Is differentiation of frequently encountered foreign bodies in corpses possible by Hounsfield density measurement?

The radiological determination of foreign objects in corpses can be difficult if they are fragmented or deformed. With multislice computed tomography, radiodensities--referred to as Hounsfield units (HU)--can be measured. We examined the possibility of differentiating 21 frequently occurring foreign bodies, such as metals, rocks, and different manmade materials by virtue of their HU values. Gold, steel, and brass showed mean HU values of 30671-30710 (upper measurable limit), mean HU values for steel, silver, copper, and limestone were 20346, 16949, 14033, and 2765, respectively. The group consisting of objects, such as aluminum, tarmac, car front-window glass, and other rocks, displayed mean HU values of 2329-2131 HU. The mean HU value of bottle glass and car side-window glass was 2088, whereas windowpane glass was 493. HU value determination may therefore help in preautopsy differentiation between case-relevant and irrelevant foreign bodies and thus be useful for autopsy planning and extraction of the objects in question.

Identification of mesenchymal stromal cells in human lung parenchyma capable of differentiating into aquaporin 5-expressing cells

The lack of effective therapies for end-stage lung disease validates the need for stem cell-based therapeutic approaches as alternative treatment options. In contrast with exogenous stem cell sources, the use of resident progenitor cells is advantageous considering the fact that the lung milieu is an ideal and familiar environment, thereby promoting the engraftment and differentiation of transplanted cells. Recent studies have shown the presence of multipotent 'mesenchymal stem cells' in the adult lung. The majority of these reports are, however, limited to animal models, and to date, there has been no report of a similar cell population in adult human lung parenchyma. Here, we show the identification of a population of primary human lung parenchyma (pHLP) mesenchymal stromal cells (MSCs) derived from intraoperative normal lung parenchyma biopsies. Surface and intracellular immunophenotyping by flow cytometry revealed that cultures do not contain alveolar type I epithelial cells or Clara cells, and are devoid of the following hematopoietic markers: CD34, CD45 and CXCR4. Cells show an expression pattern of surface antigens characteristic of MSCs, including CD73, CD166, CD105, CD90 and STRO-1. As per bone marrow MSCs, our pHLP cells have the ability to differentiate along the adipogenic, osteogenic and chondrogenic mesodermal lineages when cultured in the appropriate conditions. In addition, when placed in small airway growth media, pHLP cell cultures depict the expression of aquaporin 5 and Clara cell secretory protein, which is identified with that of alveolar type I epithelial cells and Clara cells, respectively, thereby exhibiting the capacity to potentially differentiate into airway epithelial cells. Further investigation of these resident cells may elucidate a therapeutic cell population capable of lung repair and/or regeneration.

Land use causes genetic differentiation of life-history traits in Bromus hordeaceus

There is increasing evidence that species can evolve rapidly in response to environmental change. However, although land use is one of the key drivers of current environmental change, studies of its evolutionary consequences are still fairly scarce, in particular studies that examine land-use effects across large numbers of populations, and discriminate between different aspects of land use. Here, we investigated genetic differentiation in relation to land use in the annual grass Bromus hordeaceus. A common garden study with offspring from 51 populations from three regions and a broad range of land-use types and intensities showed that there was indeed systematic population differentiation of ecologically important plant traits in relation to land use, in particular due to increasing mowing and grazing intensities. We also found strong land-use-related genetic differentiation in plant phenology, where the onset of flowering consistently shifted away from the typical time of management. In addition, increased grazing intensity significantly increased the genetic variability within populations. Our study suggests that land use can cause considerable genetic differentiation among plant populations, and that the timing of land use may select for phenological escape strategies, particularly in monocarpic plant species.

Distinct carbon sources indicate strong differentiation between tropical forest and farmland bird communities

The conversion of forest into farmland has resulted in mosaic landscapes in many parts of the tropics. From a conservation perspective, it is important to know whether tropical farmlands can buffer species loss caused by deforestation and how different functional groups of birds respond to land-use intensification. To test the degree of differentiation between farmland and forest bird communities across feeding guilds, we analyzed stable C and N isotopes in blood and claws of 101 bird species comprising four feeding guilds along a tropical forest-farmland gradient in Kenya. We additionally assessed the importance of farmland insectivores for pest control in C4 crops by using allometric relationships, C stable isotope ratios and estimates of bird species abundance. Species composition differed strongly between forest and farmland bird communities. Across seasons, forest birds primarily relied on C3 carbon sources, whereas many farmland birds also assimilated C4 carbon. While C sources of frugivores and omnivores did not differ between forest and farmland communities, insectivores used more C4 carbon in the farmland than in the forest. Granivores assimilated more C4 carbon than all other guilds in the farmland. We estimated that insectivorous farmland birds consumed at least 1,000 kg pest invertebrates km−2 year−1. We conclude that tropical forest and farmland understory bird communities are strongly separated and that tropical farmlands cannot compensate forest loss for insectivorous forest understory birds. In tropical farmlands, insectivorous bird species provide a quantitatively important contribution to pest control.

In vitro differentiation of near-unlimited numbers of functional mouse basophils using conditional Hoxb8

Background: Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought-after tool for diagnostic and drug testing purposes. Methods: A method is described to massively expand bone marrow–derived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin-3 (IL-3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression. Results: IL-3-dependent, conditional Hoxb8-immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near-unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end-differentiated and short-lived and express basophil-specific surface markers and proteases. Upon IgE- as well as C5a-mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2-type cytokines (IL-4, IL-13) and leukotriene C4. IL-3-deprivation induces apoptosis correlating with upregulation of the BH3-only proteins BCL-2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM-1). Conclusion: A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.

Enamel matrix derivative inhibits adipocyte differentiation of 3T3-L1 cells via activation of TGF-βRI kinase activity

Enamel matrix derivative (EMD), an extract of fetal porcine enamel, and TGF-β can both suppress adipogenic differentiation. However, there have been no studies that functionally link the role of EMD and TGF-β in vitro. Herein, we examined whether TGF-β signaling contributes to EMD-induced suppression of adipogenic differentiation. Adipogenesis was studied with 3T3-L1 preadipocytes in the presence of SB431542, an inhibitor of TGF-βRI kinase activity. SB431542 reversed the inhibitory effect of EMD on adipogenic differentiation, based on Oil Red O staining and mRNA expression of lipid regulated genes. SB431542 also reduced EMD-stimulated expression of connective tissue growth factor (CTGF), an autocrine inhibitor of adipogenic differentiation. Moreover, short interfering (si)RNAs for CTGF partially reversed the EMD-induced suppression of lipid regulated genes. We conclude that the TGF-βRI - CTGF axis is involved in the anti-adipogenic effects of EMD in vitro.

Influence of enamel matrix derivative on cells at different maturation stages of differentiation

Enamel matrix derivative (EMD), a porcine extract harvested from developing porcine teeth, has been shown to promote formation of new cementum, periodontal ligament and alveolar bone. Despite its widespread use, an incredibly large variability among in vitro studies has been observed. The aim of the present study was to determine the influence of EMD on cells at different maturation stages of osteoblast differentiation by testing 6 cell types to determine if cell phenotype plays a role in cell behaviour following treatment with EMD. Six cell types including MC3T3-E1 pre-osteoblasts, rat calvarial osteoblasts, human periodontal ligament (PDL) cells, ROS cells, MG63 cells and human alveolar osteoblasts were cultured in the presence or absence of EMD and proliferation rates were quantified by an MTS assay. Gene expression of collagen1(COL1), alkaline phosphate(ALP) and osteocalcin(OC) were investigated by real-time PCR. While EMD significantly increased cell proliferation of all cell types, its effect on osteoblast differentiation was more variable. EMD significantly up-regulated gene expression of COL1, ALP and OC in cells early in their differentiation process when compared to osteoblasts at later stages of maturation. Furthermore, the effect of cell passaging of primary human PDL cells (passage 2 to 15) was tested in response to treatment with EMD. EMD significantly increased cell proliferation and differentiation of cells at passages 2-5 however had completely lost their ability to respond to EMD by passages 10+. The results from the present study suggest that cell stimulation with EMD has a more pronounced effect on cells earlier in their differentiation process and may partially explain why treatment with EMD primarily favors regeneration of periodontal defects (where the periodontal ligament contains a higher number of undifferentiated progenitor cells) over regeneration of pure alveolar bone defects containing no periodontal ligament and a more limited number of osteoprogenitor cells.

Molecular phylogenetic analyses identify Alpine differentiation and dysploid chromosome number changes as major forces for the evolution of the European endemic Phyteuma (Campanulaceae)

Phyteuma is a chromosomally and ecologically diverse vascular plant genus and constitutes an excellent system for studying both the role of chromosomal change for species diversification and the evolution of high-mountain biota. This kind of research is, however, hampered by the lack of a sound phylogenetic framework exacerbated by the notoriously low predictive power of traditional taxonomy with respect to phylogenetic relationships in Campanulaceae. Based on a comprehensive taxon sampling and analyses of nuclear and plastid sequence and AFLP fingerprint data, Phyteuma is confirmed as a monophyletic group sister to the monotypic Physoplexis, which is in line with their peculiar flower morphologies. Within Phyteuma two clades, largely corresponding to previously recognized sections, are consistently found. The traditional circumscription of taxonomic series is largely rejected. Whereas distinctness of the currently recognized species is mostly corroborated, some interspecific relationships remain ambiguous due to incongruences between nuclear and plastid data. Major forces for diversification and evolution of Phyteuma are descending dysploidy (i.e., a decrease in chromosome base number) as well as allopatric and ecological differentiation within the Alps, the genus' center of species diversity. (C) 2013 Elsevier Inc. All rights reserved.

Platelets increase while serum reduces the differentiation and activity of osteoclasts in vitro

Platelets modulate formation of osteoclasts and osteoblasts, but research with different preparations of platelets remains inconclusive. Here, we assessed whether serum components modulate the effect of platelet preparations. In murine bone marrow cultures, osteoclastogenesis was investigated in the presence of platelet-released supernatant (PRS), serum containing PRS (SC-PRS), and serum. Osteoclastogenesis was quantified by the numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, TRAP activity and resorption assays. Also human osteoclastogenesis assays were performed. Viability and proliferation were tested by MTT and (3) [H]thymidine incorporation assays, respectively. Osteoblastogenesis was assessed by histochemical staining for alkaline phosphatase-of murine bone marrow cultures and human MG63 cells. We found PRS to increase the number of TRAP(+) multinucleated cells in the early phase and TRAP activity in the later phase of osteoclastogenesis. SC-PRS and serum decreased the number and activity of TRAP(+) multinucleated cells. Both serum containing preparations reduced viability and proliferation of hematopoietic progenitors. PRS decreased the numbers of alkaline phosphatase-positive colonies while SC-PRS and serum increased osteoblastmarkers in MG63. Proliferation of MG63 was stimulated by all preparations. These results show that activated platelets support osteoclastogenesis, while platelet preparations that contain serum components decrease osteoclastogenesis and increase osteoblastogenesis in vitro, suggesting that serum components modulate the effects of platelets on osteoclastogenesis and osteoblastogenesis.

Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

Thermoreversible hyaluronan-based hydrogel supports in vitro and ex vivo disc-like differentiation of human mesenchymal stem cells

BACKGROUND CONTEXT The fate of human mesenchymal stem cells (hMSCs) supplied to the degenerating intervertebral disc (IVD) is still not fully understood and can be negatively affected by low oxygen, pH, and glucose concentration of the IVD environment. The hMSC survival and yield upon injection of compromised IVD could be improved by the use of an appropriate carrier and/or by predifferentiation of hMSCs before injection. PURPOSE To optimize hMSC culture conditions in thermoreversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-pNIPAM), to achieve differentiation toward the disc phenotype in vitro, and evaluate whether preconditioning contributes to a better hMSC response ex vivo. STUDY DESIGN In vitro and ex vivo whole-organ culture of hMSCs. METHODS In vitro cultures of hMSCs were conducted in HA-pNIPAM and alginate for 1 week under hypoxia in chondropermissive medium alone and with the supplementation of transforming growth factor β1 or growth and differentiation factor 5 (GDF-5). Ex vivo, hMSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or predifferentiated with GDF-5 for 1 week in HA-pNIPAM and then supplied to the IVDs. Cell viability was evaluated by Live-Dead assay, and DNA, glycosaminoglycan (GAG), and gene expression profiles were used to assess hMSC differentiation toward the disc phenotype. RESULTS The HA-pNIPAM induced hMSC differentiation toward the disc phenotype more effectively than alginate: in vitro, higher GAG/DNA ratio and higher collagen type II, SOX9, cytokeratin-19, cluster of differentiation 24, and forkhead box protein F1 expressions were found for hMSCs cultured in HA-pNIPAM compared with those cultured in alginate, regardless of the addition of growth factors. Ex vivo, direct combination of HA-pNIPAM with the disc environment induced a stronger disc-like differentiation of hMSCs than predifferentiation of hMSCs followed by their delivery to the discs. CONCLUSIONS Hyaluronan-based thermoreversible hydrogel supports hMSC differentiation toward the disc phenotype without the need for growth factor supplementation in vitro and ex vivo. Further in vivo studies are required to confirm the suitability of this hydrogel as an effective stem cell carrier for the treatment of IVD degeneration.