Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated ß1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in orthopedic bioengineering.

Identification of virulence factors genes in Escherichia coli isolates from women with urinary tract infection in Mexico.

E coli isolates (108) from Mexican women, clinically diagnosed with urinary tract infection, were screened to identify virulence genes, phylogenetic groups, and antibiotic resistance. Isolates were identified by MicroScan4 system; additionally, the minimum inhibitory concentration (MIC) was assessed. The phylogenetic groups and 16 virulence genes encoding adhesins, toxins, siderophores, lipopolysaccharide (LPS), and invasins were identified by PCR. Phylogenetic groups distribution was as follows: B1 9.3%, A 30.6%, B2 55.6%, and D 4.6%. Virulence genes prevalence was ecp 98.1%, fimH 86.1%, traT 77.8%, sfa/focDE 74.1%, papC 62%, iutA 48.1%, fyuA 44.4%, focG 2.8%, sfaS 1.9%, hlyA 7.4%, cnf-1 6.5%, cdt-B 0.9%, cvaC 2.8%, ibeA 2.8%, and rfc 0.9%. Regarding antimicrobial resistance it was above 50% to ampicillin/sulbactam, ampicillin, piperacillin, trimethoprim/sulfamethoxazole, ciprofloxacin, and levofloxacin. Uropathogenic E. coli clustered mainly in the pathogenic phylogenetic group B2. The isolates showed a high presence of siderophores and adhesion genes and a low presence of genes encoding toxins. The high frequency of papC gene suggests that these isolates have the ability to colonize the kidneys. High resistance to drugs considered as first choice treatment such as trimethoprim/sulfamethoxazole and fluoroquinolones was consistently observed.

Identification of secretory immunoglobulin A antibody targets from human milk in cultured cells infected with enteropathogenic Escherichia coli (EPEC).

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to inject effectors into host cells and alter cellular physiology. The aim of the present study was to identify targets of human secretory immunoglobulin A (sIgA) antibodies from the proteins delivered by EPEC into HEp-2 cells after infection. Bacterial proteins delivered into EPEC-infected cells were obtained in sub-cellular fractions (cytoplasmic, membrane, and cytoskeleton) and probed with sIgA antibodies from human milk and analyzed by Western blotting. These sIgA antibodies reacted with Tir and EspB in the cytoplasmic and membrane fractions, and with intimin in the membrane fraction mainly. The sIgA also identified an EPEC surface-associated Heat-shock protein 70 (Hsp70) in HEp-2 cells infected with EPEC. Purified Hsp70 from EPEC was able to bind to HEp-2 cells, suggesting adhesive properties in this protein. EspC secreted to the medium reacted strongly with the sIgA antibodies. An EPEC 115 kDa protein, unrelated to the EspC protein, was detected in the cytoplasm of infected HEp-2 cells, suggesting that this is a new protein translocated by EPEC. The results suggest that there is a strong host antibody response to Tir and intimin, which are essential proteins for attaching and effacing (A/E) pathogen mediated disease.

Characterization of mesenchymal stem cell subpopulations from human amniotic membrane with dissimilar osteoblastic potential.

Human fetal mesenchymal stem cells can be isolated from the amniotic membrane (AM-hMSCs) by enzymatic digestion. The biological properties of this cell population have been characterized; however, few studies have focused on the presence of stem cell subpopulations and their differentiation potential. The aim of the present study was to isolate homogeneous AM-hMSC subpopulations based on the coexpression of surface markers. In addition, we aimed to characterize stem cell subpopulations through the detection of typical stem cell markers and its differentiation potential. In this study, fluorescence-activated cell sorting (FACS) was used to positively select for the surface markers CD44, CD73, and CD105. Two subpopulations were isolated: CD44+ / CD73+ / CD105+ (CD105+), and CD44+ / CD73+ / CD105- (CD105-). To characterize the cell subpopulations, the expression of pluripotency-associated markers was analyzed by reverse transcriptase-polymerase chain reaction and immunofluorescence. Our results showed positive expression of SOX2, SOX3, PAX6, OCT3/4, and NANOG in the CD105+ and CD105(-) cell subpopulations. In contrast, we did not detect expression of SSEA4 or FOXD3 in either subpopulation. Immunophenotypes, such as mesenchymal and hematopoietic markers, were studied by FACS analyses. Our data revealed the expression of the CD49a, CD49d, CD29, integrin α9ß1, CD44, CD73, and CD105 antigens in both subpopulations. In contrast, CD90, CD45, CD34, CD14, and HLA-DR expression was not detected. The ability of both subpopulations to differentiate into osteoblasts, adipocytes, and chondrocytes was evidenced using Alizarin red, Oil-Red, and Alcian blue staining, respectively. Furthermore, neuronal differentiation was demonstrated by the expression of GFAP and NEURO-D. Interestingly, we observed a dissimilar osteoblastic differentiation potential between the subpopulations. CD105- cells showed stronger expression of secreted protein acidic and rich in cysteine (SPARC) and osteonectin, which was associated with more effective calcium deposition, than CD105+ cells. In conclusion, we described a systematic method for the isolation of hMSCs that was highly reproducible and generated homogeneous cultures for osteoblast differentiation with an efficient capacity for mineralization.