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Primary sclerosing cholangitis (PSC) and Primary biliary cholangitis (PBC) are chronic inflammatory biliary diseases characterized by progressive damage of the bile ducts, resulting in hepatobiliary fibrosis and cirrhosis. Currently, specific biomarkers that allow to distinguish between PSC and PBC do not exist. In this study, we examined the salivary proteome by carrying out a comprehensive and non-invasive screening aimed at highlighting possible quali-quantitative protein deregulations that could be the starting point for the identification of effective biomarkers in future. Saliva samples collected from 6 PBC patients were analyzed using a liquid chromatography–tandem mass spectrometry technique, and the results were compared with those previously obtained in the PSC group. We identified 40 proteins as significantly deregulated in PSC patients compared to the PBC group. The Gene Ontology and pathway analyses highlighted that several proteins (e.g., small integral membrane protein 22, cofilin-1, macrophage-capping protein, plastin-2, and biliverdin reductase A) were linked to innate immune responses and actin cytoskeleton remodeling, which is a critical event in liver fibrosis and cancer progression. These findings provide new foundations for a deeper understanding of the pathophysiology of PSC and demonstrate that saliva is a suitable biological sample for obtaining proteomic fingerprints useful in the search for biomarkers capable of discriminating between the two cholestatic diseases.

Inter-instrument variations of liver stiffness measure (LSM) by transient elastography might be problematic in clinical practice. LSMs provided by 2 different systems were compared in outpatients with chronic liver disease (CLD). In 777 consecutive asymptomatic outpatients admitted at the Hepatology Unit of Pisa University Hospital the agreement of LSMs measured by FibroScan ® (Echosens, France) and FT9000 (Hisky Medical, China) (LSM Fibroscan /LSM FT9000 ) was tested using Pearson correlation and Bland-Altman analyses (BAA). Delta FT9000-FibroScan ® LSM (LSM-D) variations were analysed according to clinic-pathologic characteristics. ALT/AST/GGT and platelets-counts/portal-vein-caliber/spleen-bipolar-diameter were used as proxies of necro-inflammatory and portal hypertension, respectively. LSM FT9000 and LSM Fibroscan were highly correlated ( r  = 0.781, p  < 0.001) overall, but agreement varied according to BMI (normal-weight r  = 0.841, over-weight r  = 0.747, obese r  = 0.647, p  < 0.001). LSM showed a + 0.52 kPa bias ( p  = 0.01) with − 11/12 kPa as 95% Limit of agreement, with higher values measured by LSM FT9000 below 10 kPa and lower values above 20 kPa. In bivariate analysis LSM-D correlated with LSM Fibroscan and AST (β = 0.002, p  = 0.001 with significant interaction, p  = 0.018) and was associated with spleen-bipolar-diameter (β = 0.033, p  = 0.027) and platelets-count (β=-0.008, p  = 0.018) with significant interaction with LSM Fibroscan ( p  < 0.001). LSM Fibroscan and LSM FT9000 are strongly correlated overall with optimal agreement around 10 kPa but limited at ends of the dynamic range: LSM FT9000 provides higher values below 10 kPa whereas LSM Fibroscan higher values over 20 kPa with different measures distribution. LSM discrepancies associate with anthropometric characteristics, necro-inflammatory activity and portal hypertension proxies. These findings suggest the need of systems specific cut-offs in clinical practice.

The currently available antiviral treatments (Peg-Interferon-α and Nucleos(t)ide Analogues, NA) for chronic hepatitis B (CHB) achieve a functional cure (serum HBsAg and HDV-DNA clearance) of HBV infection in a limited number of patients. Nevertheless, the continuous pharmacological suppression of viral replication by NA halts liver disease progression lowering the risk of HCC development and improving the survival. In the near future, to fully exploit the potential of old and new drugs for HBV treatment a personalized approach to the patients will be required according to an accurate definition of their virologic, immunologic and clinical profile.

The virus/host interplay mediates liver pathology in chronic HBV infection. MiRNAs play a pivotal role in virus/host interactions and are detected in both serum and HBsAg-particles, but studies of their dynamics during chronic infection and antiviral therapy are missing. We studied serum miRNAs during different phases of chronic HBV infection and antiviral treatment. MiRNAs were profiled by miRCURY-LNA-Universal-RT-miRNA-PCR (Exiqon-A/S) and qPCR-panels-I/II-739-miRNA-assays and single-RT-q-PCRs. Two cohorts of well-characterized HBsAg-carriers were studied (median follow-up 34-52 months): a) training-panel (141 sera) and HBsAg-particles (32 samples) from 61 HBsAg-carriers and b) validation-panel (136 sera) from 84 carriers. Thirty-one miRNAs were differentially expressed in inactive-carriers (IC) and chronic-hepatitis-B (CHB) with the largest difference for miR-122-5p, miR-99a-5p and miR-192-5p (liver-specific-miRNAs), over-expressed in both sera and HBsAg-particles of CHB (ANOVA/U-test p-values: <0.000001/0.000001; <0.000001/0.000003; <0.000001/0.000005, respectively) and significantly down-regulated during- and after-treatment in sustained-virological-responders (SVR). MiRNA-profiles of IC and SVR clustered in the heatmap. Liver-miRNAs were combined with miR-335, miR-126 and miR-320a (internal controls) to build a MiR-B-Index with 100% sensitivity, 83.3% and 92.5% specificity (-1.7 cut-off) in both training and validation cohorts to identify IC. MiR-B-Index (-5.72, -20.43/14.38) correlated with ALT (49, 10/2056 U/l, ρ = -0.497, p<0.001), HBV-DNA (4.58, undetectable/>8.3 Log10 IU/mL, ρ = -0.732, p<0.001) and HBsAg (3.40, 0.11/5.49 Log10 IU/mL, ρ = -0.883, p<0.001). At multivariate analysis HBV-DNA (p = 0.002), HBsAg (p<0.001) and infection-phase (p<0.001), but not ALT (p = 0.360) correlated with MiR-B-Index. In SVR to Peg-IFN/NUCs MiR-B-Index improved during-therapy and post-treatment reaching IC-like values (5.32, -1.65/10.91 vs 6.68, 0.54/9.53, p = 0.324) beckoning sustained HBV-immune-control earlier than HBsAg-decline. Serum miRNA profile change dynamically during the different phases of chronic HBV infection. We identified a miRNA signature associated with both natural-occurring and therapy-induced immune control of HBV infection. The MiR-B-Index might be a useful biomarker for the early identification of the sustained switch from CHB to inactive HBV-infection in patients treated with antivirals.

miRNAs circulating in whole serum and HBsAg-particles are differentially expressed in chronic hepatitis B (CHB) and HBeAg-negative-HBV infection (ENI); their profiles are unknown in chronic hepatitis D (CHD). Serum- and HBsAg-associated miRNAs were analyzed in 75 subjects of 3 well-characterized groups (CHB 25, CHD 25, ENI 25) using next-generation sequencing (NGS). Overall miRNA profiles were consonant in serum and HBsAg-particles but significantly different according to the presence of hepatitis independently of Hepatitis D Virus (HDV)-co-infection. Stringent (Bonferroni Correction < 0.001) differential expression analysis showed 39 miRNAs upregulated in CHB vs. ENI and 31 of them also in CHD vs. ENI. miRNA profiles were coincident in CHB and CHD with only miR-200a-3p upregulated in CHB. Three miRNAs (miR-625-3p, miR-142-5p, and miR-223-3p) involved in immune response were upregulated in ENI. All 3 hepatocellular miRNAs of MiR-B-Index (miR-122-5p, miR-99a-5p, miR-192-5p) were overexpressed in both CHB and CHD patients. In conclusion, CHD and CHB patients showed highly similar serum miRNA profiling that was significantly different from that of individuals with HBeAg-negative infection and without liver disease.

Non invasive immunologic markers of virus-induced liver disease are unmet needs. We tested the clinical significance of quantitative total and IgM-anti-HBc in well characterized chronic-HBsAg-carriers. Sera (212) were obtained from 111 HBsAg-carriers followed-up for 52 months (28-216) during different phases of chronic-HBV-genotype-D-infection: 10 HBeAg-positive, 25 inactive-carriers (HBV-DNA≤2000IU/ml, ALT<30U/L), 66 HBeAg-negative-CHB-patients and 10 with HDV-super-infection. In 35 patients treated with Peg-IFN±nucleos(t)ide-analogues (NUCs) sera were obtained at baseline, end-of-therapy and week-24-off-therapy and in 22 treated with NUCs (for 60 months, 42-134m) at baseline and end-of-follow-up. HBsAg and IgM-anti-HBc were measured by Architect-assays (Abbott, USA); total-anti-HBc by double-antigen-sandwich-immune-assay (Wantai, China); HBV-DNA by COBAS-TaqMan (Roche, Germany). Total-anti-HBc were detectable in all sera with lower levels in HBsAg-carriers without CHB (immune-tolerant, inactive and HDV-superinfected, median 3.26, range 2.26-4.49 Log10 IU/ml) versus untreated-CHB (median 4.68, range 2.76-5.54 Log10 IU/ml), p<0.0001. IgM-anti-HBc positive using the chronic-hepatitis-cut-off\" (0.130-S/CO) were positive in 102 of 212 sera (48.1%). Overall total-anti-HBc and IgM-anti-HBc correlated significantly (p<0.001, r=0.417). Total-anti-HBc declined significantly in CHB patients with response to Peg-IFN (p<0.001) and in NUC-treated patients (p<0.001); the lowest levels (median 2.68, range 2.12-3.08 Log10 IU/ml) were found in long-term responders who cleared HBsAg subsequently. During spontaneous and therapy-induced fluctuations of CHB (remissions and reactivations) total- and IgM-anti-HBc correlated with ALT (p<0.001, r=0.351 and p=0.008, r=0.185 respectively). Total-anti-HBc qualifies as a useful marker of HBV-induced-liver-disease that might help to discriminate major phases of chronic HBV infection and to predict sustained response to antivirals.

Background: Orphan drugs are used for the diagnosis, prevention and treatment of rare diseases that, in the European Union, are defined as disorders affecting no more than 5 persons in 10,000. So far, a total of around 800 orphan medicinal products have been approved by the European Medicines Agency, however the utilization profile of orphan drugs has yet to be explored. This study aimed at assessing the utilization profile of orphan drugs authorized for marketing by the Italian Medicines Agency using population-based data. Methods: A total of 21 orphan drugs used in outpatient settings, approved in the European Union before or during the 2008–2018 period and involving 15 rare diseases, were included in the study. The monitored population included patients with one of the conditions surveilled by the population-based Tuscany Registry of Rare Diseases and diagnosed between 2000–2018. A multi-database approach was applied, by linking data from the registry with information collected in drug prescriptions databases. The prevalence and intensity of use were estimated for the selected orphan drugs and other non-orphan medications, used to treat the same rare disease and for which a change in the prevalence of use was hypothesized after authorization of the orphan drug. Results: For some diseases (acquired aplastic anemia, tuberous sclerosis complex, most metabolic diseases) a low prevalence of orphan drugs use was observed (range between 1.1–12.5%). Conversely, orphan drugs were frequently used in hemophilia B, Wilson disease and idiopathic pulmonary fibrosis (maximum of 78.3, 47.6 and 41.8%, respectively). For hemophilia B and Leber’s hereditary optic neuropathy, there are currently no other medications used in clinical practice in addition to orphan drugs. Six orphan drugs were used for the treatment of pulmonary arterial hypertension, appearing the elective therapy for this disease, albeit with different utilization profiles (range of prevalence 1.7–55.6%). Conclusion: To the best of our knowledge, this is the first study investigating the utilization profile of orphan drugs prescribed in a defined geographical area, and providing relevant information to monitor over time potential changes in the prevalence of these medications as well as in the health care decision making.

A man with hepatitis B infection was admitted to Pisa University Hospital for hepatological evaluation, which revealed multiple cystic lesions and suggested a cirrhotic evolution. Treatment with Entecavir 0.5 mg/day was started, resulting in rapid viral load suppression and alanine aminotransferase normalization. After 10 years, imaging documented a single nodule of hepatocellular carcinoma (HCC), and a robot-assisted nodule resection was performed. One year later, HCC recurrence prompted orthotopic liver transplantation, during which the patient died because of the sudden rupture of the donor’s organ and rapid multiorgan deterioration before retransplantation. During post-mortem liver examination, adult worms were evidenced within large biliary ducts, suggesting infection with Opisthorchis or Clonorchis spp. flukes. Sequencing of the ITS2 locus, following PCR amplification of DNA extracted from liver tissue, revealed 100% identity with the reference sequence of O. felineus. Infection of the patient with O. felineus was confirmed by the presence of specific IgG detected by ELISA in the patient’s sera. Two major alkaline phosphatase serum levels peaks observed during the first two years of antiviral therapy support the hypothesis that O. felineus infection worsened liver function. This case report highlights the importance of a very careful screening of parasitic infections in solid organ transplantation candidates.

ObjectiveSelected populations of patients with chronic hepatitis B (CHB) may benefit from a combined use of pegylated interferon-alpha (pegIFN-α) and nucleos(t)ides (NUCs). The aim of our study was to assess the immunomodulatory effect of pegIFN-α on T and natural killer (NK) cell responses in NUC-suppressed patients to identify cellular and/or serological parameters to predict better T cell-restoring effect and better control of infection in response to pegIFN-α for a tailored application of IFN-α add-on.Design53 HBeAg-negative NUC-treated patients with CHB were randomised at a 1:1 ratio to receive pegIFN-α-2a for 48 weeks, or to continue NUC therapy and then followed up for at least 6 months maintaining NUCs. Serum hepatitis B surface antigen (HBsAg) and hepatitis B core‐related antigen (HBcrAg) levels as well as peripheral blood NK cell phenotype and function and HBV-specific T cell responses upon in vitro stimulation with overlapping HBV peptides were measured longitudinally before, during and after pegIFN-α therapy.ResultsTwo cohorts of pegIFN-α treated patients were identified according to HBsAg decline greater or less than 0.5 log at week 24 post-treatment. PegIFN-α add-on did not significantly improve HBV-specific T cell responses during therapy but elicited a significant multispecific and polyfunctional T cell improvement at week 24 post-pegIFN-α treatment compared with baseline. This improvement was maximal in patients who had a higher drop in serum HBsAg levels and a lower basal HBcrAg values.ConclusionsPegIFN-α treatment can induce greater functional T cell improvement and HBsAg decline in patients with lower baseline HBcrAg levels. Thus, HBcrAg may represent an easily and reliably applicable parameter to select patients who are more likely to achieve better response to pegIFN-α add-on to virally suppressed patients.