised human neural stem cell line CTX0E03 is currently in a Phase 2 clinical trial (PISCES-II). Previous pre-clinical studies conducted by ReNeuron Ltd, showed evidence of functional recovery in the Bilateral Asymmetry test up to 6 weeks following transplantation into rodent brain, 4 weeks after middle cerebral artery occlusion. Resting-state fMRI is increasingly used to investigate brain function in health and disease, and may also act as a predictor of recovery due to known network changes in the post-stroke recovery period. Resting-state methods have also been applied to non-human primates and rodents which have been found to have analogous resting-state networks to humans. The sensorimotor resting-state network of rodents is impaired following experimental focal ischaemia of the middle cerebral artery territory. However, the effects of stem cell implantation on brain functional networks has not previously been investigated. Prior studies assessed sensorimotor function following sub-cortical implantation of CTX0E03 cells in the rodent post-stroke brain but with no MRI assessments of functional improvements. This thesis presents research on the effect of sub-cortical implantation of CTX0E03 cells on the resting- state sensorimotor network and sensorimotor deficits in the rat following experimental stroke, using protocols based on previous work with this cell line. The work in this thesis identified functional tests of appropriate sensitivity for long-term dysfunction suitable for this laboratory, and investigated non-invasive monitoring of physiological variables required to optimize BOLD signal stability within a high-field MRI scanner. Following experimental stroke, rats demonstrated expected sensorimotor dysfunction and changes in the resting-state sensorimotor network. CTX0E03 cells did not improve post-stroke functional outcome (compared to previous studies) and with no changes in resting-state sensorimotor network activity. However, in control animals, we observed changes in functional networks due to the stereotaxic procedure. This illustrates the sensitivity of resting-state fMRI to stereotaxic procedures. We hypothesise that the damage caused by cell or vehicle implantation may have prevented functional and network recovery which has not been previously identified due to the application of different functional tests. The findings in this thesis represent one of few pre-clinical studies in resting-state fMRI network changes post-stroke and the only to date applying this technique to evaluate functional outcomes following a clinically applicable human neural stem cell treatment for ischaemic stroke. It was found that injury caused by stereotaxic injection should be taken into account when assessing the effectiveness of treatment.

Leccinum vulpinum Watling induces DNA damage, decreases cell proliferation and induces apoptosis on the human MCF-7 breast cancer cell line

The current work aimed to study the antitumour activity of a phenolic extract of the edible mushroom Leccinum vulpinum Watling, rich essentially in hydroxybenzoic acids. In a first approach, the mushroom extract was tested against cancer cell growth by using four human tumour cell lines. Given the positive results obtained in these initial screening experiments and the evidence of some studies for an inverse relationship between mushroom consumption and breast cancer risk, a detailed study of the bioactivity of the extract was carried out on MCF-7 cells. Once the selected cell line to precede the work was the breast adenocarcinoma cell line, the human breast non-malignant cell line MCF-10A was used as control. Overall, the extract decreased cellular proliferation and induced apoptosis. Furthermore, the results also suggest that the extract causes cellular DNA damage. Data obtained highlight the potential of mushrooms as a source of biologically active compounds, particularly with antitumour activity.

Characterization of volatile compounds of Albertisia papuana Becc root extracts and cytotoxic activity in breast cancer cell line T47D

Purpose: To evaluate the cytotoxic activity of chloroform and water root extracts of Albertisia papuana Becc. on T47D cell line and identify the volatile compounds of the extracts. Methods: The plant roots were extracted with chloroform and water using maceration and boiling methods, respectively. The cytotoxicity of the extracts on T47D were determined using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Doxorubicin was used as reference drug in the cytotoxicity test while Probit analysis was used to calculate the Median Growth Inhibitory Concentration IC50 of the extracts. The volatile compounds in the chloroform and water root extracts were analyzed using Gas Chromatography-Mass Spectrophotometry GC-MS. Results: The IC50 of the chloroform and water extracts were 28.0 ± 6.0 and 88.0 ± 5.5 μg/mL, respectively whereas that of doxorubicin was 8.5 ± 0.1 μg/mL. GC-MS results showed that there were 46 compounds in the chloroform extract, out of which the five major components are ethyl linoleate (49.68 %), bicyclo (3.3.1) non-2-ene (29.29 %), ethyl palmitate (5.06 %), palmitic acid (3.67 %) and ethyl heptadecanoate (1.57 %).The water extract consisted of three compounds, butanoic acid (15.58 %); methyl cycloheptane (3.45 %), and methyl 2-O-methylpentofuranoside (80.96 %). Conclusion: The chloroform root extract of A. papuana Becc. had a fairly potent anticancer activity against breast cancer cells and may be further developed as an anticancer agent. Its major components were fatty acids and fatty acid esters.

Antitumor effect and mechanism of action of polysaccharides extracted from Polygonum perfoliatum L whole plant in human lung carcinoma A549 cell line

Purpose: To optimize the extraction conditions of polysaccharides from Polygonum perfoliatum L. (PSDP) and to evaluate their anti-tumor activities on A549 cell line. Methods: Extraction of PSDP was optimized using Box-Behnken design (BBD). Three factors of response surface methodology (RSM) including extraction time, ratio of water to raw material and number of extractions were employed to optimize the yield of PSDP. The cytotoxic effect of PSDP on human lung carcinoma A549 cell line was evaluated in vivo, while its effects on expressions of caspase3, caspase-9, Bcl-2 and Bax were determined by western blot assay. Result: BBD was significant and applicable to PSDP extraction. Based on the contour plots, response surface plots and variance analysis, it predicted that the optimum conditions for PSDP extraction were: 1.58 h (extraction time); 30.18 mL/g (ratio of water to raw material); and 2.02 (number of extractions). PSDP had significant inhibitory effect on the growth of A549 cells in a concentration- and timedependent manner (p < 0.05). After treatment with PSDP, caspase-3, caspase-9 and Bax were significantly up-regulated (p < 0.05), whereas Bcl-2 was down-regulated, all concentration-dependently. Conclusion: RSM analysis is an appropriate method to optimize PSDP extraction. The results also indicate that PSDP has significant anti-tumor effect against A549 cells, most likely via inducing mitochondria-mediated apoptosis.

Inhibition of proliferation, migration and invasion of human non-small cell lung cancer cell line A549 by phlomisoside F from Phlomis younghusbandii Mukerjee

Purpose: To determine the effect of phlomisoside F (PMF) on the proliferation, migration and invasion of human non-small cell lung cancer cell line A549 and explore the possible mechanisms. Methods: The anti-proliferative effect of PMF on A549 cells was determined by CCK-8. Subsequently, migration and invasion were evaluated by Transwell and Transwell with matrigel assays, respectively. Furthermore, cell cycle and apoptosis were assessed by flow cytometry, while the mechanisms of action were determined by Western blotting. Results: PMF exhibited significant anti-proliferative effect on A549 cells in concentration-dependent and time-dependent manners, with half maximal inhibitory concentration (IC50) of 54.51 μM. Treatment with PMF (10, 20 and 40 μM) for 48 h resulted in significantly decreased migration and invasion in A549 cells. In addition, PMF at concentrations of 25, 50 and 75 μM induced cell cycle arrest in G0/G1phase and enhanced cell apoptosis in A549 cells. Furthermore, caspase-3, caspase-9 and Bax protein expressions were up-regulated while Bacl-2 and COX-2 protein expressions were significantly downregulated at 10, 20 and 40 μM concentrations of PMF. Conclusion: PMF suppresses A549 cell growth, migration and invasion. The mechanism may be related to the induction of mitochondria-mediated apoptosis pathway via regulation of caspase-3, caspase-9, Bcl-2 and Bax expressions, and inhibition of PGE2 synthesis by reducing COX-2 expression.

Study of Bone Biomineralization in 2D and 3D Cultures of a Human Osteosarcoma Cell Line As Osteoblast-like Model

This PhD project focuses on the study of the early stages of bone biomineralization in 2D and 3D cultures of osteoblast-like SaOS-2 osteosarcoma cells, exposed to an osteogenic cocktail. The efficacy of osteogenic treatment was assessed on 2D cell cultures after 7 days. A large calcium minerals production, an overexpression of osteogenic markers and of alkaline phosphatase activity occurred in treated samples. TEM microscopy and cryo-XANES micro-spectroscopy were performed for localizing and characterizing Ca-depositions. These techniques revealed a different localization and chemical composition of Ca-minerals over time and after treatment. Nevertheless, the Mito stress test showed in treated samples a significant increase in maximal respiration levels associated to an upregulation of mitochondrial biogenesis indicative of an ongoing differentiation process. The 3D cell cultures were realized using two different hydrogels: a commercial collagen type I and a mixture of agarose and lactose-modified chitosan (CTL). Both biomaterials showed good biocompatibility with SaOS-2 cells. The gene expression analysis of SaOS-2 cells on collagen scaffolds indicated an osteogenic commitment after treatment. and Alizarin red staining highlighted the presence of Ca-spots in the differentiated samples. In addition, the intracellular magnesium quantification, and the X-ray microscopy on mineral depositions, suggested the incorporation of Mg during the early stages of bone formation process., SaOS-2 cells treated with osteogenic cocktail produced Ca mineral deposits also on CTL/agarose scaffolds, as confirmed by alizarin red staining. Further studies are underway to evaluate the differentiation also at the genetic level. Thanks to the combination of conventional laboratory methods and synchrotron-based techniques, it has been demonstrated that SaOS-2 is a suitable model for the study of biomineralization in vitro. These results have contributed to a deeper knowledge of biomineralization process in osteosarcoma cells and could provide new evidences about a therapeutic strategy acting on the reversibility of tumorigenicity by osteogenic induction.

Health-related quality of life in patients with Chagas disease: a review of the evidence

Chagas disease (ChD), a neglected tropical disease caused by infection with the parasite Trypanosoma cruzi (T. cruzi), remains a serious public health issue in Latin America and is an emerging disease in several non-endemic countries, where knowledge of the condition and experience with its clinical management are limited. Regionally, the disease is the major cause of disability secondary to tropical diseases in young adults. Health-related quality of life (HRQoL) impairment is common in patients with ChD, especially in those with Chagas dilated cardiomyopathy, the most severe manifestation of the disease, which frequently leads to heart failure. The aim of this review was to conduct a literature search for studies that have evaluated the determining factors of HRQoL in ChD patients. We included cross-sectional, case-control, cohort, and experimental studies, as well as clinical trials that evaluated the HRQoL in ChD patients aged 18 to 60 years and are presenting an explicit description of statistical analysis. Using a combination of keywords based on Descriptors in Health Sciences (DeCS) and Medical Subject Headings (MeSH) for searches in PubMed and the Scientific Electronic Library Online (SciELO), 148 studies were found. After exclusions, 12 studies were selected for analysis. Three main findings were extracted from these studies: 1) cardiac involvement is associated with a worse HRQoL in ChD patients; 2) HRQoL is associated with the patients' functional capacity; and 3) simple and inexpensive therapeutic measures are effective for improving HRQoL in ChD patients. Hence, ChD patients' functional capacity, the effectiveness of non-surgical conservative treatment, and cardiac involvement are important determining factors for the HRQoL in ChD patients.

Infección por virus pandémico (H1N1) 2009 en Andalucía

In April 2009, in response to the WHO's alert due to the existence of human infection cases with a new AH1N1 influenza virus, known as swine flu, Andalusian Health Authorities trigger an specific action plan. The surveillance actions developped provided us with appropriate clinical, epidemiological and virological characteristics of the disease. During the first few days, contingency plans were set up based on epidemiological surveillance and outbreak control measures were adopted through early alert and rapid response systems. After phase 6 was declared, influenza sentinel and severe cases surveillance were used in order to plan healthcare services, to reduce transmission and to identify and protect the most vulnerable population groups. Behaviour of pandemic influenza in Andalusia was similar to that observed in the rest of the world. Atack rate was similar to a seasonal flu and the peak was reached at the 46th/2009 week. Most of them were mild cases and affected particularly to young people. The average age of hospitalised patients was 32. Prior pulmonary disease, smoking and morbid obesity (BMI>40) were the most common pathologies and risk factors in severe cases. An impact scenario of pandemic wave in Andalusia, with an expected attack rate from 2 to 5%, was prepared considering watt observed in the southern hemisphere. Characteristics of the epidemic concerning its extent, severity and mortality rate were adjusted to this scenario.

Riesgo vascular : proceso asistencial integrado

Publicado en la página web de la Consejería de Igualdad, Salud y Políticas Sociales: www.juntadeandalucia.es/salud (Consejería de Igualdad, Salud y Políticas Sociales/ Profesionales / Nuestro Compromiso por la Calidad / Procesos Asistenciales Integrados)

Calendario vacunal 2011

Cartel y calendario de bolsillo

Prevención del Cáncer de cuello de útero: El cáncer de cuello de útero es provocado por el Virus del Papiloma Humano

Cartel y Folleto: Prevención del Cáncer de cuello de útero: El cáncer de cuello de útero es provocado por el Virus del Papiloma Humano. Díptico de citas: Prevención del Cáncer de cuello de útero. Citas para la vacunación frente al papilomavirus