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P102 α-Defensins(αDef) 1-3 are specific plasmatic markers for Crohn’s disease (CD) at diagnosis and tissue α-Def 5 methylation is a pathogenic mechanism for αDef-5 down regulation in CD.

E. Cerrillo1, I. Moret-Tatay2,3, M. Iborra1,2, E. Sáez-González1, E. Busó4, D. Ramos5, L. Tortosa1, P. Nos1,3, B. Beltrán*1,3

1Hospital Universitari La Fe, Gastroenterology, Valencia, Spain, 2Ciberehd, Gastroenterology, Valencia, Spain, 3IIS Hospital La Fe, Gastroenterology, Valencia, Spain, 4School of Medicine. Valencia University, Central Unit of Medical Research, Valencia, Spain, 5Hospital Universitari La Fe, Pathology, Valencia, Spain

Background

α-Defensines (human antimicrobial peptides) are expressed in neutrophils (αDef 1-4) and paneth cells (αDef 5-6). Ileal decreased expression of αDef5 in Crohn’s diseade CD is known. Leucocyte α–Def are increased; however, their specificity and correlation with inflammation are not clear. Aims: to characterise the concentration and utility of α-Def1-4 in plasma of CD patients and analyse the mechanism of ileal α-Def5 reduction.

Methods

Patients: CD onset patients (n = 25); CD remission patients (n = 15); control subjects, gender and age matched (n = 15); and non-CD ileitis patients (n = 8). CD activity was evaluated by Harvey–Bradshaw Index (HBI). Samples: plasma (blood extraction at CD diagnose and at remission (HBI < 4 and normal C-reactive protein [CRP]) and ileal biopsies in all groups. α-Def1-3 and 4 were detected using ELISA Kits (Hycult Biotech αDef1-3, ref HK317-01and CusabioαDef-4, Kit, ref CSB-E17958h). Immunolocalisation of α-Def 5 in Paneth cells was assessed by an anti-human α-Def 5 monoclonal antibody (8C8) (Novus Biologicals, USA) in a subset of 15 CD mucosa pairs (active and inactive) and in 12 normal control mucosa. Quantitative high-throughput methylation analysis of CpG sites in the Def5 gene was carried out using the MassARRAY® EpityperTM system (Sequenom). Statistical Mann–Whitney U and Kruskal–Wallis tests, Spearman’s rank correlation, and SPSS software were used. Statistically significant: p-value < 0.05

Table 1 Def-1-3 concentrations and CRP.

Results

In Table 1, plasma α-Def1-3 concentrations were significantly higher in patients withactive CD (aCD) and ileal involvement (L1 and L3) than in patients with inactive CD (iCD) or the control group (p < 0.01 for each). L2 CD (n = 10) patients had no different concentrations ofα-Def1-3 compared with controls (120 (104–169) vs150 (145–155), respectively (p = 0.17). α-Def1-3 concentrations were similar to controls in patients with non-CD ileitis. There was a significant positive correlation between α-Def 1-3 levels in plasma and CRP levels (Spearman’s correlation coefficient of r = 0.696 [p < 0.01]). In ileal biopsies, the immunopositivity scoring showed a significantly reduced αDef5 expression in inflamed mucosa compared with non-inflamed mucosa. CpG island 11 and 13 of α–Def5 showed a different methylation status (%) in aCD (36 ± 14; 87 ± 9) and iCD (32 ± 9; 83 ± 12) vs controls (14 ± 10; 58 ± 25) (mean+SD, p = 0.003 and p = 0.033, respectively).

Conclusion

α-Def 1-3 are specific biomarkers of ileal CD at diagnosis and correlate with the degree of inflammation. Methylation of α–Def5 gene is the epigenetic mechanism down regulating αDef5 expression.