Transient expression of Human papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector

A Tobacco mosaic virus (TMV)-derived vector was used to express a native Human papillomavirus type 16 (HPV-16) L1 gene in Nicotiana benthamiana by means of infectious in vitro RNA transcripts inoculated onto N. benthamiana plants. HPV-16 L1 protein expression was quantitated by enzyme-linked immunosorbent assays (ELISA) after concentration of the plant extract. We estimated that the L1 product yield was 20-37 μg/kg of fresh leaf material. The L1 protein in the concentrated extract was antigenically characterised using the neutralising and conformation-specific Mabs H16:V5 and H16:E70, which bound to the plant-produced protein. Particles observed by transmission electron microscopy were mainly capsomers but virus-like particles (VLPs) similar to those produced in other systems were also present. Immunisation of rabbits with the concentrated plant extract induced a weak immune response. This is the first report of the successful expression of an HPV L1 gene in plants using a plant virus vector. © 2006 Elsevier B.V. All rights reserved.

Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge : A proof-of-concept study

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Expression of HPV-11 L1 protein in transgenic Arabidopsis thaliana and Nicotiana tabacum

Background We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine. Results Transformation of plants was only achieved with the HPV-11 L1 gene with the C-terminal nuclear localization signal (NLS-) encoding region removed, and not with the full-length gene. The HPV-11 L1 NLS- gene was stably integrated and inherited through several generations of transgenic plants. Plant-derived HPV-11 L1 protein was capable of assembling into virus-like particles (VLPs), although resulting particles displayed a pleomorphic phenotype. Neutralising monoclonal antibodies binding both surface-linear and conformation-specific epitopes bound the A. thaliana-derived particles and - to a lesser degree - the N. tabacum-derived particles, suggesting that plant-derived and insect cell-derived VLPs displayed similar antigenic properties. Yields of up to 12 μg/g of HPV-11 L1 NLS- protein were harvested from transgenic A. thaliana plants, and 2 μg/g from N. tabacum plants - a significant increase over previous efforts. Immunization of New Zealand white rabbits with ∼50 μg of plant-derived HPV-11 L1 NLS- protein induced an antibody response that predominantly recognized insect cell-produced HPV-11 L1 NLS- and not NLS+ VLPs. Evaluation of the same sera concluded that none of them were able to neutralise pseudovirion in vitro. Conclusion We expressed the wild-type HPV-11 L1 NLS- gene in two different plant species and increased yields of HPV-11 L1 protein by between 500 and 1000-fold compared to previous reports. Inoculation of rabbits with extracts from both plant types resulted in a weak immune response, and antisera neither reacted with native HPV-11 L1 VLPs, nor did they neutralise HPV-11 pseudovirion infectivity. This has important and potentially negative implications for the production of HPV-11 vaccines in plants. © 2007 Kohl et al; licensee BioMed Central Ltd.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

The responsibility to protect and the Arab Spring : Libya as the exception, Syria as the norm?

On March 17 2011 the UN Security Council passed resolution 1973 authorising the use of force for civilian protection purposes in Libya.1 This resolution was hailed by many supporters of the responsibility to protect (R2P) as a crucial step towards the consolidation of the concept’s normative standing.2 Gareth Evans described the intervention as ‘a textbook case of the R2P norm working exactly as it was supposed to’.3 For Lloyd Axworthy the Libya episode signalled a move towards a ‘more humane world’.4 UN Secretary-General Ban Ki-Moon declared that it ‘affirms, clearly and unequivocally, the international community’s determination to fulfil its responsibility to protect civilians from violence perpetrated by their own government.’5 At first glance, the Security Council’s rapid, decisive response to escalating violence in Libya might well have suggested a new willingness on the part of the international community to take collective action to avert intra-state humanitarian crises. However, a closer examination of the text of resolution 1973 and statements by Security Council member states reveals a less than complete endorsement of R2P. Disagreements between states over the scope of the mandate for the use of force in Libya quickly emerged. Long-standing fears among Russia, China and other non-Western states that R2P could be used as a pretext for regime change returned to the fore as the legality and legitimacy of NATO’s military action were called into question. This post-Libya backlash against R2P has been a central factor in the international community’s subsequent inability to agree on effective civilian protection measures in Syria. Much of the optimism that surrounded R2P in the immediate aftermath of resolution 1973 has given way to a sober realization that achieving international consensus on civilian protection measures will rarely be straightforward.

Life-history constraints in grassland plant species : a growth-defence trade-off is the norm

Plant growth can be limited by resource acquisition and defence against consumers, leading to contrasting trade-off possibilities. The competition-defence hypothesis posits a trade-off between competitive ability and defence against enemies (e.g. herbivores and pathogens). The growth-defence hypothesis suggests that strong competitors for nutrients are also defended against enemies, at a cost to growth rate. We tested these hypotheses using observations of 706 plant populations of over 500 species before and following identical fertilisation and fencing treatments at 39 grassland sites worldwide. Strong positive covariance in species responses to both treatments provided support for a growth-defence trade-off: populations that increased with the removal of nutrient limitation (poor competitors) also increased following removal of consumers. This result held globally across 4 years within plant life-history groups and within the majority of individual sites. Thus, a growth-defence trade-off appears to be the norm, and mechanisms maintaining grassland biodiversity may operate within this constraint.

Epithelial-mesenchymal interconversions in normal ovarian surface epithelium and ovarian carcinomas : an exception to the norm

Cancer that arises from the ovarian surface epithelium (OSE) accounts for approximately 90% of human ovarian cancer, and is the fourth leading cause of cancer-related deaths among women in developed countries. The pathophysiology of epithelial ovarian cancer is still unclear because of the poor understanding of the complex nature of its development and the unusual mechanism(s) of disease progression. Recent studies have reported epithelial-mesenchymal transition (EMT) in cultured OSE and ovarian cancer cell lines in response to various stimuli, but our understanding of the importance of these observations for normal ovarian physiology and cancer progression is not well established. This review highlights the current literature on EMT-associated events in normal OSE and ovarian cancer cell lines, and discusses its implication for normal ovarian function as well as acquisition of neoplastic phenotypes. The pathological changes in OSE in response to EMT during neoplastic transformation and the contribution of hormones, growth factors, and cytokines that initiate and drive EMT to sustain normal ovarian function, as well as cancer development and progression are also discussed. Finally, emphasis is placed on the clinical implications of EMT and potential therapeutic opportunities that may arise from these observations have been proposed.

The BRICS and R2P : contestation, re-interpretation and norm entrepreneurship

The intra-state humanitarian crises in Libya and Syria have led to renewed debate over the content and implementation of pillar three of the responsibility to protect (R2P). This paper examines the BRICS’ (Brazil, Russia, India, China, South Africa) current perspectives on R2P and their recent efforts to shape the concept’s evolution. While Brazil’s “Responsibility while Protecting” (RwP) proposal has been widely discussed, the central focus here is on the lesser-known, semi-official Chinese idea of “Responsible Protection” (RP). Like RwP, RP proposes decision-making criteria and accountability mechanisms for UN-authorised military intervention under R2P’s third pillar. This paper argues that although RP draws heavily on previous R2P proposals such as the original 2001 ICISS report and Brazil’s RwP, by amalgamating and re-packaging these earlier ideas in a more restrictive form the Chinese initiative represents a new and distinctive interpretation of R2P. However, as it currently stands, some aspects of RP appear to be framed too strictly to provide workable guidelines for determining the permissibility of R2P military intervention, and would, therefore, benefit from clarification and refinement. Of broader significance, China’s RP and Brazil’s RwP initiatives point to the growing willingness of rising, non-Western powers to articulate and promote their own normative preferences on sovereignty, intervention and global governance. This development has potential implications both for R2P’s evolution and for the structure of the international system.

The adhesion molecule L1 regulates transendothelial migration and trafficking of dendritic cells

The adhesion molecule L1, which is extensively characterized in the nervous system, is also expressed in dendritic cells (DCs), but its function there has remained elusive. To address this issue, we ablated L1 expression in DCs of conditional knockout mice. L1-deficient DCs were impaired in adhesion to and transmigration through monolayers of either lymphatic or blood vessel endothelial cells, implicating L1 in transendothelial migration of DCs. In agreement with these findings, L1 was expressed in cutaneous DCs that migrated to draining lymph nodes, and its ablation reduced DC trafficking in vivo. Within the skin, L1 was found in Langerhans cells but not in dermal DCs, and L1 deficiency impaired Langerhans cell migration. Under inflammatory conditions, L1 also became expressed in vascular endothelium and enhanced transmigration of DCs, likely through L1 homophilic interactions. Our results implicate L1 in the regulation of DC trafficking and shed light on novel mechanisms underlying transendothelial migration of DCs. These observations might offer novel therapeutic perspectives for the treatment of certain immunological disorders.

Differences in L1 linguistic attention control between monolinguals and bilinguals

Relational elements of language (e.g. spatial prepositions) act to direct attention to aspects of an incoming message. The listener or reader must be able to use these elements to focus and refocus attention on the mental representation being constructed. Research has shown that this type of attention control is specific to language and can be distinguished from attention control for non-relational (semantic or content) elements. Twenty-two monolinguals (18–30 years) and nineteen bilinguals (18–30 years) completed two conditions of an alternating-runs task-switching paradigm in their first language. The relational condition involved processing spatial prepositions, and the non-relational condition involved processing concrete nouns and adjectives. Overall, monolinguals had significantly larger shift costs (i.e. greater attention control burden) in the relational condition than the non-relational condition, whereas bilinguals performed similarly in both conditions. This suggests that proficiency in a second language has a positive impact on linguistic attention control in one's native language.

Rationality, governmentality, natio(norm)ality: Shaping social science, scientific objects, and the invisible

The discipline of education in Anglophone-dominant contexts has always grappled with a kind of status anxiety relative to other disciplines. This is in part due to the ways in which evidence has been thought about in the theoretico-experimental sciences relative to the ethico-redemptive ones. By examining that which was considered to fall to the side of science, even of social science, this paper complexifies contemporary debates over educational science and research, including debates over evidence-based education or assumed divisions between the quantitative/qualitative and empirical/conceptual. It reapproaches historical vagaries in discourses of vision that underscore the arbitrariness of approaches to social scientific research and its objects. A less-considered set of spatializations and regionalisms in social scientific conceptions of rationality especially are exposed through a close reading of the Harvard University philosopher William James' more marginalized texts.

Blockade of PD-1/PD-L1 promotes adoptive T-Cell immunotherapy in a tolerogenic environment

Adoptive cellular immunotherapy using in vitro expanded CD8+ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The coinhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8+ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8+ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8+ T cells. Blockade of PD-1/PDL1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcmlike cells. © 2015 Blake et al.

Ratios of T-cell immune-effectors with tumour associated macrophages and PD-1/PD-L1 axis immune-checkpoint molecules, as prognosticators in diffuse large B cell lymphoma: A population-based study

Background Risk-stratification of diffuse large B-cell lymphoma (DLBCL) requires identification of patients with disease that is not cured despite initial R-CHOP. Although the prognostic importance of the tumour microenvironment (TME) is established, the optimal strategy to quantify it is unknown. Methods The relationship between immune-effector and inhibitory (checkpoint) genes was assessed by NanoString™ in 252 paraffin-embedded DLBCL tissues. A model to quantify net anti-tumoural immunity as an outcome predictor was tested in 158 R-CHOP treated patients, and validated in tissue/blood from two independent R-CHOP treated cohorts of 233 and 140 patients respectively. Findings T and NK-cell immune-effector molecule expression correlated with tumour associated macrophage and PD-1/PD-L1 axis markers consistent with malignant B-cells triggering a dynamic checkpoint response to adapt to and evade immune-surveillance. A tree-based survival model was performed to test if immune-effector to checkpoint ratios were prognostic. The CD4*CD8:(CD163/CD68)*PD-L1 ratio was better able to stratify overall survival than any single or combination of immune markers, distinguishing groups with disparate 4-year survivals (92% versus 47%). The immune ratio was independent of and added to the revised international prognostic index (R-IPI) and cell-of-origin (COO). Tissue findings were validated in 233 DLBCL R-CHOP treated patients. Furthermore, within the blood of 140 R-CHOP treated patients immune-effector:checkpoint ratios were associated with differential interim-PET/CT+ve/-ve expression.