Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease

During chronic injury a population of bipotent hepatic progenitor cells (HPCs) become activated to regenerate both cholangiocytes and hepatocytes. Here we show in human diseased liver and mouse models of the ductular reaction that Notch and Wnt signaling direct specification of HPCs via their interactions with activated myofibroblasts or macrophages. In particular, we found that during biliary regeneration, expression of Jagged 1 (a Notch ligand) by myofibroblasts promoted Notch signaling in HPCs and thus their biliary specification to cholangiocytes. Alternatively, during hepatocyte regeneration, macrophage engulfment of hepatocyte debris induced Wnt3a expression. This resulted in canonical Wnt signaling in nearby HPCs, thus maintaining expression of Numb (a cell fate determinant) within these cells and the promotion of their specification to hepatocytes. By these two pathways adult parenchymal regeneration during chronic liver injury is promoted.     

Benthic communities in the Southern Bight of the North Sea and their use in ecological monitoring

Macrobenthic and meiobenthic communities of an area off the Belgian coast of the North Sea were studied from 1970 until 1975 at 74 stations. On the basis of both macro- and meiobenthos, three zones can be distinguished in the area. The coastal zone is characterized by the macrobenthicAbra alba community, corresponding to the meiobenthicMicroarthridion littorale — Halectinosoma herdmani community, and the open sea zone by the macrobenthicVenus gallina community and the meiobenthicLeptastacus laticaudatus — Paramesochra helgolandica community. In between is a transient zone where elements of both other zones mix. The distribution of these zones is governed by the hydrodynamical regime of the region, especially by the residual and tidal current system of the Southern Bight. Within the coastal zone, the composition of the community is influenced by pollution which especially affects the epibenthic detritus-feeders of the meiobenthos. The spatial stability of parameters describing community structure can be used for monitoring changes. Temporal characteristics of these parameters could not be investigated properly, but diversity seems to be much stabler than biomass.     

In search for cross-reactivity to immunophenotype equine mesenchymal stromal cells by multicolor flow cytometry

During recent years, cell-based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate positive controls to confirm their specificity. Cross-reactivity was found and confirmed by confocal microscopy for CD45, CD73, CD79α, CD90, CD105, MHC-II, a monocyte marker, and two clones tested for CD29 and CD44. Unfortunately, none of the evaluated CD34 clones recognized the equine epitopes on positive control endothelial cells. Subsequently, umbilical cord blood-derived undifferentiated equine MSC of the fourth passage of six horses were characterized using multicolor flow cytometry based on the selected nine-marker panel of both cell surface antigens and intracytoplasmatic proteins. In addition, appropriate positive and negative controls were included, and the viable single cell population was analyzed by excluding dead cells using 7-aminoactinomycin D. Isolated equine MSC of the fourth passage were found to be CD29, CD44, CD90 positive and CD45, CD79α, MHC-II, and a monocyte marker negative. A variable expression was found for CD73 and CD105. Successful differentiation towards the osteogenic, chondrogenic, and adipogenic lineage was used as additional validation. We suggest that this selected nine-marker panel can be used for the adequate immunophenotyping of equine MSC.     

Gene expression changes in melanoma metastases in response to high-dose chemotherapy during isolated limb perfusion

Despite recent advances in melanoma therapy, disseminated melanoma still lacks effective treatment, and recurrence of the tumor frequently occurs, even after high-dose chemotherapy. The mechanisms responsible for this chemoresistance or for the formation of new relapses remain poorly understood. Using a human 'model', in which the isolated limb is perfused with high doses of the chemotherapeutic melphalan (ILP), we identified a five-gene set (ATF3, CYR61, IER5, IL6, and PTGS2) of stress-induced genes that was consistently upregulated after ILP in all in-transit metastatic melanoma samples as well as in three melphalan-treated melanoma cell lines. Early post-ILP relapses retained these elevated expressions, whereas the expression of these genes returned to their original levels in late post-ILP recurrences. In addition, we identified upregulation of these genes in the A375 cell line's side population (SP) and melanospheres, established methods to enrich for candidate cancer stem cells (CSCs), which are considered chemoresistant and tumorigenic, and thus proposed to be responsible for tumor relapse. Our data identify an immediate and short-term upregulation of early stress-responsive genes that are potentially linked to chemoresistance and CSCs.     

The Human Melanoma Side Population Displays Molecular and Functional Characteristics of Enriched Chemoresistance and Tumorigenesis

Melanoma remains the most lethal skin cancer, mainly because of high resistance to therapy. Side population (SP) cells are found in many types of cancer and are usually enriched in therapy-resistant as well as tumorigenic cells. Here, we identified a Hoechst dye-effluxing SP in a large series of human melanoma samples representing different progression phases. The SP size did not change with disease stage but was correlated with the prognostic “Breslow’s depth” in the primary (cutaneous) tumors. When injected into immunodeficient mice, the SP generated larger tumors than the bulk “main population” (MP) melanoma cells in two consecutive generations, and showed tumorigenic capacity at lower cell numbers than the MP. In addition, the SP reconstituted the heterogeneous composition of the human A375 melanoma cell line, and its clonogenic activity was 2.5-fold higher than that of the MP. Gene-expression analysis revealed upregulated expression in the melanoma SP (versus the MP) of genes associated with chemoresistance and anti-apoptosis. Consistent with these molecular characteristics, the SP increased in proportion when A375 cells were exposed to the melanoma standard chemotherapeutic agent dacarbazine, and to the aggravating condition of hypoxia. In addition, the SP showed enhanced expression of genes related to cell invasion and migration, as well as to putative (melanoma) cancer stem cells (CSC) including ABCB1 and JARID1B. ABCB1 immunoreactivity was detected in a number of tumor cells in human melanomas, and in particular in clusters at the invasive front of the primary tumors. Together, our findings support that the human melanoma SP is enriched in tumorigenic and chemoresistant capacity, considered key characteristics of CSC. The melanoma SP may therefore represent an interesting therapeutic target.     

Effect of myocardial infarction and ischemia on induction of cardiac reentries and ventricular fibrillation

The present work is aimed at investigating the effects of myocardial infarction and ischemia on induction of ventricular fibrillation. Electrophysiologic effects of global and local ischemia (variation of the dispersion of refractory periods as well as conduction velocity) on initiation of reentry mechanisms was studied by means of computer simulations based on a cellular automata model of propagation of activation wave through a ventricular surface element. A local area of ischemia where effects of the dispersion of refractory periods are investigated is then simulated. This is made using a Gaussian distribution characterized by its mean and standard deviation. These simulations show that ischemia is capable of initiating reentry phenomena which propagate through the whole ventricle; they are responsible for ventricular fibrillation which causes sudden cardiac death, even when ischemia only involves limited parts of the myocardium. Statistical study of the probability of reentries as a function of both of the size of ischemic zones and the rate of dispersion of refractory periods shows that the latter parameter is of primary importance in triggering cardiac reentries.     

Validation and usefulness of the Sperm Quality Analyzer V equine for equine semen analysis

Routine semen analysis includes evaluation of concentration combined with seminal volume, morphology and motility. Subjective analysis of these parameters is known to be inaccurate, imprecise and subject to variability. Automated semen analysis could lead to an increased standardization in and between laboratories but for that to happen automated devices need to be validated. A new device, the sperm quality analyzer V equine (SQA-Ve) version 1.00.43, was evaluated for its repeatability and agreement with light microscopy (LM), for raw and extended equine semen. Results were compared with computer assisted sperm analysis (CASA), which was also tested for its repeatability and agreement with LM. The SQA-Ve showed a good repeatability and fine agreement for assessing sperm concentration of raw semen based on scatter and Bland-Altman plots. This was in contrast with the motility parameters, which had a low repeatability. Morphology assessment with SQA-Ve was poorly repeatable as well as in poor agreement with LM. For extended semen, the findings were comparable. The SQA-Ve did well for concentration, whereas for the motility parameters repeatability was only just acceptable, with no agreement with LM. This sharply contrasted the CASA findings that were highly repeatable and almost in perfect agreement with LM. Based on these findings, the tested version of the SQA-Ve is insufficiently accurate to be used for analyzing raw or extended equine semen.     

Composition,distribution and biomass of meiobenthos in the Oosterschelde estuary (SW Netherlands)

Meiofauna composition, abundance, biomass, distribution and diversity were investigated for 31 stations in summer. The sampling covered the whole Oosterschelde and comparisons between the subtidal — intertidal and between the western-central — eastern compartment were made.Meiofauna had a community density ranging between 200 and 17 500 ind 10 cm^–2, corresponding to a dry weight of 0.2 and 8.4 gm^–2. Abundance ranged between 130 and 17 200 ind 10 cm^–2 for nematodes and between 10 and 1600 ind 10 cm^–2 for copepods. Dry weight biomass of these taxa was between 0.5–7.0 gm^–2 and 0.008–0.3 gm^–2 for nematodes and copepods respectively.The meiofauna was strongly dominated by the nematodes (36–99%), who's abundance, biomass and diversity were significantly higher intertidally than subtidally and significantly higher in the eastern part than in the western part. High numbers were positively correlated with the percentage silt and negatively with the median grain size of the sand fraction. The abundance and diversity of the copepods were highest in the subtidal, but their biomass showed an inverse trend being highest on the tidal flats.The taxa diversity of the meiofauna community and species diversity of both the nematodes and the copepods were higher in subtidal stations than on tidal flats. In the subtidal, the meiofauna and copepod diversity decreased from west to east, whereas nematode diversity increased.The vertical profile clearly reflected the sediment characteristics and could be explained by local hydrodynamic conditions.Seasonal variation was pronounced for the different taxa with peak abundance in spring, summer or autumn and minimum abundance in winter.Changes in tidal amplitude and current velocity enhanced by the storm-surge barrier will alter the meiofauna community structure. As a result meiofauna will become more important in terms of density and biomass, mainly due to increasing numbers of nematodes, increasing bioturbation, nutrient mineralisation and sustaining bacterial growth. A general decrease in meiofauna diversity is predicted. The number of copepods is expected to decrease and interstitial species will be replaced by epibenthic species, the latter being more important in terms of biomass and as food for the epibenthic macrofauna and fishes.     

Influence of different centrifugation protocols on equine semen preservation

Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600 × g for 10 min = CP1) was compared to four protocols with increasing g-force and decreased time period (600 × g, 1200 × g, 1800 × g and 2400 × g for 5 min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following centrifugation was assessed by monitoring several sperm parameters (membrane integrity using SYBR14-PI, acrosomal status using PSA-FITC, percentage total motility (TM), percentage progressive motility (PM) and beat cross frequency (BCF) obtained with computer assisted sperm analysis (CASA)) immediately after centrifugation and daily during chilled storage for 3 d. The use of CP1 resulted in a sperm loss of 22%. Increasing the centrifugation force to 1800 × g and 2400 × g for 5 min led to significantly lower sperm losses (7.4% and 2.1%, respectively; P < 0.05). Compared to the uncentrifuged samples, centrifugation of semen resulted in a better sperm quality after chilled storage. There were minimal differences between the CPs although total motility was lower for CP2 than for the other treatments (P < 0.005). In experiment 2, the centrifuged samples were cryopreserved using a standard freezing protocol and analyzed immediately upon thawing. Samples centrifuged according to CP2 resulted in a higher BCF (P < 0.005), whereas CP3 and CP5 yielded a lower BCF (P < 0.05) when compared to CP1. There were no post thaw differences between CP1 and CP4. In experiment 3, DNA integrity of the different samples was analyzed using TUNEL. Although DNA integrity decreased over time, CP had no impact. In conclusion, the loss of sperm cells in the supernatant after centrifugation can be substantially reduced by increasing the g-force up to 1800 × g or 2400 × g for a shorter period of time (5 min) compared to the standard protocol without apparent changes in semen quality, resulting in a considerable increase in the number of insemination doses per ejaculate.