Patent Application: US-80392010-A

Abstract:
ischemia - reperfusion injury involving harvested organs and allograft transplantation may be increased by stimulation of a newly described innate pro - inflammatory immune system which can cause secretion of il - 1β and subsequent neutrophilic inflammation . ischemia and hypoxia can cause metabolic acidosis and development of danger signals known to stimulate il - 1β secretion from the nalp - 3 inflammasome . based on this newly discovered mechanism causing pathobiology in iri , il - 1β targeted therapy would be capable of improving allograft tolerance , viability of harvested organs and in conditions with compromised arterial blood supply and subsequent reperfusion , such as replants , compartment syndrome , and serious vascular accidents .

Description:
harvested organs exhibit classic iri caused by systemic hypoxia from loss of arterial supply . there is concomitant breakdown of cells , nucleotides , freeing of atp , intracellular hypokalemia , buildup of urates and calcium pyrophosphates which crystallize at lower ph . fig1 illustrates an arterial blood vessel that is compromised either by partial or complete occlusion or by ablation as when an organ is harvested . the effect are hypoxia or anoxia contiguous to cells and tissue , and anaerobic acidosis as shown by a decrease in tissue ph . next to the blood vessel is an enlarged tissue cell . all of the aforementioned biochemical effects are referred to as damps ( 1 ). an enlarged cell ( 2 ) is shown with a cell membrane ( 3 ), nucleus ( 4 ), cytoplasm ( 5 ) which contains the aggregated nalp - 3 inflammasome ( 6 ). pamps ( 7 ) are shown engulfed into cytoplasm ( 5 ). the damps ( 1 ), such as crystallized urates or free atp are detected by the cell membrane ( 3 ) and / or by being partially engulfed into the cytoplasm ( 5 ) and their presence in turn stimulate the nalp - 3 inflammasome ( 6 ). il - 1β ( 8 ) is stored in the cytoplasm ( 5 ) as pro - il - 1β and stimulation of the nalp - 3 inflammasome ( 6 ) by damps ( 1 ) and pamps ( 7 ), cause the pro - il - 1β to be catalyzed to il - 1β ( 8 ), allowing it to be secreted into extracellular fluids . the secreted il - 1β ( 8 ) in turn causes attraction of neutrophils via a cascade of previously described immunologic events such as by attracting chemokines and by increased vascular adhesion molecule activation which in turn cause neutrophils to adhere to the intima of blood vessels followed by platelets and red cells accumulation , all leading to subsequent ischemia , the buildup of neutrophils causes activation of many other inflammatory events such as tissue damage by release of neutrophil proteases which damage cell and tissue integrity and by adhesion of neutrophils to blood vessel intima leading to more ischemia and hypoxia . this illustration does not demonstrate the worst case scenario , namely complete termination in arterial perfusion , such as during harvesting of organs for transplantation . there is evidence in this scenario , that harvested organs can exhibit an inflammatory cytokine burst immediately at the time of harvest due to ablation of arterial perfusion . this concept can explain why some harvested organs , such as kidneys , can begin to demonstrate signs of inflammation while they are being incubated up to 24 hours in cold fluids , and in turn explain to some degree why they are subsequently rejected following transplantation . pamps ( 7 ) with damps ( 1 ) or pamps ( 7 ) or damps ( 1 ) individually can stimulate innate immune receptors such as the nalp - 3 inflammasome ( 6 ) and even some toll - like receptors to secrete il - 1β ( 8 ). moreover the co - stimulation of the nalp3 - inflammasome ( 6 ) by damps ( 1 ) and pamps ( 7 ) together can cause synergistic il - 1β secretion , leading to greater inflammation . these aforementioned observations provide a plausible explanation for the occurrence of neutrophilic inflammation in transplantation that is not likely caused by pamps , and provides a mechanism that is operable in understanding some of the pathobiology involving harvested organs and allograft rejection . 1 ) there is evidence for the occurrence of metabolic acidosis in ir disorders caused by a combination of hypoxic induced anaerobic metabolism , lactic acidosis , atp hydrolysis and carbon dioxide retention . similar observations of tissue acidosis are likely to affect transplanted organs especially if they are harvested and preserved in cold ischemic - inducing solutions for long periods of time , such as up to 24 hours for some harvested kidneys . acidosis can encourage development of damps formation , such as crystallized uric acid / urates , as the solubility of urates is 1 to 4 mg / dl in a ph range of 3 to 6 versus 15 mg / dl or higher in ph & gt ; 7 . 0 . similarly calcium phosphate / pyrophosphates can crystallize in acidic conditions and observers have noted their deposition in mitochondria of ir models . moreover urates also crystallize optimally in cold temperatures with solubility of 4 . 5 mg / dl at 30 ° c . compared to 7 . 0 mg / dl at 37 ° c . hence both tissue acidosis and hypothermia during transplantation procedures are ideal conditions for urate / calcium pp crystallization along with formation of other previously mentioned damps from effects of acidosis , all of which are capable of stimulating il - 1β secretion from the nalp - 3 inflammasome ; 2 ) elevated uric acid has been observed in recipients of cardiac , renal and hepatic transplantation . numerous investigators have noted an unexplained association of hyperuricemia with increased risk of renal , cardiac and hepatic allograft rejection . the posited hypothesis may provide one explanation for organ rejection and dysfunction in association with elevations of this biomarker ; ( 3 ) secretion of il - 1β in ir models is well established and similar observations have been noted in transplantation studies . most of the evidence for il - 1β mediation of inflammation in transplantation is derived from studies with il - 1β tt comprising il - 1β receptor antagonists . harada et al demonstrated that gene transfection of il - 1β receptor antagonist into rats subjected to liver iri stress led to concomitant reduction in liver damage and increased survival rates in rats with gene transfection versus controls . using cultures of kidney tissue obtained from human subjects with renal graft tolerance , de oliveira et al demonstrated that an allograft tolerance factor was identifiable with the natural il - 1β receptor antagonist , further supporting the role of il - 1β as an important cytokine capable of contributing to allograft rejection . in pancreatic islet animal models , il - 1β receptor antagonist was shown to abrogate necrosis and enhance islet engraftment . a similar observation with transfected il - 1β receptor antagonist was observed protecting rodent myocardium from ir injury , and in an ex - vivo human atrial myocardial ischemia study with increased secretion of il - 1β , addition of il - 1β receptor antagonist improved myocardial contractility . finally , evaluation of gene polymorphism for il - 1β and its receptor suggest their levels can predict graft outcomes , as haplotypes with predictably low production of il - 1β receptor antagonist and high il - 1β secretion may be risk factors for renal graft rejection . several fda approved il - 1β tt biologics are commercially available , such as an il - 1β receptor blocker ( anakinra ), il - 1β trap ( rilanocept ) and a monoclonal anti - il - 1β antibody canakinumab ). the use of these biologics over a combined three to four year or more time span in caps and other il - 1β mediated disorders has been efficacious with excellent safety profiles , as there have been minimal or no reports of serious adverse events with these compounds . other caspase - 1 inhibitors not yet fda approved may also interfere in il - 1β function by inhibiting il - 1β secretion . it is again noteworthy to emphasize that there have been no known human trials or treatments with any of these biologics to preserve harvested organs and for transplantation procedures . the reticence to use il - 1 tt in humans undergoing organ transplantation should be partially overcome by the concepts promulgated in this invention . 1 . martinon f , burns k , tschopp j . the inflammasome : a molecular platform triggering activation of inflammatory caspases and processing of prol l - 1beta . mol cell 2002 ; 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