Establishment and characterization of a bladder cancer cell line with enhanced doxorubicin resistance by mevalonate pathway activation.

Resistance to chemotherapy is a major problem in the treatment of urothelial bladder cancer. Several mechanisms have been identified in resistance to doxorubicin by analysis of resistant urothelial carcinoma (UC) cell lines, prominently activation of drug efflux pumps and diminished apoptosis. We have derived a new doxorubicin-resistant cell line from BFTC-905 UC cells, designated BFTC-905-DOXO-II. A doxorubicin-responsive green fluorescent protein (GFP) reporter assay indicated that resistance in BFTC-905-DOXO-II was not due to increased drug efflux pump activity, whereas caspase-3/7 activation was indeed diminished. Gene expression microarray analysis revealed changes in proapoptotic and antiapoptotic genes, but additionally induction of the mevalonate (cholesterol) biosynthetic pathway. Treatment with simvastatin restored sensitivity of BFTC-905-DOXO-II to doxorubicin to that of the parental cell line. Induction of the mevalonate pathway has been reported as a mechanism of chemoresistance in other cancers; this is the first observation in bladder cancer. Combinations of statins with doxorubicin-containing chemotherapy regimens may provide a therapeutic advantage in such cases.

Studying longitudinal neutralising antibody levels against Equid herpesvirus 1 in experimentally infected horses using a novel pseudotype based assay.

Infection with equid herpesvirus 1 (EHV-1), a DNA virus of the Herpesviridae family represents a significant welfare issue in horses and a great impact on the equine industry. During EHV-1 infection, entry of the virus into different cell types is complex due to the presence of twelve glycoproteins (GPs) on the viral envelope. To investigate virus entry mechanisms, specific combinations of GPs were pseudotyped onto lentiviral vectors. Pseudotyped virus (PV) particles bearing gB, gD, gH and gL were able to transduce several target cell lines (HEK293T/17, RK13, CHO-K1, FHK-Tcl3, MDCK I & II), demonstrating that these four EHV-1 glycoproteins are both essential and sufficient for cell entry. The successful generation of an EHV-1 PV permitted development of a PV neutralisation assay (PVNA). The efficacy of the PVNA was tested by measuring the level of neutralising serum antibodies from EHV-1 experimentally infected horses (n = 52) sampled in a longitudinal manner. The same sera were assessed using a conventional EHV-1 virus neutralisation (VN) assay, exhibiting a strong correlation (r = 0.82) between the two assays. Furthermore, PVs routinely require -80 °C for long term storage and a dry ice cold-chain during transport, which can impede dissemination and utilisation in other stakeholder laboratories. Consequently, lyophilisation of EHV-1 PVs was conducted to address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions for different time periods. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests. Results indicated that lyophilisation could be used to stably preserve such complex herpesvirus pseudotypes, even after weeks of storage at room temperature, and that reconstituted EHV-1 PVs could be successfully employed in antibody neutralisation tests.

Genetic characterization of a new candidate hemagglutinin subtype of influenza A viruses.

ABSTRACTAvian influenza viruses (AIV) have been classified on the basis of 16 subtypes of hemagglutinin (HA) and 9 subtypes of neuraminidase. Here we describe genomic evidence for a new candidate HA subtype, nominally H19, with a large genetic distance to all previously described AIV subtypes, derived from a cloacal swab sample of a Common Pochard (Aythya ferina) in Kazakhstan, in 2008. Avian influenza monitoring in wild birds especially in migratory hotspots such as central Asia is an important approach to gain information about the circulation of known and novel influenza viruses. Genetically, the novel HA coding sequence exhibits only 68.2% nucleotide and 68.5% amino acid identity with its nearest relation in the H9 (N2) subtype. The new HA sequence should be considered in current genomic diagnostic AI assays to facilitate its detection and eventual isolation enabling further study and antigenic classification.

Serological evidence of virus infection in Eidolon helvum fruit bats: implications for bushmeat consumption in Nigeria.

Introduction: The Eidolon helvum fruit bat is one of the most widely distributed fruit bats in Africa and known to be a reservoir for several pathogenic viruses that can cause disease in animals and humans. To assess the risk of zoonotic spillover, we conducted a serological survey of 304 serum samples from E. helvum bats that were captured for human consumption in Makurdi, Nigeria. Methods: Using pseudotyped viruses, we screened 304 serum samples for neutralizing antibodies against viruses from the Coronaviridae, Filoviridae, Orthomyxoviridae and Paramyxoviridae families. Results: We report the presence of neutralizing antibodies against henipavirus lineage GH-M74a virus (odds ratio 6.23; p < 0.001), Nipah virus (odds ratio 4.04; p = 0.00031), bat influenza H17N10 virus (odds ratio 7.25; p < 0.001) and no significant association with Ebola virus (odds ratio 0.56; p = 0.375) in this bat cohort. Conclusion: The data suggest a potential risk of zoonotic spillover including the possible circulation of highly pathogenic viruses in E. helvum populations. These findings highlight the importance of maintaining sero-surveillance of E. helvum, and the necessity for further, more comprehensive investigations to monitor changes in virus prevalence, distribution over time, and across different geographic locations.

Exploiting Pan Influenza A and Pan Influenza B Pseudotype Libraries for Efficient Vaccine Antigen Selection.

We developed an influenza hemagglutinin (HA) pseudotype library encompassing Influenza A subtypes HA1-18 and Influenza B subtypes (both lineages) to be employed in influenza pseudotype microneutralization (pMN) assays. The pMN is highly sensitive and specific for detecting virus-specific neutralizing antibodies against influenza viruses and can be used to assess antibody functionality in vitro. Here we show the production of these viral HA pseudotypes and their employment as substitutes for wildtype viruses in influenza neutralization assays. We demonstrate their utility in detecting serum responses to vaccination with the ability to evaluate cross-subtype neutralizing responses elicited by specific vaccinating antigens. Our findings may inform further preclinical studies involving immunization dosing regimens in mice and may help in the creation and selection of better antigens for vaccine design. These HA pseudotypes can be harnessed to meet strategic objectives that contribute to the strengthening of global influenza surveillance, expansion of seasonal influenza prevention and control policies, and strengthening pandemic preparedness and response.

Controlling equine influenza: Traditional to next generation serological assays.

Serological assays provide an indirect route for the recognition of infectious agents via the detection of antibodies against the infectious agent of interest within serum. Serological assays for equine influenza A virus can be applied for different purposes: diagnosing infections; subtyping isolates; surveillance of circulating strains; and to evaluate the efficacy of vaccines before they reach the market. Haemagglutination inhibition (HI) and single radial haemolysis (SRH) assays are most commonly used in the equine field. This review outlines how both these assays together with virus neutralization (VN) and ELISA are performed, interpreted and applied for the control of equine influenza, giving the limitations and advantages of each. The pseudotyped virus neutralization assay (PVNA) is also discussed as a promising prospect for the future of equine influenza virus serology.

The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus.

Pseudotyped viruses (PVs) produced by co-transfecting cells with plasmids expressing lentiviral core proteins and viral envelope proteins are potentially powerful tools for studying various aspects of equine influenza virus (EIV) biology. The aim of this study was to optimise production of equine influenza PVs. Co-transfection of the HAT protease to activate the haemagglutinin (HA) yielded a higher titre PV than TMPRSS2 with the HA from A/equine/Richmond/1/2007 (H3N8), whereas for A/equine/Newmarket/79 (H3N8), both proteases resulted in equivalent titres. TMPRSS4 was ineffective with the HA of either strain. There was also an inverse relationship between the amount of protease-expression plasmids and the PV titre obtained.  Interestingly, the PV titre obtained by co-transfection of a plasmid encoding the cognate N8 NA was not as high as that generated by the addition of exogenous neuraminidase (NA) from Clostridium perfringens to allow the release of nascent PV particles. Finally, initial characterisation of the reliability of PV neutralisation tests (PVNTs) demonstrated good intra-laboratory repeatability. In conclusion, we have demonstrated that equine influenza PV production can be readily optimised to provide a flexible tool for studying EIV.

Gene therapy vectors containing CArG elements from the Egr1 gene are activated by neutron irradiation, cisplatin and doxorubicin.

Combining gene therapy with radiotherapy and chemotherapy holds potential to increase the efficacy of cancer treatment, while minimizing side effects. We tested the responsiveness of synthetic gene promoters containing CArG elements from the Early Growth Response 1 (Egr1) gene after neutron irradiation, doxorubicin and cisplatin. Human MCF-7 breast adenocarcinoma and U373-MG glioblastoma cells were transfected with plasmids containing CArG promoters controlling the expression of the green fluorescent protein (GFP). Exposing the cells to neutrons, doxorubicin or cisplatin resulted in a significant induction of transgene expression. Therapeutic advantage was demonstrated by replacing the reporter with the herpes simplex virus thymidine kinase (HSVtk), able to convert the prodrug ganciclovir (GCV) into a cytotoxin. A 1.3 Gy neutron dose caused 49% growth inhibition in MCF-7 cells, which increased to 63% in irradiated CArG-HSVtk-transfectants treated with GCV. Exposure to 0.5 microM cisplatin or 0.01 microM doxorubicin induced a growth inhibition of 25-30% in MCF-7 cells. In the presence of GCV, this value increased to 65-70% in cells transfected with the CArG promoter constructs driving the expression of HSVtk. These data indicate that combining CArG-mediated HSVtk/GCV suicide gene therapy with radio- and chemotherapy can enhance antitumor toxicity, and validates future in vivo investigations.

Dual responsive promoters to target therapeutic gene expression to radiation-resistant hypoxic tumor cells.

PURPOSE: Tumor hypoxia is unequivocally linked to poor radiotherapy outcome. This study aimed to identify enhancer sequences that respond maximally to a combination of radiation and hypoxia for use in genetic radiotherapy approaches. METHODS AND MATERIALS: The influence of radiation (5 Gy) and hypoxia (1% O2) on reporter-gene expression driven by hypoxia (HRE) and radiation (Egr-1) responsive elements was evaluated in tumor cells grown as monolayers or multicellular spheroids. Hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha protein expression was monitored in parallel. RESULTS: Of the sequences tested, an HRE from the phosphoglycerate kinase-1 gene (PGK-18[5+]) was maximally induced in response to hypoxia plus radiation in all 5 cell lines tested. The additional radiation treatment afforded a significant increase in the induction of PGK-18[5+] compared with hypoxia alone in 3 cell lines. HIF-1alpha/2alpha were induced by radiation but combined hypoxia/radiation treatment did not yield a further increase. The dual responsive nature of HREs was maintained when spheroids were irradiated after delivery of HRE constructs in a replication-deficient adenovirus. CONCLUSIONS: Hypoxia-responsive enhancer element sequences are dually responsive to combined radiation and hypoxic treatment. Their use in genetic radiotherapy in vivo could maximize expression in the most radio-resistant population at the time of radiation and also exploit microenvironmental changes after radiotherapy to yield additional switch-on.

Cancer gene therapy: 'delivery, delivery, delivery' .

Gene therapy for cancer treatment represents a promising approach that has shown selectivity and efficacy in experimental systems as well as clinical trials. Some major problems remain to be solved before this strategy becomes routinely adopted in the clinic, one of the main challenges being the improvement of gene delivery. Namely, the development of DNA vectors characterized by maximum efficiency and minimal toxicity will define the success of gene therapy and its chances of being accepted by public and clinicians. A number of issues need to be considered. The "magic" vector should be targeted, protected from degradation and immune attack, and safe for the recipient and the environment. Moreover, it should express the therapeutic gene for as long as required, in an appropriately regulated fashion. Vehicles such as retroviruses, adenoviruses and liposomes have been adopted in clinical studies, with varying results. New therapeutic modalities are also being explored in order to overcome the limitation of poor gene transfer and patient toxicity, including bacteria, adeno-associated and herpes simplex viruses, lentiviruses, cationic polymer-DNA complexes and electroporation. Some of the delivery systems tested in preclinical and clinical models are reviewed in this article, with particular attention to the targeting of the tumor environment.

Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation -assay-based diagnostic kit.

Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates -80°C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5M sucrose-PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37°C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. These results confirm the viability of a freeze-dried PVNA-based kit, which could significantly facilitate low-cost serology for a wide portfolio of emerging infectious viruses.

The human Transmembrane Protease Serine 2 is necessary for the production of Group 2 influenza A virus pseudotypes.

The monomer of influenza haemagglutinin is synthesized as a single polypeptide precursor that during maturation is cleaved by proteases into two active subunits. Other studies have demonstrated that the human Transmembrane Protease Serine 2 (TMPRSS2) can cleave the HA of human seasonal influenza viruses. Consequently, we have investigated the use of human Transmembrane Protease Serine 2 to produce high titre influenza haemmagglutinin (HA) lentiviral pseudotypes from Group 2 influenza viruses. Such pseudotypes represent powerful and safe tools to study viral entry and immune responses. Influenza pseudotype particles are obtained by co-transfecting human embryonic kidney HEK293T/17 cells using plasmids coding for the influenza HA, HIV gag-pol and a lentiviral vector incorporating firefly luciferase. However, in order to produce Group 2 pseudotypes, it was necessary to co-transfect a plasmid expressing the TMPRSS2 endoprotease, to achieve the necessary HA cleavage for infective particle generation. These lentiviral pseudotypes were shown to transduce HEK293T/17 cells with high efficiency. This demonstrates that TMPRSS2 is necessary for the functional activation, in vitro, of both the HA of human seasonal influenza and other Group 2 HA influenza strains. Additionally, we show that the Group 2 influenza pseudotype particles can be used as surrogate antigens in neutralization assays and are efficiently neutralized by corresponding influenza virus reference sera. These data demonstrate that the viral pseudotype system is a powerful method for serological surveillance of a wide range of influenza viruses.

Use of macrophages to target therapeutic adenovirus to human prostate tumors.

New therapies are required to target hypoxic areas of tumors as these sites are highly resistant to conventional cancer therapies. Monocytes continuously extravasate from the bloodstream into tumors where they differentiate into macrophages and accumulate in hypoxic areas, thereby opening up the possibility of using these cells as vehicles to deliver gene therapy to these otherwise inaccessible sites. We describe a new cell-based method that selectively targets an oncolytic adenovirus to hypoxic areas of prostate tumors. In this approach, macrophages were cotransduced with a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, whose proliferation is restricted to prostate tumor cells using prostate-specific promoter elements from the TARP, PSA, and PMSA genes. When such cotransduced cells reach an area of extreme hypoxia, the E1A/B proteins are expressed, thereby activating replication of the adenovirus. The virus is subsequently released by the host macrophage and infects neighboring tumor cells. Following systemic injection into mice bearing subcutaneous or orthotopic prostate tumors, cotransduced macrophages migrated into hypoxic tumor areas, upregulated E1A protein, and released multiple copies of adenovirus. The virus then infected neighboring cells but only proliferated and was cytotoxic in prostate tumor cells, resulting in the marked inhibition of tumor growth and reduction of pulmonary metastases. This novel delivery system employs 3 levels of tumor specificity: the natural "homing" of macrophages to hypoxic tumor areas, hypoxia-induced proliferation of the therapeutic adenovirus in host macrophages, and targeted replication of oncolytic virus in prostate tumor cells.

Inhibition of repair of radiation-induced DNA damage enhances gene expression from replication-defective adenoviral vectors.

Radiation has been shown to up-regulate gene expression from adenoviral vectors in previous studies. In the current study, we show that radiation-induced dsDNA breaks and subsequent signaling through the mitogen-activated protein kinase (MAPK) pathway are responsible, at least in part, for this enhancement of transgene expression both in vitro and in vivo. Inhibitors of ataxia-telangiectasia-mutated, poly(ADP-ribose) polymerase-mutated, and DNA-dependent protein kinase (DNA-PK)-mediated DNA repair were shown to maintain dsDNA breaks (gammaH2AX foci) by fluorescence-activated cell sorting and microscopy. Inhibition of DNA repair was associated with increased green fluorescent protein (GFP) expression from a replication-defective adenoviral vector (Ad-CMV-GFP). Radiation-induced up-regulation of gene expression was abrogated by inhibitors of MAPK (PD980059 and U0126) and phosphatidylinositol 3-kinase (LY294002) but not by p38 MAPK inhibition. A reporter plasmid assay in which GFP was under the transcriptional control of artificial Egr-1 or cytomegalovirus promoters showed that the DNA repair inhibitors increased GFP expression only in the context of the Egr-1 promoter. In vivo administration of a water-soluble DNA-PK inhibitor (KU0060648) was shown to maintain luciferase expression in HCT116 xenografts after intratumoral delivery of Ad-RSV-Luc. These data have important implications for therapeutic strategies involving multimodality use of radiation, targeted drugs, and adenoviral gene delivery and provide a framework for evaluating potential advantageous combinatorial effects.

Radiation and hypoxia inducible gene therapy systems.

Radiotherapy remains one of the primary treatment modalities for most malignancies. Biologically based improvements in the scheduling of conventional radiotherapy and treatment planning, innovations like conformal radiotherapy and intensity-modulated radiation therapy have considerably improved the targeting and effectiveness of radiation for treatment of solid tumors. These new radiotherapy technologies are also promising means of focusing the activation of anti-tumor gene therapy systems, as an approach to further improve radiotherapeutic treatment, particularly for tumors refractive to current therapies. Gene therapy vectors that express therapeutic genes following irradiation have been produced. Delivery of such vectors to the tumor allows temporal and spatial expression of the transgenes within the radiation field. Hypoxia is a physiological characteristic of solid tumors and an independent prognostic marker for poor radiation treatment outcome. Nevertheless, hypoxia has been exploited to drive therapeutic gene expression from gene therapy vectors delivered to solid tumors exhibiting significant areas of low oxygen tension. Radiation and hypoxia inducible gene therapy systems rely on the activation of gene promoters containing specific responsive elements. Recent studies have shown the potential to combine these elements, permitting either or both stimuli to drive therapeutic gene expression. Furthermore, transgene expression can be amplified and sustained using novel 'signal feedback' or recombination systems. Such innovations allow promising new strategies to improve radiation treatment outcome, particularly where tumor hypoxia is a predominant issue.

How to overcome (and exploit) tumor hypoxia for targeted gene therapy.

Tumor hypoxia has long been recognized as a critical issue in oncology. Resistance of hypoxic areas has been shown to affect treatment outcome after radiation, chemotherapy, and surgery in a number of tumor sites. Two main strategies to overcome tumor hypoxia are to increase the delivery of oxygen (or oxygen-mimetic drugs), and exploiting this unique environmental condition of solid tumors for targeted therapy. The first strategy includes hyperbaric oxygen breathing, the administration of carbogen and nicotinamide, and the delivery of chemical radiosensitizers. In contrast, bioreductive drugs and hypoxia-targeted suicide gene therapy aim at activating cytotoxic agents at the tumor site, while sparing normal tissue from damage. The cellular machinery responds to hypoxia by activating the expression of genes involved in angiogenesis, anaerobic metabolism, vascular permeability, and inflammation. In most cases, transcription is initiated by the binding of the transcription factor hypoxia-inducible factor (HIF) to hypoxia responsive elements (HREs). Hypoxia-targeting for gene therapy has been achieved by utilizing promoters containing HREs, to induce selective and efficient transgene activation at the tumor site. Hypoxia-targeted delivery and prodrug activation may add additional levels of selectivity to the treatment. In this article, the latest developments of cancer gene therapy of the hypoxic environment are discussed, with particular attention to combined protocols with ionizing radiation. Ultimately, it is proposed that by adopting specific transgene activation and molecular amplification systems, resistant hypoxic tumor tissues may be effectively targeted with gene therapy.