Viral kinetics, model fits and projected time to cure.

<p><b>(A)</b> Time (days) to reach HCV <15 IU/ml or target not detected, TND, during therapy. At end of treatment (w24) all patients but one were TND. <b>(B)</b> Observed viral kinetics and model curves in 4 representative patients (P). Filled triangles: observed HCV viral load above the limit of quantification, LOQ (>15 IU/mL); stars, observed HCV < 15 IU/mL but still detected; crossed squares, TND (arbitrary set to 1 IU/mL); solid lines, biphasic model (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187409#pone.0187409.e001" target="_blank">Eq 1</a>) best fit curves (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187409#pone.0187409.s004" target="_blank">S3 Table</a> for individual parameters). HCV viral load and fit curves of the remaining subjects are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187409#pone.0187409.s009" target="_blank">S1 Fig</a>. <b>(C)</b> Predicted treatment duration (weeks) to reach cure based on a viral cure defined as <1 virus copy in entire patient extracellular fluid (~13.5L).</p

Choline Deficiency Causes Colonic Type II Natural Killer T (NKT) Cell Loss and Alleviates Murine Colitis under Type I NKT Cell Deficiency

<div><p>Serum levels of choline and its derivatives are lower in patients with inflammatory bowel disease (IBD) than in healthy individuals. However, the effect of choline deficiency on the severity of colitis has not been investigated. In the present study, we investigated the role of choline deficiency in dextran sulfate sodium (DSS)-induced colitis in mice. Methionine-choline-deficient (MCD) diet lowered the levels of type II natural killer T (NKT) cells in the colonic lamina propria, peritoneal cavity, and mesenteric lymph nodes, and increased the levels of type II NKT cells in the livers of wild-type B6 mice compared with that in mice fed a control (CTR) diet. The gene expression pattern of the chemokine receptor CXCR6, which promotes NKT cell accumulation, varied between colon and liver in a manner dependent on the changes in the type II NKT cell levels. To examine the role of type II NKT cells in colitis under choline-deficient conditions, we assessed the severity of DSS-induced colitis in type I NKT cell-deficient (Jα18^-/-) or type I and type II NKT cell-deficient (CD1d^-/-) mice fed the MCD or CTR diets. The MCD diet led to amelioration of inflammation, decreases in interferon (IFN)-γ and interleukin (IL)-4 secretion, and a decrease in the number of IFN-γ and IL-4-producing NKT cells in Jα18^-/- mice but not in CD1d^-/- mice. Finally, adaptive transfer of lymphocytes with type II NKT cells exacerbated DSS-induced colitis in Jα18^-/- mice with MCD diet. These results suggest that choline deficiency causes proinflammatory type II NKT cell loss and alleviates DSS-induced colitis. Thus, inflammation in DSS-induced colitis under choline deficiency is caused by type II NKT cell-dependent mechanisms, including decreased type II NKT cell and proinflammatory cytokine levels.</p></div

MCD diet reduces proinflammatory cytokine levels and the number of type II NKT cells in type I NKT cell-deficient mice.

<p>(A) Experimental setup. (B) IFN-γ, (C) IL-4, and (D) IL-10 production by colonic lamina propria mononuclear cells following treatment with 100 ng/mL lipopolysaccharide (LPS) at 24 h, as analyzed by ELISA. <i>N</i> = 4, *<i>P</i> < 0.05, Student’s <i>t</i>-test. (E) Three subsets of colonic lamina propria cells separated using NK1.1 and CD3. <i>Ex vivo</i> intracellular IL-4 and IFN-γ production stimulated by LPS (1 μg/mL) or phorbol myristate acetate (PMA) + ionomycin for 5 h in NKT cells in the lamina propria in MCD vs. CTR diet-fed mice.</p

Level of CXCR6 was elevated in the liver, and the redistribution induced by the MCD diet was altered under choline-deficient conditions.

<p>(A) Experimental setup. (B) Gene expression levels of <i>CXCR6</i>, <i>CXCL16</i>, <i>EP1</i>, <i>EP2</i>, <i>EP3</i>, and <i>EP4</i> (normalized to β-actin), as assessed by quantitative PCR, in hepatic mononuclear cells and colonic lamina propria cells isolated from mice fed the MCD diet for 1 week compared with that in mice fed the CTR diet. (C-E) Representative flow cytometry plots and frequency of CXCR6⁺ population in liver and colonic lamina propria in WT mice. <i>N</i> = 4, *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 Student’s <i>t</i>-test. (F) Experimental setup. (G) Immunofluorescence DAPI staining (×40) of liver from Jα18^-/- mice with or without MCD diet at day 7 after PKH-labeled cells injection. PKH26 (red) and DAPI (blue) were expressed, respectively. PKH-labeled cells per field were enumerated. Data from 10 representative fields from four individual mice are plotted as mean ± s.e.m. ***<i>P</i> < 0.001, Student’s <i>t</i>-test. (scale bar = 50 μm) (H) Expression of NK1.1 and CD3 on PKH-labeled single live cells harvested from the liver at day 7 after injection of PKH-labeled cells. <i>N</i> = 4, ***<i>P</i> < 0.001, Student’s <i>t</i>-test.</p

Methionine-choline deficient (MCD) diet induces nonalcoholic steatohepatitis (NASH) and natural killer T (NKT) cell accumulation.

<p>(A) Representative images of hematoxylin and eosin (H & E) staining of mice fed the MCD diet for 1 week and 4 weeks or the control (CTR) diet. Scale bar, 100 μm. (B) Serum alanine aminotransferase (ALT) and triglyceride (TG) levels in MCD diet-fed mice over 8 weeks. <i>N</i> = 4–6, *<i>P</i> < 0.05 vs. MCD diet week 0, paired <i>t</i>-test. (C) Intrahepatic NK1.1⁺ T (NKT) cells were evaluated by flow cytometry in MCD diet-fed mice over 6 weeks. <i>N</i> = 5–6, **<i>P</i> < 0.01, Student’s <i>t</i>-test. (D) Intrahepatic NKT cells, natural killer (NK) cells, and NK1.1^- T (T) cells were measured by flow cytometry in MCD diet-fed mice for 1, 2, 4, 6, or 8 weeks. <i>N</i> = 2–6, *<i>P</i> < 0.05, vs. MCD 0 week, paired <i>t</i>-test. (E) Effects of 1 week of MCD diet on NKT cell subsets in mononuclear cells of the liver, colonic lamina propria, spleen, mesenteric lymph node (MLN), and intraperitoneal cavity in wild-type mice. Black and hatched bars represent NKT type I and type II cells, respectively. <i>N</i> = 4, *<i>P</i> < 0.05, **<i>P</i> < 0.01, Student’s <i>t</i>-test. (F) Effects of 1 week of MCD diet on type I and type II NKT cell subsets in the lamina propria in Jα18^-/- and CD1d^-/- mice, as measured by flow cytometry. <i>N</i> = 4, *<i>P</i> < 0.05, Student’s <i>t</i>-test. All data represent the mean ± standard error of the mean (s.e.m.).</p

The MCD diet attenuates dextran sodium sulfate (DSS)-induced colitis under type I NKT cell deficiency.

<p>(A) Experimental setup. (B) Body weight (percentage of body weight relative to initial body weight) of Jα18^-/- and CD1d^-/- mice fed the MCD and CTR diets, measured after 2.0% DSS treatment. <i>N</i> = 4–6, *<i>P</i> < 0.05 (Jα18^-/- CTR vs. Jα18^-/- MCD). (C) Representative macroscopic photographs and (D) disease activity index (DAI) scores of colons of DSS-treated Jα18^-/- and CD1d^-/- mice fed the MCD or CTR diets. <i>N</i> = 4, *<i>P</i> < 0.05, Student’s <i>t</i>-test. (E) Histological examination (H & E staining) and (F) histological scores of the colons of Jα18^-/- and CD1d^-/- mice fed the MCD or CTR diets 7 days after 2.0% DSS treatment. Original magnification × 100. All data are presented as means ± SE. <i>N</i> = 6, *<i>P</i> < 0.05, Student’s <i>t</i>-test. (G) Endoscopic evaluation and (H) endoscopic evaluation score of colorectal health in Jα18^-/- and CD1d^-/- mice. <i>N</i> = 6, *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Adaptive transfer of Jα18-CTR lamina propria cells, but not CD1d-CTR lamina propria cells, restored colonic inflammation in DSS-induced colitis.

<p>(A) Experimental setup. (B) Disease activity index (DAI) scores of colons of DSS-treated Jα18^-/- and CD1d^-/- mice fed the MCD or CTR diets, and transferred with lamina propria cells from Jα18^-/- and CD1d^-/- mice. <i>N</i> = 4, *<i>P</i> < 0.05, Student’s <i>t</i>-test. (C) Histological examination (H & E staining) and (D) histological scores of the colons of Jα18^-/- and CD1d^-/- mice fed the MCD or CTR diets 7 days after 2.0% DSS treatment. Original magnification × 100. All data are presented as means ± SE. <i>N</i> = 4, *<i>P</i> < 0.05, Student’s <i>t</i>-test.</p

Background of dynamic CT appearance of radiation injury according to previous therapy.

<p>* TACE; transcatheter arterial chemoembolization</p><p>Background of dynamic CT appearance of radiation injury according to previous therapy.</p

Identification of a Functional Variant in the <i>MICA</i> Promoter Which Regulates <i>MICA</i> Expression and Increases HCV-Related Hepatocellular Carcinoma Risk

<div><p>Hepatitis C virus (HCV) infection is the major cause of hepatocellular carcinoma (HCC) in Japan. We previously identified the association of SNP rs2596542 in the 5' flanking region of the <i>MHC class I polypeptide-related sequence A</i> (<i>MICA</i>) gene with the risk of HCV-induced HCC. In the current study, we performed detailed functional analysis of 12 candidate SNPs in the promoter region and found that a SNP rs2596538 located at 2.8 kb upstream of the <i>MICA</i> gene affected the binding of a nuclear protein(s) to the genomic segment including this SNP. By electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay, we identified that transcription factor Specificity Protein 1 (SP1) can bind to the protective G allele, but not to the risk A allele. In addition, reporter construct containing the G allele was found to exhibit higher transcriptional activity than that containing the A allele. Moreover, SNP rs2596538 showed stronger association with HCV-induced HCC (P = 1.82×10⁻⁵ and OR = 1.34) than the previously identified SNP rs2596542. We also found significantly higher serum level of soluble MICA (sMICA) in HCV-induced HCC patients carrying the G allele than those carrying the A allele (P = 0.00616). In summary, we have identified a functional SNP that is associated with the expression of MICA and the risk for HCV-induced HCC.</p> </div

A typical case belonging to Child–Pugh class A that changed from type 3 after 3 months to type 1 after 9 months (case 36).

<p>Dose distribution b) Plain c) Arterial phase d) Portal phase e) Venous phase.</p