Tological findings of the liver biopsy specimens taken from the patients with NAFLD are shown in the lower part of Table 1. The inter-observer agreement between the two pathologists for liver fibrosis stage was 90.2 .LPS-induced sCD14 in vitroFinally, to elucidate whether LPS can Title Loaded From File directly affect secretion of sCD14 from macrophages, we investigated sCD14 levels in the culture medium of RAW264.7 cells, the murine monocyte/ macrophage cell line, incubated with LPS using by Western immunoblot analysis and a sandwich enzyme-linked immunosorbent assay. LPS treatment for RAW 264.7 cells significantly increased sCD14 in the cell culture medium (Fig. 3). These results support the observation that LPS increases secretion of sCD14 from macrophages including Kupffer cells.Serum sCD14 LevelsWe measured serum sCD14 levels in the Title Loaded From File healthy controls and in the two subgroups of patients with NAFLD. We found no marked differences in the serum CD14 levels between the healthy controls and patients without NASH (Fig. 1A). However, the serum sCD14 level was markedly elevated in patients with NASH compared with that in patients without NASH (Fig. 1A). The area under the receiver operating characteristic (ROC) curve for distinguishing between not NASH and NASH using serum sCD14 level was 0.796 (Fig. 1B). Using a cutoff level of greater than 27.3 ng/ml for serum sCD14 level yielded sensitivity and specificity values of 81.5 and 72.5 , respectively. The positive and negative predictive values for the serum sCD14 level of 27.3 ng/ml were 73.6 and 80.6 , respectively.DiscussionIn the present study, we showed that serum sCD14 levels were significantly associated with diagnosis of NASH. Moreover, the increased sCD14 levels in NASH patients were highly correlated with increased hepatic CD14 expression and liver inflammation, even after adjusting for age, sex, presence of diabetes, dyslipidesCD14 and Liver Inflammation in NASHTable 1. Clinical and serological characteristics of the control and patient population.Controls Number (n) Age (years) Gender (male; female) Body mass index (kg/m2) Visceral fat area (cm2) Subcutaneous fat area (cm2) Fasting blood sugar (mg/dl) AST (IU/l) ALT (IU/l) C-reactive protein (mg/l) HOMA-IR Dyslipidemia ( ) Hypertension ( ) Steatosis grade 5?3 33?6 .66 Lobular inflammation None ,2 foci per 200x field 2? foci per 200x field .4 foci per 200x field Liver cell ballooning None Few balloon cells Many balloon cells Fibrosis stage None Perisinusoidal or periportal Perisinusoidal and portal/periportal Bridging fibrosis Cirrhosis 84.2610.1 23.864.8 22.866.2 0.2760.21 0.9660.17 0 0 21 44.869.0 14;7 21.962.Not NASH 48 50.4613.6 26;22 27.965.3 141.1634.9 191.3654.1 101.2625.1 40.1616.1 49.3627.2 0.7360.47 2.6161.39 17 (35.1) 19 (39.5)NASH 65 51.4612.8 36;29 29.165.1 151.4640.4 201.9658.1 106.2629.4 41.5617.1 54.3626.9 1.3560.94 3.6662.01 23 (47.9) 20 (41.5)P value*0.381 0.328 0.046 0.171 0.173 0.301 0.284 0.212 0.011 0.009 0.052 0.522 0.22 1923 31 11 7 x15 23 80 31 22 12 3 x30 160 47 18 3 x21 23 4 00 38 20 5Numbers represent the mean 6 SD. Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; HOMA-IR, homeostasis model for the assessment of insulin resistance. P values correspond to the comparison of the three subjects groups (not NASH, borderline NASH and definite NASH) using the Kruskal allis tests for continuous factors. doi:10.1371/journal.pone.0065211.tmia, hypertension, BMI, VFA, and SFA. To our knowledge, this is t.Tological findings of the liver biopsy specimens taken from the patients with NAFLD are shown in the lower part of Table 1. The inter-observer agreement between the two pathologists for liver fibrosis stage was 90.2 .LPS-induced sCD14 in vitroFinally, to elucidate whether LPS can directly affect secretion of sCD14 from macrophages, we investigated sCD14 levels in the culture medium of RAW264.7 cells, the murine monocyte/ macrophage cell line, incubated with LPS using by Western immunoblot analysis and a sandwich enzyme-linked immunosorbent assay. LPS treatment for RAW 264.7 cells significantly increased sCD14 in the cell culture medium (Fig. 3). These results support the observation that LPS increases secretion of sCD14 from macrophages including Kupffer cells.Serum sCD14 LevelsWe measured serum sCD14 levels in the healthy controls and in the two subgroups of patients with NAFLD. We found no marked differences in the serum CD14 levels between the healthy controls and patients without NASH (Fig. 1A). However, the serum sCD14 level was markedly elevated in patients with NASH compared with that in patients without NASH (Fig. 1A). The area under the receiver operating characteristic (ROC) curve for distinguishing between not NASH and NASH using serum sCD14 level was 0.796 (Fig. 1B). Using a cutoff level of greater than 27.3 ng/ml for serum sCD14 level yielded sensitivity and specificity values of 81.5 and 72.5 , respectively. The positive and negative predictive values for the serum sCD14 level of 27.3 ng/ml were 73.6 and 80.6 , respectively.DiscussionIn the present study, we showed that serum sCD14 levels were significantly associated with diagnosis of NASH. Moreover, the increased sCD14 levels in NASH patients were highly correlated with increased hepatic CD14 expression and liver inflammation, even after adjusting for age, sex, presence of diabetes, dyslipidesCD14 and Liver Inflammation in NASHTable 1. Clinical and serological characteristics of the control and patient population.Controls Number (n) Age (years) Gender (male; female) Body mass index (kg/m2) Visceral fat area (cm2) Subcutaneous fat area (cm2) Fasting blood sugar (mg/dl) AST (IU/l) ALT (IU/l) C-reactive protein (mg/l) HOMA-IR Dyslipidemia ( ) Hypertension ( ) Steatosis grade 5?3 33?6 .66 Lobular inflammation None ,2 foci per 200x field 2? foci per 200x field .4 foci per 200x field Liver cell ballooning None Few balloon cells Many balloon cells Fibrosis stage None Perisinusoidal or periportal Perisinusoidal and portal/periportal Bridging fibrosis Cirrhosis 84.2610.1 23.864.8 22.866.2 0.2760.21 0.9660.17 0 0 21 44.869.0 14;7 21.962.Not NASH 48 50.4613.6 26;22 27.965.3 141.1634.9 191.3654.1 101.2625.1 40.1616.1 49.3627.2 0.7360.47 2.6161.39 17 (35.1) 19 (39.5)NASH 65 51.4612.8 36;29 29.165.1 151.4640.4 201.9658.1 106.2629.4 41.5617.1 54.3626.9 1.3560.94 3.6662.01 23 (47.9) 20 (41.5)P value*0.381 0.328 0.046 0.171 0.173 0.301 0.284 0.212 0.011 0.009 0.052 0.522 0.22 1923 31 11 7 x15 23 80 31 22 12 3 x30 160 47 18 3 x21 23 4 00 38 20 5Numbers represent the mean 6 SD. Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; HOMA-IR, homeostasis model for the assessment of insulin resistance. P values correspond to the comparison of the three subjects groups (not NASH, borderline NASH and definite NASH) using the Kruskal allis tests for continuous factors. doi:10.1371/journal.pone.0065211.tmia, hypertension, BMI, VFA, and SFA. To our knowledge, this is t.