Construction of therapeutically relevant human prostate epithelial fate map by utilising miRNA and mRNA microarray expression data.

BACKGROUND: Objective identification of key miRNAs from transcriptomic data is difficult owing to the inherent inconsistencies within miRNA target-prediction algorithms and the promiscuous nature of miRNA-mRNA target relationship. METHODS: An integrated database of miRNAs and their 'relevant' mRNA targets was generated from validated miRNA and mRNA microarray data sets generated from patient-derived prostate epithelial normal and cancer stem-like cells (SCs) and committed basal (CB) cells. The effect of miR-542-5p inhibition was studied to provide proof-of-principle for database utility. RESULTS: Integration of miRNA-mRNA databases showed that signalling pathways and processes can be regulated by a single or relatively few miRNAs, for example, DNA repair/Notch pathway by miR-542-5p, P=0.008. Inhibition of miR-542-5p in CB cells (thereby achieving miR-542-5p expression levels similar to SCs) promoted efficient DNA repair and activated expression of Notch reporters, HES1 and Survivin, without inducing dedifferentiation into SCs. CONCLUSIONS: Our novel framework impartially identifies therapeutically relevant miRNA candidates from transcriptomic data sets.

Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni.

Schistosoma mansoni is the primary causative agent of schistosomiasis, which affects 200 million individuals in 74 countries. We generated 163,000 expressed-sequence tags (ESTs) from normalized cDNA libraries from six selected developmental stages of the parasite, resulting in 31,000 assembled sequences and 92% sampling of an estimated 14,000 gene complement. By analyzing automated Gene Ontology assignments, we provide a detailed view of important S. mansoni biological systems, including characterization of metazoa-specific and eukarya-conserved genes. Phylogenetic analysis suggests an early divergence from other metazoa. The data set provides insights into the molecular mechanisms of tissue organization, development, signaling, sexual dimorphism, host interactions and immune evasion and identifies novel proteins to be investigated as vaccine candidates and potential drug targets.

Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.

Notch signalling is a potential resistance mechanism of progenitor cells within patient-derived prostate cultures following ROS-inducing treatments.

Low Temperature Plasma (LTP) generates reactive oxygen and nitrogen species, causing cell death, similarly to radiation. Radiation resistance results in tumour recurrence, however mechanisms of LTP resistance are unknown. LTP was applied to patient-derived prostate epithelial cells and gene expression assessed. A typical global oxidative response (AP-1 and Nrf2 signalling) was induced, whereas Notch signalling was activated exclusively in progenitor cells. Notch inhibition induced expression of prostatic acid phosphatase (PAP), a marker of prostate epithelial cell differentiation, whilst reducing colony forming ability and preventing tumour formation. Therefore, if LTP is to be progressed as a novel treatment for prostate cancer, combination treatments should be considered in the context of cellular heterogeneity and existence of cell type-specific resistance mechanisms.

Preclinical safety assessment of Ad[I/PPT-E1A], a novel oncolytic adenovirus for prostate cancer.

Prostate cancer is the most common malignancy in the Western world. Patients can be cured only when the tumor has not metastasized outside the prostate. However, treatment with curative intent fails in a significant number of men, often resulting in untreatable progressive disease with a fatal outcome. Oncolytic adenovirus therapy may be a promising adjuvant treatment to reduce local failure or the outgrowth of micrometastatic disease. Within the European gene therapy consortium GIANT, we have developed a novel prostate-specific oncolytic adenovirus: Ad[I/PPT-E1A]. This adenovirus specifically kills prostate cells via prostate-specific replication. This article describes the clinical development of Ad[I/PPT-E1A] with particular reference to the preclinical safety assessment of this novel virus. The preclinical safety assessment involved an efficacy study in a human orthotopic xenograft mouse model, a specificity study in human primary cells, and a toxicity study in normal mice. These studies confirmed that Ad[I/PPT-E1A] efficiently kills prostate tumor cells in vivo, is not harmful to other organs, and is well tolerated in mice after systemic delivery. The safety, as well as the immunological effects of Ad[I/PPT-E1A] as a local adjuvant therapy, will now be studied in a phase I dose-escalating trial in patients with localized prostate cancer who are scheduled for curative radical prostatectomy and can be used as an updated paradigm for similar therapeutic viruses.

Adenovirus serotype 5 vectors with Tat-PTD modified hexon and serotype 35 fiber show greatly enhanced transduction capacity of primary cell cultures.

Recombinant adenovirus serotype 5 (Ad5) vectors represent one of the most efficient gene delivery vectors in life sciences. However, Ad5 is dependent on expression of the coxsackievirus-adenovirus-receptor (CAR) on the surface of target cell for efficient transduction, which limits it's utility for certain cell types. Herein we present a new vector, Ad5PTDf35, which is an Ad5 vector having serotype 35 fiber-specificity and Tat-PTD hexon-modification. This vector shows dramatically increased transduction capacity of primary human cell cultures including T cells, monocytes, macrophages, dendritic cells, pancreatic islets and exocrine cells, mesenchymal stem cells and tumor initiating cells. Biodistribution in mice following systemic administration (tail-vein injection) show significantly reduced uptake in the liver and spleen of Ad5PTDf35 compared to unmodified Ad5. Therefore, replication-competent viruses with these modifications may be further developed as oncolytic agents for cancer therapy. User-friendly backbone plasmids containing these modifications were developed for compatibility to the AdEasy-system to facilitate the development of surface-modified adenoviruses for gene delivery to difficult-to-transduce cells in basic, pre-clinical and clinical research.

Prime-boost immunisation against tropical theileriosis with two parasite surface antigens: evidence for protection and antigen synergy.

Current methods for control of tropical theileriosis in cattle suffer from several disadvantages that could be circumvented by development of an effective sub-unit vaccine. Previous work has utilised two major surface antigens (SPAG-1 and Tams1) and conventional adjuvants to provide partial protection against parasite challenge. In this study we have delivered these antigens using the prime-boost system and analysed whether a combination regime can enhance protection against lethal challenge. Delivery of the boost as recombinant protein or expressed from a recombinant MVA vector was also assessed. The results confirmed that immunisation with Tams1 alone could reduce the severity of several disease parameters compared to non-immunised controls and these effects were more marked when recombinant protein was used for boosting compared to MVA delivery. A similar outcome was obtained by immunisation with SPAG-1 alone. Significantly, delivery of SPAG-1 and Tams1 as a cocktail showed enhanced protection. This was manifest by significant improvement in a large range of clinical and parasitological parameters and, most dramatically, by the survival and recovery of 50% of the immunised animals compared to 0% of the controls. Analysis of the antibody response post-challenge showed that while there was a strong response to Tams1, no response to SPAG-1 was detected. In contrast, lymphoproliferation assays showed a significant enhancement of response at day 7 post-challenge in calves of the SPAG-1 group but a dramatic decrease of the proliferation activity in all three groups receiving Tams1. We conclude that immunisation with a cocktail of SPAG-1 and Tams1 generates a synergistic protective response that significantly improves the efficacy of recombinant vaccination against tropical theileriosis. Potential effector mechanisms that could mediate this response are discussed.