The Pathological Role Of Anti-ganglioside Antibody In Related Immune Neuropathies, And IVIg's Protection Effect In Those Diseases

Guillain-Barrésyndromes (GBS) currently are the world's leading causeof acute autoimmune neuromuscular paralysis. Gangliosides, sialicacid-bearing glycosphingolipids, are highly enriched in the vertebrate nervoussystem. Anti-ganglioside antibodies are associated with various humanneuropathies. Different lines of investigation have found strong evidence ofthe pathogenic role of anti-ganglioside antibodies in some forms of GBS.However, there're still plenty of important issues related with the role ofanti-ganglioside antibody remain unproven. For example, the nature ofantigens targeted by anti-ganglioside Abs has not been fully addressed, whichis crucial to any autoimmune disease; the experimental data for the role ofcomplement also remain inconclusive, etc. Understanding those cellular andmolecular mechanisms involved in the pathogenesis of anti-gangliosideantibody mediated injury is crucial to the development of specificimmunotherapies. A critical step in this regard is the generation of in vitromodels of such injury. Currently, lack of a robust mouse model of GBSprecludes addressing this question in vivo. To address these issues wedeveloped two ex-vitro models: (ⅰ) anti-ganglioside Ab-mediated neuronalcytotoxicity assay; (2) Organotypic spinal cord cultures system, in whichanti-ganglioside antibody caused motor nerves degeneration.Also, because of lack of a passive transfer model, the role ofanti-ganglioside antibodies in GBS continues to be debated. We recently haveraised several high-affinity monoclonal IgG anti-ganglioside antibodies. Inthis study, we try passively transfer these antibodies by intraperitonealhybridoma implantation in mice. Then, we studied the possible factors whichcould play role in the development of neuropathy.Finally, in controlled clinical studies, intravenous immunoglobulin (IVIg) has been shown to be effective as first-line therapy for both acute and chronicimmune neuropathies that include Guillain-Barre' syndrome (GBS). Whetheror not, the efficacy of different IVIg brands, or different lots of the same brand,can affect the beneficial response differently in patients remain unclear. And, ithas never been systematically examined before. To address this question, weexamined the protective efficacy of different brands of IVIg and different lotsof the two brands in the in vitro anti-ganglioside Ab-mediated neuronalcytotoxicity model.PartⅠ: Anti-ganglioside antibody-mediated neuronal cytotoxicity and itsprotection by intravenous immunoglobulin: implications for immuneneuropathiesPurpose:Study two basic issues related to antigangliosideantibody-mediated neural injury: (1) some anti-ganglioside antibodies cancross-react with glycoproteins and therefore the nature of antigens targeted bythese antibodies is not well established; and (2) although pathological studiessuggest that complement activation occurs in GBS, experimental data for therole of complement remain inconclusive.Method: we developed and characterized a simple anti-gangliosideantibody mediated cytotoxicity assay, which is based on two neuronal cell line:NG108-15 and it's mutant cell line, NG-CR-72 cell. In this assay, weadministrate both anti-gangliosides monoclonal antibodies and GBS patients'sera having anti-ganglioside activity. IVIg's (human intravenousimmunoglobulin) protection effect was also being tested in this model.Results: Our results demonstrate first, that both GBS sera containinganti-ganglioside antibodies and monoclonal anti-ganglioside antibodies causeneuronal cell lysis by targeting specific cell surface gangliosides, and secondly,that this cell lysis is complement dependent. In this assay, the GD1a cellmembrane pool appears to be more susceptible to antigangliosideantibody-mediated injury than the GM1 pool. Further, IVIg significantlydecreased cytotoxicity in this assay. Our data indicate that the mechanisms ofIVIg-mediated protection in this assay include anti-idiotypic antibodies, and down-regulation of complement activation.Conclusion: This simple cytotoxicity assay can potentially be used forscreening of (ⅰ) pathogenic anti-ganglioside antibodies in patients withimmune-mediated neuropathies; and (ⅱ) new/experimental therapies toprevent anti-ganglioside antibody-mediated neural injury.PartⅡ: An Anti-ganglioside Antibody-Secreting Hybridoma InducesNeuropathy in MicePurpose: Currently, the role of anti-ganglioside antibodies in GBScontinues to be debated because of lack of a passive transfer model. Severalpossible factors which could affect the role of anti-ganglioside antibody innerve injury were discussed in this study. We hope it can help developing thesuccessful in-vivo animal passive transfer model.Methods:We recently have raised several monoclonal IgGanti-ganglioside antibodies. We passively transfer these antibodies byintraperitoneal hybridoma implantation and by systemic administration ofpurified anti-ganglioside antibodies in mice. Finally, the pathological studywas done on those animals, and the nerve injury was evaluated. Theblood-nerve barrier in these animals was also being studied.Results:Approximately half the animals implanted with anintraperitoneal clone of anti-ganglioside antibody-secreting hybridomadeveloped a patchy, predominantly axonal neuropathy affecting a smallproportion of nerve fibers. In contrast to hybridoma implantation, passivetransfer with systemically administered anti-ganglioside antibodies did notcause nerve fiber degeneration despite high titre circulating antibodies.Blood-nerve barrier studies indicate that animals implanted with hybridomahad leaky blood-nerve barrier compared to mice that received systemicallyadministered anti-ganglioside antibodies.Conclusion: Our findings suggest that in addition to circulatingantibodies, factors such as antibody accessibility and nerve fiber resistance toantibody-mediated injury play a role in the development of neuropathy.PartⅢ: A high-affinity monoclonal anti-ganglioside antibody causes degeneration of motor nerves in vitroPurpose: To study the anti-ganglioside's role in the motor nerve injury.Methods: Recently we generated two high affinity IgG anti-GD1amonoclonal antibodies whose toxic effect on motor nerves in organotypic ratspinal cord cultures was studied.Results: Organotypic spinal cord cultures incubated with acomplement-fixing monoclonal anti-GD1a-related antibody demonstrated asignificant drop in motor neuron numbers and showed evidence ofWallerian-like degeneration of nerves. Isotype-matched sham antibody andnon-complement-fixing anti-GD1a antibody did not induce significantdegeneration of motor nerves.Conclusion: Our results indicate that the spinal cord culture system is auseful in vitro model for studying immune injury to motor neurons and nerves.The observation of in vitro cytotoxicity of anti-ganglioside antibody to motornerves supports the pathogenic role of anti-ganglioside antibodies found in theAMAN variant of GBS.PartⅣ: Comparison of different brands of IVIg in an in vitro model ofimmune neuropathyPurpose: To analyze and compare the protection efficacy of differentbrands or different lots of Intravenous immunoglobulin's (IVIg).Methods: we compared the efficacy of eight FDA approved brandsand/or lots of two brands of IVIg in a cell culture model of immuneneuropathy.Results: We report that products examined were equally effective andthere was no lot-to-lot variability in our experimental model.Conclusion: These findings support the notion that efficacy of differentIVIg products is comparable in a standardized model.