Abstract
BackgroundThere is an unmet need for novel treatments, such as drugs or vaccines, adjunctive to or replacing a burdensome life-long gluten-free diet for coeliac disease. The gold standard for successful treatment is a healed small intestinal mucosa, and therefore, the outcome measures in proof-of-concept studies should be based on evaluation of small intestine biopsies. We here evaluated morphometric, immunohistochemical and messenger RNA (mRNA) expression changes in coeliac disease patients challenged with gluten using PAXgene fixed paraffin-embedded biopsies.MethodsFifteen coeliac disease patients were challenged with 4 g of gluten per day for 10 weeks and 24 non-coeliac patients served as disease controls. A wide array of histological and immunohistochemical staining and mRNA-based gene expression tests (RT-qPCR and RNAseq) were carried out.ResultsDigital quantitative villous height: crypt depth ratio (VH: CrD) measurements revealed significant duodenal mucosal deterioration in all coeliac disease patients on gluten challenge. In contrast, the Marsh-Oberhuber class worsened in only 80% of coeliac patients. Measuring the intraepithelial CD3+ T-lymphocyte and lamina propria CD138+ plasma cell densities simultaneously proved to be a meaningful new measure of inflammation. Stainings for γδ T cells and IgA deposits, where previously frozen samples have been needed, were successful in PAXgene fixed paraffin-embedded samples. Messenger RNA extraction from the same paraffin-embedded biopsy block was successful and allowed large-scale qRT-PCR and RNAseq analyses for gene expression. Molecular morphometry, using the mRNA expression ratio of villous epithelium-specific gene APOA4 to crypt proliferation gene Ki67, showed a similar significant distinction between paired baseline and post-gluten challenge biopsies as quantitative histomorphometry.ConclusionRigorous digitally measured histologic and molecular markers suitable for gluten challenge studies can be obtained from a single paraffin-embedded biopsy specimen. Molecular morphometry seems to be a promising new tool that can be used in situations where assessing duodenal mucosal health is of paramount importance. In addition, the diagnostically valuable IgA deposits were now stained in paraffin-embedded specimens making them more accessible in routine clinics.
Highlights
There is an unmet need for novel treatments, such as drugs or vaccines, adjunctive to or replacing a burdensome life-long gluten-free diet for coeliac disease
Mucosal architecture assessments villous height (VH), crypt depth (CrD) and their ratio were similar between disease control patients and coeliac disease patients on a gluten-free diet (Fig. 2)
A significant change was seen between baseline and post gluten challenge values in PAXgene- fixed paraffin- embedded (PaxFPE) samples (p < 0.001, Fig. 2)
Summary
There is an unmet need for novel treatments, such as drugs or vaccines, adjunctive to or replacing a burdensome life-long gluten-free diet for coeliac disease. The gold standard for successful treatment is a healed small intestinal mucosa, and the outcome measures in proof-of-concept studies should be based on evaluation of small intestine biopsies. Immunohistochemical and messenger RNA (mRNA) expression changes in coeliac disease patients challenged with gluten using PAXgene fixed paraffin-embedded biopsies. The only therapeutic option is a life-long, strict gluten-free diet, which is burdensome and limits the normal dayto-day life [3]. Coeliac patients have expressed a desire to use novel drugs or vaccines as adjunctive or even alternative treatments for coeliac disease [6]. Mucosal healing is the ultimate goal in coeliac disease dietary treatment, but this is often not achieved, as reviewed by Ilus et al [7]
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