Development of a recombinant murine tumour model using hepatoma cells expressing hepatitis C virus nonstructural antigens.

Hepatitis C virus (HCV) chronically infects 2%-3% of the world's population, causing liver disease and cancer with prolonged infection. The narrow host range of the virus, being restricted largely to human hepatocytes, has made the development of relevant models to evaluate the efficacy of vaccines a challenge. We have developed a novel approach to accomplish this by generating a murine hepatoma cell line stably expressing nonstructural HCV antigens which can be used in vitro or in vivo to test HCV vaccine efficacies. These HCV-recombinant hepatoma cells formed large solid-mass tumours when implanted into syngeneic mice, allowing us to test candidate HCV vaccines to demonstrate the development of an HCV-specific immune response that limited tumour growth. Using this model, we tested the therapeutic potential of recombinant anti-HCV-specific vaccines based on two fundamentally different attenuated pathogen vaccine systems-attenuated Salmonella and recombinant adenoviral vector based vaccine. While attenuated Salmonella that secreted HCV antigens limited growth of the HCV-recombinant tumours when used in a therapeutic vaccination trial, replication-competent but noninfectious adenovirus expressing nonstructural HCV antigens showed overall greater survival and reduced weight loss compared to non-replicating nondisseminating adenovirus. Our results demonstrate a model with anti-tumour responses to HCV nonstructural (NS) protein antigens and suggest that recombinant vaccine vectors should be explored as a therapeutic strategy for controlling HCV and HCV-associated cancers.

Effects of annealing atmosphere and rGO concentration on the optical properties and enhanced photocatalytic performance of SnSe/rGO nanocomposites.

The photocatalytic properties of SnSe nanostructures (NSs) and SnSe/graphene nanocomposites with different graphene concentrations (5, 10, and 15 wt%/v) were investigated. The products were synthesized by a simple and cost-effective co-precipitation method. The samples obtained demonstrated that graphene concentration at an optimum amount was an important factor in enhancing the photocatalytic performance of the products. The graphene source was graphene oxide (GO) sheets and several characterization results indicated, which were used to remove Methylene blue (MB) dye, that the GO sheets were changed into reduced graphene oxide (rGO) sheets during the synthesis process. The optical properties of the products were studied using a room temperature photoluminescence (PL) spectrometer and it was observed that the near-band-edge (NBE) position of the samples was at the end of the red region between 729 and 756 nm of the electromagnetic spectrum, which was confirmed by a UV-vis spectrometer. The PL spectra of the samples also demonstrated three emissions from the violet, green, and orange regions of the visible spectrum, which were from different defects. The samples were annealed in a hydrogen and air atmosphere at 300 °C and it was found that defect concentrations were increased by annealing for the SnSe/rGO nanocomposites. The photocatalyst studies of the post-annealed samples revealed that the photocatalytic performance of the products was enhanced by annealing in hydrogen, while it was reduced by annealing in air. In addition to MB, the photocatalytic performance of the products for the degradation of phenol as a colorless pollutant was examined. It was observed that rGO in this process also had a significant role in the enhancement of photocatalytic performance. In fact, the electron spin resonance (ESR) test showed the role of rGO in photocatalytic activity very well.

Overview of current surgical management of fibroids: 'Organ-preserving modalities'.

Uterine fibroids are the most common solid tumours occurring in female pelvis and frequently encountered by gynaecologists. Generally about 50% remain asymptomatic and can be monitored through regular follow-up visits but symptomatic fibroids require surgical intervention at some stage. They occur in 25 - 50% of women over the age of 30 years, increasing with age and being more common in certain ethnic populations, especially the Afro-Caribbean. They have a major impact on women's health and were the most common indication for hysterectomy in England in 1993 - 1994.They have significant cost implications, the 72,362 hysterectomies performed in 1993 - 1994 costing the NHS an estimated pound70 million. Although hysterectomy is the most certain cure for women with symptomatic fibroids who do not wish to preserve fertility, an increasing number of women are choosing and looking for the options of organ-conserving surgery. The surgery can be carried out abdominally, laparoscopically and vaginally although all routes are associated with an appreciable rate of morbidity. The discussion of organ-preserving surgery includes mainly myomectomy, transcervical resection of fibroid, uterine artery embolisation (UAE) and MRI-guided laser ablation. Hysterectomy is associated with a high rate of satisfaction and is likely to relieve menstrual problems in almost all women. Much work has been undertaken on this subject so far, with a view to safe and effective surgical approaches.

Adenovirus-based libraries: efficient generation of recombinant adenoviruses by positive selection with the adenovirus protease.

Adenoviruses (Ad) deleted in the protease (PS) gene are capable of only one round of replication in non-complementing cells. This feature was exploited to develop a positive selection method for constructing adenoviral recombinants using ectopic expression of the PS gene in the E1 region. Very low levels of PS were sufficient to ensure the rescue of a PS-deleted Ad genome (Ad(Delta)PS), thereby eliminating deleterious effects PS over-expression might exert on cell or virus growth. In addition to the standard co-transfection method, an alternative protocol was developed in which the Ad5-(Delta)PS viral DNA was delivered by infection before subsequent transfection of 293 cells with the transfer vector. Under optimal conditions, at least one recombinant Ad per 10(3) cells was generated with 100% of the plaques being recombinant. Since the infection/transfection protocol is readily scalable, this represents the first method that allows for the easy construction of adenovirus vector (AdV) libraries with high diversities. This approach addresses in a novel way the bottleneck encountered when converting plasmid libraries, constructed in E. coli using a variety of well-established strategies, into corresponding AdV libraries. It maintains high diversity while generating recombinant viruses with 100% efficiency.

Induction of humoral and cellular immune responses against the nucleocapsid of bovine viral diarrhea virus by an adenovirus vector with an inducible promoter.

A new recombinant adenovirus was constructed that expressed the nucleocapsid (C protein or p14) of the bovine viral diarrhea virus (BVDV) under the control of a tetracycline-regulatable promoter. Mice covaccinated with this recombinant adenovirus, accompanied by another recombinant adenovirus expressing the trans-activator protein, induced a strong humoral immune response to the BVDV/C protein as detected by ELISA. Splenocytes from mice immunized with the recombinant adenovirus showed a specific proliferation response to both genotypes (type 1 and 2) of BVDV. High levels of IFN-gamma were detected in the supernatant of murine mononuclear cells of mice immunized by the recombinant adenovirus when stimulated in vitro by both genotypes of BVDV. These results indicate that this recombinant adenovirus is highly immunogenic and stimulates both cellular and humoral immune responses against the nucleocapsid of BVDV.

Recombinant adenoviruses expressing the E2 protein of bovine viral diarrhea virus induce humoral and cellular immune responses.

The E2 protein of bovine viral diarrhea virus (BVDV) is a major viral glycoprotein and an attractive target for BVDV vaccines. Three replication defective recombinant adenoviruses expressing the BVDV/E2 protein (rAds/E2) were constructed. Two contain a constitutive promoter, and one an inducible promoter. All three recombinant adenoviruses induced very strong BVDV specific antibody responses in a mouse model as detected by enzyme-linked immunosorbant assay (ELISA) and neutralization tests. Induction of cellular immune responses was investigated in two recombinant adenoviruses with a constitutive promoter. The mononuclear cells from the immunized mice demonstrated a proliferative response after in vitro stimulation with an homologous BVDV strain, but only one of them induced the production of IFN-gamma.

Investigation of the immunological properties of the bovine viral diarrhea virus protein NS3 expressed by an adenovirus vector in mice.

Two replication-defective human adenovirus recombinants encoding the NS3 protein (p80) of bovine viral diarrhea virus (BVDV) under the control of a modified adenovirus major later promoter (BM5), rAdBM5/NS3, and human cytomegalovirus promoter (CMV5), rAdCMV5/NS3, were constructed. These two recombinant adenoviruses were tested for their expression of the NS3 protein in vitro in three different cell lines and also in vivo for the induction of BVDV-specific immune responses in mice. The recombinant adenoviruses containing two different promoters induced different levels of humoral responses to the NS3 protein. The rAdBM5/NS3 was used to vaccinate mice in order to evaluate the ability of the NS3 protein in the induction of cellular immune responses. The rAdBM5/NS3 did not cause a stimulation of cell proliferation but caused a very strong increase in production of IFN-gamma in murine mononuclear cells stimulated in vivo by BVDV strains of genotype 1 and 2.

Antigenic variation among bovine viral diarrhea virus (BVDV) strains and the role of different cell fixation methods in immunoassays.

Antigenic variation among 13 Quebec isolates of bovine viral diarrhea virus (BVDV), 4 reference strains and 2 American isolates were studied by peroxidase-linked antibody assay (PLA assay) and neutralization test (NT). The Quebec strains consisted of 3 isolates before 1993 and 10 isolates from 1993. In the PLA assay, we compared 2 different fixatives, acetone and formalin. Acetone-fixation allowed us to identify 6 groups from amongst the viruses tested. All the Quebec isolates were different from the reference strains. In addition, antigenic variation was detected between Quebec isolates obtained before and during 1993. However, PLA assays performed after formalin fixation did not detect these antigenic variations. Neutralization tests were carried out with 2 polyclonal antibodies (PAb) and 6 monoclonal antibodies (MAb). They were used to classify BVDV strains and isolates into 4 groups and 7 subgroups respectively. In conclusion, we demonstrated that the BVDV isolates from the 1993 outbreak in Quebec are antigenically different from reference strains and from isolates existing in Quebec before 1993. In addition, we have shown that 2 internationally used fixation-methods in PLA assay give different results. The usefulness of each method is discussed.

The 5'-untranslated region sequence of a potential new genotype of bovine viral diarrhea virus.

The 5' untranslated region (UTR) of several bovine viral diarrhea virus (BVDV) isolates from the severe Quebec outbreak was amplified by polymerase chain reaction (PCR) and sequenced. Sequences revealed the loss, for the BVDV type II isolates, of an internal PstI restriction site, which is present in all known BVDV type 1 5' UTR sequences. A single restriction enzyme digestion (PstI) of an aliquot of PCR product allowed us to differentiate BVDV type I and BVDV type II.