First, IVIg fractionated over an SNA column shows increased sialylation mainly in the Fab region, as measured by glycoanalysis with high-performance liquid chromatography (HPLC), or liquid chromatographyCmass spectrometry (LC-MS)
First, IVIg fractionated over an SNA column shows increased sialylation mainly in the Fab region, as measured by glycoanalysis with high-performance liquid chromatography (HPLC), or liquid chromatographyCmass spectrometry (LC-MS). encephalomyelitis (EAE) model 6; and rheumatoid arthritis (RA) 7, using the collagen antibody-induced arthritis (CAbIA) and K/BxN models. We assessed the effects of IgG sialylation in these animal models by comparing sialic acid-enriched and -depleted IgG fractions with the untreated product. To achieve the highly sialylated IgG fraction, we used affinity chromatography with sialic acid-specific Sagglutinin (SNA) lectin. Desialylation was achieved using neuraminidase (NAse) to produce the negative material. You will find two important factors to consider when using the SNA lectin fractionation method 8. First, IVIg fractionated over an SNA column shows increased sialylation primarily in the Fab region, as measured by glycoanalysis with high-performance liquid chromatography (HPLC), or liquid chromatographyCmass spectrometry (LC-MS). The findings from both assays indicated the SNA-binding portion represents approximately 10% of the IgG molecules in IVIg. However, Fc-sialylation as assessed by LC-MS was improved in one subfraction, representing only 1C2% of the IgG molecule and only by a very small amount 8. Secondly, we have shown that lectin chromatography can result in a skewed antibody pattern of the Allopurinol sodium SNA-binding portion 8. Antigen specificity of IgG fractions was tested by enzyme-linked immunosorbent assay (ELISA) with a range of pathogen-derived and autoantigens, and shown abnormal antibody concentration patterns in the eluted material compared to the starting material. Our recent study in the antibody-induced K/BxN and CAbIA RA models in mice 7 set out to set up the part of sialylation in antibody-driven swelling. The restorative effect of IVIg and its proposed mechanisms of action were analyzed using both prophylactic and restorative protocols. Prophylactic IVIg protocols have been shown in earlier studies to protect against arthritis in the K/BxN model 1,3, and this study shown the effectiveness of prophylaxis in the CAbIA model; disease severity was reduced significantly (NAse-treated IVIg or Fc in the CAbIA Allopurinol sodium model, and confirmed that desialylation Allopurinol sodium experienced no effect on the prophylactic effectiveness of IVIg/Fc. Moreover, no enhanced effects were observed when using sialic acid-enriched (by SNA fractionation) Fc fractions prophylactically, compared to untreated Fc, in the CAbIA model. Of notice, it was observed that highly sialylated Fc certain to Protein A in a similar manner to untreated Fc, indicating that the portion had not been compromised from the SNA fractionation process 7. To further clarify our contrasting results in the CAbIA model with those reported previously in the K/BxN model 1,3, we repeated the prophylactic protocol in the K/BxN model. Our results confirmed that sialylation of IVIg or Fc was not required for suppression of swelling with this model 7. Because basophils have been suggested as important cell mediators in the anti-inflammatory mechanism of IVIg 4, as explained above, they were also examined in the CAbIA model 7. Mice were injected with basophil-depleting monoclonal antibody (mAb) MAR-1 twice daily for days 1C3 before beginning IVIg treatment (day time 5). No difference was observed during the next 7 days between the disease response of mice treated with MAR-1 and those treated with an isotype control. In the K/BxN model, this was tested having a prophylactic approach; mice were injected with MAR-1 for 10 consecutive days, beginning 2?days prior to IVIg treatment. When disease was launched at day time 0, the IVIg prophylaxis safeguarded the mice from arthritis as normal, as assessed by both medical score and caliper-measurement of ankle swelling (model of systemic lupus erythematosus (SLE) 9. These multiple mechanisms of action show that further study is required in order to fully understand the anti-inflammatory effects of IVIg. Acknowledgments F. K. and I. K. C. say thanks to Sylvia Miescher, Adrian W. Zuercher and Donald R. Branch for medical Rabbit Polyclonal to KLF11 input; we would also like to say thanks to Meridian HealthComms Ltd for providing medical writing solutions. Disclosure F. K. and I. K. C. are employees of CSL Behring AG and CSL Ltd, respectively..