A role for macrophages under cytokine control in mediating resistance to ADI-PEG20 (pegargiminase) in ASS1-deficient mesothelioma

Background Pegylated arginine deiminase (ADI-PEG20; pegargiminase) depletes arginine and improves survival outcomes for patients with argininosuccinate synthetase 1 (ASS1)-deficient malignant pleural mesothelioma (MPM). Optimisation of ADI-PEG20-based therapy will require a deeper understanding of resistance mechanisms, including those mediated by the tumor microenvironment. Here, we sought to reverse translate increased tumoral macrophage infiltration in patients with ASS1-deficient MPM relapsing on pegargiminase therapy. Methods Macrophage-MPM tumor cell line (2591, MSTO, JU77) co-cultures treated with ADI-PEG20 were analyzed by flow cytometry. Microarray experiments of gene expression profiling were performed in ADI-PEG20-treated MPM tumor cells, and macrophage-relevant genetic “hits” were validated by qPCR, ELISA, and LC/MS. Cytokine and argininosuccinate analyses were performed using plasma from pegargiminase-treated patients with MPM. Results We identified that ASS1-expressing macrophages promoted viability of ADI-PEG20-treated ASS1-negative MPM cell lines. Microarray gene expression data revealed a dominant CXCR2-dependent chemotactic signature and co-expression of VEGF-A and IL-1α in ADI-PEG20-treated MPM cell lines. We confirmed that ASS1 in macrophages was IL-1α-inducible and that the argininosuccinate concentration doubled in the cell supernatant sufficient to restore MPM cell viability under co-culture conditions with ADI-PEG20. For further validation, we detected elevated plasma VEGF-A and CXCR2-dependent cytokines, and increased argininosuccinate in patients with MPM progressing on ADI-PEG20. Finally, liposomal clodronate depleted ADI-PEG20-driven macrophage infiltration and suppressed growth significantly in the MSTO xenograft murine model. Conclusions Collectively, our data indicate that ADI-PEG20-inducible cytokines orchestrate argininosuccinate fuelling of ASS1-deficient mesothelioma by macrophages. This novel stromal-mediated resistance pathway may be leveraged to optimize arginine deprivation therapy for mesothelioma and related arginine-dependent cancers

An Optimized Chloroplast DNA Extraction Protocol for Grasses (Poaceae) Proves Suitable for Whole Plastid Genome Sequencing and SNP Detection

peer-reviewedBackground Obtaining chloroplast genome sequences is important to increase the knowledge about the fundamental biology of plastids, to understand evolutionary and ecological processes in the evolution of plants, to develop biotechnological applications (e.g. plastid engineering) and to improve the efficiency of breeding schemes. Extraction of pure chloroplast DNA is required for efficient sequencing of chloroplast genomes. Unfortunately, most protocols for extracting chloroplast DNA were developed for eudicots and do not produce sufficiently pure yields for a shotgun sequencing approach of whole plastid genomes from the monocot grasses. Methodology/Principal Findings We have developed a simple and inexpensive method to obtain chloroplast DNA from grass species by modifying and extending protocols optimized for the use in eudicots. Many protocols for extracting chloroplast DNA require an ultracentrifugation step to efficiently separate chloroplast DNA from nuclear DNA. The developed method uses two more centrifugation steps than previously reported protocols and does not require an ultracentrifuge. Conclusions/Significance The described method delivered chloroplast DNA of very high quality from two grass species belonging to highly different taxonomic subfamilies within the grass family (Lolium perenne, Pooideae; Miscanthus×giganteus, Panicoideae). The DNA from Lolium perenne was used for whole chloroplast genome sequencing and detection of SNPs. The sequence is publicly available on EMBL/GenBank

Toll-like receptor 2 expression is decreased on alveolar macrophages in cigarette smokers and COPD patients

BACKROUND: Cigarette smoke exposure including biologically active lipopolysaccharide (LPS) in the particulate phase of cigarette smoke induces activation of alveolar macrophages (AM) and alveolar epithelial cells leading to production of inflammatory mediators. This represents a crucial mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Respiratory pathogens are a major cause of exacerbations leading to recurrent cycles of injury and repair. The interaction between pathogen-associated molecular patterns and the host is mediated by pattern recognition receptors (PRR's). In the present study we characterized the expression of Toll-like receptor (TLR)- 2, TLR4 and CD14 on human AM compared to autologous monocytes obtained from patients with COPD, healthy smokers and non-smokers. METHODS: The study population consisted of 14 COPD patients without evidence for acute exacerbation, 10 healthy smokers and 17 healthy non-smokers stratified according to age. The expression of TLR2, TLR4 and CD14 surface molecules on human AM compared to autologous monocytes was assessed ex vivo using FACS analysis. In situ hybridization was performed on bronchoalveolar lavage (BAL) cells by application of the new developed HOPE-fixative. RESULTS: The expression of TLR2, TLR4 and CD14 on AM from COPD patients, smokers and non-smokers was reduced as compared to autologous monocytes. Comparing AM we detected a reduced expression of TLR2 in COPD patients and smokers. In addition TLR2 mRNA and protein expression was increased after LPS stimulation on non-smokers AM in contrast to smokers and COPD patients. CONCLUSION: Our data suggest a smoke related change in the phenotype of AM's and the cellular response to microbial stimulation which may be associated with impairment of host defenses in the lower respiratory tract

High efficiency of alphaviral gene transfer in combination with 5-fluorouracil in a mouse mammary tumor model

Copyright: Copyright 2014 Elsevier B.V., All rights reserved.Background: The combination of virotherapy and chemotherapy may enable efficient tumor regression that would be unachievable using either therapy alone. In this study, we investigated the efficiency of transgene delivery and the cytotoxic effects of alphaviral vector in combination with 5-fluorouracil (5-FU) in a mouse mammary tumor model (4 T1).Methods: Replication-deficient Semliki Forest virus (SFV) vectors carrying genes encoding fluorescent proteins were used to infect 4 T1 cell cultures treated with different doses of 5-FU. The efficiency of infection was monitored via fluorescence microscopy and quantified by fluorometry. The cytotoxicity of the combined treatment with 5-FU and alphaviral vector was measured using an MTT-based cell viability assay. In vivo experiments were performed in a subcutaneous 4 T1 mouse mammary tumor model with different 5-FU doses and an SFV vector encoding firefly luciferase.Results: Infection of 4 T1 cells with SFV prior to 5-FU treatment did not produce a synergistic anti-proliferative effect. An alternative treatment strategy, in which 5-FU was used prior to virus infection, strongly inhibited SFV expression. Nevertheless, in vivo experiments showed a significant enhancement in SFV-driven transgene (luciferase) expression upon intratumoral and intraperitoneal vector administration in 4 T1 tumor-bearing mice pretreated with 5-FU: here, we observed a positive correlation between 5-FU dose and the level of luciferase expression.Conclusions: Although 5-FU inhibited SFV-mediated transgene expression in 4 T1 cells in vitro, application of the drug in a mouse model revealed a significant enhancement of intratumoral transgene synthesis compared with 5-FU untreated mice. These results may have implications for efficient transgene delivery and the development of potent cancer treatment strategies using alphaviral vectors and 5-FU.publishersversionPeer reviewe

Recurrent respiratory papillomatosis: an overview of current thinking and treatment

Human papillomaviruses (HPV) infection in benign laryngeal papillomas is well established. The vast majority of recurrent respiratory papillomatosis lesions are due to HPV types 6 and 11. Human papillomaviruses are small non-enveloped viruses (>8 kb), that replicate within the nuclei of infected host cells. Infected host basal cell keratinocytes and papillomas arise from the disordered proliferation of these differentiating keratinocytes. Surgical debulking of papillomas is currently the treatment of choice; newer surgical approaches utilizing microdebriders are replacing laser ablation. Surgery aims to secure an adequate airway and improve and maintain an acceptable quality of voice. Adjuvant treatments currently used include cidofovir, indole-3-carbinol, ribavirin, mumps vaccine, and photodynamic therapy. The recent licensing of prophylactic HPV vaccines is a most interesting development. The low incidence of RRP does pose significant problems in recruitment of sufficient numbers to show statistical significance. Large multi-centre collaborative clinical trials are therefore required. Even so, sufficient clinical follow-up data would take several years

Bevacizumab Increases Viral Distribution in Human Anaplastic Thyroid Carcinoma Xenografts and Enhances the Effects of E1A-Defective Adenovirus d/922-947

PURPOSE: Anaplastic thyroid carcinoma is a prime target for innovative therapy because it represents one of the most lethal human neoplasms and is refractory to conventional treatments such as chemotherapy and radiotherapy. We have evaluated a novel therapeutic approach based on the oncolytic replication-selective adenovirus dl922-947. EXPERIMENTAL DESIGN: The antitumor efficacies of the E1ADeltaCR2 (dl922-947) and DeltaE1B55K (dl1520) mutants were compared in human thyroid anaplastic carcinoma cells in culture and in xenografts in vivo. To enhance the effects of dl922-947, anaplastic thyroid carcinoma tumor xenografts were treated with dl922-947 in combination with bevacizumab. RESULTS: We showed that the efficacy of dl922-947 exceeded that of dl1520 in all tested anaplastic thyroid carcinoma cells in vitro and in vivo. Furthermore, bevacizumab in combination with dl922-947 significantly reduced tumor growth compared with single treatments alone. Bevacizumab treatment significantly improved viral distribution in neoplastic tissues. CONCLUSIONS: Our data showed that dl922-947 had a higher oncolytic activity compared with dl1520 in anaplastic thyroid carcinoma cell lines and might represent a better option for virotherapy of anaplastic thyroid carcinoma. Moreover, bevacizumab increased the oncolytic effects of dl922-947 by enhancing viral distribution in tumors. The results described herein encourage the use of the dl922-947 virus in combination with bevacizumab