Obtenção e caracterização de populações de células-tronco CD200 e CD34 positivas da pele na espécie canina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Distinct mechanisms underlie adaptation of proximal tubule Na+/H+ exchanger isoform 3 in response to chronic metabolic and respiratory acidosis

The Na+/H+ exchanger isoform 3 (NHE3) is essential for HCO3- reabsorption in renal proximal tubules. The expression and function of NHE3 must adapt to acid-base conditions. The goal of this study was to elucidate the mechanisms responsible for higher proton secretion in proximal tubules during acidosis and to evaluate whether there are differences between metabolic and respiratory acidosis with regard to NHE3 modulation and, if so, to identify the relevant parameters that may trigger these distinct adaptive responses. We achieved metabolic acidosis by lowering HCO3- concentration in the cell culture medium and respiratory acidosis by increasing CO2 tension in the incubator chamber. We found that cell-surface NHE3 expression was increased in response to both forms of acidosis. Mild (pH 7.21 +/- 0.02) and severe (6.95 +/- 0.07) metabolic acidosis increased mRNA levels, at least in part due to up-regulation of transcription, whilst mild (7.11 +/- 0.03) and severe (6.86 +/- 0.01) respiratory acidosis did not up-regulate NHE3 expression. Analyses of the Nhe3 promoter region suggested that the regulatory elements sensitive to metabolic acidosis are located between -466 and -153 bp, where two consensus binding sites for SP1, a transcription factor up-regulated in metabolic acidosis, were localised. We conclude that metabolic acidosis induces Nhe3 promoter activation, which results in higher mRNA and total protein level. At the plasma membrane surface, NHE3 expression was increased in metabolic and respiratory acidosis alike, suggesting that low pH is responsible for NHE3 displacement to the cell surface.

Neurotoxicity of Anhydroecgonine Methyl Ester, a Crack Cocaine Pyrolysis Product

Smoking crack cocaine involves the inhalation of cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). Although there is evidence that cocaine is neurotoxic, the neurotoxicity of AEME has never been evaluated. AEME seems to have cholinergic agonist properties in the cardiovascular system; however, there are no reports on its effects in the central nervous system. The aim of this study was to investigate the neurotoxicity of AEME and its possible cholinergic effects in rat primary hippocampal cell cultures that were exposed to different concentrations of AEME, cocaine, and a cocaineAEME combination. We also evaluated the involvement of muscarinic cholinergic receptors in the neuronal death induced by these treatments using concomitant incubation of the cells with atropine. Neuronal injury was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The results of the viability assays showed that AEME is a neurotoxic agent that has greater neurotoxic potential than cocaine after 24 and 48 h of exposure. We also showed that incubation for 48 h with a combination of both compounds in equipotent concentrations had an additive neurotoxic effect. Although both substances decreased cell viability in the MTT assay, only cocaine increased LDH release. Caspase-3 activity was increased after 3 and 6 h of incubation with 1mM cocaine and after 6 h of 0.1 and 1.0mM AEME exposure. Atropine prevented the AEME-induced neurotoxicity, which suggests that muscarinic cholinergic receptors are involved in AEME's effects. In addition, binding experiments confirmed that AEME has an affinity for muscarinic cholinergic receptors. Nevertheless, atropine was not able to prevent the neurotoxicity produced by cocaine and the cocaineAEME combination, suggesting that these treatments activated other neuronal death pathways. Our results suggest a higher risk for neurotoxicity after smoking crack cocaine than after cocaine use alone.

First description of group A rotavirus from fecal samples of ostriches (Struthio camelus)

This study investigated the occurrence of rotavirus infections in ostriches (Struthio camelus) reared in Northern Parana, Brazil. Fecal (n = 66) and serum (n = 182) samples from nine farms located in four different cities were analyzed by silver stained-polyacrylamide gel electrophoresis (ss-PAGE), RT-PCR assay, virus isolation, and counterimmunoelectroosmophoresis (CIE). Rotavirus group A seropositivity occurred in 5.49% (10/182) of serum samples of ostriches originated from two farms. Only 9.09% (6/66) of fecal samples from ostriches with diarrhea maintained in one farm were positive by ss-PAGE, RT-PCR, and virus isolation. The G (VP7) and P (VP4) genotypes of rotavirus wild strains isolated in cell culture were determined by multiplex-nested PCR. The genotyping identified two rotavirus strains: G6P[1] and G10P[1]. In three rotavirus strains it was only possible to identify the P type; one strain being P[1] and two strains that presented the combination of P[1] + P[7]. These findings might represent the first characterization of rotavirus in ostriches, and the finding of porcine and bovine-like rotavirus genotypes in ostriches might suggest virus reassortment and possible interspecies transmission. (C) 2011 Elsevier Ltd. All rights reserved.

Does Lithium Prevent Alzheimer's Disease?

Lithium salts have a well-established role in the treatment of major affective disorders. More recently, experimental and clinical studies have provided evidence that lithium may also exert neuroprotective effects. In animal and cell culture models, lithium has been shown to increase neuronal viability through a combination of mechanisms that includes the inhibition of apoptosis, regulation of autophagy, increased mitochondrial function, and synthesis of neurotrophic factors. In humans, lithium treatment has been associated with humoral and structural evidence of neuroprotection, such as increased expression of anti-apoptotic genes, inhibition of cellular oxidative stress, synthesis of brain-derived neurotrophic factor (BDNF), cortical thickening, increased grey matter density, and hippocampal enlargement. Recent studies addressing the inhibition of glycogen synthase kinase-3 beta (GSK3B) by lithium have further suggested the modification of biological cascades that pertain to the pathophysiology of Alzheimer's disease (AD). A recent placebo-controlled clinical trial in patients with amnestic mild cognitive impairment (MCI) showed that long-term lithium treatment may actually slow the progression of cognitive and functional deficits, and also attenuate Tau hyperphosphorylation in the MCI-AD continuum. Therefore, lithium treatment may yield disease-modifying effects in AD, both by the specific modification of its pathophysiology via inhibition of overactive GSK3B, and by the unspecific provision of neurotrophic and neuroprotective support. Although the clinical evidence available so far is promising, further experimentation and replication of the evidence in large scale clinical trials is still required to assess the benefit of lithium in the treatment or prevention of cognitive decline in the elderly.

The Aurora A and B kinases are up-regulated in bone marrow-derived chronic lymphocytic leukemia cells and represent potential therapeutic targets

Background The malignant B cells in chronic lymphocytic leukemia receive signals from the bone marrow and lymph node microenvironments which regulate their survival and proliferation. Characterization of these signals and the pathways that propagate them to the interior of the cell is important for the identification of novel potential targets for therapeutic intervention. Design and Methods We compared the gene expression profiles of chronic lymphocytic leukemia B cells purified from bone marrow and peripheral blood to identify genes that are induced by the bone marrow microenvironment. Two of the differentially expressed genes were further studied in cell culture experiments and in an animal model to determine whether they could represent appropriate therapeutic targets in chronic lymphocytic leukemia. Results Functional classification analysis revealed that the majority of differentially expressed genes belong to gene ontology categories related to cell cycle and mitosis. Significantly up-regulated genes in bone marrow-derived tumor cells included important cell cycle regulators, such as Aurora A and B, survivin and CDK6. Down-regulation of Aurora A and B by RNA interference inhibited proliferation of chronic lymphocytic leukemia-derived cell lines and induced low levels of apoptosis. A similar effect was observed with the Aurora kinase inhibitor VX-680 in primary chronic lymphocytic leukemia cells that were induced to proliferate by CpG-oligonucleotides and interleukin-2. Moreover, VX-680 significantly blocked leukemia growth in a mouse model of chronic lymphocytic leukemia. Conclusions Aurora A and B are up-regulated in proliferating chronic lymphocytic leukemia cells and represent potential therapeutic targets in this disease.

Is it safe to utilize in vitro reconstituted human oral epithelium? An oncogenetic pathway study

Cell therapy is a therapeutic strategy used to replace or repair damaged tissue. The epithelium transplantation of cultivated keratinocytes has been applied to several modalities of reconstruction, like oral, urethra and ocular surface. Life and death signals work coordinately to ensure cellular quality control and the viability of an organism. The aim of this study is to verify that culture conditions did not induce genetic mutations through the analysis of the key genes: pAKT, Pten, p53 and MDM2 and investigate the presence of the related proteins in human oral keratinocytes obtained by primary culture and in vitro cultivated. Formalin fixed and paraffin embedded tissues from the oral cavity were utilized as control for normal expression of the related markers and two oral squamous cell carcinoma cell lines provided the expression pattern of the proposed markers in the event of cellular transformation. Akt, PTEN, p53 and MDM2 immunohistochemistry and Western-Blotting analyzes were performed. The results showed the expression levels and intracellular localizations of the four proteins evaluated. These analyzes confirmed that the produced in vitro epithelium is bio-compatible for its utilization as reconstruction and reparatory tissue, however further analyses and additional research on other biomarkers should be performed to analyse the long term engraftment of transplantable primary culture of oral keratinocytes and the long term resistance to cellular transformation.

Experimental infection of the tick Amblyomma cajennense, Cayenne tick, with Rickettsia rickettsii, the agent of Rocky Mountain spotted fever

In the laboratory, Amblyomma cajennense (Acari: Ixodidae) (Fabricius) larvae, nymphs and adults were exposed to Rickettsia rickettsii by feeding on needle-inoculated animals, and thereafter reared on uninfected guinea pigs or rabbits. Regardless of the tick stage that acquired the infection, subsequent tick stages were shown to be infected (confirming transstadial and transovarial transmissions) and were able to transmit R. rickettsii to uninfected animals, as demonstrated by serological and molecular analyses. However, the larval, nymphal and adult stages of A. cajennense were shown to be partially refractory to R. rickettsii infection, as in all cases, only part of the ticks became infected by this agent, after being exposed to rickettsemic animals. In addition, less than 50% of the infected engorged females transmitted rickettsiae transovarially, and when they did so, only part of the offspring became infected, indicating that vertical transmission alone is not enough to maintain R. rickettsii in A. cajennense for multiple generations. Finally, the R. rickettsii-infected tick groups had lower reproductive performance than the uninfected control group. Our results indicate that A. cajennense have a low efficiency to maintain R. rickettsii for successive generations, as R. rickettsii-infection rates should decline drastically throughout the successive tick generations.

Expression of NADPH Oxidase by Trophoblast Cells: Potential Implications for the Postimplanting Mouse Embryo

Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [ reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.

Cytotoxicity evaluation of Curcuma zedoaria (Christm.) Roscoe fluid extract used in oral hygiene products

Objective. This in vitro study evaluated the cytotoxic effects of the Curcuma zedoaria (Christm.) Roscoe (popular name: zedoary) fluid extract, as used in preparations for oral hygiene, mostly for anti-septic purposes. Materials and methods. The cell viability and cell growth were assessed by Trypan blue dye exclusion assay using the LMF cell line derived from oral mucosa. Cell viability (short-term assay) was measured 0, 6, 12 and 24 h after contact with the fluid extract. Cell growth (long-term assay) was analyzed in 1, 3, 5 and 7 days. The experimental groups were those testing the fluid extract obtained from the zedoary rhizome and the extractor liquid (ethanol 70 degrees GL) in the concentrations of 0.01-0.0001% v/v. Fresh DMEM were used in the control cultures. Results. Short-term assay-all studied cultures maintained stable cell viability; Long-term assay-there was progressive cell growth in all studied cultures. Conclusion. According to the results, the zedoary fluid extract presents low cytotoxicity and probably can be used in the oral hygiene products.

In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

Lima S.A.F., Wodewotzky T.I., Lima-Neto J.F., Beltrao-Braga P.C.B. & Alvarenga F.C.L. 2012. [In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors.] Diferenciacao in vitro de celulas-tronco mesenquimais da medula ossea de caes em precursores osteogenicos. Pesquisa Veterinaria Brasileira 32(5):463-469. Departamento de Reproducao Animal e Radiologia Veterinaria, Faculdade de Medicina Veterinaria e Zootecnia, Universidade Estadual Paulista, Campus de Botucatu, Distrito de Rubiao Junior s/n, Botucatu, SP 18618-970, Brazil. E-mail: silviavet@usp.br The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12. Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii

Introduction: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. Methods: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36 degrees C with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. Results: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075 mu M and an LLC MK2 toxicity of 2.003 mu M. Pyrimethamine was effective at an IC50 of 0.482 mu M and a toxicity of 11.178 mu M. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. Conclusions: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

Derivation and characterization of progenitor stem cells from canine allantois and amniotic fluids at the third trimester of gestation

Fetal tissues are frequently discarded before (amniocentesis) or after birth, which both facilitates stem cell access and helps to overcome ethical concerns. In the present study, we aimed to isolate and characterize stem cells from the allantoic and amniotic fluids (ALF; AMF) of third trimester canine fetuses. This gestation age has not been previously explored for stem cells isolation. The gestational age, cell culture conditions and method of isolation used in this study allowed for the establishment and efficient expansion of ALF and AMF cells. We showed that the majority of ALF and ALF cells express the stem cell markers, such as vimentin, nestin and cytokeratin 18 (CK18). Under appropriate culture conditions AMF derived cells can undergo differentiation into osteogenic, adipogenic, chondrogenic and neuron-like lineages. ALF derived cells showed adipogenic, and chondrogenic potential. Therefore, ALF and AMF cells derived at the third gestation trimester can be qualified as progenitor stem cells, accordingly referred as (alantoic fluid progenitor/stem) ALF PS cells and (amniotic fluid progenitor/stem) AMF PS cells. (C) 2012 Elsevier Ltd. All rights reserved.

Epidemiological and molecular characterization of rubella virus isolated in Sao Paulo, Brazil during 1997-2004

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as the congenital rubella syndrome (CRS). In 2003, the Pan American Health Organization (PAHO) adopted a resolution calling for the elimination of rubella and the congenital rubella syndrome (CRS) in the Americas by the year 2010. Brazil will have implemented the recommended PAHO strategy for elimination and interruption of endemic rubella virus transmission. The characterization of genotypes during the final stages of rubella elimination is important for determining whether new rubella isolates represent endemic transmission or importations. Samples (blood, urine, cerebrospinal fluid, and throat swabs) collected from patients with symptoms suggestive of rubella infection in 19972004 were isolated in cell culture and genotyped. Twenty-eight sequences were analyzed and two genotypes were identified: 1a and 1G. The information reported in this paper will contribute to understanding the molecular epidemiology of RV in Sao Paulo, Brazil. J. Med. Virol. 84:18311838, 2012. (c) 2012 Wiley Periodicals, Inc.

The influence of pore size on osteoblast phenotype expression in cultures grown on porous titanium

This study investigated the effect of pore size on osteoblastic phenotype development in cultures grown on porous titanium (Ti). Porous Ti discs with three different pore sizes, 312 mu m (Ti 312), 130 mu m (Ti 130) and 62 mu m (Ti 62) were fabricated using a powder metallurgy process. Osteoblastic cells obtained from human alveolar bone were cultured on porous Ti samples for periods of up to 14 days. Cell proliferation was affected by pore size at day 3 (p = 0.0010), day 7 (p = 0.0005) and day 10 (p = 0.0090) in the following way: Ti 62 < Ti 130 < Ti 312. Gene expression of bone markers evaluated at 14 days was affected, RUNX2 (p = 0.0153), ALP (p = 0.0153), BSP (p = 0.0156), COL (p = 0.0156), and OPN (p = 0.0156) by pore size as follows: Ti 312 < Ti 130 < Ti 62. Based on these results, the authors suggest that porous Ti surfaces with pore sizes near 62 mu m, compared with those of 312 mu m and 130 mu m, yield the highest expression of osteoblast phenotype as indicated by the lower cell proliferation rate and higher gene expression of bone markers.