High-resolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods.

Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens, identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott Real-time HCV Genotype II failed subtype 1 calling in 13 (16%) samples each and were unable to identify the HCV genotype and/or subtype in more than half of the non-genotype 1 samples. We concluded that deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections and may be more helpful with respect to informing treatment strategies with new DAA-containing regimens across all HCV subtypes.

Synergistic cytotoxicity of the poly (ADP-ribose) polymerase inhibitor ABT-888 and temozolomide in dual-drug targeted magnetic nanoparticles.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is associated with a poor prognosis because of a lack of effective treatment options. The objective of this study was to examine a new strategy for HCC treatment, namely the use of poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor (ABT-888) together with Temozolomide (TMZ) incorporated onto magnetic nanoparticles. METHODS: Magnetic Fe3 O4 /Fe cores were encapsulated within a silica shell to facilitate the simultaneous incorporation of ABT-888 and TMZ. In vitro tests were performed with HepG2, Hep3B and PLC-PRF-5 liver tumoural cell lines and with WRL-68 liver non-tumoural cells. RESULTS: The magnetic nanocarriers were loaded simultaneously with ABT-888 and TMZ. High stability and extended release were achieved in culture medium. Confocal microscopy images showed that drug-loaded particles were uptaken and accumulated into the cytoplasm of liver tumoural cells, inducing the following effects: G2/M cell cycle arrest (P < 0.05), accumulation of DNA damage (P < 0.05), mitochondrial depolarization (P < 0.01), reduction in BCL-xL, FOS, JUND gene expression (P < 0.05), PARP-1 fragmentation, Caspase-3 activation and apoptotic cell death (P < 0.05). Interestingly, drugs loaded onto nanoparticles exhibited better efficiency than free drugs (cell death triggered by drug delivery nanosystem: 53.5% vs. 34.5% by free drugs, P = 0.01). CONCLUSIONS: These magnetic nanocompounds are able to incorporate both drugs simultaneously, enter the tumour cells and release them. ABT-888/TMZ/NPs decrease the transcription of key genes involved in tumour survival and induce apoptotic cell death in a more effective manner than is achieved by free drugs.

[The value of genetics in the era of hepatitis C triple therapy]. / Valor de la genética en la era de la terapia triple frente al virus de la hepatitis C.

Despite the introduction of protease inhibitors (PI) in the treatment of hepatitis C, the sensitivity of interferon continues to be essential to achieve a sustained virological response (SVR) and to eradicate the viral infection. Currently, pegylated interferon (PEG-IFN) and ribavirin (RBV) are required to avoid selection of PI-resistance mutations. The likelihood of obtaining an SVR with dual therapy in treatment-naïve patients with genotype 1 infection varies from 40% to 50%. That is, almost half of these patients would not require a PI, thus avoiding their adverse effects and considerably reducing the cost of the treatment. Identifying which patients could potentially respond to dual therapy is one of the main challenges in clinical practice. The genetic variability of the host is one of the main factors affecting the sensitivity of PEG-IFN and therefore in the response to current treatment. Other baseline factors related to the host, the virus and, especially, to intratreatment factors such as rapid virological response (RVR) are strongly associated with the probability of achieving an SVR. The evidence on the decision to prescribe dual or triple therapy according to the factors predictive of response is based on retrospective studies or post-hoc analyses of pivotal studies on PI. Study of the polymorphisms of the IFNL3 gene (IL28B), ITPA, IFN-stimulated genes (ISGs), TT/ΔG (ss469415590; IFNL4)) and RBV transporters could help in the decision to prescribe dual or triple therapy in treatment naïve patients.

Genetic variation in interleukin 28B with respect to vertical transmission of hepatitis C virus and spontaneous clearance in HCV-infected children.

UNLABELLED: The vertical transmission of hepatitis C virus (HCV-VT) is a major route of HCV infection in children, but the risk factors remain incompletely understood. This study analyzed the role of interleukin 28B (IL28B) in HCV-VT and in the spontaneous clearance of HCV among infected infants. Between 1991 and 2009, 145 mothers were recruited for this study: 100 were HCV-RNA+ve / human immunodeficiency virus negative (HIV-ve), with 128 children, and 33 were HCV-RNA-ve/HCV antibody+ve, with 43 children. The infants were tested for HCV-RNA at birth and at regular intervals until the age of 6 years. IL28B (single nucleotide polymorphism rs12979860) was determined in the mothers and children. HCV-VT was assumed when children presented HCV-RNA+ve in two subsequent blood samples. HCV-VT-infected infants were categorized as: (1) transient viremia with posterior HCV-RNA-ve and without serum-conversion; (2) persistent infection with serum-conversion. Of the 31 mothers with CC polymorphism, 19 (61%) were HCV-RNA+ve, whereas among the 68 mothers with non-CC polymorphism, 56 (82%) were HCV-RNA+ve. In all, 26 of 128 (20%) infants born to the HCV-RNA+ve mothers acquired HCV infection, but only 9 (7%) were chronically infected. The rate of HCV-VT was higher among the mothers with higher HCV viremia. No HCV-VT was detected in the HCV-RNA-ve women. Neither the mothers' nor the childrens' IL-28 status was associated with an increased risk of HCV-VT. The factors influencing viral clearance among the infected children were genotype non-1 and genotype CC of IL28B. In logistic regression, child CC polymorphism was the only predictor of HCV-clearance in HCV genotype-1. CONCLUSION: High maternal viral load is the only predictive factor of HCV-VT. IL28B plays no role in HCV-VT, but IL28B CC child polymorphism is associated independently with the spontaneous clearance of HCV genotype-1 among infected children.

Inhibition of poly adenosine diphosphate-ribose polymerase decreases hepatocellular carcinoma growth by modulation of tumor-related gene expression.

UNLABELLED: Hepatocellular carcinoma (HCC) is associated with a poor prognosis due to a lack of effective treatment options. In HCC a significant role is played by DNA damage and the inflammatory response. Poly (ADP-ribose) polymerase-1 (PARP-1) is an important protein that regulates both these mechanisms. The objective of this study was to examine the effect of pharmacology PARP-1 inhibition on the reduction of tumor volume of HCC xenograft and on the hepatocarcinogenesis induced by diethyl-nitrosamine (DEN). Pharmacologic PARP-1 inhibition with DPQ greatly reduces tumor xenograft volume with regard to a nontreated xenograft (394 mm(3) versus 2,942 mm(3), P < 0.05). This observation was paralleled by reductions in xenograft mitosis (P = 0.02) and tumor vasculogenesis (P = 0.007, confirmed by in vitro angiogenesis study), as well as by an increase in the number of apoptotic cells in DPQ-treated mice (P = 0.04). A substantial difference in key tumor-related gene expression (transformed 3T3 cell double minute 2 [MDM2], FLT1 [vascular endothelial growth factor receptor-1, VEGFR1], epidermal growth factor receptor [EPAS1]/hypoxia-inducible factor 2 [HIF2A], EGLN1 [PHD2], epidermal growth factor receptor [EGFR], MYC, JUND, SPP1 [OPN], hepatocyte growth factor [HGF]) was found between the control tumor xenografts and the PARP inhibitor-treated xenografts (data confirmed in HCC cell lines using PARP inhibitors and PARP-1 small interfering RNA [siRNA]). Furthermore, the results obtained in mice treated with DEN to induce hepatocarcinogenesis showed, after treatment with a PARP inhibitor (DPQ), a significant reduction both in preneoplastic foci and in the expression of preneoplastic markers and proinflammatory genes (Gstm3, Vegf, Spp1 [Opn], IL6, IL1b, and Tnf), bromodeoxyuridine incorporation, and NF-kappaB activation in the initial steps of carcinogenesis (P < 0.05). CONCLUSION: This study shows that PARP inhibition is capable of controlling HCC growth and preventing tumor vasculogenesis by regulating the activation of different genes involved in tumor progression.

Seroprevalence and epidemiology of hepatitis B and C viruses in pregnant women in Spain. Risk factors for vertical transmission.

BACKGROUND & AIM: Worldwide, measures are being implemented to eradicate hepatitis B (HBV) and C (HCV) viruses, which can be transmitted from the mother during childbirth. This study aims to determine the prevalence of HBV and HCV in pregnant women in Spain, focusing on country of origin, epidemiological factors and risk of vertical transmission (VT). METHODOLOGY: Multicentre open-cohort study performed during 2015. HBV prevalence was determined in 21870 pregnant women and HCV prevalence in 7659 pregnant women. Epidemiological and risk factors for VT were analysed in positive women and differences between HBV and HCV cases were studied. RESULTS: HBV prevalence was 0.42% (91/21870) and HCV prevalence was 0.26% (20/7659). Of the women with HBV, 65.7% (44/67) were migrants. The HBV transmission route to the mother was unknown in 40.3% of cases (27/67) and VT in 31.3% (21/67). Among risk factors for VT, 67.7% (42/62) of the women had viraemia and 14.5% (9/62) tested HBeAg-positive. All of the neonates born to HBV-positive mothers received immunoprophylaxis, and none contracted infection by VT. In 80% (16/20) of the women with HCV, the transmission route was parenteral, and nine were intravenous drug users. Viraemia was present in 40% (8/20) of the women and 10% (2/20) were HIV-coinfected. No children were infected. Women with HCV were less likely than women with HBV to breastfeed their child (65% vs. 86%). CONCLUSIONS: The prevalences obtained in our study of pregnant women are lower than those previously documented for the general population. Among the women with HBV, the majority were migrants and had a maternal family history of infection, while among those with HCV, the most common factor was intravenous drug use. Despite the risk factors observed for VT, none of the children were infected. Proper immunoprophylaxis is essential to prevent VT in children born to HBV-positive women.

Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition.

ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after gamma-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced gamma-H2AX foci formation in response to gamma-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced gamma H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.

Expression and Single Nucleotide Polymorphism of Poly (ADPRibose) Polymerase-1 in Gastrointestinal Tumours: Clinical Involvement.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays a critical role in diverse cellular functions, such as DNA damage detection and repair, transcriptional regulation and cell death. Furthermore, PARP-1 has emerged as a key player in the pathogenesis of multiple inflammatory diseases and has become a promising target for the treatment of cardiovascular disorders, neurodegenerative diseases and cancer. An increasing body of evidence has linked alterations in the expression levels of PARP-1, enzymatic activity and presence of polymorphism to gastrointestinal malignancies, including oesophageal, gastric, pancreas, liver and colorectal cancers. PARP inhibition has been proposed as a valuable strategy for treating these gastrointestinal disorders. This paper summarises the most significant current literature on the involvement of PARP-1 in gastrointestinal cancer, focusing in particular on its role in the development and occurrence of tumours, providing information about clinical trials and exploring therapeutic possibilities.

Hepatitis C virus NS5A region mutation in chronic hepatitis C genotype 1 patients who are non-responders to two or more treatments and its relationship with response to a new treatment.

AIM: To determine the number of mutations in the NS5A region of the hepatitis C virus (HCV) and its relationship to the response to antiviral therapy in patients with chronic hepatitis C genotype 1 who are non-responders to two or more treatments. METHODS: Sequences within HCV NS5A [PKR binding domain (PKRBD) and the interferon-sensitivity-determining region (ISDR)] were analysed via direct sequencing in a selected cohort of 72 patients, with a total of 201 treatments [interferon-alpha (IFN-α), n = 49; IFN-α + ribavirin (RBV), n = 75; pegylated (peg) IFN-α + RBV, n = 47; first-generation direct-acting antivirals (DAAs), n = 13; and second-generation DAAs, n = 17]. Of these, 48/201 achieved a sustained virological response (SVR) and 153/201 achieved no virological response (NVR). RESULTS: For both regions, treatments resulting in SVR were associated with more baseline mutations than were treatments resulting in NVR (SVR vs NVR; PKRBD: 5.82 ± 3 vs 4.86 ± 2 mutations, P = 0.045; ISDR: 2.65 ± 2 vs 1.51 ± 1.7 mutations, P = 0.005). A decrease or no change in the number of mutations over time between treatments in the PKRBD or ISDR, as shown by sequencing, was associated with patients who usually failed to respond to treatment (PKRBD, P = 0.02; ISDR, P = 0.001). Moreover, patients showing a post-treatment baseline viral load > 600000 IU/mL and increased ISDR mutations with respect to the previous treatment were 9.21 times more likely to achieve SVR (P = 0.001). CONCLUSION: The obtained results show that among patients who have shown no response to two or more antiviral treatments, the likelihood of achieving SVR increases with the genetic variability in the ISDR region (≥ 2 mutations or number of substitutions from the HCV-J and HCV-1 prototype), especially when the viral load is greater than 600000 IU/mL.

Variation of transaminases, HCV-RNA levels and Th1/Th2 cytokine production during the post-partum period in pregnant women with chronic hepatitis C.

This study analyses the evolution of liver disease in women with chronic hepatitis C during the third trimester of pregnancy and the post-partum period, as a natural model of immune modulation and reconstitution. Of the 122 mothers recruited to this study, 89 were HCV-RNA+ve/HIV-ve and 33 were HCV-RNA-ve/HIV-ve/HCVantibody+ve and all were tested during the third trimester of pregnancy, at delivery and post-delivery. The HCV-RNA+ve mothers were categorized as either Type-A (66%), with an increase in ALT levels in the post-partum period (>40 U/L; P<0.001) or as Type-B (34%), with no variation in ALT values. The Type-A mothers also presented a significant decrease in serum HCV-RNA levels in the post-delivery period (P<0.001) and this event was concomitant with an increase in Th1 cytokine levels (INFγ, P = 0.04; IL12, P = 0.01 and IL2, P = 0.01). On the other hand, the Type-B mothers and the HCV-RNA-ve women presented no variations in either of these parameters. However, they did present higher Th1 cytokine levels in the partum period (INFγ and IL2, P<0.05) than both the Type-A and the HCV-RNA-ve women. Cytokine levels at the moment of delivery do not constitute a risk factor associated with HCV vertical transmission. It is concluded that differences in the ALT and HCV-RNA values observed in HCV-RNA+ve women in the postpartum period might be due to different ratios of Th1 cytokine production. In the Type-B women, the high partum levels of Th1 cytokines and the absence of post-partum variation in ALT and HCV-RNA levels may be related to permanent Th1 cytokine stimulation.

Valor de la genética en la era de la terapia triple frente al virus de la hepatitis C / The Value Of Genetics In The Era Of Hepatitis C Triple Therapy

A pesar de la introducción de los inhibidores de la proteasa (IP) en el tratamiento de la hepatitis C, la sensibilidad al IFN continúa siendo fundamental para alcanzar la respuesta virológica (RVS) y erradicar la infección viral. En la actualidad, el interferón pegilado (IFNpeg) y la ribavirina (RBV) son necesarios para evitar la selección de mutantes resistentes a los IP. La probabilidad de obtener la RVS con biterapia en pacientes naives infectados con genotipo 1 varía entre el 40-50%. Es decir, en casi la mitad de este grupo de pacientes no sería preciso introducir un IP, evitando así los efectos adversos del mismo y disminuyendo considerablemente el coste del tratamiento. Identificar a estos potenciales respondedores a la doble terapia es uno de los mayores retos en la práctica clínica. La variabilidad genética del huésped constituye uno de los factores fundamentales en la sensibilidad al IFNpeg y, por tanto, en la respuesta al tratamiento actual. Otros factores basales relacionados con el huésped, con el virus y, sobre todo, los factores intratratamiento como la respuesta virológica rápida (RVR), se relacionan fuertemente con la probabilidad de alcanzar la RVS. La evidencia sobre la decisión de tratar con doble o triple terapia en función de los factores predictores de respuesta se basa en estudios retrospectivos o análisis posthoc de los estudios pivotales de los IP. El estudio de los polimorfismos del gen de IFNL3 (IL28B), ITPA, genes estimulados por IFN (ISG), TT/ΔG (ss469415590; IFNL4) y de transportadores de RBV son factores genéticos importantes que nos pueden ayudar a tomar la decisión de tratar con doble o triple terapia en pacientes naives

Despite the introduction of protease inhibitors (PI) in the treatment of hepatitis C, the sensitivity of interferon continues to be essential to achieve a sustained virological response (SVR) and to eradicate the viral infection. Currently, pegylated interferon (PEG-IFN) and ribavirin (RBV) are required to avoid selection of PI-resistance mutations. The likelihood of obtaining an SVR with dual therapy in treatment-naïve patients with genotype 1 infection varies from 40% to 50%. That is, almost half of these patients would not require a PI, thus avoiding their adverse effects and considerably reducing the cost of the treatment. Identifying which patients could potentially respond to dual therapy is one of the main challenges in clinical practice. The genetic variability of the host is one of the main factors affecting the sensitivity of PEG-IFN and therefore in the response to current treatment. Other baseline factors related to the host, the virus and, especially, to intratreatment factors such as rapid virological response (RVR) are strongly associated with the probability of achieving an SVR. The evidence on the decision to prescribe dual or triple therapy according to the factors predictive of response is based on retrospective studies or post-hoc analyses of pivotal studies on PI. Study of the polymorphisms of the IFNL3 gene (IL28B), ITPA, IFN-stimulated genes (ISGs), TT/ΔG (ss469415590; IFNL4)) and RBV transporters could help in the decision to prescribe dual or triple therapy in treatment naïve patients

PARP-1 is involved in autophagy induced by DNA damage.

Autophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. PARP-1 has been implicated in different pathways leading to cell death and its inhibition potentiates chemotherapy-induced cell death. Whether PARP-1 participates in the cell's decision to commit to autophagy following DNA damage is still not known. To address this issue PARP-1 wild-type and deficient cells have been treated with a dose of doxorubicin that induces autophagy. Electron microscopy examination and GFP-LC3 transfection revealed autophagic vesicles and increased expression of genes involved in autophagy (bnip-3, cathepsin b and l and beclin-1) in wild-type cells treated with doxo but not in parp-1(-/-) cells or cells treated with a PARP inhibitor. Mechanistically the lack of autophagic features in PARP-1 deficient/PARP inhibited cells is attributed to prevention of ATP and NAD(+) depletion and to the activation of the key autophagy regulator mTOR. Pharmacological or genetical inhibition of autophagy results in increased cell death, suggesting a protective role of autophagy induced by doxorubicin. These results suggest that autophagy might be cytoprotective during the response to DNA damage and suggest that PARP-1 activation is involved in the cell's decision to undergo autophagy.