Hematopoietic reconstitution of syngeneic mice with a peripheral blood-derived, monoclonal CD34-, Sca-1+, Thy-1(low), c-kit+ stem cell line

Previous work had revealed that a CD34- fibroblast-like cell is the earliest hematopoietic progenitor population. This cell type is able to differentiate into hematopoietic progeny of all lineages and circulates in the peripheral blood, from where it can be isolated by IL-6-mediated plastic adherence. We isolated peripheral blood-derived mononuclear cells (MNC) from male CBA mice and established in vitro a fibroblast-like, adherent growing cell layer. Cells were immortalized by SV-40 transfection for cellular cloning. Monoclonal fibroblast-like cell clones were established, and the surface expression of early stem cell markers was determined by flow cytometry. Clones were CD34-, Sca-1+, Thy-1(low), and c-kit+. Lethally irradiated female CBA mice were successfully transplanted with a fibroblast-like cell clone, R-M26/2-1. After syngeneic transplantation, peripheral blood counts were back to normal in transplanted mice on days 15-20, and fluorescence in situ hybridization (FISH) revealed the sole presence of male hematopoietic cells in the BM of female recipients at weeks 7, 9, 11, and 16 after transplantation. Immunohistochemistry for the expression of CD34, Sca-1, Thy-1, and c-kit showed the presence of the phenotype of the transplanted stem cell clone along the bone spicules in the marrow cavity, giving rise to HPC of all lineages. In summary, we have shown that a CD34-, Sca-1+, Thy-1(low), and c-kit+ fibroblast-like cell is consistent with the phenotype of the earliest hematopoietic and repopulating stem cell and can be isolated from peripheral blood cells.     

G-protein-coupled receptors in normal human erythroid progenitor cells

Human erythroid progenitor cells were isolated from peripheral blood of healthy donors and amplified in a suspension culture system using recombinant growth factors (stem cell factor, interleukin-3, granulocyte-macrophage colony-stimulating factor and erythropoietin) as well as conditioned medium from a human bone marrow stroma cell line to support cell proliferation. After 6–8 days of culture, the cell population consisted mainly of erythroid colony-forming cells (burst-forming units, BFU-Es and colony-forming units, CFU-Es). In these cells, we studied ligand-induced changes in intracellular Ca ²⁺ concentration ([Ca²⁺]_i) and cAMP formation as the primary effector systems of guanine nucleotide-binding protein (G protein)-coupled receptors. The results confirmed the functional expression of receptors for adenosine (type A_2B), prostaglandin Et and isoprenaline (\-adrenoceptor), all of which stimulated adenylyl cyclase, as well as for ADP (purinoceptor types P_2T and P_2U), platelet-activating factor and thrombin all of which caused a transient increase in [Ca²⁺]_j. The efficacy of adenosine and prostaglandin El in stimulating cAMP formation was more than 5 times higher than that of isoprenaline, suggesting a low \-adrenoceptor density. The response to adenosine and isoprenaline decreased by 80 and 55% respectively during maturation into the proerythroblast stage. Similarly, thapsigargin-sensitive intracellular Ca ²⁺ stores and ligand-induced Ca ²⁺ release declined by about 60% during the CFU-E-to-erythroblast transition. The overall functional expression pattern of G protein-coupled receptors differed from that in human erythroleukaemia cell lines or from that in platelets. Primary culture systems for nontransformed cells, such as the one presented here, thus will be indispensable for the study of the functional role of G protein-dependent signalling during haematopoiesis.     

Establishment of two permanent human bone marrow stromal cell lines with long-term post irradiation feeder capacity

We describe the establishment of two permanent Simian virus 40- transformed human stromal cell lines, designated L87/4 and L88/5, derived from the bone marrow of a hematologically normal male patient. Both cell lines show a fibroblastoid morphology and do not express hematopoietic cell markers. L87/4 but not L88/5 expresses the macrophage marker CD68. The most remarkable feature of these new stromal cell lines is their ability to persist as growth-arrested adherent feeder cells after ionizing-irradiation at doses up to, and exceeding 20 Gy (L87/4). This renders them particularly useful for studying aspects of feeder dependence of hematopoietic cell development in long-term culture. Both cell lines are able to function as feeder cells, supporting the long-term proliferation of CD34+ human cord blood cells as well as the clonogenic growth of the human Burkitt lymphoma B- cell line BL70.     

Mesenchymal differentiation and organ distribution of established human stromal cell lines in NOD/SCID mice.

Two human stromal cell lines were established previously from bone marrow-derived primary long-term cultures by immortalization using the SV40 large T antigen and cellular cloning. After irradiation, the fibroblast-like cell lines L87/4 and L88/5 support hematopoietic differentiation of allogeneic cord blood cells in vitro. The stromal cells do not express CD34 and CD50, but some adhesion molecules and integrins, such as CD44, CD54 and CD58. Their expression profiles on RNA and protein levels are suggestive of their osteogenic potency. The quality and quantity of osteocalcin and osteopontin protein expression depended on the culture conditions. Expression of the osteogenic markers increased over time in culture, especially in cells growing in clusters. The stromal cells also expressed collagens I and V, but did not show any expression of collagens II and III. The potentially osteoblastic stromal cells were transplanted into NOD/ SCID recipient mice by intravenous injection and were found in various mesenchymal organs up to 10 weeks after transplantation. Osteocalcin-positive human stromal cells could be detected in the bone marrow, thymus, liver, brain and gut of the recipient animals. In summary, there is evidence that human bone-marrow-derived stromal cells have to be considered mesenchymal progenitors, persistently expressing osteogenic markers in vitro and in vivo.     

Molecular genetic findings in suicidal behavior: what is beyond the serotonergic system?

Various studies provide consistent evidence for a genetic component in suicidal behavior. First molecular genetic studies concentrated on genes of the serotonergic system based on the biochemical evidence that serotonergic neurotransmission is implicated in this behavior. Furthermore, genes of the dopaminergic and noradrenergic neurotransmitter systems have also been the subjects of investigations in this context. Some epidemical and clinical studies showed that low serum cholesterol levels are associated with suicidal behavior and genes involved in these pathways have been investigated. Microarray experiments provide the possibility of genome-wide gene expression analysis and help to investigate associated molecular mechanisms. The aim of this article is to review molecular genetic studies in suicidal behavior and to emphasize findings on new genes.     

The free choice whether or not to respond after stimulus presentation

The concept of ‘willed’ actions has attracted attention during the last few years. Free choices have been associated with activations on the medial frontal surface, the dorsolateral prefrontal cortex and the parietal lobe. Self‐paced movements and free selection between various motor responses were typically used to investigate voluntary behavior. The aim of the present study was to determine neural correlates of voluntary motor responses and the voluntary inhibition of motor responses in a group of healthy subjects. Hence, a go/nogo/voluntary selection paradigm was used. In the voluntary selection condition subjects decided freely whether or not to respond with a button press after stimulus presentation. Functional MRI data and event‐related potentials were acquired simultaneously in order to reliably investigate spatial and temporal characteristics of these responses. The results showed decision‐related enhanced neural responses predominantly in the medial frontal gyrus/supplementary motor area, lateral frontal brain regions and the inferior parietal gyrus. Additional activations associated with voluntary movements were detected in the frontal eye field as well as brain regions directly linked to motor responses (e.g. somatosensory cortical areas). Altogether, decision processes were shown to be relatively independent of the kind of response chosen. Hum Brain Mapp 2009. © 2009 Wiley‐Liss, Inc.     

Volumenberechnungen zur aktuellen intravitrealen Injektion von Lucentis®

Background

Medications for blocking vascular endothelial growth factor (VEGF) are currently used for therapy of neovascular age-related macular degeneration (AMD). Some users divide the original volume of the medication Lucentis® into smaller aliquots to be used for several patients. The aim of this study was to investigate whether the current original aliquot volume of Lucentis® can principally be subdivided to be used for treatment two patients each with a 0.05 ml injection volume.

Materials and methods

The calculation of the dead space volume of the vessels used for injection was carried out by weighing the empty and full weights using an analytical balance. The sum of all dead space volumes was subtracted from the filling volumes. Additionally, the average extractable volume was determined from 200 original aliquots of Lucentis®.

Results

Taking all necessary dead space volumes into consideration it was calculated that an extractable volume of at least 0.137 ml is necessary in order to be able to inject the necessary volume of 0.05 ml of the solution containing the active ingredient. The original volume of 0.23 ml contained in an aliquot of Lucentis® cannot therefore be divided among two patients. The average extractable volume of 0.16 ml is clearly less than the full volume of 0.23 ml.

Conclusions

The original aliquots of Lucentis® contain a full volume of 0.23 ml and are not suitable to be used to treat two patients. The volume can only be divided among several patients if several original aliquots are pooled and refilled. This however, presupposes a renewed filling of aliquots which represents a new potential source of contamination. Also legal aspects and the question of stability of the active ingredient have not been taken into consideration.