We have used total enteral nutrition (TEN) to moderately overfeed rats high-polyunsaturated fat diets to develop a model for nonalcoholic steatohepatitis (NASH). Male Sprague-Dawley rats were fed by TEN a 187 kcal.kg(-3/4).day(-1) diet containing 5% (total calories) corn oil or a 220 kcal.kg(-3/4).day(-1) diet in which corn oil constituted 5, 10, 25, 35, 40, or 70% of total calories for 21 or 65 days. Rats fed the 5% corn oil, 220 kcal.kg(-3/4).day(-1)diet had greater body weight gain (P < or = 0.05), fat mass (P < or = 0.05), and serum leptin and glucose levels (P < or = 0.05), but no liver pathology. A dose-dependent increase in hepatic triglyceride deposition occurred with increase in percent corn oil in the 220 kcal.kg(-3/4).day(-1) groups (P < or = 0.05). Steatosis, macrophage infiltration, apoptosis, and focal necrosis were present in the 70% corn oil group, accompanied by elevated serum alanine aminotransferase (ALT) levels (P < or = 0.05). An increase in oxidative stress (thiobarbituric acid-reactive substances) and TNF-alpha expression (P < or = 0.05) was observed in the 70% corn oil group, as well as an increase in hepatic CYP2E1 and CYP4A1 expression (P < or = 0.05). Significant positive correlations were observed between the level of dietary corn oil and the degree of pathology, ALTs, oxidative stress, and inflammation. Liver pathology was progressive with increased necrosis, accompanied by fibrosis, observed after 65 days of TEN. Increased expression of CD36 and l-fabp mRNA suggested development of steatosis was associated with increased fatty acid transport. These data suggest that intragastric infusion of a high-polyunsaturated fat diet at a caloric level of 17% excess total calories results in pathology similar to clinical NASH.

BACKGROUND.Pancreatic cancer (PaC) is characterized by local invasion and early metastasis. Serine proteases have been associated with invasion and metastasis of many cancers due to their ability to degrade extracellular matrix (ECM) proteins and to activate other proteases; thus, the serine proteases expressed in PaC were investigated.METHODS.An expression profile of serine proteases was generated from both normal and malignant pancreatic tissues using a polymerase chain reaction (PCR)‐based screen and differential expression of kallikrein 7 was examined by reverse‐transcriptase PCR (RT‐PCR) and immunohistochemical analyses. The ability of human kallikrein 7 (hK7) to cleave the epithelial cell adhesion molecule E‐cadherin was tested in vitro using both recombinant E‐cadherin and BxPC‐3 cells and the effects of hK7 proteolytic activity on pancreatic cell invasion and aggregation were examined.RESULTS.Expression profiling revealed that kallikrein 7 (KLK7) was overexpressed in pancreatic adenocarcinomas and its differential expression was confirmed by RT‐PCR analysis. hK7 was observed in neoplastic cells of all tumors examined with moderate‐to‐intense staining in 70% of tumors examined (16/23). In contrast, only 15% of nonmalignant tissue specimens (2/13) displayed moderate hK7 staining, whereas the remaining specimens yielded weak, if any, immunoreactivity. Using in vitro assays, hK7 was shown to cleave E‐cadherin and the soluble E‐cadherin fragment produced significantly enhanced Panc‐1 cell invasion through ECM proteins with a corresponding reduction in Panc‐1 cell aggregation.CONCLUSIONS.These results suggest that aberrant expression of KLK7 plays an important role in PaC and provides novel insight into the effects of elevated hK7 proteinase activity in this, and perhaps other, adenocarcinomas. Cancer 2007. © 2007 American Cancer Society.

A "2-hit" model for nonalcoholic steatohepatitis (NASH) has been proposed in which steatosis constitutes the "first hit" and sensitizes the liver to potential "second hits" resulting in NASH. Oxidative stress is considered a candidate for the second hit. N-acetylcysteine (NAC), an antioxidant, has been suggested as a dietary therapy for NASH. We examined the effects of NAC in a rat total enteral nutrition (TEN) model where NASH develops as the result of overfeeding dietary polyunsaturated fat. Male Sprague-Dawley rats consumed pelleted AIN-93G diets ad libitum or were overfed a 9200 kJ.kg(-0.75).d(-1) liquid diet containing 70% corn oil with or without 2 g.kg(-1).d(-1) NAC i.g. for 65 d. Hepatic steatosis was not influenced by dietary supplementation with NAC; however, the liver pathology score was lower (P

Although alcohol effects within the liver have been extensively studied, the complex mechanisms by which alcohol causes liver cancer are not well understood. It has been suggested that ethanol (EtOH) metabolism promotes tumor growth by increasing hepatocyte proliferation. In this study, we developed a mouse model of tumor promotion by chronic EtOH consumption in which EtOH feeding began 46 days post-injection of the chemical carcinogen diethylnitrosamine (DEN) and continued for 16 weeks. With a final EtOH concentration of 28% of total calories, we observed a significant increase in the total number of preneoplastic foci and liver tumors per mouse in the EtOH+DEN group compared to corresponding pair-fed (PF)+DEN and chow+DEN control groups. We also observed a 4-fold increase in hepatocyte proliferation (p<0.05) and increased cytoplasmic staining of active-β-catenin in non-tumor liver sections from EtOH+DEN mice compared to PF+DEN controls. In a rat model of alcohol-induced liver disease, we found increased hepatocyte proliferation (p<0.05); depletion of retinol and retinoic acid stores (p<0.05); increased expression of cytosolic and nuclear expression of β-catenin (p<0.05) and phosphorylated-glycogen synthase kinase 3 β (p-GSK3β, p<0.05; significant up-regulation in Wnt7a mRNA expression; and increased expression of several β-catenin targets, including, glutamine synthetase (GS), cyclin D1, Wnt1 inducible signaling pathway protein (WISP1), and matrix metalloproteinase-7 (MMP7), p<0.05. These data suggest that chronic EtOH consumption activates the Wnt/β-catenin signaling pathways to increase hepatocyte proliferation, thus promoting tumorigenesis following an initiating insult to the liver.

Background Microbubbles (MB) combined with ultrasound (US) have been shown to lyse clots without tissue plasminogen activator (tPA) both in vitro and in vivo. We evaluated sonothrombolysis with three types of MB using a rabbit embolic stroke model. Methods New Zealand White rabbits (n=74) received internal carotid angiographic embolization of single 3 day-old cylindrical clots (0.6×4.0-mm). Groups included: 1) control (n=11) embolized without treatment, 2) tPA (n=20), 3) tPA+US (n=10), 4) Perflutren Lipid MB+US (n=16), 5) albumin 3µm MB+US (n=8), and 6) tagged albumin 3µm MB+US (n=9). Treatment began 1 hour post-embolization. Ultrasound was pulsed-wave (1 MHz; 0.8 W/cm2) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Lipid MB dose was intravenous (0.16 mg/kg) over 30 minutes. Dosage of 3µm MB was 5×109 MB intravenously alone or tagged with eptifibatide and fibrin antibody over 30 minutes. Rabbits were euthanized at 24 hours. Infarct volume was determined using vital stains on brain sections. Hemorrhage was evaluated on H&E sections. Results Infarct volume percent was lower for rabbits treated with Lipid MB+US (1.0%±0.6%; P=0.013), 3µm MB+US (0.7%±0.9%; P=0.018), and tagged 3µm MB+US (0.8%±0.8%; P=0.019) compared with controls (3.5%±0.8%). The three MB types collectively had lower infarct volumes (P=0.0043) than controls. Infarct volume averaged 2.2%±0.6% and 1.7%±0.8% for rabbits treated with tPA alone and tPA+US, respectively (P=NS). Conclusions Sonothrombolysis without tPA using these MB is effective in decreasing infarct volumes. Study of human application and further MB technique development are justified.

The metastatic breast cancer cell line, 4T1, abundantly expresses the oligosaccharide sialylated Lewis x (sLe x ). SLe x oligosaccharide on tumor cells can be recognized by E-and P-selectin, contributing to tumor metastatic process. We observed that both selectins reacted with this cell line. However, contrary to the E-selectin reactivity, which was sLe x dependent, P-selectin reactivity with this cell line was sLe x -independent. The sLe x -Neg variant of the 4T1 cell line with markedly diminished expression of sLe x and lack of sLe a , provided a unique opportunity to characterize Pselectin ligands and their contribution to metastasis in the absence of overlapping selectin ligands and E-selectin binding. We observed that P-selectin binding was Ca 21-independent and sulfation-dependent. We found that P-selectin reacted primarily with cell surface chondroitin sulfate (CS) proteoglycans, which were abundantly and stably expressed on the surface of the 4T1 cell line. P-selectin binding to the 4T1 cells was inhibited by heparin and CS glycosaminoglycans (GAGs). Moreover, Heparin administration significantly inhibited experimental lung metastasis. In addition, the data suggest that surface CS GAG chains were involved in P-selectin mediated adhesion of the 4T1 cells to murine platelets and human umbilical vein endothelial cells. The data suggest that CS GAGs are also the major P-selectin-reactive ligands on the surface of human MDA-MET cells. The results warrant conducting clinical studies on the involvement of cell surface CS chains in breast cancer metastasis and evaluation of various CS types and their biosynthetic pathways as target for development of treatment strategies for antimetastatic therapy of this disease. ' 2006 Wiley-Liss, Inc.Key words: P-selectin; chondroitin sulfate; breast cancer; cell-cell adhesion Breast cancer metastasis represents a terrible milestone associated with a poor prognosis. The multistep process of metastasis includes release of malignant cells from the primary neoplasm, migration of cancer cells into the blood circulation, interaction with platelets and leukocytes in circulation, adhesion to the vascular endothelium in distant organs and growth of the disseminated cancer cells within the vessels or within the tissue following extravasation.1-3 Each step in this process requires different types of interactions between cancer cells and the host microenvironment.A widely accepted hypothesis is that cancer cells exploit the adhesion molecules used by hematopoietic cells to migrate into distant organs. 4 In particular, the selectin family of adhesion molecules whose ligands, sialyl Lewis x (sLe x ) and sialyl Lewis a (sLe a ), are expressed at elevated levels on various cancer cells of human and murine origin 5-9 and play a significant role in cancer metastasis. The oligosaccharides sLe x and sLe a are the prime ligands for P-and E-selectin and many published works underscore the relative importance of interactions between sLe x/a and these vascular receptors in the hematogenous spread ...

Abnormal distribution, modification and aggregation of transactivation response DNA-binding protein 43 (TDP-43) are the hallmarks of multiple neurodegenerative diseases, especially frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Transgenic mouse lines overexpressing wild-type or mutant TDP-43 exhibit ALS-like symptom, motor abnormalities and early paralysis followed by death. Reports on lifespan and phenotypic behaviour in Prp-TDP-43 (A315T) vary, and these animals are not fully characterized. Although it has been proposed that the approximate 20% loss of motor neurons at end stage is responsible for the severe weakness and death in TDP-43 mice, this degree of neurologic damage appears insufficient to cause death. Hence we studied these mice to further characterize and determine the reason for the death. Our characterization of TDP-43 transgenic mice showed that these mice develop ALS-like symptoms that later become compounded by gastrointestinal (GI) complications that resulted in death. This is the first report of a set of pathological evidence in the GI track that is strong indicator for the cause of death of Prp-hTDP-43 (A315T) transgenic mice.

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