Human IgM paraproteins demonstrate shared reactivity between Campylobacter jejuni lipopolysaccharides and human peripheral nerve disialylated gangliosides.

IgM paraproteins from patients with CANOMAD (chronic ataxic neuropathy, ophthalmoplegia, M-protein, agglutination, anti-disialosyl antibodies) react with NeuAc(alpha 2-8)NeuAc epitopes on a wide range of gangliosides including GQ1b, GT1a, GD1b and GD3. The tissue distribution of reactive antigens in human peripheral nerve has not been addressed in detail. In addition, the origin of these antibodies is unknown. Here we report that purified anti-disialosyl paraproteins from two affected patients bind a wide array of human peripheral nerve structures including dorsal root ganglia, dorsal and ventral root axons, femoral and oculomotor nerves. We also show that these paraproteins bind lipopolysaccharides of Campylobacter jejuni isolates from 3/3 cases of Miller Fisher syndrome, and to a less frequent extent, from cases of Guillain-Barré syndrome and enteritis controls. In conjunction with our previous studies, these data provide a possible causal link between the origin and pathogenic effects of anti-disialosyl antibodies in human paraproteinaemic neuropathy.

Semi-preparative purification and validation of monoclonal antibodies for immunotherapy in mice.

A number of rat hybridomas were adapted to grow in RPMI containing either 5% IgG-depleted FCS or 1% serum-free Nutridoma. Alternatively, protein-free Ultradoma PF was used. Growth in these media allowed purification procedures to be used that are based on tangential ultrafiltration in combination with affinity chromatography on gels linked to protein G or anti-rat L chain coupled antibodies. The isolated antibody preparations were found to be pure and to consist of monomeric intact IgG. The yield and recovery of mAb using this procedure were found to be consistently high. These antibody preparations were analyzed for endotoxin contamination. Whereas during isolation endotoxin contamination increased, the endotoxin content per mg purified protein did not. Affinity chromatography on Detoxi-gel resulted in the efficient removal of this contamination and using this protocol the antibody preparations obtained were found to be of sufficient purity, activity and low endotoxin content to permit their in vivo use in animal models of immunotherapy.

Modulation of systemic cytokine levels by implantation of alginate encapsulated cells.

The availability of cell lines that are transfected with IL-4, IL-5 and IFN-gamma cytokine genes permits the prolonged in vivo delivery of functional cytokines in relatively large doses for the modulation of specific immune responses. Often the transfected cells are xenogeneic or allogeneic to the experimental animal and have to be encapsulated in such a way that no cellular response by the host will be induced. Alginate has proven to be a simple matrix for encapsulating cells under mild conditions suitable for in vivo implantation. Encapsulated cells express the transfected IL-4 gene for at least 14 days after in vivo implantation and were shown to be functional during that period by modulating ongoing IgE responses. The application of adherent growing transfected cells permits dose-response titrations and provides an easy method for local and systemic cytokine delivery. Alternatively, hybridoma cells can be encapsulated and the secreted antibody monitored in the serum. It was found that no host immune response was triggered by alginate encapsulated cells. The efficiency of treatment by encapsulated hybridoma cells was shown to be equivalent to that of injecting purified antibodies.

Cytokine Detection and Modulation in Acute Graft vs. Host Disease in Mice.

A murine model for acute lethal graft vs. host disease (GVHD) was used to study the role that a number of cytokines play in the development of lethal GVHD. In this study we focused on the role of IL-1, IL-2, IL-4, IL-6, IFN-gamma and TNF-alpha. Lethally irradiated (C57BL x CBA)F1 mice were reconstituted either with 10(7) allogeneic BALB/c spleen cells or with a similar number of syngeneic cells, as a control. A significant rise in serum levels of IL-6, TNF-alpha and IFN-gamma levels was found in allogeneically reconstituted mice. This is in contrast to the syngeneic control group in which no rise was seen. Serum IL-2 and IL-4 levels were below the detection limit. In the supernatant of Con A stimulated spleen cells from allogeneically reconstituted mice IL-6, IFN-gamma and TNF-alpha concentrations were increased. The expression of mRNA for cytokines as detected by reverse transcription PCR was studied in spleen cells. In the allogeneic reconstituted mice the mRNA expression of IL-1alpha, IL-2, IL-6, IFN-gamma and TNF-alpha displayed faster kinetics compared with that in syngeneic reconstituted mice. The effect of treatment with recombinant cytokines, antibodies to cytokines and to cytokine receptors on the development of GVHD was investigated. Administration of recombinant IL-2 to allogeneically reconstituted mice strongly increased the morbidity and mortality whereas injection of IL-1alpha and TNF-alpha did not influence survival. Administration of antibodies against IL-2 or the IL-2 receptor decreased the morbidity and mortality. Anti-IL-6, anti-IFN-gamma, and anti-TNF-alpha mAB, on the other hand, did not affect the morbidity and mortality of GVHD. The results of this study suggest successive waves of cytokine-secreting cell populations consistent with the induction of an inflammatory response in the development of acute GVH disease.