Protein expression profiles for CD55, CD14, and CD11b following addition of LPS (1 µg/ml) to THP-1 cells pretreated with or without 100 nM 1,25-D3 (72 h).

<p>A multiplexed fluorescence-based immunoblot approach was used to determine corresponding protein expression profiles from cells whose mRNA profiles are shown in Fig. 2. A, the temporal protein induction pattern of CD55, CD11b and β-actin following a LPS-challenge (without pretreatment). CD14 levels are not shown because the apparent 50-fold induction remained below the threshold of detection (nd). B, the relative band intensities for CD55 and CD11b, normalized to β-actin, are graphically presented as the average fold-change (relative to time zero) of three independent experiments (± SD). In some instances the SD are covered by the data point symbol. C and D, the multiplex immunoblot of LPS-induced protein expression in cells pretreated for 72 h with 1,25-D3 is shown in panel C; the fold induction is graphically represented in panel D as the average fold-change (relative to time zero) of three independent experiments (± SD). In some instances the SD are covered by the data point symbol. The apparent 100-fold induction of CD14 following 1,25-D3 pretreatment but before LPS (time = 0) was within the immunoblot detection limits.</p

CD55 expression was enhanced on the surface of essentially all THP-1 cells at 24 h, following addition of LPS to 1,25-D3 pretreated cells.

<p>THP-1 cells were grown on coverslips, pretreated with 100 nM 1,25-D3 for 72 h and then stimulated with either vehicle or LPS for 24 h, as indicated. Serial 0.5 µm optical sections were obtained by confocal microscopy (40 × magnification). A, sections containing either a central (internal Z-stack) or the apical region of the cells (cell surface Z-stack) and the entire 3D image of LPS treated cells are shown. White represents CD55 staining; nucleic acid staining was omitted for clarity. B, shows a graphical representation of mean (±SD) CD55 expression levels (normalized to nucleic acid content) for three independent experiments with two fields quantified per experiment (n = 6).</p

CD55 mRNA and protein expression was not altered during the early response phase (0–24 h) following a 1,25-D3 preincubation period of 72 h with human THP-1 monocytes.

<p>THP-1 cells were cultured in complete media in the absence or presence of 100 nM 1,25-D3 (added at time = 0) and harvested at the indicated times. A, CD55, CD14 and CD11b mRNA pool sizes were determined by quantitative PCR analysis using 18S rRNA as a reference. Fold-induction is expressed relative to vehicle treated controls. Values shown are the mean +/− SD of 3 independent experiments. In some instances, the SD bars are covered by the data point symbol. Shaded data points indicate statistical differences from control values. P<0.05 was considered significant. B, protein expression was analyzed in cells at various times following addition of 1,25-D3, using a fluorescence-based immunoblot approach. Corresponding controls showed no difference over the same time period.</p

NF-κB activation was required for LPS-induced CD55 expression in 1,25-D3 pretreated THP-1 monocytes.

<p>As illustrated under panel A, at the end of the 72 h pretreatment phase with 100 nM 1,25-D3, either vehicle or one of two NF-kB inhibitors [50 µM MG132 (MG) or 30 µm parthenolide (Pa)] was added and the incubation continued for an additional 1 h. Then LPS (1 µg/ml) or vehicle was added. Cells were ultimately harvested at 2 h (mRNA) or 24 h (protein) post addition of LPS or vehicle. Quantitative PCR was used to determine CD55 (A) and CD11b (D) mRNA levels assessed at 2 h. Differences between mRNA pool sizes were determined using a two tailed student’s t test. Asterisks (Fig. 5A) indicate statistical differences between indicated treatments; *P<0.05, **P<0.01. CD55 and CD11b protein expression was assessed at 24 h following a vehicle or LPS challenge by immunoblotting (B) and quantification (C and E), respectively. Because of the semi-qualitative nature of the protein determinations, a statistical analysis was not performed on these data. Values represent means +/− SD (n = 3).</p

Pretreatment with 1,25-D3 for 72 h sustained the subsequent LPS-induced expression of CD55 mRNA in THP-1 cells, compared to cells pretreated with vehicle for 72 h.

<p>THP-1 cells were first preincubated in complete culture media with vehicle or with 100 nM 1,25-D3. Seventy-two h later (t = 0 on A–C), either vehicle or 1 µg/ml LPS was added and the incubation continued for an additional 72 h. Cells were harvested at the indicated times and quantitative PCR (n = 3) was used to determine CD55 (A), CD14 (B) and CD11b (C) mRNA pool sizes using the absolute copy number method. Mean levels are expressed in arbitrary units normalized to 18S rRNA (± SD). Differences in mRNA pool size were determined using a two tailed student’s t test. Shaded data points indicate statistical differences from corresponding controls.</p

Human alpha defensin 5 is a candidate biomarker to delineate inflammatory bowel disease

<div><p>Inability to distinguish Crohn's colitis from ulcerative colitis leads to the diagnosis of indeterminate colitis. This greatly effects medical and surgical care of the patient because treatments for the two diseases vary. Approximately 30 percent of inflammatory bowel disease patients cannot be accurately diagnosed, increasing their risk of inappropriate treatment. We sought to determine whether transcriptomic patterns could be used to develop diagnostic biomarker(s) to delineate inflammatory bowel disease more accurately. Four patients groups were assessed via whole-transcriptome microarray, qPCR, Western blot, and immunohistochemistry for differential expression of Human α-Defensin-5. In addition, immunohistochemistry for Paneth cells and Lysozyme, a Paneth cell marker, was also performed. Aberrant expression of Human α-Defensin-5 levels using transcript, Western blot, and immunohistochemistry staining levels was significantly upregulated in Crohn's colitis, <b><i>p< 0</i>.<i>0001</i></b>. Among patients with indeterminate colitis, Human α-Defensin-5 is a reliable differentiator with a positive predictive value of 96 percent. We also observed abundant ectopic crypt Paneth cells in all colectomy tissue samples of Crohn's colitis patients. In a retrospective study, we show that Human α-Defensin-5 could be used in indeterminate colitis patients to determine if they have either ulcerative colitis (low levels of Human α-Defensin-5) or Crohn's colitis (high levels of Human α-Defensin-5). Twenty of 67 patients (30 percent) who underwent restorative proctocolectomy for definitive ulcerative colitis were clinically changed to <i>de novo</i> Crohn's disease. These patients were profiled by Human α-Defensin-5 immunohistochemistry. All patients tested strongly positive. In addition, we observed by both hematoxylin and eosin and Lysozyme staining, a large number of ectopic Paneth cells in the colonic crypt of Crohn's colitis patient samples. Our experiments are the first to show that Human α-Defensin-5 is a potential candidate biomarker to molecularly differentiate Crohn's colitis from ulcerative colitis, to our knowledge. These data give us both a potential diagnostic marker in Human α-Defensin-5 and insight to develop future mechanistic studies to better understand crypt biology in Crohn's colitis.</p></div

Assessment of HD5 and Paneth cells in inflamed and normal, adjacent tissue.

<p>HD5 staining of CC inflamed and normal, adjacent tissue shows expression of HD5 in all patient samples examined (<b>Fig. 6A</b>), compared to inflamed and adjacent, normal tissue of UC patients (<b>Fig. 6B</b>). H&E stains for Paneth Cells (<b>Fig. 6C and D</b>), were negative for PCs in all tissues.</p

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.

<p>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.</p

Double stain of PCs, lyzosomes and HD5.

<p>Double staining analyses from <i>de novo</i> Crohn’s (<b>Fig. 5A</b> and <b>D</b>), and normal ileum/control (<b>Fig. 5G</b>) are presented. Image deconvolutions are displayed vertically to evaluate lysozyme-specific permanent red (<b>Fig. 5B, E</b> and <b>H</b>) and HD5α-specific DAB (<b>Fig. 5C, F</b> and <b>I</b>). The normal colon image (<b>Fig. 5J</b>), which lacks PCs, was not further processed.</p

H&E staining on parallel sections the typical morphological appearance of Paneth cell (PCs) including the presence of dense apical eosinophilic granules.

<p><b><i><u>Upper panel:</u></i></b><b>A</b>, Diverticulitis (DV, no PCs), <b>B</b>, Diverticulosis (DVL, no PCs), <b>C</b>, Normal (NL-Colon, Control, no PCs). <b><i><u>Middle panel</u></i></b><b>:</b><b>D</b>, UC (found prodromal PC in one patient, arrow). <b>E</b>, CC, demonstrate abundance of PCs allover colonic basal crypts (arrows). <b>F</b>, Normal (NL-Ileum, Control), with abundance of PCs. <b><i><u>Lower panel</u></i></b><b><u>:</u></b><b>IHC detection of Paneth cell markers α-defensin 5 (DEFA5) and lysozyme (LYZ) in the colon. G</b>, NL-Colon, <b>H</b>, CC, and <b>I</b>, NL-Ileum, Control.</p