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The kernels of the Australian blushwood tree (

The kernels of the Australian blushwood tree (Fontainea picrosperma) are the source of the veterinary anticancer drug tigilanol tiglate (2a, Stelfonta) and contain a concentration of phorboids significantly higher than croton oil, the only abundant source of these compounds previously known. The oily matrix of the blushwood kernels is composed of free fatty acids and not by glycerides as found in croton oil. By active partitioning, it was therefore possible to recover and characterize for the first time a cryptic tigliane fraction, that is, the diterpenoid fraction that, because of its lipophilicity, could not be obtained by solvent partition of crude extracts. The cryptic tigliane fraction accounted for ca. 30% of the tigliane kernel titer and was quantified by 1H NMR spectroscopy and profiled by HPLC-MS. Long-chain (linoleates and/or oleates) 20-acyl derivatives of the epoxytigliane diesters tigilanol tiglate (EBC-46, 2a), EBC-47 (4a), EBC-59 (5a), EBC-83 (6a), and EBC-177 (7a) were identified. By chemoselective acylation of EBC-46 (2a) and EBC-177 (7a) the natural triesters 2b and 7b and a selection of analogues were prepared to assist identification of the natural compounds. The presence of a free C-20 hydroxy group is a critical requirement for PKC activation by phorbol esters. The unexpected activity of 20-linoleoyl triester 2b in a cytotoxicity assay based on PKC activation was found to be related mainly to its hydrolysis to tigilanol tiglate (2a) under the prolonged conditions of the assay, while other esters were inactive. Significant differences between the esterification profile of the epoxytigliane di- and triesters exist in F. picrosperma, suggesting a precise, yet elusive, blueprint of acyl decoration for the tigliane polyol 5-hydroxyepoxyphorbol.

The adaptor protein ASC is known to facilitate caspase-1 activation essential for innate host immunity via the formation of the inflammasome complex - a multi-protein structure responsible for processing IL-1b and IL-18 to their active moieties. Here we report for the first time, a unique inflammasome-independent role for ASC in the control of transplant outcome following allogeneic bone marrow transplantation (BMT). We demonstrate that ASC-deficient donor CD8+ T cells fail to induce GVHD lethality due to an inability to differentiate into fully cytolytic effectors after BMT, with a developmental bias instead towards CD127+KLRG1- memory CD8+ T cells. Despite this, graft-versus-leukaemia effects against BCR-ABL NUP98/HOXA9 primary leukemia remained largely intact. This phenomenon was inflammasome independent, since GVHD lethality and T cell differentiation were not altered in recipients of caspase-1-deficient T cells. We also confirmed a reduced capacity for human T cells in which ASC was knocked down by shRNA to induce xenogeneic GVHD. In a model of bone marrow rejection, ASC expression in recipient CD8+ T cells profoundly impaired graft rejection and was permissive of robust engraftment across MHC barriers and long term survival. Taken together, these findings demonstrate an inflammasome-independent role for ASC in controlling GVHD and graft rejection. Thus, the inhibition of ASC in the clinic represents an important new therapeutic target to manipulate transplant outcomes.