Baseline characteristics and clinical signs of neonates treated with gentamicin.

<p>Patients are subdivided according to gestational age. Mean biomarker values presented include samples collected both on and off gentamicin treatment over the whole time course of inclusion in the study.</p

Longitudinal biomarker analysis of infants treated with multiple courses of gentamicin without a change in serum creatinine concentration.

<p>Representative figures demonstrating the longitudinal quantification of the biomarkers KIM-1 (blue; ng/mg. uCr), NGAL (green; ng/mg. uCr), NAG (yellow; IU/mg. uCr) and serum creatinine (red; µmol/L) for three infants treated with gentamicin (A–C). Gentamicin treatment episode and length of treatment (days) are indicated by the black horizontal bar on each figure for that individual patient.</p

Longitudinal biomarker analysis of infants treated with multiple courses of gentamicin with a change in serum creatinine concentration (AKI).

<p>Representative figures demonstrating the longitudinal quantification of the biomarkers KIM-1 (blue; ng/mg. uCr), NGAL (green; ng/mg. uCr), NAG (yellow; IU/mg. uCr) and serum creatinine (red; µmol/L) for three infants treated with gentamicin (A–C). Gentamicin treatment episode and length of treatment (days) are indicated by the black horizontal bar on each figure for that individual patient.</p

Association between gentamicin treatment and the change in biomarker values.

<p>The mean baseline biomarker values in the absence of any gentamicin treatment were 1.91 ng/mg uCr (95% CI 1.07, 2.76) for KIM-1, 0.13 IU/mg uCr (0.07, 0.19) for NAG, 425.4 ng/mg uCr (162.6, 688.3) for NGAL, and 62.39 µmol/l (53.1, 71.69) for creatinine.</p

S1, S2, S3 and S4 DNA are inert and inhibit HMGB1, but not LPS-induced TNF release from macrophages.

<p>A) S1–S4 DNA is inert. Primary human macrophages were incubated with HMGB1 (positive control) or S1–S4 DNA (1 µM) for 16 hours. TNF released in the supernatants was measured by ELISA. Data are mean ± SEM from 3 experiments. B) S1–S4 DNA inhibits HMGB1-induced TNF release from cultured macrophages. Primary human macrophages in 96-well culture plates were stimulated with HMGB1 and various amounts of DNA as indicated for 16 hours. TNF released in the supernatants were measured by ELISA. N = 3 independent experiments. *: p<0.05 <i>vs.</i> HMGB1 alone. C) S1–S4 DNA does not inhibit LPS-induced TNF release. Primary human macrophages in 96-well culture plates were stimulated with LPS (2 ng/ml) plus increasing amounts of S1–S4 DNA overnight at 37°C. TNF released was measured. N = 3 experiments.</p

HMGB1 causes increased intestinal LDH release; and DNA beads capture HMGB1 in biological fluids <i>ex vivo</i>.

<p>A) HMGB1 (10 and 30 µg/ml) or PBS (all in 500 µl volume) were injected into freshly isolated full length colons from mice and incubated at room temperature for 1 hour. Colonic lavage was collected and LDH levels were measured. N = 3 preparations per group. *: p<0.05, **: p<0.01 <i>vs.</i> PBS group. B) DNA beads capture HMGB1 from feces of colitis mice induced by DSS. Feces in the colon of colitis mice were gently flushed out with cold PBS containing 1× protease inhibitor, and the suspension was rotated for an hour at room temperature in the presence of gentamycin and imipenum. After centrifugation to remove fecal debris, fecal extract containing 40 µg of total protein was incubated with B1, B2 or control beads at room temperature for an hour. After washing with PBS, beads were boiled and eluate was analyzed by western blot probed with anti-HMGB1 antibodies. N = 3–4 mice per group. *: p<0.05 <i>vs.</i> DSS control. C) Female BALB/c mice (8–12 weeks old, n = 10–12 per group) were given 4% dextran sodium sulfate (DSS) dissolved in drinking water ad libitum for five days, and then switched to normal water for three days. DNA beads capture HMGB1 from inflamed colon from colitis mice. Mice were euthanized on day 9th after overnight fasting. Both B1 and B2 beads captured HMGB1 from colon culture of colitis mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103992#s2" target="_blank">Methods</a>) compared to control beads. D) DNA beads remove HMGB1 from septic mice sera. Serum (20 µl) from normal or septic mice was incubated with 50 µl of B2 or control beads at room temperature for an hour. Samples were then centrifuged at room temperature for five minutes. The binding of DNA beads with HMGB1 was measured using western blot or ELISA. Data shown are means ± SEM from 3–5 mice per group. *: p<0.05 <i>vs.</i> control beads. E) DNA beads capture HMGB1 from cell supernatants. RAW 264.7 cells in 6-well plate were stimulated with LPS (100 ng/ml) for 16 hours, and HMGB1 containing supernatant (1 ml) was collected and concentrated 10× through centrifugation with Microcon centrifugal filters. The concentrated RAW 264.7 cell supernatant was then incubated with beads (20 µl) containing control, B1 or B2 beads at room temperature for an hour with rotation. HMGB1 content in both supernatant and beads were measured by western blot. N = 2 repeats each performed in duplicate.</p

Administration of B2 beads ameliorates colitis-induced inflammation in IL-10 KO mice.

<p>Twelve weeks old female IL-10 KO mice were orally administrated with 300 µl (50% slurry, gavage) of B2 or control beads three times a week for a total of six weeks. A) Treatment with B2 beads increased body weight in mice. *: p<0.05 <i>vs.</i> control beads group. N = 5–7 mice per group. N.T. = no treatment. B) Colon and serum measurements of IL-10 KO mice treated with B2 or control beads. Colon measurements (weight, expression of IL-6 and IL-1β mRNA) and serum HMGB1 levels in IL-10 KO mice treated with B2 or control beads. *: p<0.05 <i>vs.</i> control beads group. N = 5–7 mice per group. N.T. = no treatment. C) Histological evaluation of B2 or control beads-treated IL-10 KO mice. Representative H&E staining of colon from wild type (C57BL/6) or IL-10 KO mice and histological scores (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103992#s2" target="_blank">Methods</a>) of colons are shown. *: p<0.05 <i>vs.</i> control beads group. N = 5–7 mice per group. Magnification: ×40.</p

Administration of B2 beads ameliorates inflammation in DSS-induced colitis in mice.

<p>Female BALB/c mice (10 mice per group) were given 4% DSS in drinking water to induce colitis water for 5 days to induce colitis, and then switched to normal water. Mice were orally administrated with 300 µl (50% slurry, gavage) of B2 or control beads on days 0, 2, 4 and 6 after DSS initiation and were euthanized on day 8. A) Treatment with B2 beads reduced body weight loss in DSS-induced colitis mice. *: p<0.05 <i>vs.</i> control beads group. B) Body weight change, colon measurements (weight and length) and levels of serum and fecal HMGB1 in colitis mice treated with B2 or control beads. *: p<0.05 <i>vs.</i> control beads group. C) Histological evaluation of B2 or control beads treated colitis mice. Representative H&E staining of colons from normal, control or B2 beads-treated colitis mice is shown. Histological scores of colons are shown. N = 10 mice per group. *: p<0.05 <i>vs.</i> control beads group. Magnification: ×40.</p

DNA beads are stable.

<p>A) B2 DNA beads are stable in fecal matters. Fluorescent FAM-labeled B2 beads (50 µl) was incubated with fecal extract of colitis mice (300 µl) for 2 hours at 37°C. After centrifugation, beads were washed five times with PBS and re-suspended as 50% slurry. The fluorescence intensity in both supernatants and beads (before and after the incubation) was measured by using a microplate spectrophotometer at excitation of 494 nm. N = 3 separate experiments. B) B1 and B2 beads are resistant to acidic pH, B3 is not. B1, B2 or B3 beads at the amounts indicated were incubated with HMGB1 (500 ng) in either solutions of PBS (pH 7) or HCl (pH 2 or 1) at room temperature for 2 hours. HMGB1 bound to the beads was revealed by western blot analysis. Data are representative from 3 separate experiments. C) B2 beads are resistant to degradation by DNase I. DNA beads were incubated with DNase I (40 ng/ml, a concentration observed in biological fluid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103992#pone.0103992-Macanovic1" target="_blank">[35]</a>) for 30 minutes at 37°C. The binding of HMGB1 by B2 beads with or without DNase I treatment was analyzed by western blot. Data are from 3 separate experiments.</p

DNA beads are neither cytotoxic nor able to induce type I IFN expression.

<p>A) B2 DNA beads are not cytotoxic. Caco-2 (human epithelial colorectal adenocarcinoma) cells in 24-well plates were incubated with increasing amounts of B2 or control beads overnight at 37°C in Opti-MEM medium. In time course experiments, fixed amount of B2 or control beads (5 µl) were incubated with cells for the time periods indicated. Supernatants were collected and LDH levels were analyzed by cytotoxicity kit. Triton X-100 (2%) was used as a positive control. Data are means ± SEM, N = 3 experiments. B) DNA beads do not induce type I IFN expression in macrophages. RAW 264.7 cells were incubated with LPS (2 ng/ml) or poly I∶C (1 µM, positive controls), S1, S2 or B1, B2 beads for 24 hours at 37°C. Cell total RNA was extracted; the expression IFN-β and HPRT mRNA was analyzed by quantitative PCR and presented as the ratio of IFN-β/HPRT. N = 3–4 per group. *: p<0.05 vs. medium alone.</p