Background: IgE epitope mapping of allergens reveals important information about antigen elicitors involved in allergic reactions. The peptide-based microarray immunoassay offers an advantage of scale and parallel design over previous methods of epitope mapping. It has been used to map epitopes of some food allergens but has never been used with fish allergens. Objective: We sought to develop a peptide microarray immunoassay to map allergenic fish epitopes of two isoforms of Atlantic salmon (Salmo salar) parvalbumin, Sal s 1 beta 1 and Sal s 1 beta 2. Methods: Sera from 16 fish-allergic patients with specific IgE to salmon parvalbumin were used. Twelve healthy volunteers were used as negative controls. A library of overlapping peptides was synthesized commercially, representing the primary sequence of Sal s 1 beta 1 and Sal s 1 beta 2. Peptides were used to analyze allergen-specific IgE antibodies by immunolabeling with patient sera. Results: Three antigenic regions, not previously described, were identified in Sal s 1 beta 1. Two of them correlated with those previously reported in Gad c 1, parvalbumin from Baltic cod (Gadus callarias). No allergenic regions were found in Sal s 1 beta 2. This could be explained by crucial amino acid substitutions between isoforms. Conclusions: We have identified three antigenic regions in Sal s 1 beta 1 using a peptide microarray immunoassay. These three sequential epitopes formed a unique antigenic determinant in the three-dimensional model of the protein. In addition, we proved that isoforms from the same protein might have a different allergenic behavior.

Objective—: To identify proteins related to intraluminal thrombus biological activities that could help to find novel pathological mechanisms and therapeutic targets for human abdominal aortic aneurysm (AAA). Approach and Results—: Tissue-conditioned media from patients with AAA were analyzed by a mass spectrometry–based strategy using liquid chromatography coupled to tandem mass spectrometry. Global pathway analysis by Ingenuity software highlighted the presence of several circulating proteins, among them were proteins from the complement system. Complement C3 concentration and activation were assessed in plasma from AAA patients (small AAA, AAA diameter=3–5 cm and large AAA, AAA diameter >5 cm), showing decreased C3 levels and activation in large AAA patients. No association of a combination of single-nucleotide polymorphisms in complement genes between large and small AAA patients was observed. Intense extracellular C3 inmunostaining, along with C9, was observed in AAA thrombus. Analysis of C3 in AAA tissue homogenates and tissue-conditioned media showed increased levels of C3 in AAA thrombus, as well as proteolytic fragments (C3a/C3c/C3dg), suggesting its local deposition and activation. Finally, the functional role of local complement activation in polymorphonuclear (PMN) cell activation was tested, showing that C3 blockade by anti-C3 antibody was able to decrease thrombus-induced neutrophil chemotaxis and reactive oxygen species production. Conclusions—: A decrease of systemic C3 concentration and activity in the later stages of AAA associated with local complement retention, consumption, and proteolysis in the thrombus could induce PMN chemotaxis and activation, playing a detrimental role in AAA progression.

To study the role of lipocalin-2 (Lcn2) and the effect of Lcn2 blockade via anti-Lcn2 antibody in the development of abdominal aortic aneurysm (AAA). Expression mRNA and protein levels of Lcn2 and its human orthologue neutrophil gelatinase-associated lipocalin (NGAL) in aortic wall samples from experimental mouse and human AAA samples, respectively, were analysed by real-time PCR and immunohistochemistry. Experimental AAA was induced by aortic elastase perfusion in wild-type mice (WT) and Lcn2-deficient mice (Lcn2−/−). NGAL/Lcn2 mRNA and protein levels in human and murine AAA samples were increased compared with healthy aortas. Decreased AAA incidence and reduced aortic expansion were observed in Lcn2−/− mice or mice preoperative treated with a polyclonal anti-Lcn2 antibody compared with WT mice or mice treated with control IgG, respectively, at Day 14 after elastase perfusion. Moreover, immunohistochemical analysis of AAA tissues from Lcn2−/− or anti-Lcn2-treated mice showed diminished elastin damage, reduced microvessels and polymorphonuclear neutrophil (PMN) infiltration, and enhanced preservation of vascular smooth muscle cells compared with WT aortas. Fluorescent molecular tomography revealed decreased MMP activity in AAA of Lcn2−/− mice compared with WT controls. Therapeutic administration of anti-Lcn2 antibody to WT mice 3 days after elastase perfusion decreased aortic dilatation and PMN infiltration compared with WT mice treated with control IgG. Either Lcn2 deficiency or anti-Lcn2 antibody blockade limits AAA expansion in mice by decreasing PMN infiltration in the aorta. Lcn2 modulation may therefore be a viable new therapeutic option for the treatment of AAA.