Wan-Yu Wang

Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys.

We transplanted kidneys from α1,3-galactosyltransferase knockout (GalT-KO) pigs into six baboons using two different immunosuppressive regimens, but most of the baboons died from severe acute humoral xenograft rejection. Circulating induced antibodies to non-Gal antigens were markedly elevated at rejection, which mediated strong complement-dependent cytotoxicity against GalT-KO porcine target cells. These data suggest that antibodies to non-Gal antigens will present an additional barrier to transplantation of organs from GalT-KO pigs to humans.

The role of anti-non-Gal antibodies in the development of acute humoral xenograft rejection of hDAF transgenic porcine kidneys in baboons receiving anti-Gal antibody neutralization therapy.

Background. The present study was undertaken to determine the role of preformed and induced anti-non-Gal antibodies in the rejection of hDAF pig-to-baboon kidney xenotransplants after anti-Gal antibody neutralization therapy. Methods. Seven baboons received life-supporting kidney transplants from hDAF transgenic pigs. Anti-Gal antibodies were neutralized by GAS914 or TPC (a Gal PEG glycoconjugate polymer). Group 1 (n=5) underwent a conventional immunosuppressive therapy with FK506, rabbit anti-thymocyte serum/immunoglobulin, mycophenolate mofetil, and steroids. Group 2 (n=2) received an anti-humoral immunity regimen with LF15-0195, Rituxan and cobra venom factor in addition to ATG, FK506 and steroids. Levels of anti-non-Gal antibodies and their mediated complement-dependent cytotoxic activities (CDC) were detected by flow cytometry using Gal knockout (k/o) pig lymphocytes (LC) or endothelial cells (EC) as targets. Results. Continuous infusion of GAS914/TPC significantly reduced anti-Gal antibodies. In Group 1, four of five baboons developed severe acute humoral xenograft rejection (AHXR) and the rejection was associated with either a high level of preformed anti-non-Gal IgG or a marked elevation in induced anti-non-Gal IgG and IgM. Sera collected at the time of AHXR had a high level of CDC to porcine LC/EC from Gal k/o animals. The intensive anti-humoral therapy in Group 2 completely inhibited both anti-Gal and non-Gal antibody production and prevented AHXR. However, this therapy was not well tolerated by the baboons. Conclusion. In a pig-to-baboon kidney transplant model, both preformed and induced anti-non-Gal antibodies are strongly associated with the pathogenesis of AHXR when anti-Gal antibodies are neutralized.

Complement inhibition enables renal allograft accommodation and long-term engraftment in presensitized nonhuman primates.

Protection against humoral injury mediated by donor‐specific antibodies (DSA), also known as accommodation, may allow for long‐term allograft survival in presensitized recipients. In the present study, we determined the role of complement in renal allograft accommodation in donor skin‐presensitized nonhuman primates under conventional immunosuppression. Donor skin allografts were transplanted to presensitized recipients 14 days prior to renal transplantation. Renal allografts not receiving any immunosuppressive treatment developed accelerated rejection with predominantly humoral injury, which was not prevented using conventional cyclosporine (CsA) triple therapy. Inhibition of complement activation with the Yunnan‐cobra venom factor (Y‐CVF) successfully prevented accelerated antibody‐mediated rejection and resulted in successful accommodation and long‐term renal allograft survival in most presensitized recipients. Accommodation in this model was associated with the prevention of the early antibody responses induced against donor antigens by complement inhibition. Some antiapoptotic proteins and complement regulatory proteins, including Bcl‐2, CD59, CD46 and clusterin, were upregulated in the surviving renal allografts. These results suggest that the complement inhibition‐based strategy may be valuable alternative in future clinical cross‐match positive or ABO‐incompatible transplantation.

Purification, characterization and biological activities of the L-amino acid oxidase from Bungarus fasciatus snake venom

L-amino acid oxidases (LAAOs) are widely distributed in snake venoms, which contribute to the toxicity of venoms. However, LAAO from Bungarus fasciatus (B. fasciatus) snake venom has not been isolated previously. In the present study, LAAO from B. fasciatus snake venom was purified by SP-Sepharose HP anion exchange chromatography followed by Heparin-Sepharose FF affinity chromatography procedure and the purified enzyme was named BF-LAAO. BF-LAAO presented an estimated molecular weight of 55kDa in SDS-PAGE and an apparent molecular weight of 70kDa in size-exclusion chromatography suggesting that BF-LAAO is a monomeric protein. Kinetics studies showed that BF-LAAO was very active against L-Tyr, L-Asp, L-Phe, L-Glu, L-Trp, L-His, L-Gln, L-Ile, L-Met, L-Leu and moderately active against L-Lys, L-Arg, L-Ala and L-Asn. BF-LAAO exhibited a cytotoxic effect on A549 cells and caused up to 41.2% apoptosis of A549 cells following 12h incubation period. In the mouse peritoneum, BF-LAAO provoked a marked increase in the number of neutrophils, lymphocytes and macrophages following injection. It also induced rabbit platelet aggregation in a dose-dependent manner. At 3h following injection, BF-LAAO elicited severe inflammation in the gastrocnemius muscles of mice, but failed to induce significant organ damage. In conclusion, the cytotoxic and proinflammatory activities of BF-LAAO could be the main cause of the local inflammation, which helps us to understand the pathogenesis of snakebite.

Characterization of three fibrinogenolytic enzymes from Chinese green tree viper (Trimeresurus stejnegeri) venom

From the venom of Chinese green tree viper (Trimeresurus stejnegeri), three distinct fibrinogenolytic enzymes: stejnefibrase-1, stejnefibrase-2 and stejnefibrase-3, were purified by gel filtration, ion-exchange chromatography and reverse-phase high-performance chromatography (HPLC). SDS-PAGE analysis of those three enzymes showed that they consisted of a single polypeptide chain with mol. wt of 50000, 31000 and 32000, respectively. Like TSV-PA (a specific plasminogen activator) and stejnobin (a fibrinogen-clotting enzyme) purified from the same venom, stejnefibrase-1, -2 and -3 were able to hydrolyze several chromogenic substrate. On the other hand, different from TSV-PA and stejnobin, stejnefibrase-1, -2 and -3 did not activate plasminogen and did not possess fibrinogen-clotting activity. The three purified enzymes directly degraded fibrinogen to small fragments and rendered it unclottable by thrombin. Stejnefibrase-2 degraded preferentially Bbeta-chain while stejnefibrase-1 and -3 cleaved concomitantly Aalpha and Bbeta-chains of fibrinogen. None of these proteases degraded the gamma-chain of fibrinogen. When correlated with the loss of clottability of fibrinogen, the most active enzyme was stejnefibrase-1. The activities of the three enzymes were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB), indicating that like TSV-PA and stejnobin, they are venom serine proteases.

Purification and cloning of a novel C-type lectin-like protein with platelet aggregation activity from Trimeresurus mucrosquamatus venom.

TMVA, a novel C-type lectin-like protein that induces platelet aggregation in a dose-dependent manner, was purified from the venom of Trimeresurus mucrosquamatus. It consists of two subunits, alpha (15536 Da) and beta (14873 Da). The mature amino acid sequences of the alpha (135 amino acids) and beta subunits (123 amino acids) were deduced from cloned cDNAs. Both of the sequences show great similarity to C-type lectin-like venom proteins, including a carbohydrate recognition domain. The cysteine residues of TMVA are conserved at positions corresponding to those of flavocetin-A and convulxin, including the additional Cys135 in the alpha subunit and Cys3 in the beta subunit. SDS-PAGE, mass spectrometry analysis and amino acid sequence showed that native TMVA exists as two convertible multimers of (alpha beta)(2) and (alpha beta)(4) with molecular weights of 63680 and 128518 Da, respectively. The (alpha beta)(2) complex is stabilized by an interchain disulfide bridge between the two alpha beta-heterodimers, whereas the stabilization of the (alpha beta)(4) complex seems to involve non-covalent interactions between the (alpha beta)(2) complexes.

Characterization of a thrombin-like enzyme from the venom of Trimeresurus jerdonii.

From the venom of Trimeresurus jerdonii, a distinct thrombin-like enzyme, called jerdonobin, was purified by DEAE A-25 ion-exchange chromatography, Sephadex G-75 gel filtration, and fast protein liquid chromatography (FPLC). SDS-PAGE analysis of this enzyme shows that it consists of a single polypeptide chain with a molecular weight of 38,000. The NH(2)-terminal amino acid sequence of jerdonobin has great homology with venom thrombin-like enzymes documented. Jerdonobin is able to hydrolyze several chromogenic substrates. The enzyme directly clots fibrinogen with an activity of 217 NIH units/mg. The fibrinopeptides released, identified by HPLC, consisted of fibrinopeptide A and a small amount of fibrinopepide B. The activities of the enzyme were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB). However, metal chelator (EDTA) had no effect on it, indicating it is venom serine protease.

Purification and characterization of jerdofibrase, a serine protease from the venom of Trimeresurus jerdonii snake

A fibrin(ogen)olytic serine protease from Trimeresurus jerdonii venom was identified and purified to SDS-polyacrylamide gel electrophoresis homogeneity. It is a single chain polypeptide with a molecular weight of 32kDa under reduced condition and 28kDa under non-reduced condition, respectively. The venom protease catalysed the hydrolysis of some chromogenic substrates such as S2238, S2160, S2302 and S2251. It degraded Bbeta-chain of human fibrinogen preferentially. Also the enzyme degraded fibrin directly. Its enzymatic activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF), but not affected by EDTA. That suggested it was a serine protease. N-terminal sequence of the purified component showed high homology with other snake venom serine proteases.

Characterization and cloning of a novel phospholipase A2 from the venom of Trimeresurus jerdonii snake

A phospholipase A(2) (PLA(2)), called jerdoxin, was isolated from Trimeresurus jerdonni snake venom and partially characterized. The protein was purified by three chromatographic steps. SDS-polyacrylamide gel electrophoresis in the presence or absence of dithiothreitol showed that it had a molecular mass of 15 kDa. Jerdoxin had an enzymatic activity of 39.4 micro mol/min/mg towards egg yolk phosphatidyl choline (PC). It induced edema in the footpads of mice. In addition, jerdoxin exhibited indirect hemolytic activity. About 97% hemolysis was observed when 2 micro g/ml enzyme was incubated for 90 min in the presence of PC and Ca(2+). No detectable hemolysis was noticed when PC was not added. Ca(2+) was necessary for jerdoxin to exert its hemolytic activity, since only 52% hemolysis was seen when Ca(2+) was absent in the reaction mixture. Furthermore, jerdoxin inhibited ADP induced rabbit platelet aggregation and the inhibition was dose dependent with an IC(50) of 1.0 micro M. The complete amino acid sequence of jerdoxin deduced from cDNA sequence shared high homology with other snake venom PLA(2)s, especially the D 49 PLA(2)s. Also, the residues concerned to Ca(2+) binding were conserved. This is the first report of cDNA sequence of T. jerdonii venom PLA(2).

Improved suppression of circulating complement does not block acute vascular rejection of pig-to-rhesus monkey cardiac transplants

Abstract:  At present, acute vascular rejection (AVR) remains a primary obstacle inhibiting long‐term graft survival in the pig‐to‐non‐human primate transplant model. The present study was undertaken to determine whether repetitive injection of low dose Yunnan‐cobra venom factor (Y‐CVF), a potent complement inhibitor derived from the venom of Naja kaouthia can completely abrogate hemolytic complement activity and subsequently improve the results in a pig‐to‐rhesus monkey heterotopic heart transplant model. Nine adult rhesus monkeys received a heterotopic heart transplant from wild‐type pigs and the recipients were allocated into two groups: group 1 (n = 4) received repetitive injection of low dose Y‐CVF until the end of the study and group 2 (n = 5) did not receive Y‐CVF. All recipients were treated with cyclosporine A (CsA), cyclophosphamide (CyP) and steroids. Repetitive Y‐CVF treatment led to very dramatic fall in CH50 and serum C3 levels (CH50 < 3 units/C3 remained undetectable throughout the experiment) and successfully prevented hyperacute rejection (HAR), while three of five animals in group 2 underwent HAR. However, the continuous suppression of circulating complement did not prevent AVR and the grafts in group 1 survived from 8 to 13 days. Despite undetectable C3 in circulating blood, C3 deposition was present in these grafts. The venular thrombosis was the predominant histopathologic feature of AVR. We conclude that repetitive injection of low dose Y‐CVF can be used to continuously suppress circulating complement in a very potent manner and successfully prevent HAR. However, this therapy did not inhibit complement deposition in the graft and failed to prevent AVR. These data suggest that using alternative pig donors [i.e. human decay accelerating factor (hDAF)‐transgenic] in combination with the systemic use of complement inhibitors may be necessary to further control complement activation and improve survival in pig‐to‐non‐human primate xenotransplant model.