Chordoma and its embryonic determinants

Background. Chordomas are rare malignant tumors occurring along the axial skeleton that originate from notochordal remnants. It is hypothesized that embryonic factors are involved in their aetiology. In the present study, four such factors are of interest: EVX1, which mediates posterior patterning of the embryo and is found in prostate. Wnt3a, localized to the placenta, is involved in different proliferation signaling pathways. Twist is a mediator of the epithelial-mesenchymal transition (EMT) and is expressed in the neuroblastoma cell line SHSY 5Y. Finally, brachyury is a crucial EMT mediator and involved in cell cycle regulation. Research question. The research question is tripartite. (I) It is the aim to obtain positive control tissue for validation of embryonic primers for the EVX1, Wnt3a and Twist genes. (II) Second, expression of brachyury mRNA in cultured chordoma cells will be assessed in order to validate the cell line. (III) Last, it will be investigated whether fibroblast conditioned cell culturing medium exerts a positive effect on chordoma proliferation in vitro. Materials and methods. (I) Placenta and prostate samples were obtained from patients in the Maastricht University Hospital. mRNA was isolated from prostate, placenta and SHSY 5Y cells and cDNA of the genes of interest was generated after which PCR and gel electrophoresis was employed to yield amplicon bands. (II) Chordoma cells were cultured and brachyury mRNA expression was assessed by PCR assay. (III) Cultured chordoma cells were grown in enriched DMEM or in enriched DMEM containing 33% fibroblast conditioned medium and proliferation was assessed. Results. (I) Both EVX1 and Twist are positive in prostate and SHSY 5Y respectively. Wnt3a is not positive in placenta. (II) Brachyury expression is either strongly downregulated or not expressed in two slowly growing chordoma cell cultures. (III) First data indicate enriched DMEM containing 33% fibroblast conditioned medium does not stimulate chordoma proliferation in vitro as compared to enriched DMEM without fibroblast conditioned medium

Bridging the Abyss: Law librarians come together to prepare new attorneys

This early morning session was billed as an open forum for law firm librarians and academic librarians to engage about the issue of new attorney preparation and training. We’ve all heard accounts of woefully unprepared first-year attorneys. In my experience as a law firm librarian, I’ve found that these accounts represent more of an exception than a rule. However, on numerous occasions I’ve also witnessed the rhetorical question, “What are they teaching them in law school?!” served to sympathetic colleagues with no shortage of indignant relish. So, as I roused myself from another vendor-induced slumber to sit in on this panel discussion, I hoped for a civil dialogue but prepared for a more partisan exchange

The importance of biomass net uptake for a trace metal budget in a forest stand in north-eastern France

The trace metal (TM: Cd, Cu, Ni, Pb and Zn) budget (stocks and annual fluxes) was evaluated in a forest stand (silver fir, Abies alba Miller) in north-eastern France. Trace metal concentrations were measured in different tree compartments in order to assess TM partitioning and dynamics in the trees. Inputs included bulk deposition, estimated dry deposition and weathering. Outputs were leaching and biomass exportation. Atmospheric deposition was the main input flux. The estimated dry deposition accounted for about 40% of the total trace metal deposition. The relative importance of leaching (estimated by a lumped parameter water balance model, BILJOU) and net biomass uptake (harvesting) for ecosystem exportation depended on the element. Trace metal distribution between tree compartments (stem wood and bark, branches and needles) indicated that Pb was mainly stored in the stem, whereas Zn and Ni, and to a lesser extent Cd and Cu, were translocated to aerial parts of the trees and cycled in the ecosystem. For Zn and Ni, leaching was the main output flux (N95% of the total output) and the plot budget (input–output) was negative, whereas for Pb the biomass net exportation represented 60% of the outputs and the budget was balanced. Cadmium and Cu had intermediate behaviours, with 18% and 30% of the total output relative to biomass exportation, respectively, and the budgets were negative. The net uptake by biomass was particularly important for Pb budgets, less so for Cd and Cu and not very important for Zn and Ni in such forest stands

Isolation and characterization of a new population of nasal surface macrophages and their susceptibility to PRRSV-1 subtype 1 (LV) and subtype 3 (Lena).

Sialoadhesin (Sn) and CD163 have been recognized as two important mediators for porcine reproductive and respiratory syndrome virus (PRRSV) in host macrophages. Recently, it has been demonstrated that the highly virulent Lena strain has a wider macrophage tropism than the low virulent LV strain in the nasal mucosa. Not only CD163(+)Sn(+) macrophages are infected by Lena but also CD163(+)Sn(-) macrophages. This suggests that an alternative receptor exists for binding and internalization of PRRSV Lena in the CD163(+)Sn(-) macrophages. Further investigation to find the new entry receptor was hampered by the difficulty of isolating these macrophages from the nasal mucosa. In the present study, a new population of CD163(+)Sn(-) cells has been identified that is specifically localized in the nasal lamina propria and can be isolated by an intranasal digestion approach. Isolated nasal cells were characterized using specific cell markers and their susceptibility to two different PRRSV-1 strains (LV and Lena) was tested. Upon digestion, 3.2% (flow cytometry)-6.4% (confocal microscopy) of the nasal cells were identified as CD163(+) and all (99.7%) of these CD163(+) cells were Sn-. These CD163(+)Sn(-) cells, designated as "nasal surface macrophages", showed a 4.9 times higher susceptibility to the Lena strain than to the LV strain. Furthermore, the Lena-inoculated cell cultures showed an upregulation of CD163. These results showed that our new cell isolation system is ideal for the further functional and phenotypical analysis of the new population of nasal surface macrophages and further research on the molecular pathogenesis of PRRSV in the nose

An alphaherpesvirus exploits antimicrobial beta-defensins to initiate respiratory tract infection

beta-Defensins protect the respiratory tract against the myriad of microbial pathogens entering the airways with each breath. However, this potentially hostile environment is known to serve as a portal of entry for herpesviruses. The lack of suitable respiratory model systems has precluded understanding of how herpesvirus virions overcome the abundant mucosal p-defensins during host invasion. We demonstrate how a central alphaherpesvirus, equine herpesvirus type 1 (EHV1), actually exploits p-defensins to invade its host and initiate viral spread. The equine beta-defensins (eBDs) eBD1, -2, and -3 were produced and secreted along the upper respiratory tract. Despite the marked antimicrobial action of eBD2 and -3 against many bacterial and viral pathogens, EHV1 virions were resistant to eBDs through the action of the viral glycoprotein M envelope protein. Pretreatment of EHV1 virions with eBD2 and -3 increased the subsequent infection of rabbit kidney (RK13) cells, which was dependent on viral N-linked glycans. eBD2 and -3 also caused the aggregation of EHV1 virions on the cell surface of RK13 cells. Pretreatment of primary equine respiratory epithelial cells (EREC) with eBD1, -2, and -3 resulted in increased EHV1 virion binding to and infection of these cells. EHV1-infected EREC, in turn, showed an increased production of eBD2 and -3 compared to that seen in mock- and influenza virus-infected EREC. In addition, these eBDs attracted leukocytes, which are essential for EHV1 dissemination and which serve as latent infection reservoirs. These novel mechanisms provide new insights into herpesvirus respiratory tract infection and pathogenesis. IMPORTANCE How herpesviruses circumvent mucosal defenses to promote infection of new hosts through the respiratory tract remains unknown due to a lack of hostspecific model systems. We used the alphaherpesvirus equine herpesvirus type 1 (EHV1) and equine respiratory tissues to decipher this key event in general alphaherpesvirus pathogenesis. In contrast to several respiratory viruses and bacteria, EHV1 resisted potent antimicrobial equine p-defensins (eBDs) eBD2 and eBD3 by the action of glycoprotein M. Instead, eBD2 and -3 facilitated EHV1 particle aggregation and infection of rabbit kidney (RK13) cells. In addition, virion binding to and subsequent infection of respiratory epithelial cells were increased upon preincubation of these cells with eBD1, -2, and -3. Infected cells synthesized eBD2 and -3, promoting further host cell invasion by EHV1. Finally, eBD1, -2, and -3 recruited leukocytes, which are well-known EHV1 dissemination and latency vessels. The exploitation of host innate defenses by herpesviruses during the early phase of host colonization indicates that highly specialized strategies have developed during host-pathogen coevolution

Comparison of primary virus isolation in pulmonary alveolar macrophages and four different continuous cell lines for type 1 and type 2 porcine reproductive and respiratory syndrome virus

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has a highly restricted cellular tropism. In vivo, the virus primarily infects tissue-specific macrophages in the nose, lungs, tonsils, and pharyngeal lymphoid tissues. In vitro however, the MARC-145 cell line is one of the few PRRSV susceptible cell lines that are routinely used for in vitro propagation. Previously, several PRRSV non-permissive cell lines were shown to become susceptible to PRRSV infection upon expression of recombinant entry receptors (e.g., PK15Sn-CD163, PK15S10-CD163). In the present study, we examined the suitability of different cell lines as a possible replacement of primary pulmonary alveolar macrophages (PAM) cells for isolation and growth of PRRSV. The susceptibility of four different cell lines (PK15Sn-CD163, PK15S10-CD163, MARC-145, and MARC-145Sn) for the primary isolation of PRRSV from PCR positive sera (both PRRSV1 and PRRSV2) was compared with that of PAM. To find possible correlations between the cell tropism and the viral genotype, 54 field samples were sequenced, and amino acid residues potentially associated with the cell tropism were identified. Regarding the virus titers obtained with the five different cell types, PAM gave the highest mean virus titers followed by PK15Sn-CD163, PK15S10-CD163, MARC-145Sn, and MARC-145. The titers in PK15Sn-CD163 and PK15S10-CD163 cells were significantly correlated with virus titers in PAM for both PRRSV1 (p < 0.001) and PRRSV2 (p < 0.001) compared with MARC-145Sn (PRRSV1: p = 0.22 and PRRSV2: p = 0.03) and MARC-145 (PRRSV1: p = 0.04 and PRRSV2: p = 0.12). Further, a possible correlation between cell tropism and viral genotype was assessed using PRRSV whole genome sequences in a Genome-Wide-Association Study (GWAS). The structural protein residues GP2:187L and N:28R within PRRSV2 sequences were associated with their growth in MARC-145. The GP5:78I residue for PRRSV2 and the Nsp11:155F residue for PRRSV1 was linked to a higher replication on PAM. In conclusion, PK15Sn-CD163 and PK15S10-CD163 cells are phenotypically closely related to the in vivo target macrophages and are more suitable for virus isolation and titration than MARC-145/MARC-145Sn cells. The residues of PRRSV proteins that are potentially related with cell tropism will be further investigated in the future

Role of macrophage sialoadhesin in host defense against the sialylated pathogen group B <em>Streptococcus</em>

ABSTRACT: Several bacterial pathogens decorate their surfaces with sialic acid (Sia) residues within cell wall components or capsular exopolysaccharides. Sialic acid expression can promote bacterial virulence by blocking complement activation or by engagement of inhibitory sialic acid-binding immunoglobulin-like lectins (Siglecs) on host leukocytes. Expressed at high levels on splenic and lymph node macrophages, sialoadhesin (Sn) is a unique Siglec with an elongated structure that lacks intracellular signaling motifs. Sialoadhesin allows macrophage to engage certain sialylated pathogens and stimulate inflammatory responses, but the in vivo significance of sialoadhesin in infection has not been shown. We demonstrate that macrophages phagocytose the sialylated pathogen group B Streptococcus (GBS) and increase bactericidal activity via sialoadhesin-sialic-acid-mediated recognition. Sialoadhesin expression on marginal zone metallophillic macrophages in the spleen trapped circulating GBS and restricted the spread of the GBS to distant organs, reducing mortality. Specific IgM antibody responses to GBS challenge were also impaired in sialoadhesin-deficient mice. Thus, sialoadhesin represents a key bridge to orchestrate innate and adaptive immune defenses against invasive sialylated bacterial pathogens. KEY MESSAGE: Sialoadhesin is critical for macrophages to phagocytose and clear GBS. Increased GBS organ dissemination in the sialoadhesin-deficient mice. Reduced anti-GBS IgM production in the sialoadhesin-deficient mice. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00109-014-1157-y) contains supplementary material, which is available to authorized users

Diversity and Abundance of Arbuscular–Mycorrhizal Fungus Spores in Tropical Forest and Pasture 1

As Neotropical forests are increasingly converted to agriculture (especially pastures), little is known about the impacts on microbial biodiversity. To assess such impacts, I compared spore abundance and diversity of arbuscular mycorrhizal fungi (AMF) in soils from lowland evergreen forests and pastures in Nicaragua and Costa Rica. Species composition, dominance–diversity curves, and Simpson's diversity indices were similar for both forest and pasture soils. Of 28 distinct fungus morphospecies, 11 produced more spores in pasture, while only 1 produced more spores in forest. According to species–accumulation curves, local AMF species richness did not significantly decline following conversion of forest to pasture. Because pastures contained a surprising abundance and diversity of AMF spores compared to native forest, a lack of mycorrhizal fungi is unlikely to limit plant succession, restoration, or reforestation in the pastures studied. At the regional scale, however, species-accumulation curves showed significantly greater gamma (G) diversity of spores in forest. In addition to these trends in diversity, species that sporulated more in pasture tended to have small spores, while the one species that sporulated more in forest had the largest spores. Similarly, only largespored fungi (>300 Μm) showed any seasonal variation in spore abundance, being more common in the wet season.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71602/1/j.1744-7429.2000.tb00522.x.pd

A Triple Amino Acid Substitution at Position 88/94/95 in Glycoprotein GP2a of Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV1) Is Responsible for Adaptation to MARC-145 Cells

The Meat Animal Research Center-145 (MARC-145) cell line has been proven to be valuable for viral attenuation regarding vaccine development and production. Cell-adaptation is necessary for the efficient replication of porcine reproductive and respiratory syndrome virus (PRRSV) in these cells. Multiple sequence analysis revealed consistent amino acid substitutions in GP2a (V88F, M94I, F95L) of MARC-145 cell-adapted strains. To investigate the putative effect of these substitutions, mutations at either position 88, 94, 95, and their combinations were introduced into two PRRSV1 (13V091 and IVI-1173) infectious clones followed by the recovery of viable recombinants. When comparing the replication kinetics in MARC-145 cells, a strongly positive effect on the growth characteristics of the 13V091 strain (+2.1 log10) and the IVI-1173 strain (+1.7 log10) compared to wild-type (WT) virus was only observed upon triple amino acid substitution at positions 88 (V88F), 94 (M94I), and 95 (F95L) of GP2a, suggesting that the triple mutation is a determining factor in PRRSV1 adaptation to MARC-145 cells

The M/GP5 Glycoprotein Complex of Porcine Reproductive and Respiratory Syndrome Virus Binds the Sialoadhesin Receptor in a Sialic Acid-Dependent Manner

The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP3, GP4 and GP5 envelope glycoproteins, only the M/GP5 glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP5. These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines