An exploratory study examining how nano-liquid chromatography-mass spectrometry and phosphoproteomics can differentiate patients with advanced fibrosis and higher percentage collagen in non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is among the leading causes of liver disease worldwide. It is increasingly recognized that the phenotype of NASH may involve a number of different pathways, of which each could become important therapeutic targets. The aim of this study is to use high resolution mass spectrometry (MS) and phosphoproteomics techniques to assess the serum proteome and hepatic phosphoproteome in subjects with NASH-related fibrosis. METHODS: Sixty-seven biopsy-proven NAFLD subjects with frozen sera and liver tissue were included. Reverse phase protein microarray was used to quantify the phosphorylation of key signaling proteins in liver and nano-liquid chromatography (LC)-MS was used to sequence target biomarkers in the serum. An image analysis algorithm was used to quantify the percentage of collagen (% collagen) using computer-assisted morphometry. Using multiple regression models, serum proteomes and phosphorylated hepatic proteins that were independently (p ≤ 0.05) associated with advanced fibrosis (stage ≥ 2) and higher % collagen were assessed. RESULTS: Phosphorylated signaling pathways in the liver revealed that apoptosis signal-regulating kinase 1, mitogen-activated protein kinase (ASK1-MAPK pathway involving ASK1 S38 (p < 0.02) and p38 MAPK (p = 0.0002)) activated by the inflammatory cytokine interleukin (IL-10) (p < 0.001), were independently associated with higher % collagen. LC-MS data revealed that serum alpha-2 macroglobulin (α2M) (p = 0.0004) and coagulation factor V (p = 0.0127) were independently associated with higher % hepatic collagen. CONCLUSIONS: Simultaneous profiling of serum proteome and hepatic phosphoproteome reveals that the activation of ASK1 S38, p38 MAPK in the liver, and serum α2M and coagulation factor V are independently associated with hepatic collagen deposition in patients with NASH. These data suggest the role of these pathways in the pathogenesis of NASH-related fibrosis as a potential therapeutic target.

Anti-adipocyte antibody response in patients with non-alcoholic fatty liver disease.

BACKGROUND AND AIM: A significant number of autoantibodies have been reported in patients with non-alcoholic fatty liver disease (NAFLD) patients. In the present study, our aim was to assess the role of disease and cell-specific antibodies, namely anti-adipocyte antibodies (anti-AdAb) in patients with NAFLD and non-alcoholic steatohepatitis (NASH). METHODS: Flow cytometry was used to detect the presence of anti-AdAb (immunoglobulin M [IgM] and immunoglobulin G [IgG]) in sera from patients with biopsy-proven NAFLD (n = 98) and in controls (n = 49) without liver disease. Univariate and multivariate analysis was performed to draw associations between anti-AdAb IgM and IgG levels and the different clinical variables. RESULTS: Patients with NAFLD had significantly higher levels of anti-AdAb IgM and significantly lower levels of AdAb IgG when compared with controls (P = 0.002 and P < 0.001, respectively). Patients with NASH had significantly higher levels of anti-AdAb IgM when compared with non-NASH NAFLD patients, P = 0.04. In multivariate analysis, anti-AdAb IgM was independently associated with a higher risk for NASH (odds ratio[OR]: 2.90 [confidence interval (CI) 1.18-7.16], P = 0.02). Anti-AdAb IgM was also found to be independently associated with portal inflammation in patients with NAFLD (OR: 3.01 [CI 1.15-7.90 P = 0.02]). CONCLUSIONS: Anti-AdAb IgM was independently associated with NAFLD and NASH while anti-AdAb IgG was found to be protective against NAFLD. Anti-AdAb IgM was found specifically to be associated with the inflammatory processes in NAFLD. These findings indicate that the anti-AdAb IgM and IgG may play an immunomodulatory role in the pathogenesis of NAFLD and NASH.

Major Histocompatibility Complex Class I-Related Chain A Alleles and Histology of Nonalcoholic Fatty Liver Disease.

Major histocompatibility complex class I-related chain A (MICA) is a highly polymorphic gene that modulates immune surveillance by binding to its receptor on natural killer cells, and its genetic polymorphisms have been associated with chronic immune-mediated diseases. The progressive form of nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), is characterized by accumulation of fat and inflammatory cells in the hepatic parenchyma, potentially leading to liver cell injury and fibrosis. To date, there are no data describing the potential role of MICA in the pathogenesis of NAFLD. Therefore, our aim was to assess the association between MICA polymorphism and NASH and its histologic features. A total of 134 subjects were included. DNA from patients with biopsy-proven NAFLD were genotyped using polymerase chain reaction-sequence-specific oligonucleotide for MICA alleles. Liver biopsies were assessed for histologic diagnosis of NASH and specific pathologic features, including stage of fibrosis and grade of inflammation. Multivariate analysis was performed to draw associations between MICA alleles and the different variables; P ≤ 0.05 was considered significant. Univariate analysis showed that MICA*011 (odds ratio [OR], 7.14; 95% confidence interval [CI], 1.24-41.0; P = 0.04) was associated with a higher risk for histologic NASH. Multivariate analysis showed that MICA*002 was independently associated with a lower risk for focal hepatocyte necrosis (OR, 0.24; 95% CI, 0.08-0.74; P = 0.013) and advanced fibrosis (OR, 0.11; 95% CI, 0.02-0.70; P = 0.019). MICA*017 was independently associated with a higher risk for lymphocyte-mediated inflammation (OR, 5.12; 95% CI, 1.12-23.5; P = 0.035). Conclusion: MICA alleles may be associated with NASH and its histologic features of inflammation and fibrosis. Additional research is required to investigate the potential role of MICA in increased risk or protection against NAFLD.

Pancreatic duct changes are not associated with early signs of chronic pancreatitis at magnetic resonance imaging (MRI) in patients with primary sclerosing cholangitis.

OBJECTIVE: The association between chronic pancreatitis (CP) and primary sclerosing cholangitis (PSC) has been reported previously. The aims of the present study were to evaluate the presence of early pancreatic abnormalities and duct changes, using MRCP/MRI in PSC and to evaluate possible risk factors for these changes and their clinical importance. MATERIALS AND METHODS: One hundred and three patients with PSC were identified among all MRI liver/pancreas referrals in 2001-2005. MRCP was used to grade pancreatic duct changes in three groups: grade 0 (normal), grade 1 (mild) and grade 2 (severe). For detection of early MRI signs of CP, the pancreas-spleen signal intensity ratio (SIR), the arterial and early venous phase ratio (A/PV ratio) and the age-related size of the pancreas were evaluated. RESULTS: Pancreatic duct changes were found in 24% of the PSC patients. The pancreatic duct changes were associated with extrahepatic biliary involvement and long duration of PSC but not associated with pancreas-spleen SIR, A/PV ratio, pancreas size, previous post-ERCP or acute pancreatitis. Severe pancreatic duct changes were significantly associated to abdominal pain. Clinically significant CP was seen in one PSC patient (1%). CONCLUSIONS: Pancreatic duct changes are associated with extrahepatic bile duct strictures and not with the early MRI signs of CP. Therefore, pancreatic duct changes seem to be part of the spectrum of PSC and should not be defined as CP. Pancreatic duct changes are of limited clinical importance but may contribute to abdominal pain in PSC.

Analysis of human leukocyte antigen allele polymorphism in patients with non alcoholic fatty liver disease.

The human leukocyte antigen (HLA) genes may play a role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and its progressive form, non-alcoholic steatohepatitis (NASH). The aim of this study was to assess the association of HLA class I and II alleles with NASH and its histological features.Deoxyribonucleic acid (DNA) was extracted from 140 subjects (85 biopsy-proven NAFLD and 55 controls) and genotyped for HLA (-A, -B, -C, -DR1, -DR3, -DQ, and -DP). Liver biopsies were assessed for presence of NASH, degree of fibrosis and inflammation. Multivariate analysis was performed to assess associations between HLA genes and different histologic features of NAFLD.Our data for HLA class I showed that HLA-C*4 was associated with lower risk for histologic NASH and HLA-C*6 was protective against portal fibrosis. Conversely, HLA-B*27 was associated with high-grade hepatic steatosis, while HLA-A*31 was associated with increased risk for advanced fibrosis. Among HLA class II alleles, HLA-DQA1*01 was associated with lower risk for NASH while HLA-DRB1*03 was associated with increased risk for NASH.Our findings indicate that HLA class I and II gene polymorphism may be associated with susceptibility to NASH, fibrosis and other pathologic features and may be involved in the pathogenesis of NAFLD.

The levels of monoamine neurotransmitters and measures of mental and emotional health in HCV patients treated with ledipasvir (LDV) and sofosbuvir (SOF) with or without ribavirin (RBV).

Mental and emotional health (MEH) impairment is commonly encountered in hepatitis C patients. Although the exact mechanism remains unknown, alterations in neurotransmitter and cytokine levels maybe associated with hepatitis C virus (HCV)-related MEH issues.The aim of the study was to assess association of serum biomarkers with self-reports of MEH in HCV patients before treatment and after achieving sustained virologic response (SVR).The HCV genotype-1-infected patients who achieved SVR at 12 weeks after treatment with ledipasvir (LDV)/sofosbuvir (SOF) ±â€Šribavirin (RBV) were selected. Frozen serum samples from baseline, end of treatment (EOT), and posttreatment week 4 (PTW4) were used to assay 16 cytokines and monoamine neurotransmitters. Validated self-reports were used to assess MEH.Hundred patients were evaluated. Mean age was 53 years (57% male, 86% white). Compared with baseline, emotional well-being and emotional health significantly increased by EOT, and role emotional, emotional well-being, and emotional health significantly increased at PTW4 in the RBV-containing arm (P < 0.05). In patients taking LDV/SOF + RBV, serotonin levels were significantly decreased at PTW4 compared with baseline (P = 0.046). Compared with baseline, there were significant decreases in interleukin (IL)-10 levels at EOT and PTW4 in both treatment groups. The changes in IL-8 also differed significantly between LDV/SOF + RBV and LDV/SOF groups (P < 0.05). Changes in dopamine and tryptophan levels at EOT correlated with increasing emotional health scores, whereas changes in monocyte chemoattractant protein-1 at EOT and IL-8 at PTW4 correlated with increasing mental health scores. The neurotransmitters and cytokines were found to be independent predictors of MEH scores in multiple regression analysis.Cytokine and neurotransmitter changes are associated with mental and emotional health. Patient-reported outcome scores change during and after treatment.

Expression of vascular endothelial growth factor receptor-2 or Tie-2 on peripheral blood cells defines functionally competent cell populations capable of reendothelialization.

BACKGROUND: Receptor tyrosine kinases that include vascular endothelial growth factor (VEGFR)-1, VEGFR-2, and Tie-2 regulate cardiovascular development and physiological and pathological angiogenesis. We were interested in the phenotypic and functional characterization of peripheral blood cells expressing these receptors and their therapeutic potential in vascular injury. METHODS AND RESULTS: VEGFR-1+, VEGFR-2+, and Tie-2+ cells constituted approximately 3.0+/-0.2%, 0.8+/-0.5%, and 2.0+/-0.3%, respectively, of the total population of mononuclear cells in blood. Phenotypic analysis demonstrated that all 3 cell populations mainly expressed markers of monocytic/macrophage lineage. Only VEGFR-2+ and Tie-2+ cells phenotypically, morphologically, and functionally differentiated to endothelial cells after culture, whereas VEGFR-1+ cells did not. None of the cell types proliferated in vitro. Only freshly isolated VEGFR-2+ or Tie-2+ cells but not VEGFR-2- or Tie-2- cell populations significantly contributed to efficient endothelialization of balloon-injured femoral arteries of nude mice. Furthermore, these cells also differentiated into -actin-positive smooth muscle cells. Administration of bromodeoxyuridine to animals transplanted with human endothelial progenitor cells showed that VEGFR-2+ and Tie-2+ cells proliferated in vivo. CONCLUSIONS: These data demonstrate that expression of VEGFR-2 and/or Tie-2 on peripheral blood cells defines functionally competent cell populations that proliferate in vivo and that contribute to reendothelialization. These findings may have implications for a cell-based approach in vascular diseases.

Biliary epithelial cell antibodies link adaptive and innate immune responses in primary sclerosing cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is an autoimmune liver disease with destruction of hepatic bile ducts. A high frequency of biliary epithelial cell antibodies (BEC-Ab) is present in PSC. Here, we studied the mechanisms and signaling pathways used by these Ab in causing BEC dysfunction. METHODS: Immunoassays were performed using freshly isolated BECs to study the signaling capacity of purified immunoglobulin (Ig) G and F(ab)'(2) fractions from 33 patients with PSC with anti-BEC-Ab. RESULTS: We provide evidence that stimulation of BECs with PSC IgG, but not control IgG, induced expression of Toll-like receptor (TLR) 4 and TLR9 and specific phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 as well as the transcription factors ELK-1 and nuclear factor kappaB. A specific inhibitor of ERK1/2 abrogated phosphorylation of ELK-1 and protein expression of TLR4 but not TLR9 on BECs. TLR-expressing BECs, when further stimulated with lipopolysaccharide and CpG DNA, produced high levels of interleukin-1beta, interleukin-8, interferon gamma, tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and transforming growth factor beta. Bile ducts stained positively for TLR4 and TLR9 in 58% of liver specimens taken from patients with PSC with BEC-Ab, as compared with 14% in those without BEC-Ab and also less frequently in diseased control livers. CONCLUSIONS: Our data show that binding of PSC BEC-Ab initiates ERK1/2 signaling and up-regulation of TLR, which upon ligation induces BECs to produce cytokines/chemokines, leading to the possible recruitment of inflammatory cells. Thus, in PSC, BECs are not only targets of the immune attack but may also be active participants and mediators of their own destruction. BEC-Ab may be critical regulators of cholangitis in PSC.

A novel mechanism of liver allograft rejection facilitated by antibodies to liver sinusoidal endothelial cells.

Liver sinusoidal endothelial cells (LSECs) may be implicated in the induction of liver allograft rejections. We studied the clinical consequences of LSEC-reactive antibodies and their functional capacity in modulating T-cell responses during acute rejections. Pre- and posttransplant sera and T lymphocytes from 95 liver transplant patients were used in this study. LSECs were isolated from normal healthy liver. Binding of antibodies to LSECs was detected using flow cytometry. To study whether LSEC antibodies facilitated cell-mediated immunity, a mixed cell culture (MCC) assay was used. Cytokines in the supernatants of MCC were measured by enzyme-linked immunosorbent assay. Liver biopsy sections were stained to detect the deposition of immunoglobulins in LSECs during rejections. The 2-year patient survival was 86.3%. A significantly higher number of patients with rejections had LSEC antibodies (35/50; 70%) than those without rejections (8/45; 18%) (P < .0001). Purified fractions of LSEC antibodies induced the expression of the costimulatory molecule CD86 on LSECs. A significantly higher number of patients with LSEC antibodies and rejections had an increased proliferation of T cells and markedly decreased levels of transforming growth factor beta (TGF-beta) in the MCC than those without antibodies and rejections (P < .0001, P < .0001, respectively). Deposition of antibodies in LSECs during rejection episodes was observed in the biopsies of patients with LSEC antibodies but not in those without LSEC antibodies. In conclusion, antibodies to LSECs may facilitate acute liver allograft rejections by down-regulating the immune modulating cytokine TGF-beta and thus up-regulating alloreactive T-cell proliferation.

Wegener's granulomatosis is associated with organ-specific antiendothelial cell antibodies.

BACKGROUND: Antiendothelial cell antibodies (AECA), usually detected using human umbilical vein endothelial cells (HUVEC), are frequently observed in systemic vasculitis, but their pathogenic role is unclear. Heterogeneity of endothelial cells necessitates use of clinically relevant endothelial cells for elucidation of the role of AECA in systemic vasculitis involving small blood vessels of specific organs. METHODS: Human endothelial cells were isolated from normal tissue specimens from the nose, kidney, lung, liver, and umbilical vein. Using flow cytometry, AECA were detected against both unstimulated and cytokine-stimulated [tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] endothelial cells. Functional capacity of AECA was determined by complement fixation assay. Sera from patients with Wegener's granulomatosis (16), limited Wegener's granulomatosis (8), renal limited disease (4), microscopic polyangiitis (MPA) (5), rheumatoid arthritis (10), and systemic lupus erythematosus (SLE) (9), and from healthy controls (20) were analyzed. RESULTS: Compared with controls (1) Wegener's granulomatosis is significantly associated with noncytotoxic AECA that selectively bind surface antigens on unstimulated nasal, kidney, and lung endothelial cells; (2) binding of Wegener's granulomatosis AECA to kidney and nasal endothelial cells in particular was lost upon treatment with IFN-gamma and TNF-alpha; (3) the two cytokines per se were cytotoxic (30%) to nasal and lung endothelial cells and lysis was further increased (60%) by addition of systemic vasculitis serum; and (4) Wegener's granulomatosis serum caused agglutination of cytokine-stimulated nasal endothelial cells. CONCLUSION: Based on these findings we suggest that AECA may be one factor involved in the initiation of Wegener's granulomatosis. Antigen identification and elucidation of the pathogenic roles of AECA and inflammatory cytokines in systemic vasculitis using these cells will be particularly important.