Physiology of gangliosides and the role of antiganglioside antibodies in human diseases.

Gangliosides are structurally and functionally polymorphic sialic acid containing glycosphingolipids that are widely distributed in the human body. They play important roles in protecting us against immune attacks, yet they can become targets for autoimmunity and act as receptors for microbes, like the influenza viruses, and toxins, such as the cholera toxin. The expression patterns of gangliosides vary in different tissues, during different life periods, as well as in different animals. Antibodies against gangliosides (AGA) can target immune attack e.g., against neuronal cells and neutralize their complement inhibitory activity. AGAs are important especially in acquired demyelinating immune-mediated neuropathies, like Guillain-Barré syndrome (GBS) and its variant, the Miller-Fisher syndrome (MFS). They can emerge in response to different microbial agents and immunological insults. Thereby, they can be involved in a variety of diseases. In addition, antibodies against GM3 were found in the sera of patients vaccinated with Pandemrix®, who developed secondary narcolepsy, strongly supporting the autoimmune etiology of the disease.

Autoantibodies against ganglioside GM3 are associated with narcolepsy-cataplexy developing after Pandemrix vaccination against 2009 pandemic H1N1 type influenza virus.

Following the mass vaccinations against pandemic influenza A/H1N1 virus in 2009, a sudden increase in juvenile onset narcolepsy with cataplexy (NC) was detected in several European countries where AS03-adjuvanted Pandemrix vaccine had been used. NC is a chronic neurological disorder characterized by excessive daytime sleepiness and cataplexy. In human NC, the hypocretin-producing neurons in the hypothalamus or the hypocretin signaling pathway are destroyed by an autoimmune reaction. Both genetic (e.g. HLA-DQB1*0602) and environmental risk factors (e.g. Pandemrix) contribute to the disease development, but the underlying and the mediating immunological mechanisms are largely unknown. Influenza virus hemagglutinin is known to bind gangliosides, which serve as host cell virus receptors. Anti-ganglioside antibodies have previously been linked to various neurological disorders, like the Guillain-Barré syndrome which may develop after infection or vaccination. Because of these links we screened sera of NC patients and controls for IgG anti-ganglioside antibodies against 11 human brain gangliosides (GM1, GM2, GM3, GM4, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GQ1b) and a sulfatide by using a line blot assay. Samples from 173 children and adolescents were analyzed: 48 with Pandemrix-associated NC, 20 with NC without Pandemrix association, 57 Pandemrix-vaccinated and 48 unvaccinated healthy children. We found that patients with Pandemrix-associated NC had more frequently (14.6%) anti-GM3 antibodies than vaccinated healthy controls (3.5%) (P = 0.047). Anti-GM3 antibodies were significantly associated with HLA-DQB1*0602 (P = 0.016) both in vaccinated NC patients and controls. In general, anti-ganglioside antibodies were more frequent in vaccinated (18.1%) than in unvaccinated (7.3%) individuals (P = 0.035). Our data suggest that autoimmunity against GM3 is a feature of Pandemrix-associated NC and that autoantibodies against gangliosides were induced by Pandemrix vaccination.

Characteristics of MuA transposase-catalyzed processing of model transposon end DNA hairpin substrates.

Bacteriophage Mu uses non-replicative transposition for integration into the host's chromosome and replicative transposition for phage propagation. Biochemical and structural comparisons together with evolutionary considerations suggest that the Mu transposition machinery might share functional similarities with machineries of the systems that are known to employ a hairpin intermediate during the catalytic steps of transposition. Model transposon end DNA hairpin substrates were used in a minimal-component in vitro system to study their proficiency to promote Mu transpososome assembly and subsequent MuA-catalyzed chemical reactions leading to the strand transfer product. MuA indeed was able to assemble hairpin substrates into a catalytically competent transpososome, open the hairpin ends and accurately join the opened ends to the target DNA. The hairpin opening and transposon end cleavage reactions had identical metal ion preferences, indicating similar conformations within the catalytic center for these reactions. Hairpin length influenced transpososome assembly as well as catalysis: longer loops were more efficient in these respects. In general, MuA's proficiency to utilize different types of hairpin substrates indicates a certain degree of flexibility within the transposition machinery core. Overall, the results suggest that non-replicative and replicative transposition systems may structurally and evolutionarily be more closely linked than anticipated previously.

Functional comparison of the transposition core machineries of phage Mu and Haemophilus influenzae Mu-like prophage Hin-Mu reveals interchangeable components.

Bacteriophage Mu uses DNA transposition for propagation and is a model for transposition studies in general. Recent identification of Mu-like prophages within bacterial genomes offers new material for evolutionary and comparative functional studies. One such prophage, Hin-Mu of Haemophilus influenzae Rd, was studied for its transpositional properties. The components of its transposition core machinery, the encoded transposase (MuA(Hin)) and the transposase binding sites, were evaluated for functional properties by sequence comparisons and DNase I footprinting. Transpositional activity of Hin-Mu was examined by in vitro assays directly assessing the assembly and catalytic function of the transposition core machinery. The Hin-Mu components readily assembled catalytically competent protein-DNA complexes, transpososomes. Thus, Hin-Mu encodes a functional transposase and contains critical transposase binding sites. Despite marked sequence differences, components of the Hin-Mu and Mu transposition core machineries are partially interchangeable, reflecting both conservation and flexibility in the functionally important regions within the transpososome structure.