Volume 130, Issue 6, Pages 1764-1775 (May 2006) Candida albicans Is an Immunogen for Anti–Saccharomyces cerevisiae Antibody Markers of Crohn’s Disease  Annie Standaert–Vitse, Thierry Jouault, Peggy Vandewalle, Céline Mille, Mimouna Seddik, Boualem Sendid, Jean–Maurice Mallet, Jean–Frédéric Colombel, Daniel Poulain  Gastroenterology  Volume 130, Issue 6, Pages 1764-1775 (May 2006) DOI: 10.1053/j.gastro.2006.02.009 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Relationship between generation of ASCA and anti–C albicans antibodies in patients with CD and candidiasis. (A) Serologic follow-up of 3 patients with CD (CD1, ■; CD2, •; CD3, ♦). ASCA (black symbols and continuous line) and anti-C albicans PPM antibodies (open symbols and broken line) were determined by ELISA by using S cerevisiae SU1 PPM and C albicans VW32 PPM (Platelia Candida Ab), respectively. (B) Highest levels of anti–C albicans PPM antibodies (black chart) and ASCA (gray chart) in patients with systemic candidiasis. Patients were classed according to decreasing anti–C albicans antibodies titers. The gray lines represent the cut-off for ASCA (3.12 A.U.) and the black lines the cut-off for anti–C albicans PPM antibodies (10 A.U.). Gastroenterology 2006 130, 1764-1775DOI: (10.1053/j.gastro.2006.02.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Kinetic evolution of anti-C albicans antibodies and ASCAs in patients with candidiasis. Kinetic evolution of anti–C albicans antibodies (•) and ASCAs (○) in sera from 2 patients during the time course of a C albicans systemic infection. Individual characteristics of the patients are given in Table 1. Day 0 is the time when C albicans was first isolated from 1 infecting source. The dotted line corresponds to the level of significance of anti–C albicans antibody and ASCA levels test (as established for both tests). In all patients, the anti–C albicans antibodies paralleled with ASCA, both of which regularly decreased under antifungal therapy. Gastroenterology 2006 130, 1764-1775DOI: (10.1053/j.gastro.2006.02.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Kinetic evolution of anti–C albicans antibodies (•) and ASCA (○) in rabbits injected with yeasts. Data shown are representative of the anti–C albicans (•) and anti–S cerevisiae (○) PPMs antibodies responses observed in rabbits injected intravenously with either live C albicans yeasts (106 yeasts/mL; 2 rabbits) (A) or formalin-killed C albicans yeasts (106 yeasts/mL; 2 rabbits) (B). The controls consisted of rabbits injected intravenously with live S cerevisiae yeasts (108 yeasts/mL; 2 rabbits) (C). The antibody levels are expressed as the absorbance value at serum dilution of 1/1000 as analyzed by ELISA. The times of the injections are announced with the arrows. Rabbits infected by live C albicans yeasts developed antibody responses against both C albicans and S cerevisiae PPMs. By contrast, rabbits regularly injected with formalin-killed C albicans yeasts developed a less intense humoral response, which was, at the term of the immunization procedure, restricted to the C albicans antigen. Similarly, no cross-reactivity against C albicans antigen was observed in rabbits injected with live S cerevisiae yeasts. Gastroenterology 2006 130, 1764-1775DOI: (10.1053/j.gastro.2006.02.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 ASCA specificity of antibodies obtained from rabbits infected with C albicans. Sera obtained from rabbits infected with live C albicans yeast at day 80 were incubated with increasing concentrations of ΣMan3, a chemically constructed major epitope recognized by ASCA. The reactivity of pretreated serum samples was determined by ELISA against S cerevisiase PPM. The data represent the percentage of reactivity in terms of concentration of synthetic oligomannoses compared with untreated serum. Gastroenterology 2006 130, 1764-1775DOI: (10.1053/j.gastro.2006.02.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Reactivity of purified anti-Man3 immunoglobulins against a 120-kDa mannoprotein present in C albicans and S cerevisiae. (A) Western blot analysis of C albicans or S cerevisiae whole-cell extracts incubated with affinity purified anti-ΣMan3 immunoglobulins from infected rabbit sera (lane 1) or CD patients’ sera (lanes 2–4). (B) Western blot analysis of the reactivity of purified anti-ΣMan3 Igs from a CD patient against total extracts from wild-type S cerevisiae SU1 (lane 1) or the parental strain S cerevisiae S288C (lane 2), and S cerevisiae mutant strains mnn1Δ (lane 3) and mnt4Δ (lane 4), lacking 1 α-1,3 MNT. Gastroenterology 2006 130, 1764-1775DOI: (10.1053/j.gastro.2006.02.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 PPM from C albicans grown at pH 2 mimics PPM from S cerevisiae. The reactivity of sera from patients with CD or UC displaying different levels of ASCA was determined against PPM from C albicans grown at pH 2 or pH 6 by ELISA. The data represent (A) the correlation between reactivity against S cerevisiae PPM and reactivity against C albicans PPM under acidic growth conditions and (B) the correlation between reactivity against S cerevisiae PPM and C albicans PPM grown in neutral growth conditions. Pearson’s correlation coefficients were determined with SPSS software. Gastroenterology 2006 130, 1764-1775DOI: (10.1053/j.gastro.2006.02.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 Expression of the major epitope recognized by ASCA by S cerevisiae and C albicans grown in vitro under various conditions and by C albicans infecting human tissue. (A) (1) S cerevisiae yeasts, (2) C albicans yeasts grown at 37°C or (3) 28°C, and (4) C albicans hyphal forms were incubated with purified anti-ΣMan3 immunoglobulins from CD patients. The reaction was revealed with FITC-conjugated goat anti-human immunoglobulins. (B) Immunohistochemical staining of the ASCA epitope in C albicans–infected human tissue (duodenal [1] and bronchial [2] biopsies). Tissue sections were stained with biotinylated GNL and revealed with streptavidin-labelled with FITC. Gastroenterology 2006 130, 1764-1775DOI: (10.1053/j.gastro.2006.02.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions