Biotransformation of cladribine using a stabilized biocatalyst in calcium alginate beads.

Cladribine is a nucleoside analogue widely used in the pharmaceutical industry for the treatment of several neoplasms, including hairy-cell leukemia among others. This compound has also shown efficacy in the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In this work, a green bioprocess for cladribine biosynthesis using immobilized Arthrobacter oxydans was developed. The microorganism was stabilized by entrapment immobilization in the natural matrix alginate. Different reaction parameters were optimized obtaining a biocatalyst able to achieve cladribine bioconversion values close to 85% after 1 hr, the shortest reaction times reported so far. The developed bioprocess was successfully scaled-up reaching a productivity of 138 mg L-1 hr-1 . Also, the biocatalyst was stable for 5 months in storage and in 96 hr at operational conditions.

Shiga toxin 1 induces on lipopolysaccharide-treated astrocytes the release of tumor necrosis factor-alpha that alter brain-like endothelium integrity.

The hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia, thrombocytopenia and renal dysfunction. The typical form of HUS is generally associated with infections by Gram-negative Shiga toxin (Stx)-producing Escherichia coli (STEC). Endothelial dysfunction induced by Stx is central, but bacterial lipopolysaccharide (LPS) and neutrophils (PMN) contribute to the pathophysiology. Although renal failure is characteristic of this syndrome, neurological complications occur in severe cases and is usually associated with death. Impaired blood-brain barrier (BBB) is associated with damage to cerebral endothelial cells (ECs) that comprise the BBB. Astrocytes (ASTs) are inflammatory cells in the brain and determine the BBB function. ASTs are in close proximity to ECs, hence the study of the effects of Stx1 and LPS on ASTs, and the influence of their response on ECs is essential. We have previously demonstrated that Stx1 and LPS induced activation of rat ASTs and the release of inflammatory factors such as TNF-α, nitric oxide and chemokines. Here, we demonstrate that rat ASTs-derived factors alter permeability of ECs with brain properties (HUVECd); suggesting that functional properties of BBB could also be affected. Additionally, these factors activate HUVECd and render them into a proagregant state promoting PMN and platelets adhesion. Moreover, these effects were dependent on ASTs secreted-TNF-α. Stx1 and LPS-induced ASTs response could influence brain ECs integrity and BBB function once Stx and factors associated to the STEC infection reach the brain parenchyma and therefore contribute to the development of the neuropathology observed in HUS.

Binding of galectin-1 to αIIbß3 integrin triggers "outside-in" signals, stimulates platelet activation, and controls primary hemostasis.

Understanding noncanonical mechanisms of platelet activation represents an important challenge for the identification of novel therapeutic targets in bleeding disorders, thrombosis, and cancer. We previously reported that galectin-1 (Gal-1), a ß-galactoside-binding protein, triggers platelet activation in vitro. Here we investigated the molecular mechanisms underlying this function and the physiological relevance of endogenous Gal-1 in hemostasis. Mass spectrometry analysis, as well as studies using blocking antibodies against the anti-α(IIb) subunit ofα(IIb)ß(3) integrin or platelets from patients with Glanzmann's thrombasthenia syndrome (α(IIb)ß(3) deficiency), identified this integrin as a functional Gal-1 receptor in platelets. Binding of Gal-1 to platelets triggered the phosphorylation of ß(3)-integrin, Syk, MAPKs, PI3K, PLCγ2, thromboxane (TXA(2)) release, and Ca(2+) mobilization. Not only soluble but also immobilized Gal-1 promoted platelet activation. Gal-1-deficient (Lgals1(-/-)) mice showed increased bleeding time (P<0.0002, knockout vs. wild type), which was not associated with an abnormal platelet count. Lgals1(-/-) platelets exhibited normal aggregation to PAR4, ADP, arachidonic acid, or collagen but abnormal ATP release at low collagen concentrations. Impaired spreading on fibrinogen and clot retraction with normal levels of α(IIb)ß(3) was also observed in Lgals1(-/-) platelets, indicating a failure in the "outside-in" signaling through this integrin. This study identifies a noncanonical mechanism, based on galectin-integrin interactions, for regulating platelet activation.