The online version of this article (https://doi.org/10.1186/s12950-017-0178-z) contains supplementary material, which is available to authorized users.
Gastrointestinal acute graft-versus-host disease (GI aGVHD) is a lethal complication following allogeneic hematopoietic stem cell transplantation (HSCT). However, it is still very difficult to make a diagnosis of GI aGVHD in practice. To date, no consensus plasma biomarker of GI aGVHD can be used to help make a diagnosis. Here, we attempted to identify GI aGVHD associated plasma proteins in murine model, which can help make a diagnosis of GI aGVHD.
We used 8-plex isobaric tags for relative and absolute quantitation (8-plex iTRAQ) to screen out proteins in plasma samples taken from murine models before and after allogeneic HSCT. Next mRNA expressions were validated by quantitative real-time polymerase chain reaction in mouse intestinal epithelial samples.
We found that five proteins were increased at least 2-fold in the allogeneic group at day 7 compared with days 0, 3 and 15 (after Cyclosporin A treatment) and the syngeneic group at day 7. These 5 proteins were VNN3, ZNF746, C4BP, KNG1 and FETUB, and they were consistent with results from negative labeling with 8-plex iTRAQ. Furthermore, increase in mRNA level of VNN3 was confirmed in murine intestinal epithelial samples with aGVHD.
Our results demonstrate that plasma VNN3 protein is associated with GI aGVHD in murine model.
Additional file 1: aGVHD was verified by histological changes in allogeneic recipients. (A and B) Donor-derived cells in the allogeneic group were measured by flow cytometry at day 7 to confirm implantation of donor. (C) H&E staining of mouse small intestine tissues in the allogeneic group on day 7. Small intestinal mucous villi degenerate and denude. (D) H&E staining of mouse small intestinal tissues of mice in the syngeneic group on day 7. Small intestinal mucosa had no obvious pathological changes. n=5 mice per time point per group. (DOC 3783 kb)12950_2017_178_MOESM1_ESM.doc
Additional file 2: Positive iTRAQ label of proteins with increased levels in day 7 in allogeneic group with aGVHD.8-plex iTRAQ approach is that the relative quantification is achieved via the difference in abundance of the reporter product ions (i.e., m/z 113, 114, 115, 116, 117, 118, 119, 121). For positive labeling, 115 was day 0, 114 was day 3 (allogeneic group), 116 was day 7 (syngeneic group), 117 was day 7 (allogeneic group), 118 was day 11(syngeneic group ip PBS), 119 was day 11 (syngeneic group ip LPS), and 121 was day 15 (after CsA treatment in allogeneic group), 113 was labeled day 22 (syngeneic group). Differential protein levels between aGVHD+ and aGVHD- from eight time points were evaluated for 117/115, 117/114, 117/116, 117/121, 117/118, 117/119 and 117/113 sets, respectively, with the ratio more than 2.0 defined as increased. (DOC 48 kb)12950_2017_178_MOESM2_ESM.doc
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