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Human aging is associated with a progressive loss of muscle mass and strength and a concomitant fat accumulation in form of inter-muscular adipose tissue, causing skeletal muscle function decline and immobilization. Fat accumulation can also occur as intra-muscular triglycerides (IMTG) deposition in lipid droplets, which are associated with perilipin proteins, such as Perilipin2 (Plin2). It is not known whether Plin2 expression changes with age and if this has consequences on muscle mass and strength. We studied the expression of Plin2 in the vastus lateralis (VL) muscle of both healthy subjects and patients affected by lower limb mobility limitation of different age. We found that Plin2 expression increases with age, this phenomenon being particularly evident in patients. Moreover, Plin2 expression is inversely correlated with quadriceps strength and VL thickness. To investigate the molecular mechanisms underpinning this phenomenon, we focused on IGF-1/p53 network/signalling pathway, involved in muscle physiology. We found that Plin2 expression strongly correlates with increased p53 activation and reduced IGF-1 expression. To confirm these observations made on humans, we studied mice overexpressing muscle-specific IGF-1, which are protected from sarcopenia. These mice resulted almost negative for the expression of Plin2 and p53 at two years of age. We conclude that fat deposition within skeletal muscle in form of Plin2-coated lipid droplets increases with age and is associated with decreased muscle strength and thickness, likely through an IGF-1- and p53-dependent mechanism. The data also suggest that excessive intramuscular fat accumulation could be the initial trigger for p53 activation and consequent loss of muscle mass and strength.

We present a multi-technique study on in vitro epithelial-mesenchymal transition (EMT) in human MCF-7 cells cultured on electrospun scaffolds of poly(l-lactic acid) (PLA), with random and aligned fiber orientations. Our aim is to investigate the morphological and genetic characteristics induced by extracellular matrix in tumor cells cultured in different 3D environments, and at different time points. Cell vitality was assessed with AlamarBlue at days 1, 3, 5 and 7. Scanning electron microscopy was performed at culture days 3 and 7. Immunohistochemistry (for E-cadherin, β-catenin, cytokeratins, nucleophosmin, tubulin, Ki-67 and vimentin), immunofluorescence (for F-actin) western blot (for E-cadherin, β-catenin and vimentin) and transmission electron microscopy were carried out at day 7. An EMT gene array followed by PCR analysis confirmed the regulation of selected genes. At day 7, scanning electron microscopy on aligned-PLA revealed spindle-shaped cells gathered in buds and ribbon-like structures, with a higher nucleolar/nuclear ratio and a loss in E-cadherin and β-catenin at immunohistochemistry and western blot. An up-regulation of SMAD2, TGF-β2, TFPI2 and SOX10 was found in aligned-PLA compared to random-PLA cultured cells. The topography of the extracellular matrix has a role in tumor EMT, and a more aggressive phenotype characterizes MCF-7 cells cultured on aligned-PLA scaffold., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: While the role of serum HCV RNA quantitation in hepatitis C virus recurrence after liver transplantation is well established, the meaning of HCV RNA tissue quantitation is largely unclear, and no correlations with recipient outcome have been investigated yet., Aims: To assess the predictive value, and a possible prognostic role, of tissue and serum HCV RNA in first post-transplant biopsies., Methods: We retrospectively reviewed the first post-transplant biopsies of 83 recipients. Tissue and serum HCV RNA was quantitated by RT-PCR, and compared with serum, clinical and histological data., Results: HCV RNA quantitation allowed us to categorise recipients into three different risk groups: (1) tissue HCV RNA ≤ 1.5 IU/ng with any serum HCV RNA; (2) tissue HCV RNA>1.5 IU/ng and serum HCV RNA < 40 × 10(6)copies/mL; (3) tissue HCV RNA>1.5 IU/ng and serum HCV RNA ≥ 40 × 10(6)copies/mL. Hepatitis C virus recurrence rates in the three groups were 68%, 91% and 100% (P=0.004); hepatitis C virus-related mortality was 0%, 14% and 45% respectively (P<0.001)., Conclusions: This preliminary study on serum and tissue HCV RNA quantitation allows recipient "stratification" in prognostic groups, which could be applicable in the future for timely antiviral treatment and/or immunosuppression modulation., (Copyright © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Primary liver sarcomatous carcinomas (PLSCs) are very aggressive tumors. They are characterized by a fast clinical course, and therefore need a prompt histological diagnosis. Here, we report two cases of PLSC. One arises in a non-cirrhotic liver and the other in cirrhosis, with differences in onset and histological features. Special emphasis is put on the expression of albumin and HCC markers, and their possible usefulness in the diagnosis. The English literature of the last 20 years was revised (92 cases). Immunohistochemistry was performed manually or automatically; in situ hybridization (ISH) technique for albumin mRNA detection was carried out. The sarcomatoid components in both cases were immunoreactive for K8/18, Glutamine Synthetase and EZH2, and negative for Glypican 3, SMA, caldesmon, desmin, DOG-1, CD34, CD31, CD117, CD56, and alpha-fetoprotein. The detection of albumin mRNA by ISH was negative in the sarcomatoid component in both cases. PLSC represents a diagnostic challenge for pathologists, especially in its "pure" form: neither albumin mRNA detection nor HCC markers are useful for the diagnosis: positivity for K8/18 and the negativity for the mesenchymal markers seem to represent the main tools for the histological diagnosis., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The organic anion transporter peptides (OATP) 1B1 and 1B3 are hepatocytic-specific transporters determinant for the uptake of the contrast media Gd-EOB-DTPA during magnetic resonance, but variably lost in hepatocellular carcinoma (HCC). Here, we studied a series of HCCs from livers that underwent liver transplantation (OLT) and correlated the expression of OATP 1B1/1B3 with HCC morphological features and the expression of the biliary-type keratins K7 and K19, the latter previously correlated with a worse prognosis after OLT. Seventy-five HCCs from 69 OLT patients were evaluated by histology and immunohistochemistry with monoclonal antibodies against OATP 1B1/1B3, K7, and K19. Histopathological and immunohistochemical features were therefore compared to recipient follow-up data. Thirty-four (45%) HCCs were completely OATP-, and 18 (24%) showed positivity for K7 and/or K19. We observed a significant inverse correlation between OATP and K7/19 expression (P < 0.001): all OATP+ cases were K7/19-, while all K7+ and/or K19+ cases were OATP-. Sixteen cases were negative for all antibodies. No correlation was found between histopathological features and immunohistochemistry. Twenty-five recipients experienced HCC recurrence, and ten died from neoplastic recurrence. Neither OATP nor keratin expressions were correlated with HCC recurrence, while OATP negativity significantly correlated with HCC-related death after recurrence (P = 0.036). In conclusion, HCCs show a progressive loss in OATP immunoreactivity that correlates with the gain of a biliary phenotype. Although further studies are required to define these findings better, our results support the idea that OATP could be used together with K7/19 to identify a phenotypical "spectrum" in HCC progression.

Despite the central role of proteasomes in relevant physiological pathways and pathological processes, this topic is unexpectedly largely unexplored in human liver. Here we present data on the presence of proteasome and immunoproteasome in human livers from normal adults, fetuses and patients affected by major hepatic diseases such as cirrhosis and chronic active hepatitis. Immunohistochemistry for constitutive (alpha4 and beta1) and inducible (LMP2 and LMP7) proteasome subunits, and for the PA28alphabeta regulator, was performed in liver samples from 38 normal subjects, 6 fetuses, 2 pediatric cases, and 19 pathological cases (10 chronic active hepatitis and 9 cirrhosis). The immunohistochemical data have been validated and quantified by Western blotting analysis. The most striking result we found was the concomitant presence in hepatocyte cytoplasm of all healthy subjects, including the pediatric cases, of constitutive proteasome and immunoproteasome subunits, as well as PA28alphabeta. At variance, immunoproteasome was not present in hepatocytes from fetuses, while a strong cytoplasmic and nuclear positivity for LMP2 and LMP7 was found in pathological samples, directly correlated to the histopathological grade of inflammation. At variance from other organs such as the brain, immunoproteasome is present in livers from normal adult and pediatric cases, in apparent absence of pathological processes, suggesting the presence of a peculiar regulation of the proteasome/immunoproteasome system, likely related to the physiological stimuli derived from the gut microbiota after birth. Other inflammatory stimuli contribute in inducing high levels of immunoproteasome in pathological conditions, where its role deserve further attention., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The assessment of hepatitis C virus (HCV) RNA in liver tissues is clinically relevant in cases where histology, liver function tests, and HCV serology are not sufficient for a definitive diagnosis of HCV-related hepatitis. We analyzed 215 formalin-fixed, paraffin-embedded liver needle biopsies from patients infected with HCV genotypes 1b and 2. HCV RNA extracted from paraffin sections were quantified by means of a TaqMan real-time reverse transcription-polymerase chain reaction method. The quantification of HCV RNA in liver tissue was correlated with the amount of HCV detected by immunohistochemistry (IHC) on paired frozen biopsies, the HCV RNA load in the serum, and the main serum tests of liver function and cholestasis. HCV RNA was detected by real-time reverse transcription-polymerase chain reaction in 169 liver biopsies (78.6%) with a mean value of 13.59+/-37.25 IU/ng. Tissue HCV RNA levels strongly correlated with the IHC results (P<0.001, Spearman test), HCV serum load (P<0.001), aspartate aminotransferase (P=0.001), gamma-glutamyl transpeptidase (P=0.012), and aspartate aminotransferase/alanine aminotransferase ratio (P=0.029). HCV RNA was amplified in up to 7-year-old archival tissue samples. Real-time HCV RNA quantification on archival liver tissue may be clinically relevant in case of "occult" HCV infection or for the diagnosis of patients with known HCV infection and hepatic dysfunction but seronegative for HCV RNA. The assessment of the levels of HCV RNA in the liver might also be important for monitoring the effectiveness of antiviral therapy and the progression of disease in patients with chronic HCV hepatitis.

We compared tissue hepatitis C virus (HCV) RNA polymerase chain reaction quantification and HCV immunohistochemistry (IHC) to histology in biopsy tissues in order to differentiate between acute rejection and HCV hepatitis recurrence early after orthotopic liver transplantation (OLT). We analyzed the first biopsy performed because of alteration of serum aminotransferases in 65 consecutive OLT patients with HCV genotype 1b. In the histological analysis, we quantified the portal tracts, Councilman bodies, Councilman body/portal tract (CP) ratio, steatosis, and Knodell and Ishak scores. The 52 patients (80%) with histological HCV recurrence [recurrence-positive (Rec+)] were separated from the 6 (9%) with acute rejection and the 7 (11%) with undetermined pathological features [recurrence-negative (Rec-)]. HCV RNA strongly correlated with HCV IHC, regardless of the histological diagnosis (P < 0.001). Both HCV RNA and HCV IHC were significantly associated with CP ratio (P = 0.041 and P = 0.008). No statistical correlation was found between HCV RNA, HCV IHC, and the other histopathologic features or the hepatitis scores. HCV RNA, HCV IHC, and CP ratio were the only variables able to discriminate between Rec+ and Rec- patients (Mann-Whitney test P < 0.001, P < 0.001, P = 0.014). In conclusion, a combined evaluation of histology, tissue HCV RNA, and HCV IHC significantly discriminated between OLT patients with or without HCV recurrence.