PMC

░ Percentage of sera from ACD and different groups of CCD patients reactive with rGal-1. Analysis of IgE (□), IgM () and IgG (▪) isotypes.***P < 0·0001 (IgE isotype ACD versus controls); **P = 0·004 (IgM isotype ACD versus controls); *P = 0·024 (IgG isotype CCD versus controls).

IgE reactivity against rGal-1 in sera from controls, ACD and different groups of CCD patients. ELISA: each symbol represents the mean value of duplicates, obtained with each serum assayed at a 1 : 100 dilution. The horizontal line indicates the mean +2 SD, obtained with control sera. *P < 0·0001 (ACD versus controls).

Anti-Gal-1 antibodies eluted from ACD patients sera do not cross-react with SAPA. Sera from three patients (▪) or the eluted anti-Gal-1 antibodies (□) purified by immunoadsorption were tested against (a) SAPA and (b) rGal-1 by ELISA. Immunoreactivities were detected using a peroxidase-conjugated antihuman specific IgM antibody. Similar results were obtained using an alkaline phosphatase-conjugated anti human IgE antibody (data not shown).

IgM reactivity against rGal-1 in sera from controls, ACD and different groups of CCD. (a) ELISA: each symbol represents the mean value of duplicates, obtained with each serum assayed at a 1:100 dilution. The horizontal line indicates the mean +2 SD, obtained with control sera. *P = 0·005 (ACD versus controls). (b) Western blot: IgM immunoreactive profile of sera from ACD patients (1:200 dilution) against rGal-1 (lanes 1 and 2). Sera from control individuals were processed in parallel (lane 3). Molecular weight markers are indicated on the left.

Galectin-1 is differentially expressed in heart tissue from patients with CCD. (a) Heart tissue extracts corresponding to patients with CCD (lanes 3 and 4:50 and 10 µg, respectively) and to control patients (lanes 1 and 2:50 and 10 µg, respectively) were prepared and analysed by SDS-PAGE on a 15% polyacrylamide slab gel. Then, proteins were transferred onto nitrocellulose membranes and immunoblotted with a rabbit antihuman rGal-1 polyclonal antibody (1:500). The immunoreactive monomeric and dimeric forms of Gal-1 are indicated by the arrows. Results are representative of three independent experiments. (b) Gels were also stained by Coomassie blue to show the equal protein loading in comparable lanes.

IgG reactivity against rGal-1 in sera from controls, ACD and different groups of CCD patients. (a) ELISA: each symbol represents the mean value of duplicates, obtained with each serum assayed at a 1:100 dilution. The horizontal line indicates the mean +2 SD, obtained with control sera. *P = 0·05 (CCD GIII versus controls); *P = N.S. (ACD versus controls). (b) Western blot: IgG immunoreactive profile of sera from CCD patients (GI, GII and GIII) against rGal-1 (lanes 4, 5 and 6, respectively). Recombinant Gal-1 was resolved on a 15% polyacrylamide slab gel, transferred to nitrocellulose membranes and tested with a 1:100 dilution of each serum. Sera from control individuals were assayed at the same dilution (lane 7). The immunoreactive protein bands are indicated by the arrows. Positive controls were assayed by blotting rGal-1 (5, 1 and 0·5 µg) with the rabbit antihuman Gal-1 antibody (lanes 1, 2 and 3, respectively).