High degree of mitochondrial gene heterogeneity in the bat tick species Ixodes vespertilionis, I. ariadnae and I. simplex from Eurasia

Background: Phylogeographical studies allow precise genetic comparison of specimens, which were collected over large geographical ranges and belong to the same or closely related animal species. These methods have also been used to compare ticks of veterinary-medical importance. However, relevant data are missing in the case of ixodid ticks of bats, despite (1) the vast geographical range of both Ixodes vespertilionis and Ixodes simplex, and (2) the considerable uncertainty in their taxonomy, which is currently unresolvable by morphological clues. Methods: In the present study 21 ticks were selected from collections or were freshly removed from bats or cave walls in six European and four Asian countries. The DNA was extracted and PCRs were performed to amplify part of the cytochrome oxidase I (COI), 16S and 12S rDNA genes, followed by sequencing for identification and molecular-phylogenetic comparison. Results: No morphological differences were observed between Ixodes vespertilionis specimens from Spain and from other parts of Europe, but corresponding genotypes had only 94.6 % COI sequence identity. An I. vespertilionis specimen collected in Vietnam was different both morphologically and genetically (i.e. with only 84.1 % COI sequence identity in comparison with I. vespertilionis from Europe). Two ticks (collected in Vietnam and in Japan) formed a monophyletic clade and shared morphological features with I. ariadnae, recently described and hitherto only reported in Europe. In addition, two Asiatic specimens of I. simplex were shown to differ markedly from European genotypes of the same species. Phylogenetic relationships of ticks showed similar clustering patterns with those of their associated bat host species. Conclusions: Although all three ixodid bat tick species evaluated in the present study appear to be widespread in Eurasia, they exhibit pronounced genetic differences. Data of this study also reflect that I. vespertilionis may represent a species complex

Alginate hydrogel enriched with enamel matrix derivative to target osteogenic cell differentiation in TiO2 scaffolds

The purpose of bone tissue engineering is to employ scaffolds, cells, and growth factors to facilitate healing of bone defects. The aim of this study was to assess the viability and osteogenic differentiation of primary human osteoblasts and adipose tissue–derived mesenchymal stem cells from various donors on titanium dioxide (TiO2) scaffolds coated with an alginate hydrogel enriched with enamel matrix derivative. Cells were harvested for quantitative reverse transcription polymerase chain reaction on days 14 and 21, and medium was collected on days 2, 14, and 21 for protein analyses. Neither coating with alginate hydrogel nor alginate hydrogel enriched with enamel matrix derivative induced a cytotoxic response. Enamel matrix derivative–enriched alginate hydrogel significantly increased the expression of osteoblast markers COL1A1, TNFRSF11B, and BGLAP and secretion of osteopontin in human osteoblasts, whereas osteogenic differentiation of human adipose tissue–derived mesenchymal stem cells seemed unaffected by enamel matrix derivative. The alginate hydrogel coating procedure may have potential for local delivery of enamel matrix derivative and other stimulatory factors for use in bone tissue engineering

Identification of an Effective Early Signaling Signature during Neo-Vasculogenesis <i>In Vivo</i> by <i>Ex Vivo</i> Proteomic Profiling

<div><p>Therapeutic neo-vasculogenesis <i>in vivo</i> can be achieved by the co-transplantation of human endothelial colony-forming progenitor cells (ECFCs) with mesenchymal stem/progenitor cells (MSPCs). The underlying mechanism is not completely understood thus hampering the development of novel stem cell therapies. We hypothesized that proteomic profiling could be used to retrieve the <i>in vivo</i> signaling signature during the initial phase of human neo-vasculogenesis. ECFCs and MSPCs were therefore either transplanted alone or co-transplanted subcutaneously into immune deficient mice. Early cell signaling, occurring within the first 24 hours <i>in vivo</i>, was analyzed using antibody microarray proteomic profiling. Vessel formation and persistence were verified in parallel transplants for up to 24 weeks. Proteomic analysis revealed significant alteration of regulatory components including caspases, calcium/calmodulin-dependent protein kinase, DNA protein kinase, human ErbB2 receptor-tyrosine kinase as well as mitogen-activated protein kinases. Caspase-4 was selected from array results as one therapeutic candidate for targeting vascular network formation <i>in vitro</i> as well as modulating therapeutic vasculogenesis <i>in vivo</i>. As a proof-of-principle, caspase-4 and general caspase-blocking led to diminished endothelial network formation <i>in vitro</i> and significantly decreased vasculogenesis <i>in vivo</i>. Proteomic profiling <i>ex vivo</i> thus unraveled a signaling signature which can be used for target selection to modulate neo-vasculogenesis <i>in vivo</i>.</p></div

Overview of the project strategy.

<p>Autologous pairs of mesenchymal stem/progenitor cells (MSPC) and endothelial colony-forming progenitor cells (ECFC) were isolated, purified and expanded from the same human umbilical cord as described (<a href="http://www.jove.com/author/Andreas_Reinisch" target="_blank">http://www.jove.com/author/Andreas_Reinisch</a><i>)</i> (<b>A</b>)<b>.</b> Cells were combined with matrix and injected subcutaneously in four 300 µL implants per animal into the flank of immune compromised NSG mice as specified (<b>B</b>). Plugs were explanted 24 hours after implantation (<b>C</b>) to detect the signaling signature operative during the early phase of therapeutic vasculogenesis using proteomic profiling on customized antibody microarrays comparing either co-transplants of ECFC+MSPC with transplants containing sole ECFC, MSPC or Matrix <i>in vivo</i> (groups I – III), or co-transplants <i>in vivo</i> with a mixture of ECFC+MSPC created <i>in vitro</i> (group IV) (<b>E</b>)<b>.</b> Macroscopic view of plugs explanted after 2, 8 and 24 weeks (<b>C and D</b>). Histology and morphometry were performed to visualize vessel formation and stability in plugs in a time course (for up to 24 weeks) (<b>F and G</b>)<b>.</b> Small molecule inhibition of selected targets (from the array analysis) was used as a proof-of-principle confirming drugability of significantly regulated proteins within the early vasculogenesis signaling signature.</p

Macroscopic and microscopic features of the plugs two and eight weeks after implantation.

<p>Plugs were harvested two (<b>A</b>) and eight weeks (<b>B</b>) after subcutaneous co-transplantation of ECFC+MSPC (ratio 80∶ 20, left column), MSPC only (2^nd column), ECFC only (3^rd column), and cell-free matrix (right column). Representative macroscopic pictures of plugs in subcutaneous location (upper rows), micrographs of hematoxylin and eosin staining (HE, middle rows) and immune histochemistry combining human CD31 colored brown with hematoxylin counter-stain (huCD31, bottom rows) are depicted. Total cell number within the plugs was 2×10⁶ per 300 µL matrix (n = 3 per group and time course).</p

Vessel formation is impaired upon caspase-4 and pan-caspase inhibition.

<p>Mean number of vessels created by ECFC+MSPC co-transplantation of MSPC+ECFC pairs derived from three different human organs (for primary data see Figures S3 and S4: umbilical cord-derived ECFC+umbilical cord-derived MSPC; white adipose tissue-derived ECFC+umbilical cord blood-derived MSPC; umbilical cord blood-derived ECFC+bone marrow-derived MSPC) comparing untreated progenitor cells with those pretreated either with DMSO <b>(A and B, left panels),</b> Caspase-4 inhibitor <b>(A, right panels)</b> or pan-caspase inhibitor <b>(B, right panel)</b> (** p<0.001, *** p≤0.0001; n = 6 mice and 6 plugs per pretreatment; regardless of the cell source and combination).</p

Effect of caspase inhibitors on ECFC network formation<b><i>in vitro</i></b><b>.</b>

<p>ECFCs were tested un-treated (<b>A</b>) and upon eight hour pretreatment with either DMSO (<b>B</b>), Caspase-4 inhibitor Z-LEVD-FMK (<b>C</b>) or pan-caspase inhibitor Q-VD-OPh (<b>D</b>) in a 24 hour angiogenesis assay <i>in vitro</i>. Quantification of cell network branching points and corresponding un-paired t-test results (*** p≤0.0001) are depicted (<b>E</b>). Each field represents an area of 9.45 mm². Scale bar: 1000 µm, n = 3 per condition.</p

Lipophilic components of diesel exhaust particles induce pro-inflammatory responses in human endothelial cells through AhR dependent pathway(s)

Abstract Background Exposure to traffic-derived particulate matter (PM), such as diesel exhaust particles (DEP), is a leading environmental cause of cardiovascular disease (CVD), and may contribute to endothelial dysfunction and development of atherosclerosis. It is still debated how DEP and other inhaled PM can contribute to CVD. However, organic chemicals (OC) adhered to the particle surface, are considered central to many of the biological effects. In the present study, we have explored the ability of OC from DEP to reach the endothelium and trigger pro-inflammatory reactions, a central step on the path to atherosclerosis. Results Exposure-relevant concentrations of DEP (0.12 μg/cm2) applied on the epithelial side of an alveolar 3D tri-culture, rapidly induced pro-inflammatory and aryl hydrocarbon receptor (AhR)-regulated genes in the basolateral endothelial cells. These effects seem to be due to soluble lipophilic constituents rather than particle translocation. Extractable organic material of DEP (DEP-EOM) was next fractionated with increasing polarity, chemically characterized, and examined for direct effects on pro-inflammatory and AhR-regulated genes in human microvascular endothelial (HMEC-1) cells and primary human endothelial cells (PHEC) from four healthy donors. Exposure-relevant concentrations of lipophilic DEP-EOM (0.15 μg/cm2) induced low to moderate increases in IL-1α, IL-1β, COX2 and MMP-1 gene expression, and the MMP-1 secretion was increased. By contrast, the more polar EOM had negligible effects, even at higher concentrations. Use of pharmacological inhibitors indicated that AhR and protease-activated receptor-2 (PAR-2) were central in regulation of EOM-induced gene expression. Some effects also seemed to be attributed to redox-responses, at least at the highest exposure concentrations tested. Although the most lipophilic EOM, that contained the majority of PAHs and aliphatics, had the clearest low-concentration effects, there was no straight-forward link between chemical composition and biological effects. Conclusion Lipophilic and semi-lipophilic chemicals seemed to detach from DEP, translocate through alveolar epithelial cells and trigger pro-inflammatory reactions in endothelial cells at exposure-relevant concentrations. These effects appeared to be triggered by AhR agonists, and involve PAR-2 signaling

Additional file 1: of Lipophilic components of diesel exhaust particles induce pro-inflammatory responses in human endothelial cells through AhR dependent pathway(s)

Figure S1. In a 3D tri-culture, exposure to SiNP on the epithelial side, induced COX-2 on the epithelial side, but not in the endothelial cells. Furthermore EAhy.926 endothelial cells exposed directly to SiNP up-regulated COX-2. Figure S2. The amount of volatile/semi-volatile compounds extracted decreased according to polarity of the solvents. Figure S3. Cytotoxicity of DEP-EOM in HMEC-1 and PHEC. Figure S4. Lipophilic DEP-EOMs cause CXCL8 secretion in HMEC-1 cells. Figure S5. PHEC were 99% CD31-positive. Figure S6 Size distribution, DEP and SiNP. Table S1. GC-MS quantified compounds with corresponding MS ions and calibration standards. (DOCX 2036 kb