Evaluating the Relationship Between Metabolic Syndrome and Liver Biopsy-Proven Non-Alcoholic Steatohepatitis in China: A Multicenter Cross-Sectional Study Design

<p><strong>Article full text</strong></p> <p><br> The full text of this article can be found <a href="https://link.springer.com/article/10.1007/s12325-016-0416-4"><b>here</b>.</a><br> <br> <strong>Provide enhanced digital features for this article</strong><br> If you are an author of this publication and would like to provide additional enhanced digital features for your article then please contact <u>[email protected]</u>.<br> <br> The journal offers a range of additional features designed to increase visibility and readership. All features will be thoroughly peer reviewed to ensure the content is of the highest scientific standard and all features are marked as ‘peer reviewed’ to ensure readers are aware that the content has been reviewed to the same level as the articles they are being presented alongside. Moreover, all sponsorship and disclosure information is included to provide complete transparency and adherence to good publication practices. This ensures that however the content is reached the reader has a full understanding of its origin. No fees are charged for hosting additional open access content.<br> <br> Other enhanced features include, but are not limited to:<br> • Slide decks<br> • Videos and animations<br> • Audio abstracts<br> • Audio slides<u></u></p

Additional file 1 of Repopulating Kupffer cells originate directly from hematopoietic stem cells

Additional file 1. Fig S1. Intraperitoneal injection with 20mg/kg clodronate-liposomes did not deplete bone marrow macrophages and did not trigger liver inflammation. A Flow cytometric analysis of bone marrow-derived mononuclear phagocytic system (BM MPS) cells of C57/BL mice received indicated dose of clodronate-liposomes injection (n = 5/group). B Cell count of BM MPS of C57/BL mice treated with the indicated dose of clodronate-liposomes analyzed in A. C Liver tissue from all normal-saline treatment mice at each time point revealed normal cellular architecture (n = 5). Liver tissue from the clodronate-liposomes group revealed no damage to liver cells and inflammatory cells infiltration (n = 5). Liver tissue from the carbon tetrachloride treatment group revealed some damage to liver cells, inflammatory cells infiltration, fatty changes, and centrilobular necrosis (n = 5), scale bar = 50 μm. D Serum alanine aminotransferase of mice from the carbon tetrachloride group was significantly increased at 24 hours posttreatment, and returned to normal level at 96 hours (n = 5/group). In contrast, serum alanine aminotransferase of mice from the clodronate-liposomes group and normal saline group was remained unchanged, at the meantime. Fig S2. Analysis of kupffer cells (KCs) from C57BL/6 mice following intraperitoneal injection with 20mg/kg clodronate-liposomes. A GFP expression on CD68+ and CD68− KCs from E8.5 pulsed Cre mice at 8 weeks after birth. B Flow-cytometric analysis of KCs from C57BL/6 mice 24 hours after being treated with intraperitoneal injection of clodronate-liposomes of indicated dose (n = 6/group). C Percentage of KCs from C57BL/6 mice treated with intraperitoneal injection of clodronate-liposomes of indicated dose analyzed in B. Fig S3. Proliferating ration of bone-marrow-cells (BMCs) and labeled/unlabeled kupffer cells (KCs) from E8.5-pulsed Csf1RCreERT2; RosamT/mG mice at 10 day and 90 day post-intraperitoneal injection with 20mg/kg of control-liposomes. A Representative results of percentage of EdU+ BMCs and labeled/unlabeled KCs. B Percentage of EdU+ BMCs or labeled/unlabeled KCs from indicated mice analyzed in A. Values are the means ± SEM from 6 samples. ***P < 0.001, N.S No significant difference between each Edu/no-Edu group by t-test. Fig S4. Flow cytometric analysis of GFP expression of hematopoietic stem cells and blood leukocytes within purified GFP+ HSC-chimeric Kitw/Kitwv mice. Fig S5. Flow cytometric analysis of YFP expression of indicated cells within adult pulsed Cx3cr1CreERT2; RosaYFP mice (Cre) or Cx3cr1wt; RosaYFP mice (No cre). (A) Flow-cytometric analysis of bone marrow monocytic cells and blood MO from adult pulsed Cx3cr1wt; RosaYFP or Cx3cr1CreERT2; RosaYFP mice at indicated time point post pulse (n = 5/group). (B) Gating strategy of intra-splenic MO. Dot plots are gated on viable single splenic cells. Intra-splenic MO are defined as Ly6C+ cells. (C) Flow cytometric analysis of YFP expression on intra-splenic MO and KCs within the same adult pulsed Csf1rMeriCreMer; RosaYFP mice at indicated time point post intraperitoneal injection of 20mg/kg Clo (n = 4/group). (D) Flow-cytometric analysis of blood leukocytes and KCs from adult pulsed Csf1rMeriCreMer; RosaYFP mice at 25-day post pulse (n = 5/group). (E) Flow-cytometric analysis of blood leukocytes and KCs from adult pulsed Csf1rwt; RosaYFP mice at 25-days post pulse (n = 5/group). (F), (G), (H), (I) Labeling index of intra-splenic MO and KCs at indicated time point post intraperitoneal injection of 20mg/kg Clo, analyzed in (C). *** P < 0.001. Fig S6. Flow-cytometric analysis of bone-marrow hematopoietic stem cells from C57BL/6 mice at indicated time point post intraperitoneal injection with 20gm/kg clodronate-liposomes (n = 5/group). Fig S7. Flow-cytometric analysis of blood hematopoietic stem cells (HSCs) from C57BL/6 mice at indicated time point post intraperitoneal injection with 20mg/kg Clo (n = 5/group). HSCs were defined as LinnegSca-1+c-kit+CD34+CD135− cells. Fig S8. Flow cytometric analysis of GFP liver non-parenchymal cells in kupffer cells-depleted mice revived GFP+ hematopoietic stem cells engraftment. Fig S9. Percent of GFP kupffer cells (KCs) from KC-depleted and purified GFP+ hematopoietic stem cellsengrafted, KC-depleted and purified GFP multipotent progenitors engrafted, or KC-depleted and purified GFP+ monocytic cells engrafted C57BL/6 mice at 90-day post-engraftment. Values are the means ± SEM from 6 samples. ***P < 0.001 between groups by ANOVA. Fig S10. Gating strategies of kupffer cells (KCs), bone marrow-derived mononuclear phagocytic system cells, macrophages, and blood leukocytes. A Gating strategy of non-parenchymal liver cells (NPCs). Dot plots are gated on total liver NPCs, 7-AAD+ dead cells, and doublets were excluded from the analysis and sorting. B Gating strategy of KCs. Dot plots are gated on viable single liver NPCs. KCs are defined as F4/80+, CD45+ C, CD11b+ D, and Ly6C− E cells. F Gating strategy of bone marrow hematopoietic stem cells. 7-AAD+ dead cells and doublets were excluded from the analysis and sorting. Dot plots are gated on all bone marrow cells, then on Lineage-cells, then on Sca-1+ and c-kit+ cells, and finally on CD34− CD135− cells. Hematopoietic stem cells are defined as CD34− and CD135− CD48− CD150+ LSK cells. LT-HSC is defined CD34− and CD135− CD48− CD150− LSK cells, ST-HSC is defined as CD34+ and CD135− CD48− CD150+ LSK cells, MMP2 is defined as CD135− CD34− CD150+ CD48+ LSK cells, MMP3 is defined as CD135− CD34+ CD150− CD48−, MMP4 is defined as CD135+ CD34+ CD150− CD48+ LSK cells. G Gating strategy of bone marrow monocytic progenitors. H Gating strategy of bone marrow macrophages cells. I Gating strategy of blood B cells. J Gating strategy of blood T cells. K Gating strategy of blood monocytic cells. Dot plots are gated on blood cells in the monocyte region. Monocytic cells is defined as CD115+ cells. M Gating strategy of blood neutrophilia granulocyte

Mutation Inactivation of Nijmegen Breakage Syndrome Gene (<i>NBS1</i>) in Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma

<div><p>Nijmegen breakage syndrome (NBS) with <i>NBS1</i> germ-line mutation is a human autosomal recessive disease characterized by genomic instability and enhanced cancer predisposition. The <i>NBS1</i> gene codes for a protein, Nbs1(p95/Nibrin), involved in the processing/repair of DNA double-strand breaks. Hepatocellular carcinoma (HCC) is a complex and heterogeneous tumor with several genomic alterations. Recent studies have shown that heterozygous <i>NBS1</i> mice exhibited a higher incidence of HCC than did wild-type mice. The objective of the present study is to assess whether <i>NBS1</i> mutations play a role in the pathogenesis of human primary liver cancer, including HBV-associated HCC and intrahepatic cholangiocarcinoma (ICC). Eight missense <i>NBS1</i> mutations were identified in six of 64 (9.4%) HCCs and two of 18 (11.1%) ICCs, whereas only one synonymous mutation was found in 89 control cases of cirrhosis and chronic hepatitis B. Analysis of the functional consequences of the identified <i>NBS1</i> mutations in Mre11-binding domain showed loss of nuclear localization of Nbs1 partner Mre11, one of the hallmarks for Nbs1 deficiency, in one HCC and two ICCs with <i>NBS1</i> mutations. Moreover, seven of the eight tumors with <i>NBS1</i> mutations had at least one genetic alteration in the <i>TP53</i> pathway, including <i>TP53</i> mutation, <i>MDM2</i> amplification, <i>p14ARF</i> homozygous deletion and promoter methylation, implying a synergistic effect of Nbs1 disruption and p53 inactivation. Our findings provide novel insight on the molecular pathogenesis of primary liver cancer characterized by mutation inactivation of <i>NBS1</i>, a DNA repair associated gene.</p> </div

Endogenous expression of Nbs1 in HCC or ICC cell lines.

<p>Western blots analysis of endogenous expression of Nbs1 in three HCC cell lines (HepG2, Hep-3B and HepG2.2.15 ) and one ICC cell line(HCCC-9810).β-actin was used as protein loading control.</p

Distribution and type of <i>NBS1</i> mutations in HCC and ICC.

<p>Mutations are located preferentially in exon 11/12 (Mre11-binding domain), but also in or close to other functional domains (FHA domain; second BRCT (BRCT2) domain; ATM phosphorylated sites, Ser278/Ser343/Ser397/Ser615). </p

Representative DNA sequencing of <i>NBS1</i> mutations in HCC and ICC.

<p>(A) Missense NBS1 mutation at codon 638 (TCT→CCT, Ser→Pro) was identified in a case of ICC, but not in the adjacent non-tumorous tissue, indicating that the mutation was somatic. (B) Missense NBS1 mutation at codon 603 (TTC→TTA, Phe→Leu) in a case of HCC. The reverse complementary sequence is shown. (C) Synonymous NBS1 mutation at codon 90 (ACT→ACG, Thr→Thr) in a case of HBV-associated cirrhosis.</p

Increasing Newly Diagnosed Rate and Changing Risk Factors of HCV in Yanbian Prefecture, a High Endemic Area in China

<div><p>Background</p><p>The newly diagnosed rate of HCV infection is increasing in China. However, the risk factors have not been fully identified. Here, a survey was performed in Yanbian Prefecture, a high-endemic area in China.</p><p>Methods</p><p>We identified newly diagnosed HCV infection in 2007–2011, using the local National Disease Supervision Information Management System from the Chinese Center for Disease Control and Prevention. We determined the risk factors using a case-control survey by questionnaire.</p><p>Results</p><p>Yanbian Prefecture had a rapid increase in the yearly newly diagnosed rate of HCV infection from 32.6 to 72.1/100.000 from the year 2007 to 2011. People aged 50–64 years had a high HCV infection of 43.4%, but only 0.3% of cases were reported in those aged less than 20 years. Cosmetic treatment, family history, blood transfusion, and dental treatment were independent risk factors for HCV infection. Unexpectedly, cosmetic treatments [odd ratio (OR) = 5.15, 95% confidence interval (CI) = 2.31–11.48, <i>P</i> = 0.00] and family history (OR = 4.68, 95% CI = 2.67–8.75, <i>P</i> = 0.00) showed a higher risk than the conventional risk factors of blood transfusion (OR = 4.49, 95% CI = 1.95–10.37, <i>P</i> = 0.001) and dental treatment (OR = 2.98, 95% CI = 1.42–6.25, <i>P</i> = 0.00). To further analyze the intrafamilial transmission, we found that spouses of HCV patients had an increased risk for acquiring HCV (OR = 5.75, 95% CI: 1.94–17.07), without significant association between either HCV RNA viral load (<i>P</i> = 0.29) or genotype (<i>P</i> = 0.43).</p><p>Conclusions</p><p>HCV infection was increased in Yanbian Prefecture. Cosmetic treatment was a higher risk factor than medical procedure. HCV infection had a clear family clustering phenomenon, especially between spouses.</p></div