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

A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes

BACKGROUND: TGM1(transglutaminase 1) is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment. METHODS: In vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes. RESULTS: In vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA) and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the transcriptional activity. CONCLUSIONS: A distal region of the TGM1 gene promoter, containing AP1 and Sp1 binding sites, is evolutionarily conserved and responsible for high level expression in transgenic mice and in transfected keratinocyte cultures

CXCL1 can be regulated by IL-6 and promotes granulocyte adhesion to brain capillaries during bacterial toxin exposure and encephalomyelitis

<p>Abstract</p> <p>Background</p> <p>Granulocytes generally exert protective roles in the central nervous system (CNS), but recent studies suggest that they can be detrimental in experimental autoimmune encephalomyelitis (EAE), the most common model of multiple sclerosis. While the cytokines and adhesion molecules involved in granulocyte adhesion to the brain vasculature have started to be elucidated, the required chemokines remain undetermined.</p> <p>Methods</p> <p>CXCR2 ligand expression was examined in the CNS of mice suffering from EAE or exposed to bacterial toxins by quantitative RT-PCR and <it>in situ </it>hybridization. CXCL1 expression was analyzed in IL-6-treated endothelial cell cultures by quantitative RT-PCR and ELISA. Granulocytes were counted in the brain vasculature after treatment with a neutralizing anti-CXCL1 antibody using stereological techniques.</p> <p>Results</p> <p>CXCL1 was the most highly expressed ligand of the granulocyte receptor CXCR2 in the CNS of mice subjected to EAE or infused with lipopolysaccharide (LPS) or pertussis toxin (PTX), the latter being commonly used to induce EAE. IL-6 upregulated CXCL1 expression in brain endothelial cells by acting transcriptionally and mediated the stimulatory effect of PTX on CXCL1 expression. The anti-CXCL1 antibody reduced granulocyte adhesion to brain capillaries in the three conditions under study. Importantly, it attenuated EAE severity when given daily for a week during the effector phase of the disease.</p> <p>Conclusions</p> <p>This study identifies CXCL1 not only as a key regulator of granulocyte recruitment into the CNS, but also as a new potential target for the treatment of neuroinflammatory diseases such as multiple sclerosis.</p

Health Conditions and Their Impact among Adolescents and Young Adults with Down Syndrome

Objective: To examine the prevalence of medical conditions and use of health services among young adults with Down syndrome and describe the impact of these conditions upon their lives. Methods: Using questionnaire data collected in 2011 from parents of young adults with Down syndrome we investigated the medical conditions experienced by their children in the previous 12 months. Univariate, linear and logistic regression analyses were performed. Results: We found that in addition to the conditions commonly experienced by children with Down syndrome, including eye and vision problems (affecting 73%), ear and hearing problems (affecting 45%), cardiac (affecting 25%) and respiratory problems (affecting 36%), conditions also found to be prevalent within our young adult cohort included musculoskeletal conditions (affecting 61%), body weight (affecting 57%), skin (affecting 56%) and mental health (affecting 32%) conditions and among young women menstrual conditions (affecting 58%). Few parents reported that these conditions had no impact, with common impacts related to restrictions in opportunities to participate in employment and community leisure activities for the young people, as well as safety concerns. Conclusion: There is the need to monitor, screen and provide appropriate strategies such as through the promotion of healthy lifestyles to prevent the development of comorbidities in young people with Down syndrome and, where present, to reduce their impact

Reductive stress selectively disrupts collagen homeostasis and modifies growth factor-independent signalling through the MAPK/Akt pathway in human dermal fibroblasts

Redox stress is a well-known contributor to ageing and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide bonds. However, the quantitative response of the cellular proteome to reductants has not been explored, particularly in cells such as fibroblasts that produce extracellular matrix proteins. Here, we have used a robust, unbiased, label-free SWATH-MS proteomic approach to quantitate the response of skin fibroblast cells to DTT in the presence or absence of the growth factor PDGF. Of the 4487 proteins identified, only 42 proteins showed a statistically significant change of 2-fold or more with reductive stress. Our proteomics data show that reductive stress results in the loss of a small subset of reductant-sensitive proteins (including the collagens COL1A1/2 and COL3A1, and the myopathy-associated collagens COL6A1/2/3), and the downregulation of targets downstream of the MAPK pathway. We show that a reducing environment alters signalling through the PDGF-associated MAPK/Akt pathways, inducing chronic dephosphorylation of ERK1/2 at Thr202/Tyr204 and phosphorylation of Akt at Ser473 in a growth factor-independent manner. Our data highlights collagens as sentinel molecules for redox stress downstream of MAPK/Akt, and identifies intervention points to modulate the redox environment to target skin diseases and conditions associated with erroneous matrix deposition

Gene Therapy Approaches Using Reproducible and Fully Penetrant Lentivirus-Mediated Endogenous Glioma Models

Animal models have proven invaluable for progress toward greater understanding of the etiology, pathogenesis, and genetics of a wide range of human diseases. The development of relevant brain tumor animal models is a critical resource for building our understanding of cancers that arise within the brain and for the development of novel therapies. The central role of these models is particularly apparent for gliomas, which are common and devastating primary brain tumors. Effective models accurately demonstrate pathological features and behavior that are analogous to the human disease. Models aim to develop tumors with high penetrance and low latency, features that are ideal for preclinical therapeutic development. Lentiviral vector-induced models fulfill these requirements while giving investigators excellent control over the genetic profile of resulting tumors. This flexibility is especially relevant in the context of recent advances in the understanding of the genetic lesions found in human grade IV glioma, glioblastoma multiforme (GBM). Further, these endogenous tumor models would be ideal for the testing of novel gene therapy strategies which could potentially be implemented in Phase 1 clinical trials for these devastating human brain cancers.Fil: Lynes, John. University Of Michigan Medical School; Estados UnidosFil: Koschmann, Carl. University Of Michigan Medical School; Estados UnidosFil: Wibowo, Mia. University Of Michigan Medical School; Estados UnidosFil: Saxena, Vandana. University Of Michigan Medical School; Estados UnidosFil: Candolfi, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Moreno Ayala, Mariela Alejandra. University Of Michigan Medical School; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Castro, Maria Graciela. University Of Michigan Medical School; Estados UnidosFil: Lowenstein, Pedro R.. University Of Michigan Medical School; Estados Unido