The roles of the preproghrelin-derived peptides - ghrelin, desacyl ghrelin and obestatin - in prostate cancer

Prostate cancer is the second most common cause of cancer related deaths in Western men. Despite the significant improvements in current treatment techniques, there is no cure for advanced metastatic, castrate-resistant disease. Early detection and prevention of progression to a castrate-resistant state may provide new strategies to improve survival. A number of growth factors have been shown to act in an autocrine/paracrine manner to modulate prostate cancer tumour growth. Our laboratory has previously shown that ghrelin and its receptors (the functional GHS-R1a and the non-functional GHS-R1b) are expressed in prostate cancer specimens and cell lines. We have shown that ghrelin increases cell proliferation in the PC3 and LNCaP prostate cancer cell lines through activation of ERK1/2, suggesting that ghrelin could regulate prostate cancer cell growth and play a role in the progression of the disease. Ghrelin is a 28 amino-acid peptide hormone, identified to be the natural ligand of the growth hormone secretagogue receptor (GHS-R1a). It is well characterised as a growth hormone releasing and as an orexigenic peptide that stimulates appetite and feeding and regulates energy expenditure and bodyweight. In addition to its orexigenic properties, ghrelin has been shown to play a regulatory role in a number of systems, including the reproductive, immune and cardiovascular systems and may play a role in a number of pathological conditions such as chronic heart failure, anorexia, cachexia, obesity, diabetes and cancer. In cancer, ghrelin and its receptor are expressed in a range of tumours and cancer cell lines and ghrelin has been demonstrated to modulate cell proliferation, apoptosis, migration and invasion in some cell types. The ghrelin gene (GHRL) encodes preproghrelin peptide, which is processed to produce three currently known functional peptides - ghrelin, desacyl ghrelin and obestatin. Prohormone convertases (PCs) have been shown to cleave the preproghrelin peptide into two primary products - the 28 amino acid peptide, ghrelin, and the remaining 117 amino acid C-terminal peptide, C-ghrelin. C-ghrelin can then be further processed to produce the 23 amino acid peptide, obestatin. Ghrelin circulates in two different forms - an octanoylated form (known as ghrelin) and a non-octanoylated form, desacyl ghrelin. The unique post-translational addition of octanoic acid to the serine 3 residue of the propeptide chain to form acylated ghrelin is catalysed by ghrelin O-acyltransferase (GOAT). This modification is necessary for binding of ghrelin to its only known functional receptor, the GHS-R1a. As desacyl ghrelin cannot bind and activate the GHS-R1a, it was initially thought to be an inactive peptide, despite the fact that it circulates at much higher levels than ghrelin. Further research has demonstrated that desacyl ghrelin is biologically active and shares some of the actions of ghrelin, as well as having some opposing and distinct roles. Interestingly, both ghrelin and desacyl ghrelin have been shown to modulate apoptosis, cell differentiation and proliferation in some cell types, and to stimulate cell proliferation through activation of ERK1/2 and PI3K/Akt pathways. The third known peptide product of the ghrelin preprohormone, obestatin, was initially thought to oppose the actions of ghrelin in appetite regulation and food intake and to mediate its effects through the G protein-coupled receptor 39 (GPR39). Subsequent research failed to reproduce the initial findings, however, and the possible anorexigenic effects of obestatin, as well as the identity of its receptor, remain unclear. Obestatin plays some important physiological roles, including roles in improving memory, the inhibition of thirst and anxiety, increased secretion of pancreatic juice, and regulation of cell proliferation, survival, apoptosis and differentiation. Preliminary studies have also shown that obestatin stimulates cell proliferation in some cell types through activation of ERK1/2, Akt and PKC pathways. Overall, however, at the commencement of this PhD project, relatively little was known regarding the functions and mechanisms of action of the preproghrelin-derived functional peptides in modulating prostate cancer cell proliferation. The roles of obestatin, and desacyl ghrelin as potential growth factors had not previously been investigated, and the potential expression and regulation of the preproghrelin processing enzymes, GOAT and prohormone convertases was unknown in prostate cancer cell lines. Therefore, the overall objectives of this study were to: 1. investigate the effects of obestatin on cell proliferation and signaling in prostate cancer cell lines 2. compare the effects of desacyl ghrelin and ghrelin on cell proliferation and signaling in prostate cancer cell lines 3. investigate whether prostate cancer cell lines possess the necessary enzymatic machinery to produce ghrelin and desacyl ghrelin and if these peptides can regulate GOAT expression Our laboratory has previously shown that ghrelin stimulates cell proliferation in the PC3 and LNCaP prostate cancer cell line through activation of the ERK1/2 pathway. In this study it has been demonstrated that treatments with either ghrelin, desacyl ghrelin or obestatin over 72 hours significantly increased cell proliferation in the PC3 prostate cancer cell line but had no significant effect in the RWPE-1 transformed normal prostate cell line. Ghrelin (1000nM) stimulated cell proliferation in the PC3 prostate cancer cell line by 31.66 6.68% (p<0.01) with the WST-1 method, and 13.55 5.68% (p<0.05) with the CyQUANT assay. Desacyl ghrelin (1000nM) increased cell proliferation in PC3 cells by 21.73 2.62% (p<0.01) (WST-1), and 15.46 7.05% (p<0.05) (CyQUANT) above untreated control. Obestatin (1000nM) induced a 28.37 7.47% (p<0.01) (WST-1) and 12.14 7.47% (p<0.05) (CyQUANT) significant increase in cell proliferation in the PC3 prostate cancer cell line. Ghrelin and desacyl ghrelin treatments stimulated Akt and ERK phosphorylation across a range of concentrations (p<0.01). Obestatin treatment significantly stimulated Akt, ERK and PKC phosphorylation (p<0.05). Through the use of specific inhibitors, the MAPK inhibitor U0126 and the Akt1/2 kinase inhibitor, it was demonstrated that ghrelin- and obestatin-induced cell proliferation in the PC3 prostate cancer cell line is mediated through activation of ERK1/2 and Akt pathways. Although desacyl ghrelin significantly stimulated Akt and ERK phosphorylation, U0126 failed to prevent desacyl ghrelin-induced cell proliferation suggesting ghrelin and desacyl ghrelin might act through different mechanisms to increase cell proliferation. Ghrelin and desacyl ghrelin have shown a proliferative effect in osteoblasts, pancreatic -cells and cardiomyocytes through activation of ERK1/2 and PI3K/Akt pathways. Here it has been shown that ghrelin and its non-acylated form exert the same function and stimulate cell proliferation in the PC3 prostate cancer cell line through activation of the Akt pathway. Ghrelin-induced proliferation was also mediated through activation of the ERK1/2 pathway, however, desacyl ghrelin seems to stimulate cell proliferation in an ERK1/2-independent manner. As desacyl ghrelin does not bind and activate GHSR1a, the only known functional ghrelin receptor, the finding that both ghrelin and desacyl ghrelin stimulate cell proliferation in the PC3 cell line suggests that these peptides could be acting through the yet unidentified alternative ghrelin receptor in this cell type. Obestatin treatment also stimulated PKC phosphorylation, however, a direct role for this pathway in stimulating cell proliferation could not be proven using available PKC pathway inhibitors, as they caused significant cell death over the extended timeframe of the cell proliferation assays. Obestatin has been shown to stimulate cell proliferation through activation of PKC isoforms in human retinal epithelial cells and in the human gastric cancer cell line KATO-III. We have demonstrated that all of the prostate-derived cell lines examined (PC3, LNCaP, DU145, 22Rv1, RWPE-1 and RWPE-2) expressed GOAT and at least one of the prohormone convertases, which are known to cleave the proghrelin peptide, PC1/3, PC2 and furin, at the mRNA level. These cells, therefore, are likely to possess the necessary machinery to cleave the preproghrelin protein and to produce the mature ghrelin and desacyl ghrelin peptides. In addition to prohormone convertases, the presence of octanoic acid is essential for acylated ghrelin production. In this study octanoic acid supplementation significantly increased cell proliferation in the PC3 prostate cancer cell line by over 20% compared to untreated controls (p<0.01), but surprisingly, not in the DU145, LNCaP or 22Rv1 prostate cancer cell lines or in the RWPE-1 and RWPE-2 prostate-derived cell lines. In addition, we demonstrated that exogenous ghrelin induced a statistically significant two-fold decrease in GOAT mRNA expression in the PC3 cell line (p<0.05), suggesting that ghrelin could pontentially downregulate its own acylation and, therefore, regulate the balance between ghrelin and desacyl ghrelin. This was not observed, however, in the DU145 and LNCaP prostate cancer cell lines. The GOAT-ghrelin system represents a direct link between ingested nutrients and regulation of ghrelin production and the ghrelin/desacyl ghrelin ratio. Regulation of ghrelin acylation is a potentially attractive and desirable tool for the development of better therapies for a number of pathological conditions where ghrelin has been shown to play a key role. The finding that desacyl ghrelin stimulates cell proliferation in the PC3 prostate cancer cell line, and responds to ghrelin in the same way, suggests that this cell line expresses an alternative ghrelin receptor. Although all the cell lines examined expressed both GHS-R1a and GHS-R1b mRNA, it remains uncertain whether these cell lines express the unidentified alternative ghrelin receptor. It is possible that the varied responses seen could be due to the expression of different ghrelin receptors in different cell lines. In addition to GOAT, prohormone convertases and octanoic acid availability may regulate the production of different peptides from the ghrelin preprohormone. The studies presented in this thesis provide significant new information regarding the roles and mechanisms of action of the preproghrelin-derived peptides, ghrelin, desacyl ghrelin and obestatin, in modulating prostate cancer cell line proliferation. A number of key questions remain to be resolved, however, including the identification of the alternative ghrelin/desacyl ghrelin receptor, the identification of the obestatin receptor, a clarification of the signaling mechanisms which mediate cell proliferation in response to obestatin treatment and a better understanding of the regulation at both the gene and post-translational levels of functional peptide generation. Further studies investigating the role of the ghrelin axis using in vivo prostate cancer models may be warranted. Until these issues are determined, the potential for the ghrelin axis, to be recognised as a novel useful target for therapy for cancer or other pathologies will be uncertain.

Fixed bed pyrolysis of date seed waste for liquid oil production

The conversion of biomass waste in the form of date seed into pyrolysis oil by fixed bed pyrolysis reactor has been taken into consideration in this study. A fixed bed pyrolysis has been designed and fabricated for obtaining liquid fuel from these date seeds. The major component of the system are fixed bed pyrolysis reactor, liquid condenser and liquid collector. The date seed in particle form is pyrolysed in an externally heated 7.6 cm diameter and 46 cm high fixed bed reactor with nitrogen as the carrier gas. The reactor is heated by means of a biomass source cylindrical heater from 4000C to 6000C. The products are oil, char and gas. The reactor bed temperature, running time and feed particle size are considered as process parameters. The parameters are found to influence the product yield significantly. A maximum liquid yield of 50 wt.% is obtained at a reactor bed temperature of 5000 C for a feed size volume of 0.11- 0.20 cm3 with a running time of 120 minutes. The pyrolysis oil obtained at this optimum process conditions are analyzed for some fuel properties and compared with some other biomass derived pyrolysis oils and also with conventional fuels. The oil is found to possess favorable flash point and reasonable density and viscosity. The higher calorific value is found to be 28.636 MJ/kg which is significantly higher than other biomass derived pyrolysis oils.

The creative cities discourse : production and/or consumption?

The already considerable debate about what constitutes a 'creative' city becomes ever more critical as the world urbanizes at a rapid pace. In this chapter the author argues that the key tensions in discussions over what makes cities more conducive to and supportive of creativity revolve around perspectives that are either production-centric or consumption-centric. Scholars are increasing prepared to claim priority for the city-region over the nation-state as an economic and cultural agent in the contemporary world, but are they ready to deal with major changes in the nature of cultural production and consumption themselves? A number of examples of new challenges for the creative cities 'discourse' rounds out the chapter.

Design and construction of fixed bed pyrolysis system and plum seed pyrolysis for bio-oil production

This work investigated the production of bio oil from plum seed (Zyziphus jujuba) by fixed bed pyrolysis technology. A fixed bed pyrolysis system has been designed and fabricated for production of bio oil. The major components of the system are: fixed bed reactor, liquid condenser and liquid collector. Nitrogen gas was used to maintain the inert atmosphere in the reactor where the pyrolysis reaction takes place. The feedstock considered in this study is plum seed as it is available waste material in Bangladesh. The reactor is heated by means of a cylindrical biomass external heater. Rice husk was used as the energy source. The products are oil, char and gas. The parameters varied are reactor bed temperature, running time and feed particle size. The parameters are found to influence the product yields significantly. The maximum liquid yield of 39 wt% at 5200C for a feed particle size of 2.36-4.75 mm and a gas flow rate of 8 liter/min with a running time of 120 minute. The pyrolysis oil obtained at these optimum process conditions are analyzed for some of their properties as an alternative fuel. The density of the liquid was closer with diesel. The viscosity of the plum seed liquid was lower than that of the conventional fuels. The calorific value of the pyrolysis oil is one half of the diesel fuel.

Loss to assets : production of power from speed breaker

In this work, prospect and feasibility of power generation by using speed breakers has been investigated. In this project a mechanism to generate power by converting the potential energy generated by a vehicle going up on a speed breaker into kinetic energy. This arrangement is made one rotation as soon as the vehicle moves over the speed breaker and has been increased using gears. After the production electricity, a storing unit has been used to hoard the generated electricity during the day and will be used during the night. Two prototypes have made using rack and pinion gear, spur gear, springs and generator .From which a considerable amount of energy is obtained. Nonetheless the cost of the prototype was inexpensive which proves the feasibility of this project and the idea can be applied on heavy traffic roads. Further investigation is being carried on to introduce the technology for practical approach.

Fixed bed pyrolysis of pulm seed waste for liquid oil production

Among various thermo-chemical conversion processes, pyrolysis is considered as an emerging technology for liquid oil production. The conversion of biomass waste in the form of plum seed into pyrolysis oil by fixed bed pyrolysis reactor has been taken into consideration in this study. A fixed bed pyrolysis has been designed and fabricated for obtaining liquid fuel from this plum seeds. The major component of the system are fixed bed pyrolysis reactor, liquid condenser and liquid collectors. The plum seed in particle form is pyrolysed in an externally heated 7.6 cm diameter and 46 cm high fixed bed reactor with nitrogen as the carrier gas. The reactor is heated by means of a biomass source cylindrical heater from 4000C to 6000C. The products are oil, char and gas. The reactor bed temperature, running time and feed particle size are considered as process parameters. The parameters are found to influence the product yield significantly. A maximum liquid yield of 39 wt% of biomass feed is obtained with particle size of 2.36-4.75 mm at a reactor bed temperature of 520oC with a running time of 120 minutes. The pyrolysis oil obtained at this optimum process conditions are analyzed for some fuel properties and compared with some other biomass derived pyrolysis oils and conventional fuels. The oil is found to possess favorable flash point and reasonable density and viscosity. The higher calorific value is found to be 22.39 MJ/kg which is higher than other biomass derived pyrolysis oils.

Production of liquid fuel and activated carbon from mahogany seed by using pyrolysis technology

The renovation of biomass waste in the form of Mahogany seed waste into bio-fuel as well as activated carbon by fixed bed pyrolysis reactor has been taken into consideration in this study. The mahogany seed in particle form is pyrolyzed in an enormously heated fixed bed reactor with nitrogen as the carrier gas. The reactor is heated from 4000C to 6000C using a external heater in which rice husk and charcoal are used as the heater biomass fuel. Reactor bed temperature, running time and feed particle size have been varied to get the optimum operating conditions of the system. The parameters are found to influence the product yields to a large extent. A maximum liquid and char yield are 49 wt. % and 35 wt. % respectively obtained at a reactor bed temperature 5000C when the running time is 90 minutes. Acquired pyrolyzed oil at these optimal process conditions were analyzed for some of their properties as an alternative fuel. The oil possesses comparable flame temperature, favorable flash point and reasonable viscosity along with somewhat higher density. The kinematic viscosity of the derived fuel is 3.8 cSt and density is 1525 kg/m3. The higher calorific value is found 32.4 MJ/kg which is significantly higher than other biomass derived fuel. Moderate adsorption capacity of the prepared activated carbon in case of methyl blue & tea water was also revealed.

Cyclic Imide and Open-Chain Amide Carboxylic Acid Derivatives from the Facile Reaction of cis-Cyclohexane-1,2-dicarboxylic Anhydride with Three Substituted Anilines

The structures of the compounds from the reaction of cis-cyclohexane-1,2-dicarboxylic anhydride with 4-chloroaniline [rac-N-(4-chlorophenyl)-2-carboxycycloclohexane-1-carboxamide] (1), 4-bromoaniline [2-(4-bromophenyl)-perhydroisoindolyl-1,3-dione] (2) and 3-hydroxy-4-carboxyaniline (5-aminosalicylic acid) [2-(3-hydroxy-4-carboxyphenyl)-perhydroisoindolyl-1,3-dione] (3) have been determined at 200 K. Crystals of the open-chain amide carboxylic acid 1 are orthorhombic, space group Pbcn, with unit cell dimensions a = 20.1753(10), b = 8.6267(4), c = 15.9940(9) Å, and Z = 8. Compounds 2 and 3 are cyclic imides, with 1 monoclinic having space group P21 and cell dimensions a = 11.5321(3), b = 6.7095(2), c = 17.2040(5) Å, β = 102.527(3)o. Compound 3 is orthorhombic with cell dimensions a = 6.4642(3), b = 12.8196(5), c = 16.4197(7) Å. Molecules of 1 form hydrogen-bonded cyclic dimers which are extended into a two-dimensional layered structure through amide-group associations: 3 forms into one-dimensional zigzag chains through carboxylic acid…imide O-atom hydrogen bonds, while compound 2 is essentially unassociated. With both cyclic imides 2 and 3, disorder is found which involves the presence of partial enantiomeric replacement of the cis-cyclohexane-1,2-substituted ring systems.

Effect of lipopolysaccharide from periodontal pathogens on the production of tissue plasminogen activator and plasminogen activator inhibitor 2 by human gingival fibroblasts.

Both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) are important proteolysis factors present in inflamed human periodontal tissues. The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) on the synthesis of t-PA and PAI-2 by human gingival fibroblasts (HGF). LPS from different periodontal pathogens including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum were extracted by the hot phenol water method. The levels of t-PA and PAI-2 secreted into the cell culture media were measured by enzyme-linked immunosorbent assays (ELISA). The mRNA for t-PA and PAI-2 were measured by RT-PCR. The results showed t-PA synthesis was increased in response to all types of LPS studied and PAI-2 level was increased by LPS from A. actinomycetemcomitans and F. nucleatum, but not P. gingivalis. When comparing the effects of LPS from non-periodontal bacteria (Escherichia coli and Salmonella enteritidis) with the LPS from periodontal pathogens, we found that the ratio of t-PA to PAI-2 was greater following exposure of the cells to LPS from periodontal pathogens. The highest ratio of t-PA to PAI-2 was found in those cells exposed to LPS from P. gingivalis. These results indicate that LPS derived from periodontal pathogens may cause unbalanced regulation of plasminogen activator and plasminogen activator inhibitor by HGF and such an effect may, in part, contribute to the destruction of periodontal connective tissue through dysregulated pericellular proteolysis.

Project 27 : insights into a production-centred study on art direction and love

This paper offers insight into the development of a PhD in advertising art direction. For over half a century art directors within the advertising industry have been adapting to the changes occurring in media, culture and the corporate sector, toward enhancing professional performance and competitiveness. These professionals seldom offer explicit justification about the role images play in effective communication. It is uncertain how this situation affects advertising performance, because advertising has, nevertheless, evolved in parallel to this as an industry able to fabricate new opportunities for itself. However, uncertainties in the formalization of art direction knowledge restrict the possibilities of knowledge transfer in higher education. The theoretical knowledge supporting advertising art direction has been adapted spontaneously from disciplines that rarely focus on specific aspects related to the production of advertising content, like, for example: marketing communication, design, visual communication, or visual art. Meanwhile, in scholarly research, vast empirical knowledge has been generated about advertising images, but often with limited insight into production expertise. Because art direction is understood as an industry practice and not as an academic discipline, an art direction perspective in scholarly contributions is rare. Scholarly research that is relevant to art direction seldom offers viewpoints to help understand how it is that research outputs may specifically contribute to art direction practices. There is a need to formally understanding the knowledge underlying art direction and using it to explore models for visual analysis and knowledge transfer in higher education. This paper provides insight into the development of a thesis that explored this need. The PhD thesis to which this paper refers is Strategic Aesthetics in Advertising Campaigns: Implications for Art Direction Education.

Acquisition modelling for heavy engineering assets in mining industry

Each year, organizations in Australian mining industry (asset intensive industry) spend substantial amount of capital (A$86 billion in 2009-10) (Statistics, 2011) in acquiring engineering assets. Engineering assets are put to use in operations to generate value. Different functions (departments) of an organization have different expectations and requirements from each of the engineering asset e.g. return on investment, reliability, efficiency, maintainability, low cost of running the asset, low or nil environmental impact and easy of disposal, potential salvage value etc. Assets are acquired from suppliers or built by service providers and or internally. The process of acquiring assets is supported by procurement function. One of the most costly mistakes that organizations can make is acquiring the inappropriate or non-conforming assets that do not fit the purpose. The root cause of acquiring non confirming assets belongs to incorrect acquisition decision and the process of making decisions. It is very important that an asset acquisition decision is based on inputs and multi-criteria of each function within the organization which has direct or indirect impact on the acquisition, utilization, maintenance and disposal of the asset. Literature review shows that currently there is no comprehensive process framework and tool available to evaluate the inclusiveness and breadth of asset acquisition decisions that are taken in the Mining Organizations. This thesis discusses various such criteria and inputs that need to be considered and evaluated from various functions within the organization while making the asset acquisition decision. Criteria from functions such as finance, production, maintenance, logistics, procurement, asset management, environment health and safety, material management, training and development etc. need to be considered to make an effective and coherent asset acquisition decision. The thesis also discusses a tool that is developed to be used in the multi-criteria and cross functional acquisition decision making. The development of multi-criteria and cross functional inputs based decision framework and tool which utilizes that framework to formulate cross functional and integrated asset acquisition decisions are the contribution of this research.

Marginal reversible jump Markov chain Monte Carlo with application to motor unit number estimation

Motor unit number estimation (MUNE) is a method which aims to provide a quantitative indicator of progression of diseases that lead to loss of motor units, such as motor neurone disease. However the development of a reliable, repeatable and fast real-time MUNE method has proved elusive hitherto. Ridall et al. (2007) implement a reversible jump Markov chain Monte Carlo (RJMCMC) algorithm to produce a posterior distribution for the number of motor units using a Bayesian hierarchical model that takes into account biological information about motor unit activation. However we find that the approach can be unreliable for some datasets since it can suffer from poor cross-dimensional mixing. Here we focus on improved inference by marginalising over latent variables to create the likelihood. In particular we explore how this can improve the RJMCMC mixing and investigate alternative approaches that utilise the likelihood (e.g. DIC (Spiegelhalter et al., 2002)). For this model the marginalisation is over latent variables which, for a larger number of motor units, is an intractable summation over all combinations of a set of latent binary variables whose joint sample space increases exponentially with the number of motor units. We provide a tractable and accurate approximation for this quantity and also investigate simulation approaches incorporated into RJMCMC using results of Andrieu and Roberts (2009).

Setting up a platform for plant-based influenza virus vaccine production in South Africa

Background During a global influenza pandemic, the vaccine requirements of developing countries can surpass their supply capabilities, if these exist at all, compelling them to rely on developed countries for stocks that may not be available in time. There is thus a need for developing countries in general to produce their own pandemic and possibly seasonal influenza vaccines. Here we describe the development of a plant-based platform for producing influenza vaccines locally, in South Africa. Plant-produced influenza vaccine candidates are quicker to develop and potentially cheaper than egg-produced influenza vaccines, and their production can be rapidly upscaled. In this study, we investigated the feasibility of producing a vaccine to the highly pathogenic avian influenza A subtype H5N1 virus, the most generally virulent influenza virus identified to date. Two variants of the haemagglutinin (HA) surface glycoprotein gene were synthesised for optimum expression in plants: these were the full-length HA gene (H5) and a truncated form lacking the transmembrane domain (H5tr). The genes were cloned into a panel of Agrobacterium tumefaciens binary plant expression vectors in order to test HA accumulation in different cell compartments. The constructs were transiently expressed in tobacco by means of agroinfiltration. Stable transgenic tobacco plants were also generated to provide seed for stable storage of the material as a pre-pandemic strategy. Results For both transient and transgenic expression systems the highest accumulation of full-length H5 protein occurred in the apoplastic spaces, while the highest accumulation of H5tr was in the endoplasmic reticulum. The H5 proteins were produced at relatively high concentrations in both systems. Following partial purification, haemagglutination and haemagglutination inhibition tests indicated that the conformation of the plant-produced HA variants was correct and the proteins were functional. The immunisation of chickens and mice with the candidate vaccines elicited HA-specific antibody responses. Conclusions We managed, after synthesis of two versions of a single gene, to produce by transient and transgenic expression in plants, two variants of a highly pathogenic avian influenza virus HA protein which could have vaccine potential. This is a proof of principle of the potential of plant-produced influenza vaccines as a feasible pandemic response strategy for South Africa and other developing countries.

Optimization of chimeric HIV-1 virus-like particle production in a baculovirus-insect cell expression system

A baculovirus-insect cell expression system potentially provides the means to produce prophylactic HIV-1 virus-like particle (VLP) vaccines inexpensively and in large quantities. However, the system must be optimized to maximize yields and increase process efficiency. In this study, we optimized the production of two novel, chimeric HIV-1 VLP vaccine candidates (GagRT and GagTN) in insect cells. This was done by monitoring the effects of four specific factors on VLP expression: these were insect cell line, cell density, multiplicity of infection (MOI), and infection time. The use of western blots, Gag p24 ELISA, and four-factorial ANOVA allowed the determination of the most favorable conditions for chimeric VLP production, as well as which factors affected VLP expression most significantly. Both VLP vaccine candidates favored similar optimal conditions, demonstrating higher yields of VLPs when produced in the Trichoplusia ni Pro insect cell line, at a cell density of 1 × 106 cells/mL, and an infection time of 96 h post infection. It was found that cell density and infection time were major influencing factors, but that MOI did not affect VLP expression significantly. This work provides a potentially valuable guideline for HIV-1 protein vaccine optimization, as well as for general optimization of a baculovirus-based expression system to produce complex recombinant proteins. © 2009 American Institute of Chemical Engineers.

Human papillomavirus vaccines in plants

Human papillomaviruses are the etiological agents of cervical cancer, one of the two most prevalent cancers in women in developing countries. Currently available prophylactic vaccines are based on the L1 major capsid protein, which forms virus-like particles when expressed in yeast and insect cell lines. Despite their recognized efficacy, there are significant shortcomings: the vaccines are expensive, include only two oncogenic virus types, are delivered via intramuscular injection and require a cold chain. Plant expression systems may provide ways of overcoming some of these problems, in particular the expense. In this article, we report recent promising advances in the production of prophylactic and therapeutic vaccines against human papillomavirus by expression of the relevant antigens in plants, and discuss future prospects for the use of such vaccines. © 2010 Expert Reviews Ltd.