MiR-126 and miR-126* regulate shear-resistant firm leukocyte adhesion to human brain endothelium

Leukocyte adhesion to brain endothelial cells, the blood-brain barrier main component, is a critical step in the pathogenesis of neuroinflammatory diseases such as multiple sclerosis (MS). Leukocyte adhesion is mediated mainly by selectins, cell adhesion molecules and chemokines induced by pro-inflammatory cytokines such as TNFα and IFNγ, but the regulation of this process is not fully clear. This study investigated the regulation of firm leukocyte adhesion to human brain endothelium by two different brain endothelial microRNAs (miRs), miR-126 and miR-126*, that are downregulated by TNFα and IFNγ in a human brain endothelial cell line, hCMEC/D3. Using a leukocyte adhesion in vitro assay under shear forces mimicking blood flow, we observed that reduction of endothelial miR-126 and miR-126* enhanced firm monocyte and T cell adhesion to hCMEC/D3 cells, whereas their increased expression partially prevented THP1, Jurkat and primary MS patient-derived PBMC firm adhesion. Furthermore, we observed that miR-126* and miR-126 downregulation increased E-selectin and VCAM1, respectively, while miR-126 overexpression reduced VCAM1 and CCL2 expression by hCMEC/D3 cells, suggesting that these miRs regulate leukocyte adhesion by modulating the expression of adhesion-associated endothelial mRNA targets. Hence, human brain endothelial miR-126 and miR-126* could be used as a therapeutic tool to reduce leukocyte adhesion and thus reduce neuroinflammation

DIFFERENTIAL EXPRESSION OF GENES UNDER CONTROL OF THE MATING-TYPE GENES IN THE SECONDARY MYCELIUM OF SCHIZOPHYLLUM-COMMUNE

The Schizophyllum commune SC3 gene, which encodes a hydrophobin that coats aerial hyphae, is expressed in both monokaryons and dikaryons. The dikaryons were formed by mating two monokaryons with different MATA and MATB genes, leading to activation of the MATA- and MATB-controlled pathways (MATA-on and MATB-on). In contrast to the monokaryons, the dikaryons also expressed other hydrophobin genes (SC1, SC4) as well as a gene (SC7) encoding a hydrophilic wall protein. None of these four genes was expressed in MATA-off MATB-on mycelia, indicating that MATB-on represses SC3 and that both MATA-on and MATB-on are required for activation of SC1, SC4 and SC7. In fruiting dikaryons, immunolabelling revealed that SC3p was produced by aerial hyphae but not by hyphae that constitute the fruit-body tissue. In contrast to aerial hyphae, the latter produced dikaryon-specific transcripts and secreted SC7p into the extracellular matrix of the tissue. This suggests that in the aerial hyphae of the dikaryon the MATB-on pathway was not effective (MATB-off). We observed that in these aerial hyphae the two nuclei were wider apart than in a typical dikaryon. Although other explanations are not ruled out, we tentatively propose that effective interaction of different MATB genes requires proximity of the two nuclei containing these genes, and that disruption of this binucleate state represents a novel mechanism of gene control for spatial cell differentiation in the secondary mycelium

Cellular handling of a dexamethasone-anti-E-selectin immunoconjugate by activated endothelial cells: Comparison with free dexamethasone

Purpose. For selective inhibition of endothelial cell activation in chronic inflammation, we have developed a dexamethasone-anti-E-selectin immunoconjugate. The present study was performed to evaluate the cellular handling of this immunoconjugate by activated primary endothelial cells and to compare its drug delivery capacity with free dexamethasone. Methods. The binding, uptake, and degradation of I-125-radiolabeled dexamethasone-anti-E-selectin immunoconjugate by TNFalpha-activated endothelial cells were studied for different time periods and at different concentrations, as well as in the presence of inhibitors for E-selectin binding and lysosomal degradation. Its drug delivery capacity was compared with the uptake of unconjugated H-3-labeled dexamethasone. Results. The immunoconjugate was internalized by E-selectin expressing activated endothelial cells and degraded in the lysosomal compartment. The receptor-mediated binding and uptake was saturable, implying a maximal attainable intracellular concentration of the drug. In contrast, free dexamethasone entered both resting and activated endothelial cells by passive diffusion. Conclusions. The dexamethasone-anti-E-selectin immunoconjugate is capable of selective delivering the coupled drug into activated endothelial cells. This targeting concept enables disease-induced drug delivery in which intracellular concentrations can be reached comparable with those obtained after incubation with 3 muM dexamethasone

Hydrophobin Genes Involved in Formation of Aerial Hyphae and Fruit Bodies in Schizophyllum.

Fungi typically grow by apical extension of hyphae that penetrate moist substrates. After establishing a branched feeding mycelium, the hyphae differentiate and grow away from the substrate into the air where they form various structures such as aerial hyphae and mushrooms. In the basidiomycete species Schizophyllum commune, we previously identified a family of homologous genes that code for small cysteine-rich hydrophobic proteins. We now report that the encoded hydrophobins are excreted in abundance into the culture medium by submerged feeding hyphae but form highly insoluble complexes in the walls of emerging hyphae. The Sc3 gene encodes a hydrophobin present in walls of aerial hyphae. The homologous Sc1 and Sc4 genes, which are regulated by the mating-type genes, encode hydrophobins present in walls of fruit body hyphae. The hydrophobins are probably instrumental in the emergence of these aerial structures

THE FUNGAL HYDROPHOBIN SC3P SELF-ASSEMBLES AT THE SURFACE OF AERIAL HYPHAE AS A PROTEIN MEMBRANE CONSTITUTING THE HYDROPHOBIC RODLET LAYER

The Schizophyllum commune hydrophobin Sc3p is a small, hydrophobic, cysteine-rich protein involved in the formation of aerial hyphae. Using an antibody against purified Sc3p we found that the hydrophobin is secreted into the medium at the spices of growing submerged hyphae but in emerging aerial hyphae it accumulates at the hyphal surface. Here, the hydrophobin self-assembles at the wall/air interface into an SDS-insoluble protein membrane, at the aerial site very hydrophobic and with the appearance of a mosaic of 10 nn spaced parallel rodlets. Interfacial self-assembly of the hydrophobin also occurs in vitro. When solutions containing various concentrations of purified Sc3p were dried down onto a glass surface, the amount of assembled Sc3p depended on the area of the interface. Surplus of Sc3p remained in the monomeric form, apparently because formation of a monolayer of assembled Sc3p abolishes the hydrophilic/hydrophobic interface. The 10 nm thick layer of assembled Sc3p at the surface of aerial hyphae thus probably represents a monolayer of the protein