BackgroundHepatocellular carcinoma is the third cause of cancer related death for which new treatment strategies are needed. Targeting DNA repair pathways to sensitize tumor cells to chemo- or radiotherapy is under investigation for the treatment of several cancers with poly(ADP-ribose) polymerase (PARP) inhibitors showing great potential. The aim of this preclinical study was to evaluate the expression of PARP and PARG genes in a panel of liver cancer cell lines and primary human hepatocytes, their DNA repair capacity and assess the impact on cell survival of PARP inhibitors alone and in combination with radiotherapy.MethodsQuantitative PCR was used to measure PARP-1, -2, -3 and PARG mRNA levels and western blotting for PARP-1 protein expression and ADP-ribose polymer formation after exposure of cells to doxorubicin, a topoisomerase II poison. DNA repair capacity was assessed using an in vitro DNA lesion excision/synthesis assay and the effects on cell killing of the PARP inhibitor ABT-888 alone and in combination with ionizing radiation using clonogenic survival.ResultsAlthough a wide range in expression of the PARPs and PARG was found correlations between PARP-1 and PARP-2 mRNA levels and PARP-1 mRNA and protein levels were noted. However these expression profiles were not predictive of PARP activity in the different cell lines that also showed variability in excision/synthesis repair capacity. 4 of the 7 lines were sensitive to ABT-888 alone and the two lines tested showed enhanced radiosensitivity in the presence of ABT-888.ConclusionsPARP inhibitors combined with radiotherapy show potential as a therapeutic option for hepatocellular carcinoma.

Fibroblasts are known to be present in variable amounts in human breast adenocarcinoma tissue. In order to investigate if they influence in some way the proliferation rate of the carcinoma cells, we developed a coculture model in which cells of well characterized breast epithelial cell lines were seeded and grown in microchamber slides along with fibroblasts derived from breast tumor biopsies. As representatives of hormone dependent and independent tumor cells, we used MCF-7 and BT-20 cell lines. A third line, NPM-21T, derived from non proliferating mastopathy cells immortalized by SV-40 T DNA transfection, was representative of non tumor epithelial cells. The proliferation rate of the adenocarcinoma and epithelial cells was assessed by measurement of the BrdU labeling index, the cells being identified by specific beta-actin immunostaining. It was found that the proliferation of the adenocarcinoma cells was significantly increased in the presence of fibroblasts, while that of immortalized cells was not. Moreover, 1,25(OH)2D3, which was known to be a negative regulator of carcinoma cell growth, was found to be able also to blunt the overgrowth in the presence of fibroblasts. The absence of response of NPM-21T cells to the presence of fibroblasts suggests that the tumor cells could be the origin of their own overgrowth, through an indirect mechanism mediated by the fibroblasts. The factors which are involved and the 1,25(OH)2D3 mechanism of action are not yet identified.

Background & Aims: Although HBV is a major cause of death in Africa, its genetic variability has been poorly documented. This study aimed to address whether HBV genotype and surface gene variants are associated with HBV-related liver disease in The Gambia. Methods: We conducted a case-control study nested in the Prevention of Liver Fibrosis and Cancer in Africa programme. Consecutive treatment-naive patients with chronic HBV infection and detectable viral load were recruited: 211 controls with no significant liver disease and 91 cases (56 cirrhosis and 35 HCC cases). HBV genotypes and surface gene variants were determined by Sanger sequencing or next-generation sequencing (NGS) in serum DNA. Aflatoxin B1 (AFB1)-specific codon 249 TP53 mutation was determined by NGS in circulating cell-free plasma DNA. Results: In phylogenetic analysis, 85% of individuals carried HBV genotype E, 14% genotype A, and 1% A/E recombinant viruses. Surface gene variants were more frequently observed in cases (43% and 57% in cirrhosis and HCC cases, respectively) than controls (25%; p <0.001), with preS2 deletions between nucleotides 38-55 (preS2D38-55) being the main genetic variant detected. In multivariable analysis, HBeAg seropositivity, low HBsAg levels, and HDV seropositivity were significantly associated with cirrhosis and HCC, whilst older age, higher viral load, genotype A, preS2D38-55, and AFB1 exposure were only associated with HCC. There was a multiplicative joint effect of preS2D38-55 variants with HBeAg seropositivity (odds ratio [OR] 43.1 [10.4-177.7]), high viral load >2,000 IU/ml (OR 22.7 [8.0-64.9]), HBsAg levels <10,000 IU/ml (OR 19.0 [5.5-65.3]), and AFB1 exposure (OR 29.3 [3.7-230.4]) on HCC risk. Conclusions: This study identified a hotspot for HBV preS2 deletions as a strong independent factor for HCC in The Gambia, with HBV genotypes and AFB1 exposure contributing to the high liver cancer risk.

Hepatitis B virus (HBV) is a major cause of chronic liver disease worldwide. HBV infection is diagnosed by serological tests, while real-time polymerase chain reaction (qRT-PCR) assays are used to quantify viral load, which is a crucial parameter to determine viral replication and to monitor antiviral treatments. However, measuring viral load in resource-limited countries remains nonsystematic, due to the high cost of commercial kits. Here, we describe the development, validation and implementation of a low-cost, in-house qRT-PCR assay to monitor HBV viral load in chronic carriers enrolled in the PROLIFICA programme in the Gambia and Senegal. Over 1500 HBsAg-positive patients, including 210 chronically infected HBV patients, who were given antiviral treatment (tenofovir), were monitored by qRT-PCR using the SYBR Green- and HBV-specific primers. Twenty-four tenofovir-treated patients were followed up and their viral load was tested every 3 months over the 12-month experimental time course. Compared to commercial assays, our in-house assay was shown to be (i) highly reliable, with good intra- and interassay reproducibility over a wide range (45-4.5 × 10 copies mL ), (ii) very similar in the viral loads detected (R = .90), (iii) highly sensitive, as it detected loads as low as 30 copies mL (~5 IU mL ), (iv) cheaper (2- to 3-fold), (v) easier to implement and (vi) more rapid. Based on our experience, we recommend this assay as a reliable alternative to commercial assays, for monitoring HBV viraemia in resource-limited, highly endemic countries to reduce the cost and technical obstacles associated with commercial kits.

Hepatitis B virus (HBV) infection remains a major public health concern worldwide with 240 million individuals chronically infected and at risk of developing cirrhosis and hepatocellular carcinoma. Current treatments rarely cure chronic hepatitis B infection, highlighting the need for new anti-HBV drugs. Nucleic acid polymers (NAPs) are phosphorothioated oligonucleotides that have demonstrated a great potential to inhibit infection with several viruses. In chronically infected human patients, NAPs administration lead to a decline of blood HBsAg and HBV DNA and to HBsAg seroconversion, the expected signs of functional cure. NAPs have also been shown to prevent infection of duck hepatocytes with the Avihepadnavirus duck hepatitis B virus (DHBV) and to exert an antiviral activity against established DHBV infection in vitro and in vivo.In this study, we investigated the specific anti-HBV antiviral activity of NAPs in the HepaRG human hepatoma cell line and primary cultures of human hepatocytes. NAPs with different chemical features (phosphorothioation, 2’O-methyl ribose, 5-methylcytidine) were assessed for antiviral activity when provided at the time of HBV inoculation or post-inoculation. NAPs dose-dependently inhibited HBV entry in a phosphorothioation-dependent, sequence-independent and size-dependent manner. This inhibition of HBV entry by NAPs was impaired by 2’O-methyl ribose modification. NAP treatment after viral inoculation did not elicit any antiviral activity.

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days from 314 in 2013 to 252 in 2018 was measured. The main decreases were observed for amphotericin B (ratio=0.3), voriconazole (ratio=0.50) and caspofungin (ratio=0.59). The most important increases have been shown for: flucytosine (ratio=10.25), micafungin (ratio=2.73) and fluconazole (ratio=1.22). Fluctuation in consumption is linked to several factors: drug shortages, evolution in recommendations and patient profiles. French drug market supplies break of oxacillin/penicillin M increases first-and second-generation cephalosporin prescriptions. A local guideline for transplant patients recently replaces fluconazole by mycafungin in antifungal prophylaxis. Conclusion Both the overall numbers of antibiotics and antifungals DDD/1000 beds-days decrease over the 5 year study period. A multidisplinary analysis comprehends the consumption evolution in our paediatric ICU. It should be monitored on a continuous basis by pharmacists in healthcare settings.