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Vaccines and first-generation antiviral therapeutics have provided important protection against COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there remains a need for additional therapeutic options that provide enhanced efficacy and protection against potential viral resistance. The SARS-CoV-2 papain-like protease (PL pro ) is one of the two essential cysteine proteases involved in viral replication. While inhibitors of the SARS-CoV-2 main protease have demonstrated clinical efficacy, known PL pro inhibitors have, to date, lacked the inhibitory potency and requisite pharmacokinetics to demonstrate that targeting PL pro translates to in vivo efficacy in a preclinical setting. Here, we report the machine learning-driven discovery of potent, selective, and orally available SARS-CoV-2 PL pro inhibitors, with lead compound PF-07957472 ( 4 ) providing robust efficacy in a mouse-adapted model of COVID-19 infection.

Background: Neoangiogenesis plays a crucial role in the progression of hepatocellular carcinoma (HCC), and concerns have been raised about the role of neoangiogenesis on the effectiveness of transarterial chemoembolization (TACE)., Aim: In this study, we aimed to evaluate Vascular Endothelial Growth Factor (VEGF) and Hypoxia-Inducible Factor-1α (HIF-1α) as circulating prognostic biomarkers in HCC patients treated with TACE., Methods: Blood samples were collected from 163 patients before (t 0 ) and four weeks after TACE (t 1 )., Results: Higher levels of VEGF after TACE were demonstrated (264.0 [78.7-450.8] vs. 278.6 [95.0-576.6] pg/mL; p < 0.0001). Responders to TACE had lower levels of VEGF than non-responders both at t 0 (200.0 [58.9-415.8] vs. 406.6 [181.4-558.6] pg/mL; p = 0.006) and at t 1 (257.3 [68.5-528.6] vs. 425.9 [245.2-808.3] pg/mL; p = 0.003), and in both groups there was an increase in VEGF compared to measurements before treatment (p = 0.001 and p = 0.005, respectively). VEGF was not associated with overall survival (OS), while patients with HIF-1α ≤ 0.49 ng/mL showed better prognosis (median OS 28.0 months [95% CI 19.7-36.3] vs. 17.0 months [95% CI 11.1-22.9]; p = 0.01). Moreover, HIF-1α was identified as an independent prognostic parameter., Conclusions: VEGF and HIF-1α can be considered useful prognostic biomarkers in HCC patients treated with TACE., Competing Interests: Conflict of Interest The Authors declare no potential conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

MicroRNAs (miRNAs) are small, non-coding RNA molecules involved in regulating gene expression. Many studies, mostly conducted on pediatric patients, suggested that oxidative stress and several miRNAs may play an important role in coeliac disease (CeD) pathogenesis. However, the interplay between oxidative stress and miRNA regulatory functions in CeD remains to be clarified. In this review, we aimed to perform a literature review on the role of miRNAs and oxidative stress in adult CeD patients and to analyze their potential interactions. In this direction, we also reported the preliminary results of a pilot study we recently performed., Competing Interests: Matteo Fassan has a consulting or advisory role for Astellas Pharma, GlaxoSmithKline, Roche, MSD Oncology, Amgen, Lilly, Incyte, Novartis, AstraZeneca, and Pierre Fabre, and research funding for Astellas Pharma, QED Therapeutics, Macrophage Pharma, and Diaceutics. Fabiana Zingone has served as a speaker for Werfen, EG Stada Group, Fresenius Kabi, Kedrion, Janssen, Pfizer, Takeda, Unifarco, Malesci, and Galapagos and has served as a consultant for Galapagos and Takeda. Filippo Pelizzaro served as a consultant for EISAI and MSD. Romilda Cardin, Giulia Sarasini, Milena Minotto, Chiara Carlotto, Michela Palo, and Fabio Farinati have no conflicts of interest to declare., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Microsampling, a reduced volume sampling method, has successfully gained attention at the International Conference on Harmonization (ICH) level and established benefits support its use in Toxicokinetic (TK) studies. These improved sampling techniques are less invasive and in large animal species improve animal welfare (refinement). To evaluate if the plasma concentrations of drugs were influenced by the blood sampling method, the traditional method from femoral vein and microsampling from tail vein in Cynomolgus monkeys were compared. The pharmacokinetic parameters (C max , T max and AUC) of four drugs (selected based on acid-base and volume of distribution properties) in non-human primate were correlated. The plasma samples were quantified using standard LC-MS/MS methods, qualified to evaluate the precision and accuracy before the analysis of real samples. The results reported in this work demonstrated the suitability of microsampling in supporting PK/TK studies in non-human primates. The data show that the exposure of drugs tested after blood collection using standard procedure from femoral vein and microsampling from tail vein is correlated and is not influenced by acid-base characteristics and volume of distribution., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to represent a global health emergency as a highly transmissible, airborne virus. An important coronaviral drug target for treatment of COVID-19 is the conserved main protease (M pro ). Nirmatrelvir is a potent M pro inhibitor and the antiviral component of Paxlovid. The significant viral sequencing effort during the ongoing COVID-19 pandemic represented a unique opportunity to assess potential nirmatrelvir escape mutations from emerging variants of SARS-CoV-2. To establish the baseline mutational landscape of M pro prior to the introduction of M pro inhibitors, M pro sequences and its cleavage junction regions were retrieved from ~4,892,000 high-quality SARS-CoV-2 genomes in the open-access Global Initiative on Sharing Avian Influenza Data (GISAID) database. Any mutations identified from comparison to the reference sequence (Wuhan-Hu-1) were catalogued and analyzed. Mutations at sites key to nirmatrelvir binding and protease functionality (e.g., dimerization sites) were still rare. Structural comparison of M pro also showed conservation of key nirmatrelvir contact residues across the extended Coronaviridae family (α-, β-, and γ-coronaviruses). Additionally, we showed that over time, the SARS-CoV-2 M pro enzyme remained under purifying selection and was highly conserved relative to the spike protein. Now, with the emergency use authorization (EUA) of Paxlovid and its expected widespread use across the globe, it is essential to continue large-scale genomic surveillance of SARS-CoV-2 M pro evolution. This study establishes a robust analysis framework for monitoring emergent mutations in millions of virus isolates, with the goal of identifying potential resistance to present and future SARS-CoV-2 antivirals. IMPORTANCE The recent authorization of oral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antivirals, such as Paxlovid, has ushered in a new era of the COVID-19 pandemic. The emergence of new variants, as well as the selective pressure imposed by antiviral drugs themselves, raises concern for potential escape mutations in key drug binding motifs. To determine the potential emergence of antiviral resistance in globally circulating isolates and its implications for the clinical response to the COVID-19 pandemic, sequencing of SARS-CoV-2 viral isolates before, during, and after the introduction of new antiviral treatments is critical. The infrastructure built herein for active genetic surveillance of M pro evolution and emergent mutations will play an important role in assessing potential antiviral resistance as the pandemic progresses and M pro inhibitors are introduced. We anticipate our framework to be the starting point in a larger effort for global monitoring of the SARS-CoV-2 M pro mutational landscape.

IOA-289 is a novel small molecule inhibitor of autotaxin developed as a first-in-class therapy of fibrotic pathologies including cancer. A method for quantitation of IOA-289 in human plasma was developed using a stable isotope labeled compound ([ 13 C 4 ]IOA-289) as internal standard. The analytes were extracted from human plasma by protein precipitation and the analysis was performed by liquid chromatography coupled with tandem mass spectrometric detection (LCMS/MS). The chromatographic separation was performed with a gradient elution from a BEH C18 column and under these conditions the retention time and the run time were 1 and 2 min, respectively. The assay was fully validated over the range 3-3000 ng/mL, proved to be accurate, precise and selective and was successfully applied to quantitate IOA-289 in plasma samples from subjects in a first-in-humanclinical trial., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Introduction: MicroRNAs (miRNAs) have been proposed as diagnostic markers, biomarkers of neoplastic progression, and possible therapeutic targets in several immune-mediated diseases. We aimed to analyze the expression profile of selected miRNAs (miR21, miR142, miR223, miR155) in patients with autoimmune atrophic gastritis (AAG), patients with non-autoimmune multifocal atrophic gastritis (MAG), and healthy control subjects (HC)., Materials and Methods: A total of 103 patients with AAG were consecutively recruited for this study among those attending our gastroenterology outpatient clinic. Participating patients were divided into two groups: primary, not Helicobacter pylori (HP)-associated related AAG (n=57, P-AAG) and HP-associated AAG (n=46, HP-AAG); this subgroup included HP-positive patients, patients with previously reported HP infection, and patients harboring antral atrophy, considered as a stigma of HP infection. We also included 20 sex-age-matched MAG patients and 10 HC. Upper endoscopy with gastric biopsies were performed on each AAG and MAG patient. Circulating levels of miR21-5p, miR142-3p, miR223-3p, and miR155-5p were measured by RT-PCR in all groups., Results: MiR-21 was over-expressed in P-AAG (p=0.02), HP-AAG (p = 0.04), and MAG (p=0.03) compared with HC. By contrast, miR-142 was more expressed in HC than in HP-AAG (p=0.04) and MAG (p=0.03). MiR-155 showed no significant differences among the four subgroups, while, unexpectedly, miR-223 was overexpressed in HC compared to P-AAG (p=0.01), HP-AAG (p=0.003), and MAG (p<0.001), and was higher in P-AAG than in MAG (p=0.05)., Conclusions: MiR-21 was over-expressed in patients with gastric precancerous conditions irrespective of etiology, while in the same subgroups miR-142 and miR-223 were under-expressed compared to healthy controls. Controlling miRNAs up- or downregulation could lead to a breakthrough in treating chronic autoimmune diseases and potentially interfere with the progression to cancer., Competing Interests: FF received commercial research funding from Gilead and Bayer, MF received commercial research funding from Astellas Farmas and QED. FZ received commercial research funding from Takeda, Janssen, EG, Sofar, Norgine. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zingone, Pilotto, Cardin, Maddalo, Orlando, Fassan, Marsilio, Collesei, Pelizzaro and Farinati.)

We aimed to explore the activation of monoacylglycerol lipase (MAGL)/cyclooxygenase-2 (COX-2)/prostaglandin E 2 (PGE2) axis in hepatocellular carcinoma (HCC), evaluating circulating PGE2 as prognostic biomarker in HCC patients. PGE2 levels were measured in blood samples from 24 cirrhotics, and 34 HCC patients were consecutively collected between January 2016 and December 2017. In a subgroup of patients, tissue expression of MAGL mRNA and immunohistochemistry for MAGL and COX-2 were obtained. Despite tumor tissues showing overexpression of MAGL mRNA and higher levels of both MAGL and COX-2 at immunohistochemistry, PGE2 levels were not significantly different in HCC and cirrhotics. HCC patients with circulating PGE2 levels > 14 pg/mL had a significantly shorter overall survival (19.4 vs. 49.9 months; p = 0.03), the finding being confirmed by the multivariate analysis (HR 3.37 [95% CI 1.00-11.60]; p = 0.05). The MAGL/COX-2/PGE2 axis is activated in HCC, and circulating PGE2 proved to be a potential prognostic biomarker., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: MicroRNAs (miRNAs) have been proposed as biomarkers in hepatocellular carcinoma (HCC). We aim at evaluating miR-21 and miR-122 in HCC patients treated with drug-eluting beads transarterial chemoembolization (DEB-TACE) as prognostic biomarkers and investigating their correlation with hypoxia inducible factor-1α (HIF-1α) serum levels., Methods: In this retrospective study, 12 healthy subjects, 28 cirrhotics, and 54 HCC patients (tested before and four weeks after DEB-TACE) were included. Whole blood miR-21 and miR-122 levels were measured by quantitative real time (qRT)-PCR, while serum HIF-1α was assessed by an enzyme-linked immunosorbent assay (ELISA) test., Results: The highest level of miR-21 was found in cirrhotics, while HCC patients had the highest level of miR-122 (which was even higher in "viral" HCC, p = 0.006). miR-21 ratio (after/before DEB-TACE) and miR-122 below their respective cut-offs identified patients with longer progression-free survival ( p = 0.0002 and p = 0.02, respectively). The combined assessment of alpha-fetoprotein and miR-21 ratio, both independent prognostic predictors, identified early progressors among patients with complete or partial radiological response. miR-21 levels positively correlated with HIF-1α before ( p = 0.045) and after DEB-TACE ( p = 0.035)., Conclusions: miR-21 ratio and miR-122 are useful prognostic markers after DEB-TACE. miR-21 correlates with HIF-1α and probably has a role in modulating angiogenesis in HCC.

Background: Current literature still lacks studies evaluating the effectiveness and safety of switching from Infliximab originator to SB2 biosimilar in Inflammatory Bowel Diseases (IBDs). We aimed to verify the ability of SB2 to maintain the clinical and biochemical response induced by originator after switching. As secondary outcome, we aimed to verify safety, tolerability and immunogenicity of SB2 biosimilar compared with its IFX originator., Methods: We prospectively enrolled all patients who switched from originator to SB2 at three Italian IBD Units from August 2018 to April 2020. We collected clinical and biochemical data at the time of switch (T0), and at the first (T1) and the second (T2) visits after switching (mean time from switching: 135 and 329 days, respectively). In addition, data regarding therapeutic drug monitoring at T0 and T1 were recorded., Results: Eighty-five IBD patients (28 with Ulcerative Colitis and 57 with Crohn's Disease) were included in the study. At T1, we observed statistically significant modifications in clinical activity of disease (70 patients were in clinical remission at baseline and 60 at T1 p = 0.02), but not at T2 (p = 0.3). Fecal calprotectin values were not different both at T1 and T2 (both p = 0.9) as well as the rate of concomitant treatment with steroids (p = 0.2 and p = 0.1) or immunosuppressants (p = 0.1 and p = 1.0). Moreover, the need for therapeutic optimization from T0 to T1 and from T1 to T2 was found significant (both p = 0.01). No anti-drug antibodies were identified at T1, and no serious adverse events were recorded., Conclusions: Overall, our data show that most of the patients switching from Infliximab originator to SB2 maintain the clinical and biochemical remission for at least 1 year. Further data are necessary to understand the clinical implications of these findings in the long term., Competing Interests: Conflict of interest statement: - Edoardo Vincenzo Savarino served as a speaker, consultant, and advisory board member for AbbVie, MSD, Takeda, Ferring. - Renata D’Incà served as a speaker, consultant, and advisory board member for AbbVie, MSD, Takeda, Ferring. - The other authors have no conflict of interest., (© The Author(s), 2021.)

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related death worldwide. Diagnostic, prognostic, and predictive biomarkers are urgently needed in order to improve patient survival. Indeed, the most widely used biomarkers, such as alpha-fetoprotein (AFP), have limited accuracy as both diagnostic and prognostic tests. Liver biopsy provides an insight on the biology of the tumor, but it is an invasive procedure, not routinely used, and not representative of the whole neoplasia due to the demonstrated intra-tumoral heterogeneity. In recent years, liquid biopsy, defined as the molecular analysis of cancer by-products, released by the tumor in the bloodstream, emerged as an appealing source of new biomarkers. Several studies focused on evaluating extracellular vesicles, circulating tumor cells, cell-free DNA and non-coding RNA as novel reliable biomarkers. In this review, we aimed to provide a comprehensive overview on the most relevant available evidence on novel circulating biomarkers for early diagnosis, prognostic stratification, and therapeutic monitoring. Liquid biopsy seems to be a very promising instrument and, in the near future, some of these new non-invasive tools will probably change the clinical management of HCC patients.

Anti-drug antibodies can interfere with the activity of anti-tumor necrosis factor (TNF) agents by increasing drug clearance via direct neutralization. The presence of anti-drug antibodies is clinically relevant when trough drug concentrations are undetectable or sub-therapeutic. However, traditional immunoassay is not easily and rapidly accessible, making the translation of the results into treatment adjustment difficult. The availability of a point-of-care (POC) test for therapeutic drug monitoring (TDM) might represent an important step forward for improving the management of inflammatory bowel disease (IBD) patients in clinical practice. In this pilot study, we compared the results obtained with POC tests with those obtained by enzyme-linked immunosorbent assay (ELISA) in a group of IBD patients treated with Infliximab (IFX). We showed that POC test can reliably detect presence of antibody-to-IFX with 100% of specificity and 76% sensitivity, in strong agreement with the ELISA test ( k -coefficient = 0.84)., Competing Interests: Conflict of interest statement: The authors declare that there is no conflict of interest., (© The Author(s), 2021.)

Background:  Portal vein thrombosis (PVT) is the most common thrombotic complication in cirrhosis; however, local risk factors involved in its pathogenesis are still not fully investigated. The aim of the study was to evaluate hemostasis and endothelial damage in the portal vein in patients with cirrhosis and portal hypertension., Methods:  Adult cirrhotics undergoing transjugular intrahepatic portosystemic shunt were consecutively enrolled. Rotational thromboelastometry (ROTEM), dosage of total circulating glycosaminoglycans (GAGs), and endotoxemia levels (lipopolysaccharide [LPS]), along with evaluation of endothelial dysfunction by quantification of circulating endothelial microparticles (MPs), were performed on citrated peripheric and portal venous blood samples from each enrolled patient., Results:  Forty-five cirrhotics were enrolled. ROTEM analysis revealed the presence of a significant heparin-like effect in portal blood (median ɑ angle NATEM 50° vs. HEPTEM 55°, p  = 0.027; median coagulation time NATEM 665 s vs. HEPTEM 585 s, p  = 0.006), which was not detected in peripheral blood, and was associated with a higher concentration of circulating GAGs. Even though total annexin V-MP circulating MPs were less concentrated in the splanchnic district, the proportion of MPs of endothelial origin, with respect to annexin V-MP, was significantly increased in the portal district ( p  = 0.036). LPS concentration was higher in portal (197 pg/mL) compared with peripheral blood (165 pg/mL) ( p  < 0.001)., Conclusion:  Evidences of a damage of glycocalyx along with increased concentration of endothelial MPs suggest the presence of a significant endothelial alteration in the portal vein with respect to peripheral veins. Portal site-specific endothelial damage could hamper its antithrombotic properties and may represent an important local risk factor in the pathogenesis of PVT., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Aim: Squamous cell carcinoma antigen immune complexed with immunoglobulin M (SCCA-IgM) is a useful but not completely satisfactory biomarker of hepatocellular carcinoma (HCC). Considering its gender-specific behavior in preclinical models, we investigated gender-related differences of SCCA-IgM as a prognostic marker in HCC. Patients & methods: Two hundred and eight prospectively recruited patients treated with transarterial chemoembolization in a single tertiary care hospital were retrospectively evaluated. Correlations between SCCA-IgM levels, clinical characteristics and survival were assessed according to gender. Results: When the disease was advanced, SCCA-IgM was higher in males and lower in females. Levels below 130 AU/ml predicted a significantly longer survival in males (p = 0.007) and a shorter survival in females (p = 0.01). Conclusion: In predicting the prognosis of HCC patients, the interpretation of SCCA-IgM should consider gender as a relevant variable.

Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae are classified as critical priority pathogens, with extended-spectrum β-lactamases (ESBLs) as principal resistance determinants. Enmetazobactam (formerly AAI101) is a novel ESBL inhibitor developed in combination with cefepime for empirical treatment of serious Gram-negative infections in settings where ESBLs are prevalent. Cefepime-enmetazobactam has been investigated in a phase 3 trial in patients with complicated urinary tract infections or acute pyelonephritis. This study examined pharmacokinetic-pharmacodynamic (PK-PD) relationships of enmetazobactam, in combination with cefepime, for ESBL-producing isolates of Klebsiella pneumoniae in 26-h murine neutropenic thigh infection models. Enmetazobactam dose fractionation identified the time above a free threshold concentration ( fT > C T ) as the PK-PD index predictive of efficacy. Nine ESBL-producing isolates of K. pneumoniae , resistant to cefepime and piperacillin-tazobactam, were included in enmetazobactam dose-ranging studies. The isolates encoded CTX-M-type, SHV-12, DHA-1, and OXA-48 β-lactamases and covered a cefepime-enmetazobactam MIC range from 0.06 to 2 μg/ml. Enmetazobactam restored the efficacy of cefepime against all isolates tested. Sigmoid curve fitting across the combined set of isolates identified enmetazobactam PK-PD targets for stasis and for a 1-log 10 bioburden reduction of 8% and 44% fT  > 2 μg/ml, respectively, with a concomitant cefepime PK-PD target of 40 to 60% fT > cefepime-enmetazobactam MIC. These findings support clinical dose selection and breakpoint setting for cefepime-enmetazobactam., (Copyright © 2020 American Society for Microbiology.)

Background and Aims: Inflammatory bowel diseases (IBDs) are treated with anti-TNF agents. Strategies to monitor response to therapy may improve clinical control of the disease and reduce economical costs. Previous evidence suggests cleavage of infliximab (IFX) by Matrix Metalloproteinase 3 (MMP3) as a mechanism leading to loss of response. Our study aimed to evaluate if MMP3 serum levels could be considered an early marker of anti-TNF nonresponse and to analyze the correlation with other biochemical markers of treatment failure such as IFX trough levels and anti-IFX antibodies, inflammatory markers, and albumin levels., Methods: Retrospectively, 73 IBD patients who had received IFX for at least 1 year were enrolled: 35 patients were responders and 38 were nonresponders at 52 weeks. Clinical and biochemical data (Harvey-Bradshaw index [HBI], Mayo score, body mass index [BMI], C-reactive protein [CRP], fecal calprotectin and albumin levels), MMP3 serum levels, and drug monitoring were assessed at baseline, postinduction, and 52 weeks., Results: The MMP3 levels were similar at baseline (19.83 vs 17.92 ng/mL), but at postinduction, patients who failed to respond at 1 year had significantly higher levels than patients who responded (26.09 vs 8.68 ng/mL, P < 0.001); the difference was confirmed at week 52 (29.56 vs 11.48 ng/mL, P < 0.001). The MMP3 levels tended to be higher in patients without antidrug antibodies than in patients with antidrug antibodies at postinduction and 52 weeks., Conclusions: The MMP3 serum determination may represent an early marker of response to infliximab., (© 2019 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Defects in Cockayne syndrome type A (CSA), a gene involved in nucleotide excision repair, cause an autosomal recessive syndrome characterized by growth failure, progressive neurological dysfunction, premature aging, and skin photosensitivity and atrophy. Beyond its role in DNA repair, the CSA protein has additional functions in transcription and oxidative stress response, which are not yet fully elucidated. Here, we investigated the role of CSA protein in primary human keratinocyte senescence. Primary keratinocytes from three patients with CS-A displayed premature aging features, namely premature clonal conversion, high steady-state levels of reactive oxygen species and 8-OH-hydroxyguanine, and senescence-associated secretory phenotype. Stable transduction of CS-A keratinocytes with the wild-type CSA gene restored the normal cellular sensitivity to UV irradiation and normal 8-OH-hydroxyguanine levels. Gene correction was also characterized by proper restoration of keratinocyte clonogenic capacity and expression of clonal conversion key regulators (p16 and p63), decreased NF-κB activity and, in turn, the expression of its targets (NOX1 and MnSOD), and the secretion of senescence-associated secretory phenotype mediators. Overall, the CSA protein plays an important role in protecting cells from senescence by facilitating DNA damage processing, maintaining physiological redox status and keratinocyte clonogenic ability, and reducing the senescence-associated secretory phenotype-mediated inflammatory phenotype., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: The histological intrahepatic microvasculature lesions have not been deeply investigated outside the setting of portal hypertension. The aim of this study was to analyse the type and the prevalence of microvasculature abnormalities and their correlation with inflammatory activity, fibrosis stage and tissue markers of fibrogenesis, angiogenesis and oxidative DNA damage in liver biopsies obtained from patients with chronic viral hepatitis., Methods: Seventy-four liver biopsies from untreated patients affected by hepatitis B (22 cases) and C (52 cases) were included. The presence of microvascular changes was correlated with (i) the severity of the activity and fibrosis; (ii) immunohistochemical markers of angiogenesis (CD34) and hepatic stellate cells activation (alpha-smooth muscle actin); (iii) a tissue marker of oxidative damage (8-OHdG adducts)., Results: Sixty-five out of 74 biopsies (87.8%) showed vascular lesions. Portal angiomatosis was the most prevalent (62.2%) and it was associated with, on 1 side, the fibrosis stage at both univariate (P < .0001) and multivariate analysis (P = .01, OR = 9.4 [1.6-54]) and, on the other, with angiogenesis (P = .05) and hepatic stellate cells activation (P = .002). Interestingly, 36/46 cases with portal angiomatosis were at early/intermediate fibrosis stage. The hepatic stellate cells activation was also associated with the presence of aberrant periportal vessels (P = .01)., Conclusions: The histological alterations of intrahepatic microvasculature, usually seen in cirrhosis and portal hypertension, occur in chronic viral hepatitis even at early/intermediate fibrosis stages. Their correlation with angiogenesis and fibrogenesis supports a possible involvement in disease progression., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Goal: To evaluate the potential role of the determination of the immunocomplexed form of squamous cell carcinoma antigen [SCCA-immunoglobulin (Ig)M] for the screening of Barrett esophagus (BE) and esophageal adenocarcinoma (EAC)., Background: The cost-effectiveness of surveillance in BE is still debated and the use of biomarkers in screening and surveillance still not recommended. No information is available regarding SCCA-IgM determination in BE., Study: SCCA-IgM levels were determined (enzyme-linked immunosorbent assay) in 231 patients prospectively recruited, 71 with BE, 53 with EAC, and 107 controls, including 42 blood donors and 65 patients with gastroesophageal reflux. SCCA-IgM cutoffs between BE/EAC and controls and for BE "at risk" versus short nondysplastic BE were calculated by receiver operating characteristic curves. Immunostaining for SCCA-IgM was obtained in a subgroup of patients., Results: Median SCCA-IgM values were significantly higher in BE and EAC than in controls (P=0.0001). Patients with SCCA-IgM levels above the cutoff had a 33 times higher relative risk of harboring BE or EAC (P=0.0001). Patients "at risk," with long or dysplastic BE had SCCA-IgM levels significantly higher than those with short nondysplastic BE (P=0.035) and patients with SCCA-IgM above the cutoff had a 8 times higher relative risk of having BE "at risk." SCCA was expressed in Barrett mucosa but not in cardiac metaplasia., Conclusions: Serum SCCA-IgM determination allows the identification of patients at risk for BE/EAC and the stratification of BE patients in subgroups with different cancer risk. Because of the still limited number of controls, large, prospective studies are required to confirm this evidence.

Herpesviruses have coevolved with their hosts over hundreds of millions of years and exploit fundamental features of their biology. Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells, and it has been hypothesized that systemic dissemination arises from infected stem cells in bone marrow. However, poor CMV transfer by stem cell transplantation argues against this being the main reservoir. To identify alternative pathways for CMV spread, we tracked murine CMV (MCMV) colonization after mucosal entry. We show that following intranasal MCMV infection, lung CD11c + dendritic cells (DC) migrated sequentially to lymph nodes (LN), blood, and then salivary glands. Replication-deficient virus followed the same route, and thus, DC infected peripherally traversed LN to enter the blood. Given that DC are thought to die locally following their arrival and integration into LN, recirculation into blood represents a new pathway. We examined host and viral factors that facilitated this LN traverse. We show that MCMV-infected DC exited LN by a distinct route to lymphocytes, entering high endothelial venules and bypassing the efferent lymph. LN exit required CD44 and the viral M33 chemokine receptor, without which infected DC accumulated in LN and systemic spread was greatly reduced. Taken together, our studies provide the first demonstration of virus-driven DC recirculation. As viruses follow host-defined pathways, high endothelial venules may normally allow DC to pass from LN back into blood. IMPORTANCE Human cytomegalovirus (HCMV) causes devastating disease in the unborn fetus and in the immunocompromised. There is no licensed vaccine, and preventive measures are impeded by our poor understanding of early events in host colonization. HCMV and murine CMV (MCMV) both infect blood-borne myeloid cells. HCMV-infected blood cells are thought to derive from infected bone marrow stem cells. However, infected stem cells have not been visualized in vivo nor shown to produce virus ex vivo , and hematopoietic transplants poorly transfer infection. We show that MCMV-infected dendritic cells in the lungs reach the blood via lymph nodes, surprisingly migrating into high endothelial venules. Dissemination did not require viral replication. It depended on the constitutively active viral chemokine receptor M33 and on the host hyaluronan receptor CD44. Thus, viral chemokine receptors are a possible target to limit systemic CMV infections., (Copyright © 2017 Farrell et al.)

Cytomegaloviruses (CMVs) establish persistent, systemic infections and cause disease by maternal-foetal transfer, suggesting that their dissemination is a key target for antiviral intervention. Late clinical presentation has meant that human CMV (HCMV) dissemination is not well understood. Murine CMV (MCMV) provides a tractable model. Whole mouse imaging of virus-expressed luciferase has proved a useful way to track systemic infections. MCMV, in which the abundant lytic gene M78 was luciferase-tagged via a self-cleaving peptide (M78-LUC), allowed serial, unbiased imaging of systemic and peripheral infection without significant virus attenuation. Ex vivo luciferase imaging showed greater sensitivity than plaque assay, and revealed both well-known infection sites (the lungs, lymph nodes, salivary glands, liver, spleen and pancreas) and less explored sites (the bone marrow and upper respiratory tract). We applied luciferase imaging to tracking MCMV lacking M33, a chemokine receptor conserved in HCMV and a proposed anti-viral drug target. M33-deficient M78-LUC colonized normally in peripheral sites and local draining lymph nodes but spread poorly to the salivary gland, suggesting a defect in vascular transport consistent with properties of a chemokine receptor.

In viral hepatitis, inflammation is correlated with chronic oxidative stress, one of the biological events leading to DNA damage and hepatocellular carcinoma (HCC) development. Aim of this study was to investigate the complex molecular network linking oxidative damage to telomere length and telomerase activity and regulation in hepatitis C and B virus-related liver carcinogenesis. We investigated 142 patients: 21 with HCC (in both tumor and peritumor tissues) and 121 with chronic viral hepatitis in different stages. We evaluated 8-hydroxydeoxyguanosine (8-OHdG), marker of oxidative DNA damage, OGG1 gene polymorphism, telomere length, telomerase activity, TERT promoter methylation, and mitochondrial TERT localization. In hepatitis C-related damage, 8-OHdG levels increased since the early disease stages, whereas hepatitis B-related liver disease was characterized by a later and sharper 8-OHdG accumulation (P = 0.005). In C virus-infected patients, telomeres were shorter (P = 0.03), whereas telomerase activity was higher in tumors than that in the less advanced stages of disease in both groups (P = 0.0001, P = 0.05), with an earlier increase in hepatitis C. Similarly, TERT promoter methylation was higher in tumor and peritumor tissues in both groups (P = 0.02, P = 0.0001). Finally, TERT was localized in mitochondria in tumor and peritumor samples, with 8-OHdG levels significantly lower in mitochondrial than those in genomic DNA (P = 0.0003). These data describe a pathway in which oxidative DNA damage accumulates in correspondence with telomere shortening, telomerase activation, and TERT promoter methylation with a different time course in hepatitis B and C virus-related liver carcinogenesis. Finally, TERT localizes in mitochondria in HCC, where it lacks a canonical function., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background & Aims: MicroRNAs (miRNAs) have been involved in hepatocarcinogenesis, but little is known on their role in the progression of chronic viral hepatitis. Aim of this study was to identify miRNA signatures associated with stages of disease progression in patients with chronic viral hepatitis., Methods: MiRNA expression profile was investigated in liver biopsies from patients with chronic viral hepatitis and correlated with clinical, virological and histopathological features. Relevant miRNAs were further investigated., Results: Most of the significant changes in miRNA expression were associated with liver fibrosis stages and included the significant up-regulation of a group of miRNAs that were demonstrated to target the master regulators of epithelial-mesenchymal transition ZEB1 and ZEB2 and involved in the preservation of epithelial cell differentiation, but also in cell proliferation and fibrogenesis. In agreement with miRNA data, immunostaining of liver biopsies showed that expression of the epithelial marker E-cadherin was maintained in severe fibrosis/cirrhosis while expression of ZEBs and other markers of epithelial-mesenchymal transition were low or absent. Severe liver fibrosis was also significantly associated with the down-regulation of miRNAs with antiproliferative and tumour suppressor activity. Similar changes in miRNA and target gene expression were demonstrated along with disease progression in a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis, suggesting that they might represent a general response to liver injury., Conclusion: Chronic viral hepatitis progression is associated with the activation of miRNA pathways that promote cell proliferation and fibrogenesis, but preserve the differentiated hepatocyte phenotype., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background and Aim: The serpin squamous cell carcinoma antigen complexed with IgM (SCCA-IgM) has been reported as a promising serological marker for hepatocellular carcinoma (HCC). We aimed to further evaluate SCCA-IgM diagnostic accuracy and to determine its prognostic role., Methods: SCCA-IgM levels were determined in 327 sera obtained from 81 HCC patients, 206 cirrhotics and 40 healthy blood donors (controls). Sensitivity, specificity, correlation with clinical and tumor parameters and with survival were evaluated., Results: HCC patients had SCCA-IgM levels significantly higher than controls and cirrhotics (P < 0.0001). Sensitivity, specificity, positive and negative predictive values for HCC were 89%, 50%, 41% and 92%, respectively. In comparison, sensitivity and specificity for alphafetoprotein were 48% and 85%. SCCA-IgM levels were not significantly correlated with clinical or biological variables. With a cut-off of 130 AU/mL (receiver operating characteristic curves), SCCA-IgM proved efficient in the prediction of prognosis, identifying the patients with long overall survival (efficiency validated in the homogenous subgroup of patients with intermediate-stage HCC undergoing transarterial chemoembolization) and predicting progression-free survival. A Cox multivariate analysis confirmed SCCA-IgM predictive value, identifying tumor size and SCCA-IgM levels as independent predictors of survival. A reduction in SCCA-IgM levels correlated with response to treatment., Conclusions: SCCA-IgM is a sensitive marker of HCC in patients with cirrhosis even though lacking in specificity. The determination of the levels of the marker in HCC patients is highly efficient in predicting the patients' prognosis, identifying those with long overall and progression-free survival and the responders and should be introduced in the clinical practice., (© 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.)

The histo-pathologic and molecular mechanisms leading to initiation and progression of hepatocellular carcinoma (HCC) are still ill-defined; however, there is increasing evidence that the gradual accumulation of mutations, genetic and epigenetic changes which occur in preneoplastic hepatocytes results in the development of dysplastic foci, nodules, and finally, overt HCC. As well as many other neoplasias, liver cancer is considered an "inflammatory cancer", arising from a context of inflammation, and characterized by inflammation-related mechanisms that favor tumor cell survival, proliferation, and invasion. Molecular mechanisms that link inflammation and neoplasia have been widely investigated, and it has been well established that inflammatory cells recruited at these sites with ongoing inflammatory activity release chemokines that enhance the production of reactive oxygen species. The latter, in turn, probably have a major pathogenic role in the continuum starting from hepatitis followed by chronic inflammation, and ultimately leading to cancer. The relationship amongst chronic liver injury, free radical production, and development of HCC is explored in the present review, particularly in the light of the complex network that involves oxidative DNA damage, cytokine synthesis, telomere dysfunction, and microRNA regulation.

Background: Barrett esophagus develops in a scenario of chronic inflammation, linked to free radical formation and oxidative DNA damage. Eight-hydroxydeoxyguanosine, the main oxidative DNA adduct, is partially repaired by a glycosylase (OGG1) whose polymorphism is associated to a reduced repair capacity. Telomeres are particularly prone to oxidative damage, which leads to shortening and cell senescence, while elongation, by telomerase activity, is linked to cell immortalization and cancer. Limited data are available on this point with respect to Barrett esophagus. This study aimed to evaluate the link among 8-hydroxydeoxyguanosine, OGG1 polymorphism, telomerase activity, telomere length, and p53 mutation in Barrett progression., Methods: Forty consecutive patients with short- and long-segment Barrett esophagus and 20 controls with gastroesophageal reflux disease without Barrett esophagus were recruited. Analysis of biopsy samples was undertaken to study 8-hydroxydeoxyguanosine levels, OGG1 polymorphism, telomerase activity, and telomere length. Serum samples were obtained for p53 mutation., Results: Controls had significantly lower levels of 8-hydroxydeoxyguanosine and telomerase activity, with normal telomere length and no p53 mutation. In short-segment Barrett esophagus, 8-hydroxydeoxyguanosine levels were higher and telomeres underwent significant shortening, with stimulation of telomerase activity but no p53 mutations. In long-segment Barrett esophagus, 8-hydroxydeoxyguanosine reached maximal levels, with telomere elongation, and 42 % of the patients showed p53 mutation., Conclusions: In Barrett patients, with disease progression, oxidative DNA damage accumulates, causing telomere instability, telomerase activation, and, in a late phase, mutations in the p53 gene, thus abrogating its activity as the checkpoint of proliferation and apoptosis, and facilitating progression to cancer.

Purpose: Circulating free DNA (cfDNA) is an extracellular DNA released in the blood by tumor apoptotic/necrotic cells. cfDNA determination has been proposed as a non-invasive and sensitive marker in the diagnosis of cancer. Our aim was to validate the quantification of cfDNA as a diagnostic and prognostic tool in hepatocellular carcinoma (HCC)., Methods: cfDNA was quantified by real-time PCR amplification of the hTERT gene in 142 plasma samples obtained from 66 patients with HCC, 35 with cirrhosis (CIRR) and 41 with advanced HCV-related chronic hepatitis (CH)., Results: cfDNA was documented in the plasma of 22 % of the CH patients, 57 % of those with CIRR and 61 % of HCC patients. Its concentration was lower in CH with respect to CIRR and HCC (p = 0.02). A cutoff value in the diagnosis of HCC was calculated by the ROC method (area under the curve 0.69, 91 % sensitivity, 43 % specificity) considering HCC versus CH/CIRR, taken together. Patients with multinodular HCC showed significantly higher levels of cfDNA (p = 0.05). A cutoff value for cfDNA was also calculated for discriminating patients with long or short survival. Survival was significantly longer in patients with cfDNA below than in those above the cutoff value (37 vs. 24 months, p = 0.03). Similar results were obtained in the subgroups of patients with viral or with HCV-only etiology, with slightly higher overall diagnostic accuracy., Conclusions: The role of the quantitative analysis of cfDNA as a diagnostic test is debatable, but cfDNA levels discriminate patients with more advanced stages of disease, demonstrating a prognostic relevance in patients with HCC.

Aim: To compare the mucosal concentrations of 5-aminosalicylic acid (5-ASA) resulting from different pharmaceutical formulations and analyse the influence of inflammation on the mucosal concentrations., Methods: The study included 130 inflammatory bowel disease (IBD) patients receiving 5-ASA as pH-dependent-release formulations (73 patients), time-dependent-release formulations (11 patients), or pro-drugs (18 patients). In addition, 28 patients were receiving topical treatment (2-4 g/d) with pH-dependent-release formulations. Endoscopic biopsies were obtained from the sigmoid region during the colonoscopy. The 5-ASA concentrations (ng/mg) were measured in tissue homogenates using high-pressure liquid chromatography with electrochemical detection. The t test and Mann-Whitney test, when appropriate, were used for statistical analysis., Results: Patients receiving pH-dependent-release formulations showed significantly higher mucosal concentrations of 5-ASA (51.75 ± 5.72 ng/mg) compared with patients receiving pro-drugs (33.35 ± 5.78 ng/mg, P = 0.01) or time-dependent-release formulations (38.24 ± 5.53 ng/mg, P = 0.04). Patients with endoscopic remission had significantly higher mucosal concentrations of 5-ASA than patients with active disease (60.14 ± 7.95 ng/mg vs 35.66 ± 5.68 ng/mg, P = 0.02). Similar results were obtained when we compared patients with the histological appearance of remission and patients with active histological inflammation (67.53 ± 9.22 ng/mg vs 35.53 ± 5.63 ng/mg, P < 0.001). Significantly higher mucosal concentrations of 5-ASA were detected in patients treated with both oral and topical treatments in combination compared with patients who received oral treatment with pH-dependent-release formulations alone (72.33 ± 11.23 ng/mg vs 51.75 ± 5.72 ng/mg, P = 0.03)., Conclusion: IBD patients showed significant variability in mucosal 5-ASA concentrations depending on the type of formulation, and the highest mean concentration was achieved using pH-dependent-release formulations.

Background: Coffee is associated with a reduced risk of hepatocellular carcinoma in patients with chronic C hepatitis. This prospective trial was aimed at assessing the mechanisms underlying coffee-related protective effects., Methods: Forty patients with chronic hepatitis C were randomized into two groups: the first consumed 4 cups of coffee/day for 30 days, while the second remained coffee "abstinent". At day 30, the groups were switched over for a second month., Results: At baseline, aspartate aminotransferase and alanine aminotransferase were lower in patients drinking 3-5 (Group B) than 0-2 cups/day (Group A) (56 ± 6 vs 74 ± 11/60 ± 3 vs 73 ± 7 U/L p=0.05/p=0.04, respectively). HCV-RNA levels were significantly higher in Group B [(6.2 ± 1.5) × 10(5)vs (3.9 ± 1.0) × 10(5)UI/mL, p=0.05]. During coffee intake, 8-hydroxydeoxyguanosine and collagen levels were significantly lower than during abstinence (15 ± 3 vs 44 ± 16 8-hydroxydeoxyguanosine/10(5)deoxyguanosine, p=0.05 and 56 ± 9 vs 86 ± 21 ng/mL, p=0.04). Telomere length was significantly higher in patients during coffee intake (0.68 ± 0.06 vs 0.48 ± 0.04 Arbitrary Units, p=0.006). Telomere length and 8-hydroxydeoxyguanosine were inversely correlated., Conclusion: In chronic hepatitis C coffee consumption induces a reduction in oxidative damage, correlated with increased telomere length and apoptosis, with lower collagen synthesis, factors that probably mediate the protection exerted by coffee with respect to disease progression., (Copyright © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Immunotherapeutic herpes simplex virus 2 (HSV-2) vaccine efficacy depends upon the promotion of antigen-specific immune responses that inhibit reactivation or reactivated virus, thus controlling both recurrent lesions and viral shedding. In the present study, a candidate subunit vaccine, GEN-003/MM-2, was evaluated for its ability to induce a broad-spectrum immune response in mice and therapeutic efficacy in HSV-2-infected guinea pigs. GEN-003 is comprised of HSV-2 glycoprotein D2 (gD2ΔTMR340-363) and a truncated form of infected cell polypeptide 4 (ICP4383-766), formulated with Matrix M-2 (MM-2) adjuvant (GEN-003/MM-2). In addition to eliciting humoral immune responses, CD4(+) and CD8(+) T cells characterized by the secretion of multiple cytokines and cytolytic antigen-specific T cell responses that were able to be recalled at least 44 days after the last immunization were induced in immunized mice. Furthermore, vaccination with either GEN-003 or GEN-003/MM-2 led to significant reductions in both the prevalence and severity of lesions in HSV-2-infected guinea pigs compared to those of phosphate-buffered saline (PBS) control-vaccinated animals. While vaccination with MM-2 adjuvant alone decreased recurrent disease symptoms compared to the PBS control group, the difference was not statistically significant. Importantly, the frequency of recurrent viral shedding was considerably reduced in GEN-003/MM-2-vaccinated animals but not in GEN-003- or MM-2-vaccinated animals. These findings suggest a possible role for immunotherapeutic GEN-003/MM-2 vaccination as a viable alternative to chronic antiviral drugs in the treatment and control of genital herpes disease.

Background: CD80 has been thought to play an active role in immunosurveillance as it has been found to be up-regulated in ulcerative colitis (UC) patients with dysplasia. The aim of the present study was to analyse early events in UC-related and non-inflammatory carcinogenesis with reference to CD80 expression to clarify what stimuli are involved in its up-regulation in these patients., Patients and Methods: Sixty-two patients affected with UC, UC with dysplasia, UC and cancer, colonic adenoma, or colonic cancer and 11 healthy subjects were enroled in our study. Tissue samples were taken from surgical specimens during colonic resection or during colonoscopy. Mucosal mRNA expression of Toll-like receptor-4 (TLR4) and nuclear factor-kappaB (NF-κB) was quantified with Real Time RT-PCR. TLR4, β-catenin and p53 expressions were analysed by immunohistochemistry. Mucosal levels of activated NF-κB were measured with immunometric assays while 8-Hydroxydeoxyguanosine (8-OHdG) levels were quantified by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Non-parametric tests were used for statistical analysis., Results: 8-OHdG mucosal levels were higher in the patients with UC + dysplasia with respect to those in the patients with UC only (p=0.03). CD80 mRNA mucosal levels were directly correlated with 8-OHdG mucosal levels (τ=0.26, p=0.04), TLR4 protein expression (τ=0.45, p<0.01) and NF-κB mRNA expression and activity (τ=0.24, p=0.02; τ=0.34, p=0.02, respectively). CD80 protein expression, instead, was directly correlated with 8-OHdG mucosal levels (τ=0.19, p=0.05) and inversely correlated with TLR4 mRNA expression (τ=-0.25, p=0.03)., Conclusion: Oxidative DNA damage peaked in UC-related dysplasia and was found to be directly correlated to CD80 expression. The direct correlation between TLR4 protein expression and CD80 mRNA and the indirect correlation between CD80 protein and TLR4 mRNA expressions give substance to the hypothesis that they play a role in immunosurveillance. No significant correlations between CD80 expression and p53 and β-catenin accumulation during oncogenesis were, instead, observed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Gastric cancer (GC) is still a leading cause of cancer-related death worldwide, and environmental, genetic, and epigenetic DNA changes are involved in the process of gastric carcinogenesis. The objective of this study was to establish the extent of DNA methylation at various CpG islands in GC and in precancerous changes [gastric noninvasive neoplasia (NIN)]. Eighty-one gastric samples were analyzed using methylation-specific PCR at several CpG islands. Thirty-eight samples were obtained at surgery [19 neoplastic (GC) and 19 nonneoplastic cancer-surrounding tissues (sGC)] and 43 at endoscopy (biopsies in 23 NIN patients and 20 controls). Hypermethylation of TPEF (a growth inhibitor), PTGER3 (a prostaglandin receptor isoform), and MINT31 (a promoter locus regulating calcium channels that is involved in p53 mutation) discriminated NIN and GC from normal mucosa, suggesting an early role as initiating events, whereas hypermethylation at ARGHAP20 developed with the progression from NIN to GC. MINT31 hypermethylation predicted persistence or worsening of NIN and cancer development. In conclusion, these data support a progressive accumulation of aberrant methylations in NIN and GC at various CpG islands with distinct time courses. With hypermethylation, the genes involved in regulating the balance between apoptosis and cell proliferation may become silenced and trigger gastric tumorigenesis. Hypermethylation of MINT31 predicted NIN persistence, as well as progression to higher grade or to GC, and might be used as a marker of GC risk.

Background: Dysregulation of autophagy is important in the pathogenesis of many diseases, including cancer. Several aspects of the biological role of autophagy are however still unclear and the relationship between apoptosis and autophagy, particularly in the liver has yet to be thoroughly explored. In this study we evaluated the expression of Beclin 1 (one of the main autophagocytic agents, which bridges autophagy, apoptosis and both differentiation), and both pro- (Bad, Bax) and anti-apoptotic (Bcl-2, Bcl-xL) factors in liver samples from patients with different stages of liver disease., Methods: The study concerned 93 patients from 49 cases of chronic hepatitis (CH) (30 HCV and 19 HBV-related), 13 of cirrhosis (CIRR) (10 HCV and 3 HBV-related), 21 of hepatocellular carcinoma (both HCC and peritumoral tissues [PHCC]), and 10 controls (CONTR). Real-time PCR and Western blotting were used to measure mRNA and protein expression levels., Results: Beclin 1 mRNA levels were lower in HCC than in CH (P = 0.010) or CIRR (P = 0.011), and so were the Bcl-xL transcripts (P < 0.0001). Bad mRNA levels were higher in CH and CIRR than in CONTR, while Bax transcripts were increased in all tissues (P = 0.036). PHCC expressed the highest Bcl-2 mRNA levels. HBV-related CH tissues showed significantly higher Bcl-xL and Bad mRNA levels than HCV-related CH (P = 0.003 and P = 0.016, respectively)., Conclusions: High Beclin 1, Bcl-xL and Bad levels in CH and CIRR tissues suggest an interaction between autophagy and apoptosis in the early and intermediate stages of viral hepatitis. In HCC these processes seem to be downregulated, probably enabling the survival and growth of neoplastic hepatocytes.

Background: MicroRNAs expression has been extensively studied in hepatocellular carcinoma but little is known regarding the relationship, if any, with inflammation, production of reactive oxygen species (ROS), host's repair mechanisms and cell immortalization. This study aimed at assessing the extent of oxidative DNA damage (8-hydroxydeoxyguanosine - 8-OHdG) in different phases of the carcinogenetic process, in relation to DNA repair gene polymorphism, telomeric dysfunction and to the expression of several microRNAs, non-coding genes involved in post-transcriptional regulation, cell proliferation, differentiation and death., Methods: Tissue samples obtained either at surgery, [neoplastic (HCC) and adjacent non-cancerous cirrhotic tissues (NCCT)] at percutaneous or laparoscopic biopsy (patients with HCV or HBV-related hepatitis or patients undergoing cholecystectomy) were analysed for 8-OHdG (HPLC-ED), OGG1 (a DNA repair gene) polymorphism (PCR-RFLP), telomerase activity, telomere length (T/S, by RT-PCR), Taqman microRNA assay and Bad/Bax mRNA (RT-PCR). Fifty-eight samples from 29 HCC patients (obtained in both neoplastic and peritumoral tissues), 22 from chronic hepatitis (CH) and 10 controls (cholecystectomy patients - CON) were examined., Results: Eight-OHdG levels were significantly higher in HCC and NCCT than in CH and CON (p=0.001). Telomerase activity was significantly higher in HCC than in the remaining subgroups (p=0.002); conversely T/S was significantly lower in HCC (p=0.05). MiR-199a-b, -195, -122, -92a and -145 were down-regulated in the majority of HCCs while miR-222 was up-regulated. A positive correlation was observed among 8-OHdG levels, disease stage, telomerase activity, OGG1 polymorphisms and ALT/GGT levels. In HCC, miR-92 expression correlated positively with telomerase activity, 8-OHdG levels and Bad/Bax mRNA., Conclusions: The above findings confirm the accumulation, in the progression of chronic liver damage to HCC, of a ROS-mediated oxidative DNA damage, and suggest that this correlates with induction of telomerase activity and, as a novel finding, with over-expression of miR-92, a microRNA that plays a role in both the apoptotic process and in cellular proliferation pathways.

Background: Low temperature plasmas have been proposed in medicine as agents for tissue disinfection and have received increasing attention due to the frequency of bacterial resistance to antibiotics. This study explored whether atmospheric-pressure cold plasma (APCP) generated by a new portable device that ionizes a flow of helium gas can inactivate ocular pathogens without causing significant tissue damage., Methodology/principal Findings: We tested the APCP effects on cultured Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Herpes simplex virus-1, ocular cells (conjunctival fibroblasts and keratocytes) and ex-vivo corneas. Exposure to APCP for 0.5 to 5 minutes significantly reduced microbial viability (colony-forming units) but not human cell viability (MTT assay, FACS and Tunel analysis) or the number of HSV-1 plaque-forming units. Increased levels of intracellular reactive oxygen species (ROS) in exposed microorganisms and cells were found using a FACS-activated 2',7'-dichlorofluorescein diacetate probe. Immunoassays demonstrated no induction of thymine dimers in cell cultures and corneal tissues. A transient increased expression of 8-OHdG, genes and proteins related to oxidative stress (OGG1, GPX, NFE2L2), was determined in ocular cells and corneas by HPLC, qRT-PCR and Western blot analysis., Conclusions: A short application of APCP appears to be an efficient and rapid ocular disinfectant for bacteria and fungi without significant damage on ocular cells and tissues, although the treatment of conjunctival fibroblasts and keratocytes caused a time-restricted generation of intracellular ROS and oxidative stress-related responses.

Recent evidence in multiple sclerosis (MS) suggests that active CMV infection may result in more benign clinical disease. The goal of this pilot study was to determine whether underlying murine CMV (MCMV) infection affects the course of the Theiler's murine encephalitis virus (TMEV) induced murine model of MS. A group of eight TMEV-infected mice were co-infected with MCMV at 2 weeks prior to TMEV infection while a second group of TMEV-infected mice received MCMV two weeks post TMEV. We also used 2 control groups, where at the above time points MCMV was replaced with PBS. Outcome measures included (1) monthly monitoring of disability via rotarod for 8 months; (2) in vivo MRI for brain atrophy studies and (3) FACS analysis of brain infiltrating lymphocytes at 8 months post TMEV infection. Co-infection with MCMV influenced the disease course in mice infected prior to TMEV infection. In this group, rotarod detectable motor performance was significantly improved starting 3 months post-infection and beyond (p≤0.024). In addition, their brain atrophy was close to 30% reduced at 8 months, but this was only present as a trend due to low power (p = 0.19). A significant reduction in the proportion of brain infiltrating CD3+ cells was detected in this group (p = 0.026), while the proportion of CD45+ Mac1+ cells significantly increased (p = 0.003). There was also a strong trend for a reduced proportion of CD4+ cells (p = 0.17) while CD8 and B220+ cell proportion did not change. These findings support an immunomodulatory effect of MCMV infection in this MS model. Future studies in this co-infection model will provide insight into mechanisms which modulate the development of demyelination and may be utilized for the development of novel therapeutic strategies.

Aim: To evaluate whether combination therapy with anti-tumour necrosis factor α (TNFα) antibody and Zn acetate is beneficial in dextran sodium sulphate (DSS) colitis., Methods: Colitis was induced in CD1-Swiss mice with 5% DSS for 7 d. The experimental mice were then randomised into the following subgroups: standard diet + DSS treated (induced colitis group); standard diet + DSS + subcutaneous 25 μg anti-TNFα treated group; Zn acetate treated group + DSS + subcutaneous 25 μg anti-TNFα; standard diet + DSS + subcutaneous 6.25 μg anti-TNFα treated group and Zn acetate treated group + DSS + subcutaneous 6.25 μg anti-TNFα. Each group of mice was matched with a similar group of sham control animals. Macroscopic and histological features were scored blindly. Homogenates of the colonic mucosa were assessed for myeloperoxidase activity as a biochemical marker of inflammation and DNA adducts (8OH-dG) as a measure of oxidative damage., Results: DSS produced submucosal erosions, ulcers, inflammatory cell infiltration and cryptic abscesses which were reduced in both groups of mice receiving either anti-TNFα alone or combined with zinc. The effect was more pronounced in the latter group (vs Zn diet, P < 0.02). Myeloperoxidase activity (vs controls, P < 0.02) and DNA adducts, greatly elevated in the DSS fed colitis group (vs controls, P < 0.05), were significantly reduced in the treated groups, with a more remarkable effect in the group receiving combined therapy (vs standard diet, P < 0.04)., Conclusion: DSS induces colonic inflammation which is modulated by the administration of anti-TNFα. Combining anti-TNFα with Zn acetate offers marginal benefit in colitis severity.

The performance of two field probes (YSI 6600 and TriOS), used for the measurement of in vivo phycocyanin fluorescence, was compared and validated in the laboratory in 2008 and 2009 with cultures of Microcystis aeruginosa and field samples. The background noise of the two probes was low and the detection limits were estimated at 1500 cells mL(-1) for the YSI and 0.69 µg PC L(-1) for the TriOS. The linearity and repeatability of both probes have been excellent. Strong relationships were observed between the in vivo fluorescence and the total cyanobacterial biovolume (R(2) = 0.82 YSI; 0.83 TriOS) or the abundance (R(2) = 0.71 YSI; 0.75 TriOS) of cyanobacteria. However, the difference between cell densities determined by microscopy and measured by the YSI can be very large and has been associated to the variability of cell volume among cyanobacteria. This last observation makes the YSI a qualitative tool if a post-calibration is not done. The analysis of filtrated samples showed that dissolved phycocyanin (extracellular) may represent a significant fluorescence signal. No relationship could be established between the abundance, the total cyanobacterial biovolume or the in vivo fluorescence of phycocyanin and the concentrations of cyanotoxins (R(2) ≤ 0.22).

Herpes simplex virus (HSV) infections are common but there is no vaccine available. We evaluated cationic liposome-DNA complexes (CLDC) as an adjuvant for an HSV gD2 vaccine and compared it to an MPL/Alum adjuvant in a guinea pig model of genital herpes. The addition of CLDC to the gD2 vaccine significantly decreased acute and recurrent disease and most importantly the number of days with recurrent virus shedding compared to gD2 alone. Reductions in these outcomes were also detected when gD2+CLDC was compared to gD2+MPL/Alum. When the vaccine and adjuvants were evaluated as therapeutic vaccines, they were ineffective. CLDC enhanced protection compared to MPL/Alum and is the first vaccine to reduce recurrent virus shedding, a key to decreasing the spread of HSV-2.

Background: Bax inhibitor-1 (BI-1) is an evolutionary conserved endoplasmic reticulum protein that, when overexpressed in mammalian cells, suppresses the apoptosis induced by Bax, a pro-apoptotic member of the Bcl-2 family. The aims of this study were: (1) to clarify the role of intrinsic anti- and pro-apoptotic mediators, evaluating Bax and BI-1 mRNA and protein expressions in liver tissues from patients with different degrees of liver damage; (2) to determine whether HCV and HBV infections modulate said expression., Methods: We examined 62 patients: 39 with chronic hepatitis (CH) (31 HCV-related and 8 HBV-related); 7 with cirrhosis (6 HCV-related and 1 HBV-related); 13 with hepatocellular carcinoma (HCC) [7 in viral cirrhosis (6 HCV- and 1 HBV-related), 6 in non-viral cirrhosis]; and 3 controls. Bax and BI-1 mRNAs were quantified by real-time PCR, and BI-1 protein expression by Western blot., Results: CH tissues expressed significantly higher BI-1 mRNA levels than cirrhotic tissues surrounding HCC (P < 0.0001) or HCC (P < 0.0001). Significantly higher Bax transcripts were observed in HCV-genotype-1-related than in HCV-genotype-3-related CH (P = 0.033). A positive correlation emerged between BI-1 and Bax transcripts in CH tissues, even when HCV-related CH and HCV-genotype-1-related CH were considered alone (P = 0.0007, P = 0.0005 and P = 0.0017, respectively)., Conclusions: BI-1 expression is down-regulated as liver damage progresses. The high BI-1 mRNAs levels observed in early liver disease may protect virus-infected cells against apoptosis, while their progressive downregulation may facilitate hepatocellular carcinogenesis. HCV genotype seems to have a relevant role in Bax transcript expression.

Background: Chronic inflammation is linked to an increased risk of cancer. The molecular mechanisms underlying this correlation have been long investigated and it is well known that the inflammatory cells recruited in the inflamed tissues release chemical mediators, in particular reactive oxygen species (ROS). With respect to digestive systems, ROS have been implicated in a number of pathologies, including Helicobacter pylori-related gastritis, Barrett's esophagus, inflammatory disease of the lower gastrointestinal tract, alcoholic liver disease and several other types of toxic and virus-mediated liver injury. ROS levels within cells and tissues are controlled by numerous antioxidant defense mechanisms, but in inflammation, ROS overproduction exceeds defenses and damage intracellular macromolecules, including nucleic acids, with formation of potentially mutagenic and carcinogenic DNA adducts., Aims: This paper summarizes our own experience investigating the link between inflammation, ROS production and oxidative DNA damage as well as the impact of the above events on cytokine and growth factor release, oncogene activation, telomere instability and microRNA in H. pylori-related gastritis, Barrett's esophagus and, in particular, hepatitis C virus-related liver disease. The paper also describes, at least in part, the complex scenario involving nitric oxide production and its impact in some gastrointestinal diseases, as well as a number of other molecular and biochemical changes related to ROS production and inflammation., Conclusions: The paper falls obviously short of being an exhaustive summary of our understanding, but the data reported are intended as a stimulus to broaden the knowledge on the topic, also in view of the possible therapeutic implications of any advance obtained., (Copyright © 2010 S. Karger AG, Basel.)

IL-10 plays a central role in restraining the vigor of inflammatory responses, but the critical cellular sources of this counter-regulatory cytokine remain speculative in many disease models. Using a novel IL-10 transcriptional reporter mouse, we found an unexpected predominance of B cells (including plasma cells) among IL-10-expressing cells in peripheral lymphoid tissues at baseline and during diverse models of in vivo immunological challenge. Use of a novel B cell-specific IL-10 knockout mouse revealed that B cell-derived IL-10 nonredundantly decreases virus-specific CD8(+) T cell responses and plasma cell expansion during murine cytomegalovirus infection and modestly restrains immune activation after challenge with foreign Abs to IgD. In contrast, no role for B cell-derived IL-10 was evident during endotoxemia; however, although B cells dominated lymphoid tissue IL-10 production in this model, myeloid cells were dominant in blood and liver. These data suggest that B cells are an underappreciated source of counter-regulatory IL-10 production in lymphoid tissues, provide a clear rationale for testing the biological role of B cell-derived IL-10 in infectious and inflammatory disease, and underscore the utility of cell type-specific knockouts for mechanistic limning of immune counter-regulation.

G Protein-coupled receptors (GPCRs) selectivity is an important aspect of drug discovery process, and distinguishing between related receptor subtypes is often the key to therapeutic success. Nowadays, very few valuable computational tools are available for the prediction of receptor subtypes selectivity. In the present study, we present an alternative application of the Support Vector Machine (SVM) and Support Vector Regression (SVR) methodologies to simultaneously describe both A(2A)R versus A(3)R subtypes selectivity profile and the corresponding receptor binding affinities. We have implemented an integrated application of SVM-SVR approach, based on the use of our recently reported autocorrelated molecular descriptors encoding for the Molecular Electrostatic Potential (autoMEP), to simultaneously discriminate A(2A)R versus A(3)R antagonists and to predict their binding affinity to the corresponding receptor subtype of a large dataset of known pyrazolo-triazolo-pyrimidine analogs. To validate our approach, we have synthetized 51 new pyrazolo-triazolo-pyrimidine derivatives anticipating both A(2A)R/A(3)R subtypes selectivity and receptor binding affinity profiles.

Cathepsin B (CATB) and urokinase-type plasminogen activator (UPA) play an important part in cancer invasion and metastasis. The behavior of CATB and UPA has not been evaluated in the same experimental setting in different gastrointestinal tumors and in precancerous lesions. Serum CATB and plasma UPA levels were determined by enzyme-linked immunoadsorbent assay and their sensitivity, specificity, and diagnostic accuracy have been calculated in patients with colorectal (n=72), gastric (n=30), hepatocellular (n=28), and pancreatic cancer (n=15) as well as in gastric epithelial dysplasia (n=25), colorectal adenomas (n=30), and tumor-free control patients (n=44). Serum CATB and plasma UPA antigen concentrations were significantly higher in patients with cancer than in controls. When all tumors were considered, the sensitivity, specificity, and diagnostic accuracy of CATB (89, 86, and 89%) were higher than that of UPA (76, 70, and 74%). CATB demonstrated in all types of tumors a better diagnostic accuracy than UPA. The positive predictive values of CATB (95%) and UPA (89%) may suggest their use in the evaluation of patients with a suspicion of malignancy. CATB and UPA were significantly higher in patients with gastric epithelial dysplasia and colorectal adenomas than in controls. Antigen levels of CATB and UPA were significantly correlated in both cancers and precancerous lesions. At the time of clinical presentation, serum CATB and plasma UPA antigen levels are sensitive indicators of gastrointestinal malignancies. Determination of serum CATB and plasma UPA levels may be useful to identify patients at a higher risk for progression to cancer, who could be subjected to a more strict follow-up protocol.

Phosphorothioated oligonucleotides have a sequence-independent antiviral activity as amphipathic polymers (APs). The activity of these agents against herpesvirus infections in vitro and in vivo was investigated. The previously established sequence-independent, phosphorothioation-dependent antiviral activity of APs was confirmed in vitro by showing that a variety of equivalently sized homo- and heteropolymeric AP sequences were similarly active against herpes simplex virus type 1 (HSV-1) infection in vitro compared to the 40mer degenerate parent compound (REP 9), while the absence of phosphorothioation resulted in the loss of antiviral activity. In addition, REP 9 demonstrated in vitro activity against a broad spectrum of other herpesviruses: HSV-2 (50% effective concentration [EC(50)], 0.02 to 0.06 microM), human cytomegalovirus (EC(50), 0.02 to 0.13 microM), varicella zoster virus (EC(50), <0.02 microM), Epstein-Barr virus (EC(50), 14.7 microM) and human herpesvirus types 6A/B (EC(50), 2.9 to 10.2 microM). The murine microbicide model of genital HSV-2 was then used to evaluate in vivo activity. REP 9 (275 mg/ml) protected 75% of animals from disease and infection when provided 5 or 30 min prior to vaginal challenge. When an acid-stable analog (REP 9C) was used, 75% of mice were protected when treated with 240 mg/ml 5 min prior to infection (P < 0.001), while a lower dose (100 mg/ml) protected 100% of the mice (P < 0.001). The acid stable REP 9C formulation also provided protection at 30 min (83%, P < 0.001) and 60 min (50%, P = 0.07) against disease. These observations suggest that APs may have microbicidal activity and potential as broad-spectrum antiherpetic agents and represent a novel class of agents that should be studied further.

Background: Cathepsin B and L (CATB, CATL), urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 play an important role in colorectal cancer invasion. The tumor marker utility and prognostic relevance of these proteases have not been evaluated in the same experimental setting and compared with that of CEA and CA-19-9., Methods: Protease, CEA and CA 19-9 serum or plasma levels were determined in 56 patients with colorectal cancer, 25 patients with ulcerative colitis, 26 patients with colorectal adenomas and 35 tumor-free control patients. Protease, CEA, CA 19-9 levels have been determined by ELISA and electrochemiluminescence immunoassay, respectively; their sensitivity, specificity, diagnostic accuracy have been calculated and correlated with clinicopathological staging., Results: The protease antigen levels were significantly higher in colorectal cancer compared with other groups. Sensitivity of PAI-1 (94%), CATB (82%), uPA (69%), CATL (41%) were higher than those of CEA or CA 19-9 (30% and 18%, respectively). PAI-1, CATB and uPA demonstrated a better accuracy than CEA or CA 19-9. A combination of PAI-1 with CATB or uPA exhibited the highest sensitivity value (98%). High CATB, PAI-1, CEA and CA 19-9 levels correlated with advanced Dukes stages. CATB (P = 0.0004), CATL (P = 0.02), PAI-1 (P = 0.01) and CA 19-9 (P = 0.004) had a significant prognostic impact. PAI-1 (P = 0.001), CATB (P = 0.04) and CA 19-9 (P = 0.02) proved as independent prognostic variables., Conclusion: At the time of clinical detection proteases are more sensitive indicators for colorectal cancer than the commonly used tumor markers. Determinations of CATB, CATL and PAI-1 have a major prognostic impact in patients with colorectal cancer.

Oxidative DNA damage is thought to play an important part in the pathogenesis of H. pylori-induced mucosal damage. 8-OHdG is a sensitive marker of DNA oxidation and is repaired by a polymorphic glycosylase (OGG1) more effectively than by OGG1-Cys(326). The aims of this study were to ascertain the respective roles of H. pylori, cagA status and OGG1 polymorphism in determining 8-OHdG levels in benign and premalignant stomach diseases and in gastric cancer (GC). The study involved 50 GC patients (for whom both neoplastic tissue and surrounding mucosa were available), 35 with intestinal metaplasia and atrophy (IMA) and 43 controls. H. pylori and cagA status were determined by histology and polymerase chain reaction for urease and cagA. 8-OHdG was assayed using HPLC with an electrochemical detector (HPLC-ED). The OGG1 1245C-->G transversion was identified using RFLP analyses. 8-OHdG levels were significantly higher in GC, with no differences in relation to H. pylori or cagA status. OGG1 polymorphism was documented in 34% of GC (15 Ser/Cys, 2 Cys/Cys). OGG1 1245C-->G polymorphism was detected in 54% of IMA patients, but only 16% of controls (p = 0.0004) and coincided with significantly higher 8-OHdG levels. In the multivariate analysis, 8-OHdG levels were predicted by histotype and OGG1 status. OGG1 1245C-->G polymorphism was common in both GC and IMA, but very rare in controls, and correlated more closely with 8-OHdG levels than do H. pylori infection or cagA status., ((c) 2008 Wiley-Liss, Inc.)

The Fas / Fas-ligand (FasL) system is an important death signal pathway in the liver. An enhanced local inflammatory response prompted by FasL expression, which contributes to neutrophil recruitment and interleukin-1 beta (IL-1beta) release, seems to be crucial to chronic liver damage, persistence of viral infections, and probably initiation and / or promotion of HCC. In order to evaluate the expression of Fas, FasL, and IL-1beta in different stages of human liver disease and to determine whether hepatitis B virus (HBV) and hepatitis C virus (HCV) infections modulate their expression, also in relation to apoptosis, we examined 87 liver samples obtained from patients with: chronic hepatitis (CH) (n.42), cirrhosis (n.9) and hepatocellular carcinoma (HCC) (n.16) and corresponding peritumoural tissues (n.16); histologically-normal liver (n.4) as controls. Fas, FasL and IL-1beta mRNA were quantified using reverse transcriptase-polymerase chain reaction. The apoptotic index was evaluated by TUNEL analysis. Our data showed a progressive Fas / FasL increase from CH to cirrhosis followed by a decline from the latter to HCC. In histological sections apoptosis was detected in HCC. A significant difference emerged between HCV and HBV-related disease for IL-1beta expression only in CH. A significant positive correlation between IL-1beta and FasL in HCV-related disease (P = 0.014) and an inverse correlation between IL-1beta and Fas in HBV-related disease (P = 0.021) were observed. The different pattern of IL-1beta, Fas and FasL expression found in HCV- and HBV-mediated liver disease, points to a different modulation of immune response B and C virus induced, while the decline in Fas / FasL expression in HCC may be related to defence mechanisms adopted by HCC cells against the immune system.