Methods: Experiments were performed with CN var. grubii H99 (serotype A) grown in liquid Sabouraud media at 30ºC. First, we established that human IgM (Sigma Aldrich) binds H99 and Laminarin (a polymer consisting primarily of β (1-3) glucan with occasional β (1-6) branching (Sigma Aldrich) by ELISA using Goat Anti-Human IgM-AP Then, we cultured CN in titan cell medium (TCM, 5% Sabouraud and 5% Fetal bovine serum diluted in MOPS 50 mM at pH 7.3 plus 15 µM sodium azide) at 37ºC with CO2 for 18 hours with and without human IgM or IgG (Sigma Aldrich), after which cell size was evaluated using India Ink in a Zeiss microscope.
Results: We found that IgM-treated cells exhibited a significant reduction in CN capsule size and titan cell formation (total cell size) compared to controls without IgM or with IgG. Median total cell size (µm) were: IgM (15.04), IgG (20) and PBS (22.24), p<0.05 using the Kolmogorov-Smirnow test to estimate normality and one-way ANOVA to compare between groups. There were no statistical differences in cell size after incubation with human IgG or PBS. To gain insight into how IgM may mediate its effect, we demonstrated that it bound mainly to the CN cell wall with some diffuse punctuate to the capsule by immunofluorescence.
Conclusion: Our results reveal that natural IgM has the ability to inhibit CN titan cell formation in cultured cells. Given the importance of titan cell formation in virulence, our results suggest that direct effects of natural antibody on CN biology may contribute to human resistance to CD. This hypothesis is under investigation in our laboratory.
N. Trevijano Contador,