Invariant NKT cells are more abundant in peanut-allergic adults and a subset of CD8+ iNKT cells are depleted after peanut oil exposure.

Introduction: Peanut allergy is one of the most prevalent food allergies globally. Currently, most research into the mechanisms involved in protein allergy focuses on the protein allergens under investigation, and information on the function of accompanying compounds, such as lipids, is scarce. Thus, this research investigates the role of peanut-associated lipids and invariant natural killer T (iNKT) cells in peanut allergy using a novel, human, in vitro assay. Methods: PBMCs from non-allergic and peanut-allergic subjects were stimulated with the glycolipid, α-Galactosylceramide (α-GalCer), over 14 days for iNKT cell expansion. Autologous dendritic cells (DCs) were stimulated with either peanut oil, the lipid-binding peanut allergen, Ara h 8, or both peanut oil and Ara h 8. The expanded iNKT cells were then immunomagnetically isolated and co-cultured for 5 h with autologous DCs, and cytokine expression was measured by flow cytometry. Results: A 5-fold higher iNKT cell population was observed in peanut-allergic subject peripheral blood compared to non-allergic controls. In all subjects, conventional flow analysis highlighted iNKTs co-cultured with autologous α-GalCer-pulsed DCs displayed increased IL-4 and IFN-y secretion within 5 hours of co-culture. A 10-parameter unsupervised clustering analysis of iNKT phenotype found significantly more CD3+CD8+CD25+IL-4+IL-5+IL-10+IFNγ+ cells in non-allergic adults following culture with peanut oil. Conclusion: For the first time, we show iNKT cells are more abundant in peanut-allergic adults compared to non-allergic adults, and peanut lipid-exposed iNKT cells resulted in the identification of a subset of CD8+ iNKT cells which was significantly lower in peanut-allergic adults. Thus, this study proposes a role for iNKT cells and peanut allergen-associated lipids in peanut allergy.

PBMC-derived extracellular vesicles in a smoking-related inflammatory disease model.

Extracellular vesicles (EVs) function as mediators of intercellular communication and as such influence the recipient cell function. EVs derived from immune cells can carry out many of the same functions as their parental cells, as they carry costimulatory molecules, antigens, and antigen-MHC complexes. As a result, there is a strong interest in understanding the composition and origin of immune cell-derived EVs in order to understand their role in the pathogenesis of diseases. This study aimed to optimize methodologies to study immune cell-derived EVs. Peripheral blood mononuclear cell-derived small EVs were isolated and observed using conventional transmission electron microscopy and sized by nanoparticle tracking analysis. They were then enumerated and profiled using imaging flow cytometry and were further characterized using a flow cytometric multiplex bead assay. These techniques were then applied to our current research, namely smoking-related inflammatory disease. We present here a comprehensive approach to analyze PBMC-derived small EVs in smoking-related inflammatory disease following the Minimal Information for Studies of Extracellular Vesicle 2018 guidelines.

A mechanoresponsive nano-sized carrier achieves intracellular release of drug on external ultrasound stimulus.

Control over intracellular release of therapeutic compounds incorporated into nano-carriers will open new possibilities for targeted treatments of various diseases including cancer, and viral and bacterial infections. Here we report our study on mechanoresponsive nano-sized liposomes which, following internalization by cells, achieve intracellular delivery of encapsulated cargo on application of external ultrasound stimulus. This is demonstrated in a bespoke cell reporter system designed to assess free drug in cytoplasm. Biophysical analyses show that drug release is attributable to the action of a mechanoresponsive spiropyran-based compound embedded in the liposomal lipid membrane. Exposure to external ultrasound stimulus results in opening of the molecular structure of the embedded spiropyran, a consequent increase in liposomal lipid membrane fluidity, and size-dependent release of encapsulated model drugs, all pointing to lipid bilayer perturbation. The study hence illustrates feasibility of the proposed concept where intracellular drug release from mechanoresponsive liposomes can be triggered on demand by external ultrasound stimulus.

ABCB1 inhibition provides a novel therapeutic target to block TWIST1-induced migration in medulloblastoma.

BACKGROUND: Therapeutic intervention in metastatic medulloblastoma is dependent on elucidating the underlying metastatic mechanism. We investigated whether an epithelial-mesenchymal transition (EMT)-like pathway could drive medulloblastoma metastasis. METHODS: A 3D Basement Membrane Extract (3D-BME) model was used to investigate medulloblastoma cell migration. Cell line growth was quantified with AlamarBlue metabolic assays and the morphology assessed by time-lapse imaging. Gene expression was analyzed by qRT-PCR and protein expression by immunohistochemistry of patient tissue microarrays and mouse orthotopic xenografts. Chromatin immunoprecipitation was used to determine whether the EMT transcription factor TWIST1 bound to the promoter of the multidrug pump ABCB1. TWIST1 was overexpressed in MED6 cells by lentiviral transduction (MED6-TWIST1). Inhibition of ABCB1 was mediated by vardenafil, and TWIST1 expression was reduced by either Harmine or shRNA. RESULTS: Metastatic cells migrated to form large metabolically active aggregates, whereas non-tumorigenic/non-metastatic cells formed small aggregates with decreasing metabolic activity. TWIST1 expression was upregulated in the 3D-BME model. TWIST1 and ABCB1 were significantly associated with metastasis in patients (P = .041 and P = .04, respectively). High nuclear TWIST1 expression was observed in the invasive edge of the MED1 orthotopic model, and TWIST1 knockdown in cell lines was associated with reduced cell migration (P < .05). TWIST1 bound to the ABCB1 promoter (P = .03) and induced cell aggregation in metastatic and TWIST1-overexpressing, non-metastatic (MED6-TWIST1) cells, which was significantly attenuated by vardenafil (P < .05). CONCLUSIONS: In this study, we identified a TWIST1-ABCB1 signaling axis during medulloblastoma migration, which can be therapeutically targeted with the clinically approved ABCB1 inhibitor, vardenafil.

Corrigendum to: ABCB1 inhibition provides a novel therapeutic target to block TWIST1-induced migration in medulloblastoma.

[This corrects the article DOI: 10.1093/noajnl/vdab030.].

Metabolism-based isolation of invasive glioblastoma cells with specific gene signatures and tumorigenic potential.

BACKGROUND: Glioblastoma (GBM) is a highly aggressive brain tumor with rapid subclonal diversification, harboring molecular abnormalities that vary temporospatially, a contributor to therapy resistance. Fluorescence-guided neurosurgical resection utilizes the administration of 5-aminolevulinic acid (5-ALA) generating individually fluorescent tumor cells within a background population of non-neoplastic cells in the invasive tumor region. The aim of the study was to specifically isolate and interrogate the invasive GBM cell population using a novel 5-ALA-based method. METHODS: We have isolated the critical invasive GBM cell population by developing 5-ALA-based metabolic fluorescence-activated cell sorting. This allows purification and study of invasive cells from GBM without an overwhelming background "normal brain" signal to confound data. The population was studied using RNAseq, real-time PCR, and immunohistochemistry, with gene targets functionally interrogated on proliferation and migration assays using siRNA knockdown and known drug inhibitors. RESULTS: RNAseq analysis identifies specific genes such as SERPINE1 which is highly expressed in invasive GBM cells but at low levels in the surrounding normal brain parenchyma. siRNA knockdown and pharmacological inhibition with specific inhibitors of SERPINE1 reduced the capacity of GBM cells to invade in an in vitro assay. Rodent xenografts of 5-ALA-positive cells were established and serially transplanted, confirming tumorigenicity of the fluorescent patient-derived cells but not the 5-ALA-negative cells. CONCLUSIONS: Identification of unique molecular features in the invasive GBM population offers hope for developing more efficacious targeted therapies compared to targeting the tumor core and for isolating tumor subpopulations based upon intrinsic metabolic properties.

A 3D Heterotypic Breast Cancer Model Demonstrates a Role for Mesenchymal Stem Cells in Driving a Proliferative and Invasive Phenotype.

Previous indirect 2D co-culture studies have demonstrated that mesenchymal stem cells (MSCs) promote breast cancer (BC) progression through secretion of paracrine factors including growth factors, cytokines and chemokines. In order to investigate this aspect of the tumour microenvironment in a more relevant 3D co-culture model, spheroids incorporating breast cancer cells (BCCs), both cell lines and primary BCCs expanded as patient-derived xenografts, and MSCs were established. MSCs in co-cultures were shown to enhance proliferation of estrogen receptor (ER)/progesterone receptor (PR)-positive BCCs. In addition, co-culture resulted in downregulation of E-cadherin in parallel with upregulation of the epithelial-mesenchymal transition (EMT)-relation transcription factor, SNAIL. Cytoplasmic relocalization of ski-related novel protein N (SnON), a negative regulator of transforming growth factor-beta (TGF-ß) signalling, and of ß-catenin, involved in a number of pathways including Wnt signalling, was also observed in BCCs in co-cultures in contrast to monocultures. In addition, the ß-catenin inhibitor, 3-[[(4-methylphenyl)sulfonyl]amino]-benzoic acid methyl ester (MSAB), mediated reduced growth and invasion in the co-cultures. This study highlights the potential role for SnON as a biomarker for BC invasiveness, and the importance of interactions between TGF-ß and Wnt signalling, involving SnON. Such pathways may contribute towards identifying possible targets for therapeutic intervention in BC patients.

Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines.

Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.

Intracellular processing of silica-coated superparamagnetic iron nanoparticles in human mesenchymal stem cells.

Silica-coated superparamagnetic iron nanoparticles (SiMAGs) are an exciting biomedical technology capable of targeted delivery of cell-based therapeutics and disease diagnosis. However, in order to realise their full clinical potential, their intracellular fate must be determined. The analytical techniques of super-resolution fluorescence microscopy, particle counting flow cytometry and pH-sensitive nanosensors were applied to elucidate mechanisms of intracellular SiMAG processing in human mesenchymal stem cell (hMSCs). Super-resolution microscopy showed SiMAG fluorescently-tagged nanoparticles are endocytosed and co-localised within lysosomes. When exposed to simulated lysosomal conditions SiMAGs were solubilised and exhibited diminishing fluorescence emission over 7 days. The in vitro intracellular metabolism of SiMAGs was monitored in hMSCs using flow cytometry and co-localised pH-sensitive nanosensors. A decrease in SiMAG fluorescence emission, which corresponded to a decrease in lysosomal pH was observed, mirroring ex vivo observations, suggesting SiMAG lysosomal exposure degrades fluorescent silica-coatings and iron cores. These findings indicate although there is a significant decrease in intracellular SiMAG loading, sufficient particles remain internalised (>50%) to render SiMAG treated cells amenable to long-term magnetic cell manipulation. Our analytical approach provides important insights into the understanding of the intracellular fate of SiMAG processing, which could be readily applied to other particle therapeutics, to advance their clinical translation.

Multicomponent analysis of the tumour microenvironment reveals low CD8 T cell number, low stromal caveolin-1 and high tenascin-C and their combination as significant prognostic markers in non-small cell lung cancer.

The complex interplay of the tumour microenvironment (TME) and its role in disease progression and response to therapy is poorly understood. The majority of studies to date focus on individual components or molecules within the TME and so lack the power correlative analysis. Here we have performed a multi-parameter analysis of the TME in 62 resectable non-small cell lung cancer (NSCLC) specimens detailing number and location of immune infiltrate, assessing markers of cancer-associated fibroblasts, caveolin-1 and tenascin-C, and correlating with clinicopathological details, as well as markers of disease progression such as epithelial-to-mesenchymal transition (EMT). The influence of individual parameters on overall survival was determined in univariate and multivariate analysis and the combination of risk factors and interplay between components analysed. Low numbers of CD8 T cells, low stromal levels of caveolin-1 or high levels of tenascin-C were significant prognostic markers of decreased overall survival in both univariate and multivariate analysis. Patients with two or more risk factors had dramatically reduced overall survival and those with all three a median survival of just 7.5 months. In addition, low levels of tumour E-cadherin correlated with reduced immune infiltrate into the tumour nests, possibly linking EMT to the avoidance of CD8 T cell control. The multicomponent approach has allowed identification of the dominant influences on overall survival, and exploration of the interplay between different components of the TME in NSCLC.

Individual patient oesophageal cancer 3D models for tailored treatment.

BACKGROUND: A model to predict chemotherapy response would provide a marked clinical benefit, enabling tailored treatment of oesophageal cancer, where less than half of patients respond to the routinely administered chemotherapy. METHODS: Cancer cells were established from tumour biopsies taken from individual patients about to undergo neoadjuvant chemotherapy. A 3D-tumour growth assay (3D-TGA) was developed, in which cancer cells were grown with or without supporting mesenchymal cells, then subjected to chemo-sensitivity testing using the standard chemotherapy administered in clinic, and a novel emerging HDAC inhibitor, Panobinostat. RESULTS: Individual patient's cancer cells could be expanded and screened within a clinically applicable timescale of 3 weeks. Incorporating mesenchymal support within the 3D-TGA significantly enhanced both the growth and drug resistance profiles of the patient's cancer cells. The ex vivo drug response in the presence, but not absence, of mesenchymal cells accurately reflected clinical chemo-sensitivity, as measured by tumour regression grade. Combination with Panobinostat enhanced response and proved efficacious in otherwise chemo-resistant tumours. CONCLUSIONS: This novel method of establishing individual patient oesophageal cancers in the laboratory, from small endoscopic biopsies, enables clinically-relevant chemo-sensitivity testing, and reduces use of animals by providing more refined in vitro models for pre-screening of drugs. The 3D-TGA accurately predicted chemo-sensitivity in patients, and could be developed to guide tailored patient treatment. The incorporation of mesenchymal cells as the stromal cell component of the tumour micro-environment had a significant effect upon enhancing chemotherapy drug resistance in oesophageal cancer, and could prove a useful target for future drug development.

3-Dimensional Patient-Derived Lung Cancer Assays Reveal Resistance to Standards-of-Care Promoted by Stromal Cells but Sensitivity to Histone Deacetylase Inhibitors.

There is a growing recognition that current preclinical models do not reflect the tumor microenvironment in cellular, biological, and biophysical content and this may have a profound effect on drug efficacy testing, especially in the era of molecular-targeted agents. Here, we describe a method to directly embed low-passage patient tumor-derived tissue into basement membrane extract, ensuring a low proportion of cell death to anoikis and growth complementation by coculture with patient-derived cancer-associated fibroblasts (CAF). A range of solid tumors proved amenable to growth and pharmacologic testing in this 3D assay. A study of 30 early-stage non-small cell lung cancer (NSCLC) specimens revealed high levels of de novo resistance to a large range of standard-of-care agents, while histone deacetylase (HDAC) inhibitors and their combination with antineoplastic drugs displayed high levels of efficacy. Increased resistance was seen in the presence of patient-derived CAFs for many agents, highlighting the utility of the assay for tumor microenvironment-educated drug testing. Standard-of-care agents showed similar responses in the 3D ex vivo and patient-matched in vivo models validating the 3D-Tumor Growth Assay (3D-TGA) as a high-throughput screen for close-to-patient tumors using significantly reduced animal numbers. Mol Cancer Ther; 15(4); 753-63. ©2016 AACR.

Quantum dot labelling of adenovirus allows highly sensitive single cell flow and imaging cytometry.

A quantum dot method for highly efficient labelling of single adenoviral particles is developed. The technique has no impact on viral fitness and allows the imaging and tracking of virus binding and internalisation events using a variety of techniques including imaging cytometry and confocal microscopy. The method is applied to characterise the tropism of different adenoviral vectors.

HLA-peptide multimer selection of adenovirus-specific T cells for adoptive T-cell therapy.

Adenovirus (Ad) infection is a cause of significant morbidity and mortality in hematopoietic stem cell transplant recipients and virus-specific immunotherapy is one option for improved control. Cellular immunity is an important component in suppression of Ad replication but the frequency and population distribution of Ad-specific CD8 T cells has not been systematically investigated. This is an important question in relation to the potential use of these cells for adoptive transfer. To address this question, HLA-peptide multimers were generated for 8 HLA class I-restricted Ad epitopes, which are highly conserved across Ad species. Epitope-specific CD8 T cells from healthy donors were identified by tetramer staining and HLA class I A*01-restricted TDL peptide staining T cells were characterized in relation to frequency, phenotype, and function. The cells demonstrated a minimally differentiated central memory phenotype (CD45RA, CD45RO, CCR7, CD62L, CD27, CD28, and CD57) and were able to produce IFN-γ and proliferate extensively upon antigen stimulation in vitro. After proliferation, the phenotype switched to CD45RO, although it is interesting to note that CCR7 expression was retained. Despite their low frequency, tetramer-staining cells could be enriched with magnetic bead technology. Their characteristics should permit rapid establishment in vivo post adoptive transfer, increasing therapeutic options for patients with Ad infection. This is the first reported characterization of Ad-specific tetramer-staining T cells with a view to adoptive transfer to hematopoietic stem cell transplant patients with Ad infection. The efficacy of these cells needs to be further evaluated in the setting of a clinical trial.

Elevated SP-1 transcription factor expression and activity drives basal and hypoxia-induced vascular endothelial growth factor (VEGF) expression in non-small cell lung cancer.

VEGF plays a central role in angiogenesis in cancer. Non-small cell lung cancer (NSCLC) tumors have increased microvascular density, localized hypoxia, and high VEGF expression levels; however, there is a lack of understanding of how oncogenic and tumor microenvironment changes such as hypoxia lead to greater VEGF expression in lung and other cancers. We show that NSCLC cells secreted higher levels of VEGF than normal airway epithelial cells. Actinomycin D inhibited all NSCLC VEGF secretion, and VEGF minimal promoter-luciferase reporter constructs were constitutively active until the last 85 base pairs before the transcription start site containing three SP-1 transcription factor-binding sites; mutation of these VEGF promoter SP-1-binding sites eliminated VEGF promoter activity. Furthermore, dominant negative SP-1, mithramycin A, and SP-1 shRNA decreased VEGF promoter activity, whereas overexpression of SP-1 increased VEGF promoter activity. Chromatin immunoprecipitation assays demonstrated SP-1, p300, and PCA/F histone acetyltransferase binding and histone H4 hyperacetylation at the VEGF promoter in NSCLC cells. Cultured NSCLC cells expressed higher levels of SP-1 protein than normal airway epithelial cells, and double-fluorescence immunohistochemistry showed a strong correlation between SP-1 and VEGF in human NSCLC tumors. In addition, hypoxia-driven VEGF expression in NSCLC cells was SP-1-dependent, with hypoxia increasing SP-1 activity and binding to the VEGF promoter. These studies are the first to demonstrate that overexpression of SP-1 plays a central role in hypoxia-induced VEGF secretion.

Adenovirus vector-specific T cells demonstrate a unique memory phenotype with high proliferative potential and coexpression of CCR5 and integrin alpha4beta7.

BACKGROUND: The Step Study was a randomized trial to reduce HIV infection through vaccination with an adenovirus type 5 (Ad5)-based gag/pol/nef construct; analysis following early cessation of the trial revealed an excess of HIV seroconversion in Ad5 seropositive men. This led to the suggestion that the Ad based vector may boost the number of CD4 chemokine receptor 5 (CCR5) T cells, target cells for HIV infection. OBJECTIVES: We sought to determine the immunophenotype and proliferative capacity of Ad5-specific T cells in the peripheral blood of adult donors to determine whether stimulation with replication defective Ad5 vectors could result in the significant expansion of a CD4 CCR5 T-cell subset. METHODS: Ad5-specific T cells were identified in the peripheral blood of healthy donors by interferon-gamma secretion assay and proliferative response was measured by carboxyfluorescein succinimidyl ester labelling. Cells were analyzed by flow cytometry to determine T-cell differentiation marker, CCR5 and alpha4beta7 expression on memory and proliferated cells. RESULTS: Ad5-specific CD4 T cells within healthy adult donors exhibit a unique minimally differentiated memory phenotype with coexpression of CD45RA, CD45RO and CCR7. Stimulation with Ad vector leads to rapid expansion in vitro and a switch to an effector memory phenotype. Both short-term reactivated and proliferating Ad5-specific CD4 T cells express the HIV coreceptor CCR5 and the HIV gp120-binding integrin alpha4beta7. CONCLUSION: Ad5-specific T cells demonstrate a phenotype and proliferative potential that would support HIV infection; these results are pertinent to the findings of the Step Study and future use of Ad5 as a vaccine vector.

Antivector and tumor immune responses following adenovirus-directed enzyme prodrug therapy for the treatment of prostate cancer.

We have completed a phase I/II suicide gene therapy clinical trial in patients with prostate cancer, using an E1/E3-deleted replication-deficient adenovirus (CTL102) encoding the bacterial nitroreductase enzyme in combination with prodrug CB1954. This study has provided an opportunity to monitor and characterize vector- and tumor-specific adaptive immunity before and after single or repeat injections of adenovirus. Here we report robust vector-specific humoral and cellular immune responses in all patients monitored. However, we found no correlation between preexisting immunity or the magnitude of the immune response to vector and the clinical outcome as measured by changes in serum prostate-specific antigen (PSA) level. Increased frequency of T cells recognizing prostate-specific antigens PSA or prostate-specific membrane antigen (PSMA) was detected in 3 of 11 patients after therapy, suggesting that this direct cytotoxic strategy can also stimulate tumor-specific immunity.

The CD4+ T-cell response to adenovirus is focused against conserved residues within the hexon protein.

Adenovirus is a significant pathogen in immunocompromised patients and is widely utilized as a gene delivery vector, so a detailed understanding of the human immune response to adenovirus infection is critical. This study characterized the adenovirus-specific CD4(+) T-cell response of healthy donors by incubation with whole virus or with individual hexon and fiber proteins. Adenovirus-specific CD4(+) T cells averaged 0.26 % of the CD4(+) T-cell pool and were detectable in all donors. T cells recognizing the highly conserved hexon protein accounted for 0.09 %, whereas no response was observed against the fiber protein. A panel of hexon-specific CD4(+) T-cell clones was generated and shown to lyse targets infected with adenovirus from different serotypes and species. Three CD4 T-cell epitopes are described, which map to highly conserved regions of the hexon protein.

Adenovirus type 5 interactions with human blood cells may compromise systemic delivery.

Intravenous delivery of adenovirus vectors requires that the virus is not inactivated in the bloodstream. Serum neutralizing activity is well documented, but we show here that type 5 adenovirus also interacts with human blood cells. Over 90% of a typical virus dose binds to human (but not murine) erythrocytes ex vivo, and samples from a patient administered adenovirus in a clinical trial showed that over 98% of viral DNA in the blood was cell associated. In contrast, nearly all viral genomes in the murine bloodstream are free in the plasma. Adenovirus bound to human blood cells fails to infect A549 lung carcinoma cells, although dilution to below 1.7 x 10(7) blood cells/ml relieves this inhibition. Addition of blood cells can prevent infection by adenovirus that has been prebound to A549 cells. Adenovirus also associates with human neutrophils and monocytes ex vivo, particularly in the presence of autologous plasma, giving dose-dependent transgene expression in CD14-positive monocytes. Finally, although plasma with a high neutralizing titer (defined on A549 cells) inhibits monocyte infection, weakly neutralizing plasma can actually enhance monocyte transduction. This may increase antigen presentation following intravenous injection, while blood cell binding may both decrease access of the virus to extravascular targets and inhibit infection of cells to which the virus does gain access.

Viral gene therapy strategies: from basic science to clinical application.

A major impediment to the successful application of gene therapy for the treatment of a range of diseases is not a paucity of therapeutic genes, but the lack of an efficient non-toxic gene delivery system. Having evolved to deliver their genes to target cells, viruses are currently the most effective means of gene delivery and can be manipulated to express therapeutic genes or to replicate specifically in certain cells. Gene therapy is being developed for a range of diseases including inherited monogenic disorders and cardiovascular disease, but it is in the treatment of cancer that this approach has been most evident, resulting in the recent licensing of a gene therapy for the routine treatment of head and neck cancer in China. A variety of virus vectors have been employed to deliver genes to cells to provide either transient (eg adenovirus, vaccinia virus) or permanent (eg retrovirus, adeno-associated virus) transgene expression and each approach has its own advantages and disadvantages. Paramount is the safety of these virus vectors and a greater understanding of the virus-host interaction is key to optimizing the use of these vectors for routine clinical use. Recent developments in the modification of the virus coat allow more targeted approaches and herald the advent of systemic delivery of therapeutic viruses. In the context of cancer, the ability of attenuated viruses to replicate specifically in tumour cells has already yielded some impressive results in clinical trials and bodes well for the future of this approach, particularly when combined with more traditional anti-cancer therapies.