Towards tolerance in liver transplantation.

Life-long immunosuppression has always been considered the key in managing liver graft protection from recipient rejection. However, it is associated with severe adverse effects that lead to increased morbidity and mortality, including infections, cardiovascular diseases, kidney failure, metabolic disorders and de novo malignancies. This explains the great interest that has developed in the concept of tolerance in recent years. The liver, thanks to its marked tolerogenicity, is to be considered a privileged organ: up to 60% of selected patients undergoing liver transplantation could safely withdraw immunosuppression.

Ab initio Everolimus-based Versus Standard Calcineurin Inhibitor Immunosuppression Regimen in Liver Transplant Recipients.

AIM: We designed a retrospective case-control study to determine the efficacy and feasibility of everolimus (EVR) combined with low-dose tacrolimus (Tac) ab initio versus standard-dose Tac after liver transplantation (LT). METHODS: Seventy-one adult LT patients, receiving EVR and low-dose Tac without corticosteroids or induction therapy from postoperative day 1 (EVR group) were compared with a well-matched control group of 61 recipients treated with standard-dose Tac in association with antimetabolite. RESULTS: Baseline characteristics for the two groups were comparable. The overall patient and graft survival rates were similar (P = .908). Liver function was stable during the follow-up. In the EVR group, biopsy-proven acute rejection occurred in two cases (2.8%), whereas chronic rejection occurred in one (1.4%). The EVR group experienced a better renal function already after 2 weeks (estimated glomerular filtration rate: 89.85 [36.46 to 115.3] mL/min/1.73 m2 vs. 68.77 [16.11 to 115.42] mL/min/1.73 m2; P = .013), which was also observed after a median time of 27 months (range, 0 to 82 months) from LT (estimated glomerular filtration rate: 80 [45 to 118.3] mL/min/1.73 m2 vs. 70.9 [45 to 88.4] mL/min/1.73 m2; P = .04). After a median time of 27 months, the EVR group showed lower incidence of arterial hypertension and insulin-dependent diabetes mellitus. CONCLUSION: Ab initio EVR-based immunosuppression could be a valid option immediately after surgery in recipients at high-risk for post-LT renal impairment.

Long-term outcomes of direct acting antivirals in post-transplant advanced hepatitis C virus recurrence and fibrosing cholestatic hepatitis.

Long-term functional outcomes of sofosbuvir-based antiviral treatment were evaluated in a cohort study involving 16 Italian centres within the international compassionate use programme for post-transplant hepatitis C virus (HCV) recurrence. Seventy-three patients with cirrhosis (n=52) or fibrosing cholestatic hepatitis (FCH, n=21) received 24-week sofosbuvir with ribavirin±pegylated interferon or interferon-free sofosbuvir-based regimen with daclatasvir/simeprevir+ribavirin. The patients were observed for a median time of 103 (82-112) weeks. Twelve of 73 (16.4%) died (10 non-FCH, 2 FCH) and two underwent re-LT. Sustained virological response was achieved in 46 of 66 (69.7%): 31 of 47 (66%) non-FCH and 15 of 19 (79%) FCH patients. All relapsers were successfully retreated. Comparing the data of baseline with last follow-up, MELD and Child-Turcotte-Pugh scores improved both in non-FCH (15.3±6.5 vs 10.5±3.8, P<.0001 and 8.4±2.1 vs 5.7±1.3, P<.0001, respectively) and FCH (17.3±5.9 vs 10.1±2.8, P=.001 and 8.2±1.6 vs 5.5±1, P=.001, respectively). Short-treatment mortality was higher in patients with baseline MELD≥25 than in those with MELD<25 (42.9% vs 4.8%, P=.011). Long-term mortality was 53.3% among patients with baseline MELD≥20 and 7.5% among those with MELD<20 (P<.0001). Among deceased patients 75% were Child-Turcotte-Pugh class C at baseline, while among survivors 83.9% were class A or B (P<.0001). Direct acting antivirals-based treatments for severe post-transplant hepatitis C recurrence, comprising fibrosing cholestatic hepatitis, significantly improve liver function, even without viral clearance and permit an excellent long-term survival. The setting of severe HCV recurrence may require the identification of "too-sick-to-treat patients" to avoid futile treatments.

Optimal efficacy of interferon-free HCV retreatment after protease inhibitor failure in real life.

OBJECTIVES: First-generation protease-inhibitors (PIs) have suboptimal efficacy in GT-1 patients with advanced liver disease, and patients experiencing treatment failure may require urgent retreatment. Our objective was to analyse the real-life efficacy of interferon (IFN)-free retreatment after PI-failure, and the role of genotypic-resistance-testing (GRT) in guiding retreatment choice. METHODS: In this multi-centre observational study, patients retreated with IFN-free regimens after first-generation PI-failure (telaprevir-boceprevir-simeprevir) were included. Sustained-virological-response (SVR) was evaluated at week 12 of follow-up. GRT was performed by population-sequencing. RESULTS: After PI-failure, 121 patients (cirrhotic=86.8%) were retreated following three different strategies: A) with 'GRT-guided' regimens (N=18); B) with 'AASLD/EASL recommended, not GRT-guided' regimens (N=72); C) with 'not recommended, not GRT-guided' regimens (N=31). Overall SVR rate was 91%, but all 18 patients treated with 'GRT-guided' regimens reached SVR (100%), despite heterogeneity in treatment duration, use of PI and ribavirin, versus 68/72 patients (94.4%) receiving 'AASLD/EASL recommended, not GRT-guided' regimens. SVR was strongly reduced (77.4%) among the 31 patients who received a 'not recommended, not GRT-guided regimen' (p <0.01). Among 37 patients retreated with a PI, SVR rate was 89.2% (33/37). Four GT-1a cirrhotic patients failed an option (C) simeprevir-containing treatment; three out of four had a baseline R155K NS3-RAS. All seven patients treated with paritaprevir-containing regimens reached SVR, regardless of treatment duration and performance of a baseline-GRT. CONCLUSION: Retreatment of PI-experienced patients can induce maximal SVR rates in real life. Baseline-GRT could help to optimize retreatment strategy, allowing PIs to be reconsidered when chosen after a RASs evaluation.

HCV NS3 sequencing as a reliable and clinically useful tool for the assessment of genotype and resistance mutations for clinical samples with different HCV-RNA levels.

OBJECTIVES: This study aims to evaluate the reliability and clinical utility of NS3 sequencing in hepatitis C virus (HCV) 1-infected patients who were candidates to start a PI-containing regimen. METHODS: NS3 protease sequencing was performed by in-house-developed HCV-1 subtype-specific protocols. Phylogenetic analysis was used to test sequencing reliability and concordance with previous genotype/subtype assignment by commercial genotyping assays. RESULTS: Five hundred and sixty-seven HCV plasma samples with quantifiable HCV-RNA from 326 HCV-infected patients were collected between 2011 and 2014. Overall, the success rate of NS3 sequencing was 88.9%. The success rate between the two subtype protocols (HCV-1a/HCV-1b) was similarly high for samples with HCV-RNA >3 log IU/mL (>92% success rate), while it was slightly lower for HCV-1a samples with HCV-RNA ≤3 log IU/mL compared with HCV-1b samples. Phylogenetic analysis confirmed the genotype/subtype given by commercial genotyping assays in 92.9% (303/326) of cases analysed. In the remaining 23 cases (7.1%), 1 was HCV-1g (previously defined as subtype 1a), 1 was HCV-4d (previously defined as genotype 1b) and 1 was HCV-1b (previously defined as genotype 2a/2c). In the other cases, NS3 sequencing precisely resolved the either previous undetermined/discordant subtype 1 or double genotype/subtype assignment by commercial genotyping assays. Resistance-associated variants (RAVs) to PI were detected in 31.0% of samples. This prevalence changed according to PI experience (17.1% in PI-naive patients versus 79.2% in boceprevir/telaprevir/simeprevir-failing patients). Among 96 patients with available virological outcome following boceprevir/telaprevir treatment, a trend of association between baseline NS3 RAVs and virological failure was observed (particularly for HCV-1a-infected patients: 3/21 failing patients versus 0/22 achieving sustained virological response; P = 0.11). CONCLUSIONS: HCV-NS3 sequencing provides reliable results and at the same time gives two clinically relevant pieces of information: a correct subtype/genotype assignment and the detection of variants that may interfere with the efficacy of PI.

Safety of Complete and Sustained Prophylaxis Withdrawal in Patients Liver-transplanted for Hepatitis B Virus-related Cirrhosis at Low Risk of Hepatitis B Virus Recurrence.

BACKGROUND: HBV (hepatitis B virus) reactivation after liver transplantation may be related to persistence of covalently closed circular (ccc) DNA. We investigated the safety of HBV prophylaxis withdrawal in selected HBV transplanted patients. METHODS: Thirty patients transplanted 64-195 months earlier (23 males, median age 56 years), HBsAg-positive, HBeAg, and HBV-DNA-negative at transplant (43% HCV/HDV coinfected), with undetectable intrahepatic total and ccc-DNA were enrolled. All patients underwent HBIg withdrawal and continued lamivudine with monthly HBsAg and HBV-DNA monitoring and sequential liver biopsies. Those with confirmed intrahepatic total and ccc-DNA undetect-ability 24 weeks after stopping HBIg also underwent lamivudine withdrawal and were followed up without prophylaxis. RESULTS: Twenty-five patients did not exhibit signs of HBV recurrence after prophylaxis withdrawal (median follow-up 28.7 months, range 22-42). Five patients became HBsAg-positive: one early after HBIg withdrawal, the other four after HBIg and lamivudine withdrawal. None of these patients experienced clinically relevant events. In the first patient, HBIg were reinstituted with prompt HBsAg negativization. Of the other four, one remained HBsAg-positive with detectable HBV-DNA and mild alanine transaminase elevation and was successfully treated with tenofovir. In the remaining three, HBsAg positivity was transient and followed by anti-HBs se-roconversion; thus no antiviral treatment was needed. CONCLUSION: Patients with undetectable HBV viremia at transplant and no evidence of intrahepatic total and ccc-DNA may safely undergo cautious weaning of prophylaxis, showing the low rate of HBV recurrence after a 2-year follow-up. Undetectability of intrahepatic ccc-DNA may help to identify patients at low risk of recurrence; yet studies with longer follow-up are needed.

In vitro activity of Tachyplesin III alone and in combination with terbinafine against clinical isolates of dermatophytes.

Aim of our study was to investigate the in vitro effects of Tachyplesin III (TP), a potent disulfide-linked peptide, in dermatophytes infections, with respect to or in combination with terbinafine (TERB), against 20 clinical isolates of dermatophytes belonging to four species. A broth microdilution method following the CLSI recommendations (M38-A) was used for testing drugs alone and in combination. TERB MICs were significantly lower than those observed for TP (p<0.001). Testing for antifungal agents in combination was performed for TERB with TP for all the 20 isolates. TERB activity in combination with TP showed indifferent activity for 14 of the 20 isolates (70%); synergic activity for 6 of the 20 isolates (30%); no antagonistic activity was observed. Further experiments were conducted with Microsporum canis 133, Trichophyton rubrum 62 and Trichophyton mentagrophytes 91 for fungal biomass. TP and TERB did not show a significant growth reduction compared to the control against T. mentagrophytes and T. rubrum. A significant difference of growth reduction both for TP and TERB compared to controls was observed for M. canis (p<0.01). In conclusion our study demonstrated that Tachyplesin III has potential activity against dermatophytes. In addition, we observed that the in vitro activity of Tachyplesin III can be enhanced upon combination with terbinafine. Synergy could permit lower doses of the individual antifungal agents to be used more effectively and/or safely.

Recurrent myocardial ischaemia during combination antiviral therapy in a patient with chronic hepatitis C and normal aminotransferase levels.

A 63-year-old man with chronic hepatitis C and persistently normal alanine aminotransferase levels was treated with peginterferon alpha-2a (180 microg weekly) and ribavirin (800 mg daily). Hepatitis C virus-ribonucleic acid was negative at week 4. After 12 weeks of therapy, haemoglobin levels had decreased by 3.5mg/dL, and he developed a syncope episode with electrocardiographic signs of myocardial ischaemia. Antiviral treatment was stopped and the ischaemia-like electrocardiographic changes resolved completely within 2 months. Eight months later, because of the previous rapid virological response and patient motivation, he was treated again with peginterferon and ribavirin. Baseline and weekly electrocardiographic recordings were obtained during treatment. At week 4 hepatitis C virus-ribonucleic acid was negative. At week 8, when the haemoglobin levels had decreased by 3.4 mg/dL, the patient developed the same ischaemia-like changes that occurred during the previous treatment. Antiviral therapy was stopped and the electrocardiographic ischaemia-like changes disappeared after 1 month. The patient neither had a history of previous cardiovascular diseases, nor evidence of current disease at myocardial scintigraphy. However, a coronary microvessel spasm, possibly related to drug-toxicity and/or anaemia could not be excluded. This case indicates the need of strict electrocardiographic monitoring in elderly patients undergoing treatment with peginterferon and ribavirin.

Extended double-dosage HBV vaccination after liver transplantation is ineffective, in the absence of lamivudine and prior wash-out of human Hepatitis B immunoglobulins.

BACKGROUND: The recommended prophylaxis against hepatitis B virus recurrence after liver transplantation based on hepatitis B immunoglobulins and lamivudine is highly expensive. A recent study reported a significant anti-HBs (antibodies against hepatitis B surface antigen) response after a reinforced vaccination against hepatitis B virus, a result not confirmed in a study from our group. Concomitant lamivudine treatment and the achievement of complete washout of anti-hepatitis B-specific immunoglobulin prior to vaccination in our study could explain the contradiction. AIMS: To test the efficacy of a reinforced anti-hepatitis B virus vaccination schedule without lamivudine and without previous anti-hepatitis B-specific immunoglobulin washout. METHODS: A double reinforced course of S-recombinant hepatitis B virus vaccination was given to seven male patients who were transplanted for hepatitis B virus-related cirrhosis. Vaccination consisted of two cycles of three intramuscular double doses (40 microg), given at month 0, 1, 2, and 3, 4, 5, respectively. The first dose was given 2 weeks after stopping lamivudine and the intravenous administration of anti-HBs immunoglobulins. The latter was continued throughout the study and follow-up period to maintain an anti-HBs titre >100 IU/L. RESULTS: At the end of both the first and the second vaccination cycle none of the patients developed an anti-HBs titre greater than the basal anti-HBs titre. CONCLUSION: These data confirm and expand our previous data on the lack of effectiveness of conventional recombinant hepatitis B virus vaccination in liver transplant recipients.

Extended HBV vaccination in liver transplant recipients for HBV-related cirrhosis: report of two successful cases.

The effectiveness of hepatitis B virus vaccination in liver transplant recipients for hepatitis B virus-related end-stage liver disease is controversial. We report two successful cases, who developed sustained protection after long-term vaccination. Case 1. A 58-year-old male, transplanted 9 years earlier, received three intramuscular monthly doses of 40 microg of recombinant S vaccine and developed an anti-hepatitis B surface titre of 154 IU/L. After an additional 40 microg dose, he reached an anti-hepatitis B surface peak of 687 IU/L and then maintained a "protective" titre (>100 IU/L) without further vaccinations for the next 40 months. At this time, revaccination with three monthly doses of 40 microg resulted in an anti-hepatitis B surface titre greater than 25,000 IU/L, sustained over time. Case 2. A 56-year-old woman, transplanted 8 years earlier, first received three intramuscular monthly doses of 40 microg of S vaccine without developing any detectable anti-HBs. She was then given multiple intradermal vaccine doses which resulted in a titre of 37 IU/L. Next, after readministration of three 40 microg intramuscular monthly doses, she developed an anti-HBs titre of 280 IU/L. In the following 4 years, the anti-HBs titre dropped below 100 IU/L four times (at month 20, 30, 38 and 44) and readministration of single 40 microg doses of vaccine was always sufficient to restore a protective titre. Conclusion. Extended HBV vaccination may afford valid protection against HBV recurrence in selected liver transplant recipients.