Increased Diacylglycerols Characterize Hepatic Lipid Changes in Progression of Human Nonalcoholic Fatty Liver Disease; Comparison to a Murine Model

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and progression to cirrhosis. While differences in liver lipids between disease states have been reported, precise composition of phospholipids and diacylglycerols (DAG) at a lipid species level has not been previously described. The goal of this study was to characterize changes in lipid species through progression of human NAFLD using advanced lipidomic technology and compare this with a murine model of early and advanced NAFLD.Utilizing mass spectrometry lipidomics, over 250 phospholipid and diacylglycerol species (DAGs) were identified in normal and diseased human and murine liver extracts.Significant differences between phospholipid composition of normal and diseased livers were demonstrated, notably among DAG species, consistent with previous reports that DAG transferases are involved in the progression of NAFLD and liver fibrosis. In addition, a novel phospholipid species (ether linked phosphatidylinositol) was identified in human cirrhotic liver extracts.Using parallel lipidomics analysis of murine and human liver tissues it was determined that mice maintained on a high-fat diet provide a reproducible model of NAFLD in regards to specificity of lipid species in the liver. These studies demonstrated that novel lipid species may serve as markers of advanced liver disease and importantly, marked increases in DAG species are a hallmark of NAFLD. Elevated DAGs may contribute to altered triglyceride, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) levels characteristic of the disease and specific DAG species might be important lipid signaling molecules in the progression of NAFLD

Differing Endoplasmic Reticulum Stress Response to Excess Lipogenesis versus Lipid Oversupply in Relation to Hepatic Steatosis and Insulin Resistance

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been implicated in hepatic steatosis and insulin resistance. The present study investigated their roles in the development of hepatic steatosis and insulin resistance during de novo lipogenesis (DNL) compared to extrahepatic lipid oversupply. Male C57BL/6J mice were fed either a high fructose (HFru) or high fat (HFat) diet to induce DNL or lipid oversupply in/to the liver. Both HFru and HFat feeding increased hepatic triglyceride within 3 days (by 3.5 and 2.4 fold) and the steatosis remained persistent from 1 week onwards (p<0.01 vs Con). Glucose intolerance (iAUC increased by ∼60%) and blunted insulin-stimulated hepatic Akt and GSK3β phosphorylation (∼40–60%) were found in both feeding conditions (p<0.01 vs Con, assessed after 1 week). No impairment of mitochondrial function was found (oxidation capacity, expression of PGC1α, CPT1, respiratory complexes, enzymatic activity of citrate synthase & β-HAD). As expected, DNL was increased (∼60%) in HFru-fed mice and decreased (32%) in HFat-fed mice (all p<0.05). Interestingly, associated with the upregulated lipogenic enzymes (ACC, FAS and SCD1), two (PERK/eIF2α and IRE1/XBP1) of three ER stress pathways were significantly activated in HFru-fed mice. However, no significant ER stress was observed in HFat-fed mice during the development of hepatic steatosis. Our findings indicate that HFru and HFat diets can result in hepatic steatosis and insulin resistance without obvious mitochondrial defects via different lipid metabolic pathways. The fact that ER stress is apparent only with HFru feeding suggests that ER stress is involved in DNL per se rather than resulting from hepatic steatosis or insulin resistance

An autologous dendritic cell vaccine polarizes a Th-1 response which is tumoricidal to patient-derived breast cancer cells.

Breast cancer remains one of the leading causes of cancer-associated death worldwide. Conventional treatment is associated with substantial toxicity and suboptimal efficacy. We, therefore, developed and evaluated the in vitro efficacy of an autologous dendritic cell (DC) vaccine to treat breast cancer. We recruited 12 female patients with stage 1, 2, or 3 breast cancer and matured their DCs with autologous tumour-specific lysate, a toll-like receptor (TLR)-3 and 7/8 agonist, and an interferon-containing cocktail. The efficacy of the vaccine was evaluated by its ability to elicit a cytotoxic T-lymphocyte response to autologous breast cancer cells in vitro. Matured DCs (≥ 60% upregulation of CD80, CD86, CD83, and CCR7) produced high levels of the Th1 effector cytokine, IL12-p70 (1.2 ng/ml; p < 0.0001), compared to DCs pulsed with tumour lysate, or matured with an interferon-containing cocktail alone. We further showed that matured DCs enhance antigen-specific CD8 + T-cell responses to HER-2 (4.5%; p < 0.005) and MUC-1 (19%; p < 0.05) tetramers. The mature DCs could elicit a robust and dose-dependent antigen-specific cytotoxic T-lymphocyte response (65%) which was tumoricidal to autologous breast cancer cells in vitro compared to T-lymphocytes that were primed with autologous lysate loaded-DCs (p < 0.005). Lastly, we showed that the mature DCs post-cryopreservation maintained high viability, maintained their mature phenotype, and remained free of endotoxins or mycoplasma. We have developed a DC vaccine that is cytotoxic to autologous breast cancer cells in vitro. The tools and technology generated here will now be applied to a phase I/IIa clinical trial

Hepatocellular proliferation correlates with inflammatory cell and cytokine changes in a murine model of nonalchoholic fatty liver disease.

Nonalchoholic fatty liver disease (NAFLD) is a problem of increasing prevalence and clinical significance worldwide and is associated with increased risk of development of end stage liver disease and cirrhosis, and can be complicated by hepatocellular carcinoma (HCC). NAFLD is characterized by physical and molecular changes in the liver microenvironment which include an influx of inflammatory cell populations, fibrosis, changes in gene expression, and cytokine production. To better understand changes to the liver in the setting of steatosis, we used a murine model of diet induced hepatic steatosis and corroborated our results with human patient samples of NAFLD. Among the cellular changes, we identified a significant increase in hepatocellular proliferation in the setting of steatosis as compared to controls. Analysis of inflammatory cell populations revealed increased infiltration of CD11b positive myeloid and CD3 positive lymphocytic cell populations in steatotic livers compared to normal livers. Resident Kupffer cells of the liver comprise the largest percentage of these myeloid cells and appear to be responsible for important cytokine alterations impacting proliferation of cells in the liver microenvironment. Significant alterations in cytokine profiles in the plasma and liver tissue lysates from normal and steatotic mice were detected including leptin, CXCL1, CXCL2, and CXCL16 that were further shown to directly increase hepatocyte proliferation in vitro. This increased hepatocellular proliferation and turnover in the setting of steatosis may play important roles in the progression and complications of NAFLD

Increased metastases are associated with inflammation and matrix metalloproteinase-9 activity at incision sites in a murine model of peritoneal dissemination of colorectal cancer

Pro-inflammatory processes associated with the early postoperative state are known to contribute to peritoneal metastases in patients with advanced diseases. This study aimed to determine whether the wound healing response after an abdominal incision leads to increased matrix metalloproteinase (MMP)-9 activity locally, contributing to peritoneal metastasis. Metastatic tumors were initiated in C57bl/6J male mice (8wk of age) using a peritoneal injection model with syngeneic MC38 murine colon cancer cells; appropriate control mice also were studied. Injections were performed into the peritoneum in the right lower quadrant. We then observed the occurrence and rate of peritoneal metastasis for each group. By making an incision into the abdominal wall of mice, an inflammatory response was induced at the wound site. The inflammatory response initiated by the wound, in turn, increased the proliferation of mesothelial cells and increased inflammatory cell numbers locally, which contributed to an increase in parietal peritoneal metastases. In addition, the wound healing process increased the expression of pro-inflammatory cytokines and the number of inflammatory cells in the peritoneum. Moreover, MMP-9 in the modeled postoperative injury setting increased the number and severity of peritoneal metastases. Thus, we conclude that wound-associated inflammation enhances pro-MMP-9 expression, which plays a key role in the growth and progression of cancer cells associated with peritoneal metastases

Quantification of inflammatory cell populations in human liver samples.

<p>Immunohistochemical staining was used to detect and quantify changes in inflammatory cell populations between normal and steatotic human liver samples. Positive immunoreactive staining (dark brown) was calculated as a percentage of total area for (A) CD45 positive inflammatory cells, (B) CD68 positive macrophages (C) CD3 positive T lymphocytes (D) CD8 positive cytotoxic T cells and (E) CD56 positive NK cells in frozen sections of normal and steatotic human livers. Results demonstrate a significant increase in the number of CD45 and CD3 positive cells in the steatotic livers when compared to normal liver samples. CD68 macrophages, CD8 cytotoxic T cells, and CD56 NK cells were not significantly altered between samples. Images are 20x.</p