Targeting p16INK4a Promotes Lipofibroblasts and Alveolar Regeneration after Early-Life Injury.

Rationale: Promoting endogenous pulmonary regeneration is crucial after damage to restore normal lungs and prevent the onset of chronic adult lung diseases.Objectives: To investigate whether the cell-cycle inhibitor p16INK4a limits lung regeneration after newborn bronchopulmonary dysplasia (BPD), a condition characterized by the arrest of alveolar development, leading to adult sequelae.Methods: We exposed p16INK4a-/- and p16INK4aATTAC (apoptosis through targeted activation of caspase 8) transgenic mice to postnatal hyperoxia, followed by pneumonectomy of the p16INK4a-/- mice. We measured p16INK4a in blood mononuclear cells of preterm newborns, 7- to 15-year-old survivors of BPD, and the lungs of patients with BPD.Measurements and Main Results: p16INK4a concentrations increased in lung fibroblasts after hyperoxia-induced BPD in mice and persisted into adulthood. p16INK4a deficiency did not protect against hyperoxic lesions in newborn pups but promoted restoration of the lung architecture by adulthood. Curative clearance of p16INK4a-positive cells once hyperoxic lung lesions were established restored normal lungs by adulthood. p16INK4a deficiency increased neutral lipid synthesis and promoted lipofibroblast and alveolar type 2 (AT2) cell development within the stem-cell niche. Besides, lipofibroblasts support self-renewal of AT2 cells into alveolospheres. Induction with a PPARγ (peroxisome proliferator-activated receptor γ) agonist after hyperoxia also increased lipofibroblast and AT2 cell numbers and restored alveolar architecture in hyperoxia-exposed mice. After pneumonectomy, p16INK4a deficiency again led to an increase in lipofibroblast and AT2 cell numbers in the contralateral lung. Finally, we observed p16INK4a mRNA overexpression in the blood and lungs of preterm newborns, which persisted in the blood of older survivors of BPD.Conclusions: These data demonstrate the potential of targeting p16INK4a and promoting lipofibroblast development to stimulate alveolar regeneration from childhood to adulthood.

Small bowel in vivo bioengineering using an aortic matrix in a porcine model.

OBJECTIVE: To evaluate the feasibility of an in vivo small bowel bioengineering model using allogeneic aortic grafts in pigs. BACKGROUND: The best treatment for short bowel syndrome is still unclear. Intestinal transplantation, as well as lifelong parenteral nutrition is associated with a 5-year survival rate of less than 50 %. We have already used allogeneic arterial segments to replace the upper airway in sheep. The results were encouraging with an induced transformation of the aortic wall into tracheo-bronchial bronchial-type tissue. METHODS: Seven young mini-pigs were used. A 10-cm-diameter, allogeneic, aortic graft was interposed in an excluded small bowel segment and wrapped by the neighboring omentum. Animals were autopsied at 1 (n = 2), 3 (n = 3), and 6 months (n = 2), respectively. Specimens were examined macroscopically and microscopically. RESULTS: The overall survival rate of the animals was 71.4 %. No anastomotic leak occurred. Histologic analysis revealed intestinal-like wall transformation of the aortic graft in the surviving animals. CONCLUSION: Aortic-enteric anastomosis is feasible in a porcine model. Moreover, in vivo, bioengineered, intestinal-like transformation of the vascular wall was identified.

Liver resection for hepatocellular carcinoma in 313 Western patients: tumor biology and underlying liver rather than tumor size drive prognosis.

BACKGROUND & AIMS: Treatment decisions for hepatocellular carcinoma are mostly guided by tumor size. The aim of this study was to analyze resection outcomes according to tumor size and characterize prognostic factors. METHODS: Patients resected at a Western center between 1989 and 2010 were grouped by largest tumor size: <50mm, 50-100mm, and >100mm. The primary end points were overall- and recurrence-free survival. Univariate associations with primary endpoints were entered into a Cox proportional hazard regression model. RESULTS: Three hundred thirteen patients underwent resection: 111 (36%) had tumors <50mm, 113 (36%) had tumors between 50 and 100mm, and 89 (28%) had tumors >100mm. Five-year overall and disease-free survival rates for the three groups were 67%, 46%, and 34%, and 32%, 27%, and 27%, respectively. Thirty-five patients, mostly from <50mm group, underwent transplantation which was associated with a 91% 5 year survival rate. Tumor size was not an independent predictor of overall or recurrence-free survival on multivariate analyses. Independent predictors of decreased overall survival were: intraoperative transfusion (HR=2.60), cirrhosis (HR=2.42), poorly differentiated tumor (HR=2.04), satellite lesions (HR=1.69), alpha-fetoprotein >200 (HR=1.53), and microvascular invasion (HR=1.48). The use of salvage transplantation was an independent predictor of improved survival (HR=0.21). Recurrence-free survival was predicted by intraoperative transfusion (HR=2.15), poorly differentiated tumor (HR=1.87), microvascular invasion (HR=1.71) and cirrhosis (HR=1.69). CONCLUSION: By studying a large group of patients across a distribution of tumor sizes and background liver diseases, it is demonstrated that size alone is a limited prognostic factor. Tumor biology and condition of the underlying liver are better prognosticators and should be given closer attention. Although hampered by recurrence rates, resection is safe and offers good overall survival. In addition, it may allow for better selection for salvage transplantation after consideration of histopathological risk factors.

Histopathologic comparison of monopolar versus no-touch multipolar radiofrequency ablation to treat hepatocellular carcinoma within Milan criteria.

PURPOSE: To compare histopathologically the completeness of radiofrequency (RF) ablation to treat hepatocellular carcinoma (HCC) with monopolar or multipolar technique. MATERIALS AND METHODS: Thirty-five consecutive patients (mean age, 59 y) with cirrhosis and HCC (n = 59) within Milan criteria received RF ablation and subsequently underwent liver transplantation (LT) for tumor progression or liver failure. Data were extracted retrospectively from a prospective database. Thirty nodules were treated with a monopolar device with internally cooled (n = 17) or perfused (n = 13) electrodes, and 29 were treated with a multipolar technique with internally cooled electrodes based on the "no-touch" concept. This consisted of inserting two or three straight electrodes around the nodule to avoid intratumor puncture to the greatest extent possible. Effectiveness of the three devices was compared by histopathologic examination of explants. Fisher exact and χ(2) tests and multivariate logistic regression analysis were performed. RESULTS: Mean sizes of nodules ablated (25, 22, and 21.6 mm) and median times from ablation to LT (11, 7.5, and 8.4 months) for patients treated with the monopolar internally cooled electrode device (MoICD), monopolar perfused electrode device (MoPED), and multipolar internally cooled electrode device (MuICD), respectively, were similar (P = .8 and P = .9, respectively). Pathologic examination showed complete necrosis for eight of 17 and six of 13 nodules treated with the MoICD and MoPED, respectively, versus 26 of 29 treated with the MuICD (P = .0019). In multivariate analysis, RF technique remained the predictive factor for complete necrosis (P = .005). CONCLUSIONS: Ablation of small HCCs with multipolar RF ablation based on the no-touch concept improves the rate of complete necrosis during pathologic examination compared with monopolar techniques.

Intravoxel incoherent motion (IVIM) MR imaging of colorectal liver metastases: are we only looking at tumor necrosis?

PURPOSE: To determine if intra-voxel incoherent motion diffusion-weighted imaging (IVIM-DWI) parameters, including free molecular-based (D) and perfusion-related (D*, f) diffusion parameters, correlate with the degree of tumor necrosis and viable tumor in colo-rectal cancer (CRC) metastasis. MATERIALS AND METHODS: Fifteen patients referred for resection of liver metastases from CRC were retrospectively included in this Institutional Review Board approved study. An IVIM-DWI sequence was performed on a 1.5 Tesla MR imaging system, with 10 b factors (0, 10, 20, 30, 50, 80, 100, 200, 400 and 800 s/mm(2) ). Mean D, D*, f and apparent diffusion coefficient (ADC) values were determined in metastases with a longest diameter above 10 mm. Correlations between the diffusion parameters and the degree of liver tumor necrosis and viable tissue were determined (Spearman). RESULTS: Correlation between diffusion parameters and histopathological findings was performed in 35 hepatic metastases with a diameter of more than 10 mm (mean size of 17.9 mm; range, 1-68 mm). Both D (r = 0.36; P = 0.035) and ADC (r = 0.4; P = 0.02) correlated with the degree of tumor necrosis but not with viable tumor. CONCLUSION: ADC variation observed in CRC metastases following systemic chemotherapy reflects a specific increase in free-molecular diffusion (D), in itself correlated to the degree of metastasis necrosis.

Acute respiratory failure in patients with toxic epidermal necrolysis: clinical features and factors associated with mechanical ventilation.

OBJECTIVES: Stevens-Johnson syndrome and toxic epidermal necrolysis are severe adverse cutaneous drug reactions characterized by widespread skin and mucous membrane detachments, including bronchial mucosa, which may be associated with respiratory failure requiring mechanical ventilation. The presentation and outcome of patients requiring mechanical ventilation and the characteristics of bronchial epithelial lesions among ventilated patients are reported. Predictors of mechanical ventilation available on hospital admission were identified using univariate and multivariate logistic regressions. DESIGN: Retrospective cohort study. SETTING: Medical ICU and dermatology department of a tertiary care hospital, which hosts the French national referral center for toxic epidermal necrolysis. PATIENTS: Patients admitted for Stevens-Johnson syndrome/toxic epidermal necrolysis over a 14-year period were included. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of the 221 patients included in the study, 56 patients (25.3%) required mechanical ventilation. None of the patients received noninvasive ventilation. Patients requiring mechanical ventilation had a larger baseline detached body surface area, higher Logistic Organ Dysfunction score, and Simplified Acute Physiology Score II, and they presented more often with shock, pulmonary infiltrates, and renal dysfunction (p < 0.0001 for all comparisons). Among patients receiving mechanical ventilation, 57% of the patients died; those having bronchial epithelial lesions (22 of 56) required intubation earlier than others (1 [1-4] vs 4 [1-6] d after hospital admission; p = 0.027). Variables associated with mechanical ventilation in multivariate analysis included serum bicarbonates less than 20 mM (odds ratio, 4.9 [95% CI, 1.1-22.7]; p = 0.041), serum urea greater than 10 mM (odds ratio, 7.0 [95% CI, 2.2-22.8]; p < 0.001), a detached body surface area between 10% and 29% (odds ratio, 3.7 [95% CI, 1.0-13.8]; p = 0.048) or greater than or equal to 30% (odds ratio, 19.7 [95% CI, 4.4-87.4]; p < 0.0001), WBCs more than 12,000/mm3 (odds ratio, 11.6 [95% CI, 2.8-48.1]; p < 0.001), blood hemoglobin less than 8 g/dL (odds ratio, 8.1 [95% CI, 1.2-55.2]; p = 0.032), and more extensive pulmonary infiltrates (odds ratio, 9.7 [95% CI, 3.6-25.9]; p < 0.0001). CONCLUSIONS: Mechanical ventilation is required in one of four Stevens-Johnson syndrome/toxic epidermal necrolysis patients and is associated with a poor outcome. Prompt identification of Stevens-Johnson syndrome/toxic epidermal necrolysis patients at higher risk of intubation could help guide their early management, particularly for those having bronchial epithelial lesions.

Microbial dysbiosis and colon carcinogenesis: could colon cancer be considered a bacteria-related disease?

Colorectal cancer (CRC) is posing an increasingly important burden on the health care system, with western countries seeing a growing incidence of the disease. Except for germline DNA mutations which have been attributed to less than 5% of patients, little is known about the main causes of CRC. However, environment factors such as food, lifestyle and medication are now suspected to have a major influence on inducing cancers. Today, exhaustive quantitative and qualitative evaluation of all environmental factors is not possible. Various environment-induced diseases have been characterized based on colon microflora, also called microbiota, analyses. Growing data have shown specific changes in microflora (i.e. dysbiosis) in the stools of patients with colon cancer or those adherent to the colonic mucosa. Thus, it appears that microbiota may be considered a platform offering host and environment interactions for studying CRCs. The hypothesis that colon cancer might be a bacteria-related disease is suggested and perspectives are discussed.

Intratracheally administered titanium dioxide or carbon black nanoparticles do not aggravate elastase-induced pulmonary emphysema in rats.

BACKGROUND: Titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) have biological effects that could aggravate pulmonary emphysema. The aim of this study was to evaluate whether pulmonary administration of TiO2 or CB NPs in rats could induce and/or aggravate elastase-induced emphysema, and to investigate the underlying molecular mechanisms. METHODS: On day 1, Sprague-Dawley rats were intratracheally instilled with 25 U kg⁻¹ pancreatic porcine elastase or saline. On day 7, they received an intratracheal instillation of TiO2 or CB (at 100 and 500 µg) dispersed in bovine serum albumin or bovine serum albumin alone. Animals were sacrificed at days 8 or 21, and bronchoalveolar lavage (BAL) cellularity, histological analysis of inflammation and emphysema, and lung mRNA expression of heme oxygenase-1 (HO-1), interleukin-1ß (IL-1ß), macrophage inflammatory protein-2, monocyte chemotactic protein-1, and matrix metalloprotease (MMP)-1, and -12 were measured. In addition, pulmonary MMP-12 expression was also analyzed at the protein level by immunohistochemistry. RESULTS: TiO2 NPs per se did not modify the parameters investigated, but CB NPs increased perivascular/peribronchial infiltration, and macrophage MMP-12 expression, without inducing emphysema. Elastase administration increased BAL cellularity, histological inflammation, HO-1, IL-1ß and macrophage MMP-12 expression and induced emphysema. Exposure to TiO2 NPs did not modify pulmonary responses to elastase, but exposure to CB NPs aggravated elastase-induced histological inflammation without aggravating emphysema. CONCLUSIONS: TiO2 and CB NPs did not aggravate elastase-induced emphysema. However, CB NPs induced histological inflammation and MMP-12 mRNA and protein expression in macrophages.

In vivo cellular imaging pinpoints the role of reactive oxygen species in the early steps of adult hematopoietic reconstitution.

Few techniques are available to characterize in vivo the early cellular dynamics of long-term reconstitution of hematopoiesis after transplantation of hematopoietic stem cells (HSCs) after lethal irradiation. Using a fiber-optic imaging system, we track the early steps of in vivo recruitment and proliferation of Lin(-)Sca-1(+)c-Kit(+)CD34(-) (LSKCD34(-)) HSCs highly enriched in HSCs and transplanted into lethally irradiated mice. Recruitment of the transplanted LSKCD34(-) hematopoietic cells first occurs in the femoral head and is continuous during 24 hours. Quantification of the fluorescence emitted by the transplanted hematopoietic cells shows that proliferation of LSKCD34(-) hematopoietic cells in the femoral head was potent 3 days after transplantation. Using a development of this fiber-optic imaging system, we show that the transplanted LSKCD34(-) hematopoietic cells are associated with vascularized structures as early as 5 hours after transplantation. This early association is dependent on reactive oxygen species (ROS) partly through the regulation of vascular cell adhesion molecule-1 expression on endothelial cells and is followed by a ROS-dependent proliferation of LSKCD34(-) hematopoietic cells. This new in vivo imaging technique permits the observation of the early steps of hematopoietic reconstitution by HSCs in long bones and shows a new role of ROS in the recruitment of HSCs by bone marrow endothelial cells.

Liver resection for transplantable hepatocellular carcinoma: long-term survival and role of secondary liver transplantation.

BACKGROUND/PURPOSE: Liver transplantation (LT) is the best theoretical treatment of hepatocellular carcinoma (HCC) fulfilling the Milan criteria (TNM stages 1-2). However, LT is limited by organ availability and tumor progression on the waiting list. Liver resection (LR) may represent an alternative in these patients. The aim of this study is to report the results of LR in transplantable patients. PATIENTS: From 1990 to 2007, 274 patients underwent liver resection for HCC. Sixty-seven (24%) met the Milan criteria on pathologic study of the specimen. Ten were TNM stage 1 and 57 stage 2 and all had chronic liver disease. There were 56 men and 11 women with a mean age of 63. LR included 12 major hepatectomies, 14 bisegmentectomies, 14 segmentectomies, and 27 nonanatomic resections. Thirty-seven resections were performed through a laparoscopic approach and there were only 8 open resections since 1998. RESULTS: Three patients died postoperatively (4.5%), none after laparoscopic resection. Morbidity rate was 34%. After a mean follow-up of 4.8 years, 36 patients (54%) developed intrahepatic tumor recurrence. Twenty-eight (77%) were again transplantable of which 16 (44%) were transplanted. Two additional patients underwent pre-emptive LT (ie before recurrence). When considering 44 patients <65 years at the time of resection (ie upper age limit for LT), the rates of recurrence, transplantable recurrence, and intention to treat salvage transplantation (patients with transplantable recurrence actually transplanted) were 59%, 80%, and 61%, respectively. Overall and disease free 5-year survival rates were 72% and 44%, respectively. Survival was not influenced by TNM stage 1 or 2, AFP level, tumor differentiation, or the presence microscopic vascular invasion. Survival after salvage LT was 70% and 87% when calculated from the date of LT and LR, respectively. CONCLUSION: LR for small solitary HCC in compensated cirrhosis yields an overall survival rate comparable to upfront LT. Despite a significant recurrence rate, close imaging monitoring after resection allows salvage LT in 61% of patients with recurrence on intention to treat analysis.

Liver resection for hepatocellular carcinoma.

The indications and the results for liver resection for hepatocellular cancer (HCC) depend on the stage of the tumor at diagnosis, the functional reserve of the liver, and the use of suitably adapted surgical techniques. This article briefly discusses liver resection for HCC in patients who do not have chronic liver disease and then discusses liver resection for HCC in patients who have chronic liver disease.

Association of CYP1B1 germ line mutations with hepatocyte nuclear factor 1alpha-mutated hepatocellular adenoma.

Biallelic somatic mutations of TCF1 coding for hepatocyte nuclear factor 1alpha (HNF1alpha) are found in 50% of the hepatocellular adenoma (HCA) cases usually associated with oral contraception. In rare cases, HNF1alpha germ line mutations could also predispose to familial adenomatosis. In order to identify new genetic factors predisposing to HNF1alpha-mutated HCA, we searched for mutations in genes involved in the metabolism of estrogen. For 10 genes (CYP1A1, CYP1A2, CYP3A4, CYP3A5, COMT, UGT2B7, NQO1, GSTM1, GSTP1, and GSTT1), we did not find mutations nor differences in the allele distribution among 32 women presenting HNF1alpha-mutated adenomas compared with 58 controls. In contrast, we identified a CYP1B1 germ line heterozygous mutation in 4 of 32 women presenting HNF1alpha-mutated adenomas compared with none in 58 controls. We confirmed these results with the identification of four additional CYP1B1 mutations in a second series of 26 cases. No mutations were found in the control group, which was extended to 98 individuals, and only a known rare genetic variant was observed in two controls (P = 0.0003). We did an ethoxyresorufin O-deethylase assay to evaluate the functional consequence of the CYP1B1 mutations. We found reduced enzymatic activity in each CYP1B1 variant. In addition, an E229K CYP1B1 mutation was found in a woman with a germ line HNF1alpha mutation in a familial adenomatosis context. In this large family, all three patients with adenomatosis bore both HNF1 and CYP1B1 germ line mutations. In conclusion, our data suggested that CYP1B1 germ line-inactivating mutations might increase the incidence of HCA in women with HNF1alpha mutations.

Fibrolamellar hepatocellular carcinoma: a case report with cytological features in a sixteen-year-old girl.

We report a case of a 16-yr-old girl with a liver tumor revealed by thrombophlebitis of the left leg. On physical examination the patient was found to have painless hepatomegaly. Ultrasound and CAT scan showed a large tumor of the left portion of the liver, measuring 14 cm in diameter. Cytological preparations were touch imprints of the biopsy fragments obtained under ultrasound guidance. Cytological examination using May-Grünwald Giemsa stain revealed highly cellular smears containing large tumor cells with a round nucleus, prominent nucleoli, and abundant granular basophilic cytoplasm. Cytological features were those of fibrolamellar hepatocellular carcinoma, confirmed by histological examination of the biopsy sample as well as the surgical specimen obtained after wide excision of the lesion following ineffective neoadjuvant chemotherapy.

Laparoscopic liver resection for peripheral hepatocellular carcinoma in patients with chronic liver disease: midterm results and perspectives.

OBJECTIVE: Report the midterm results of laparoscopic resection for hepatocellular in chronic liver disease (CLD). SUMMARY BACKGROUND DATA: Surgical resection for hepatocellular carcinoma (HCC) in chronic liver disease (CLD) remains controversial because of high morbidity and recurrence rates. Laparoscopic resection of liver tumors has recently been developed and could reduce morbidity. METHODS: From 1998 to 2003, patients with HCC and CLD were considered for laparoscopic liver resection. Inclusion criteria were chronic hepatitis or Child's A cirrhosis, solitary tumor < or =5 cm in size, and location in peripheral segments of the liver. Mortality, morbidity, recurrence rates, and survival were analyzed. RESULTS: A total of 27 patients were included. Liver resections included anatomic resection in 17 cases and non anatomic resection in 10. Seven conversions to laparotomy (26%) occurred for moderate hemorrhage in 5 cases and technical difficulties in 2 cases. Mortality and morbidity rates were 0% and 33%, respectively. Postoperative ascites and encephalopathy occurred in 2 patients (7%) who both had undergone conversion to laparotomy. Mean surgical margin was 11 mm (range, 1-47 mm). After a mean follow-up of 2 years (range, 1.1-4.7), 8 patients (30%) developed intrahepatic tumor recurrence of which one died. Treatment of recurrence was possible in 4 patients (50%), including orthotopic liver transplantation, right hepatectomy, radiofrequency ablation, and chemoembolization in 1 case each. There were no adhesions in the 2 reoperated patients. Overall and disease-free 3-year survival rates were 93% and 64%, respectively. CONCLUSION: Our study shows that laparoscopic liver resection for HCC in selected patients is a safe procedure with very good midterm results. This approach could have an impact on the therapeutic strategy of HCC complicating CLD as a treatment with curative intent or as a bridge to liver transplantation.

Antifibrogenic role of the cannabinoid receptor CB2 in the liver.

BACKGROUND & AIMS: Hepatic myofibroblasts are central for the development of liver fibrosis associated with chronic liver diseases, and blocking their accumulation may prevent fibrogenesis. Cannabinoids are the active components of marijuana and act via 2 G-protein-coupled receptors, CB1 and CB2. Here, we investigated whether liver fibrogenic cells are a target of cannabinoids. METHODS: CB2 receptors were characterized in biopsy specimens of normal human liver and active cirrhosis by immunohistochemistry, and in cultures of hepatic stellate cells and hepatic myofibroblasts by reverse-transcription polymerase chain reaction (RT-PCR), immunocytochemistry, and GTPgammaS assays. Functional studies were performed in cultured hepatic myofibroblasts and activated hepatic stellate cells. Carbon tetrachloride-induced liver fibrosis was studied in mice invalidated for CB2 receptors. RESULTS: In liver biopsy specimens from patients with active cirrhosis of various etiologies, CB2 receptors were expressed in nonparenchymal cells located within and at the edge of fibrous septa in smooth muscle alpha-actin-positive cells. In contrast, CB2 receptors were not detected in normal human liver. CB2 receptors were also detected in cultured hepatic myofibroblasts and in activated hepatic stellate cells. Their activation triggered potent antifibrogenic effects, namely, growth inhibition and apoptosis. Growth inhibition involved cyclooxygenase-2, and apoptosis resulted from oxidative stress. Finally, mice invalidated for CB2 receptors developed enhanced liver fibrosis following chronic carbon tetrachloride treatment as compared with wild-type mice. CONCLUSIONS: These data constitute the first demonstration that CB2 receptors are highly up-regulated in the cirrhotic liver, predominantly in hepatic fibrogenic cells. Moreover, this study also highlights the antifibrogenic role of CB2 receptors during chronic liver injury.

Low-grade steatosis and major changes in portal flow as new prognostic factors in steroid-treated alcoholic hepatitis.

The aim of this study was to assess the prevalence and prognostic value of major alterations of portal flow in patients with steroid-treated alcoholic hepatitis. Fifty patients with severe, histologically proven alcoholic hepatitis were enrolled. Clinical data, liver test results, and hepatic Doppler ultrasound findings were collected at inclusion and at month 2. Patients were followed for 1 year or until death. Major changes in portal flow were defined as reversed or alternating flow in the portal trunk and/or in intrahepatic portal branches. Changes in portal flow were observed in 24 (48.0%) of 50 and 17 (39.5%) of 43 patients at inclusion and month 2, respectively. Univariate analysis showed that age older than 50 years, steatosis less than 20% on initial liver biopsy, presence of major changes in portal flow, Child-Turcotte-Pugh score higher than 12, factor V level higher than 45%, and hepatofugal splenic blood flow were associated with a lower 1-year survival. Cox regression analysis showed that steatosis < 20% (relative hazard [RH] = 9.3, P = .0009) and major changes in portal flow (RH = 3.1, P = .04), were independently associated with poor survival. In conclusion, major changes in portal flow are frequent in patients with severe alcoholic hepatitis. Altered portal flow and steatosis < 20% are new prognostic factors in steroid-treated alcoholic hepatitis and must be taken into account in patient management.

Activated protein C resistance acquired through liver transplantation and associated with recurrent venous thrombosis.

We report a new case of recurrent, extra-hepatic, deep vein thrombosis occurring after orthotopic liver transplantation for hepatocellular carcinoma complicating 'mixed' alcoholic and post-hepatitic C cirrhosis. Coagulation tests showed activated protein C resistance. The patient's genomic DNA was negative for the factor V Leiden mutation. Analysis of the grafted liver DNA showed that the donor was a heterozygous carrier of the factor V Leiden mutation and that the recipient's activated protein C resistance was acquired through the transplantation. Screening of candidate liver donors for a prothrombotic tendency is controversial. However, this case suggests that patients who develop venous thrombosis after liver transplantation should be screened for thrombophilic abnormalities, bearing in mind that genetic abnormalities which do not affect clotting test results, such as the G20210A mutation in the factor II gene, can only be diagnosed by testing the donor or graft.

Altered expression of E-cadherin in hepatocellular carcinoma: correlations with genetic alterations, beta-catenin expression, and clinical features.

E-cadherin is a key cell adhesion protein implicated as a tumor/invasion suppressor in human carcinomas and a binding partner of beta-catenin, which plays a critical role in Wnt signaling and in tumorigenesis. Here we report genetic and expression studies of E-cadherin and beta-catenin in hepatocellular carcinoma (HCC). Immunohistochemical analysis of E-cadherin expression in 37 HCCs and adjacent nontumor tissues revealed important variations among tumor samples, ranging from complete or heterogeneous down-regulation in 35% of cases to marked overexpression in 40% of tumors. Loss of E-cadherin expression was closely associated with loss of heterozygosity (LOH) at the E-cadherin locus and methylation of CpG islands in the promoter region (P <.002), predominantly in hepatitis B virus (HBV)-related tumors (P <.005). No mutation of the E-cadherin gene could be detected in the tumors examined, suggesting the requirement for reversible mechanisms of E-cadherin down-regulation. In most HCCs, including E-cadherin-positive and -negative cases, beta-catenin was strongly expressed at the cell membrane and nuclear accumulation of the protein was correlated with the presence of mutations in the beta-catenin gene itself, but not with E-cadherin loss. At difference with a number of epithelial cancers, vascular invasion was frequently noted in HCCs showing enforced expression of the membranous E-cadherin/beta-catenin complex. In conclusion, these data support the notion that E-cadherin might play diverse and seemingly paradoxic roles in HCC, reflecting specific requirements for tumor growth and spread in the liver environment.

Transcriptional activation of interleukin-8 by beta-catenin-Tcf4.

Nuclear translocation of beta-catenin and its association with Tcf/Lef factors are key steps in transduction of the Wnt signal, which is aberrantly activated in a variety of human cancers. In a search for new beta-catenin-Tcf target genes, we analyzed beta-catenin-induced alterations of gene expression in primary human hepatocytes, after transduction of either dominant stable beta-catenin or its truncated, transactivation-deficient counterpart by means of a lentiviral vector. cDNA microarray analysis revealed a limited set of up-regulated genes, including known Wnt targets such as matrilysin and keratin-1. In this screen, we identified the CXC chemokine interleukin 8 (IL-8) as a direct target of beta-catenin-Tcf4. IL-8 is constitutively expressed in various cancers, and it has been implicated in tumor progression through its mitogenic, motogenic, and angiogenic activities. The IL-8 promoter contains a unique consensus Tcf/Lef site that is critical for IL-8 activation by beta-catenin. We show here that the p300 coactivator was required for efficient transactivation of beta-catenin on this promoter. Ectopic expression of beta-catenin in hepatoma cells promoted IL-8 secretion, which stimulated endothelial cell migration. These data define IL-8 as a Wnt target and suggest that IL-8 induction by beta-catenin might be implicated in developmental and tumorigenic processes.