Fig 1.
Diagnostic uncertainty and inaccuracy in IBD clinical setting.
A, Indeterminant colitis. Twenty-one IC patients were followed for approximately ten years. At the end of the 10 year period, 28.5% of the patients could still not be delineated into a precise diagnosis of either UC or CC. B, Crohn’s as Ulcerative colitis. Sixty-seven UC RPC operated patients were followed for re-evaluated after a mean follow-up of 9.4 (range, 8–13) years for changing course of diagnosis. Thirty percent of these patients required a change of diagnosis to de novo Crohn’s disease.
Table 1.
Abbreviated list of targets from affymetrix cDNA microarray.
A total of 484 genes were highlighted in the microarray as potential markers for distinguishing UC from CC. The gene showing the largest fold change between the two diseases was Human Defensin 5 (HD5).
Table 2.
Full list of targets from NanoString Human Inflammation PCR array.
16 inflammatory genes were changed in this subset of samples. HD5 was the only gene to appear in both the microarray and the NanoString PCR array.
Fig 2.
Aberrantly expressed of HD5 in IBD.
A, Transcript levels of HD5 in moderate UC and CC. qRT-PCR confirms an increase in HD5 levels in moderate CC compared to moderate UC (p<0.05). Each point represents one patient sample. B, Representative HD5 Western Blot. HD5 levels (top) are higher in moderate and severe CC levels compared to all other disease states. β-actin loading control is shown on bottom. C, Graphical representation (densitometry) of HD5 Levels. Band intensities were measured for all samples and graphed as a ratio to β-actin loading control. Each point represents an individual sample. Moderate and severe CC levels of HD5 are both significantly higher than all other disease states (p<0.0001). D-G Representative IHC images of HD5 staining. D, Diverticulosis (DVL) no primary antibody control. E, Diverticulitis (DV). F, UC. G, CC (positive, arrow). H, HD5 staining counts. Each point represents one patient sample.
Table 3.
IHC staining for HD5 agrees with final diagnostic outcome in a sample of IC patients even when there was no agreement with the attending physician.
Fig 3.
It is possible to use HD5 to determine patient candidacy for IPAA.
A. Representative results from a RPC-operated patient that did not change the diagnosis after surgery and was molecularly tested using HD5 IHC. B, Representative results from a UC RPC and IPAA operated patients that did change the diagnosis from UC to de novo Crohn’s was molecularly tested using HD5 IHC. C. NL-Ileum, control. D., Quantification of NEARAS HD5 IHC staining spot counts for UC RPC and IPAA-operated patients who did not have their original diagnosis changed versus those who did change from UC to de novo Crohn's (Fig 2A vs. 2B). (Ctrl 1 —staining control, UC—Ulcerative Colitis, CC—Crohn’s Colitis, DV—Diverticulitis, DVL—Diverticulosis).
Fig 4.
H&E staining on parallel sections the typical morphological appearance of Paneth cell (PCs) including the presence of dense apical eosinophilic granules.
Upper panel: A, Diverticulitis (DV, no PCs), B, Diverticulosis (DVL, no PCs), C, Normal (NL-Colon, Control, no PCs). Middle panel: D, UC (found prodromal PC in one patient, arrow). E, CC, demonstrate abundance of PCs allover colonic basal crypts (arrows). F, Normal (NL-Ileum, Control), with abundance of PCs. Lower panel: IHC detection of Paneth cell markers α-defensin 5 (DEFA5) and lysozyme (LYZ) in the colon. G, NL-Colon, H, CC, and I, NL-Ileum, Control.
Fig 5.
Double stain of PCs, lyzosomes and HD5.
Double staining analyses from de novo Crohn’s (Fig. 5A and D), and normal ileum/control (Fig. 5G) are presented. Image deconvolutions are displayed vertically to evaluate lysozyme-specific permanent red (Fig. 5B, E and H) and HD5α-specific DAB (Fig. 5C, F and I). The normal colon image (Fig. 5J), which lacks PCs, was not further processed.
Fig 6.
Assessment of HD5 and Paneth cells in inflamed and normal, adjacent tissue.
HD5 staining of CC inflamed and normal, adjacent tissue shows expression of HD5 in all patient samples examined (Fig. 6A), compared to inflamed and adjacent, normal tissue of UC patients (Fig. 6B). H&E stains for Paneth Cells (Fig. 6C and D), were negative for PCs in all tissues.