Circadian rhythmic parameters of 5-methylcytosine content, SAH concentration, SAM/SAH ratio and DNMT gene transcriptions in livers of WT and <i>Per1^-/-Per2^-/-</i> DKO mice.

<p>Data represent means ± S.E.M. (n = 3–4).</p><p>*<i>p</i> < 0.05,</p><p>**<i>p</i> < 0.01 compared with WT.</p><p>Circadian rhythmic parameters of 5-methylcytosine content, SAH concentration, SAM/SAH ratio and DNMT gene transcriptions in livers of WT and <i>Per1^-/-Per2^-/-</i> DKO mice.</p

Primer sequences for real-time RT-PCR analysis.

<p>Primer sequences for real-time RT-PCR analysis.</p

Effect of administration of 5′-AMP on global DNA methylation.

<p>HPLC analysis for the level of (A) adenosine, (B) SAH, (C) SAM/SAH ratio at 1 h after i.p. injection of 5′-AMP (0.5μmol/g and 1μmol/g body weight). (D) Genomic 5-mC content at 1 h and 4 h after 5′-AMP treatment. Data represent means ± S.E.M. (n = 4). *<i>p</i> < 0.05, **<i>p</i> < 0.01 compared with saline control.</p

Hepatic expression of <i>Dnmts</i> and <i>Tets</i> in WT mice.

<p>The mRNA expression of (A) <i>Dnmt1</i>, (B) <i>Dnmt3a</i>, (C) <i>Dnmt3b</i>, (D) <i>Tet2</i> and (E) <i>Tet3</i> in WT livers. One-way ANOVA showed significant variations of the expression of <i>Dnmt3a</i>, <i>Dnmt3b</i>, <i>Tet2</i> and <i>Tet3</i> over time in liver (<i>p</i> < 0.05) and no significant variations in <i>Dnmt1</i> (<i>p</i> > 0.05). Data represent means ± S.E.M. (n = 3–4). *<i>p</i> < 0.05, **<i>p</i> < 0.01 for LSD post hoc test compared with ZT13. (F) The hepatic <i>Dnmt3a</i> mRNA levels in mice under LD and DD. Data represent means ± S.E.M. (n = 4). (G) Western blotting analysis for DNMT3A protein levels. The signal intensities of DNMT3A were normalized to the intensities of β-actin. Data represent means ± S.E.M. from three independent experiments. *<i>p</i> < 0.05 compared with ZT1, ^#<i>p</i> < 0.05 compared with CT1. ZT: zeitgeber time, CT: circadian time.</p

Daily changes of SAH concentration and SAM/SAH ratio in WT liver.

<p>One-way ANOVA showed that both (A) SAH and (B) SAM/SAH ratio displayed a clear diurnal variation (<i>p</i> < 0.01). Data represent means ± S.E.M. (n = 4). *<i>p</i> < 0.05, **<i>p</i> < 0.01 for LSD post hoc test compared with ZT17.</p

Daily variation of global DNA 5-methylcytosine content in WT mice.

<p>(A) One-way ANOVA showed significant variation of genomic 5-mC content over time in livers of WT mice (<i>p</i> = 0.048). Data represent means ± S.E.M. (n = 4). *<i>p</i> < 0.05, **<i>p</i> < 0.01 for LSD post hoc test compared with ZT1. (B) Genomic 5-mC content was analyzed at CT1 and CT13 in DD cycles. Data represent means ± S.E.M. (n = 4). **<i>p</i> < 0.01 compared with ZT1, ^##<i>p</i> < 0.01 compared with CT1. (C) Increased 5-methylcytosine content in LINE-1 at ZT13 vs. ZT1 in WT mice. 64–326 of the consensus sequence in LINE-1 containing 15 CpG dinucleotides were analyzed. Each line represents a unique DNA clone; filled and open circles represent methylated and unmethylated CpGs, respectively. ZT: zeitgeber time, CT: circadian time.</p

Effect of <i>Per1</i>^<i>-/-</i><i>Per2</i>^<i>-/-</i> DKO on the patterns of daily variations.

<p>(A) Genomic 5-mC content, (B) SAH, (C) SAM/SAH ratio, and the expression of (D) <i>Dnmt1</i>, (E) <i>Dnmt3a</i> and (F) <i>Dnmt3b</i> were analyzed by single cosinor method in WT and DKO mice. Data represent means ± S.E.M. (n = 3–4). <i>P</i> value from time effect by one-way ANOVA analysis of variance. *<i>p</i> < 0.05, **<i>p</i> < 0.01 by two-way ANOVA comparing WT and DKO mice. M: mesor.</p

Partial Depletion of Regulatory T Cells Does Not Influence the Inflammation Caused by High Dose Hemi-Body Irradiation

<div><p>There is clinical interest in the modulation of regulatory T cells for cancer therapy. The safety of these therapies in combination with conventional anti-cancer therapies, including radiation therapy, can be studied in animal models. The effects of partial depletion of regulatory T (Treg) cells with an anti-CD25 antibody in conjunction with ionizing radiation on inflammation and tissue injury were analyzed in C57BL/6 mice. An anti-CD25 antibody (PC61) was administered 3 days prior to 13 Gy lower-half hemi-body irradiation (HBI). The blood, spleen, mesenteric lymph nodes (mLNs) and inguinal lymph nodes (iLNs) were harvested at various times thereafter. Alterations in the proportion of leukocyte subsets including CD4⁺ T cells, CD8⁺ T cells, Treg cells, B cells, NK cells, NK1.1⁺ T cells, macrophages and granulocytes were analyzed by FACS. The lungs, liver, pancreas, stomach, jejunum, duodenum, ileum, colon and kidney were harvested and studied by H&E staining. Expression of inflammatory mediators in plasma and tissue were investigated by ELISA. HBI significantly decreased the leukocyte pool though the various leukocyte subsets had different sensitivities to HBI. The administration of PC61 significantly decreased the proportion of Treg cells in spleen, iLN, mLN and blood (reduction of approximately 60%). Irradiation significantly increased the proportion of Treg cells in the spleen, iLN and mLN. HBI induced a systemic inflammatory reaction as demonstrated by increased plasma levels of IL-6, KC/CXCL1 and circulating granulocytes in the blood. Neutrophils also infiltrated the small bowel. The same general patterns were observed whether or not Treg cells were partially depleted with PC61 prior to HBI. These data demonstrate that partial depletion of Treg cells in these mice does not influence HBI-induced inflammatory response and tissue injury, and that combining anti-CD25 therapy with radiation may be safe and well tolerated in a clinical setting.</p> </div

Inflammatory mediators in plasma.

<p>Plasma levels of (A) IL-6 and (B) KC/CXCL1 were determined at different time points after treatment. Data are shown as Mean±SD (n = 4). Two way ANOVA showed significant interactions over time between treatments (all p<0.01). No differences were detected between PC61+irradiation compared to Rat IgG+irradiation treatment group profiles over time in the levels of IL-6 (p≥0.099) and KC/CXCL1 (p≥0.475). No significant differences were found between the PC61+sham irradiation compared to Rat IgG+sham irradiation treatment group profiles over time (all p>0.05). Differences between all other pair-wise comparisons of treatment group profiles over time were significant (p<0.05). All post hoc pairwise comparisons were performed with Tukey's multiple comparisons. This is one representative experiment of three performed.</p

The absolute number of CD4⁺FoxP3⁺ Treg cells in the spleens and LNs.

<p>Both PC61 administration and irradiation reduced the number of CD4⁺FoxP3⁺ Treg cells in spleen, iLN and mLN. Irradiation resulted in a more rapid restoration of Treg cells after day 7. Data are shown as Mean±SD (n = 4). Two way ANOVA showed significant interactions over time between treatments in the Treg absolute numbers in spleen, iLN and mLN (all p<0.01). Differences between all pairs (PC61+sham irradiation <i>vs</i> Rat IgG+sham irradiation, PC61+irradiation <i>vs</i> PC61+sham irradiation, Rat IgG+sham irradiation compare to Rat IgG+irradiation, Rat IgG+sham irradiation compare to PC61+irradiation, PC61+sham irradiation compared to Rat IgG+irradiation and PC61+sham irradiation compared to PC61+irradiation treatment groups) of treatment group profiles over time were significant (p<0.05). All post hoc pairwise comparisons were performed with Tukey's multiple comparisons. This is one representative experiment of three performed.</p