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.

sTRAIL coupled to liposomes improves its pharmacokinetic profile and overcomes neuroblastoma tumour resistance in combination with Bortezomib.

Neuroblastoma (NB), the most common and deadly extracranial solid tumour of childhood, represents a challenging in paediatric oncology. Soluble tumour necrosis factor (TNF)-related apoptosis-inducing ligand (sTRAIL) is a cancer cell-specific molecule exerting remarkable anti-tumour activities against paediatric malignancies both in vitro and in preclinical settings. However, due to its too fast elimination and to the undesired related side effects, the improvement of sTRAIL in vivo bioavailability and the specific delivery to the tumour is mandatory for increasing its therapeutic efficacy. In this manuscript, we developed an innovative pegylated liposomal formulation carrying the sTRAIL at the outer surface (sTRAIL-SL) with the intent to improve its serum half-life and increase its efficacy in vivo, while reducing side effects. Furthermore, the possibility to combine sTRAIL-SL with the proteasome inhibitor Bortezomib (BTZ) was investigated, being BTZ able to sensitize tumour cells toward TRAIL-induced apoptosis. We demonstrated that sTRAIL preserved and improved its anti-tumour activity when coupled to nanocarriers. Moreover, sTRAIL-SL ameliorated its PK profile in blood allowing sTRAIL to exert a more potent anti-tumour activity, which led, upon BTZ priming, to a statistically significant enhanced life spans in two models of sTRAIL-resistant NB-bearing mice. Finally, mechanistic studies indicated that the combination of sTRAIL with BTZ sensitized sTRAIL-resistant NB tumour cells to sTRAIL-induced cell death, both in vitro and in vivo, through the Akt/GSK3/ß-catenin axis-dependent mechanism. In conclusion, our results suggest that sTRAIL-SL might be an efficient vehicle for sTRAIL delivery and that its use in clinic, in combination with BTZ, might represent an adjuvant strategy for the treatment of stage IV, sTRAIL-resistant, NB patients.

Liver transplantation for hepatitis B and C virus-related cirrhosis: mid-term results.

Hepatitis C virus (HCV) recurrence after orthotopic liver transplantation (OLT) is almost universal; cirrhosis develops in up to 30% of cases. Currently there is interest in the midterm outcomes of HCV patients with concomitant hepatitis B virus (HBV) infection among OLT recipients. We therefore retrospectively analyzed our database of patients who underwent OLT for HCV-HBV-related cirrhosis. Between April 1992 and December 2008, 350 patients underwent OLT, including 20 (5.7%) transplanted for HBV-HCV cirrhosis. We assessed patient and graft survivals at 1 and 5 years, as well as the progression of fibrosis. Protocol liver biopsies were available yearly after OLT. The survival curves were analyzed by the Kaplan-Meier approach and chronic hepatitis evaluated according to the Ishak scoring system. At a median follow-up of 68.4 +/- 53 months, the 1- and 5-year patient and graft survival rates were 80% and 70%, respectively. The 5-year fibrosis progression rate was 0.17 +/- 0.08 units of fibrosis. The only patient who developed histologic cirrhosis within 10 years of follow-up showed a lamivudine-resistant HBV recurrence. Patients transplanted for HBV-HCV coinfection showed a lower fibrosis progression rate compared with HCV monoinfected subjects.

Recent advances in targeted anti-vasculature therapy: the neuroblastoma model.

Novel anti-vasculature strategies that are emerging for the treatment of cancer and for the inhibition of angiogenesis may be a promising new tool for the adjuvant therapy of malignant tumours. Over the last fifteen years, several reports have been published concerning the relationship between tumour progression and angiogenesis in experimental models of neuroblastoma in vitro and in vivo. Moreover, a high vascular index in neuroblastoma correlates with poor prognosis, suggesting dependence of aggressive tumour growth on active angiogenesis. Here, we present an overview of the most recent advances in anti-vasculature therapy of neuroblastoma, and describe some preclinical results as well as future perspectives.

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.

13C-methacetin breath test for monitoring hepatic function in cirrhotic patients before and after liver transplantation.

BACKGROUND: In patients with chronic liver disease, the measurement of liver function is critical for monitoring disease progression, predicting the prognosis and choosing therapeutic strategies. The 13C-methacetin breath test is a simple, non-invasive diagnostic tool based on an inexpensive, non-toxic substance, which allows the accurate measurement of liver functional reserve. AIM: To investigate the 13C-methacetin breath test as a tool to monitor hepatic function in liver transplant candidates and recipients. METHODS: Twenty-eight cirrhotic patients listed for orthotopic liver transplantation and 10 healthy controls were studied. The 13C-methacetin breath test (75 mg per os) was performed at baseline and at 12-week intervals. Intra-operative measurements were obtained during the liver transplantation procedure in nine patients. Results were expressed as the 13C-methacetin cumulative oxidation percentage 45 min after substrate ingestion. RESULTS: The mean 13C-methacetin cumulative oxidation at 45 min was 16.4 +/- 3.5% in healthy controls and 5.4 +/- 4.2% in cirrhotic patients at the time of listing. In 11 patients who underwent successful liver transplantation, mean oxidation increased from 3.3 +/- 1.6% before transplantation to 17.0 +/- 5.2% at 6 months of follow-up. Variations in methacetine oxidation were closely related to the recovery of liver function. The mean intra-operative 13C-methacetin cumulative oxidation increased from 0.1% during the anhepatic phase to 3.7 +/- 2.0% 2 h after reperfusion. CONCLUSIONS: The 13C-methacetin breath test is a simple and potentially useful tool for monitoring hepatic function in cirrhotic patients listed for liver transplantation, and during the intra-operative and post-operative phases.

Protective role of tauroursodeoxycholate during harvesting and cold storage of human liver: a pilot study in transplant recipients.

BACKGROUND: Ischemia-reperfusion injury is a major cause of early graft dysfunction after liver transplantation. Tauroursodeoxycholic acid (TUDCA), a natural amidated hydrophilic bile salt, protects from cholestasis and hepatocellular damage in a variety of experimental models, as well as from ischemia-reperfusion injury. We investigated in the human liver transplantation setting the effect of the addition of TUDCA at time of liver harvesting and cold storage on the intra- and postoperative enzyme release and liver histopathology at the end of cold storage, at reperfusion, and 7 days after transplantation. METHODS: Eighteen patients undergoing elective liver transplantation were studied, including 6 serving as controls. In six patients, TUDCA was added to the University of Wisconsin solution used during harvesting and cold storage, to reach final concentrations of 2 mM. In three of these patients, TUDCA (3 g) was infused in the portal vein of the donor before organ explantation; in the other three cases, TUDCA was given through both routes. RESULTS: The use of TUDCA did not cause adverse events. The release of aspartate aminotransferase in the inferior vena cava blood during liver flushing was significantly lower (P=0.05) in TUDCA-treated than in control grafts, as were cytolytic enzyme levels in peripheral blood during the first postoperative week (P<0.02). At electron microscopy, an overt endothelial damage (cytoplasmic vacuolization, cell leakage, and destruction with exposure of hepatocytes to the sinusoidal lumen) was invariably found in control grafts, both at reperfusion and at day 7 after transplant. These features were significantly ameliorated by TUDCA (P<0.001). Several ultrastructural cytoplasmic abnormalities of hepatocytes were seen. Among these, damage to mitochondria matrix and crystae was significantly reduced in TUDCA-treated versus control grafts (P<0.01). Mild to severe damage of bile canaliculi was a constant feature in control biopsies, with dilatation of canalicular lumen and loss of microvilli. Both these abnormalities were markedly ameliorated (P<0.001 by TUDCA). The best preservation was observed when TUDCA was given through both routes. CONCLUSIONS: The use of TUDCA during harvesting and cold storage of human liver is associated with significant protection from ischemia-reperfusion injury. The clinical significance of this findings must be studied.