High frequency of multiclass HCV resistance-associated mutations in patients failing direct-acting antivirals: real-life data.

BACKGROUND: Direct-acting antiviral (DAA) therapy has dramatically increased sustained virological response rates in HCV-infected patients. However, resistance-associated substitutions (RAS) interfering with NS3- and NS5A-targeted therapy, still emerge. This real-life study analysed the type and frequency of RAS in rare cases of patients failing DAA regimens in 12 clinical centres in Israel. METHODS: Blood samples and clinical data from 49 patients who failed various DAAs were collected. RAS identified in the NS3 and NS5A regions by population (Sanger) and next-generation sequencing (NGS) were compared by treatment regimen and HCV subtypes. RESULTS: The majority (71.4%, 35/49) of patients were infected with the genotype (GT)1b strain, while 12.2% (6/49) carried GT1a and 14.3% GT3a/b (7), GT4a (1) and GT1b/GT3a. RAS were identified in 85.7% (42/49) of failures, of which 90.5% (38/42) were clinically relevant RAS (known to be associated with a specific GT and DAA in patients failing therapy or those with more than twofold change in in vitro replicon assays). The most abundant RAS were 168A/E/Q/G/N/V (32.6%, 16/49) identified in NS3, and 93H/N (61.2%, 30/49), 31I/M/V (34.7%, 17/49) and 30R/H/K (12.2%, 6/49), identified in NS5A. Significantly more clinically relevant RAS were identified in NS5A (82.2%, 37/45) than in NS3 (35.7%, 10/28; P<0.01). While RAS were identified in all GT1a, GT3b and GT4a failures (100%, 10/10), only 71.8% (28/39) of GT1b or GT3a failures had RAS (P=0.09). In four cases, NGS identified additional clinically relevant RAS and in one patient, NGS deciphered coexistence of GT3a and GT1b infections. CONCLUSIONS: Our findings, together with additional real-life data, will contribute to the optimization of retreatment in DAA failure, when cost-related and suboptimal regimens must be employed.

Primary sclerosing cholangitis is associated with abnormalities in CFTR.

BACKGROUND: The etiology of primary sclerosing cholangitis (PSC) is unknown. PSC and Cystic Fibrosis related liver disease have common features: chronic inflammation, biliary damage and similar cholangiographic findings. It is unknown whether or not PSC is related to cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. We hypothesize that a sub-group of PSC patients may be a "single-organ" presentation of CF. METHODS: Patients with PSC underwent nasal potential difference (NPD) measurement, sweat chloride measurement and complete CFTR sequencing by new generation sequencing. RESULTS: 6/32 patients aged 46 ±â€¯13 yrs. had CFTR causing mutations on one allele and 19 had CFTR polymorphisms; 6/23 tested had abnormal and 21 had intermediate sweat tests; 4/32 patients had abnormal NPD. One patient had chronic pancreatitis and was infertile. CONCLUSIONS: 19% of PSC patients had features of CFTR related disorder, 19% carry CFTR mutations and 50% had CFTR polymorphisms. In some patients, PSC may be a single organ presentation of CF or a CFTR-related disorder.

MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth.

Some solid tumors have reduced posttranscriptional RNA editing by adenosine deaminase acting on RNA (ADAR) enzymes, but the functional significance of this alteration has been unclear. Here, we found the primary RNA-editing enzyme ADAR1 is frequently reduced in metastatic melanomas. In situ analysis of melanoma samples using progression tissue microarrays indicated a substantial downregulation of ADAR1 during the metastatic transition. Further, ADAR1 knockdown altered cell morphology, promoted in vitro proliferation, and markedly enhanced the tumorigenicity in vivo. A comparative whole genome expression microarray analysis revealed that ADAR1 controls the expression of more than 100 microRNAs (miRNAs) that regulate many genes associated with the observed phenotypes. Importantly, we discovered that ADAR1 fundamentally regulates miRNA processing in an RNA binding­dependent, yet RNA editing­independent manner by regulating Dicer expression at the translational level via let-7. In addition, ADAR1 formed a complex with DGCR8 that was mutually exclusive with the DGCR8-Drosha complex that processes pri-miRNAs in the nucleus. We found that cancer cells silence ADAR1 by overexpressing miR-17 and miR-432, which both directly target the ADAR1 transcript. We further demonstrated that the genes encoding miR-17 and miR-432 are frequently amplified in melanoma and that aberrant hypomethylation of the imprinted DLK1-DIO3 region in chromosome 14 can also drive miR-432 overexpression.

Mucosal immunization against hepatitis A: antibody responses are enhanced by co-administration of synthetic oligodeoxynucleotides and a novel cationic lipid.

Hepatitis A caused by hepatitis A virus (HAV) transmitted by the fecal-oral route, results in considerable morbidity and economic loss. Mucosal immunization can be more effective than conventional injection at inducing both local and systemic immunity to HAV. Here we show that co-administration of killed HAV with synthetic oligodeoxynucleotides (ODNs) containing CpG sequences, and a novel polycationic sphingolipid (CCS)/cholesterol liposomal delivery system, markedly enhances the HAV-specific antibody response at the intestinal interface, particularly when delivered intrarectally or intranasally, to Balb/c mice at low HAV doses. A mucosally delivered, antigen-sparing HAV vaccine that is easily administered without specialized equipment or personnel, is an attractive alternative for facilitating mass immunization in hepatitis A outbreaks.