Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

A pterocarpan from the seeds of Bituminaria morisiana

A new prenylated pterocarpan, named morisianine, was isolated together with the known secondary metabolites erybraedin C, psoralen and angelicin from the seeds of Bituminaria morisiana. The structures of the compounds were elucidated mainly by 1D and 2D NMR experiments as well as mass spectrometry. The new compound was subjected to cytotoxicity screening against a panel of human cancer cells.

Protein overexpression following lentiviral infection of primary mature neutrophils is due to pseudotransduction

Neutrophils are terminally differentiated cells with a short life-span due to constitutive apoptosis. Because of these characteristics, genetic manipulation of neutrophils has been difficult, although it is highly desired given the importance of neutrophils in the immune system. Here we demonstrate that transduction of primary human mature neutrophils with enhanced green fluorescent protein (eGFP)-encoding lentiviral particles results in GFP-containing cells as previously reported. Yet, our data further show that GFP expression in neutrophils upon transduction is largely due to protein transfer, a process called lentiviral pseudotransduction, and not due to bona fide transduction. Thus, inhibition of viral genome integration by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) or of protein biosynthesis by cycloheximide (CHX) did not abolish GFP levels in transduced neutrophils. Importantly, lentiviral pseudotransduction of the enzyme death-associated protein kinase 2 (DAPK2) into primary human mature neutrophils resulted in increased protein levels, but not enzymatic functionality. Based on our data and previous reports of unspecific viral effects on immune cells following lentiviral transduction, we discourage scientists to use lentiviral transduction methods to manipulate primary mature neutrophils.

SerpinB1 protects the mature neutrophil reserve in the bone marrow

SerpinB1 is among the most efficient inhibitors of neutrophil serine proteases--NE, CG, and PR-3--and we investigated here its role in neutrophil development and homeostasis. We found that serpinB1 is expressed in all human bone marrow leukocytes, including stem and progenitor cells. Expression levels were highest in the neutrophil lineage and peaked at the promyelocyte stage, coincident with the production and packaging of the target proteases. Neutrophil numbers were decreased substantially in the bone marrow of serpinB1(-/-) mice. This cellular deficit was associated with an increase in serum G-CSF levels. On induction of acute pulmonary injury, neutrophils were recruited to the lungs, causing the bone marrow reserve pool to be completely exhausted in serpinB1(-/-) mice. Numbers of myeloid progenitors were normal in serpinB1(-/-) bone marrow, coincident with the absence of target protease expression at these developmental stages. Maturation arrest of serpinB1(-/-) neutrophils was excluded by the normal CFU-G growth in vitro and the normal expression in mature neutrophils of early and late differentiation markers. Normal absolute numbers of proliferating neutrophils and pulse-chase kinetic studies in vivo showed that the bone marrow deficit in serpinB1(-/-) mice was largely restricted to mature, postmitotic neutrophils. Finally, upon overnight culture, apoptosis and necrosis were greater in purified bone marrow neutrophils from serpinB1(-/-) compared with WT mice. Collectively, these findings demonstrate that serpinB1 sustains a healthy neutrophil reserve that is required in acute immune responses.

CLLU1 expression distinguishes chronic lymphocytic leukemia from other mature B-cell neoplasms

The distinction of CLL from other mature B-cell neoplasms, especially from leukemic forms of mantle cell lymphoma or splenic marginal zone lymphoma, can be difficult but has important prognostic and therapeutic implications. We measured CLLU1 (CLL upregulated gene1) mRNA by qPCR and found a highly significant difference between CLL and other lymphoid neoplasms (AUC 0.96, 95%CI 0.93-0.99). Based on our cut-off values we can predict CLL and other mature B-cell neoplasms with high probability (PPV 99% and 94%). Analysis of CLLU1 expression is a rapid and reliable tool that may facilitate the diagnosis of mature B-cell neoplasms especially in inconclusive cases.

Irrigants for non-surgical root canal treatment in mature permanent teeth

Root canal treatment is carried out on teeth in which irreversible pulpitis has led to necrosis of the dental pulp. As a treatment option it is an alternative to dental extraction. Mechanical preparation and irrigation with antiseptic or antibacterial solutions destroys bacteria and cleans the infected root canal. Irrigants should be effective in deactivating bacteria in the entire root canal space without causing any adverse tissue reactions. Sodium hypochlorite (NaOCl) and chlorhexidine are commonly used but there is uncertainty as to which solution, concentration or combination is the most effective.

Expression of CD95 on mature leukocytes of MRL/lpr mice after transplantation of genetically modified bone marrow stem cells

Bone marrow transplantation (BMT) is commonly used for the treatment of severe haematological and immunological diseases. For instance, the autoimmune lymphoproliferative syndrome (ALPS) caused by a complete expression defect of CD95 (Fas, APO-1) can be cured by allogeneic BMT. However, since this therapy may not generate satisfactory results when only partially compatible donors are available, we were interested in the development of a potential alternative treatment by using lentiviral gene transfer of a normal copy of CD95 cDNA in hematopoietic stem cells. Here, we show that this approach applied to MRL/lpr mice results in the expression of functional CD95 receptors on the surface of lymphocytes, monocytes, and granulocytes. This suggests that correction of CD95 deficiency can be achieved by gene therapy.

Tissue neogenesis and STRO-1 expression in immature and mature articular cartilage

This study determined the potential for neotissue formation and the role of STRO-1+ cells in immature versus mature articular cartilage. Cartilage explants from immature and mature bovine knee joints were cultured for up to 12 weeks and stained with safranin-O, for type II collagen and STRO-1. Bovine chondrocyte pellet cultures and murine knee joints at the age of 2 weeks and 3 months, and surgically injured cartilage, were analyzed for changes in STRO-1 expression patterns. Results show that immature explants contained more STRO-1+ cells than mature explants. After 8 weeks in culture, immature explants showed STRO-1+ cell proliferation and newly formed tissue, which contained glycosaminoglycan and type II collagen. Mature cartilage explants showed only minimal cell expansion and neotissue formation. Pellet cultures with chondrocytes from immature cartilage showed increased glycosaminoglycan synthesis and STRO-1+ staining, as compared to pellets with mature chondrocytes. The frequency of STRO-1+ cells in murine knee joints significantly declined with joint maturation. Following surgical injury, immature explants had higher potential for tissue repair than mature explants. In conclusion, these findings suggest that the high percentage of STRO-1+ cells in immature cartilage changes with joint maturation. STRO-1+ cells have the potential to form new cartilage spontaneously and after tissue injury. (c) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Gastropod Seed Dispersal: An Invasive Slug Destroys Far More Seeds in Its Gut than Native Gastropods

Seed dispersal is one of the most important mechanisms shaping biodiversity, and animals are one of the key dispersal vectors. Animal seed dispersal can directly or indirectly be altered by invasive organisms through the establishment of new or the disruption of existing seed dispersal interactions. So far it is known for a few gastropod species that they ingest and defecate viable plant seeds and consequently act as seed dispersers, referred to as gastropodochory. In a multi-species experiment, consisting of five different plant species and four different gastropod species, we tested with a fully crossed design whether gastropodochory is a general mechanism across native gastropod species, and whether it is altered by the invasive alien slug species Arion lusitanicus. Specifically, we hypothesized that a) native gastropod species consume the seeds from all tested plant species in equal numbers (have no preference), b) the voracious invasive alien slug A. lusitanicus – similarly to its herbivore behaviour – consumes a higher amount of seeds than native gastropods, and that c) seed viability is equal among different gastropod species after gut passage. As expected all tested gastropod species consumed all tested plant species. Against our expectation there was a difference in the amount of consumed seeds, with the largest and native mollusk Helix pomatia consuming most seeds, followed by the invasive slug and the other gastropods. Seed damage and germination rates did not differ after gut passage through different native species, but seed damage was significantly higher after gut passage through the invasive slug A. lusitanicus, and their germination rates were significantly reduced.

Do mature pulmonary lobes grow after transplantation into an immature recipient?

BACKGROUND The use of reduced-size adult lung transplants could help solve the profound pediatric donor lung shortage. However, adequate long-term function of the mature grafts requires growth in proportion to the recipient's development. METHODS Mature left lower lobes from adult mini-pigs (age: 7 months; mean body weight: 30 kg) were transplanted into 14-week-old piglets (mean body weight: 15 kg). By the end of the 14-week holding period, lungs of the recipients (n = 4) were harvested. After volumetric measurements, the lung morphology was studied using light microscopy, scanning, and transmission electron microscopy. Changes of alveolar airspace volume were determined using a computer aided image analysis system. Comparisons were made to age- and weight-matched controls. RESULTS Volumetric studies showed no significant differences (p = 0.49) between the specific volume (mL/kg body weight) of lobar grafts and left lower lobes of adult controls. Morphologic studies showed marked structural differences between the grafts and the right native lungs of the recipients, with increased average alveolar diameter of the grafts. On light microscopy and scanning electron microscopy, alveoli appeared dilated and rounded compared to the normal polygonal shape in the controls. The computer generated semi-quantitative data of relative alveolar airspace volume tended to be higher in transplanted lobes. CONCLUSIONS The mature pulmonary lobar grafts have filled the growing left hemithorax of the developing recipient. Emphysema-like alterations of the grafts were observed without evidence of alveolar growth in the mature lobar transplants. Thus, it can be questioned whether mature pulmonary grafts can guarantee sufficient long-term gas exchange in growing recipients.

Intra-annual dynamics of non-structural carbohydrates in the cambium of mature conifer trees reflects radial growth demands

The presence of soluble carbohydrates in the cambial zone, either from sugars recently produced during photosynthesis or from starch remobilized from storage organs, is necessary for radial tree growth. However, considerable uncertainties on carbohydrate dynamics and the consequences on tree productivity exist. This study aims to better understand the variation in different carbon pools at intra-annual resolution by quantifying how cambial zone sugar and starch concentrations fluctuate over the season and in relation to cambial phenology. A comparison between two physiologically different species growing at the same site, i.e., the evergreen Picea abies Karst. and the deciduous Larix decidua Mill., and between L. decidua from two contrasting elevations, is presented to identify mechanisms of growth limitation. Results indicate that the annual cycle of sugar concentration within the cambial zone is coupled to the process of wood formation. The highest sugar concentration is observed when the number of cells in secondary wall formation and lignification stages is at a maximum, subsequent to most radial growth. Starch disappears in winter, while other freeze-resistant non-structural carbohydrates (NSCs) increase. Slight differences in NSC concentration between species are consistent with the differing climate sensitivity of the evergreen and deciduous species investigated. The general absence of differences between elevations suggests that the cambial activity of trees growing at the treeline was not limited by the availability of carbohydrates at the cambial zone but instead by environmental controls on the growing season duration.