CD271 as a marker to identify mesenchymal stem cells from diverse sources before culture

Mesenchymal stem cells, due to their characteristics are ideal candidates for cellular therapy. Currently, in culture these cells are defined by their adherence to plastic, specific surface antigen expression and multipotent differentiation potential. However, the in vivo identification of mesenchymal stem cells, before culture, is not so well established. Pre-culture identification markers would ensure higher purity than that obtained with selection based on adherence to plastic. Up until now, CD271 has been described as the most specific marker for the characterization andpurification of human bone marrow mesenchymal stem cells. This marker has been shown to be specifically expressed by these cells. Thus, CD271 has been proposed as a versatile marker to selectively isolated and expand multipotent mesenchymal stem cells with both immunosuppressive and lymphohematopoietic engraftment-promoting properties. This review focuses on this marker, specifically on identification of mesenchymal stem cells from different tissues. Literature revision suggests that CD271 should not be defined as a universal marker to identify mesenchymal stem cells before culture from different sources. In the case of bone marrow or adipose tissue, CD271 could be considered a quite suitable marker; however this marker seems to be inadequate for the isolation of mesenchymal stem cells from other tissues such as umbilical cord blood or wharton’s jelly among others.     

Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing

A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.     

Seasonal diet changes in elephant and impala in mopane woodland

Elephant and impala as intermediate feeders, having a mixed diet of grass and browse, respond to seasonal fluctuations of forage quality by changing their diet composition. We tested the hypotheses that (1) the decrease in forage quality is accompanied by a change in diet from more monocots in the wet season to more dicots in the dry season and that that change is more pronounced and faster in impala than in elephant; (2) mopane (Colophospermum mopane), the most abundant dicot species, is the most important species in the elephant diet in mopane woodland, whereas impala feed relatively less on mopane due to the high condensed tannin concentration; and (3) impala on nutrient-rich soils have a diet consisting of more grass and change later to diet of more browse than impala on nutrient-poor soils. The phosphorus content and in vitro digestibility of monocots decreased and the NDF content increased significantly towards the end of the wet season, whereas in dicots no significant trend could be detected. We argue that this decreasing monocot quality caused elephant and impala to consume more dicots in the dry season. Elephant changed their diet gradually over a 16-week period from 70% to 25% monocots, whereas impala changed diets rapidly (2?C4?weeks) from 95% to 70% monocots. For both elephants and impala, there was a positive correlation between percentage of monocots and dicots in the diet and the in vitro digestibility of these forage items. Mopane was the most important dicot species in the elephant diet and its contribution to the diet increased significantly in the dry season, whereas impala selected other dicot species. On nutrient-rich gabbroic soils, impala ate significantly more monocots than impala from nutrient-poor granitic soils, which was related to the higher in vitro digestibility of the monocots on gabbroic soil. Digestibility of food items appears to be an important determinant of diet change from the wet to the dry season in impala and elephants.     

Pseudomonas aeruginosa flagellin and alginate elicit very distinct gene expression patterns in airway epithelial cells: implications for cystic fibrosis disease

Infection with the opportunistic pathogen Pseudomonas aeruginosa remains a major health concern. Two P. aeruginosa phenotypes relevant in human disease include motility and mucoidy. Motility is characterized by the presence of flagella and is essential in the establishment of acute infections, while mucoidy, defined by the production of the exopolysaccharide alginate, is critical in the development of chronic infections, such as the infections seen in cystic fibrosis patients. Indeed, chronic infection of the lung by mucoid P. aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. We have used Calu-3 human airway epithelial cells to investigate global responses to infection with motile and mucoid P. aeruginosa. The response of airway epithelial cells to exposure to P. aeruginosa motile strains is characterized by a specific increase in gene expression in pathways controlling inflammation and host defense. By contrast, the response of airway epithelia to the stimuli presented by mucoid P. aeruginosa is not proinflammatory and, hence, may not be conducive to the effective elimination of the pathogen. The pattern of gene expression directed by flagellin, but not alginate, includes innate host defense genes, proinflammatory cytokines, and chemokines. By contrast, infection with alginate-producing P. aeruginosa results in an overall attenuation of host responses and an antiapoptotic effect.     

Flavopiridol induces mitochondrial-mediated apoptosis in murine glioma GL261 cells via release of cytochrome c and apoptosis inducing factor

Glioblastoma (GBM) remains one of the most challenging solid cancers to treat due to its highly proliferative, angiogenic and invasive nature. The small molecule CDK inhibitor, flavopiridol, has demonstrated antitumor activity in human xenograft models and is currently in clinical trials showing efficacy in patients with advanced disease. We have developed an experimental animal model using the murine glioma GL261 cells as a novel in vivo system to screen potential therapeutic agents for GBM. Results of in vitro testing demonstrate that flavopiridol has several relevant clinical characteristics such as its ability to: 1. inhibit cell growth; 2. inhibit cell migration; 3. decrease expression of cyclin D1, CDK4 and p21; 4. induce apoptosis in cells with high levels of p27 expression; and 5. decrease the expression of the anti-apoptotic protein Bcl-2. The mechanism by which flavopiridol induces apoptosis is mitochondrial-mediated. We demonstrate by electron microscopy and immunohistochemistry that drug treatment induces mitochondrial damage that was accompanied by the release of cytochrome c into the cytosol together with the translocation of apoptosis inducing factor (AIF) into the nucleus. This finding in murine glioma cells differs from the mechanism of flavopiridolinduced cell death reported by us for human glioma cells (Alonso et al., Mol Cancer Ther 2003; 2:139) where drug treatment induced a caspase- and cytochrome c-independent pathway in the absence of detectable damage to mitochondria. In apoptotic human glioma cells only translocation of AIF into the nucleus occurred. Thus, the same drug kills different types of glioma cells by different mitochondrial-dependent pathways.