Article Figures & Data
Figures
- FIG 1
Different inflammatory biomarkers detected in the clinical stool samples examined. Stools (300 mg) from 100 antibiotic-associated diarrheal patients (50 CDI positive and 50 CDI negative) were evaluated for the presence of 36 inflammatory-associated proteins using the Proteome Profiler human cytokine array panel A kit (R&D Systems, Minneapolis, MN). Abbreviations: Pos. Ctl, positive control; Neg. Ctl, negative control; CD40L, CD40 ligand; IFN-γ, gamma interferon, IL-1α, interleukin-1α; IL-1β, interleukin-1β; IL-1ra, interleukin-1 receptor antagonist; IL-8, interleukin-8, IL-23; interleukin-23; MIF, macrophage migration inhibitory factor; PAI-1, plasminogen activator inhibitor 1.
- FIG 2
Fold change in the amount of inflammatory markers detected in CDI-positive and CDI-negative stools. Stools (300 mg) from 100 antibiotic-associated diarrheal patients (50 CDI positive and 50 CDI negative) were evaluated for the presence of 36 inflammatory biomarkers using a Proteome Profiler human cytokine array panel A kit (R&D Systems, Minneapolis, MN). Band intensities were determined and converted into pixel densities using ImageJ (National Institutes of Health, Bethesda, MD). Error bars represent the standard deviations from the mean values for each inflammatory biomarker. Significant differences between CDI-positive and CDI-negative stools are denoted by asterisks. The P values, based on a nonparametric t test, were 0.011 for C5a, 0.042 for CD40L, 0.045 for G-CSF, 0.030 for I-309, 0.001 for IL-13; 0.031 for IL-16, 0.027 for IL-27, 0.013 for MCP-1, 0.021 for TNF-α, 0.004 for IL-8, and 0.002 for IL-23. Abbreviations: GM-CSF, granulocyte colony-stimulating factor; GRO-α, growth-regulated oncogene-alpha; sICAM-1, soluble intercellular adhesion molecule 1; IP-10, interferon gamma-induced protein 10; I-TAC, interferon-inducible T cell alpha chemoattractant; MIP-1α and MIP-1β, macrophage inflammatory proteins alpha and beta, respectively; SDF-1, stromal cell-derived factor 1; sTREM-1, soluble triggering receptor expressed on myeloid cells 1; MIF, macrophage migration inhibitory factor; PAI-1, plasminogen activator inhibitor 1. The other abbreviations are defined in the text.
- FIG 3
IL-8 and IL-23 were present in the majority of CDI-positive and CDI-negative stools. (A) IL-8 and IL-23 were detected in a greater number of CDI-positive stools than CDI-negative stools. Stools (300 mg) from 100 antibiotic-associated diarrheal patients (50 CDI positive and 50 CDI negative) were evaluated for the presence of 36 inflammation-associated proteins using a Proteome Profiler human cytokine array panel A kit (R&D Systems, Minneapolis, MN). Stools from the hospitalized patients (controls) without diarrhea were not evaluated by this initial assay but were included retrospectively in the quantitative ELISA for comparison. (B) CDI-positive stools contain large amounts of IL-8 and relatively small amounts of IL-23 compared to the amounts in stools from CDI-negative diarrheal patients and hospitalized patients without diarrhea. The concentrations of IL-8 and IL-23 were determined in stools from 50 CDI-positive patients, 50 CDI-negative patients, and 45 nondiarrheal controls by quantitative ELISA (R&D Systems, Minneapolis, MN). A Mann-Whitney two-tailed test showed significant differences between CDI-positive patients and nondiarrheal controls (P = 0.0001), CDI-positive patients and CDI-negative patients with diarrhea (P = 0.002), and CDI-negative patients and nondiarrheal controls (P = 0.006) for IL-8 and CDI-positive patients and nondiarrheal controls (P = 0.001), CDI-positive patients and CDI-negative patients with diarrhea (P = 0.003), and CDI-negative patients and nondiarrheal controls (P = 0.0001) for IL-23. Horizontal bars, mean concentrations.
- FIG 4
Comparison of Th1 and Th2 cytokines in CDI-positive and CDI-negative stools. (A) Fold change in amounts of Th1 cytokines (IFN-γ, IL-2, IL-12, TNF-α) and Th2 cytokines (IL-4, IL-5, IL-6, IL-10, IL-13) obtained from the initial Proteome Profiler human cytokine array assay. Stools (300 mg) from 100 antibiotic-associated diarrheal patients (50 CDI positive and 50 CDI negative) were evaluated for the presence of 36 inflammatory proteins using a Proteome Profiler human cytokine array panel A kit (R&D Systems, Minneapolis, MN). Data are expressed as the mean of the relative band intensity of each cytokine. Stools from the hospitalized patients (controls) without diarrhea were not evaluated by this initial assay but were included retrospectively in the quantitative ELISA for comparison. Error bars represent the standard error of measurement between two replicates per sample. *, P < 0.05. (B) Concentrations of IFN-γ, TNF-α, and IL-13 in CDI-positive stools and CDI-negative stools from diarrheal patients and stools from hospitalized controls without diarrhea determined by quantitative ELISA (R&D Systems, Minneapolis, MN). The Kruskal-Wallis test showed significant differences between the means (P < 0.0001). Horizontal bars, mean concentrations.
- FIG 5
CDI-positive stools contain larger amounts of lactoferrin and calprotectin than CDI-negative diarrheal stools and stools from hospitalized patients (controls) without diarrhea. The concentrations of lactoferrin and calprotectin were measured in stools from 50 CDI-positive and CDI-negative patients and hospitalized patients without diarrhea. Fecal lactoferrin and calprotectin concentrations were determined using an IBD-SCAN test (Techlab, Blacksburg, VA) and an HK325 human calprotectin ELISA kit (Hycult Biotech, Plymouth Meeting, PA), respectively. Median lactoferrin concentrations were 31.4 μg/ml (CDI-positive patient stools), 6.3 μg/ml (CDI-negative patient stools), and 5.6 μg/ml (stools from hospitalized patients without diarrhea). Median calprotectin concentrations were 18.0 μg/ml (CDI-positive patient stools), 6.5 μg/ml (CDI-negative patient stools), and 8.7 μg/ml (stools from hospitalized patients without diarrhea). One-way analysis of variance showed significant differences between the means of the CDI-positive patients, CDI-negative patients, and nondiarrheal controls for both lactoferrin and calprotectin (P < 0.0001).
Tables
- TABLE 1
Characteristics of study populationa
Characteristic CDI-positive patients (n = 50) CDI-negative patients (n = 50) Control patients with no diarrhea (n = 45) Avg (range) age (yr) 58 (2–91) 56 (21–83) 63 (22–85) No. of patients by: Ethnicity Asian 1 1 0 B/AA 10 8 11 H/L 4 6 9 W/C 28 29 25 Other 7 6 Gender Female 27 24 19 Male 23 26 26 ↵a CDI, patients with C. difficile infection; B/AA, black/African-American; H/L, Hispanic/Latino; W/C, white/Caucasian.
- TABLE 2
Fecal concentrations of IL-13, IL-8, IL-23, TNF-α, IFN-γ, lactoferrin, and calprotectin from the CDI-positive and CDI-negative diarrheal stools and nondiarrheal stools evaluateda
Patient group Concn (pg/ml) Concn (μg/ml) IL-13 IL-8 IL-23 TNF-α IFN-γ Lactoferrin Calprotectin CDI positive (n = 50) Mean 323.8 318.2 722 59.8 69.7 43.4 23.8 Median 277.4 242 390.5 33.7 58 31.4 18 Range 35.7–1,150 13.1–1,529 110.0–7,069 2.7–163.6 8.7–225.7 3.0–155.2 2.8–70.2 P value 0.01 0.022 0.001 0.078 0.067 0.019 0.029 CDI negative (n = 50) Mean 141.1 84.7 946.7 31.2 48.3 24.8 8.8 Median 138.8 83.3 932.6 25.7 31.5 6.3 6.5 Range 14.3–319.3 8.5–191.6 185.5–2,016 0.6–177.7 0.04–204.3 0.6–140.3 2.0–31.0 P value 0.024 0.034 0.0032 0.055 0.088 0.028 0.054 Nondiarrheal control (n = 45) Mean 234.3 79.8 1,617 22.3 57.6 6.8 10.2 Median 203.5 52 768 18 50.2 5.6 8.7 Range 19.2–701.7 21.4–295.7 489.0–6,810 0.3–83.3 10.9–143.3 0.5–35.0 1.8–33.2 P value 0.06 0.01 0.041 0.092 0.002 0.069 0.073 ↵a Concentrations were determined by quantitative ELISA (R&D Systems, Minneapolis, MN).
