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Supplementary Tables S1 & S2 from Epidermal Growth Factor Receptor Signaling Is Up-regulated in Human Colonic Aberrant Crypt Foci

Aberrant crypt foci (ACF) are collections of abnormal colonic crypts with heterogeneous molecular and pathologic characteristics. Large and dysplastic ACF are putative precursors of colon cancer with neoplastic risk related to increased proliferation. In this study, we examined the role of epidermal growth factor receptor (EGFR) signaling in regulating ACF proliferation. Using magnification chromoendoscopy, we collected large ACF with endoscopic features of dysplasia and separately biopsied adjacent mucosa. Transcript levels were measured by real-time PCR, proteins were assessed by Western blotting, and levels were expressed as fold changes of adjacent mucosa. K-ras and B-Raf mutations were assessed by PCR and Ras activation by the ratio Ras-GTP / (Ras-GTP + Ras-GDP). At the RNA level, 38% of ACF were hyperproliferative, with proliferating cell nuclear antigen (PCNA) mRNA ≥2-fold of adjacent mucosa. Hyperproliferative ACF had significantly increased mRNA levels of EGFR (6.0 ± 1.7–fold), transforming growth factor-α (14.4 ± 5.0–fold), heparin-binding EGF-like growth factor (4.5 ± 1.4–fold), cyclin D1 (4.6 ± 0.7–fold), and cyclooxygenase-2 (COX-2; 9.3 ± 4.2–fold; P < 0.05). At the protein level, 46% of ACF were hyperproliferative (PCNA, 3.2 ± 1.2–fold). In hyperproliferative ACF, 44% possessed significant increases in four EGFR signaling components: EGFR (9.5 ± 1.3–fold), phosphoactive ErbB2 (2.6 ± 0.4–fold), phosphoactive extracellular signal-regulated kinase (3.7 ± 1.1–fold), and cyclin D1 (3.4 ± 0.8–fold; P < 0.05). Ras was activated in 46% of ACF (3.2 ± 0.4–fold; P < 0.05), but K-ras mutations were present in only 7% of ACF. In contrast to COX-2 mRNA, the protein was not increased in hyperproliferative ACF. In summary, we have shown that ACF with up-regulated PCNA possess increased EGFR signaling components that likely contribute to the enhanced proliferative state of dysplastic-appearing ACF. (Cancer Res 2006; 66(11): 5656-64)

The most frequent chromosomal structural loss in hepatocellular carcinoma (HCC) is of the short arm of chromosome 8 (8p). Genes on the remaining homologous chromosome, however, are not recurrently mutated, and the identity of key 8p tumor-suppressor genes (TSG) is unknown. In this work, analysis of minimal commonly deleted 8p segments to identify candidate TSG implicated GATA4, a master transcription factor driver of hepatocyte epithelial lineage fate. In a murine model, liver-conditional deletion of 1 Gata4 allele to model the haploinsufficiency seen in HCC produced enlarged livers with a gene expression profile of persistent precursor proliferation and failed hepatocyte epithelial differentiation. HCC mimicked this gene expression profile, even in cases that were morphologically classified as well differentiated. HCC with intact chromosome 8p also featured GATA4 loss of function via GATA4 germline mutations that abrogated GATA4 interactions with a coactivator, MED12, or by inactivating mutations directly in GATA4 coactivators, including ARID1A. GATA4 reintroduction into GATA4-haploinsufficient HCC cells or ARID1A reintroduction into ARID1A-mutant/GATA4-intact HCC cells activated hundreds of hepatocyte genes and quenched the proliferative precursor program. Thus, disruption of GATA4-mediated transactivation in HCC suppresses hepatocyte epithelial differentiation to sustain replicative precursor phenotype.

Background While non-alcoholic fatty liver disease (NAFLD) has been well characterised in patients with diabetes mellitus (DM), less is known about NAFLD in non-DM patients. We investigated the clinical characteristics of NAFLD patients with and without DM and accuracy of the NAFLD fibrosis score (NFS) in these two NAFLD groups. Methods Clinical, biochemical and histological variables were evaluated in this prospective cross-sectional study of 503 patients with biopsy proven NAFLD. Comparisons between patients with and without DM were analysed. NFS was correlated with liver histology to assess its robustness in patients with and without DM. Results There were 503 biopsy proven NAFLD patients with 48% of the cohort being diabetic. Relative to patients without DM, patients with DM were older (52 vs. 46 years, p, Highlights • Patients with diabetes have more severe NAFLD based on histology. • Severe NAFLD can occur in a considerable proportion of non-diabetic NAFLD patients. • The NAFLD fibrosis score may be less accurate in non-diabetics.

Background Roux-en-Y gastric bypass (RYGB) improves the pathophysiology that contributes to obesity-related nonalcoholic steatohepatitis (NASH). Whether obesity-related fibrosis improves is unclear. We hypothesized that RYGB reverses NASH and fibrosis, and indocyanine green (ICG) clearance provides a sensitive measure for detecting asymptomatic fatty liver disease. Methods One hundred six obese adults scheduled for RYGB had preoperative liver function assessed using standard tests and ICG clearance and core liver biopsies obtained during RYGB. Once patients lost 60% of their preoperative weight or weight loss plateaued, liver function was reassessed. Repeat liver biopsies were obtained on patients with NASH at the time of RYGB. Results RYGB improved steatosis, lobular inflammation, hepatocyte ballooning and fibrosis. Serum albumin, AST, and ALT decreased the most in patients with NASH and NASH plus fibrosis. Twenty seven (26%) patients had normal baseline liver histology and 45 (43%) had NASH or NASH plus fibrosis. Nine of 13 patients with substantial fatty liver had normalized histology after weight loss, while severity of disease in the rest had stabilized or was reduced. Mean ICG clearance in patients with normal/mild fatty liver disease and those with histological fatty livers did not differ significantly. Conclusions RYGB surgery reverses NASH and liver fibrosis. Underlying mechanisms that facilitate improvement remain unclear.

Oxidative stress is a core abnormality responsible for disease progression in nonalcoholic fatty liver disease (NAFLD). By employing a highly sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) approach we recently were able to define the circulating profile of bioactive lipid peroxidation products characteristic of patients with nonalcoholic steatohepatitis (NASH) and developed the OxNASH score for NASH diagnosis. The aims of this study were to assess the utility of OxNASH as a predictor of NASH and study the association between OxNASH and specific histologic features of NAFLD.Our cohort consisted of 122 patients undergoing liver biopsy for clinical suspicion of NAFLD. The NAFLD activity score (NAS) was calculated for each patient. Levels of fatty acid oxidation products were quantified using stable isotope dilution LC/MS/MS, and OxNASH was calculated.The mean age of our patients was 49.3 (±11.6) years, and the mean body mass index was 31.5 (±4.8) kg/m(2). The majority of patients were Caucasian (82 %) and 48 % were female. OxNASH correlated with NAS and with the individual histologic features of NAFLD, namely, steatosis, inflammation, and ballooning (P0.05), with the strongest association being with inflammation [rho (ρ) 0.40, 95 % confidence interval 0.23, 0.57, P0.001]. There was also a correlation between the stage of fibrosis and OxNASH (P = 0.001). These associations remained statistically significant after adjustment for multiple confounders.Based on our results, in adult patients with NAFLD, OxNASH correlates with histologic features of NASH and appears to be a promising noninvasive marker.

The growing demand for liver transplantation and the concomitant scarcity of cadaveric livers have increased the need for living donor liver transplantation (LDLT). Ensuring the safety of donors and recipients is critical. The preoperative identification of the vascular and biliary tract anatomy with 3-dimensional (3D) printing may allow better preoperative surgical planning, avert unnecessary surgery in patients with potentially unsuitable anatomy, and thereby decrease the complications of liver transplant surgery. We developed a protocol and successfully 3D-printed synthetic livers (along with their complex networks of vascular and biliary structures) replicating the native livers of 6 patients: 3 living donors and 3 respective recipients who underwent LDLT. To our knowledge, these are the first complete 3D-printed livers. Using standardized preoperative, intraoperative, and postoperative assessments, we demonstrated identical anatomical and geometrical landmarks in the 3D-printed models and native livers.

Context.—High-resolution scanning technology provides an opportunity for pathologists to make diagnoses directly from whole slide images (WSIs), but few studies have attempted to validate the diagnoses so obtained.Objective.—To compare WSI versus microscope slide diagnoses of previously interpreted cases after a 1-year delayed re-review (“wash-out”) period.Design.—An a priori power study estimated that 450 cases might be needed to demonstrate noninferiority, based on a null hypothesis: “The true difference in major discrepancies between WSI and microscope slide review is greater than 4%.” Slides of consecutive cases interpreted by 2 pathologists 1 year prior were retrieved from files, and alternate cases were scanned at original magnification of ×20. Each pathologist reviewed his or her cases using either a microscope or imaging application. Independent pathologists identified and classified discrepancies; an independent statistician calculated major and minor discrepancy rates for both WSI and microscope slide review of the previously interpreted cases.Results.—The 607 cases reviewed reflected the subspecialty interests of the 2 pathologists. Study limitations include the lack of cytopathology, hematopathology, or lymphoid cases; the case mix was not enriched with difficult cases; and both pathologists had interpreted several hundred WSI cases before the study to minimize the learning curve. The major and minor discrepancy rates for WSI were 1.65% and 2.31%, whereas rates for microscope slide reviews were 0.99% and 4.93%.Conclusions.—Based on our assumptions and study design, diagnostic review by WSI was not inferior to microscope slide review (P < .001).

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease (NAFLD) and is the fourth most common indication for liver transplantation. Risk factors for NAFLD can persist and even worsen after liver transplantation. However, the risk and significance of NAFLD recurrence remain unclear. Reported posttransplant NAFLD and NASH recurrence rates vary widely across studies. There is little information detailing the histological evolution of NAFLD recurrence, and the long-term natural history of NAFLD recurrence is unclear. In this review, we summarize the findings of studies on the prevalence of recurrent NAFLD and its risk factors in the posttransplant setting, and we explore reasons for the discrepant reported recurrence rates. On the basis of currently available data, the relatively low rates of advanced fibrosis and NAFLD-associated graft loss and the comparability of the survival rates for these patients and patients undergoing transplantation for other diseases suggest that although NAFLD or NASH can recur, the clinical significance of disease recurrence for graft or patient survival may be small.

Glypican-3 (GPC3) is an oncofetal protein that has been demonstrated to be a useful diagnostic immunomarker for hepatocellular carcinoma and hepatoblastoma. Its expression in mesenchymal tumors of the liver, particularly undifferentiated embryonal sarcoma (UES) and mesenchymal hamartoma (MH), has not been investigated. In this study, a total of 24 UESs and 18 MHs were immunohistochemically stained for GPC3 expression. The results showed cytoplasmic staining for GPC3 in 14 (58%) UESs, of which 6 exhibited diffuse immunoreactivity and the remaining 8 showed focal positivity. The patients with GPC3-positive UES tended to be younger (mean 18 years; median 11 years) than those with GPC3-negative tumors (mean 39.4 years; median 27 years), although the difference did not reach statistical significance (P = .06). Eight MHs also exhibited GPC3 immunoreactivity (44%; 4 diffuse and 4 focal). Positive staining in all 8 cases was primarily seen in entrapped nonlesional hepatocytes with a canalicular and cytoplasmic staining pattern. In only 4 cases (22%) was GPC3 immunoreactivity also observed in the mesenchymal component. The patients with positive staining also tended to be younger (mean 2.6 years; median 1.1 years) compared with those with negative staining (mean 16.3 years; median 4.5 years), but the difference was not statistically significant (P = .15). Our data demonstrate that GPC3 is expressed in a subset of UES and MH of the liver. Caution should thus be exercised when evaluating a GPC3-expressing hepatic neoplasm, particularly on a needle biopsy when the differential diagnosis includes poorly differentiated hepatocellular carcinoma or hepatoblastoma.

A possible association between nonalcoholic fatty liver disease (NAFLD) and hypothyroidism has been suggested. The recognized link between hypothyroidism and elements of the metabolic syndrome may explain this association. The purpose of this study was to determine the prevalence of hypothyroidism in a cohort of patients with NAFLD and analyze the potential factors associated with hypothyroidism in this patient population. Two hundred forty-six patients with biopsy-proven NAFLD attending hepatology clinics at the Cleveland Clinic between October 2006 and June 2009, and 430 age-, gender-, race- and BMI-matched control subjects seen in the general internal medicine clinic were included. Patients with a clinical diagnosis of hypothyroidism who were on thyroid replacement therapy were considered to be hypothyroid. Hypothyroidism was more frequent among patients with NAFLD (21% vs. 9.5%; P

Approximately 60 million adults in the United States have nonalcoholic fatty liver disease (NAFLD)1. The histologic spectrum of NAFLD spans from simple steatosis to nonalcoholic steatohepatitis (NASH), characterized by hepatocellular injury, inflammation, and fibrosis that can eventually progress to cirrhosis2,3.. Until recently, no therapy had been definitely proven beneficial for patients with NASH. A recent study showed some benefits of therapy with vitamin E in a proportion of patients with NASH4, however, there is still a need for additional and more effective therapies for patients with NASH. Multiple factors and pathways are involved in the pathogenesis and progression of NAFLD and NASH. Imbalances in inflammatory cytokines, oxidative stress, and insulin resistance are some of the proposed mechanisms involved in NASH pathogenesis and progression5–8. Cytokines including tumor necrosis factor alpha (TNF-alpha)9,10, a pro-inflammatory cytokine, and adiponectin, an anti-inflammatory cytokine9, are believed to play an important role in hepatocellular damage, inflammation and fibrogenesis in NASH11. In mice, fatty liver disease is improved by inhibition of hepatic TNF-alpha production12 and by infusion of anti-TNF-alpha neutralizing antibody13,14. Pentoxifylline (PTX) is a methylxanthine derivative known to increase red blood cell flexibility, reduce blood viscosity, and decrease platelet aggregation15,16. There is evidence supportive of a potential role for PTX in NASH. PTX inhibits a number of pro-inflammatory cytokines including TNF alpha17–19. In addition, PTX may have hepatoprotective effects17,20. It increases hepatic glutathione levels in mice with steatohepatitis induced by a methionine choline deficient diet17 and reduces the production of oxygen radicals induced by prolonged ischemia time in rat livers21. Potential antifibrogenic effects of PTX have been suggested by in vitro studies on hepatic stellate cells22,23, and in a rat model of biliary duct occlusion24. Therefore, PTX has a number of mechanisms that may provide therapeutic benefit to NASH patients. Pilot studies of PTX in patients with NASH have been conducted and their results have suggested possible benefits of PTX in NASH25,26. However, to date, published studies have had significant limitations, predominantly uncontrolled design, small sample sizes, and lack of histological follow up evaluation. The primary aim of this study was to compare the effectiveness of PTX over one year with placebo in patients with NASH. The primary outcome measure was defined as an improvement of 2 or more points in the NAFLD activity score (NAS)27. Other aims were to compare the effects of PTX compared to placebo on serum transaminases; to assess the effect of PTX on insulin sensitivity; to assess the effect of PTX on hepatocyte apoptosis; and to assess the effect of therapy with PTX on serum levels of TNF alpha and adiponectin. We also aimed to compare the rate of adverse events in patients with NASH receiving PTX compared with placebo.

The extrinsic death receptor-mediated pathway of apoptosis is involved in nonalcoholic steatohepatitis (NASH) development. Our aims were to create and validate a noninvasive prediction model for NASH diagnosis based on specific circulating markers of apoptosis.Our initial cohort consisted of 95 consecutive patients undergoing a liver biopsy for clinically suspected NASH. Blood was obtained from each patient at the time of liver biopsy. Plasma caspase 3 generated cytokeratin-18 fragments (CK-18), soluble Fas (sFas), and soluble Fas ligand (sFasL) were measured. Histology was assessed by an experienced hepatopathologist. The validation cohort consisted of 82 consecutive patients that underwent liver biopsy at the time of bariatric surgery.Patients with NASH had significantly higher levels of CK-18 and sFas than patients in the "not NASH" group [median (25th, 75th percentile): 508 (280, 846) U/L versus 176 (131, 224) U/L (p0.001), and 11.8 (7.8, 12.5) ng/ml versus 5.9 (4.8, 8.3) ng/ml (p0.001), respectively]. A significant positive correlation was revealed between the apoptosis markers and liver histopathology independent of other metabolic factors. A prediction model was generated including CK-18 fragments and sFas levels that showed an AUC of 0.93 and 0.79 in the initial and validation cohorts, respectively. A cutoff value using this model predicted NASH with a sensitivity and specificity of 88% and 89%, respectively.Quantification of circulating levels of two apoptotic markers accurately predicts the presence of NASH, supporting the potential usefulness of these markers in clinical practice for noninvasive diagnosis of NASH.

Although nonalcoholic fatty liver disease (NAFLD) is frequent in obesity, the metabolic determinants of advanced liver disease remain unclear. Adipokines reflect inflammation and insulin resistance associated with obesity and may identify advanced NAFLD. At the time of obesity surgery, 142 consecutive patients underwent liver biopsy and had their preoperative demographic and clinical data obtained. Liver histology was scored by the NAFLD activity score, and patients subdivided into four groups. Concentrations of retinol-binding protein 4 (RBP4), adiponectin, tumor necrosis factor-alpha (TNF-alpha), and leptin were determined approximately 1 week prior to surgery and results were related to liver histology. The prevalence of no NAFLD was 30%, simple steatosis 23%, borderline nonalcoholic steatohepatitis (NASH) 28%, and definitive NASH 18%. Type 2 diabetes mellitus (T2DM) and metabolic syndrome (MS) prevalence were 39 and 75%, respectively, and did not differ across the four histological groups (P = NS). Triglyceride (TG) and alanine transaminase (ALT) levels, strongly associated with advanced stages of NAFLD and NASH (P = 0.04). TG levels >150 mg/dl, increased the likelihood of NASH 3.4-fold, whereas high-density lipoprotein (HDL) levels predicted no NAFLD (P < 0.01). Concentrations of TNF-alpha, leptin, and RBP4 did not differ among histological groups and thus did not identify NASH; however, there was a trend for adiponectin to be lower in NASH vs. no NAFLD (P = 0.061). In summary, both TG and ALT levels assist in identification of NASH in an obesity surgery cohort. These findings underscore the importance of fatty acid delivery mechanisms to NASH development in severely obese individuals.

Nonalcoholic fatty liver disease is the most common form of chronic liver disease in both children and adults.1,2 It is estimated that nonalcoholic fatty liver disease (NAFLD) affects about 20% to 30% of adults and 10% of children in the United States.3,4 NAFLD represents a wide spectrum of conditions ranging from fatty liver to nonalcoholic steatohepatitis (NASH) to advanced fibrosis and cirrhosis.5 Steatosis without significant liver cell injury or fibrosis is the most common form of NAFLD in both adults and children.1,2 Studies in the adult population have variably suggested that steatosis is a benign nonprogressive condition, and NASH is recognized as a potentially serious condition with significant risk of morbidity and mortality.6-9 Liver biopsy remains the standard for establishing the diagnosis of NASH.10,11 The histological criteria for NASH in the adult population have evolved over the last two decades since the seminal publication of Ludwig et al.12 The principal histological features of adult NASH include the presence of macrovesicular steatosis (characterized by a single or a few large droplets of fat and displacement of the nucleus), ballooning degeneration of hepatocytes, and a mixed lobular inflammation with or without a degree of portal inflammation.10,11 Other features that are commonly present include zone 3 perisinusoidal–pericellular fibrosis; Mallory-Denk bodies (Mallory's hyaline); megamitochondria; acidophil bodies; and iron in hepatocytes, sinusoidal lining cells, or both. A growing body of evidence suggests that children with NASH frequently show histopathological features that differ from those of adults characterized mainly by lack of zone 3 accentuation and the predominance of portal-based inflammation and fibrosis.13 Schwimmer et al.14 identified this recently as type 2 NASH, with the aforementioned features in adults as type 1 NASH. The prevalence of this pattern in a wide range of pediatric cases as well as other other histopathological lesions and their relevance and prognostic significance in children with NAFLD remains to be determined. Thus, we conducted this study of biopsies and clinical information from five North American centers to document the histological features of pediatric NAFLD, determine the frequency and prognostic value of these features, and perform clinico-pathological correlations.

Hepatocellular carcinoma (HCC) is a highly vascular tumor. Angiogenesis in HCC is mediated at least in part by vascular endothelial growth factor (VEGF), which is expressed in HCC and surrounding cirrhotic tissue. VEGF mediates its angiogenic effects through multiple receptors including VEGF receptor 2 (VEGFr2, KDR/FLK-1), The distribution and clinical significance of VEGFr2 expression in HCC and cirrhotic liver in the setting of liver transplantation have not been tissue site specific evaluated. Immunohistochemical staining for VEGFr2 was performed in 78 liver explants from patients with HCC undergoing liver transplantation. VEGFr2 levels in HCC were significantly increased compared to adjacent, nontumorous cirrhotic liver areas (P < 0.05). VEGFr2 levels were significantly higher in the veins and sinusoids of poorly differentiated tumors (P < 0.05). VEGFr2 levels in the tumors were not significantly different between patients within and beyond Milan criteria. However, VEGFr2 levels were significantly higher in the arteries of non-tumorous liver in patients beyond Milan criteria (P < 0.05). No significant association was observed between VEGFr2 levels and the presence of tumor vascular invasion or recurrence post transplantation. These findings suggest that VEGFr2 up-regulation is a feature of poor differentiation and tumor progression. Further investigation is needed to assess the value of angiogenesis modulation in preventing tumor formation and/or progression in cirrhotic patients.

Although hyperinsulinemia and its associated metabolic syndrome (MS) have been implicated in the progression of hepatic fibrosis in hepatitis C virus (HCV) patients, little is known about the consequences of MS after orthotopic liver transplantation (OLT). The aim of this study was to assess the association between MS and fibrosis progression in patients with recurrent HCV after OLT. We identified all OLT/HCV patients (1998-2005) with at least 2 post-OLT liver biopsies. MS was defined with Adult Treatment Panel III criteria at 1 year post-OLT. The Ludwig-Batts scoring system was used to stage all biopsies (408 biopsies from 95 patients). The first biopsy that showed progression post-OLT was used for the time-to-progression analysis. Univariable and multivariable logistic regression analysis was performed to identify factors associated with fibrosis progression. MS was present in 50% of patients. Average follow-up to last available biopsy was 24 ± 17 months, during which 72% of subjects had fibrosis progression. The overall median rate of fibrosis progression was 0.08 units per month (Q25, Q75: 0.0, 0.17). By univariable analysis, high HCV RNA at 4 months post-OLT (P < 0.001), diabetes (P = 0.046), cytomegalovirus infection (P = 0.006), and MS (P = 0.049) were associated with progression of fibrosis. In multivariable analysis, MS was independently associated with progression of fibrosis beyond 1 year after OLT (odds ratio = 6.3, P = 0.017). A high viral load at 4 months post-OLT (odds ratio = 1.1, P = 0.004) and steroid therapy for acute rejection (odds ratio = 1.9, P = 0.05) were independently associated with fibrosis progression. In conclusion, MS, a potentially modifiable disease, is common and is strongly associated with long-term fibrosis progression in the setting of recurrent HCV after OLT. Liver Transpl 14:1287–1293, 2008. © 2008 AASLD.

The metabolic syndrome (MS) is a unique condition in which the underlying mechanism is related to insulin resistance. In hepatitis C virus (HCV) patients, insulin resistance has been linked to treatment failure. The aim of this study was to estimate the prevalence of MS in HCV patients undergoing antiviral therapy and to assess its predictive value in treatment outcome.All HCV treatment-naive patients who met the inclusion/exclusion criteria were studied (n = 228). MS was defined using the National Cholesterol Education Program Adult Treatment Panel III criteria. A logistic regression analysis was performed to study multivariable associations. The final model contained sex, ethnicity, body mass index, viral load, genotype, steatosis, fibrosis stage, and MS.MS was present in 59 of 228 (26%) patients. Genotype 1 (P = .002) and presence of steatosis (P.001) were found to be associated significantly with MS. Overall, sustained virologic response (SVR) was achieved in 108 of 228 (47%) patients. Male sex, non-Caucasian ethnicity, higher body mass index, high viral load, genotype 1, higher fibrosis stage, and MS were associated significantly with a lack of SVR. After adjusting for confounding variables, MS remained independently associated with a lack of SVR (P.01). Specifically, subjects with MS were 3.8 (95% confidence interval, 1.4-10.5) times more likely to fail treatment than those without MS.MS is seen frequently in patients with chronic HCV and is associated independently to lack of SVR. These findings support the concept that an aggressive intervention approach comprising lifestyle modification alone or in combination with drug treatment of the MS components may play an important role in improving antiviral responses in these patients.

Background:Nonalcoholic steatohepatitis (NASH) is common and severe in patients with diabetes mellitus. Although, there are no effective treatments for NASH in diabetic patients, preliminary reports suggest that polyunsaturated fatty acids (PUFA) may be beneficial in these patients.Aim:A prospective

Aberrant crypt foci (ACF) are collections of abnormal colonic crypts with heterogeneous molecular and pathologic characteristics. Large and dysplastic ACF are putative precursors of colon cancer with neoplastic risk related to increased proliferation. In this study, we examined the role of epidermal growth factor receptor (EGFR) signaling in regulating ACF proliferation. Using magnification chromoendoscopy, we collected large ACF with endoscopic features of dysplasia and separately biopsied adjacent mucosa. Transcript levels were measured by real-time PCR, proteins were assessed by Western blotting, and levels were expressed as fold changes of adjacent mucosa. K-ras and B-Raf mutations were assessed by PCR and Ras activation by the ratio Ras-GTP / (Ras-GTP + Ras-GDP). At the RNA level, 38% of ACF were hyperproliferative, with proliferating cell nuclear antigen (PCNA) mRNA ≥2-fold of adjacent mucosa. Hyperproliferative ACF had significantly increased mRNA levels of EGFR (6.0 ± 1.7–fold), transforming growth factor-α (14.4 ± 5.0–fold), heparin-binding EGF-like growth factor (4.5 ± 1.4–fold), cyclin D1 (4.6 ± 0.7–fold), and cyclooxygenase-2 (COX-2; 9.3 ± 4.2–fold; P < 0.05). At the protein level, 46% of ACF were hyperproliferative (PCNA, 3.2 ± 1.2–fold). In hyperproliferative ACF, 44% possessed significant increases in four EGFR signaling components: EGFR (9.5 ± 1.3–fold), phosphoactive ErbB2 (2.6 ± 0.4–fold), phosphoactive extracellular signal-regulated kinase (3.7 ± 1.1–fold), and cyclin D1 (3.4 ± 0.8–fold; P < 0.05). Ras was activated in 46% of ACF (3.2 ± 0.4–fold; P < 0.05), but K-ras mutations were present in only 7% of ACF. In contrast to COX-2 mRNA, the protein was not increased in hyperproliferative ACF. In summary, we have shown that ACF with up-regulated PCNA possess increased EGFR signaling components that likely contribute to the enhanced proliferative state of dysplastic-appearing ACF. (Cancer Res 2006; 66(11): 5656-64)

Objectives Duplicate laboratory tests that are unwarranted increase unnecessary phlebotomy, which contributes to iatrogenic anemia, decreased patient satisfaction, and increased health care costs. Materials and Methods We employed a clinical decision support tool (CDST) to block unnecessary duplicate test orders during the computerized physician order entry (CPOE) process. We assessed laboratory cost savings after 2 years and searched for untoward patient events associated with this intervention. Results This CDST blocked 11,790 unnecessary duplicate test orders in these 2 years, which resulted in a cost savings of $183,586. There were no untoward effects reported associated with this intervention. Conclusions The movement to CPOE affords real-time interaction between the laboratory and the physician through CDSTs that signal duplicate orders. These interactions save health care dollars and should also increase patient satisfaction and well-being.

Background & aims: Diabetes mellitus is a risk factor for advanced fibrosis in non-alcoholic fatty liver disease. However, not all non-alcoholic fatty liver disease patients with diabetes develop advanced fibrosis. We hypothesised that prescription medications used by these patients influence the development of advanced fibrosis. We investigated the association of commonly used medications and advanced fibrosis in non-alcoholic fatty liver disease patients with diabetes. Methods: Clinical information including demographics, medical history, medication history, biochemical and histologic variables were ascertained in 459 patients with biopsy proven non-alcoholic fatty liver disease. We compared characteristics of patients with and without diabetes and explored potential associations between classes of drugs as risk factors and advanced fibrosis among the diabetic patients with NAFLD. Results: Presence of diabetes was an independent risk factor for advanced fibrosis. In diabetic patients, age (OR 1.09; 95%CI 1.04-1.15, p=0.000) and grade of ballooning (OR 5.59; 95%CI 2.69-11.61, p=0.000) had a positive relationship with advanced fibrosis. The use of insulin (OR 4.95; 95%CI 1.65-14.88, p=0.004) and sulfonylurea (OR 5.07; 95%CI 1.87-13.75, p=0.001) were positively associated while statin use (OR 0.31; 95%CI 0.12-0.78, p=0.013) was negatively associated with advanced fibrosis. Conclusion: Among non-alcoholic fatty liver disease patients with diabetes, the prevalence of advanced fibrosis was higher in patients treated with insulin and sulfonylurea, but lower in patients on statins. These findings provide support for a greater role of statin use in non-alcoholic fatty liver disease patients with diabetes while limiting the use of insulin and sulfonylurea.

Complement 4d (C4d) is a marker of complement activation that has been used to evaluate humoral rejection in renal and heart allografts. Studies suggested a role for C4d detection in liver allografts in diagnosing acute cellular and humoral rejection but none correlated this with the pre-transplant liver disease. Our study analyzed the association of C4d deposition in liver allografts with the pre-transplant liver disease. C4d deposition was evaluated by indirect immunofluorescence and correlated with lymphocytotoxic crossmatch results, post-transplant clinicopathological diagnosis and type of pre-transplant liver disease. Allograft biopsies were classified by the native liver disease. After excluding 20 patients with rare liver diseases; C4d deposition was evaluated in 506 biopsies from 310 patients including 25 with PSC, 198 with viral hepatitis and 87 with other diseases. C4d immunereactivity distribution was not different among biopsies from patients with different lymphocytotoxic crossmatch results. Sinusoidal C4d deposition was noted in 11.9% of biopsies and 15.2% of patients (in one or more biopsies). 26% (9/35) of biopsies from patients with PSC had sinusoidal C4d deposition; more frequently than from patients with viral hepatitis 12% (43/348) (p=0.04) and other liver diseases 7% (8/123) (p=0.005). In conclusion, C4d deposition in liver allografts is independent of the crossmatch results. It occurs with a variety of pathologic abnormalities and underlying liver diseases; but is more frequent in patients with PSC. This suggests that mechanisms other than allo-immunity activate complement. The mechanisms and clinical significance of C4d deposition in liver allografts in patients with PSC remain to be determined.

Background: Autoimmune pancreatitis (AIP) is a recently characterized disease with specific clinical, radiographic, and histological features. These diagnostic features have been codified in the recently revised HISORt criteria. The aim of this study was to determine how the recognition and management of AIP has evolved at our center since the publication of the HISORt criteria in 2006. Methods: We conducted a historical cohort study consisting of patients with AIP based on the revised HISORt criteria seen at our tertiary care center since 1990. Cases were identified from pathology, laboratory, and pancreas clinic databases. The medical records were reviewed to ascertain demographic and clinical characteristics, radiologic and laboratory results, and patient outcomes. When available, prior images and pathology slides were retrospectively reviewed. The clinical outcomes of the patients were assessed following surgical or medical treatment, and compared based on the calendar year of presentation (before or after 2006). Results: Forty-seven cases were identified based on the revised HISORt criteria. Of these, 22 were evaluated before and 25 after January 1, 2006. In the early cohort, the diagnosis was frequently missed, including 15 patients that underwent surgical resections. None from the early cohort had a serum IgG4 drawn or mention of possible AIP in the imaging reports. When histology was obtained, the surgical pathologist did not perform IgG4 or Movat stain to allow a histological diagnosis of AIP. Several patients developed diabetes (n=3), calcific pancreatitis with exocrine insufficiency (n=3), proximal biliary strictures (n=7), and pancreatic cancer (n=1) during follow-up. In contrast, patients in the late cohort were less likely to undergo a surgical resection that the early cohort (36% vs. 68%, p=0.042). They were more likely to have a serum IgG4 drawn (80% vs. 0%) and to undergo a corticosteroid trial (44% vs. 0%, p=0.0003). 10/11 patients (92%) who underwent corticosteroid trials had resolution of their symptoms and improvement in structural abnormalities on imaging. Conclusion: A growing multidisciplinary awareness of AIP has led to improved diagnostic evaluation, prompter diagnosis, fewer surgical resections, and more frequent corticosteroid trials.

Gastrointestinal polyposis is a common clinical problem, yet there is no consensus on how to best manage patients with moderate-load polyposis. Identifying genetic features of this disorder could improve management and especially surveillance of these patients. We sought to determine the prevalence of hamartomatous polyposis-associated mutations in the susceptibility genes PTEN, BMPR1A, SMAD4, ENG, and STK11 in individuals with ≥5 gastrointestinal polyps, including at least 1 hamartomatous or hyperplastic/serrated polyp.We performed a prospective, referral-based study of 603 patients (median age: 51 years; range, 2-89 years) enrolled from June 2006 through January 2012. Genomic DNA was extracted from peripheral lymphocytes and analyzed for specific mutations and large rearrangements in PTEN, BMPR1A, SMAD4, and STK11, as well as mutations in ENG. Recursive partitioning analysis was used to determine cutoffs for continuous variables. The prevalence of mutations was compared using Fisher's exact test. Logistic regression analyses were used to determine univariate and multivariate risk factors.Of 603 patients, 119 (20%) had a personal history of colorectal cancer and most (n = 461 [76%]) had30 polyps. Seventy-seven patients (13%) were found to have polyposis-associated mutations, including 11 in ENG (1.8%), 13 in PTEN (2.2%), 13 in STK11 (2.2%), 20 in BMPR1A (3.3%), and 21 in SMAD4 (3.5%). Univariate clinical predictors for risk of having these mutations included age at presentation younger than 40 years (19% vs 10%; P = .008), a polyp burden of ≥30 (19% vs 11%; P = .014), and male sex (16% vs 10%; P = .03). Patients who had ≥1 ganglioneuroma (29% vs 2%; P.001) or presented with polyps of ≥3 histologic types (20% vs 2%; P = .003) were more likely to have germline mutations in PTEN.Age younger than 40 years, male sex, and specific polyp histologies are significantly associated with risk of germline mutations in hamartomatous-polyposis associated genes. These associations could guide clinical decision making and further investigations.

Colonic inertia is a frustrating motility disorder to patients, clinicians, and pathologists. The pathogenesis is largely unknown. The aims of this study were to: (1) characterize the expression of smoothelin, a novel smooth muscle-specific contractile protein expressed only by terminally differentiated smooth muscle cells, in the normal gastrointestinal (GI) tract; and (2) determine whether smoothelin is aberrantly expressed in patients with colonic inertia. A total of 57 resections of the normal GI tract (distal esophagus to left colon) were obtained from patients without GI motor dysfunction. Sixty-one colon resections were obtained from patients with a clinical diagnosis of colonic inertia. Smoothelin immunostaining was conducted on full-thickness tissue sections. In the nondysmotile controls, strong and diffuse cytoplasmic staining for smoothelin was observed in both the inner circular and outer longitudinal layers of the muscularis propria (MP) throughout the entire GI tract. The muscularis mucosae (MM) and muscular vessel walls were either completely negative or only patchily and weakly stained. The 1 exception to this pattern was observed in the distal esophagus, in which the MM was also diffusely and strongly stained. In cases with colonic inertia, a moderate to marked reduction of smoothelin immunoreactivity was observed in 15 of 61 (24.6%) colon resections, selectively seen in the outer layer of the MP. The data demonstrate that smoothelin is differentially expressed in the MP and MM of the normal GI tract and suggest that defective smoothelin expression may play a role in the pathogenesis of colonic inertia in a subset of patients. Copyright © 2012 by Lippincott Williams & Wilkins.

Lean methodologies have been applied in many industries to reduce waste. We applied Lean techniques to redesign laboratory workstations with the aim of reducing the number of times employees must leave their workstations to complete their tasks. At baseline in 68 workflows (aggregates or sequence of process steps) studied, 251 (38%) of 664 tasks required workers to walk away from their workstations. After analysis and redesign, only 59 (9%) of the 664 tasks required technologists to leave their workstations to complete these tasks. On average, 3.4 travel events were removed for each workstation. Time studies in a single laboratory section demonstrated that workers spend 8 to 70 seconds in travel each time they step away from the workstation. The redesigned workstations will allow employees to spend less time travelling around the laboratory. Additional benefits include employee training in waste identification, improved overall laboratory layout, and identification of other process improvement opportunities in our laboratory.

Pentoxifylline (PTX) improved the histological features of nonalcoholic steatohepatitis (NASH) in a recent randomized placebo-controlled trial. However, the underlying mechanism responsible for the beneficial effects of PTX in NASH remains unidentified. A key role of lipid oxidation in the pathogenesis and progression of NASH has been established. PTX is known to decrease free-radical-mediated oxidative stress and inhibit lipid oxidation. The primary aim of this study was to evaluate the effects of PTX on levels of lipid oxidation products in patients with NASH. Levels of multiple structurally specific oxidized fatty acids including hydroxy-octadecadienoic acids (HODEs), oxo-octadecadienoic acids (oxoODEs), and hydroxy-eicosatetraenoic acids (HETEs) were quantified by mass spectrometry in plasma obtained at baseline and at study completion in patients who completed 1 year of therapy with PTX or placebo in a randomized controlled trial. Therapy with PTX resulted in significant decreases in 9-HODE and 13-oxoODE, oxidized lipid products of linoleic acid (LA) linked to histological severity in nonalcoholic fatty liver disease. Similarly, PTX therapy was associated with significant decreases in 8-HETE, 9-HETE, and 11-HETE compared to placebo. Statistically significant correlations were demonstrated between the decrease in HODEs and oxoODEs and improved histological scores of fibrosis and between the decrease in HETEs and improved lobular inflammation.Therapy with PTX compared to placebo was associated with a significant reduction of oxidized fatty acids. This novel evidence supports that the beneficial effects of PTX in patients with NASH are likely partly mediated through decreasing lipid oxidation, largely free-radical-mediated lipid oxidation. Additionally, this is the first report on the link between decreased oxidized lipid products and improved histological disease in the setting of a therapeutic trial in NASH.

Background & Aims Esophageal adenocarcinoma (EA) is increasingly common among patients with Barrett's esophagus (BE). We aimed to provide consensus recommendations based on the medical literature that clinicians could use to manage patients with BE and low-grade dysplasia, high-grade dysplasia (HGD), or early-stage EA. Methods We performed an international, multidisciplinary, systematic, evidence-based review of different management strategies for patients with BE and dysplasia or early-stage EA. We used a Delphi process to develop consensus statements. The results of literature searches were screened using a unique, interactive, Web-based data-sifting platform; we used 11,904 papers to inform the choice of statements selected. An a priori threshold of 80% agreement was used to establish consensus for each statement. Results Eighty-one of the 91 statements achieved consensus despite generally low quality of evidence, including 8 clinical statements: (1) specimens from endoscopic resection are better than biopsies for staging lesions, (2) it is important to carefully map the size of the dysplastic areas, (3) patients that receive ablative or surgical therapy require endoscopic follow-up, (4) high-resolution endoscopy is necessary for accurate diagnosis, (5) endoscopic therapy for HGD is preferred to surveillance, (6) endoscopic therapy for HGD is preferred to surgery, (7) the combination of endoscopic resection and radiofrequency ablation is the most effective therapy, and (8) after endoscopic removal of lesions from patients with HGD, all areas of BE should be ablated. Conclusions We developed a data-sifting platform and used the Delphi process to create evidence-based consensus statements for the management of patients with BE and early-stage EA. This approach identified important clinical features of the diseases and areas for future studies.

Oxidative stress is a core abnormality responsible for disease progression in nonalcoholic fatty liver disease (NAFLD). However, the pathways that contribute to oxidative damage in vivo are poorly understood. Our aims were to define the circulating profile of lipid oxidation products in NAFLD patients, the source of these products, and assess whether their circulating levels reflect histological changes in the liver. The levels of multiple structurally specific oxidized fatty acids, including individual hydroxy-eicosatetraenoic acids (HETE), hydroxy-octadecadenoic acids (HODE), and oxo-octadecadenoic acids (oxoODE), were measured by mass spectrometry in plasma at time of liver biopsy in an initial cohort of 73 and a validation cohort of 49 consecutive patients. Of the markers monitored, 9- and 13-HODEs and 9- and 13-oxoODEs, products of free radical-mediated oxidation of linoleic acid (LA), were significantly elevated in patients with nonalcoholic steatohepatitis (NASH), compared with patients with steatosis. A strong correlation was revealed between these oxidation products and liver histopathology (inflammation, fibrosis, and steatosis). Further analyses of HODEs showed equivalent R and S chiral distribution. A risk score for NASH (oxNASH) was developed in the initial clinical cohort and shown to have high diagnostic accuracy for NASH versus steatosis in the independent validation cohort. Subjects with elevated oxNASH levels (top tertile) were 9.7-fold (P < 0.0001) more likely to have NASH than those with low levels (bottom tertile). Collectively, these findings support a key role for free radical-mediated linoleic acid oxidation in human NASH and define a risk score, oxNASH, for noninvasive detection of the presence of NASH.

Objective: Esophageal high-grade dysplasia/tumor in situ (HGD/Tis) management is in evolution. However, treatment decisions must be made on clinical staging,which may notreflect pathologic staging. Long-termrandomized trial information, large treatmentseries, and cancer registry data do not exist to guide treatment decisions. This evaluation of esophagectomy for clinically diagnosed HGD (cHGD) serves as a reference point for future therapies. Methods: From a 1296-patient prospective esophagectomy database, 134 patients were diagnosed with cHGD (HGD without detectable mass at biopsy) before esophagectomy (mean age 60 10 years, 120 [90%] male, and 132 [99%] Caucasian). Median follow-up was 7.1 years. Results: Histopathologic cell type was adenocarcinoma in 124 (93%) patients. Pathologic T (tumor) classification (pT) was 77 (57%) pHGD, 46 (34%) pT1a, eight (6%) pT1b, and one each (1%) indefinite for dysplasia, low-grade dysplasia, and pT2. Three (2%) had regional lymph node metastases (pT1N1M0). There was one hospital death (0.7%) and four deaths from recurrent cancer. Survival at 1 month, 6 months, and 5, 10, and 15 years was 99%, 97%, 96%, 94%, 82%, and 75%, respectively. Survival was at least that of a matched population. Older age and poor lung function predicted worse survival. Sixteen patients developed nonesophageal cancers, 6.1 times greater than expected. Conclusions: Despite clinical staging errors, survival following esophagectomy for cHGD is excellent. The diagnosis of cHGD does not alter survival referenced to the matched general population; however, cHGD patients appear to be at increased risk of second nonesophageal primarycancers. Therapy for cHGD should be patient specific, because patient and not cancer characteristics determine survival.

Gastroesophageal reflux disease (GERD) is one of the most common problems in clinical practice today. It is widely believed that functional and structural abnormalities of the gastroesophageal junction as well as an abnormal exposure to gastroduodenal contents are the main contributors to its pathogenesis. Novel findings of the inflammatory process in GERD suggest a far more complex process involving multifaceted inflammatory mechanisms. This review summarizes knowledge about the expression of inflammatory mediators in GERD and their potential cellular sources and provides an integrated concept of disease pathogenesis. In addition we evaluate the contribution of inflammatory mediators to well-known complications of GERD, namely motility abnormalities, fibrosis, and carcinogenesis. Novel findings regarding the pathophysiology of esophageal inflammation should enhance our understanding of GERD and its complications and provide new treatment insights.

The vast majority of clinical tissue samples are formalin-fixed and paraffin-preserved. This type of preservation has been considered an obstacle to protein extraction from these tissues. However, these are the very tissue samples that have associated patient histories, diagnoses and outcomes - ideal samples in the quest to translate bench research into clinical applications. Thus, until recently, these valuable specimens have been unavailable for proteomic analysis.Over the last decade, researchers have been exploring efficient methods to undo protein cross-linking caused by standard tissue fixatives and extract proteins from archived tissue specimens. These methods have been applied in different clinical proteomic studies. In this report, we attempt to review the development of these techniques, summarize the proteomic findings, and discuss the impact on future clinical proteomics.

The aims of this study were to determine whether combining features of a western lifestyle in mice with trans fats in a high-fat diet, high-fructose corn syrup in the water, and interventions designed to promote sedentary behavior would cause the hepatic histopathological and metabolic abnormalities that characterize nonalcoholic steatohepatitis (NASH). Male C57BL/6 mice fed ad libitum high-fat chow containing trans fats (partially hydrogenated vegetable oil) and relevant amounts of a high-fructose corn syrup (HFCS) equivalent for 1–16 wk were compared with mice fed standard chow or mice with trans fats or HFCS omitted. Cage racks were removed from western diet mice to promote sedentary behavior. By 16 wk, trans fat-fed mice became obese and developed severe hepatic steatosis with associated necroinflammatory changes. Plasma alanine aminotransferase levels increased, as did liver TNF-α and procollagen mRNA, indicating an inflammatory and profibrogenic response to injury. Glucose intolerance and impaired fasting glucose developed within 2 and 4 wk, respectively. Plasma insulin, resistin, and leptin levels increased in a profile similar to that seen in patients with NASH. The individual components of this diet contributed to the phenotype independently; isocaloric replacement of trans fats with lard established that trans fats played a major role in promoting hepatic steatosis and injury, whereas inclusion of HFCS promoted food consumption, obesity, and impaired insulin sensitivity. Combining risk factors for the metabolic syndrome by feeding mice trans fats and HFCS induced histological features of NASH in the context of a metabolic profile similar to patients with this disease. Because dietary trans fats promoted liver steatosis and injury, their role in the epidemic of NASH needs further evaluation.

Background/Aims Hepatic cavernous hemangioma is the second most common liver tumor after metastases. Vascular endothelial growth factor (VEGF) is recognized as an essential regulator of blood vessel growth . High VEGF expression leads to increased angiogenic activity in cavernous hemangioma endothelial cells. The use of specific antibodies directed against VEGF abolishes this vascular endothelial growth-promoting activity in vitro . Bevacizumab is a recombinant humanized monoclonal antibody directed against VEGF which is used for the treatment of metastatic colorectal cancer in combination with 5-fluorouracil-based regimens. Methods We report a patient with invasive colorectal adenocarcinoma and suspected liver metastasis on radiological examination, who showed a significant decrease in the size of his liver lesions after bevacizumab treatment. Histology of the liver lesions revealed hemangioma with a strong staining for VEGF and anti-VEGFr2 antibody in the hemangioma endothelial cells. To date, surgical resection provides the only consistently effective method for treatment of hepatic hemangioma. Conclusions This is the first documented case of hepatic hemangioma responsive to antiangiogenic therapy, suggesting a possible use for these agents in treating symptomatic patients without surgery. VEGF-signaling blockade including bevacizumab use poses a potential new treatment modality for vascular neoplasms in the liver and other sites.

Nonalcoholic fatty liver disease (NAFLD) is extremely common among morbidly obese patients. We assessed the usefulness of plasma caspase-generated cytokeratin 18 (CK-18) fragments as a novel marker for NAFLD in a bariatric cohort.The cohort consisted of 99 consecutive patients who underwent liver biopsy at the time of bariatric surgery. CK-18 levels were measured by using an enzyme-linked immunosorbent assay before and 6 months after surgery. Patients were subdivided into 4 histologic groups: not NAFLD (normal liver biopsy), nonalcoholic fatty liver (NAFL), borderline diagnosis, and definitive nonalcoholic steatohepatitis (NASH).CK-18 levels were significantly higher in subjects with NASH compared with those with not NAFLD, NAFL, or borderline diagnosis (median [25th quartile, 75th quartile], 389 U/L [275, 839] vs 196 U/L [158, 245], vs 217 U/L [154, 228], or vs 200 U/L [176, 274], respectively; P.0001). CK-18 levels were significantly higher in subjects with moderate to severe fibrosis versus those with no or mild fibrosis (334.5 U/L [240.5, 896] vs 207 U/L [175, 275], respectively; P = .007). A significant decrease in CK-18 levels was observed in most patients 6 months postoperatively. The area under the receiver operating characteristic curve for NASH diagnosis was estimated to be 0.88 (95% confidence interval, 0.77-0.99). The values with the best combination of sensitivity and specificity were 252 U/L (sensitivity, 82%; specificity, 77%) and 275 U/L (sensitivity, 77%; specificity, 100%).These results support the potential utility of this test for diagnosis and staging of NAFLD before bariatric surgery.

Backgrounds/aims De-novo autoimmune hepatitis (AIH) after liver transplantation (LT) is rising. Yet there is scarcity of data on the characteristics and the long-term outcomes of this condition. The aim of this study is to investigate the clinical characteristics and long-term outcomes of patients with de novo AIH following LT.Methods Using transplant liver biopsy database, we identified all patients with de-novo AIH following LT at our institution between 2008 and 2013. Patients with hepatitis C virus infection were excluded. The diagnosis of de-novo AIH was made according to the classical and simplified criteria defined by the International Autoimmune Hepatitis Group. Clinical information was gathered from electronic medical records. H&E stained sections and histochemical stains from the liver biopsies revealed findings compatible with AIH. Results A total of nineteen patients with de-novo AIH were identified (58% female, median age of 46 years), with mean international autoimmune hepatitis score of 12.1. Underlying liver disease were primary sclerosing cholangitis (n=4), primary biliary cirrhosis (n=3), biliary atresia (n=3), drug induced liver failure (n=2), alcoholic hepatitis (n=2) and others (n=5). The interval period from the LT to diagnosis of de-novo AIH was 19.6 months (1.6-197.8), during which 11 (58%) patients developed at least one episode of acute cellular rejection prior to the diagnosis of de-novo AIH. All patients were successfully treated with corticosteroids and incremental dose in immunosuppression. All showed complete biochemical response to treatment but 9 (47.4%) patients relapsed upon tapering down corticosteroids. Patients were followed over 6.7 year (1.5-17) years post-LT. Eight (42%) patients progressed to cirrhosis of whom 3 (15%) patients expired and 2 (10%) required second LT due to complications of end stage liver disease. Conclusion The present study shows the long term clinical outcomes of the patients with de-novo AIH post-LT. Although most patients exhibit a good initial response to medical therapy, de-novo AIH post-LT is likely to recur and progress to liver cirrhosis. Therefore, we should consider denovo AIH in patients who show abnormal liver tests or graft dysfunction after LT.

In patients with nonalcoholic fatty liver disease (NAFLD), a liver biopsy remains the only reliable way to differentiate simple steatosis from nonalcoholic steatohepatitis (NASH). Noninvasive methods are urgently needed. Increasing evidence suggests hepatocyte apoptosis is a key mediator of liver injury in NAFLD. The aim of this study was to quantify hepatocyte apoptosis in plasma from patients with NAFLD and correlate it with histological severity. Plasma was obtained from 44 consecutive patients with suspected NAFLD at the time of liver biopsy. Histology was assessed blindly. Caspase-3– generated cytokeratin-18 fragments were measured in situ via immunohistochemistry and in vivo using a novel enzyme-linked immunosorbent assay. Plasma cytokeratin-18 fragments were markedly increased in patients with NASH compared with patients with simple steatosis or normal biopsies (median [interquartile range]: 765.7 U/L [479.6-991.1], 202.4 U/L [160.4-258.2], 215.5 U/L [150.2-296.2], respectively; P < .001). Cytokeratin-18 fragment levels independently predicted NASH (OR 1.95; 95% CI 1.18-3.22; P .009 for every 50 U/L increase). A cutoff value of 395 U/L calculated using the receiver operating characteristic curve approach showed a specificity of 99.9%, a sensitivity of 85.7%, and positive and negative predictive values of 99.9% and 85.7%, respectively, for the diagnosis of NASH. In conclusion, these findings strongly suggest that determination of hepatocyte caspase activation in the blood is a strong and independent predictor of NASH in human subjects. These data highlight the potential usefulness of this test as a noninvasive diagnostic means of determining histological disease severity in patients with NAFLD. (HEPATOLOGY 2006;44:27-33.)

A number of studies have suggested B7-H1, a B7 family member, inhibits T cell responses. Therefore, its expression on nonlymphoid tissues has been proposed to prevent T cell-mediated tissue destruction. To test this hypothesis, we generated transgenic mice that expressed B7-H1 on pancreatic islet beta cells. Surprisingly, we observed accelerated rejection of transplanted allogeneic B7-H1-expressing islet beta cells. Furthermore, transgenic B7-H1 expression broke immune tolerance, as some of the mice spontaneously developed T cell-dependent autoimmune diabetes. In addition, B7-H1 expression increased CD8+ T cell proliferation and promoted autoimmunity induction in a T cell adoptive transfer model of diabetes. Consistent with these findings, B7-H1.Ig fusion protein augmented naive T cell priming both in vitro and in vivo. Our results demonstrate that B7-H1 can provide positive costimulation for naive T cells to promote allograft rejection and autoimmune disease pathogenesis.

Hepatocellular carcinoma is a common malignancy causing significant morbidity and mortality worldwide. In this study we use expression microarray technology to identify novel genes that consistently displayed altered expression levels in the earliest identifiable precursors to hepatocellular carcinoma, dysplastic and macroregenerative nodules. The gene expression profiles from nine patients with end-stage hepatitis C cirrhosis that contained a combined 11 dysplastic or macroregenerative nodules were compared to the patient's matched cirrhotic liver tissue. A total of 53 genes were consistently dysregulated in the patient liver specimens. Six of seven genes were validated by quantitative real-time reverse transcriptase-polymerase chain reaction, or by immunohistochemical studies performed on an independent set of lesions. The novel genes, including caveolin-1, semaphorin E, and FMS-like tyrosine kinase 3 ligand, have putative roles in carcinogenesis but have not been reported in hepatocellular carcinogenesis. Microarray expression analysis of dysplastic and macroregenerative liver nodules provide insight into the earliest changes in hepatocellular carcinogenesis.

Background: In patients with NASH, progression of liver fibrosis is the central determinant of poor liver related outcomes. Therefore, there is a critical need for treatment that improves or attenuates fibrosis in NASH. Recently, a RCT of pentoxifylline (PTX) in 55 patients with NASH showed greater decrease in fibrosis score among patients on PTX compared to placebo [Zein CO et al, Hepatology 2011]. In a smaller RCT of PTX in NASH hepatic expression of fibrosis genes was reduced by PTX [Van Wagner LB et al, Ann Hepatol 2011]. Liver biopsy is invasive, risky, and limited by discordance of fibrosis staging related to both sampling and inter-observer variability, thus accurate prediction of fibrosis using non-invasive markers is attractive. Thus far, studies of serum fibrosis markers in NASH have been few and limited to cross-sectional design. Aims: To compare levels of serum markers of fibrosis: TIMP-1, hyaluronic acid (HA) and amino-terminal propeptide of type 3 collagen (P3NP) in patients treated with PTX or placebo for one year. And, to correlate changes in these markers with the changes in fibrosis on liver biopsy before and after treatment. Methods: 80 patients treated with PTX (n=44) or placebo (n=36) who completed one year of therapy as part of two recently published RCTs were included. Biopsies of subjects in both trials were reviewed and scored by a single blinded pathologist. TIMP-1, HA, and P3NP were measured and a European Liver Fibrosis (ELF) panel score calculated using stored blood samples collected at entry and end of study. Changes from baseline were compared between treatment arms and correlated with histologic fibrosis stage. Results: After one year, patients treated with PTX had a greater decrease in all measured markers of fibrosis compared to those on placebo: TIMP-1 [mean change -14 (+/-41) vs +12.5 (+/-34), p=0.005]; HA [-15.7 (+/-50.7) vs +8.9 (+/-42.5), p=0.03]; P3NP [-1.3 (+/-2.3) vs +0.29 (+/-2), p=0.005]; and calculated ELF score [-0.18 (+/-0.4) vs +0.12 (+/-0.45), p=0.005]. In addition, subjects with improved or stable fibrosis on biopsy had significant decreases from baseline in levels of TIMP-1 (p=0.045), P3NP (p=0.033) and ELF calculation (p=0.041) compared to subjects with progression of fibrosis on histology. Conclusions: TIMP-1, HA, P3NP, and calculated ELF score decreased in patients with NASH treated with PTX compared to placebo. Furthermore, changes from baseline on TIMP-1, P3NP, and calculated ELF score correlated with non-progression or progression of fibrosis on follow up biopsy. These results not only further substantiate that PTX appears to have a favorable effect on fibrosis in patients with NASH, but also provide novel information on the performance of serum markers of fibrosis in relation to histological changes over time in the setting of a therapeutic intervention in NASH.