Copolymer 1 (Cop 1) is a synthetic basic random copolymer of amino acids that has been shown to be effective in suppression of experimental allergic encephalomyelitis and is being tested as a
Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complementmediated intravascular hemolysis because of the lack from erythrocyte surface of the complement regulators CD55 and CD59, with subsequent uncontrolled continuous spontaneous activation of the complement alternative pathway (CAP), and at times of the complement classic pathway. Here we investigate in an in vitro model the effect on PNH erythrocytes of a novel therapeutic strategy for membranetargeted delivery of a CAP inhibitor. TT30 is a 65 kDa recombinant human fusion protein consisting of the iC3b/C3d-binding region of complement receptor 2 (CR2) and the inhibitory domain of the CAP regulator factor H (fH). TT30 completely inhibits in a dose-dependent manner hemolysis of PNH erythrocytes in a modified extended acidified serum assay, and also prevents C3 fragment deposition on surviving PNH erythrocytes. The efficacy of TT30 derives from its direct binding to PNH erythrocytes; if binding to the erythrocytes is disrupted, only partial inhibition of hemolysis is mediated by TT30 in solution, which is similar to that produced by the fH moiety of TT30 alone, or by intact human fH. TT30 is a membranetargeted selective CAP inhibitor that may prevent both intravascular and C3-mediated extravascular hemolysis of PNH erythrocytes and warrants consideration for the treatment of PNH patients. (Blood.
2012;119(26):6307-6316) IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is a blood disorder clinically characterized by intravascular hemolysis, thrombophilia, and bone marrow failure. 1-3 A unique feature of PNH is the presence of clonal populations of blood cells that are defective in glycosylphosphatidylinositol (GPI)-anchor biosynthesis, 4 because they derive from stem cells harboring an acquired somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene. 5,6 GPIlinked surface proteins include CD55 (also known as decayaccelerating factor [DAF]) 7,8 and CD59 (or membrane inhibitor of reactive lysis [MIRL]), 9,10 2 major complement regulators on the cell surface. Because of the lack of these 2 regulators, PNH erythrocytes (red blood cells [RBCs]) are exquisitely vulnerable to complement activation. Indeed, the main mechanism of hemolysis in PNH is the intravascular destruction of CD59 deficient RBCs by the membrane attack complex (MAC; supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). 11 MAC formation in patients can be abrupt and massive when complement is triggered by specific conditions, as with an infection, explaining the paroxysms of hemoglobinuria that have given PNH its name. However, at a lower rate, hemolysis in PNH is continuous, and it is accounted for by the so-called tickover of the complement alternative pathway (CAP). Tickover occurs through spontaneous hydrolysis of C3, binding of factor B to this form of C3, and subsequent formation of a C3 cleavage and activating multiprotein complex designated a C3 convertase (supplemental Figure 1). 12,1...
K1H 8L6 y5 T cells congregate in MS plaques and react to heat shock proteins expressed by human oligodendrocytes. We have previously demonstrated that peripheral blood (PB) derived y5 T cells readily lyse human oligodendrocytes in vitro. We now examine the mechanism underlying this cytotoxicity using y5 T cells derived from both MS PB and cerebrospinal fluid (CSF), well characterized target cell lines, and freshly derived human oligodendrocytes. y5 T cells are expanded in vitro by stimulation with anti-y5 TCR monoclonal antibody and supplementation with IL-2 and IL-4 (CSF) or IL-2 alone (PB). The purity of resulting populations was assessed by flow cytometry and contaminating cell subsets (ap T cells) were further eliminated by C'-lysis. Human oligodendrocytes were isolated from brain specimens obtained during surgery for intractable epilepsy and were kindly provided by Dr. J. Antel (MNI). Cytotoxicity was measured by a 5hr ~ICrrelease assay. To differentiate between the two major pathways for cytotoxicity (Fas-mediated or release of granzymes or perforin) a series of specific inhibitors were used. Physical interference with y5 T cell-target cell interaction in transwells, or pre-incubation of y8 T cells for 2 hr with the calcium chelator Mg2EDTA (2.5-7mM), which blocks granule exocytosis and perforin function, or with concanamycin A (CMA), (1-2,OOOnM), an H+-ATPase inhibitor that raises pH within granules leading to accelerated perforin degradation, completely abrogated lysis of target cells with little Fas expression (Daudi, U937). The addition of brefeldin A, an inhibitor of the cell surface transportation of glycoprotein (leading to low expression of Fas Ligand on y8 T cells) was required however, to completely abolish the lysis of Fas-expressing targets (Jurkat, oligodendrocytes). The inhibition of MS CSF or PB derived y5 T cell-induced lysis of oligodendrocytes with CMA alone was 60-90%. No differences in the magnitude of killing was observed among y5 T cells derived from MS or non-MS patients, CSF or PB. Pre-treating target cells with anti-Fas monoclonal antibody ZB4 (up to 3pg/ml) or addition of isocoumarins, that interfere with granzyme activity, had little effect in blocking cytolysis. These results demonstrate that human oligodendrocytes are highly susceptible to lysis via the perforin-based mechanism used by y5 T cells. Blocking this cytotoxic mechanism in vivo, may thus represent a novel strategy for the protection of oligodendrocytes from cytotoxic T cell-mediated damage in MS.
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