S. Iacob

Role of microRNA-199a-5p and discoidin domain receptor 1 in human hepatocellular carcinoma invasion

BackgroundMicro-ribonucleic acid (miRNA)-199a-5p has been reported to be decreased in hepatocellular carcinoma (HCC) compared to normal tissue. Discoidin domain receptor-1 (DDR1) tyrosine kinase, involved in cell invasion-related signaling pathway, was predicted to be a potential target of miR-199a-5p by the use of miRNA target prediction algorithms. The aim of this study was to investigate the role of miR-199a-5p and DDR1 in HCC invasion.MethodsMature miR-199a-5p and DDR1 expression were evaluated in tumor and adjacent non-tumor liver tissues from 23 patients with HCC undergoing liver resection and five hepatoma cell lines by the use of real-time quantitative RT-PCR (qRT-PCR) analysis. The effect of aberrant miR-199a-5p expression on cell invasion was assessed in vitro using HepG2 and SNU-182 hepatoma cell lines. Luciferase reporter assay was employed to validate DDR1 as a putative miR-199a-5p target gene. Regulation of DDR1 expression by miR-199a-5p was assessed by the use qRT-PCR and western blotting analysis.ResultsA significant down-regulation of miR-199a-5p was observed in 65.2% of HCC tissues and in four of five cell lines. In contrast, DDR1 expression was significantly increased in 52.2% of HCC samples and in two of five cell lines. Increased DDR1 expression in HCC was associated with advanced tumor stage. DDR1 was shown to be a direct target of miR-199a-5p by luciferase reporter assay. Transfection of miR-199a-5p inhibited invasion of HepG2 but not SNU-182 hepatoma cells.ConclusionsDecreased expression of miR-199a-5p contributes to increased cell invasion by functional deregulation of DDR1 activity in HCC. However, the effect of miR-199a-5p on DDR1 varies among individuals and hepatoma cell lines. These findings may have significant translational relevance for development of new targeted therapies as well as prognostic prediction for patients with HCC.

MicroRNA-101 inhibits human hepatocellular carcinoma progression through EZH2 downregulation and increased cytostatic drug sensitivity

BACKGROUND & AIMS Oncogene polycomb group protein enhancer of zeste homolog 2 (EZH2) has been proposed to be a target gene of putative tumor suppressor microRNA-101 (miR-101). The aim of our study was to investigate the functional role of both miR-101 and EZH2 in human hepatocellular carcinoma (HCC). METHODS MiR-101 and EZH2 expressions were evaluated in tumor tissues of 99 HCC patients and 7 liver cancer cell lines by real-time PCR. Luciferase reporter assay was employed to validate whether EZH2 represents a target gene of miR-101. The effect of miR-101 on HCC growth as well as programmed cell death was studied in vitro and in vivo. RESULTS MiR-101 expression was significantly downregulated in most of HCC tissues and all cell lines, whereas EZH2 was significantly overexpressed in most of HCC tissues and all cell lines. There was a negative correlation between expression levels of miR-101 and EZH2. Luciferase assay results confirmed EZH2 as a direct target gene of miR-101, which negatively regulates EZH2 expression in HCC. Ectopic overexpression of miR-101 dramatically repressed proliferation, invasion, colony formation as well as cell cycle progression in vitro and suppressed tumorigenicity in vivo. Furthermore, miR-101 inhibited autophagy and synergized with either doxorubicin or fluorouracil to induce apoptosis in tumor cells. CONCLUSION Tumor suppressor miR-101 represses HCC progression through directly targeting EZH2 oncogene and sensitizes liver cancer cells to chemotherapeutic treatment. Our findings provide significant insights into molecular mechanisms of hepatocarcinogenesis and may have clinical relevance for the development of novel targeted therapies for HCC.

Efficacy, safety, and immunosuppressant adherence in stable liver transplant patients converted from a twice-daily tacrolimus-based regimen to once-daily tacrolimus extended-release formulation

The aim of this study was to determine the efficacy, safety, and immunosuppressant adherence in 125 stable liver transplant (LT) patients converted from twice‐daily tacrolimus (TAC BID) to once‐daily TAC (TAC OD). Tacrolimus trough levels, laboratory parameters, metabolic disorders, selected patient reported outcomes, and adverse events were assessed. Mean TAC trough level concentration was 6.1 ± 2.3 ng/ml at study entry, decreased to 5.5 ± 2.1 ng/ml (P = 0.016) and 5.5 ± 2.2 ng/ml (P = 0.019) after 1 and 2 weeks, respectively, and tended to equal the baseline value during further follow‐up. At week 1, TAC concentrations were lower in 62.4% of patients and higher in 36.0% when compared with baseline. Renal and cardiovascular risk factors remained stable and no rejection episodes occurred over 12 months. Adverse events were consistent with the safety profile known from previous studies with TAC BID. Nonadherence measured by the “Basel Assessment of Adherence Scale to Immunosuppressives” was evident in 66.4% at study entry and decreased to 30.9% postconversion (P < 0.0001). Prevalence of nonadherence at baseline was significantly higher in patients converted >2 years after LT and in those ≤60 years of age. Conversion to TAC OD is safe, enhances immunosuppressant adherence and should be accompanied by a close TAC level monitoring during the initial period.

Epidemiology of inflammatory bowel disease in adults who refer to gastroenterology care in Romania: a multicentre study.

Aim The Romanian Society of Gastrointestinal Endoscopy developed a multicentre, prospective study to evaluate the frequency and epidemiological features of inflammatory bowel disease (IBD) in an adult population that refers to gastroenterology centres in Romania. Material and methods Eighteen centres with adequate diagnostic and therapeutic facilities uniformly distributed across Romania reported through a proforma the new and old IBD cases between 1 June 2002 and 1 June 2003. The rates of incidence and prevalence of ulcerative colitis (UC) and Crohns disease (CD) have been reported per 100 000 inhabitants. Epidemiological features and disease characteristics were analysed. Results During the study, 407 cases of UC (163 incident cases) and 254 cases of CD (85 incident cases) were identified. The incidence in the referral population was 0.97/100 000 and 0.50/100 000 for UC and CD, respectively, whereas the prevalences was 2.42/100 000 and 1.51/100 000 for UC and CD, respectively. A slight male predominance, wider age distribution and predominant urban residence were the main demographic features of incident cases; smoking and appendectomy/appendicitis were infrequent in UC, while a higher proportion of CD patients were smokers. Lower rates of severe, extensive or complicated IBD were noted as compared with the literature. Conclusion Incidence and prevalence rates of IBD in Romania are low. Predominant male involvement and a wider distribution of incident cases were the main demographic features in our population. IBD in our patients had a more ‘benign’ course as compared with the literature, with lower rates of severe, extensive or complicated disease and a small proportion of patients who need surgery.

Assessment of allograft fibrosis by transient elastography and noninvasive biomarker scoring systems in liver transplant patients.

Background. This prospective, monocentric study was designed to assess the efficacy of transient elastography (TE), biochemical tests, and more complex scores in determining fibrosis stage in 157 patients transplanted for hepatitis C virus (HCV) infection or non–HCV-related liver diseases. Methods and Results. The optimal TE cutoff values for HCV patients and non-HCV patients were 4.7 and 5.0 kPa for F≥1, 7.1 and 7.3 kPa for F≥2, 10.9 kPa and 9.9 kPa for F≥3, and 17.3 and 12.6 kPa for F=4, respectively. The corresponding area under the receiver operating characteristic (AUROC) curves for F≥1, F≥2, F≥3, and F=4 were 0.95 and 0.86, 0.89 and 0.85, 0.97 and 0.88, and 0.99 and 0.97 for HCV and non-HCV patients, respectively. On the basis of the logistic regression equation, we created a model (FibroTransplant score) to identify advanced fibrosis (F≥3). The accuracy of this model was tested in a validation group (n=74). AUROCs for diagnosis of F≥3 in HCV patients and non-HCV patients of the training group were 0.89 and 0.83 (FibroTransplant score), 0.86 and 0.66 (Benlloch score), 0.81 and 0.71 (aspartate aminotransferase-to-platelet ratio index), 0.80 and 0.77 (Hepascore), 0.79 and 0.70 (FibroTest), 0.78 and 0.71 (FIB-4), 0.75 and 0.60 (Forns index), 0.73 and 0.69 (FibroIndex), and 0.70 and 0.59 (Lok score). Among the validation group, AUROCs of the FibroTransplant score for F≥3 were 0.90 and 0.91, respectively. Conclusions. TE and the FibroTransplant score can be reliably used for diagnosing advanced fibrosis in transplanted patients.

Frequency and predictive factors for overlap syndrome between autoimmune hepatitis and primary cholestatic liver disease.

Objectives To evaluate the frequency of cholestatic pattern in patients with autoimmune hepatitis (AIH) and to identify predictive factors associated with the development of the overlap syndrome. Methods Eighty-two consecutive patients diagnosed with AIH at the referral centre between January 1998 and June 2002 were included in the study. The new scoring system modified by the International Autoimmune Hepatitis Group was used to classify patients as definite/probable. Overlap syndrome was considered when the patient had clinical, serological and histological characteristics of two conditions: AIH and primary biliary cirrhosis (PBC) or AIH and primary sclerosing cholangitis (PSC). Results From the 82 AIH patients (76 female and six male), 84.1% presented definite AIH (> 15 points) and 15.9% probable AIH (10–15 points). The frequency of the overlap syndrome was 20%: 13% with PBC and 7% with PSC. In the univariate analysis the overlap syndrome was associated with male gender (P = 0.01), age < 35 years (P < 0.0001), histopathological aspect of cholestasis (P < 0.0001), suboptimal response to treatment (P < 0.0001) and probable AIH (P < 0.0001). Age < 35 years, probable AIH and the absence of anti-nuclear antibody (ANA) have been identified as independent indicators of the overlap diagnosis by the logistic regression analysis. Conclusion Patients with overlap syndrome between AIH and primary cholestatic liver disease are frequently diagnosed in clinical practice, representing 20% of AIH cases in our study. The independent predictive factors associated with the diagnosis of overlap syndrome are young age, ANA(−) profile, and probable diagnosis according with the scoring system for AIH.

Predictors of graft and patient survival in hepatitis C virus (HCV) recipients : Model to predict HCV cirrhosis after liver transplantation

Background. Hepatitis C virus (HCV) recurrence after liver transplantation (LT) is almost universal, but the natural history of recurrent HCV in the allograft is highly variable. Our study had two aims: 1) to assess the impact of different pre- and postLT factors on graft and patient survival in HCV transplant recipients and 2) to create a model which may predict the patients at risk for HCV-related graft cirrhosis at 5 years postLT. Methods. A total of 168 LTs were considered for this study. Univariate and multivariate Cox proportional hazards regression model was used, as well as logistic regression analysis to create a model of prediction of HCV cirrhosis within 5 years after LT. Results. Predictive factors for both decreased graft and patient survival included patients recently transplanted (2000–2004), induction without azathioprine, short-term therapy with mycophenolate mofetil and prednisone (≤6 months), presence of early cholestasis, histologically proven early recurrence of hepatitis C. Recipient human leukocyte antigen DR3 positivity, presence of early cholestasis, and donor age >50 years were identified as independent predictors of graft cirrhosis within 5 years. A predictive model was established in order to calculate at 6 months a risk score for graft HCV cirrhosis within 5 years postLT using a formula that included the identified independent predictors. The area under receiver operating characteristic curve was 0.83, indicating a good ability to predict medium-term HCV allograft cirrhosis. Conclusion. This model may be a useful tool for better identifying high-risk HCV patients who should be selected for early initiation of antiviral therapy.

Genetic, immunological and clinical risk factors for biliary strictures following liver transplantation

Biliary strictures after liver transplantation (LT) are a major cause of morbidity and reduced graft survival.

Donor-Specific Anti-HLA Antibodies and Endothelial C4d Deposition-Association With Chronic Liver Allograft Failure.

Background The significance of humoral immune response for allograft survival after liver transplantation (LT) is still a matter of debate. The aim of this cross-sectional study was to assess immunological and clinical factors associated with advanced fibrosis (F3-F4) and chronic graft failure in LT recipients. Methods Serum samples from 174 patients prospectively enrolled and followed up for 12 months were tested for anti-HLA antibodies and compared against donor HLA types. Immunohistochemical C4d staining was performed on formalin-fixed, paraffin-embedded liver tissue. Results Mean time period from LT to enrollment was 66.9 ± 51.9 months. Independent predictive factors for graft failure included donor-positive cytomegalovirus serostatus (P = 0.02), donor-specific antibodies (DSA) against HLA class II (P = 0.03), donor age (P = 0.01), hepatitis C virus allograft reinfection (P = 0.0008), and biliary complications (P = 0.003). HLA class II DSA and HLA class I antibody positivity, hepatitis C virus reinfection, and mycophenolate mofetil-free regimens were significant risk factors for advanced fibrosis after LT. There was a significant association between C4d deposition on allograft endothelial cells and presence of class II DSA (P < 0.0001). Patients with C4d deposits had a 4.3 times higher risk of graft failure than those with negative staining and a significantly lower median time to graft failure (94.6 months [range, 3.6-158.9 months] vs 176.4 months [range, 9.4-217.8 months], P < 0.0001). Conclusions Screening for HLA DSA might be useful for early identification of LT recipients at increased risk of graft failure who could benefit from closer surveillance and tailored immunosuppressive regimens.

Prevalence of hepatitis C in Romania: different from European rates?

(13%) would result. Actually, the majority of cases without dilatation of HA (using both Caselitz and Buscarini’s thresholds) had both color-spots and hypervascularization, thus further supporting our statement that intra-hepatic parameters have a better sensitivity and accuracy than extrahepatic ultrasonography parameters due to their ability to permit the diagnosis of even very small AVMs in their early stage of development. Evidently the presence of more severe vascular involvement determines an angiodynamic remodelling and gross abnormalities which can be detected on B-mode ultrasonographic study (mainly enlargement of HA in the extrahepatic tract and ‘double channel aspect’ in the portal spaces). In conclusion, in this first controlled, prospective study, we have demonstrated that the diagnosis of HAVMs in HHT can reliably be made by merely using intra-hepatic parameters and does not require evidence of extrahepatic abnormalities. The latter are useful to grade the haemodynamic impact of HAVMs and the possible effect on liver angioarchitecture and clinical significance. We disagree with Dr. Buscarini’s final comment in which she states that ‘‘Doppler US diagnosis of liver VMs in HHT requires a combination of extrahepatic and intra-hepatic findings, which can provide a diagnostic accuracy ranging between 95% and 99% for different observers” [7] because in the absence of a standard reference technique, data on sensitivity and accuracy cannot be considered reliable.