Human mesenchymal stem cell proliferation and osteogenic differentiation during long-term ex vivo cultivation is not age dependent.

Mesenchymal stem cells (MSCs) are of major clinical interest for the development of cell-based strategies to treat musculoskeletal diseases including critical-size bone defects caused by trauma, degenerative disorders, or infections. Elderly people mainly suffer from critical-size bone defects from the rising incidence of trauma, osteoporosis, and arthroplasties. In this study we investigated the influence of donor age on proliferation and osteogenic differentiation in long-term ex vivo cultures of primary human MSCs from patients in different age groups. Fifteen patients (8 men/7 women) comprised three age groups: (I) <50 years, (II) 50-65 years, and (III) >65 years. MSCs harvested from bone marrow derived from routine surgical procedures were isolated and cultured in standard medium over eight passages. Osteogenic differentiation was induced by dexamethasone (10 nM), ascorbic acid (300 µM), and ß-glycerophosphate (3.5 mM). Osteogenic differentiation capacity of MSCs was quantified by alkaline phosphatase (ALP) activity, fluorescence-activated cell sorting (FACS) analysis of the surface markers CD9, CD90, CD54, CD166, CD105, CD44, and CD73, and RT-PCR for Coll I and II, Cbfa 1, ALP, OC, BSP1, and GAPDH genes characterized the phenotypic changes during monolayer expansion. In vitro chondrogenic differentiation was analyzed by immunohistochemistry and RT-PCR. Progenitor cells could be expanded in the long term from all bone marrow donations. FACS single staining analysis from MSCs showed no significant difference between the age groups. The surface antigen CD166 was predominantly found in all cell cultures independently of differentiation stage. Comparison of expanded and differentiated MSCs within a single age group showed that undifferentiated MSCs had higher CD44 levels. Osteogenic stimulation of MSCs was confirmed by measuring ALP activity. The highest ALP activity was found in probands of the age group >65 years. Additionally, we observed a tendency toward male-specific ALP increase during differentiation. Osteogenic marker gene expression in MSCs was detected by RT-PCR. No significant expression differences were detected between the three donor age groups. Micromass culture of MSCs resulted histologically and immunohistologically in a chondrogenic phenotype. Elderly osteoprogenitor cell donors are a highly clinically relevant patient population. In summary, cultivation leads to a reduced osteogenic differentiation capacity regardless of age. Because donor age does not affect osteogenic differentiation potential, it should not be used as an exclusion criterion for autologous transplantation of human adult MSCs.

Legionella dresdenensis sp. nov., isolated from river water.

Legionella-like isolates, strains W03-356(T), W03-357 and W03-359, from three independent water samples from the river Elbe, Germany, were analysed by using a polyphasic approach. Morphological and biochemical characterization revealed that they were Gram-negative, aerobic, non-spore-forming bacilli with a cut glass colony appearance that grew only on L-cysteine-supplemented buffered charcoal yeast extract agar. Phylogenetic analysis based on sequence comparisons of the 16S rRNA, macrophage infectivity potentiator (mip), gyrase subunit A (gyrA), ribosomal polymerase B (rpoB) and RNase P (rnpB) genes confirmed that the three isolates were distinct from recognized species of the genus Legionella. Phenotypic characterization of strain W03-356(T) based on fatty acid profiles confirmed that it was closely related to Legionella rubrilucens ATCC 35304(T) and Legionella pneumophila ATCC 33152(T), but distinct from other species of the genus Legionella. Serotyping of the isolates showed that they were distinct from all recognized species of the genus Legionella. Strains W03-356(T), W03-357 and W03-359 are thus considered to represent a novel species of the genus Legionella, for which the name Legionella dresdenensis sp. nov. is proposed. The type strain is W03-356(T) (=DSM 19488(T)=NCTC 13409(T)).

Assessing the risk of transmission of viral diseases in flooded areas: viral load of the River Elbe in Dresden during the flood of August 2002.

In order to assess the risk of transmission of viral diseases during floods, the viral burden in flooded areas of the city of Dresden (Germany) in August 2002 was investigated. Water samples were collected from 9 sampling sites and tested for the presence of 11 enteric viral pathogens. As a control, water samples from the same sites were analyzed in seasonal intervals over the following year. A total of 36 samples were collected, 92% (33/36) being positive for at least one virus. Adenovirus type 40/41 was the most frequently detected (53%), followed by astrovirus (50%) and enterovirus (50%). In all samples, low levels of bacteriophages were detected with no specificity as to sampling site and season, indicating a moderate river contamination with wastewater. A striking association between water temperature and viral genome detection was observed, as illustrated in August 2002 (mean water temperature of 17.8 degrees, 8 sites positive for 17 viruses), in comparison to November 2002 (mean water temperature of 7.6 degrees, 9 sites positive for 45 viruses). Importantly, hepatitis A viral RNA was not detected in the flooded area. In conclusion, our results indicate no increased risk for transmission of viral diseases through water contact in flooded areas.