α-Catulin downregulates E-cadherin and promotes melanoma progression and invasion.

Metastasis is associated with poor prognosis for melanoma responsible for about 90% of skin cancer-related mortality. To metastasize, melanoma cells must escape keratinocyte control, invade across the basement membrane and survive in the dermis by resisting apoptosis before they can intravasate into the circulation. α-Catulin (CTNNAL1) is a cytoplasmic molecule that integrates the crosstalk between nuclear factor-kappa B and Rho signaling pathways, binds to ß-catenin and increases the level of both α-catenin and ß-catenin and therefore has potential effects on inflammation, apoptosis and cytoskeletal reorganization. Here, we show that α-catulin is highly expressed in melanoma cells. Expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of expression of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. Knockdown of α-catulin promoted adhesion to and inhibited migration away from keratinocytes in an E-cadherin-dependent manner and decreased the transmigration through a keratinocyte monolayer, as well as in Transwell assays using collagens, laminin and fibronectin coating. Moreover, knockdown promoted homotypic spheroid formation and concomitantly increased E-cadherin expression along with downregulation of transcription factors implicated in its repression (Snail/Slug, Twist and ZEB). Consistent with the molecular changes, α-catulin provoked invasion of melanoma cells in a three-dimensional culture assay by the upregulation of matrix metalloproteinases 2 and 9 and the activation of ROCK/Rho. As such, α-catulin may represent a key driver of the metastatic process, implicating potential for therapeutic interference.

Endothelial cell-based methods for the detection of cyanobacterial anti-inflammatory and wound-healing promoting metabolites.

Acute lung injury is accompanied by an increased endothelial chemokine production and adhesion molecule expression, which may result in an extensive neutrophil infiltration. Moreover, a destruction of the alveolar epithelium and capillary endothelium may result in permeability edema. As such, the search for novel anti-inflammatory substances, able to downregulate these parameters as well as the tissue damage holds therapeutic promise. We therefore describe here the use of human endothelial cell-based in vitro assays for the detection of anti-inflammatory and wound-healing metabolites from cyanobacteria.

Development of cellulose-DNA immunoadsorbent.

The aim of this study was to prepare a DNA immunoadsorbent for the specific, extracorporeal removal of anti-DNA antibodies from the blood of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Two kinds of cellulose beads were applied as a carrier. Calf thymus DNA was covalently coupled to the carrier using the epichlorohydrin method. Efforts were focused on optimization of conditions for activation and coupling, trying to couple as much DNA as possible to a certain amount of carrier. It was found that the activation level increased with the increase of NaOH concentration and the amount of epichlorohydrin used. The mole of epichlorohydrin must be in excess of that of NaOH because excess NaOH could react further with the epoxy groups in the beads resulting in a decrease of activation level. High activation level could be obtained in a medium of 3.0 M NaOH. The DNA coupling was found to be mainly temperature and pH dependent. Using 0.1 M Tris-HCl buffer, pH 8 at a temperature of 50-90 degrees C, more than 3 mg of DNA could be coupled to 1 ml of wet beads. Prolonging the coupling reaction under 50 degrees C to 72 h resulted in the same coupling capacity as that obtained under 90 degrees C. To evaluate the adsorption ability for anti-DNA of this immunoadsorbent, batch and circulation tests were applied using SLE patient plasma. The immunoadsorbents showed excellent adsorption capacity, especially the cellulose with smaller size (200-300 microm). The incubation of 20 ml of patient's plasma with 1 ml of adsorbent resulted in an 80% decline in the anti-DNA antibody level. In the circulation tests, 30 ml of plasma was circulated through a column containing 3 ml of adsorbent. The maximum decline in anti-DNA level, 80%, was obtained after 60 min. Such high adsorption capacity and high adsorption rate suggest this immunoadsorbent may be used for treatment. For comparison, 1,4-butanediol diglycidyl ether activation method and other DNA sources were tested with the same protocol.

Selective removal of circulating immune complexes from patient plasma.

The principle of a patient-specific immunoadsorber (PsIA) is demonstrated. Studies with model systems (HSA/anti-HSA) on immobilization, stability, and leakage form the basis for the presented fast-performance liquid chromatography (FPLC) and batch experiments, which were conducted using two different protein A adsorbers and autologous and heterologous PsIA systems. Experiments to determine the binding capacity of protein A adsorbers and PsIAs are described. In all experiments, the adsorption of plasma IgG, total protein, and C1q and C3d circulating immune complexes were measured. Plasma of patients with autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus) was investigated. Analysis was performed in both the initial plasma and the flow-through or supernatant. Results of the investigations using FPLC and batch experiments were compared. Autologous PsIA systems are suitable for the selective removal of elevated levels of circulating immune complexes in the plasma.