Hematopoietic stem cell and mesenchymal stem cell population size in bone marrow samples depends on patient’s age and harvesting technique

Mesenchymal stem cells (MSCs) are heterogeneous population of cells with great potential for regenerative medicine. MSCs are relatively easy to expand in a cell culture, however determination of their concentration in harvested tissue is more complex and is not implemented as routine procedure. To identify MSCs collected from bone marrow we have used two combinations of cell markers (CD45^?/CD73⁺/CD90⁺/CD105⁺ and CD45^?/CD271⁺) and fibroblast colony-forming unit (CFU-F) assay. Further, in donors of various ages, mesenchymal stem cell concentration was compared with the result of CFU-F assay and with hematopoietic stem cell concentration, determined by a standardized flow cytometric assay. A positive correlation of MSC populations to the CFU-F numbers is observed, the population of the CD45^?/CD271⁺ cells correlates better with CFU-F numbers than the population of the CD45^?/CD73⁺/CD90⁺/CD105⁺ cells. The relationship between the hematopoietic CD45^dim/CD34⁺ cell concentration and mesenchymal CFU-Fs or CD45^?/CD271⁺ cells shows a positive linear regression. An age-related quantitative reduction of hematopoietic CD45^dim/CD34⁺, mesenchymal CD45^?/CD73⁺/CD90⁺/CD105⁺ and CD45^?/CD271⁺ stem cells, and CFU-F numbers were noted. Additionally, statistically significant higher CFU-F numbers were observed when bone marrow samples were harvested from three different sites from the anterior iliac crest instead of harvesting the same sample amount only from one site.     

Comparative study of the AT(1) receptor prodrug antagonist candesartan cilexetil with other sartans on the interactions with membrane bilayers

Drug-membrane interactions of the candesartan cilexetil (TCV-116) have been studied on molecular basis by applying various complementary biophysical techniques namely differential scanning calorimetry (DSC), Raman spectroscopy, small and wide angle X-ray scattering (SAXS and WAXS), solution (1)H and (13)C nuclear magnetic resonance (NMR) and solid state (13)C and (31)P (NMR) spectroscopies. In addition, (31)P cross polarization (CP) NMR broadline fitting methodology in combination with ab initio computations has been applied. Finally molecular dynamics (MD) was applied to find the low energy conformation and position of candesartan cilexetil in the bilayers. Thus, the experimental results complemented with in silico MD results provided information on the localization, orientation, and dynamic properties of TCV-116 in the lipidic environment. The effects of this prodrug have been compared with other AT(1) receptor antagonists hitherto studied. The prodrug TCV-116 as other sartans has been found to be accommodated in the polar/apolar interface of the bilayer. In particular, it anchors in the mesophase region of the lipid bilayers with the tetrazole group oriented toward the polar headgroup spanning from water interface toward the mesophase and upper segment of the hydrophobic region. In spite of their localization identity, their thermal and dynamic effects are distinct pointing out that each sartan has its own fingerprint of action in the membrane bilayer, which is determined by the parameters derived from the above mentioned biophysical techniques.     

Breast MRI in nonpalpable breast lesions: a randomized trial with diagnostic and therapeutic outcome – MONET – study

Background

In orthodontic treatment, anchorage control is a fundamental aspect. Usually conventional mechanism for orthodontic anchorage control can be either extraoral or intraoral that is headgear or intermaxillary elastics. Their use are combined with various side effects such as tipping of occlusal plane or undesirable movements of teeth. Especially in cases, where key-teeth are missing, conventional anchorage defined as tooth-borne anchorage will meet limitations. Therefore, the use of endosseous implants for anchorage purposes are increasingly used to achieve positional stability and maximum anchorage.

Methods/Design

The intended study is designed as a prospective, multicenter randomized controlled trial (RCT), comparing and contrasting the effect of early loading of palatal implant therapy versus implant loading after 12 weeks post implantation using the new ortho-implant type II anchor system device (Orthosystem Straumann, Basel, Switzerland). 124 participants, mainly adult males or females, whose diagnoses require temporary stationary implant-based anchorage treatment will be randomized 1:1 to one of two treatment groups: group 1 will receive a loading of implant standard therapy after a healing period of 12 week (gold standard), whereas group 2 will receive an early loading of orthodontic implants within 1 week after implant insertion. Participants will be at least followed for 12 months after implant placement. The primary endpoint is to investigate the behavior of early loaded palatal implants in order to find out if shorter healing periods might be justified to accelerate active orthodontic treatment. Secondary outcomes will focus e.g. on achievement of orthodontic treatment goals and quantity of direct implant-bone interface of removed bone specimens. As tertiary objective, a histologic and microtomography evaluation of all retrieved implants will be performed to obtain data on the performance of the SLA surface in human bone evaluation of all retrieved implants. Additionally, resonance frequency analysis (RFA, Osstell? mentor) will be used at different times for clinically monitoring the implant stability and for histological comparison in order to measure the reliability of the resonance frequency measuring device.

Trial registration

Current Controlled Trials ISRCTN97142521.     

p85alpha regulates osteoblast differentiation by cross-talking with the MAPK pathway

Class IA phosphoinositide 3-kinase (PI3K) is involved in regulating many cellular functions including cell growth, proliferation, cell survival, and differentiation. The p85 regulatory subunit is a critical component of the PI3K signaling pathway. Mesenchymal stem cells (MSC) are multipotent cells that can be differentiated into osteoblasts (OBs), adipocytes, and chondrocytes under defined culture conditions. To determine whether p85α subunit of PI3K affects biological functions of MSCs, bone marrow-derived wild type (WT) and p85α-deficient (p85α(-/-)) cells were employed in this study. Increased cell growth, higher proliferation rate and reduced number of senescent cells were observed in MSCs lacking p85α compare with WT MSCs as evaluated by CFU-F assay, thymidine incorporation assay, and β-galactosidase staining, respectively. These functional changes are associated with the increased cell cycle, increased expression of cyclin D, cyclin E, and reduced expression of p16 and p19 in p85α(-/-) MSCs. In addition, a time-dependent reduction in alkaline phosphatase (ALP) activity and osteocalcin mRNA expression was observed in p85α(-/-) MSCs compared with WT MSCs, suggesting impaired osteoblast differentiation due to p85α deficiency in MSCs. The impaired p85α(-/-) osteoblast differentiation was associated with increased activation of Akt and MAPK. Importantly, bone morphogenic protein 2 (BMP2) was able to intensify the differentiation of osteoblasts derived from WT MSCs, whereas this process was significantly impaired as a result of p85α deficiency. Addition of LY294002, a PI3K inhibitor, did not alter the differentiation of osteoblasts in either genotype. However, application of PD98059, a Mek/MAPK inhibitor, significantly enhanced osteoblast differentiation in WT and p85α(-/-) MSCs. These results suggest that p85α plays an essential role in osteoblast differentiation from MSCs by repressing the activation of MAPK pathway.     

Rat spermatogenesis in vitro traced by quantitative flow cytometry

In vitro differentiation of germ cells in rat seminiferous tubule segments at stages II-III of the epithelial cycle was studied. DNA flow cytometry was used for quantitation of absolute cell numbers from the cultured tubule segments that were compared to freshly isolated stages of the cycle, as identified by transillumination stereomicroscopy of the seminiferous tubules and phase-contrast microscopy of live cell squashes. Spermatogonia and spermatocytes from stages II-III showed normal morphological differentiation during 7 days in vitro. Round spermatids differentiated to Step 7 of spermiogenesis but Step 16 spermatids failed to develop. Acid phosphatase activity in the spermatogenic cells changed normally during the culture. As compared with freshly isolated control tubule segments, 35% of round spermatids and 42% of pachytene spermatocytes were present in culture after 7 days. The cell numbers recovered from defined stages by DNA flow cytometry were close to those found in morphometric studies. Flow cytometry is an efficient quantitation method for cells liberated from seminiferous epithelium. Spermatogonia, spermatocytes, and early spermatids are able to differentiate in vitro, but spermatids approaching the elongation (acrosome) phase, and particularly the maturation phase, fail to differentiate under present culture conditions.     

Rationale and design of the SMART Heart study

Left ventricular hypertrophy (LVH) is an independent risk factor for the development of heart failure, coronary heart disease and stroke. LVH develops in response to haemodynamic overload, e.g. hypertension. LVH was originally thought to start as an adaptive and beneficial response required to normalise wall stress. However, this concept has been challenged by recent animal experiments suggesting that any degree of LVH is detrimental for the preservation of cardiac function and survival. If confirmed in humans, these findings imply that an increase in LV mass should be prevented, e.g. by lifestyle or pharmacological interventions. To facilitate and optimise interventions, the SMART Heart study was recently set up to develop a prediction model, also involving single nucleotide polymorphism data, for the identification of subjects at high risk of developing LVH in hypertension. For this purpose 1000 subjects with chronic hypertension will undergo cardiac MR imaging. In addition, this study allows the extrapolation of animal experimental genetic research into the human situation. (Neth Heart J 2007;15:295-8).     

Spectrum of <Emphasis Type="Italic">CREBBP</Emphasis> mutations in Indian patients with Rubinstein-Taybi syndrome

Rubinstein-Taybi syndrome (RSTS), a developmental disorder comprising abnormalities that include mental retardation, an unusual facial appearance, broad thumbs and big toes is frequently associated with molecular lesions in the CREB-binding protein gene, CREBBP. The objective of the present study was to identify and analyse CREBBP mutations in Indian RSTS patients on which there are no data. Direct sequencing of CREBBP performed in 13 RSTS patients identified the three zinc fingers (CH1, CH2, CH3) and HAT domain as mutational hotspots in which ten novel pathogenic mutations were localized. Functional analysis revealed that three of these mutations affecting amino acids Glu1459, Leu1668 and Glu1724 were critical for histone acetyltransferase activity. Twenty-eight novel CREBBP single-nucleotide polymorphisms (SNPs) were also identified in the Indian population. Linkage disequilibrium studies revealed associations between (i) SNP (rs129974/c.3836-206G>C) and mutation (p.Asp1340Ala); (ii) (rs130002) with mutation (p.Asn435Lys) and (iii) SNPs rs129974, rs130002 and SNP (c.3836-206G>C) signifying a disease affection status. In conclusion, the present study reports the highest detection rate of CREBBP mutations (76.9%) in RSTS patients to date, of which ten are predicted to be pathogenic and three critical for histone acetyltransferase activity. Moreover, identification of the association of CREBBP polymorphisms with disease susceptibility could be an important risk factor for the pathogenesis of RSTS.     

Simulations of the NK cell immune synapse reveal that activation thresholds can be established by inhibitory receptors acting locally

NK cell activation is regulated by a balance between activating and inhibitory signals. To address the question of how these signals are spatially integrated, we created a computer simulation of activating and inhibitory NK cell immunological synapse (NKIS) assembly, implementing either a "quantity-based" inhibition model or a "distance-based" inhibition model. The simulations mimicked the observed molecule distributions in inhibitory and activating NKIS and yielded several new insights. First, the total signal is highly influenced by activating complex dissociation rates but not by adhesion and inhibitory complex dissociation rates. Second, concerted motion of receptors in clusters significantly accelerates NKIS maturation. Third, when the potential of a cis interaction between Ly49 receptors and MHC class I on murine NK cells was added to the model, the integrated signal as a function of receptor and ligand numbers was only slightly increased, at least up to the level of 50% cis-bound Ly49 receptors reached in the model. Fourth, and perhaps most importantly, the integrated signal behavior obtained when using the distance-based inhibition signal model was closer to the experimentally observed behavior, with an inhibition radius of the order 3-10 molecules. Microscopy to visualize Vav activation in NK cells on micropatterned surfaces of activating and inhibitory strips revealed that Vav is only locally activated where activating receptors are ligated within a single NK cell contact. Taken together, these data are consistent with a model in which inhibitory receptors act locally; that is, that every bound inhibitory receptor acts on activating receptors within a certain radius around it.