Sample records for zebrafish xenograft model

  1. A novel zebrafish human tumor xenograft model validated for anti-cancer drug screening.

    PubMed

    Jung, Da-Woon; Oh, Eun-Sang; Park, Si-Hwan; Chang, Young-Tae; Kim, Cheol-Hee; Choi, Seok-Yong; Williams, Darren R

    2012-07-01

    The development of a relatively simple, reliant and cost-effective animal test will greatly facilitate drug development. In this study, our goal was the establishment of a rapid, simple, sensitive and reproducible zebrafish xenograft model for anti-cancer drug screening. We optimized the conditions for the cancer cell xenograft in terms of injected cell numbers, incubation temperature and time. A range of human carcinoma cell types were stained with a fluorescent dye prior to injection into the fish larvae. Subsequent cancer cell dissemination was observed under fluorescent microscopy. Differences in injected cell numbers were reflected in the rate of dissemination from the xenograft site. Paclitaxel, known as a microtubule stabilizer, dose-dependently inhibited cancer cell dissemination in our zebrafish xenograft model. An anti-migratory drug, LY294002 (phosphatidylinositol 3-kinase inhibitor) also decreased the cancer cell dissemination. Chemical modifications to increase cancer drug pharmacokinetics, such as increased solubility (17-DMAG compared to geldanamycin) could also be assessed in our xenograft model. In addition to testing our new model using known anti-cancer drugs, we carried out further validation by screening a tagged triazine library. Two novel anti-cancer drug candidates were discovered. Therefore, our zebrafish xenograft model provides a vertebrate animal system for the rapid screening and pre-clinical testing of novel anti-cancer agents, prior to the requirement for testing in mammals. Our model system should greatly facilitate drug development for cancer therapy because of its speed, simplicity and reproducibility. PMID:22569777

  2. Enrichment of human prostate cancer cells with tumor initiating properties in mouse and zebrafish xenografts by differential adhesion

    PubMed Central

    Bansal, Nitu; Davis, Stephani; Tereshchenko, Irina; Budak-Alpdogan, Tulin; Zhong, Hua; Stein, Mark N.; Kim, Isaac Yi; DiPaola, Robert S.; Bertino, Joseph R.; Sabaawy, Hatem E.

    2014-01-01

    BACKGROUND Prostate tumor-initiating cells (TICs) have intrinsic resistance to current therapies. TICs are commonly isolated by cell sorting or dye exclusion, however, isolating TICs from limited primary prostate cancer (PCa) tissues is inherently inefficient. We adapted the collagen adherence feature to develop a combined immunophenotypic and time-of-adherence assay to identify human prostate TICs. METHODS PCa cells from multiple cell lines and primary tissues were allowed to adhere to several matrix molecules, and fractions of adherent cells were examined for their TIC properties. RESULTS Collagen-I rapidly-adherent PCa cells have significantly higher clonogenic, migration, and invasion abilities, and initiated more tumor xenografts in mice when compared to slowly-adherent and no-adherent cells. To determine the relative frequency of TICs among PCa cell lines and primary PCa cells, we utilized zebrafish xenografts to define the tumor initiation potential of serial dilutions of rapidly-adherent ?2?1hi/CD44hi cells compared to non-adherent cells with ?2?1low/CD44low phenotype. Tumor initiation from rapidly-adherent ?2?1hi/CD44hi TICs harboring the TMPRSS2:ERG fusion generated xenografts comprising of PCa cells expressing Erg, AMACR, and PSA. Moreover, PCa-cell dissemination was consistently observed in the immune-permissive zebrafish microenvironment from as-few-as 3 rapidly-adherent ?2?1hi/CD44hi cells. In zebrafish xenografts, self-renewing prostate TICs comprise 0.02–0.9% of PC3 cells, 0.3–1.3% of DU145 cells, and 0.22–14.3% of primary prostate adenocarcinomas. CONCLUSION Zebrafish PCa xenografts were used to determine that the frequency of prostate TICs varies among PCa cell lines and primary PCa tissues. These data support a paradigm of utilizing zebrafish xenografts to evaluate novel therapies targeting tumor initiating cells in prostate cancer. PMID:24154958

  3. Orthotopic models of pediatric brain tumors in zebrafish.

    PubMed

    Eden, C J; Ju, B; Murugesan, M; Phoenix, T N; Nimmervoll, B; Tong, Y; Ellison, D W; Finkelstein, D; Wright, K; Boulos, N; Dapper, J; Thiruvenkatam, R; Lessman, C A; Taylor, M R; Gilbertson, R J

    2015-03-26

    High-throughput screens (HTS) of compound toxicity against cancer cells can identify thousands of potential new drug-leads. But only limited numbers of these compounds can progress to expensive and labor-intensive efficacy studies in mice, creating a 'bottle neck' in the drug development pipeline. Approaches that triage drug-leads for further study are greatly needed. Here we provide an intermediary platform between HTS and mice by adapting mouse models of pediatric brain tumors to grow as orthotopic xenografts in the brains of zebrafish. Freshly isolated mouse ependymoma, glioma and choroid plexus carcinoma cells expressing red fluorescence protein were conditioned to grow at 34?°C. Conditioned tumor cells were then transplanted orthotopically into the brains of zebrafish acclimatized to ambient temperatures of 34?°C. Live in vivo fluorescence imaging identified robust, quantifiable and reproducible brain tumor growth as well as spinal metastasis in zebrafish. All tumor xenografts in zebrafish retained the histological characteristics of the corresponding parent mouse tumor and efficiently recruited fish endothelial cells to form a tumor vasculature. Finally, by treating zebrafish harboring ERBB2-driven gliomas with an appropriate cytotoxic chemotherapy (5-fluorouracil) or tyrosine kinase inhibitor (erlotinib), we show that these models can effectively assess drug efficacy. Our data demonstrate, for the first time, that mouse brain tumors can grow orthotopically in fish and serve as a platform to study drug efficacy. As large cohorts of brain tumor-bearing zebrafish can be generated rapidly and inexpensively, these models may serve as a powerful tool to triage drug-leads from HTS for formal efficacy testing in mice. PMID:24747973

  4. Zebrafish as a Model for the Study of Human Myeloid Malignancies

    PubMed Central

    Lu, Jeng-Wei; Hsieh, Meng-Shan; Liao, Heng-An; Yang, Yi-Ju; Ho, Yi-Jung; Lin, Liang-In

    2015-01-01

    Myeloid malignancies are heterogeneous disorders characterized by uncontrolled proliferation or/and blockage of differentiation of myeloid progenitor cells. Although a substantial number of gene alterations have been identified, the mechanism by which these abnormalities interact has yet to be elucidated. Over the past decades, zebrafish have become an important model organism, especially in biomedical research. Several zebrafish models have been developed to recapitulate the characteristics of specific myeloid malignancies that provide novel insight into the pathogenesis of these diseases and allow the evaluation of novel small molecule drugs. This report will focus on illustrative examples of applications of zebrafish models, including transgenesis, zebrafish xenograft models, and cell transplantation approaches, to the study of human myeloid malignancies.

  5. Zebrafish: Modeling for Herpes Simplex Virus Infections

    PubMed Central

    Antoine, Thessicar Evadney; Jones, Kevin S.; Dale, Rodney M.; Shukla, Deepak

    2014-01-01

    Abstract For many years, zebrafish have been the prototypical model for studies in developmental biology. In recent years, zebrafish has emerged as a powerful model system to study infectious diseases, including viral infections. Experiments conducted with herpes simplex virus type-1 in adult zebrafish or in embryo models are encouraging as they establish proof of concept with viral-host tropism and possible screening of antiviral compounds. In addition, the presence of human homologs of viral entry receptors in zebrafish such as 3-O sulfated heparan sulfate, nectins, and tumor necrosis factor receptor superfamily member 14-like receptor bring strong rationale for virologists to test their in vivo significance in viral entry in a zebrafish model and compare the structure–function basis of virus zebrafish receptor interaction for viral entry. On the other end, a zebrafish model is already being used for studying inflammation and angiogenesis, with or without genetic manipulations, and therefore can be exploited to study viral infection-associated pathologies. The major advantage with zebrafish is low cost, easy breeding and maintenance, rapid lifecycle, and a transparent nature, which allows visualizing dissemination of fluorescently labeled virus infection in real time either at a localized region or the whole body. Further, the availability of multiple transgenic lines that express fluorescently tagged immune cells for in vivo imaging of virus infected animals is extremely attractive. In addition, a fully developed immune system and potential for receptor-specific knockouts further advocate the use of zebrafish as a new tool to study viral infections. In this review, we focus on expanding the potential of zebrafish model system in understanding human infectious diseases and future benefits. PMID:24266790

  6. Zebrafish models of cerebrovascular disease

    PubMed Central

    Walcott, Brian P; Peterson, Randall T

    2014-01-01

    Perturbations in cerebral blood flow and abnormalities in blood vessel structure are the hallmarks of cerebrovascular disease. While there are many genetic and environmental factors that affect these entities through a heterogeneous group of disease processes, the ultimate final pathologic insult in humans is defined as a stroke, or damage to brain parenchyma. In the case of ischemic stroke, blood fails to reach its target destination whereas in hemorrhagic stroke, extravasation of blood occurs outside of the blood vessel lumen, resulting in direct damage to brain parenchyma. As these acute events can be neurologically devastating, if not fatal, development of novel therapeutics are urgently needed. The zebrafish (Danio rerio) is an attractive model for the study of cerebrovascular disease because of its morphological and physiological similarity to human cerebral vasculature, its ability to be genetically manipulated, and its fecundity allowing for large-scale, phenotype-based screens. PMID:24517974

  7. Next-Generation Sequence Analysis of Cancer Xenograft Models

    PubMed Central

    Rossello, Fernando J.; Tothill, Richard W.; Britt, Kara; Marini, Kieren D.; Falzon, Jeanette; Thomas, David M.; Peacock, Craig D.; Marchionni, Luigi; Li, Jason; Bennett, Samara; Tantoso, Erwin; Brown, Tracey; Chan, Philip; Martelotto, Luciano G.; Watkins, D. Neil

    2013-01-01

    Next-generation sequencing (NGS) studies in cancer are limited by the amount, quality and purity of tissue samples. In this situation, primary xenografts have proven useful preclinical models. However, the presence of mouse-derived stromal cells represents a technical challenge to their use in NGS studies. We examined this problem in an established primary xenograft model of small cell lung cancer (SCLC), a malignancy often diagnosed from small biopsy or needle aspirate samples. Using an in silico strategy that assign reads according to species-of-origin, we prospectively compared NGS data from primary xenograft models with matched cell lines and with published datasets. We show here that low-coverage whole-genome analysis demonstrated remarkable concordance between published genome data and internal controls, despite the presence of mouse genomic DNA. Exome capture sequencing revealed that this enrichment procedure was highly species-specific, with less than 4% of reads aligning to the mouse genome. Human-specific expression profiling with RNA-Seq replicated array-based gene expression experiments, whereas mouse-specific transcript profiles correlated with published datasets from human cancer stroma. We conclude that primary xenografts represent a useful platform for complex NGS analysis in cancer research for tumours with limited sample resources, or those with prominent stromal cell populations. PMID:24086345

  8. Zebrafish models of dyslipidemia: Relevance to atherosclerosis and angiogenesis

    PubMed Central

    Fang, Longhou; Liu, Chao; Miller, Yury I.

    2013-01-01

    Lipid and lipoprotein metabolism in zebrafish and in humans are remarkably similar. Zebrafish express all major nuclear receptors, lipid transporters, apolipoproteins and enzymes involved in lipoprotein metabolism. Unlike mice, zebrafish express cetp and the Cetp activity is detected in zebrafish plasma. Feeding zebrafish a high cholesterol diet, without any genetic intervention, results in significant hypercholesterolemia and robust lipoprotein oxidation, making zebrafish an attractive animal model to study mechanisms relevant to early development of human atherosclerosis. These studies are facilitated by the optical transparency of zebrafish larvae and the availability of transgenic zebrafish expressing fluorescent proteins in endothelial cells and macrophages. Thus, vascular processes can be monitored in live animals. In this review article we discuss recent advances in using dyslipidemic zebrafish in atherosclerosis-related studies. We also summarize recent work connecting lipid metabolism with regulation of angiogenesis, the work that considerably benefited from using the zebrafish model. These studies uncovered the role of aibp, abca1, abcg1, mtp, apoB and apoC2 in regulation of angiogenesis in zebrafish and paved the way for future studies in mammals, which may suggest new therapeutic approaches to modulation of excessive or diminished angiogenesis contributing to the pathogenesis of human disease. PMID:24095954

  9. Using the zebrafish model for Alzheimer’s disease research

    PubMed Central

    Newman, Morgan; Ebrahimie, Esmaeil; Lardelli, Michael

    2014-01-01

    Rodent models have been extensively used to investigate the cause and mechanisms behind Alzheimer’s disease. Despite many years of intensive research using these models we still lack a detailed understanding of the molecular events that lead to neurodegeneration. Although zebrafish lack the complexity of advanced cognitive behaviors evident in rodent models they have proven to be a very informative model for the study of human diseases. In this review we give an overview of how the zebrafish has been used to study Alzheimer’s disease. Zebrafish possess genes orthologous to those mutated in familial Alzheimer’s disease and research using zebrafish has revealed unique characteristics of these genes that have been difficult to observe in rodent models. The zebrafish is becoming an increasingly popular model for the investigation of Alzheimer’s disease and will complement studies using other models to help complete our understanding of this disease. PMID:25071820

  10. Adult Zebrafish model of streptococcal infection

    PubMed Central

    Phelps, Hilary A.; Runft, Donna L.

    2009-01-01

    Streptococcal pathogens cause a wide array of clinical syndromes in humans, including invasive systemic infections resulting in high mortality rates. Many of these pathogens are human specific, and therefore difficult to analyze in vivo using typical animal models, as these models rarely replicate what is observed in human infections. This unit describes the use of the zebrafish (Danio rerio) as an animal model for streptococcal infection to analyze multiple disease states. This model closely mimics the necrotizing fasciitis/myositis pathology observed in humans from a Streptococcus pyogenes infection. The use of a zoonotic pathogen, Streptococcus iniae, which replicates systemic infections caused by many streptococcal pathogens, including dissemination to the brain, is also described. Included protocols describe both intraperitoneal and intramuscular infections, as well as methods for histological and quantitative measurements of infection. PMID:19412913

  11. The zebrafish as a model for complex tissue regeneration.

    PubMed

    Gemberling, Matthew; Bailey, Travis J; Hyde, David R; Poss, Kenneth D

    2013-11-01

    For centuries, philosophers and scientists have been fascinated by the principles and implications of regeneration in lower vertebrate species. Two features have made zebrafish an informative model system for determining mechanisms of regenerative events. First, they are highly regenerative, able to regrow amputated fins, as well as a lesioned brain, retina, spinal cord, heart, and other tissues. Second, they are amenable to both forward and reverse genetic approaches, with a research toolset regularly updated by an expanding community of zebrafish researchers. Zebrafish studies have helped identify new mechanistic underpinnings of regeneration in multiple tissues and, in some cases, have served as a guide for contemplating regenerative strategies in mammals. Here, we review the recent history of zebrafish as a genetic model system for understanding how and why tissue regeneration occurs. PMID:23927865

  12. REVIEW: Zebrafish: A Renewed Model System For Functional Genomics

    NASA Astrophysics Data System (ADS)

    Wen, Xiao-Yan

    2008-01-01

    In the post genome era, a major goal in molecular biology is to determine the function of the many thousands of genes present in the vertebrate genome. The zebrafish (Danio rerio) provides an almost ideal genetic model to identify the biological roles of these novel genes, in part because their embryos are transparent and develop rapidly. The zebrafish has many advantages over mouse for genome-wide mutagenesis studies, allowing for easier, cheaper and faster functional characterization of novel genes in the vertebrate genome. Many molecular research tools such as chemical mutagenesis, transgenesis, gene trapping, gene knockdown, TILLING, gene targeting, RNAi and chemical genetic screen are now available in zebrafish. Combining all the forward, reverse, and chemical genetic tools, it is expected that zebrafish will make invaluable contribution to vertebrate functional genomics in functional annotation of the genes, modeling human diseases and drug discoveries.

  13. The emerging use of zebrafish to model metabolic disease

    PubMed Central

    Seth, Asha; Stemple, Derek L.; Barroso, Inês

    2013-01-01

    The zebrafish research community is celebrating! The zebrafish genome has recently been sequenced, the Zebrafish Mutation Project (launched by the Wellcome Trust Sanger Institute) has published the results of its first large-scale ethylnitrosourea (ENU) mutagenesis screen, and a host of new techniques, such as the genome editing technologies TALEN and CRISPR-Cas, are enabling specific mutations to be created in model organisms and investigated in vivo. The zebrafish truly seems to be coming of age. These powerful resources invoke the question of whether zebrafish can be increasingly used to model human disease, particularly common, chronic diseases of metabolism such as obesity and type 2 diabetes. In recent years, there has been considerable success, mainly from genomic approaches, in identifying genetic variants that are associated with these conditions in humans; however, mechanistic insights into the role of implicated disease loci are lacking. In this Review, we highlight some of the advantages and disadvantages of zebrafish to address the organism’s utility as a model system for human metabolic diseases. PMID:24046387

  14. Conservation and Early Expression of Zebrafish Tyrosine Kinases Support the Utility of Zebrafish as a Model for Tyrosine Kinase Biology

    PubMed Central

    Challa, Anil Kumar

    2013-01-01

    Abstract Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome. PMID:23234507

  15. Modeling tuberculous meningitis in zebrafish using Mycobacterium marinum

    PubMed Central

    van Leeuwen, Lisanne M.; van der Kuip, Martijn; Youssef, Sameh A.; de Bruin, Alain; Bitter, Wilbert; van Furth, A. Marceline; van der Sar, Astrid M.

    2014-01-01

    Tuberculous meningitis (TBM) is one of the most severe extrapulmonary manifestations of tuberculosis, with a high morbidity and mortality. Characteristic pathological features of TBM are Rich foci, i.e. brain- and spinal-cord-specific granulomas formed after hematogenous spread of pulmonary tuberculosis. Little is known about the early pathogenesis of TBM and the role of Rich foci. We have adapted the zebrafish model of Mycobacterium marinum infection (zebrafish–M. marinum model) to study TBM. First, we analyzed whether TBM occurs in adult zebrafish and showed that intraperitoneal infection resulted in granuloma formation in the meninges in 20% of the cases, with occasional brain parenchyma involvement. In zebrafish embryos, bacterial infiltration and clustering of infected phagocytes was observed after infection at three different inoculation sites: parenchyma, hindbrain ventricle and caudal vein. Infection via the bloodstream resulted in the formation of early granulomas in brain tissue in 70% of the cases. In these zebrafish embryos, infiltrates were located in the proximity of blood vessels. Interestingly, no differences were observed when embryos were infected before or after early formation of the blood-brain barrier (BBB), indicating that bacteria are able to cross this barrier with relatively high efficiency. In agreement with this observation, infected zebrafish larvae also showed infiltration of the brain tissue. Upon infection of embryos with an M. marinum ESX-1 mutant, only small clusters and scattered isolated phagocytes with high bacterial loads were present in the brain tissue. In conclusion, our adapted zebrafish–M. marinum infection model for studying granuloma formation in the brain will allow for the detailed analysis of both bacterial and host factors involved in TBM. It will help solve longstanding questions on the role of Rich foci and potentially contribute to the development of better diagnostic tools and therapeutics. PMID:24997190

  16. A jump persistent turning walker to model zebrafish locomotion.

    PubMed

    Mwaffo, Violet; Anderson, Ross P; Butail, Sachit; Porfiri, Maurizio

    2015-01-01

    Zebrafish are gaining momentum as a laboratory animal species for the investigation of several functional and dysfunctional biological processes. Mathematical models of zebrafish behaviour are expected to considerably aid in the design of hypothesis-driven studies by enabling preliminary in silico tests that can be used to infer possible experimental outcomes without the use of zebrafish. This study is motivated by observations of sudden, drastic changes in zebrafish locomotion in the form of large deviations in turn rate. We demonstrate that such deviations can be captured through a stochastic mean reverting jump diffusion model, a process that is commonly used in financial engineering to describe large changes in the price of an asset. The jump process-based model is validated on trajectory data of adult subjects swimming in a shallow circular tank obtained from an overhead camera. Through statistical comparison of the empirical distribution of the turn rate against theoretical predictions, we demonstrate the feasibility of describing zebrafish as a jump persistent turning walker. The critical role of the jump term is assessed through comparison with a simplified mean reversion diffusion model, which does not allow for describing the heavy-tailed distributions observed in the fish turn rate. PMID:25392396

  17. A multi-scale model for correlation in B cell VDJ usage of zebrafish

    Microsoft Academic Search

    Keyao Pan; Michael W. Deem

    2011-01-01

    The zebrafish (Danio rerio) is one of the model animals used for the study of immunology because the dynamics in the adaptive immune system of zebrafish are similar to that in higher animals. In this work, we built a multi-scale model to simulate the dynamics of B cells in the primary and secondary immune responses of zebrafish. We use this

  18. Improvement of Radiation-Mediated Immunosuppression of Human NSCLC Tumour Xenografts in a Nude Rat Model

    PubMed Central

    Tokalov, Sergey V.; Enghardt, Wolfgang; Abolmaali, Nasreddin

    2010-01-01

    Human tumour xenografts in a nude rat model have consistently been used as an essential part of preclinical studies for anticancer drugs activity in human. Commonly, these animals receive whole body irradiation to assure immunosuppression. But whole body dose delivery might be inhomogeneous and the resulting incomplete bone marrow depletion may modify tumour behaviour. To improve irradiation-mediated immunosuppression of human non-small cell lung cancer (NSCLC) xenografts in a nude rat model irradiation (2 + 2 Gy) from opposite sides of animals has been performed using a conventional X-ray tube. The described modification of whole body irradiation improves growth properties of human NSCLC xenografts in a nude rat model. The design of the whole body irradiation mediated immunosuppression described here for NSCLC xenografts may be useful for research applications involving other types of human tumours. PMID:20368799

  19. Improvement of radiation-mediated immunosuppression of human NSCLC tumour xenografts in a nude rat model.

    PubMed

    Tokalov, Sergey V; Enghardt, Wolfgang; Abolmaali, Nasreddin

    2010-01-01

    Human tumour xenografts in a nude rat model have consistently been used as an essential part of preclinical studies for anticancer drugs activity in human. Commonly, these animals receive whole body irradiation to assure immunosuppression. But whole body dose delivery might be inhomogeneous and the resulting incomplete bone marrow depletion may modify tumour behaviour. To improve irradiation-mediated immunosuppression of human non-small cell lung cancer (NSCLC) xenografts in a nude rat model irradiation (2 + 2 Gy) from opposite sides of animals has been performed using a conventional X-ray tube. The described modification of whole body irradiation improves growth properties of human NSCLC xenografts in a nude rat model. The design of the whole body irradiation mediated immunosuppression described here for NSCLC xenografts may be useful for research applications involving other types of human tumours. PMID:20368799

  20. Analysis of the Retina in the Zebrafish Model

    PubMed Central

    Avanesov, Andrei; Malicki, Jarema

    2014-01-01

    The zebrafish is one of the leading models for the analysis of the vertebrate visual system. A wide assortment of molecular, genetic, and cell biological approaches is available to study zebrafish visual system development and function. As new techniques become available, genetic analysis and imaging continue to be the strengths of the zebrafish model. In particular, recent developments in the use of transposons and zinc finger nucleases to produce new generations of mutant strains enhance both forward and reverse genetic analysis. Similarly, the imaging of developmental and physiological processes benefits from a wide assortment of fluorescent proteins and the ways to express them in the embryo. The zebrafish is also highly attractive for high-throughput screening of small molecules, a promising strategy to search for compounds with therapeutic potential. Here we discuss experimental approaches used in the zebrafish model to study morpho?genetic transformations, cell fate decisions, and the differentiation of fine morphological features that ultimately lead to the formation of the functional vertebrate visual system. PMID:21111217

  1. Zebrafish: A Model for the Study of Addiction Genetics

    PubMed Central

    Klee, Eric W; Schneider, Henning; Clark, Karl; Cousin, Margot; Ebbert, Jon; Hooten, Michael; Karpyak, Victor; Warner, David; Ekker, Stephen

    2013-01-01

    Drug abuse and dependence are multifaceted disorders with complex genetic underpinnings. Identifying specific genetic correlates is challenging and may be more readily accomplished by defining endophenotypes specific for addictive disorders. Symptoms and syndromes, including acute drug response, consumption, preference, and withdrawal, are potential endophenotypes characterizing addiction that have been investigated using model organisms. We present a review of major genes involved in serotonergic, dopaminergic, GABAergic, and adrenoreceptor signaling that are considered to be directly involved in nicotine, opioid, cannabinoid, and ethanol use and dependence. The zebrafish genome encodes likely homologs of the vast majority of these loci. We also review the known expression patterns of these genes in zebrafish. The information presented in this review provides support for the use of zebrafish as a viable model for studying genetic factors related to drug addiction. Expansion of investigations into drug response using model organisms holds the potential to advance our understanding of drug response and addiction in humans. PMID:22207143

  2. Zebrafish: a model for the study of addiction genetics.

    PubMed

    Klee, Eric W; Schneider, Henning; Clark, Karl J; Cousin, Margot A; Ebbert, Jon O; Hooten, W Michael; Karpyak, Victor M; Warner, David O; Ekker, Stephen C

    2012-06-01

    Drug abuse and dependence are multifaceted disorders with complex genetic underpinnings. Identifying specific genetic correlates is challenging and may be more readily accomplished by defining endophenotypes specific for addictive disorders. Symptoms and syndromes, including acute drug response, consumption, preference, and withdrawal, are potential endophenotypes characterizing addiction that have been investigated using model organisms. We present a review of major genes involved in serotonergic, dopaminergic, GABAergic, and adrenoreceptor signaling that are considered to be directly involved in nicotine, opioid, cannabinoid, and ethanol use and dependence. The zebrafish genome encodes likely homologs of the vast majority of these loci. We also review the known expression patterns of these genes in zebrafish. The information presented in this review provides support for the use of zebrafish as a viable model for studying genetic factors related to drug addiction. Expansion of investigations into drug response using model organisms holds the potential to advance our understanding of drug response and addiction in humans. PMID:22207143

  3. Zebrafish: A Model System to Study Heritable Skin Diseases

    PubMed Central

    Li, Qiaoli; Frank, Michael; Thisse, Christine; Thisse, Bernard; Uitto, Jouni

    2012-01-01

    Heritable skin diseases represent a broad spectrum of clinical manifestations due to mutations in ~500 different genes. A number of model systems have been developed to advance our understanding of the pathomechanisms of genodermatoses. Zebrafish (Danio rerio), a freshwater vertebrate, has a well-characterized genome, the expression of which can be easily manipulated. The larvae develop rapidly, with all major organs having developed by 5–6 days post fertilization, including the skin, consisting of the epidermis comprising two cell layers and separated from the dermal collagenous matrix by a basement membrane. This perspective highlights the morphologic and ultrastructural features of zebrafish skin, in the context of cutaneous gene expression. These observations suggest that zebrafish provides a useful model system to study molecular aspects of skin development, as well as the pathogenesis and treatment of select heritable skin diseases. PMID:21191402

  4. A zebrafish model for hepatoerythropoietic porphyria

    Microsoft Academic Search

    Han Wang; Qiaoming Long; Scott D. Marty; Shigeru Sassa; Shuo Lin

    1998-01-01

    Defects in the enzymes involved in the haem biosynthetic pathway can lead to a group of human diseases known as the porphyrias. yquem (yqe tp61) is a zebrafish mutant with a photosensitive porphyria syndrome. Here we show that the porphyric phenotype is due to an inherited homozygous mutation in the gene encoding uroporphyrinogen decarboxylase (UROD); a homozygous deficiency of this

  5. A Murine Xenograft Model of Spontaneous Metastases of Human Lung Adenocarcinoma1

    PubMed Central

    Harris, James E.; Shin, James; Lee, Beverly; Pelosky, Kristen; Hooker, Craig M.; Harbom, Kirsten; Hulbert, Alicia; Zahnow, Cynthia; Yang, Stephen C.; Baylin, Stephen; Brayton, Cory; Brock, Malcolm V.

    2012-01-01

    Background The flank is commonly used for primary xenografts in mice, but it is rare for these tumors to metastasize. Tail vein injection creates a pattern of metastases, but is artificial. We hypothesized that the liver is a convenient alternative xenograft site and that metastases would gradually proceed spontaneously. Materials and Methods Using 15 NOD.CB17-Prkdcscid/NcrCrl (NOD/SCID) mice, 10,000 A549 cells were xenografted into the liver while a second group of five mice were xenografted in the flank with 100,000 A549 cells as a control. Mice were euthanized and grossly dissected at 7 wk. A third group of seven mice received liver xenografts with A549 and a mouse each week was euthanized for 7 wk and evaluated. The liver, lung, and spleen were examined histologically. Results At 7 wk, 15/15 liver xenografted mice had gross primary tumor in the liver. Histologic review confirmed multiple microscopic foci of metastatic disease in all mice (15/15) throughout the lungs, mediastinal nodes, and spleen. The control group had primary tumor in the flank (4/5), but none had histologic evidence of metastases. Serially euthanized liver xenografted mice revealed evidence of a gradual spontaneous metastatic model system with the first histologic findings of micrometastases appearing in the lungs by wk 5, which became wide spread by wk 7. Splenic and mediastinal lymph node metastases developed in wk 6 and 7. Conclusions Liver xenografting of A549 cells into NOD/SCID mice is a reliable way of developing widespread micrometastases. This model allows the study of a gradually developing solid tumor with subsequent metastatic spread. PMID:21872887

  6. Developing an Experimental Model of Vascular Toxicity in Embryonic Zebrafish

    EPA Science Inventory

    Developing an Experimental Model of Vascular Toxicity in Embryonic Zebrafish Tamara Tal, Integrated Systems Toxicology Division, U.S. EPA Background: There are tens of thousands of chemicals that have yet to be fully evaluated for their toxicity by validated in vivo testing ...

  7. The zebrafish as a model to study polycystic liver disease.

    PubMed

    Tietz Bogert, Pamela S; Huang, Bing Q; Gradilone, Sergio A; Masyuk, Tetyana V; Moulder, Gary L; Ekker, Stephen C; Larusso, Nicholas F

    2013-06-01

    In the polycystic liver diseases (PLD), genetic defects initiate the formation of cysts in the liver and kidney. In rodent models of PLD (i.e., the PCK rat and Pkd2(WS25/-) mouse), we have studied hepatorenal cystic disease and therapeutic approaches. In this study, we employed zebrafish injected with morpholinos against genes involved in the PLD, including sec63, prkcsh, and pkd1a. We calculated the liver cystic area, and based on our rodent studies, we exposed the embryos to pasireotide [1??M] or vitamin K3 [100??M] and assessed the endoplasmic reticulum (ER) in cholangiocytes in embryos treated with 4-phenylbutyrate (4-PBA). Our results show that (a) morpholinos against sec63, prkcsh, and pkd1a eliminate expression of the respective proteins; (b) phenotypic body changes included curved tail and the formation of hepatic cysts in zebrafish larvae; (c) exposure of embryos to pasireotide inhibited hepatic cystogenesis in the zebrafish models; and (d) exposure of embryos to 4-PBA resulted in the ER in cholangiocytes resolving from a curved to a smooth appearance. Our results suggest that the zebrafish model of PLD may provide a means to screen drugs that could inhibit hepatic cystogenesis. PMID:23668934

  8. Systematic Analysis of a Xenograft Mice Model for KSHV+ Primary Effusion Lymphoma (PEL)

    PubMed Central

    Dai, Lu; Trillo-Tinoco, Jimena; Bai, Lihua; Kang, Baoli; Xu, Zengguang; Wen, Xiaofei; Valle, Luis Del; Qin, Zhiqiang

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus is the causative agent of primary effusion lymphoma (PEL), which arises preferentially in the setting of infection with human immunodeficiency virus (HIV). Even with standard cytotoxic chemotherapy, PEL continues to cause high mortality rates, requiring the development of novel therapeutic strategies. PEL xenograft models employing immunodeficient mice have been used to study the in vivo effects of a variety of therapeutic approaches. However, it remains unclear whether these xenograft models entirely reflect clinical presentations of KSHV+ PEL, especially given the recent description of extracavitary solid tumor variants arising in patients. In addition, effusion and solid tumor cells propagated in vivo exhibit unique biology, differing from one another or from their parental cell lines propagated through in vitro culture. Therefore, we used a KSHV+ PEL/BCBL-1 xenograft model involving non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice, and compared characteristics of effusion and solid tumors with their parent cell culture-derived counterparts. Our results indicate that although this xenograft model can be used for study of effusion and solid lymphoma observed in patients, tumor cells in vivo display unique features to those passed in vitro, including viral lytic gene expression profile, rate of solid tumor development, the host proteins and the complex of tumor microenvironment. These items should be carefully considered when the xenograft model is used for testing novel therapeutic strategies against KSHV-related lymphoma. PMID:24587336

  9. Systematic analysis of a xenograft mice model for KSHV+ primary effusion lymphoma (PEL).

    PubMed

    Dai, Lu; Trillo-Tinoco, Jimena; Bai, Lihua; Kang, Baoli; Xu, Zengguang; Wen, Xiaofei; Del Valle, Luis; Qin, Zhiqiang

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus is the causative agent of primary effusion lymphoma (PEL), which arises preferentially in the setting of infection with human immunodeficiency virus (HIV). Even with standard cytotoxic chemotherapy, PEL continues to cause high mortality rates, requiring the development of novel therapeutic strategies. PEL xenograft models employing immunodeficient mice have been used to study the in vivo effects of a variety of therapeutic approaches. However, it remains unclear whether these xenograft models entirely reflect clinical presentations of KSHV(+) PEL, especially given the recent description of extracavitary solid tumor variants arising in patients. In addition, effusion and solid tumor cells propagated in vivo exhibit unique biology, differing from one another or from their parental cell lines propagated through in vitro culture. Therefore, we used a KSHV(+) PEL/BCBL-1 xenograft model involving non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice, and compared characteristics of effusion and solid tumors with their parent cell culture-derived counterparts. Our results indicate that although this xenograft model can be used for study of effusion and solid lymphoma observed in patients, tumor cells in vivo display unique features to those passed in vitro, including viral lytic gene expression profile, rate of solid tumor development, the host proteins and the complex of tumor microenvironment. These items should be carefully considered when the xenograft model is used for testing novel therapeutic strategies against KSHV-related lymphoma. PMID:24587336

  10. A zebrafish model for subgenomic hepatitis C virus replication.

    PubMed

    Ding, Cun-Bao; Zhao, Ye; Zhang, Jing-Pu; Peng, Zong-Gen; Song, Dan-Qing; Jiang, Jian-Dong

    2015-03-01

    Persistent infection with hepatitis C virus (HCV) is a major risk factor in the development of hepatocellular carcinoma. The elucidation of the pathogenesis of HCV-associated liver disease is hampered by the absence of an appropriate small animal model. Zebrafish exhibits high genetic homology to mammals, and is easily manipulated experimentally. In this study, we describe the use of a zebrafish model for the analysis of HCV replication mechanisms. As the 5' untranslated region (UTR), the core protein, the non-structural protein 5B (NS5B) and the 3'UTR are essential for HCV replication, we constructed a HCV sub-replicon gene construct including the 4 gene sequences and the enhanced green fluorescent protein (EGFP) reporter gene; these genes were transcribed through the mouse hepatocyte nuclear factor 4 (mHNF4) promoter. By microinjection of the subgenomic replicon vector into zebrafish larvae, the virus was easily detected by observing EGFP fluorescence in the liver. The positive core and NS5B signals showed positive expression of the HCV gene construct in zebrafish by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Importantly, the negative strand sequence of the HCV subgenomic RNA was detected by RT-PCR and hybridization in situ, demonstrating that the HCV sub-replicon has positive replication activity. Furthermore, the hybridization signal mainly appeared in the liver region of larvae, as detected by the sense probe of the core protein or NS5B, which confirmed that the sub-replicon amplification occurred in the zebrafish liver. The amplification of the sub-replicon caused alterations in the expression of certain genes, which is similar to HCV infection in human liver cells. To verify the use of this zebrafish model in drug evaluation, two drugs against HCV used in clinical practice, ribavirin and oxymatrine, were tested and these drugs showed significant inhibition of replication of the HCV sub-replicon in the larvae. In conclusion, this zebrafish model of HCV may prove to be a novel and simple in vivo model for the study of the mechanisms of HCV replication and may also prove useful in the disovery of new anti-HCV drugs. PMID:25572289

  11. Genetic modeling of Li-Fraumeni syndrome in zebrafish

    PubMed Central

    Parant, John M.; George, Stephen A.; Holden, Joseph A.; Yost, H. Joseph

    2010-01-01

    SUMMARY Li-Fraumeni syndrome (LFS) is a highly penetrant, autosomal dominant, human familial cancer predisposition. Although a key role for the tumor suppressor p53 has been implicated in LFS, the genetic and cellular mechanisms underpinning this disease remain unknown. Therefore, modeling LFS in a vertebrate system that is accessible to both large-scale genetic screens and in vivo cell biological studies will facilitate the in vivo dissection of disease mechanisms, help identify candidate genes, and spur the discovery of therapeutic compounds. Here, we describe a forward genetic screen in zebrafish embryos that was used to identify LFS candidate genes, which yielded a p53 mutant (p53I166T) that as an adult develops tumors, predominantly sarcomas, with 100% penetrance. As in humans with LFS, tumors arise in heterozygotes and display loss of heterozygosity (LOH). This report of LOH indicates that Knudson’s two-hit hypothesis, a hallmark of human autosomal dominant cancer syndromes, can be modeled in zebrafish. Furthermore, as with some LFS mutations, the zebrafish p53I166T allele is a loss-of-function allele with dominant-negative activity in vivo. Additionally, we demonstrate that the p53 regulatory pathway, including Mdm2 regulation, is evolutionarily conserved in zebrafish, providing a bona fide biological context in which to systematically uncover novel modifier genes and therapeutic agents for human LFS. PMID:20075382

  12. A new model to study visual attention in zebrafish.

    PubMed

    Braida, Daniela; Ponzoni, Luisa; Martucci, Roberta; Sala, Mariaelvina

    2014-12-01

    The major part of cognitive tasks applied to zebrafish has not fully assessed their attentional ability, a process by which the nervous system learns, organizes sensory input and generates coordinated behaviour. In an attempt to maximize the value of zebrafish as an animal model of cognition, we tested the possibility to apply a modified version of novel object recognition test named virtual object recognition test (VORT) using 2D geometrical shapes (square, triangle, circle, cross, etc.) on two iPod 3.5-inch widescreen displays, located on two opposite walls of the water tank. Each fish was subjected to a familiarization trial (T1), and after different time delays (from 5 min to 96 h) to a novel shape recognition trial (T2). A progressive decrease, across time, of memory performance, in terms of mean discrimination index and mean exploration time, was shown. The predictive validity was tested using cholinergic drugs. Nicotine (0.02 mg/kg intraperitoneally, IP) significantly increased, while scopolamine (0.025 mg/kg IP) and mecamylamine decreased, mean discrimination index. Zebrafish discriminated different movements (vertical, horizontal, oblique) and the discrimination index increased significantly when moving poorly discriminated shapes were presented, thus increasing visual attention. Taken together these findings demonstrate that VORT is a viable, fast and useful model to evaluate sustained attention in zebrafish and for predicting the efficacy of pharmacotherapies for cognitive disorders. PMID:24681194

  13. An Individual-Based Model of Zebrafish Population Dynamics Accounting for Energy Dynamics

    PubMed Central

    Beaudouin, Rémy; Goussen, Benoit; Piccini, Benjamin; Augustine, Starrlight; Devillers, James; Brion, François; Péry, Alexandre R. R.

    2015-01-01

    Developing population dynamics models for zebrafish is crucial in order to extrapolate from toxicity data measured at the organism level to biological levels relevant to support and enhance ecological risk assessment. To achieve this, a dynamic energy budget for individual zebrafish (DEB model) was coupled to an individual based model of zebrafish population dynamics (IBM model). Next, we fitted the DEB model to new experimental data on zebrafish growth and reproduction thus improving existing models. We further analysed the DEB-model and DEB-IBM using a sensitivity analysis. Finally, the predictions of the DEB-IBM were compared to existing observations on natural zebrafish populations and the predicted population dynamics are realistic. While our zebrafish DEB-IBM model can still be improved by acquiring new experimental data on the most uncertain processes (e.g. survival or feeding), it can already serve to predict the impact of compounds at the population level. PMID:25938409

  14. Zebrafish as animal model for aquaculture nutrition research.

    PubMed

    Ulloa, Pilar E; Medrano, Juan F; Feijoo, Carmen G

    2014-01-01

    The aquaculture industry continues to promote the diversification of ingredients used in aquafeed in order to achieve a more sustainable aquaculture production system. The evaluation of large numbers of diets in aquaculture species is costly and requires time-consuming trials in some species. In contrast, zebrafish (Danio rerio) can solve these drawbacks as an experimental model, and represents an ideal organism to carry out preliminary evaluation of diets. In addition, zebrafish has a sequenced genome allowing the efficient utilization of new technologies, such as RNA-sequencing and genotyping platforms to study the molecular mechanisms that underlie the organism's response to nutrients. Also, biotechnological tools like transgenic lines with fluorescently labeled neutrophils that allow the evaluation of the immune response in vivo, are readily available in this species. Thus, zebrafish provides an attractive platform for testing many ingredients to select those with the highest potential of success in aquaculture. In this perspective article aspects related to diet evaluation in which zebrafish can make important contributions to nutritional genomics and nutritional immunity are discussed. PMID:25309575

  15. The Developing Utility of Zebrafish Models for Cognitive Enhancers Research

    PubMed Central

    Stewart, Adam Michael; Kalueff, Allan V

    2012-01-01

    Whereas cognitive impairment is a common symptom in multiple brain disorders, predictive and high-throughput animal models of cognition and behavior are becoming increasingly important in the field of translational neuroscience research. In particular, reliable models of the cognitive deficits characteristic of numerous neurobehavioral disorders such as Alzheimer’s disease and schizophrenia have become a significant focus of investigation. While rodents have traditionally been used to study cognitive phenotypes, zebrafish (Danio rerio) are gaining popularity as an excellent model to complement current translational neuroscience research. Here we discuss recent advances in pharmacological and genetic approaches using zebrafish models to study cognitive impairments and to discover novel cognitive enhancers and neuroprotective mechanisms. PMID:23449968

  16. Zebrafish: a new companion for translational research in oncology.

    PubMed

    Barriuso, Jorge; Nagaraju, Raghavendar; Hurlstone, Adam

    2015-03-01

    In an era of high-throughput "omic" technologies, the unprecedented amount of data that can be generated presents a significant opportunity but simultaneously an even greater challenge for oncologists trying to provide personalized treatment. Classically, preclinical testing of new targets and identification of active compounds against those targets have entailed the extensive use of established human cell lines, as well as genetically modified mouse tumor models. Patient-derived xenografts in zebrafish may in the near future provide a platform for selecting an appropriate personalized therapy and together with zebrafish transgenic tumor models represent an alternative vehicle for drug development. The zebrafish is readily genetically modified. The transparency of zebrafish embryos and the recent development of pigment-deficient zebrafish afford researchers the valuable capacity to observe directly cancer formation and progression in a live vertebrate host. The zebrafish is amenable to transplantation assays that test the serial passage of fluorescently labeled tumor cells as well as their capacity to disseminate and/or metastasize. Progress achieved to date in genetic engineering and xenotransplantation will establish the zebrafish as one of the most versatile animal models for cancer research. A model organism that can be used in transgenesis, transplantation assays, single-cell functional assays, and in vivo imaging studies make zebrafish a natural companion for mice in translational oncology research. PMID:25573382

  17. Zebrafish: an in vivo model for nano EHS studies

    PubMed Central

    Zhao, Yan; Nel, André E.; Lin, Shuo

    2014-01-01

    To assure a responsible and sustainable growth of nanotechnology, the environmental health and safety (EHS) aspect of engineered nanomaterials and nano-related products needs to be addressed at a rate commensurate with the expansion of nanotechnology. Zebrafish has been demonstrated as a correlative in vivo vertebrate model for such task, and the current advances of using zebrafish for nano EHS studies are summarized here. In addition to morphological and histopathological observations, the accessibility of gene manipulation would greatly empower such a model for detailed mechanistic studies of any nanoparticles of interest. The potential for establishing high-throughput screening platforms to facilitate the nano EHS studies is highlighted, and a discussion is presented on how toxicogenomics approaches represent a future direction to guide the identification of toxicity pathways. PMID:23208995

  18. Zebrafish: A Model System for the Study of Eye Genetics

    PubMed Central

    Fadool, James M.; Dowling, John E.

    2008-01-01

    Over the last decade, the use of the zebrafish as a genetic model has moved beyond the proof-of-concept for the analysis of vertebrate embryonic development to demonstrated utility as a mainstream model organism for the understanding of human disease. The initial identification of a variety of zebrafish mutations affecting the eye and retina, and the subsequent cloning of mutated genes have revealed cellular, molecular and physiological processes fundamental to visual system development. With the increasing development of genetic manipulations, sophisticated techniques for phenotypic characterization, behavioral approaches and screening strategies, the identification of novel genes or novel gene functions will have important implications for our understanding of human eye diseases, pathogenesis, and treatment. PMID:17962065

  19. Antitumor efficacy of a novel polymerpeptidedrug conjugate in human tumor xenograft models

    E-print Network

    Chau, Ying

    Antitumor efficacy of a novel polymer­peptide­drug conjugate in human tumor xenograft models Ying in the presence of matrix-metalloproteinase-2 (MMP-2) and matrix-metalloprotei- nase-9 (MMP-9), 2 important tumor-associated); tumor-asso- ciated enzymes; polymer-drug conjugate; cancer targeting Polymer­drug conjugates have shown

  20. Computational Graph Theoretical Model of the Zebrafish Sensorimotor Pathway

    NASA Astrophysics Data System (ADS)

    Peterson, Joshua M.; Stobb, Michael; Mazzag, Bori; Gahtan, Ethan

    2011-11-01

    Mapping the detailed connectivity patterns of neural circuits is a central goal of neuroscience and has been the focus of extensive current research [4, 3]. The best quantitative approach to analyze the acquired data is still unclear but graph theory has been used with success [3, 1]. We present a graph theoretical model with vertices and edges representing neurons and synaptic connections, respectively. Our system is the zebrafish posterior lateral line sensorimotor pathway. The goal of our analysis is to elucidate mechanisms of information processing in this neural pathway by comparing the mathematical properties of its graph to those of other, previously described graphs. We create a zebrafish model based on currently known anatomical data. The degree distributions and small-world measures of this model is compared to small-world, random and 3-compartment random graphs of the same size (with over 2500 nodes and 160,000 connections). We find that the zebrafish graph shows small-worldness similar to other neural networks and does not have a scale-free distribution of connections.

  1. Zebrafish as a novel vertebrate model to dissect enterococcal pathogenesis.

    PubMed

    Prajsnar, Tomasz K; Renshaw, Stephen A; Ogryzko, Nikolay V; Foster, Simon J; Serror, Pascale; Mesnage, Stéphane

    2013-11-01

    Enterococcus faecalis is an opportunistic pathogen responsible for a wide range of life-threatening nosocomial infections, such as septicemia, peritonitis, and endocarditis. E. faecalis infections are associated with a high mortality and substantial health care costs and cause therapeutic problems due to the intrinsic resistance of this bacterium to antibiotics. Several factors contributing to E. faecalis virulence have been identified. Due to the variety of infections caused by this organism, numerous animal models have been used to mimic E. faecalis infections, but none of them is considered ideal for monitoring pathogenesis. Here, we studied for the first time E. faecalis pathogenesis in zebrafish larvae. Using model strains, chosen isogenic mutants, and fluorescent derivatives expressing green fluorescent protein (GFP), we analyzed both lethality and bacterial dissemination in infected larvae. Genetically engineered immunocompromised zebrafish allowed the identification of two critical steps for successful establishment of disease: (i) host phagocytosis evasion mediated by the Epa rhamnopolysaccharide and (ii) tissue damage mediated by the quorum-sensing Fsr regulon. Our results reveal that the zebrafish is a novel, powerful model for studying E. faecalis pathogenesis, enabling us to dissect the mechanism of enterococcal virulence. PMID:24002065

  2. Modelling Delta-Notch perturbations during zebrafish somitogenesis.

    PubMed

    Murray, Philip J; Maini, Philip K; Baker, Ruth E

    2013-01-15

    The discovery over the last 15 years of molecular clocks and gradients in the pre-somitic mesoderm of numerous vertebrate species has added significant weight to Cooke and Zeeman's 'clock and wavefront' model of somitogenesis, in which a travelling wavefront determines the spatial position of somite formation and the somitogenesis clock controls periodicity (Cooke and Zeeman, 1976). However, recent high-throughput measurements of spatiotemporal patterns of gene expression in different zebrafish mutant backgrounds allow further quantitative evaluation of the clock and wavefront hypothesis. In this study we describe how our recently proposed model, in which oscillator coupling drives the propagation of an emergent wavefront, can be used to provide mechanistic and testable explanations for the following observed phenomena in zebrafish embryos: (a) the variation in somite measurements across a number of zebrafish mutants; (b) the delayed formation of somites and the formation of 'salt and pepper' patterns of gene expression upon disruption of oscillator coupling; and (c) spatial correlations in the 'salt and pepper' patterns in Delta-Notch mutants. In light of our results, we propose a number of plausible experiments that could be used to further test the model. PMID:23085238

  3. A two-scale model for correlation between B cell VDJ usage in zebrafish

    NASA Astrophysics Data System (ADS)

    Pan, Keyao; Deem, Michael

    2011-03-01

    The zebrafish (Danio rerio) is one of the model animals for study of immunology. The dynamics of the adaptive immune system in zebrafish is similar to that in higher animals. In this work, we built a two-scale model to simulate the dynamics of B cells in primary and secondary immune reactions in zebrafish and to explain the reported correlation between VDJ usage of B cell repertoires in distinct zebrafish. The first scale of the model consists of a generalized NK model to simulate the B cell maturation process in the 10-day primary immune response. The second scale uses a delay ordinary differential equation system to model the immune responses in the 6-month lifespan of zebrafish. The generalized NK model shows that mature B cells specific to one antigen mostly possess a single VDJ recombination. The probability that mature B cells in two zebrafish have the same VDJ recombination increases with the B cell population size or the B cell selection intensity and decreases with the B cell hypermutation rate. The ODE model shows a distribution of correlation in the VDJ usage of the B cell repertoires in two six-month-old zebrafish that is highly similar to that from experiment. This work presents a simple theory to explain the experimentally observed correlation in VDJ usage of distinct zebrafish B cell repertoires after an immune response.

  4. Human skeletal muscle xenograft as a new preclinical model for muscle disorders.

    PubMed

    Zhang, Yuanfan; King, Oliver D; Rahimov, Fedik; Jones, Takako I; Ward, Christopher W; Kerr, Jaclyn P; Liu, Naili; Emerson, Charles P; Kunkel, Louis M; Partridge, Terence A; Wagner, Kathryn R

    2014-06-15

    Development of novel therapeutics requires good animal models of disease. Disorders for which good animal models do not exist have very few drugs in development or clinical trial. Even where there are accepted, albeit imperfect models, the leap from promising preclinical drug results to positive clinical trials commonly fails, including in disorders of skeletal muscle. The main alternative model for early drug development, tissue culture, lacks both the architecture and, usually, the metabolic fidelity of the normal tissue in vivo. Herein, we demonstrate the feasibility and validity of human to mouse xenografts as a preclinical model of myopathy. Human skeletal muscle biopsies transplanted into the anterior tibial compartment of the hindlimbs of NOD-Rag1(null) IL2r?(null) immunodeficient host mice regenerate new vascularized and innervated myofibers from human myogenic precursor cells. The grafts exhibit contractile and calcium release behavior, characteristic of functional muscle tissue. The validity of the human graft as a model of facioscapulohumeral muscular dystrophy is demonstrated in disease biomarker studies, showing that gene expression profiles of xenografts mirror those of the fresh donor biopsies. These findings illustrate the value of a new experimental model of muscle disease, the human muscle xenograft in mice, as a feasible and valid preclinical tool to better investigate the pathogenesis of human genetic myopathies and to more accurately predict their response to novel therapeutics. PMID:24452336

  5. Toward developmental models of psychiatric disorders in zebrafish

    PubMed Central

    Norton, William H. J.

    2013-01-01

    Psychiatric disorders are a diverse set of diseases that affect all aspects of mental function including social interaction, thinking, feeling, and mood. Although psychiatric disorders place a large economic burden on society, the drugs available to treat them are often palliative with variable efficacy and intolerable side-effects. The development of novel drugs has been hindered by a lack of knowledge about the etiology of these diseases. It is thus necessary to further investigate psychiatric disorders using a combination of human molecular genetics, gene-by-environment studies, in vitro pharmacological and biochemistry experiments, animal models, and investigation of the non-biological basis of these diseases, such as environmental effects. Many psychiatric disorders, including autism spectrum disorder, attention-deficit/hyperactivity disorder, mental retardation, and schizophrenia can be triggered by alterations to neural development. The zebrafish is a popular model for developmental biology that is increasingly used to study human disease. Recent work has extended this approach to examine psychiatric disorders as well. However, since psychiatric disorders affect complex mental functions that might be human specific, it is not possible to fully model them in fish. In this review, I will propose that the suitability of zebrafish for developmental studies, and the genetic tools available to manipulate them, provide a powerful model to study the roles of genes that are linked to psychiatric disorders during neural development. The relative speed and ease of conducting experiments in zebrafish can be used to address two areas of future research: the contribution of environmental factors to disease onset, and screening for novel therapeutic compounds. PMID:23637652

  6. Hematein, a casein kinase II inhibitor, inhibits lung cancer tumor growth in a murine xenograft model

    PubMed Central

    HUNG, MING-SZU; XU, ZHIDONG; CHEN, YU; SMITH, EMMANUEL; MAO, JIAN-HUA; HSIEH, DAVID; LIN, YU-CHING; YANG, CHENG-TA; JABLONS, DAVID M.; YOU, LIANG

    2013-01-01

    Casein kinase II (CK2) inhibitors suppress cancer cell growth. In this study, we examined the inhibitory effects of a novel CK2 inhibitor, hematein, on tumor growth in a murine xenograft model. We found that in lung cancer cells, hematein inhibited cancer cell growth, Akt/PKB Ser129 phosphorylation, the Wnt/TCF pathway and increased apoptosis. In a murine xenograft model of lung cancer, hematein inhibited tumor growth without significant toxicity to the mice tested. Molecular docking showed that hematein binds to CK2? in durable binding sites. Collectively, our results suggest that hematein is an allosteric inhibitor of protein kinase CK2 and has antitumor activity to lung cancer. PMID:24008396

  7. The interpretation of marker protein assays: a critical appraisal in clinical studies and a xenograft model.

    PubMed

    Raghavan, D; Gibbs, J; Nogueira Costa, R; Kohn, J; Orr, A H; Barrett, A; Peckham, M J

    1980-04-01

    Although useful in the management of malignant testicular tumours, alphafoetoprotein and human chromonic gonadotrophin are not perfect parameters of tumour bulk. In a series of 65 patients with marker-positive advanced disease, 23 showed the phenomenon of discordance of markers or dissociation from tumour response. Transplantable human malignant teratoma xenografts were established in immune-suppressed mice as models of the human disease. In the model system, it was demonstrated that tumour AFP concentration was reflected by AFP titres in the blood, but that tumour AFP (or blood AFP) did not correlate well with tumour size. One xenograft line illustrated the evolution of marker-negative tumour cells from a patient who was initially AFP-positive. PMID:6158975

  8. The interpretation of marker protein assays: a critical appraisal in clinical studies and a xenograft model.

    PubMed Central

    Raghavan, D.; Gibbs, J.; Nogueira Costa, R.; Kohn, J.; Orr, A. H.; Barrett, A.; Peckham, M. J.

    1980-01-01

    Although useful in the management of malignant testicular tumours, alphafoetoprotein and human chromonic gonadotrophin are not perfect parameters of tumour bulk. In a series of 65 patients with marker-positive advanced disease, 23 showed the phenomenon of discordance of markers or dissociation from tumour response. Transplantable human malignant teratoma xenografts were established in immune-suppressed mice as models of the human disease. In the model system, it was demonstrated that tumour AFP concentration was reflected by AFP titres in the blood, but that tumour AFP (or blood AFP) did not correlate well with tumour size. One xenograft line illustrated the evolution of marker-negative tumour cells from a patient who was initially AFP-positive. PMID:6158975

  9. The T61 human breast cancer xenograft: An experimental model of estrogen therapy of breast cancer

    Microsoft Academic Search

    Nils Briinner I; Mogens Spang-Thomsen; Kevin Cullen

    1996-01-01

    Summary Endocrine therapy is one of the principal treatment modalities of breast cancer, both in an adjuvant setting and in advanced disease. The T61 breast cancer xenograft described here provides an experimental model of the effects of estrogen treatment at a molecular level. T61 is an estrogen receptor positive tumor which was originally derived from a T1N0M0 invasive ductal cancer

  10. A multi-scale model for correlation in B cell VDJ usage of zebrafish

    NASA Astrophysics Data System (ADS)

    Pan, Keyao; Deem, Michael W.

    2011-10-01

    The zebrafish (Danio rerio) is one of the model animals used for the study of immunology because the dynamics in the adaptive immune system of zebrafish are similar to that in higher animals. In this work, we built a multi-scale model to simulate the dynamics of B cells in the primary and secondary immune responses of zebrafish. We use this model to explain the reported correlation between VDJ usage of B cell repertoires in individual zebrafish. We use a delay ordinary differential equation (ODE) system to model the immune responses in the 6-month lifespan of a zebrafish. This mean field theory gives the number of high-affinity B cells as a function of time during an infection. The sequences of those B cells are then taken from a distribution calculated by a 'microscopic' random energy model. This generalized NK model shows that mature B cells specific to one antigen largely possess a single VDJ recombination. The model allows first-principle calculation of the probability, p, that two zebrafish responding to the same antigen will select the same VDJ recombination. This probability p increases with the B cell population size and the B cell selection intensity. The probability p decreases with the B cell hypermutation rate. The multi-scale model predicts correlations in the immune system of the zebrafish that are highly similar to that from experiment.

  11. A multi-scale model for correlation in B cell VDJ usage of zebrafish

    PubMed Central

    Pan, Keyao; Deem, Michael W

    2015-01-01

    The zebrafish (Danio rerio) is one of the model animals for study of immunology because the dynamics in the adaptive immune system of zebrafish are similar to that in higher animals. In this work, we built a multi-scale model to simulate the dynamics of B cells in the primary and secondary immune responses of zebrafish. We use this model to explain the reported correlation between VDJ usage of B cell repertoires in individual zebrafish. We use a delay ordinary differential equation (ODE) system to model the immune responses in the 6-month lifespan of a zebrafish. This mean field theory gives the number of high affinity B cells as a function of time during an infection. The sequences of those B cells are then taken from a distribution calculated by a “microscopic” random energy model. This generalized NK model shows that mature B cells specific to one antigen largely possess a single VDJ recombination. The model allows first-principles calculation of the probability, p, that two zebrafish responding to the same antigen will select the same VDJ recombination. This probability p increases with the B cell population size and the B cell selection intensity. The probability p decreases with the B cell hypermutation rate. The multi-scale model predicts correlations in the immune system of the zebrafish that are highly similar to that from experiment. PMID:21832808

  12. Zebrafish as a model to investigate CNS myelination.

    PubMed

    Preston, Marnie A; Macklin, Wendy B

    2015-02-01

    Myelin plays a critical role in proper neuronal function by providing trophic and metabolic support to axons and facilitating energy-efficient saltatory conduction. Myelination is influenced by numerous molecules including growth factors, hormones, transmembrane receptors and extracellular molecules, which activate signaling cascades that drive cellular maturation. Key signaling molecules and downstream signaling cascades controlling myelination have been identified in cell culture systems. However, in vitro systems are not able to faithfully replicate the complex in vivo signaling environment that occurs during development or following injury. Currently, it remains time-consuming and expensive to investigate myelination in vivo in rodents, the most widely used model for studying mammalian myelination. As such, there is a need for alternative in vivo myelination models, particularly ones that can test molecular mechanisms without removing oligodendrocyte lineage cells from their native signaling environment or disrupting intercellular interactions with other cell types present during myelination. Here, we review the ever-increasing role of zebrafish in studies uncovering novel mechanisms controlling vertebrate myelination. These innovative studies range from observations of the behavior of single cells during in vivo myelination as well as mutagenesis- and pharmacology-based screens in whole animals. Additionally, we discuss recent efforts to develop novel models of demyelination and oligodendrocyte cell death in adult zebrafish for the study of cellular behavior in real time during repair and regeneration of damaged nervous systems. PMID:25263121

  13. Emergence of zebrafish models in oncology for validating novel anticancer drug targets and nanomaterials

    PubMed Central

    Mimeault, Murielle; Batra, Surinder K.

    2013-01-01

    The in vivo zebrafish models have recently attracted great attention in molecular oncology to investigate multiple genetic alterations associated with the development of human cancers and validate novel anticancer drug targets. Particularly, the transparent zebrafish models can be used as a xenotransplantation system to rapidly assess the tumorigenicity and metastatic behavior of cancer stem and/or progenitor cells and their progenies. Moreover, the zebrafish models have emerged as powerful tools for an in vivo testing of novel anticancer agents and nanomaterials for counteracting tumor formation and metastases and improving the efficacy of current radiation and chemotherapeutic treatments against aggressive, metastatic and lethal cancers. PMID:22903142

  14. Zebrafish stripes as a model for vertebrate colour pattern formation.

    PubMed

    Singh, Ajeet Pratap; Nüsslein-Volhard, Christiane

    2015-01-19

    Colour patterns are prominent features of many animals and have important functions in communication, such as camouflage, kin recognition and mate choice. As targets for natural as well as sexual selection, they are of high evolutionary significance. The molecular mechanisms underlying colour pattern formation in vertebrates are not well understood. Progress in transgenic tools, in vivo imaging and the availability of a large collection of mutants make the zebrafish (Danio rerio) an attractive model to study vertebrate colouration. Zebrafish display golden and blue horizontal stripes that form during metamorphosis as mosaics of yellow xanthophores, silvery or blue iridophores and black melanophores in the hypodermis. Lineage tracing revealed the origin of the adult pigment cells and their individual cellular behaviours during the formation of the striped pattern. Mutant analysis indicated that interactions between all three pigment cell types are required for the formation of the pattern, and a number of cell surface molecules and signalling systems have been identified as mediators of these interactions. The understanding of the mechanisms that underlie colour pattern formation is an important step towards deciphering the genetic basis of variation in evolution. PMID:25602311

  15. A Renewable Tissue Resource of Phenotypically Stable, Biologically and Ethnically Diverse, Patient-derived Human Breast Cancer Xenograft (PDX) Models

    PubMed Central

    Zhang, Xiaomei; Claerhout, Sofie; Pratt, Aleix; Dobrolecki, Lacey E.; Petrovic, Ivana; Lai, Qing; Landis, Melissa D.; Wiechmann, Lisa; Schiff, Rachel; Giuliano, Mario; Wong, Helen; Fuqua, Suzanne W.; Contreras, Alejandro; Gutierrez, Carolina; Huang, Jian; Mao, Sufeng; Pavlick, Anne C.; Froehlich, Amber M.; Wu, Meng-Fen; Tsimelzon, Anna; Hilsenbeck, Susan G.; Chen, Edward S.; Zuloaga, Pavel; Shaw, Chad A.; Rimawi, Mothaffar F.; Perou, Charles M.; Mills, Gordon B.; Chang, Jenny C.; Lewis, Michael T.

    2013-01-01

    Breast cancer research is hampered by difficulties in obtaining and studying primary human breast tissue, and by the lack of in vivo preclinical models that reflect patient tumor biology accurately. To overcome these limitations, we propagated a cohort of human breast tumors grown in the epithelium-free mammary fat pad of SCID/Beige and NOD/SCID/IL2?-receptor null (NSG) mice, under a series of transplant conditions. Both models yielded stably transplantable xenografts at comparably high rates (~21% and ~19%, respectively). Of the conditions tested, xenograft take rate was highest in the presence of a low-dose estradiol pellet. Overall, 32 stably transplantable xenograft lines were established, representing 25 unique patients. Most tumors yielding xenografts were “triple-negative” (ER-PR-HER2+) (n=19). However, we established lines from three ER-PR-HER2+ tumors, one ER+PR-HER2?, one ER+PR+HER2? and one “triple-positive” (ER+PR+HER2+) tumor. Serially passaged xenografts show biological consistency with the tumor of origin, are phenotypically stable across multiple transplant generations at the histologic, transcriptomic, proteomic, and genomic levels, and show comparable treatment responses as those observed clinically. Xenografts representing 12 patients, including two ER+ lines, showed metastasis to the mouse lung. These models thus serve as a renewable, quality-controlled tissue resource for preclinical studies investigating treatment response and metastasis. PMID:23737486

  16. Hierarchical Models for Tumor Xenograft Experiments in Drug Development

    Microsoft Academic Search

    Hong-Bin Fang; Guo-Liang Tian; Ming Tan

    2004-01-01

    In cancer drug development, demonstrated anticancer activity in animal models is an important step to bring a promising compound to clinic. Proper design and analysis of experiments using laboratory animals have received increasing attention recently. These experiments involve informatively censored longitudinal data with small samples. The problem is further complicated because of order constraints due to the intrinsic growth of

  17. Quaternary and tertiary aldoxime antidotes for organophosphate exposure in a zebrafish model system.

    PubMed

    Schmidt, Hayden R; Radi?, Zoran; Taylor, Palmer; Fradinger, Erica A

    2015-04-15

    The zebrafish is rapidly becoming an important model system for screening of new therapeutics. Here we evaluated the zebrafish as a potential pharmacological model for screening novel oxime antidotes to organophosphate (OP)-inhibited acetylcholinesterase (AChE). The ki values determined for chlorpyrifos oxon (CPO) and dichlorvos (DDVP) showed that CPO was a more potent inhibitor of both human and zebrafish AChE, but overall zebrafish AChE was less sensitive to OP inhibition. In contrast, aldoxime antidotes, the quaternary ammonium 2-PAM and tertiary amine RS-194B, showed generally similar overall reactivation kinetics, kr, in both zebrafish and human AChE. However, differences between the Kox and k2 constants suggest that zebrafish AChE associates more tightly with oximes, but has a slower maximal reactivation rate than human AChE. Homology modeling suggests that these kinetic differences result from divergences in the amino acids lining the entrance to the active site gorge. Although 2-PAM had the more favorable in vitro reactivation kinetics, RS-194B was more effective antidote in vivo. In intact zebrafish embryos, antidotal treatment with RS-194B rescued embryos from OP toxicity, whereas 2-PAM had no effect. Dechorionation of the embryos prior to antidotal treatment allowed both 2-PAM and RS-194B to rescue zebrafish embryos from OP toxicity. Interestingly, RS-194B and 2-PAM alone increased cholinergic motor activity in dechorionated embryos possibly due to the reversible inhibition kinetics, Ki and ?Ki, of the oximes. Together these results demonstrate that the zebrafish at various developmental stages provides an excellent model for investigating membrane penetrant antidotes to OP exposure. PMID:25701203

  18. Development and rescue of human familial hypercholesterolaemia in a xenograft mouse model.

    PubMed

    Bissig-Choisat, Beatrice; Wang, Lili; Legras, Xavier; Saha, Pradip K; Chen, Leon; Bell, Peter; Pankowicz, Francis P; Hill, Matthew C; Barzi, Mercedes; Leyton, Claudia Kettlun; Leung, Hon-Chiu Eastwood; Kruse, Robert L; Himes, Ryan W; Goss, John A; Wilson, James M; Chan, Lawrence; Lagor, William R; Bissig, Karl-Dimiter

    2015-01-01

    Diseases of lipid metabolism are a major cause of human morbidity, but no animal model entirely recapitulates human lipoprotein metabolism. Here we develop a xenograft mouse model using hepatocytes from a patient with familial hypercholesterolaemia caused by loss-of-function mutations in the low-density lipoprotein receptor (LDLR). Like familial hypercholesterolaemia patients, our familial hypercholesterolaemia liver chimeric mice develop hypercholesterolaemia and a 'humanized' serum profile, including expression of the emerging drug targets cholesteryl ester transfer protein and apolipoprotein (a), for which no genes exist in mice. We go on to replace the missing LDLR in familial hypercholesterolaemia liver chimeric mice using an adeno-associated virus 9-based gene therapy and restore normal lipoprotein profiles after administration of a single dose. Our study marks the first time a human metabolic disease is induced in an experimental animal model by human hepatocyte transplantation and treated by gene therapy. Such xenograft platforms offer the ability to validate human experimental therapies and may foster their rapid translation into the clinic. PMID:26081744

  19. Developmental Exposure to Estrogen Alters Differentiation and Epigenetic Programming in a Human Fetal Prostate Xenograft Model

    PubMed Central

    Saffarini, Camelia M.; McDonnell-Clark, Elizabeth V.; Amin, Ali; Huse, Susan M.; Boekelheide, Kim

    2015-01-01

    Prostate cancer is the most frequent non-cutaneous malignancy in men. There is strong evidence in rodents that neonatal estrogen exposure plays a role in the development of this disease. However, there is little information regarding the effects of estrogen in human fetal prostate tissue. This study explored early life estrogen exposure, with and without a secondary estrogen and testosterone treatment in a human fetal prostate xenograft model. Histopathological lesions, proliferation, and serum hormone levels were evaluated at 7, 30, 90, and 200-day time-points after xenografting. The expression of 40 key genes involved in prostatic glandular and stromal growth, cell-cycle progression, apoptosis, hormone receptors and tumor suppressors was evaluated using a custom PCR array. Epigenome-wide analysis of DNA methylation was performed on whole tissue, and laser capture-microdissection (LCM) isolated epithelial and stromal compartments of 200-day prostate xenografts. Combined initial plus secondary estrogenic exposures had the most severe tissue changes as revealed by the presence of hyperplastic glands at day 200. Gene expression changes corresponded with the cellular events in the KEGG prostate cancer pathway, indicating that initial plus secondary exposure to estrogen altered the PI3K-Akt signaling pathway, ultimately resulting in apoptosis inhibition and an increase in cell cycle progression. DNA methylation revealed that differentially methylated CpG sites significantly predominate in the stromal compartment as a result of estrogen-treatment, thereby providing new targets for future investigation. By using human fetal prostate tissue and eliminating the need for species extrapolation, this study provides novel insights into the gene expression and epigenetic effects related to prostate carcinogenesis following early life estrogen exposure. PMID:25799167

  20. Developmental exposure to estrogen alters differentiation and epigenetic programming in a human fetal prostate xenograft model.

    PubMed

    Saffarini, Camelia M; McDonnell-Clark, Elizabeth V; Amin, Ali; Huse, Susan M; Boekelheide, Kim

    2015-01-01

    Prostate cancer is the most frequent non-cutaneous malignancy in men. There is strong evidence in rodents that neonatal estrogen exposure plays a role in the development of this disease. However, there is little information regarding the effects of estrogen in human fetal prostate tissue. This study explored early life estrogen exposure, with and without a secondary estrogen and testosterone treatment in a human fetal prostate xenograft model. Histopathological lesions, proliferation, and serum hormone levels were evaluated at 7, 30, 90, and 200-day time-points after xenografting. The expression of 40 key genes involved in prostatic glandular and stromal growth, cell-cycle progression, apoptosis, hormone receptors and tumor suppressors was evaluated using a custom PCR array. Epigenome-wide analysis of DNA methylation was performed on whole tissue, and laser capture-microdissection (LCM) isolated epithelial and stromal compartments of 200-day prostate xenografts. Combined initial plus secondary estrogenic exposures had the most severe tissue changes as revealed by the presence of hyperplastic glands at day 200. Gene expression changes corresponded with the cellular events in the KEGG prostate cancer pathway, indicating that initial plus secondary exposure to estrogen altered the PI3K-Akt signaling pathway, ultimately resulting in apoptosis inhibition and an increase in cell cycle progression. DNA methylation revealed that differentially methylated CpG sites significantly predominate in the stromal compartment as a result of estrogen-treatment, thereby providing new targets for future investigation. By using human fetal prostate tissue and eliminating the need for species extrapolation, this study provides novel insights into the gene expression and epigenetic effects related to prostate carcinogenesis following early life estrogen exposure. PMID:25799167

  1. Large-Scale Assessment of the Zebrafish Embryo as a Possible Predictive Model in Toxicity Testing

    Microsoft Academic Search

    Shaukat Ali; Harald G. J. Van Mil; Michael K. Richardson; Ferenc Mueller

    2011-01-01

    BackgroundIn the drug discovery pipeline, safety pharmacology is a major issue. The zebrafish has been proposed as a model that can bridge the gap in this field between cell assays (which are cost-effective, but low in data content) and rodent assays (which are high in data content, but less cost-efficient). However, zebrafish assays are only likely to be useful if

  2. Modeling pegylated liposomal doxorubicin-induced hand-foot syndrome and intestinal mucositis in zebrafish

    PubMed Central

    Chen, Yau-Hung; Lee, Ya-Ting; Wen, Chi-Chung; Chen, Yun-Chen; Chen, Yu-Jen

    2014-01-01

    Pegylated liposomal doxorubicin (PLD) has been widely used to treat cancer. The adverse effects of PLD noted in clinical practice, especially hand-foot syndrome (HFS), are regarded as unique, and the management methods for them remain limited. This study was aimed at developing a feasible experimental model for translational medicine to solve this clinical issue by using skin fluorescent transgenic zebrafish. We established an optimal protocol for the administration of Lipo-Dox™, a PLD in current clinical use, to the Tg(k18:dsred) zebrafish line expressing red fluorescence in keratinocytes. We made use of bodyweight, survival rate, gross observation, flssuorescent microscopic assessment, and pathological examination of the zebrafish to assess this model. The consecutive administration protocol of PLD resulted in growth retardation of the zebrafish embryo and survival impairment, indicating establishment of a significant toxicity. We observed fin necrosis and keratinocyte dissociation phenotypes in the PLD-treated fish after consecutive administration. The skin toxicity induced by the Lipo-Dox injection was subsequently reversible, which might be compatible with a clinical course of skin recovery after discontinuation of Lipo-Dox administration. Furthermore, we found that the number of intestinal goblet cells, an important marker of intestinal inflammation, in the Lipo-Dox-injected zebrafish was markedly increased, accompanied by impaired mucosal integrity. The intestinal inflammation induced by Lipo-Dox resembled the intestinal mucositis the clinical patients suffered from after the administration of PLD. In conclusion, we established a zebrafish model for PLD-induced HFS. The intestinal mucositis simultaneously noted in the PLD-treated zebrafish validated the similarity of clinical courses after administration of PLD. This model is easily assessable, efficient, and worthy for use in developing a new therapeutic protocol for prevention or treatment of HFS as well as intestinal mucositis. Further clinical investigations to validate the correlation between human and zebrafish data are warranted. PMID:25061318

  3. Small-sample inference for incomplete longitudinal data with truncation and censoring in tumor xenograft models.

    PubMed

    Tan, Ming; Fang, Hong-Bin; Tian, Guo-Liang; Houghton, Peter J

    2002-09-01

    In cancer drug development, demonstrating activity in xenograft models, where mice are grafted with human cancer cells, is an important step in bringing a promising compound to humans. A key outcome variable is the tumor volume measured in a given period of time for groups of mice given different doses of a single or combination anticancer regimen. However, a mouse may die before the end of a study or may be sacrificed when its tumor volume quadruples, and its tumor may be suppressed for some time and then grow back. Thus, incomplete repeated measurements arise. The incompleteness or missingness is also caused by drastic tumor shrinkage (<0.01 cm3) or random truncation. Because of the small sample sizes in these models, asymptotic inferences are usually not appropriate. We propose two parametric test procedures based on the EM algorithm and the Bayesian method to compare treatment effects among different groups while accounting for informative censoring. A real xenograft study on a new antitumor agent, temozolomide, combined with irinotecan is analyzed using the proposed methods. PMID:12229996

  4. Microbial fingerprinting detects intestinal microbiota dysbiosis in Zebrafish models with chemically-induced enterocolitis

    PubMed Central

    2013-01-01

    Background Inflammatory bowel disease (IBD) involves a breakdown in interactions between the host immune response and the resident commensal microbiota. Recent studies have suggested gut physiology and pathology relevant to human IBD can be rapidly modeled in zebrafish larvae. The aim of this study was to investigate the dysbiosis of intestinal microbiota in zebrafish models with IBD-like enterocolitis using culture-independent techniques. Results IBD-like enterocolitis was induced by exposing larval zebrafish to trinitrobenzenesulfonic acid (TNBS). Pathology was assessed by histology and immunofluorescence. Changes in intestinal microbiota were evaluated by denaturing gradient gel electrophoresis (DGGE) and the predominant bacterial composition was determined with DNA sequencing and BLAST and confirmed by real-time polymerase chain reaction. Larval zebrafish exposed to TNBS displayed intestinal-fold architecture disruption and inflammation reminiscent of human IBD. In this study, we defined a reduced biodiversity of gut bacterial community in TNBS-induced coliitis. The intestinal microbiota dysbiosis in zebrafish larvae with IBD-like colitis was characterized by an increased proportion of Proteobacteria (especially Burkholderia) and a decreased of Firmicutes(Lactobacillus group), which were significantly correlated with enterocolitis severity(Pearson correlation p < 0.01). Conclusions This is the first description of intestinal microbiota dysbiosis in zebrafish IBD-like models, and these changes correlate with TNBS-induced enterocolitis. Prevention or reversal of this dysbiosis may be a viable option for reducing the incidence and severity of human IBD. PMID:24325678

  5. An adult zebrafish model for Laribacter hongkongensis infection: Koch's postulates fulfilled

    PubMed Central

    Xie, Jun; He, Jia-Bei; Shi, Jia-Wei; Xiao, Qiang; Li, Ling; Woo, Patrick CY

    2014-01-01

    Laribacter hongkongensis is a gram-negative emerging bacterium associated with invasive bacteremic infections in patients with liver disease and fish-borne community-acquired gastroenteritis and traveler's diarrhea. Although the complete genome of L. hongkongensis has been sequenced, no animal model is available for further study of its pathogenicity mechanisms. In this study, we showed that adult zebrafish infected with L. hongkongensis by immersion following dermal abrasion or intraperitoneal injection suffered mortality in a dose-dependent manner, with lethal dose 50 (LD50) of 2.1×104 and 1.9×104?colony-forming units (CFU)/mL, respectively. All mortalities occurred in the first four days post-infection. Zebrafish that died showed characteristic clinicopathological features: swimming near water surface, marked lethargy and sidestroke; abdominal hemorrhage, ulcers and marked swelling with ascites; and hydropic degeneration and necrosis of hepatocytes around central vein and inflammatory cells infiltration. L. hongkongensis was recovered from the ascitic fluid and tissues of zebrafish that died. Of the 30 zebrafish infected with 2.1×104?CFU/mL (LD50) L. hongkongensis isolated from dead zebrafish using the immersion following dermal abrasion method, 18 (60%) died. All zebrafish that died also showed the characteristic clinical and pathological features. Histopathological studies also showed dilation of hepatic central vein and hydropic degeneration. L. hongkongensis was isolated from the zebrafish that died. The Koch's postulates for L. hongkongensis as an infectious agent have been fulfilled. This highly reproducible and effective zebrafish model is of crucial importance for future studies on virulence factors for L. hongkongensis infection. PMID:26038498

  6. Curcumin induces apoptosis and inhibits growth of human Burkitt's lymphoma in xenograft mouse model.

    PubMed

    Li, Zai-xin; Ouyang, Ke-qing; Jiang, Xv; Wang, Dong; Hu, Yinghe

    2009-03-31

    Curcumin, a natural compound extracted from rhizomes of curcuma Curcuma species, has been shown to possess potent anti-inflammatory, anti-tumor and anti-oxidative properties. However, the mechanism of action of the compound remains poorly understood. In this report, we have analyzed the effects of curcumin on the cell proliferation of Burkitt's lymphoma Raji cells. The results demonstrated that curcumin could effectively inhibit the growth of Raji cells in a dose- and time-dependent manner. Further studies indicated that curcumin treatment resulted in apoptosis of cells. Biochemical analysis showed that the expression of Bax, Bid and cytochrome C were up-regulated, while the expression of oncogene c-Myc was down regulated after curcumin treatment. Furthermore, poly (ADP-ribose) polymerase (PARP) cleavage was induced by the compound. Interestingly, the antiapoptotic Bcl-2 expression was not significantly changed in Raji cells after curcumin treatment. These results suggested that the mechanism of action of curcumin was to induce mitochondrial damage and therefore led to Raji cell apoptosis. We further investigated the in vivo effects of curcumin on the growth of xenograft tumors in nude mice. The results showed that curcumin could effectively inhibit tumor growth in the xenograft mouse model. The overall results showed that curcumin could suppress the growth of Burkitt's lymphoma cells in both in vitro and in vitro systems. PMID:19326074

  7. Prolonged exposure to acetaminophen reduces testosterone production by the human fetal testis in a xenograft model.

    PubMed

    van den Driesche, Sander; Macdonald, Joni; Anderson, Richard A; Johnston, Zoe C; Chetty, Tarini; Smith, Lee B; McKinnell, Chris; Dean, Afshan; Homer, Natalie Z; Jorgensen, Anne; Camacho-Moll, Maria E; Sharpe, Richard M; Mitchell, Rod T

    2015-05-20

    Most common male reproductive disorders are linked to lower testosterone exposure in fetal life, although the factors responsible for suppressing fetal testosterone remain largely unknown. Protracted use of acetaminophen during pregnancy is associated with increased risk of cryptorchidism in sons, but effects on fetal testosterone production have not been demonstrated. We used a validated xenograft model to expose human fetal testes to clinically relevant doses and regimens of acetaminophen. Exposure to a therapeutic dose of acetaminophen for 7 days significantly reduced plasma testosterone (45% reduction; P = 0.025) and seminal vesicle weight (a biomarker of androgen exposure; 18% reduction; P = 0.005) in castrate host mice bearing human fetal testis xenografts, whereas acetaminophen exposure for just 1 day did not alter either parameter. Plasma acetaminophen concentrations (at 1 hour after the final dose) in exposed host mice were substantially below those reported in humans after a therapeutic oral dose. Subsequent in utero exposure studies in rats indicated that the acetaminophen-induced reduction in testosterone likely results from reduced expression of key steroidogenic enzymes (Cyp11a1, Cyp17a1). Our results suggest that protracted use of acetaminophen (1 week) may suppress fetal testosterone production, which could have adverse consequences. Further studies are required to establish the dose-response and treatment-duration relationships to delineate the maximum dose and treatment period without this adverse effect. PMID:25995226

  8. Predictive Markers of Efficacy for an Angiopoietin-2 Targeting Therapeutic in Xenograft Models

    PubMed Central

    Triana-Baltzer, Gallen; Pavlicek, Adam; Goulart, Ariadne; Huang, Hanhua; Pirie-Shepherd, Steven; Levin, Nancy

    2013-01-01

    The clinical efficacy of anti-angiogenic therapies has been difficult to predict, and biomarkers that can predict responsiveness are sorely needed in this era of personalized medicine. CVX-060 is an angiopoietin-2 (Ang2) targeting therapeutic, consisting of two peptides that bind Ang2 with high affinity and specificity, covalently fused to a scaffold antibody. In order to optimize the use of this compound in the clinic the construction of a predictive model is described, based on the efficacy of CVX-060 in 13 cell line and 2 patient-derived xenograft models. Pretreatment size tumors from each of the models were profiled for the levels of 27 protein markers of angiogenesis, SNP haplotype in 5 angiogenesis genes, and somatic mutation status for 11 genes implicated in tumor growth and/or vascularization. CVX-060 efficacy was determined as tumor growth inhibition (TGI%) at termination of each study. A predictive statistical model was constructed based on the correlation of these efficacy data with the marker profiles, and the model was subsequently tested by prospective analysis in 11 additional models. The results reveal a range of CVX-060 efficacy in xenograft models of diverse tissue types (0-64% TGI, median = 27%) and define a subset of 3 proteins (Ang1, EGF, Emmprin), the levels of which may be predictive of TGI by Ang2 blockade. The direction of the associations is such that better efficacy correlates with high levels of target and low levels of compensatory/antagonizing molecules. This effort has revealed a set of candidate predictive markers for CVX-060 efficacy that will be further evaluated in ongoing clinical trials. PMID:24244628

  9. Dietary protein restriction inhibits tumor growth in human xenograft models of prostate and breast cancer

    PubMed Central

    Rastelli, Antonella L.; Miles, Kiersten Marie; Ciamporcero, Eric; Longo, Valter D.; Nguyen, Holly; Vessella, Robert; Pili, Roberto

    2013-01-01

    Purpose: Data from epidemiological and experimental studies suggest that dietary protein intake may play a role in inhibiting prostate and breast cancer by modulating the IGF/AKT/mTOR pathway. In this study we investigated the effects of diets with different protein content or quality on prostate and breast cancer. Experimental Design: To test our hypothesis we assessed the inhibitory effect of protein diet restriction on prostate and breast cancer growth, serum PSA and IGF-1 concentrations, mTOR activity and epigenetic markers, by using human xenograft cancer models. Results: Our results showed a 70% inhibition of tumor growth in the castrate-resistant LuCaP23.1 prostate cancer model and a 56% inhibition in the WHIM16 breast cancer model fed with a 7% protein diet when compared to an isocaloric 21% protein diet. Inhibition of tumor growth correlated, in the LuCaP23.1 model, with decreased serum PSA and IGF-1 levels, down-regulation of mTORC1 activity, decreased cell proliferation as indicated by Ki67 staining, and reduction in epigenetic markers of prostate cancer progression, including the histone methyltransferase EZH2 and the associated histone mark H3K27me3. In addition, we observed that modifications of dietary protein quality, independently of protein quantity, decreased tumor growth. A diet containing 20% plant protein inhibited tumor weight by 37% as compared to a 20% animal dairy protein diet. Conclusions: Our findings suggest that a reduction in dietary protein intake is highly effective in inhibiting tumor growth in human xenograft prostate and breast cancer models, possibly through the inhibition of the IGF/AKT/mTOR pathway and epigenetic modifications. PMID:24353195

  10. A Novel Ovarian Xenografting Model to Characterize the Impact of Chemotherapy Agents on Human Primordial Follicle Reserve

    Microsoft Academic Search

    Ozgur Oktem; Kutluk Oktay

    2007-01-01

    Many chemotherapeutic agents, especially of the alkylating family, alter fertility in premenopausal females. However, it is not practically possible to quantify and characterize the impact of cancer drugs on ovarian reserve in a clinical setting. Thus, our specific aim was to develop a xenograft model to characterize the in vivo impact of chemotherapy agents on human ovary. Ovarian pieces from

  11. Inflammation precedes the development of human malignant mesotheliomas in a SCID mouse xenograft model

    PubMed Central

    Hillegass, Jedd M.; Shukla, Arti; Lathrop, Sherrill A.; MacPherson, Maximilian B.; Beuschel, Stacie L.; Butnor, Kelly J.; Testa, Joseph R.; Pass, Harvey I.; Carbone, Michele; Steele, Chad; Mossman, Brooke T.

    2010-01-01

    Asbestos fibers cause chronic inflammation that may be critical to the development of malignant mesothelioma (MM). Two human MM cell lines (Hmeso, PPM Mill) were used in a SCID mouse xenograft model to assess time-dependent patterns of inflammation and tumor formation. After intraperitoneal (IP) injection of MM cells, mice were euthanized at 7, 14, and 30 days, and peritoneal lavage fluid (PLF) was examined for immune cell profiles and human and mouse cytokines. Increases in human MM-derived IL-6, IL-8, bFGF, and VEGF were observed in mice at 7 days postinjection of either MM line, and a striking neutrophilia was observed at all time points. Free-floating tumor spheroids developed in mice at 14 days, and both spheroids and adherent MM tumor masses occurred in all mice at 30 days. Results suggest that inflammation and cytokine production precede and may be critical to the development of MMs. PMID:20716277

  12. Calculated and TLD-based absorbed dose estimates for I-131-labeled 3F8 monoclonal antibody in a human neuroblastoma xenograft nude mouse model

    Microsoft Academic Search

    Omer Ugur; Andrew M. Scott; Lale Kostakoglu; T. Edmond Hui; Mary E. Masterson; Robert Febo; George Sgouros; Eddie Rosa; Bipin M. Mehta; Darrell R. Fisher; Nai-Kong V. Cheung; Steven M. Larson

    1995-01-01

    Preclinical evaluation of the therapeutic potential of radiolabeled antibodies is commonly performed in a xenografted nude mouse model. To assess therapeutic efficacy it is important to estimate the absorbed dose to the tumor and normal tissues of the nude mouse. The current study was designed to accurately measure radiation does to human neuroblastoma xenografts and normal organs in nude mice

  13. CysLT1R Antagonists Inhibit Tumor Growth in a Xenograft Model of Colon Cancer

    PubMed Central

    Savari, Sayeh; Liu, Minghui; Zhang, Yuan; Sime, Wondossen; Sjölander, Anita

    2013-01-01

    The expression of the inflammatory G-protein coupled receptor CysLT1R has been shown to be upregulated in colon cancer patients and associated with poor prognosis. The present study investigated the correlation between CysLT1R and colon cancer development in vivo using CysLT1R antagonists (ZM198,615 or Montelukast) and the nude mouse xenograft model. Two drug administration regimens were established. The first regimen was established to investigate the importance of CysLT1R in tumor initiation. Nude mice were inoculated with 50 µM CysLT1R antagonist-pretreated HCT-116 colon cancer cells and received continued treatment (5 mg/kg/day, intraperitoneally). The second regimen aimed to address the role of CysLT1R in tumor progression. Nude mice were inoculated with non-pretreated HCT-116 cells and did not receive CysLT1R antagonist treatment until recordable tumor appearance. Both regimens resulted in significantly reduced tumor size, attributed to changes in proliferation and apoptosis as determined by reduced Ki-67 levels and increased levels of p21WAF/Cip1 (P<0.01), cleaved caspase 3, and the caspase-cleaved product of cytokeratin 18. Decreased levels of VEGF (P<0.01) and reduced vessel size (P<0.05) were also observed, the latter only in the ZM198,615-pretreatment group. Furthermore, we performed a series of in vitro studies using the colon cancer cell line HCT-116 and CysLT1R antagonists. In addition to significant reductions in cell proliferation, adhesion and colony formation, we observed induction of cell cycle arrest and apoptosis in a dose-dependent manner. The ability of Montelukast to inhibit growth of human colon cancer xenograft was further validated by using two additional colon cancer cell lines, SW-480 and HT-29. Our results demonstrate that CysLT1R antagonists inhibit growth of colon cancer xenografts primarily by reducing proliferation and inducing apoptosis of the tumor cells. PMID:24039952

  14. Treatment of malignant effusion by oncolytic virotherapy in an experimental subcutaneous xenograft model of lung cancer

    PubMed Central

    2013-01-01

    Background Malignant pleural effusion (MPE) is associated with advanced stages of lung cancer and is mainly dependent on invasion of the pleura and expression of vascular endothelial growth factor (VEGF) by cancer cells. As MPE indicates an incurable disease with limited palliative treatment options and poor outcome, there is an urgent need for new and efficient treatment options. Methods In this study, we used subcutaneously generated PC14PE6 lung adenocarcinoma xenografts in athymic mice that developed subcutaneous malignant effusions (ME) which mimic pleural effusions of the orthotopic model. Using this approach monitoring of therapeutic intervention was facilitated by direct observation of subcutaneous ME formation without the need of sacrificing mice or special imaging equipment as in case of MPE. Further, we tested oncolytic virotherapy using Vaccinia virus as a novel treatment modality against ME in this subcutaneous PC14PE6 xenograft model of advanced lung adenocarcinoma. Results We demonstrated significant therapeutic efficacy of Vaccinia virus treatment of both advanced lung adenocarcinoma and tumor-associated ME. We attribute the efficacy to the virus-mediated reduction of tumor cell-derived VEGF levels in tumors, decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of the blood vessel-invading tumor cells. Moreover, we showed that the use of oncolytic Vaccinia virus encoding for a single-chain antibody (scAb) against VEGF (GLAF-1) significantly enhanced mono-therapy of oncolytic treatment. Conclusions Here, we demonstrate for the first time that oncolytic virotherapy using tumor-specific Vaccinia virus represents a novel and promising treatment modality for therapy of ME associated with advanced lung cancer. PMID:23635329

  15. Antitumor activity of IHL-305, a novel pegylated liposome containing irinotecan, in human xenograft models.

    PubMed

    Matsuzaki, Takeshi; Takagi, Akimitsu; Furuta, Tomio; Ueno, Satoshi; Kurita, Akinobu; Nohara, Gou; Kodaira, Hiroshi; Sawada, Seigo; Hashimoto, Shusuke

    2012-01-01

    The antitumor effect of IHL-305, a novel pegylated liposome containing irinotecan, was investigated in human xenograft models. After subcutaneous transplantation of several human cancer cell lines (colorectal, non-small cell lung, small cell lung, prostate, ovarian and gastric cancer cells) to nude mice, IHL-305 or CPT-11 was administered intravenously 3 times at 4-day intervals. In all xenograft models tested, IHL-305 showed superior antitumor activity to that of CPT?11 and a comparable tumor-growth-inhibitory effect at one-eighth or less of the dose of CPT-11, even against HT-29 colorectal and NCI-H460 non-small cell lung cancer cell lines, which show intrinsic resistance to CPT-11. A single injection or 2 injections of IHL-305 on several dosing schedules also resulted in a significant antitumor effect compared to that of vehicle control in a dose-dependent manner and showed comparable antitumor activity at about one-fifth the dose of the maximum tolerated dose of CPT-11. The analysis of the concentrations of irinotecan and SN-38, an active metabolite of CPT-11, in plasma and tumors revealed that irinotecan was maintained at high concentrations, and the prolonged presence of SN-38 in plasma and tumors in IHL-305 treated mice compared with CPT-11-treated mice. Therefore, the stronger tumor inhibitory effect of IHL-305, as compared to CPT-11, was associated with the difference in the concentration of irinotecan in plasma or tumors after each agent was administered and with the maintainance of a higher concentration of SN-38. These results indicate that IHL-305 demonstrated superior antitumor activity against a wide range of tumors at lower doses than CPT-11. PMID:21935577

  16. Host response to xenograft ECM implantation is not different between the shoulder and body wall sites in the rat model

    PubMed Central

    Leigh, Diane R.; Mesiha, Mena; Baker, Andrew R.; Walker, Esteban; Derwin, Kathleen A.

    2012-01-01

    Summary In the context of tendon and ligament repair, mechanical loading and the presence of joint synovial fluid are known to profoundly influence the form and function of the repair tissue and potentially the host response to biomaterials. Previously we demonstrated that a xenograft extra cellular matrix (ECM) scaffold implanted in the rat shoulder elicited a unique host response from that seen in the body wall. However, the host response to xenografts implanted in shoulders with a tendon/capsule injury was not different from xenografts implanted in shoulders with no injury. In the current study, we hypothesized that varying clinically relevant surgical and environmental factors would introduce significant differences in host response to xenograft implantation at the shoulder. Contrary to our hypothesis, we found no significant differences in host response between any shoulder implantation conditions or between shoulder and body wall implantation in the rat model. These findings suggest that there is no advantage to using an orthotopic shoulder model to investigate the host response to rotator cuff scaffold materials in the rat model, and due to the insensitivity of its host response to various clinically relevant surgical conditions, may suggest that the rat does not provide a surrogate for directly translating the host response to biomaterials to the human application. PMID:22618690

  17. Optimization of arterial spin labeling MRI for quantitative tumor perfusion in a mouse xenograft model.

    PubMed

    Rajendran, Reshmi; Liang, Jieming; Tang, Mei Yee Annie; Henry, Brian; Chuang, Kai-Hsiang

    2015-08-01

    Perfusion is an important biomarker of tissue function and has been associated with tumor pathophysiology such as angiogenesis and hypoxia. Arterial spin labeling (ASL) MRI allows noninvasive and quantitative imaging of perfusion; however, the application in mouse xenograft tumor models has been challenging due to the low sensitivity and high perfusion heterogeneity. In this study, flow-sensitive alternating inversion recovery (FAIR) ASL was optimized for a mouse xenograft tumor. To assess the sensitivity and reliability for measuring low perfusion, the lumbar muscle was used as a reference region. By optimizing the number of averages and inversion times, muscle perfusion as low as 32.4 ± 4.8 (mean ± standard deviation) ml/100 g/min could be measured in 20 min at 7 T with a quantification error of 14.4 ± 9.1%. Applying the optimized protocol, heterogeneous perfusion ranging from 49.5 to 211.2 ml/100 g/min in a renal carcinoma was observed. To understand the relationship with tumor pathology, global and regional tumor perfusion was compared with histological staining of blood vessels (CD34), hypoxia (CAIX) and apoptosis (TUNEL). No correlation was observed when the global tumor perfusion was compared with these pathological parameters. Regional analysis shows that areas of high perfusion had low microvessel density, which was due to larger vessel area compared with areas of low perfusion. Nonetheless, these were not correlated with hypoxia or apoptosis. The results suggest that tumor perfusion may reflect certain aspect of angiogenesis, but its relationship with other pathologies needs further investigation. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26104980

  18. Survivin Antisense Oligonucleotides Effectively Radiosensitize Colorectal Cancer Cells in Both Tissue Culture and Murine Xenograft Models

    SciTech Connect

    Roedel, Franz [Department of Radiation Therapy and Oncology, University of Frankfurt/Main, Frankfurt (Germany)], E-mail: franz.roedel@kgu.de; Frey, Benjamin [Department of Radiation Oncology, University of Erlangen, Erlangen (Germany); Leitmann, Werner M.S. [Lilly Deutschland GmbH, Bad Homburg (Germany); Capalbo, Gianni; Weiss, Christian; Roedel, Claus [Department of Radiation Therapy and Oncology, University of Frankfurt/Main, Frankfurt (Germany)

    2008-05-01

    Purpose: Survivin shows a radiation resistance factor in colorectal cancer. In the present study, we determined whether survivin messenger RNA levels in patients with rectal cancer predict tumor response after neoadjuvant radiochemotherapy and whether inhibition of survivin by the use of antisense oligonucleotides (ASOs) enhances radiation responses. Methods and Materials: SW480 colorectal carcinoma cells were transfected with survivin ASO (LY2181308) and irradiated with doses ranging from 0-8 Gy. Survivin expression, cell-cycle distribution, {gamma}H2AX fluorescence, and induction of apoptosis were monitored by means of immunoblotting, flow cytometry, and caspase 3/7 activity. Clonogenic survival was determined by using a colony-forming assay. An SW480 xenograft model was used to investigate the effect of survivin attenuation and irradiation on tumor growth. Furthermore, survivin messenger RNA levels were studied in patient biopsy specimens by using Affymetrix microarray analysis. Results: In the translational study of 20 patients with rectal cancer, increased survivin levels were associated with significantly greater risk of local tumor recurrence (p = 0.009). Treatment of SW480 cells with survivin ASOs and irradiation resulted in an increased percentage of apoptotic cells, caspase 3/7 activity, fraction of cells in the G{sub 2}/M phase, and H2AX phosphorylation. Clonogenic survival decreased compared with control-treated cells. Furthermore, treatment of SW480 xenografts with survivin ASOs and irradiation resulted in a significant delay in tumor growth. Conclusion: Survivin appears to be a molecular biomarker in patients with rectal cancer. Furthermore, in vitro and in vivo data suggest a potential role of survivin as a molecular target to improve treatment response to radiotherapy in patients with rectal cancer.

  19. A Post-Developmental Genetic Screen for Zebrafish Models of Inherited Liver Disease

    PubMed Central

    Kim, Seok-Hyung; Wu, Shu-Yu; Baek, Jeong-In; Choi, Soo Young; Su, Yanhui; Flynn, Charles R.; Gamse, Joshua T.; Ess, Kevin C.; Hardiman, Gary; Lipschutz, Joshua H.; Abumrad, Naji N.; Rockey, Don C.

    2015-01-01

    Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease such as simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis and fibrosis. However, the molecular pathogenesis and genetic variations causing NAFLD are poorly understood. The high prevalence and incidence of NAFLD suggests that genetic variations on a large number of genes might be involved in NAFLD. To identify genetic variants causing inherited liver disease, we used zebrafish as a model system for a large-scale mutant screen, and adopted a whole genome sequencing approach for rapid identification of mutated genes found in our screen. Here, we report on a forward genetic screen of ENU mutagenized zebrafish. From 250 F2 lines of ENU mutagenized zebrafish during post-developmental stages (5 to 8 days post fertilization), we identified 19 unique mutant zebrafish lines displaying visual evidence of hepatomegaly and/or steatosis with no developmental defects. Histological analysis of mutants revealed several specific phenotypes, including common steatosis, micro/macrovesicular steatosis, hepatomegaly, ballooning, and acute hepatocellular necrosis. This work has identified multiple post-developmental mutants and establishes zebrafish as a novel animal model for post-developmental inherited liver disease. PMID:25950913

  20. Rapid adaptation of molecular resources from zebrafish and medaka to develop an estuarine/marine model.

    PubMed

    Chen, Xueping; Li, Li; Wong, Chris Kong Chu; Cheng, Shuk Han

    2009-05-01

    Many estuary and coastal waters are highly threatened by heavy anthropogenic pollutants. Oryzias melastigma, also called O. dancena, a marine medaka that showed sensitive response to hypoxia and estrogenic endocrine disruptors in previous studies, is becoming a sentinel species for marine ecotoxicology studies. However, the lack of strong molecular foundation and knowledge of early developmental stages hampers its practical applications. Combining our research strength on zebrafish embryos, this study revealed both morphological and molecular (at mRNA and protein levels) development of embryos of this emergent model. Whole mount immunostaining technique specific for O. melastigma was successfully developed based on zebrafish standard protocols. We demonstrated that 17 out of 61 primary antibodies, which were previously tested in zebrafish, showed specific immunoreactivity with O. melastigma. These antibodies clearly illustrated the embryonic development of target tissues (principally neurons) in this medaka. Additionally, partial cDNA fragments of 11 organ-specific marker genes were isolated according to genomic resources of zebrafish, Japanese medaka and other fishes. Of the 11 genes, 8 are widely used as organ markers and their expression patterns were remarkably similar to their homologues in zebrafish and Japanese medaka. The expression profiles of the remaining 3 genes in fish are reported for the first time. These molecular markers (17 antibodies and 11 mRNA probes) can be used as responsive indicators in environmental toxicity evaluation. Moreover, this study brought forward and demonstrated the advantage of transferring techniques and resources from one model to another to hasten the research of interest. PMID:19302835

  1. Development of novel combined anticalcification protocols including immunologic modification for prolonged durability of cardiac xenograft: preclinical study using large-animal long-term circulatory models.

    PubMed

    Lim, Hong-Gook; Jeong, Saeromi; Shin, Jun-Seop; Park, Chung-Gyu; Kim, Yong Jin

    2015-01-01

    Cardiac xenografts are conventionally cross-linked with glutaraldehyde (GA) to impart tissue stability, reduce antigenicity, and maintain tissue sterility. However, GA-fixed xenografts are prone to calcification after long-term implantation in humans, because of phospholipids, free aldehyde groups, and residual antigenicity. We evaluated preclinical safety and efficacy using large-animal long-term circulatory models for our novel combined anticalcification protocol including immunological modification, which had been proven effective in small animal experiments. Bovine/porcine xenografts were treated with decellularization, immunological modification with ?-galactosidase, GA fixation with organic solvent, and detoxification with glycine. Valve conduits made of these xenografts were transplanted into the pulmonary root of goats, and hemodynamic, radiological, immunohistopathological, and biochemical results were obtained for 12 months after implantation. Evaluation of echocardiography and cardiac catheterization demonstrated good hemodynamic status and function of the pulmonary xenograft valves. Durability of the xenografts was well preserved without calcification by specimen radiography and immunohistopathological examination. The calcium concentrations of the explanted xenografts were lower than the control xenografts. This preclinical study using large-animal long-term circulatory models demonstrated that our synergistic and simultaneous employment of multiple anticalcification therapies and novel tissue treatments, including immunological modifications, have promising safety and efficacy and should be examined further in future clinical studies. PMID:25303800

  2. Gene knockdown by morpholino-modified oligonucleotides in the zebrafish model: applications for developmental toxicology

    PubMed Central

    Timme-Laragy, Alicia R.; Karchner, Sibel I.; Hahn, Mark E.

    2014-01-01

    Summary The zebrafish (Danio rerio) has long been used as a model for developmental biology, making it an excellent model to use also in developmental toxicology. The many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos. They can be easily manipulated genetically through the use of transgenic technology and gene knock-down via morpholino-modified antisense oligonucleotides (MOs). Knocking down specific genes to assess their role in the response to toxicant exposure provides a way to further our knowledge of how developmental toxicants work on a molecular and mechanistic level, while establishing a relationship between these molecular events and morphological, behavioral, and/or physiological effects (i.e. phenotypic anchoring). In this chapter we address important considerations for using MOs to study developmental toxicology in zebrafish embryos and provide a protocol for their use. PMID:22669659

  3. Establishment, Maintenance and in vitro and in vivo Applications of Primary Human Glioblastoma Multiforme (GBM) Xenograft Models for Translational Biology Studies and Drug Discovery

    PubMed Central

    Carlson, Brett L.; Pokorny, Jenny L.; Schroeder, Mark A.; Sarkaria, Jann N.

    2011-01-01

    Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. One model that has gained wide acceptance in the neuro-oncology community is the primary xenograft model. This model entails the engraftment of patient tumor specimens into the flank of nude mice and subsequent serial passage of these tumors in the flank of mice. These tumors then can be used to establish short-term explant cultures or intracranial xenografts. The focus of this manuscript is to review the procedures associated with the establishment, maintenance and utilization of a primary GBM xenograft panel. PMID:21743824

  4. The State of the Art of the Zebrafish Model for Toxicology and Toxicologic Pathology Research—Advantages and Current Limitations

    PubMed Central

    Spitsbergen, Jan M.; Kent, Michael L.

    2007-01-01

    The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1–2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology. PMID:12597434

  5. Generation and validation of a zebrafish model of EAST (epilepsy, ataxia, sensorineural deafness and tubulopathy) syndrome

    PubMed Central

    Mahmood, Fahad; Mozere, Monika; Zdebik, Anselm A.; Stanescu, Horia C.; Tobin, Jonathan; Beales, Philip L.; Kleta, Robert; Bockenhauer, Detlef; Russell, Claire

    2013-01-01

    SUMMARY Recessive mutations in KCNJ10, which encodes an inwardly rectifying potassium channel, were recently identified as the cause of EAST syndrome, a severe and disabling multi-organ disorder consisting of epilepsy, ataxia, sensorineural deafness and tubulopathy that becomes clinically apparent with seizures in infancy. A Kcnj10 knockout mouse shows postnatal mortality and is therefore not suitable for drug discovery. Because zebrafish are ideal for in vivo screening for potential therapeutics, we tested whether kcnj10 knockdown in zebrafish would fill this need. We cloned zebrafish kcnj10 and demonstrated that its function is equivalent to that of human KCNJ10. We next injected splice- and translation-blocking kcnj10 antisense morpholino oligonucleotides and reproduced the cardinal symptoms of EAST syndrome – ataxia, epilepsy and renal tubular defects. Several of these phenotypes could be assayed in an automated manner. We could rescue the morphant phenotype with complementary RNA (cRNA) encoding human wild-type KCNJ10, but not with cRNA encoding a KCNJ10 mutation identified in individuals with EAST syndrome. Our results suggest that zebrafish will be a valuable tool to screen for compounds that are potentially therapeutic for EAST syndrome or its individual symptoms. Knockdown of kcnj10 represents the first zebrafish model of a salt-losing tubulopathy, which has relevance for blood pressure control. PMID:23471908

  6. Host immune response and acute disease in a zebrafish model of Francisella pathogenesis.

    PubMed

    Vojtech, Lucia N; Sanders, George E; Conway, Carla; Ostland, Vaughn; Hansen, John D

    2009-02-01

    Members of the bacterial genus Francisella are highly virulent and infectious pathogens. New models to study Francisella pathogenesis in evolutionarily distinct species are needed to provide comparative insight, as the mechanisms of host resistance and pathogen virulence are not well understood. We took advantage of the recent discovery of a novel species of Francisella to establish a zebrafish/Francisella comparative model of pathogenesis and host immune response. Adult zebrafish were susceptible to acute Francisella-induced disease and suffered mortality in a dose-dependent manner. Using immunohistochemical analysis, we localized bacterial antigens primarily to lymphoid tissues and livers of zebrafish following infection by intraperitoneal injection, which corresponded to regions of local cellular necrosis. Francisella sp. bacteria replicated rapidly in these tissues beginning 12 h postinfection, and bacterial titers rose steadily, leveled off, and then decreased by 7 days postinfection. Zebrafish mounted a significant tissue-specific proinflammatory response to infection as measured by the upregulation of interleukin-1beta (IL-1beta), gamma interferon, and tumor necrosis factor alpha mRNA beginning by 6 h postinfection and persisting for up to 7 days postinfection. In addition, exposure of zebrafish to heat-killed bacteria demonstrated that the significant induction of IL-1beta was highly specific to live bacteria. Taken together, the pathology and immune response to acute Francisella infection in zebrafish share many features with those in mammals, highlighting the usefulness of this new model system for addressing both general and specific questions about Francisella host-pathogen interactions via an evolutionary approach. PMID:19047404

  7. Gucy2f zebrafish knockdown – a model for Gucy2d-related leber congenital amaurosis

    PubMed Central

    Stiebel-Kalish, Hadas; Reich, Ehud; Rainy, Nir; Vatine, Gad; Nisgav, Yael; Tovar, Anna; Gothilf, Yoav; Bach, Michael

    2012-01-01

    Mutations in retinal-specific guanylate cyclase (Gucy2d) are associated with Leber congenital amaurosis-1 (LCA1). Zebrafish offer unique advantages relative to rodents, including their excellent color vision, precocious retinal development, robust visual testing strategies, low cost, relatively easy transgenesis and shortened experimental times. In this study we will demonstrate the feasibility of using gene-targeting in the zebrafish as a model for the photoreceptor-specific GUCY2D-related LCA1, by reporting the visual phenotype and retinal histology resulting from Gucy2f knockdown. Gucy2f zebrafish LCA-orthologous cDNA was identified and isolated by PCR amplification. Its expression pattern was determined by whole-mount in-situ hybridization and its function was studied by gene knockdown using two different morpholino-modified oligos (MO), one that blocks translation of Gucy2f and one that blocks splicing of Gucy2f. Visual function was assessed with an optomotor assay on 6-days-post-fertilization larvae, and by analyzing changes in retinal histology. Gucy2f knockdown resulted in significantly lower vision as measured by the optomotor response compared with uninjected and control MO-injected zebrafish larvae. Histological changes in the Gucy2f-knockdown larvae included loss and shortening of cone and rod outer segments. A zebrafish model of Gucy2f-related LCA1 displays early visual dysfunction and photoreceptor layer dystrophy. This study serves as proof of concept for the use of zebrafish as a simple, inexpensive model with excellent vision on which further study of LCA-related genes is possible. PMID:22378290

  8. Nilotinib potentiates anticancer drug sensitivity in murine ABCB1-, ABCG2-, and ABCC10-multidrug resistance xenograft models

    PubMed Central

    Tiwari, Amit K.; Sodani, Kamlesh; Dai, Chun-ling; Abuznait, Alaa H.; Singh, Satyakam; Xiao, Zhi-Jie; Patel, Atish; Talele, Tanaji T.; Fu, Liwu; Kaddoumi, Amal; Gallo, James M.; Chen, Zhe-Sheng

    2012-01-01

    A panel of clinically used tyrosine kinase inhibitors was compared and nilotinib was found to most potently sensitize specific anticancer agents by blocking the functions of ABCB1/P-glycoprotein, ABCG2/BCRP and ABCC10/MRP7 transporters involved in multi-drug resistance. Nilotinib appreciably enhanced the antitumor response of 1) paclitaxel in the ABCB1- and novel ABCC10-xenograft models, and 2) doxorubicin in a novel ABCG2-xenograft model. With no apparent toxicity observed in the above models, nilotinib attenuated tumor growth synergistically and increased paclitaxel concentrations in ABCB1-overexpressing tumors. The beneficial actions of nilotinib warrant consideration as viable combinations in the clinic with agents that suffer from MDR-mediated insensitivity. PMID:23063650

  9. Residual Disease in a Novel Xenograft Model of RUNX1-Mutated, Cytogenetically Normal Acute Myeloid Leukemia

    PubMed Central

    Sivagnanalingam, Umayal; Balys, Marlene; Eberhardt, Allison; Wang, Nancy; Myers, Jason R.; Ashton, John M.; Becker, Michael W.; Calvi, Laura M.; Mendler, Jason H.

    2015-01-01

    Cytogenetically normal acute myeloid leukemia (CN-AML) patients harboring RUNX1 mutations have a dismal prognosis with anthracycline/cytarabine-based chemotherapy. We aimed to develop an in vivo model of RUNX1-mutated, CN-AML in which the nature of residual disease in this molecular disease subset could be explored. We utilized a well-characterized patient-derived, RUNX1-mutated CN-AML line (CG-SH). Tail vein injection of CG-SH into NOD scid gamma mice led to leukemic engraftment in the bone marrow, spleen, and peripheral blood within 6 weeks. Treatment of leukemic mice with anthracycline/cytarabine-based chemotherapy resulted in clearance of disease from the spleen and peripheral blood, but persistence of disease in the bone marrow as assessed by flow cytometry and secondary transplantation. Whole exome sequencing of CG-SH revealed mutations in ASXL1, CEBPA, GATA2, and SETBP1, not previously reported. We conclude that CG-SH xenografts are a robust, reproducible in vivo model of CN-AML in which to explore mechanisms of chemotherapy resistance and novel therapeutic approaches. PMID:26177509

  10. Imaging Tumor Variation in Response to Photodynamic Therapy in Pancreatic Cancer Xenograft Models

    SciTech Connect

    Samkoe, Kimberley S., E-mail: samkoe@dartmouth.ed [Thayer School of Engineering, Dartmouth College, Hanover, NH (United States); Chen, Alina [Thayer School of Engineering, Dartmouth College, Hanover, NH (United States); Rizvi, Imran [Thayer School of Engineering, Dartmouth College, Hanover, NH (United States); Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA (United States); O'Hara, Julia A. [Thayer School of Engineering, Dartmouth College, Hanover, NH (United States); Hoopes, P. Jack [Thayer School of Engineering, Dartmouth College, Hanover, NH (United States); Department of Surgery, Dartmouth Medical School, Hanover, NH (United States); Pereira, Stephen P. [Institute of Hepatology, University College London Medical School, London (United Kingdom); Hasan, Tayyaba [Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA (United States); Pogue, Brian W. [Thayer School of Engineering, Dartmouth College, Hanover, NH (United States); Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA (United States); Department of Surgery, Dartmouth Medical School, Hanover, NH (United States)

    2010-01-15

    Purpose: A treatment monitoring study investigated the differential effects of orthotopic pancreatic cancer models in response to interstitial photodynamic therapy (PDT), and the validity of using magnetic resonance imaging as a surrogate measure of response was assessed. Methods and Materials: Different orthotopic pancreatic cancer xenograft models (AsPC-1 and Panc-1) were used to represent the range of pathophysiology observed in human beings. Identical dose escalation studies (10, 20, and 40J/cm) using interstitial verteporfin PDT were performed, and magnetic resonance imaging with T2-weighted and T1-weighted contrast were used to monitor the total tumor volume and the vascular perfusion volume, respectively. Results: There was a significant amount of necrosis in the slower-growing Panc-1 tumor using high light dose, although complete necrosis was not observed. Lower doses were required for the same level of tumor kill in the faster-growing AsPC-1 cell line. Conclusions: The tumor growth rate and vascular pattern of the tumor affect the optimal PDT treatment regimen, with faster-growing tumors being relatively easier to treat. This highlights the fact that therapy in human beings shows a heterogeneous range of outcomes, and suggests a need for careful individualized treatment outcomes assessment in clinical work.

  11. Evaluation of zebrafish as a model to study the pathogenesis of the opportunistic pathogen Cronobacter turicensis

    PubMed Central

    Fehr, Alexander; Eshwar, Athmanya K; Neuhauss, Stephan CF; Ruetten, Maja; Lehner, Angelika; Vaughan, Lloyd

    2015-01-01

    Bacteria belonging to the genus Cronobacter spp. have been recognized as causative agents of life-threatening systemic infections, primarily in premature, low-birth weight and/or immune-compromised neonates. Knowledge remains scarce regarding the underlying molecular mechanisms of disease development. In this study, we evaluated the use of a zebrafish model to study the pathogenesis of Cronobacter turicensis LMG 23827T, a clinical isolate responsible for two fatal sepsis cases in neonates. Here, the microinjection of approximately 50 colony forming units (CFUs) into the yolk sac resulted in the rapid multiplication of bacteria and dissemination into the blood stream at 24 h post infection (hpi), followed by the development of a severe bacteremia and larval death within 3 days. In contrast, the innate immune response of the embryos was sufficiently developed to control infection after the intravenous injection of up to 104 CFUs of bacteria. Infection studies using an isogenic mutant devoid of surviving and replicating in human macrophages (?fkpA) showed that this strain was highly attenuated in its ability to kill the larvae. In addition, the suitability of the zebrafish model system to study the effectiveness of antibiotics to treat Cronobacter infections in zebrafish embryos was examined. Our data indicate that the zebrafish model represents an excellent vertebrate model to study virulence-related aspects of this opportunistic pathogen in vivo. PMID:26060602

  12. Evaluation of zebrafish as a model to study the pathogenesis of the opportunistic pathogen Cronobacter turicensis.

    PubMed

    Fehr, Alexander; Eshwar, Athmanya K; Neuhauss, Stephan Cf; Ruetten, Maja; Lehner, Angelika; Vaughan, Lloyd

    2015-05-01

    Bacteria belonging to the genus Cronobacter spp. have been recognized as causative agents of life-threatening systemic infections, primarily in premature, low-birth weight and/or immune-compromised neonates. Knowledge remains scarce regarding the underlying molecular mechanisms of disease development. In this study, we evaluated the use of a zebrafish model to study the pathogenesis of Cronobacter turicensis LMG 23827(T), a clinical isolate responsible for two fatal sepsis cases in neonates. Here, the microinjection of approximately 50 colony forming units (CFUs) into the yolk sac resulted in the rapid multiplication of bacteria and dissemination into the blood stream at 24 h post infection (hpi), followed by the development of a severe bacteremia and larval death within 3 days. In contrast, the innate immune response of the embryos was sufficiently developed to control infection after the intravenous injection of up to 10(4) CFUs of bacteria. Infection studies using an isogenic mutant devoid of surviving and replicating in human macrophages (?fkpA) showed that this strain was highly attenuated in its ability to kill the larvae. In addition, the suitability of the zebrafish model system to study the effectiveness of antibiotics to treat Cronobacter infections in zebrafish embryos was examined. Our data indicate that the zebrafish model represents an excellent vertebrate model to study virulence-related aspects of this opportunistic pathogen in vivo. PMID:26060602

  13. Monitoring the development of xenograft triple negative breast cancer models using diffusion-weighted magnetic resonance imaging

    PubMed Central

    Stephen, Renu M; Pagel, Mark D; Brown, Kathy; Baker, Amanda F; Meuillet, Emmanuelle J; Gillies, Robert J

    2014-01-01

    Evaluations of tumor growth rates and molecular biomarkers are traditionally used to assess new mouse models of human breast cancers. This study investigated the utility of diffusion weighted (DW)-magnetic resonance imaging (MRI) for evaluating cellular proliferation of new tumor models of triple negative breast cancer, which may augment traditional analysis methods. Eleven human breast cancer cell lines were used to develop xenograft tumors in severe combined immunodeficient mice, with two of these cell lines exhibiting sufficient growth to be serially passaged. DW-MRI was performed to measure the distributions of the apparent diffusion coefficient (ADC) in these two tumor xenograft models, which showed a correlation with tumor growth rates and doubling times during each passage. The distributions of the ADC values were also correlated with expression of Ki67, a biomarker of cell proliferation, and HIF-1? and VEGFR2 that are essential proteins involved in regulating aerobic glycolysis and angiogenesis that support tumor cell proliferation. Although PTEN levels were different between the two xenograft models, AKT levels did not differ nor did they correlate with tumor growth. This last result demonstrates the complexity of signaling protein pathways and the difficulty in interpreting the effects of protein expression on tumor cell proliferation. In contrast, DW-MRI may be a more direct assessment of tumor growth and cancer cell proliferation. PMID:23239438

  14. Inhalation Delivery and Antitumor Activity of Celecoxib in Human Orthotopic Non-Small Cell Lung Cancer Xenograft Model

    Microsoft Academic Search

    Suniket V. Fulzele; Abhijit Chatterjee; Madhu Sudhan Shaik; Tanise Jackson; Mandip Singh

    2006-01-01

    Purpose  To determine the in vivo anti-tumor effect of aerosolized Celecoxib (Cxb) in combination with i.v Docetaxel (Doc) and compare the anti-tumor effect with oral Cxb combined with i.v Doc in human orthotopic non-small cell lung cancer (NSCLC) xenograft model.Materials and Methods  Female Nu\\/Nu mice were implanted with orthotopic tumors by injecting A549 cells into the lung parenchyma. Seven day after tumor

  15. p202, an Interferon-inducible Protein, Mediates Multiple Antitumor Activities in Human Pancreatic Cancer Xenograft Models1

    Microsoft Academic Search

    Yong Wen; Duen-Hwa Yan; Bailiang Wang; Bill Spohn; Yi Ding; Ruping Shao; Yiyu Zou; Keping Xie; Mien-Chie Hung

    p202, an IFN-inducible protein, interacts with certain transcriptional activators leading to transcriptional repression. p202 expression has been associated with inhibition of cancer cell growth in vitro and in vivo .T o examine a potential p202-mediated antitumor activity in pancreatic can- cer, we used both ectopic and orthotopic xenograft models and demon- strated that p202 expression is associated with multiple antitumor

  16. Antitumor Activity of Folate Receptor-Targeted Liposomal Doxorubicin in a KB Oral Carcinoma Murine Xenograft Model

    Microsoft Academic Search

    Xing Q. Pan; Huaqing Wang; Robert J. Lee

    2003-01-01

    Purpose. The expression of folate receptor (FR) is amplified in many types of human cancers. Previously, FR-targeted liposomal doxorubicin (f-L-DOX) has been shown to exhibit superior and selective cytotoxicity against FR(+) tumor cells in vitro compared to nontargeted liposomal doxorubicin (L-DOX). This study further investigates f-L-DOX for its antitumor efficacy in vivo using a murine tumor xenograft model.

  17. Cellular characterization of ultrasound-stimulated microbubble radiation enhancement in a prostate cancer xenograft model

    PubMed Central

    Al-Mahrouki, Azza A.; Iradji, Sara; Tran, William Tyler; Czarnota, Gregory J.

    2014-01-01

    Tumor radiation resistance poses a major obstacle in achieving an optimal outcome in radiation therapy. In the current study, we characterize a novel therapeutic approach that combines ultrasound-driven microbubbles with radiation to increase treatment responses in a prostate cancer xenograft model in mice. Tumor response to ultrasound-driven microbubbles and radiation was assessed 24 hours after treatment, which consisted of radiation treatments alone (2 Gy or 8 Gy) or ultrasound-stimulated microbubbles only, or a combination of radiation and ultrasound-stimulated microbubbles. Immunohistochemical analysis using in situ end labeling (ISEL) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) revealed increased cell death within tumors exposed to combined treatments compared with untreated tumors or tumors exposed to radiation alone. Several biomarkers were investigated to evaluate cell proliferation (Ki67), blood leakage (factor VIII), angiogenesis (cluster of differentiation molecule CD31), ceramide-formation, angiogenesis signaling [vascular endothelial growth factor (VEGF)], oxygen limitation (prolyl hydroxylase PHD2) and DNA damage/repair (?H2AX). Results demonstrated reduced vascularity due to vascular disruption by ultrasound-stimulated microbubbles, increased ceramide production and increased DNA damage of tumor cells, despite decreased tumor oxygenation with significantly less proliferating cells in the combined treatments. This combined approach could be a feasible option as a novel enhancing approach in radiation therapy. PMID:24487407

  18. Cellular characterization of ultrasound-stimulated microbubble radiation enhancement in a prostate cancer xenograft model.

    PubMed

    Al-Mahrouki, Azza A; Iradji, Sara; Tran, William Tyler; Czarnota, Gregory J

    2014-03-01

    Tumor radiation resistance poses a major obstacle in achieving an optimal outcome in radiation therapy. In the current study, we characterize a novel therapeutic approach that combines ultrasound-driven microbubbles with radiation to increase treatment responses in a prostate cancer xenograft model in mice. Tumor response to ultrasound-driven microbubbles and radiation was assessed 24 hours after treatment, which consisted of radiation treatments alone (2 Gy or 8 Gy) or ultrasound-stimulated microbubbles only, or a combination of radiation and ultrasound-stimulated microbubbles. Immunohistochemical analysis using in situ end labeling (ISEL) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) revealed increased cell death within tumors exposed to combined treatments compared with untreated tumors or tumors exposed to radiation alone. Several biomarkers were investigated to evaluate cell proliferation (Ki67), blood leakage (factor VIII), angiogenesis (cluster of differentiation molecule CD31), ceramide-formation, angiogenesis signaling [vascular endothelial growth factor (VEGF)], oxygen limitation (prolyl hydroxylase PHD2) and DNA damage/repair (?H2AX). Results demonstrated reduced vascularity due to vascular disruption by ultrasound-stimulated microbubbles, increased ceramide production and increased DNA damage of tumor cells, despite decreased tumor oxygenation with significantly less proliferating cells in the combined treatments. This combined approach could be a feasible option as a novel enhancing approach in radiation therapy. PMID:24487407

  19. A patient tumor-derived orthotopic xenograft mouse model replicating the group 3 supratentorial primitive neuroectodermal tumor in children

    PubMed Central

    Liu, Zhigang; Zhao, Xiumei; Wang, Yue; Mao, Hua; Huang, Yulun; Kogiso, Mari; Qi, Lin; Baxter, Patricia A.; Man, Tsz-Kwong; Adesina, Adekunle; Su, Jack M.; Picard, Daniel; Ching Ho, King; Huang, Annie; Perlaky, Laszlo; Lau, Ching C.; Chintagumpala, Murali; Li, Xiao-Nan

    2014-01-01

    Background Supratentorial primitive neuroectodermal tumor (sPNET) is a malignant brain tumor with poor prognosis. New model systems that replicate sPNET's molecular subtype(s) and maintain cancer stem cell (CSC) pool are needed. Methods A fresh surgical specimen of a pediatric sPNET was directly injected into the right cerebrum of Rag2/SCID mice. The xenograft tumors were serially sub-transplanted in mouse brains, characterized histopathologically, and subclassified into molecular subtype through qRT-PCR and immunohistochemical analysis. CSCs were identified through flow cytometric profiling of putative CSC markers (CD133, CD15, CD24, CD44, and CD117), functional examination of neurosphere forming efficiency in vitro, and tumor formation capacity in vivo. To establish a neurosphere line, neurospheres were propagated in serum-free medium. Results Formation of intracerebral xenograft tumors was confirmed in 4 of the 5 mice injected with the patient tumor. These xenograft tumors were sub-transplanted in vivo 5 times. They replicated the histopathological features of the original patient tumor and expressed the molecular markers (TWIST1 and FOXJ1) of group 3 sPNET. CD133+ and CD15+ cells were found to have strong neurosphere-forming efficiency in vitro and potent tumor-forming capacity (with as few as 100 cells) in vivo. A neurosphere line BXD-2664PNET-NS was established that preserved stem cell features and expressed group 3 markers. Conclusion We have established a group 3 sPNET xenograft mouse model (IC-2664PNET) with matching neurosphere line (BXD-2664PNET-NS) and identified CD133+ and CD15+ cells as the major CSC subpopulations. This novel model system should facilitate biological studies and preclinical drug screenings for childhood sPNET. PMID:24470556

  20. Tumour bed irradiation of human tumour xenografts in a nude rat model using a common X-ray tube.

    PubMed

    Tokalov, S V; Enghardt, W; Abolmaali, N

    2010-06-01

    Studies that investigate the radiation of human tumour xenografts require an appropriate radiation source and highly standardized conditions during radiation. This work reports on the design of a standardized irradiation device using a commercially available X-ray tube with a custom constructed lead collimator with two circular apertures and an animal bed plate, permitting synchronous irradiation of two animals. Dosimetry and the corresponding methodology for radiotherapy of human non-small cell lung cancer xenograft tumours transplanted to and growing subcutaneously on the right lower limb in a nude rat model were investigated. Procedures and results described herein prove the feasibility of use of the device, which is applicable for any investigation involving irradiation of non-tumorous and tumorous lesions in small animals. PMID:20689176

  1. Zebrafish forebrain and temporal conditioning

    PubMed Central

    Cheng, Ruey-Kuang; Jesuthasan, Suresh J.; Penney, Trevor B.

    2014-01-01

    The rise of zebrafish as a neuroscience research model organism, in conjunction with recent progress in single-cell resolution whole-brain imaging of larval zebrafish, opens a new window of opportunity for research on interval timing. In this article, we review zebrafish neuroanatomy and neuromodulatory systems, with particular focus on identifying homologies between the zebrafish forebrain and the mammalian forebrain. The neuroanatomical and neurochemical basis of interval timing is summarized with emphasis on the potential of using zebrafish to reveal the neural circuits for interval timing. The behavioural repertoire of larval zebrafish is reviewed and we demonstrate that larval zebrafish are capable of expecting a stimulus at a precise time point with minimal training. In conclusion, we propose that interval timing research using zebrafish and whole-brain calcium imaging at single-cell resolution will contribute to our understanding of how timing and time perception originate in the vertebrate brain from the level of single cells to circuits. PMID:24446496

  2. A tissue-engineered humanized xenograft model of human breast cancer metastasis to bone

    PubMed Central

    Thibaudeau, Laure; Taubenberger, Anna V.; Holzapfel, Boris M.; Quent, Verena M.; Fuehrmann, Tobias; Hesami, Parisa; Brown, Toby D.; Dalton, Paul D.; Power, Carl A.; Hollier, Brett G.; Hutmacher, Dietmar W.

    2014-01-01

    ABSTRACT The skeleton is a preferred homing site for breast cancer metastasis. To date, treatment options for patients with bone metastases are mostly palliative and the disease is still incurable. Indeed, key mechanisms involved in breast cancer osteotropism are still only partially understood due to the lack of suitable animal models to mimic metastasis of human tumor cells to a human bone microenvironment. In the presented study, we investigate the use of a human tissue-engineered bone construct to develop a humanized xenograft model of breast cancer-induced bone metastasis in a murine host. Primary human osteoblastic cell-seeded melt electrospun scaffolds in combination with recombinant human bone morphogenetic protein 7 were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. The tissue-engineered constructs led to the formation of a morphologically intact ‘organ’ bone incorporating a high amount of mineralized tissue, live osteocytes and bone marrow spaces. The newly formed bone was largely humanized, as indicated by the incorporation of human bone cells and human-derived matrix proteins. After intracardiac injection, the dissemination of luciferase-expressing human breast cancer cell lines to the humanized bone ossicles was detected by bioluminescent imaging. Histological analysis revealed the presence of metastases with clear osteolysis in the newly formed bone. Thus, human tissue-engineered bone constructs can be applied efficiently as a target tissue for human breast cancer cells injected into the blood circulation and replicate the osteolytic phenotype associated with breast cancer-induced bone lesions. In conclusion, we have developed an appropriate model for investigation of species-specific mechanisms of human breast cancer-related bone metastasis in vivo. PMID:24713276

  3. The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): Implications for the zebrafish as a model organism.

    PubMed

    Spagnoli, Sean; Xue, Lan; Kent, Michael L

    2015-09-15

    The zebrafish's potential as a model for human neurobehavioral research appears nearly limitless despite its relatively recent emergence as an experimental organism. Since the zebrafish has only been part of the research community for a handful of decades, pathogens from its commercial origins continue to plague laboratory stocks. One such pathogen is Pseudoloma neurophilia, a common microparasite in zebrafish laboratories world-wide that generally produces subclinical infections. Given its high prevalence, its predilection for the host's brain and spinal cord, and the delicate nature of neurobehavioral research, the behavioral consequences of subclinical P. neurophilia infection must be explored. Fish infected via cohabitation were tested for startle response habituation in parallel with controls in a device that administered ten taps over 10min along with taps at 18 and 60min to evaluate habituation extinction. After testing, fish were euthanized and evaluated for infection via histopathology. Infected fish had a significantly smaller reduction in startle velocity during habituation compared to uninfected tankmates and controls. Habituation was eliminated in infected and control fish at 18min, whereas exposed negative fish retained partial habituation at 18min. Infection was also associated with enhanced capture evasion: Despite the absence of external symptoms, infected fish tended to be caught later than uninfected fish netted from the same tank. The combination of decreased overall habituation, early extinction of habituation compared to uninfected cohorts, and enhanced netting evasion indicates that P. neurophilia infection is associated with a behavioral phenotype distinct from that of controls and uninfected cohorts. Because of its prevalence in zebrafish facilities, P. neurophilia has the potential to insidiously influence a wide range of neurobehavioral studies if these associations are causative. Rigorous health screening is therefore vital to the improvement of the zebrafish as a translational model for human behavior. PMID:26028515

  4. Adult zebrafish as a model system for cutaneous wound-healing research.

    PubMed

    Richardson, Rebecca; Slanchev, Krasimir; Kraus, Christopher; Knyphausen, Philipp; Eming, Sabine; Hammerschmidt, Matthias

    2013-06-01

    Upon injury, the skin must quickly regenerate to regain its barrier function. In mammals, wound healing is rapid and scar free during embryogenesis, whereas in adults it involves multiple steps including blood clotting, inflammation, re-epithelialization, vascularization, and granulation tissue formation and maturation, resulting in a scar. We have established a rapid and robust method to introduce full-thickness wounds onto the flank of adult zebrafish, and show that apart from external fibrin clot formation, all steps of adult mammalian wound repair also exist in zebrafish. Wound re-epithelialization is extremely rapid and initiates with no apparent lag phase, subsequently followed by the immigration of inflammatory cells and the formation of granulation tissue, consisting of macrophages, fibroblasts, blood vessels, and collagen. The granulation tissue later regresses, resulting in minimal scar formation. Studies after chemical treatment or with transgenic fish further suggest that wound re-epithelialization occurs independently of inflammation and fibroblast growth factor signaling, whereas both are essential for fibroblast recruitment and granulation tissue formation. Together, these results demonstrate that major steps and principles of cutaneous wound healing are conserved among adult mammals and adult zebrafish, making zebrafish a valuable model for studying vertebrate skin repair. PMID:23325040

  5. Combination of Vandetanib, Radiotherapy, and Irinotecan in the LoVo Human Colorectal Cancer Xenograft Model

    SciTech Connect

    Wachsberger, Phyllis, E-mail: Phyllis.wachsberger@jeffersonhospital.or [Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, PA (United States); Burd, Randy [Department of Nutritional Sciences, University of Arizona, Tucson, AZ (United States); Ryan, Anderson [AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield (United Kingdom); Daskalakis, Constantine [Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson University, Philadelphia, PA (United States); Dicker, Adam P. [Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, PA (United States)

    2009-11-01

    Purpose: The tumor growth kinetics of the human LoVo colorectal xenograft model was assessed in response to vandetanib, an orally available receptor tyrosine kinase inhibitor, radiotherapy (RT), or irinotecan (CPT-11), as single therapies and in combination. Methods and Materials: LoVo cells were injected subcutaneously into the right hind limb (5x10{sup 6} cells in 100muL phosphate-buffered saline) of athymic NCR NUM mice and tumors were grown to a volume of 200-300 mm{sup 3} before treatment. Vandetanib was administered at 50 mg/kg daily orally for 14 days starting on Day 1. RT was given as three fractions (3x3 Gy) on Days 1, 2, and 3. CPT-11 was given at 15 mg/kg intraperitoneally on Days 1 and 3. Tumor volumes were measured on a daily basis and calculated by measuring tumor diameters with digital calipers in two orthogonal dimensions. Results: All three single treatments (vandetanib, CPT-11, and radiation) significantly slowed LoVo colorectal tumor growth. Vandetanib significantly increased the antitumor effects of CPT-11 and radiation when given in combination with either of these treatments. These treatment combinations resulted in a slow tumor growth rate during the 2 weeks of vandetanib administration. The triple combination of vandetanib, CPT-11, and radiation produced the most marked improvement in response as observed by measurable shrinkage of tumors during the first week of treatment. Conclusions: The tumor growth delay kinetics observed in this study of the LoVo colorectal model suggest concurrent and sustained post-sequencing of vandetanib with cytotoxic therapy may be beneficial in tumors of this type.

  6. NAD+ Biosynthesis Ameliorates a Zebrafish Model of Muscular Dystrophy

    PubMed Central

    Goody, Michelle F.; Kelly, Meghan W.; Reynolds, Christine J.; Khalil, Andre; Crawford, Bryan D.; Henry, Clarissa A.

    2012-01-01

    Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex– or integrin alpha7–deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction with integrin alpha6 to reduce muscle degeneration. Taken together, these results define a novel cell adhesion pathway that may have future therapeutic relevance for a broad spectrum of muscular dystrophies. PMID:23109907

  7. Danio Rerio, the Zebrafish

    PubMed Central

    2000-01-01

    The zebrafish has long been a favourite model for the study of vertebrate development. Here we provide an overview of the current state of knowledge and resources for the study of this fish, with comments on the future direction of zebrafish genomics from Professor Mark Fishman and Dr Stephen Wilson. PMID:11025533

  8. Endogenous retrovirus induces leukemia in a xenograft mouse model for primary myelofibrosis

    PubMed Central

    Triviai, Ioanna; Ziegler, Marion; Bergholz, Ulla; Oler, Andrew J.; Stübig, Thomas; Prassolov, Vladimir; Fehse, Boris; Kozak, Christine A.; Kröger, Nicolaus; Stocking, Carol

    2014-01-01

    The compound immunodeficiencies in nonobese diabetic (NOD) inbred mice homozygous for the Prkdcscid and Il2rgnull alleles (NSG mice) permit engraftment of a wide-range of primary human cells, enabling sophisticated modeling of human disease. In studies designed to define neoplastic stem cells of primary myelofibrosis (PMF), a myeloproliferative neoplasm characterized by profound disruption of the hematopoietic microenvironment, we observed a high frequency of acute myeloid leukemia (AML) in NSG mice. AML was of mouse origin, confined to PMF-xenografted mice, and contained multiple clonal integrations of ecotropic murine leukemia virus (E-MuLV). Significantly, MuLV replication was not only observed in diseased mice, but also in nontreated NSG controls. Furthermore, in addition to the single ecotropic endogenous retrovirus (eERV) located on chromosome 11 (Emv30) in the NOD genome, multiple de novo germ-line eERV integrations were observed in mice from each of four independent NSG mouse colonies. Analysis confirmed that E-MuLV originated from the Emv30 provirus and that recombination events were not necessary for virus replication or AML induction. Pathogenicity is thus likely attributable to PMF-mediated paracrine stimulation of mouse myeloid cells, which serve as targets for retroviral infection and transformation, as evidenced by integration into the Evi1 locus, a hotspot for retroviral-induced myeloid leukemia. This study thus corroborates a role of paracrine stimulation in PMF disease progression, underlines the importance of target cell type and numbers in MuLV-induced disease, and mandates awareness of replicating MuLV in NOD immunodeficient mice, which can significantly influence experimental results and their interpretation. PMID:24912157

  9. Endogenous retrovirus induces leukemia in a xenograft mouse model for primary myelofibrosis.

    PubMed

    Triviai, Ioanna; Ziegler, Marion; Bergholz, Ulla; Oler, Andrew J; Stübig, Thomas; Prassolov, Vladimir; Fehse, Boris; Kozak, Christine A; Kröger, Nicolaus; Stocking, Carol

    2014-06-10

    The compound immunodeficiencies in nonobese diabetic (NOD) inbred mice homozygous for the Prkdc(scid) and Il2rg(null) alleles (NSG mice) permit engraftment of a wide-range of primary human cells, enabling sophisticated modeling of human disease. In studies designed to define neoplastic stem cells of primary myelofibrosis (PMF), a myeloproliferative neoplasm characterized by profound disruption of the hematopoietic microenvironment, we observed a high frequency of acute myeloid leukemia (AML) in NSG mice. AML was of mouse origin, confined to PMF-xenografted mice, and contained multiple clonal integrations of ecotropic murine leukemia virus (E-MuLV). Significantly, MuLV replication was not only observed in diseased mice, but also in nontreated NSG controls. Furthermore, in addition to the single ecotropic endogenous retrovirus (eERV) located on chromosome 11 (Emv30) in the NOD genome, multiple de novo germ-line eERV integrations were observed in mice from each of four independent NSG mouse colonies. Analysis confirmed that E-MuLV originated from the Emv30 provirus and that recombination events were not necessary for virus replication or AML induction. Pathogenicity is thus likely attributable to PMF-mediated paracrine stimulation of mouse myeloid cells, which serve as targets for retroviral infection and transformation, as evidenced by integration into the Evi1 locus, a hotspot for retroviral-induced myeloid leukemia. This study thus corroborates a role of paracrine stimulation in PMF disease progression, underlines the importance of target cell type and numbers in MuLV-induced disease, and mandates awareness of replicating MuLV in NOD immunodeficient mice, which can significantly influence experimental results and their interpretation. PMID:24912157

  10. Mitotane effects in a H295R xenograft model of adjuvant treatment of adrenocortical cancer.

    PubMed

    Lindhe, O; Skogseid, B

    2010-09-01

    Adrenocortical cancer is one of the most aggressive endocrine malignancies. Growth through the capsule or accidental release of cancer cells during surgery frequently results in metastatic disease. We investigated the antitumoral effect of 2 adrenocorticolytic compounds, O, P'-DDD and MeSO2-DDE, in the adrenocortical cell line H295R both in vitro and as a xenograft model in vivo. H295R cells were injected s. c. in nude mice. O, P'-DDD, MeSO2-DDE, or oil (control) was administered i. p., either simultaneously with cell injection at day 0 (mimicking adjuvant treatment), or at day 48 (established tumors). Accumulation of PET tracers [ (11)C]methionine (MET), [ (11)C] metomidate (MTO), 2-deoxy-2-[ (18)F]fluoro-d-glucose (FDG), and [ (18)F]-l-tyrosine (FLT) in the aggregates were assessed +/- drug treatment in vitro. Tumor growth was significantly inhibited when O, P'-DDD was given at the same time as injection of tumor cells. No significant growth inhibition was observed after treatment with O, P'-DDD at day 48. A significant reduction in FLT uptake and an increased FDG uptake, compared to control, were observed following treatment with 15 microM O, P'-DDD (p<0.01) in vitro. MeSO2-DDE (15 microM) treatment gave rise to a reduced MET and an increased FLT uptake (p<0.01). Both compounds reduced the uptake of MTO compared to control (p<0.01). Treatment with O, P'-DDD simultaneously to inoculation of H295R cells in mice, imitating release of cells during surgery, gave a markedly better effect than treatment of established H295R tumors. We suggest that FLT may be a potential PET biomarker when assessing adrenocortical cancer treatment with O,P'-DDD. Further studies in humans are needed to investigate this. PMID:20665429

  11. Appropriateness of Using Patient-Derived Xenograft Models for Pharmacologic Evaluation of Novel Therapies for Esophageal/Gastro-Esophageal Junction Cancers

    PubMed Central

    Dodbiba, Lorin; Teichman, Jennifer; Fleet, Andrew; Thai, Henry; Starmans, Maud H. W.; Navab, Roya; Chen, Zhuo; Girgis, Hala; Eng, Lawson; Espin-Garcia, Osvaldo; Shen, Xiaowei; Bandarchi, Bizhan; Schwock, Joerg; Tsao, Ming-Sound; El-Zimaity, Hala; Der, Sandy D.; Xu, Wei; Bristow, Robert G.; Darling, Gail E.; Boutros, Paul C.

    2015-01-01

    The high morbidity and mortality of patients with esophageal (E) and gastro-esophageal junction (GEJ) cancers, warrants new pre-clinical models for drug testing. The utility of primary tumor xenografts (PTXGs) as pre-clinical models was assessed. Clinicopathological, immunohistochemical markers (p53, p16, Ki-67, Her-2/neu and EGFR), and global mRNA abundance profiles were evaluated to determine selection biases of samples implanted or engrafted, compared with the underlying population. Nine primary E/GEJ adenocarcinoma xenograft lines were further characterized for the spectrum and stability of gene/protein expression over passages. Seven primary esophageal adenocarcinoma xenograft lines were treated with individual or combination chemotherapy. Tumors that were implanted (n=55) in NOD/SCID mice had features suggestive of more aggressive biology than tumors that were never implanted (n=32). Of those implanted, 21/55 engrafted; engraftment was associated with poorly differentiated tumors (p=0.04) and older patients (p=0.01). Expression of immunohistochemical markers were similar between patient sample and corresponding xenograft. mRNA differences observed between patient tumors and first passage xenografts were largely due to loss of human stroma in xenografts. mRNA patterns of early vs late passage xenografts and of small vs large tumors of the same passage were similar. Complete resistance was present in 2/7 xenografts while the remaining tumors showed varying degrees of sensitivity, that remained constant across passages. Because of their ability to recapitulate primary tumor characteristics during engraftment and across serial passaging, PTXGs can be useful clinical systems for assessment of drug sensitivity of human E/GEJ cancers. PMID:25826681

  12. Blood vessel hyperpermeability and pathophysiology in human tumour xenograft models of breast cancer: a comparison of ectopic and orthotopic tumours

    PubMed Central

    2012-01-01

    Background Human tumour xenografts in immune compromised mice are widely used as cancer models because they are easy to reproduce and simple to use in a variety of pre-clinical assessments. Developments in nanomedicine have led to the use of tumour xenografts in testing nanoscale delivery devices, such as nanoparticles and polymer-drug conjugates, for targeting and efficacy via the enhanced permeability and retention (EPR) effect. For these results to be meaningful, the hyperpermeable vasculature and reduced lymphatic drainage associated with tumour pathophysiology must be replicated in the model. In pre-clinical breast cancer xenograft models, cells are commonly introduced via injection either orthotopically (mammary fat pad, MFP) or ectopically (subcutaneous, SC), and the organ environment experienced by the tumour cells has been shown to influence their behaviour. Methods To evaluate xenograft models of breast cancer in the context of EPR, both orthotopic MFP and ectopic SC injections of MDA-MB-231-H2N cells were given to NOD scid gamma (NSG) mice. Animals with matched tumours in two size categories were tested by injection of a high molecular weight dextran as a model nanocarrier. Tumours were collected and sectioned to assess dextran accumulation compared to liver tissue as a positive control. To understand the cellular basis of these observations, tumour sections were also immunostained for endothelial cells, basement membranes, pericytes, and lymphatic vessels. Results SC tumours required longer development times to become size matched to MFP tumours, and also presented wide size variability and ulcerated skin lesions 6 weeks after cell injection. The 3 week MFP tumour model demonstrated greater dextran accumulation than the size matched 5 week SC tumour model (for P?model 3 weeks after cell injection. Both the MFP and SC tumours showed evidence of insufficient lymphatic drainage, as many fluid-filled and collagen IV-lined spaces were observed, which likely contain excess interstitial fluid. Conclusions Dextran accumulation and immunostaining results suggest that small MFP tumours best replicate the vascular permeability required to observe the EPR effect in vivo. A more predictable growth profile and the absence of ulcerated skin lesions further point to the MFP model as a strong choice for long term treatment studies that initiate after a target tumour size has been reached. PMID:23217114

  13. Model organisms in the fight against muscular dystrophy: lessons from drosophila and Zebrafish.

    PubMed

    Plantié, Emilie; Migocka-Patrza?ek, Marta; Daczewska, Ma?gorzata; Jagla, Krzysztof

    2015-01-01

    Muscular dystrophies (MD) are a heterogeneous group of genetic disorders that cause muscle weakness, abnormal contractions and muscle wasting, often leading to premature death. More than 30 types of MD have been described so far; those most thoroughly studied are Duchenne muscular dystrophy (DMD), myotonic dystrophy type 1 (DM1) and congenital MDs. Structurally, physiologically and biochemically, MDs affect different types of muscles and cause individual symptoms such that genetic and molecular pathways underlying their pathogenesis thus remain poorly understood. To improve our knowledge of how MD-caused muscle defects arise and to find efficacious therapeutic treatments, different animal models have been generated and applied. Among these, simple non-mammalian Drosophila and zebrafish models have proved most useful. This review discusses how zebrafish and Drosophila MD have helped to identify genetic determinants of MDs and design innovative therapeutic strategies with a special focus on DMD, DM1 and congenital MDs. PMID:25859781

  14. Xmrk, Kras and Myc Transgenic Zebrafish Liver Cancer Models Share Molecular Signatures with Subsets of Human Hepatocellular Carcinoma

    PubMed Central

    Zheng, Weiling; Li, Zhen; Nguyen, Anh Tuan; Li, Caixia; Emelyanov, Alexander; Gong, Zhiyuan

    2014-01-01

    Previously three oncogene transgenic zebrafish lines with inducible expression of xmrk, kras or Myc in the liver have been generated and these transgenic lines develop oncogene-addicted liver tumors upon chemical induction. In the current study, comparative transcriptomic approaches were used to examine the correlation of the three induced transgenic liver cancers with human liver cancers. RNA profiles from the three zebrafish tumors indicated relatively small overlaps of significantly deregulated genes and biological pathways. Nevertheless, the three transgenic tumor signatures all showed significant correlation with advanced or very advanced human hepatocellular carcinoma (HCC). Interestingly, molecular signature from each oncogene-induced zebrafish liver tumor correlated with only a small subset of human HCC samples (24–29%) and there were conserved up-regulated pathways between the zebrafish and correlated human HCC subgroup. The three zebrafish liver cancer models together represented nearly half (47.2%) of human HCCs while some human HCCs showed significant correlation with more than one signature defined from the three oncogene-addicted zebrafish tumors. In contrast, commonly deregulated genes (21 up and 16 down) in the three zebrafish tumor models generally showed accordant deregulation in the majority of human HCCs, suggesting that these genes might be more consistently deregulated in a broad range of human HCCs with different molecular mechanisms and thus serve as common diagnosis markers and therapeutic targets. Thus, these transgenic zebrafish models with well-defined oncogene-induced tumors are valuable tools for molecular classification of human HCCs and for understanding of molecular drivers in hepatocarcinogenesis in each human HCC subgroup. PMID:24633177

  15. Efficacy of an Fc-modified anti-CD123 antibody (CSL362) combined with chemotherapy in xenograft models of acute myelogenous leukemia in immunodeficient mice

    PubMed Central

    Lee, Erwin M.; Yee, Dean; Busfield, Samantha J.; McManus, Julie F.; Cummings, Nik; Vairo, Gino; Wei, Andrew; Ramshaw, Hayley S.; Powell, Jason A.; Lopez, Angel F.; Lewis, Ian D.; McCall, Martin N.; Lock, Richard B.

    2015-01-01

    The prognosis of older patients with acute myelogenous leukemia is generally poor. The interleukin-3 receptor ?-chain (CD123) is highly expressed on the surface of acute leukemia cells compared with normal hematopoietic stem cells. CSL362 is a fully humanized, CD123-neutralizing monoclonal antibody containing a modified Fc structure, which enhances human natural killer cell antibody-dependent cell-mediated cytotoxicity. Six continuous acute myelogenous leukemia xenografts established from patient explants and characterized by cell and molecular criteria, produced progressively lethal disease 42-202 days after transplantation. CSL362 alone reduced engraftment of one of four and three of four acute myelogenous leukemia xenografts in the bone marrow and peripheral organs, respectively. A cytarabine and daunorubicin regimen was optimized using this model to identify potentially synergistic interactions with CSL362. Cytarabine/daunorubicin improved the survival of mice engrafted with four of four acute myelogenous leukemia xenografts by 31–41 days. Moreover, CSL362 extended the survival of cytarabine/daunorubicin-treated mice for two of two acute myelogenous leukemia xenografts, while augmentation of natural killer cell-deficient NSG mice with adoptively transferred human natural killer cells improved survival against a single xenograft. Interestingly, this enhanced CSL362 efficacy was lost in the absence of chemotherapy. This study shows that acute myelogenous leukemia xenografts provide a platform for the evaluation of new therapeutics, simulating complex in vivo interactions, and that the in vivo efficacy of CSL362 supports continued clinical development of this drug. PMID:26130514

  16. Efficacy of an Fc-modified anti-CD123 antibody (CSL362) combined with chemotherapy in xenograft models of acute myelogenous leukemia in immunodeficient mice.

    PubMed

    Lee, Erwin M; Yee, Dean; Busfield, Samantha J; McManus, Julie F; Cummings, Nik; Vairo, Gino; Wei, Andrew; Ramshaw, Hayley S; Powell, Jason A; Lopez, Angel F; Lewis, Ian D; McCall, Martin N; Lock, Richard B

    2015-07-01

    The prognosis of older patients with acute myelogenous leukemia is generally poor. The interleukin-3 receptor ?-chain (CD123) is highly expressed on the surface of acute leukemia cells compared with normal hematopoietic stem cells. CSL362 is a fully humanized, CD123-neutralizing monoclonal antibody containing a modified Fc structure, which enhances human natural killer cell antibody-dependent cell-mediated cytotoxicity. Six continuous acute myelogenous leukemia xenografts established from patient explants and characterized by cell and molecular criteria, produced progressively lethal disease 42-202 days after transplantation. CSL362 alone reduced engraftment of one of four and three of four acute myelogenous leukemia xenografts in the bone marrow and peripheral organs, respectively. A cytarabine and daunorubicin regimen was optimized using this model to identify potentially synergistic interactions with CSL362. Cytarabine/daunorubicin improved the survival of mice engrafted with four of four acute myelogenous leukemia xenografts by 31-41 days. Moreover, CSL362 extended the survival of cytarabine/daunorubicin-treated mice for two of two acute myelogenous leukemia xenografts, while augmentation of natural killer cell-deficient NSG mice with adoptively transferred human natural killer cells improved survival against a single xenograft. Interestingly, this enhanced CSL362 efficacy was lost in the absence of chemotherapy. This study shows that acute myelogenous leukemia xenografts provide a platform for the evaluation of new therapeutics, simulating complex in vivo interactions, and that the in vivo efficacy of CSL362 supports continued clinical development of this drug. PMID:26130514

  17. Neutrophil migration: moving from zebrafish models to human autoimmunity.

    PubMed

    Shelef, Miriam A; Tauzin, Sebastien; Huttenlocher, Anna

    2013-11-01

    There has been a resurgence of interest in the neutrophil's role in autoimmune disease. Classically considered an early responder that dies at the site of inflammation, new findings using live imaging of embryonic zebrafish and other modalities suggest that neutrophils can reverse migrate away from sites of inflammation. These 'inflammation-sensitized' neutrophils, as well as the neutrophil extracellular traps and other products made by neutrophils in general, may have many implications for autoimmunity. Here, we review what is known about the role of neutrophils in three different autoimmune diseases: rheumatoid arthritis, systemic lupus erythematosus, and small vessel vasculitis. We then highlight recent findings related to several cytoskeletal regulators that guide neutrophil recruitment including Lyn, Rac2, and SHIP. Finally, we discuss how our improved understanding of the molecules that control neutrophil chemotaxis may impact our knowledge of autoimmunity. PMID:24117827

  18. Pazopanib, a Receptor Tyrosine Kinase Inhibitor, Suppresses Tumor Growth through Angiogenesis in Dedifferentiated Liposarcoma Xenograft Models123

    PubMed Central

    Li, Haifu; Wozniak, Agnieszka; Sciot, Raf; Cornillie, Jasmien; Wellens, Jasmien; Van Looy, Thomas; Vanleeuw, Ulla; Stas, Marguerite; Hompes, Daphne; Debiec-Rychter, Maria; Schöffski, Patrick

    2014-01-01

    INTRODUCTION: The rarity of dedifferentiated liposarcoma (DDLPS) and the lack of experimental DDLPS models limit the development of novel therapeutic strategies. Pazopanib (PAZ) is a tyrosine kinase inhibitor that is approved for the treatment of non-adipocytic advanced soft tissue sarcoma. The activity of this agent has not yet been properly explored in preclinical liposarcoma models nor in a randomized phase ? clinical trial in this entity. The aim of the present study was to investigate whether PAZ had antitumor activity in DDLPS models in vivo. MATERIAL AND METHODS: We established two patient-derived DDLPS xenograft models (UZLX-STS3 and UZLX-STS5) through implantation of tumor material from sarcoma patients in athymic nude NMRI mice. An animal model of the SW872 liposarcoma cell line was also used. To investigate the efficacy of PAZ in vivo, mice bearing tumors were treated for 2 weeks with sterile water, doxorubicin (1.2 mg/kg, intraperitoneally, twice per week), PAZ [40 mg/kg, orally (p.o.), twice per day], or PAZ plus doxorubicin (same schedules as for single treatments). RESULTS: Patient-derived xenografts retained the histologic and molecular features of DDLPS. PAZ significantly delayed tumor growth by decreasing proliferation and inhibited angiogenesis in all models tested. Combining the angiogenesis inhibitor with an anthracycline did not show superior efficacy. CONCLUSION: These results suggest that PAZ has potential antitumor activity in DDLPS primarily through antiangiogenic effects and therefore should be explored in clinical trials. PMID:25500074

  19. Use of TSH?:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals

    SciTech Connect

    Ji, Cheng [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China) [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Graduate University of Chinese Academy of Sciences, Beijing (China); Jin, Xia; He, Jiangyan [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China)] [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Yin, Zhan, E-mail: zyin@ihb.ac.cn [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China)] [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China)

    2012-07-15

    Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit ? (TSH?) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSH? in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSH? promoter, the similar responsive patterns between the expression levels of TSH?:EGFP and endogenous TSH? mRNA in the pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSH?:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system. Highlights: ? The promoter of zebrafish TSH? gene has been identified. ? The stable TSH?:EGFP transgenic zebrafish reporter germline has been generated. ? The EGFP in the transgenic fish recapitulated the pattern of pituitary TSH? mRNA. ? The transgenic zebrafish may be an in vivo model for EDC assessment.

  20. A robust and rapid xenograft model to assess efficacy of chemotherapeutic agents for human acute myeloid leukemia.

    PubMed

    Saland, E; Boutzen, H; Castellano, R; Pouyet, L; Griessinger, E; Larrue, C; de Toni, F; Scotland, S; David, M; Danet-Desnoyers, G; Vergez, F; Barreira, Y; Collette, Y; Récher, C; Sarry, J-E

    2015-01-01

    Relevant preclinical mouse models are crucial to screen new therapeutic agents for acute myeloid leukemia (AML). Current in vivo models based on the use of patient samples are not easy to establish and manipulate in the laboratory. Our objective was to develop robust xenograft models of human AML using well-characterized cell lines as a more accessible and faster alternative to those incorporating the use of patient-derived AML cells. Five widely used AML cell lines representing various AML subtypes were transplanted and expanded into highly immunodeficient non-obese diabetic/LtSz-severe combined immunodeficiency IL2R?c(null) mice (for example, cell line-derived xenografts). We show here that bone marrow sublethal conditioning with busulfan or irradiation has equal efficiency for the xenotransplantation of AML cell lines. Although higher number of injected AML cells did not change tumor engraftment in bone marrow and spleen, it significantly reduced the overall survival in mice for all tested AML cell lines. On the basis of AML cell characteristics, these models also exhibited a broad range of overall mouse survival, engraftment, tissue infiltration and aggressiveness. Thus, we have established a robust, rapid and straightforward in vivo model based on engraftment behavior of AML cell lines, all vital prerequisites for testing new therapeutic agents in preclinical studies. PMID:25794133

  1. A robust and rapid xenograft model to assess efficacy of chemotherapeutic agents for human acute myeloid leukemia

    PubMed Central

    Saland, E; Boutzen, H; Castellano, R; Pouyet, L; Griessinger, E; Larrue, C; de Toni, F; Scotland, S; David, M; Danet-Desnoyers, G; Vergez, F; Barreira, Y; Collette, Y; Récher, C; Sarry, J-E

    2015-01-01

    Relevant preclinical mouse models are crucial to screen new therapeutic agents for acute myeloid leukemia (AML). Current in vivo models based on the use of patient samples are not easy to establish and manipulate in the laboratory. Our objective was to develop robust xenograft models of human AML using well-characterized cell lines as a more accessible and faster alternative to those incorporating the use of patient-derived AML cells. Five widely used AML cell lines representing various AML subtypes were transplanted and expanded into highly immunodeficient non-obese diabetic/LtSz-severe combined immunodeficiency IL2R?cnull mice (for example, cell line-derived xenografts). We show here that bone marrow sublethal conditioning with busulfan or irradiation has equal efficiency for the xenotransplantation of AML cell lines. Although higher number of injected AML cells did not change tumor engraftment in bone marrow and spleen, it significantly reduced the overall survival in mice for all tested AML cell lines. On the basis of AML cell characteristics, these models also exhibited a broad range of overall mouse survival, engraftment, tissue infiltration and aggressiveness. Thus, we have established a robust, rapid and straightforward in vivo model based on engraftment behavior of AML cell lines, all vital prerequisites for testing new therapeutic agents in preclinical studies. PMID:25794133

  2. Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia.

    PubMed

    Liu, Chao; Gates, Keith P; Fang, Longhou; Amar, Marcelo J; Schneider, Dina A; Geng, Honglian; Huang, Wei; Kim, Jungsu; Pattison, Jennifer; Zhang, Jian; Witztum, Joseph L; Remaley, Alan T; Dong, P Duc; Miller, Yury I

    2015-08-01

    Apolipoprotein C-II (APOC2) is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases. PMID:26044956

  3. Invasiveness and metastasis of retinoblastoma in an orthotopic zebrafish tumor model

    PubMed Central

    Chen, Xiaoyun; Wang, Jian; Cao, Ziquan; Hosaka, Kayoko; Jensen, Lasse; Yang, Huasheng; Sun, Yuping; Zhuang, Rujie; Liu, Yizhi; Cao, Yihai

    2015-01-01

    Retinoblastoma is a highly invasive malignant tumor that often invades the brain and metastasizes to distal organs through the blood stream. Invasiveness and metastasis of retinoblastoma can occur at the early stage of tumor development. However, an optimal preclinical model to study retinoblastoma invasiveness and metastasis in relation to drug treatment has not been developed. Here, we developed an orthotopic zebrafish model in which retinoblastoma invasion and metastasis can be monitored at a single cell level. We took the advantages of immune privilege and transparent nature of developing zebrafish embryos. Intravitreal implantation of color-coded retinoblastoma cells allowed us to kinetically monitor tumor cell invasion and metastasis. Further, interactions between retinoblastoma cells and surrounding microvasculatures were studied using a transgenic zebrafish that exhibited green fluorescent signals in blood vessels. We discovered that tumor cells invaded neighboring tissues and blood stream when primary tumors were at the microscopic sizes. These findings demonstrate that retinoblastoma metastasis occurs at the early stage and antiangiogenic drugs such as Vegf morpholino and sunitinib could potentially interfere with tumor invasiveness and metastasis. Thus, this orthotopic retinoblastoma model offers a new and unique opportunity to study the early events of tumor invasion, metastasis and drug responses. PMID:26169357

  4. Spotlight on Zebrafish: Translational Impact

    PubMed Central

    Patton, E. Elizabeth; Dhillon, Paraminder; Amatruda, James F.; Ramakrishnan, Lalita

    2014-01-01

    In recent years, the zebrafish has emerged as an increasingly prominent model in biomedical research. To showcase the translational impact of the model across multiple disease areas, Disease Models & Mechanisms has compiled a Special Issue that includes thought-provoking reviews, original research reporting new and important insights into disease mechanisms, and novel resources that expand the zebrafish toolkit. This Editorial provides a summary of the issue’s contents, highlighting the diversity of zebrafish disease models and their clinical applications. PMID:24973741

  5. Transcriptome analysis of anti-fatty liver action by Campari tomato using a zebrafish diet-induced obesity model

    PubMed Central

    2011-01-01

    Background High dietary intake of vegetable products is beneficial against obesity and its related diseases such as dyslipidemia, nonalcoholic fatty liver disease, and cancer. We previously developed a diet-induced obesity model of zebrafish (DIO-zebrafish) that develops visceral adiposity, dyslipidemia, and liver steatosis. Zebrafish is a polyphagous animal; thus we hypothesized that DIO-zebrafish could be used for transcriptome analysis of anti-obesity effects of vegetables. Results Each vegetable exhibited different effects against obesity. We focused on "Campari" tomato, which suppressed increase of body weight, plasma TG, and lipid droplets in livers of DIO-zebrafish. Campari tomato decreased srebf1 mRNA by increase of foxo1 gene expression, which may depend on high contents of ?-carotene in this strain. Conclusions Campari tomato ameliorates diet-induced obesity, especially dyslipidemia and liver steatosis via downregulation of gene expression related to lipogenesis. DIO-zebrafish can discriminate the anti-obesity effects of different strains of vegetables, and will become a powerful tool to assess outcomes and find novel mechanisms of anti-obesity effects of natural products. PMID:22152339

  6. MEDI3617, a human anti-angiopoietin 2 monoclonal antibody, inhibits angiogenesis and tumor growth in human tumor xenograft models.

    PubMed

    Leow, Ching Ching; Coffman, Karen; Inigo, Ivan; Breen, Shannon; Czapiga, Meggan; Soukharev, Serguei; Gingles, Neill; Peterson, Norman; Fazenbaker, Christine; Woods, Rob; Jallal, Bahija; Ricketts, Sally-Ann; Lavallee, Theresa; Coats, Steve; Chang, Yong

    2012-05-01

    Angiopoietin 2 (Ang2) is an important regulator of angiogenesis, blood vessel maturation and integrity of the vascular endothelium. The correlation between the dynamic expression of Ang2 in tumors with regions of high angiogenic activity and a poor prognosis in many tumor types makes Ang2 an ideal drug target. We have generated MEDI3617, a human anti-Ang2 monoclonal antibody that neutralizes Ang2 by preventing its binding to the Tie2 receptor in vitro, and inhibits angiogenesis and tumor growth in vivo. Treatment of mice with MEDI3617 resulted in inhibition of angiogenesis in several mouse models including: FGF2-induced angiogenesis in a basement extract plug model, tumor and retinal angiogenesis. In xenograft tumor models, treatment with MEDI3617 resulted in a reduction in tumor angiogenesis and an increase in tumor hypoxia. The administration of MEDI3617 as a single agent to mice bearing human tumor xenografts resulted in tumor growth inhibition against a broad spectrum of tumor types. Combining MEDI3617 with chemotherapy or bevacizumab resulted in a delay in tumor growth and no body weight loss was observed in the combination groups. These results, combined with pharmacodynamic studies, demonstrate that treatment of tumor-bearing mice with MEDI3617 significantly inhibited tumor growth as a single agent by blocking tumor angiogenesis. Together, these data show that MEDI3617 is a robust antiangiogenic agent and support the clinical evaluation and biomarker development of MEDI3617 in cancer patients. PMID:22327175

  7. Xenograft and genetically engineered mouse model systems of osteosarcoma and Ewing's sarcoma: tumor models for cancer drug discovery

    PubMed Central

    Sampson, Valerie B; Kamara, Davida F; Kolb, E Anders

    2014-01-01

    Introduction There are > 75 histological types of solid tumors that are classified into two major groups: bone and soft-tissue sarcomas. These diseases are more prevalent in children, and pediatric sarcomas tend to be highly aggressive and rapidly progressive. Sarcomas in adults may follow a more indolent course, but aggressive tumors are also common. Sarcomas that are metastatic at diagnosis, or recurrent following therapy, remain refractory to current treatment options with dismal overall survival rates. A major focus of clinical trials, for patients with sarcoma, is to identify novel and more effective therapeutic strategies targeted to genomic or proteomic aberrations specific to the malignant cells. Critical to the understanding of the potential for targeted therapies are models of disease that are representative of clinical disease and predictive of relevant clinical responses. Areas covered In this article, the authors discuss the use of mouse xenograft models and genetically engineered mice in cancer drug discovery. The authors provide a special focus on models for the two most common bone sarcomas: osteosarcoma (OS) and Ewing's sarcoma (ES). Expert opinion Predicting whether a new anticancer agent will have a positive therapeutic index in patients with OS and ES remains a challenge. The use of mouse sarcoma models for understanding the mechanisms involved in the response of tumors to new treatments is an important step in the process of drug discovery and the development of clinically relevant therapeutic strategies for these diseases. PMID:23844615

  8. Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis.

    PubMed

    Thienpont, Benedicte; Tingaud-Sequeira, Angèle; Prats, Eva; Barata, Carlos; Babin, Patrick J; Raldúa, Demetrio

    2011-09-01

    Thyroxine-immunofluorescence quantitative disruption test (TIQDT) was designed to provide a simple, rapid, alternative bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function. This study demonstrated that zebrafish eleutheroembryos provided a suitable vertebrate model, not only for screening the potential thyroid disrupting effect of molecules, but also for estimating the potential hazards associated with exposure to chemicals directly impairing thyroxine (T4) synthesis. Amitrole, potassium perchlorate, potassium thiocyanate, methimazole (MMI), phloroglucinol, 6-propyl-2-thiouracil, ethylenethiourea, benzophenone-2, resorcinol, pyrazole, sulfamethoxazole, sodium bromide, mancozeb, and genistein were classified as thyroid gland function disruptors. Concordance between TIQDT on zebrafish and mammalian published data was very high and the physiological relevance of T4-intrafollicular content was clearly higher than regulation at the transcriptional level of tg or slc5a5. Moreover, concentration-response analysis provided information about the thyroid disrupting potency and hazard of selected positive compounds. Finally, the effect of perchlorate, but not MMI, was completely rescued by low-micromolar amounts of iodide. TIQDT performed on zebrafish eleutheroembryos is an alternative whole-organism screening assay that provides relevant information for environmental and human risk assessments. PMID:21800831

  9. Mind the fish: zebrafish as a model in cognitive social neuroscience

    PubMed Central

    Oliveira, Rui F.

    2013-01-01

    Understanding how the brain implements social behavior on one hand, and how social processes feedback on the brain to promote fine-tuning of behavioral output according to changes in the social environment is a major challenge in contemporary neuroscience. A critical step to take this challenge successfully is finding the appropriate level of analysis when relating social to biological phenomena. Given the enormous complexity of both the neural networks of the brain and social systems, the use of a cognitive level of analysis (in an information processing perspective) is proposed here as an explanatory interface between brain and behavior. A conceptual framework for a cognitive approach to comparative social neuroscience is proposed, consisting of the following steps to be taken across different species with varying social systems: (1) identification of the functional building blocks of social skills; (2) identification of the cognitive mechanisms underlying the previously identified social skills; and (3) mapping these information processing mechanisms onto the brain. Teleost fish are presented here as a group of choice to develop this approach, given the diversity of social systems present in closely related species that allows for planned phylogenetic comparisons, and the availability of neurogenetic tools that allows the visualization and manipulation of selected neural circuits in model species such as the zebrafish. Finally, the state-of-the art of zebrafish social cognition and of the tools available to map social cognitive abilities to neural circuits in zebrafish are reviewed. PMID:23964204

  10. Using Zebrafish as a Model System for Studying the Transgenerational Effects of Dioxin

    PubMed Central

    Baker, Tracie R.; Peterson, Richard E.; Heideman, Warren

    2014-01-01

    2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) has been associated with many disease states in humans. A rising concern is that exposure early in life can lead to adult toxicity and toxicity in subsequent generations. Juvenile zebrafish exposed to TCDD (50 pg/ml in water; 1 h exposure) at 3 and 7 weeks post fertilization showed toxicity only later in adulthood. We have maintained the offspring of these exposed F0 fish to determine whether we could find adverse affects in the next two generations of F1 and F2 offspring. TCDD exposure produced a significantly higher female:male ratio in all three generations. Scoliosis-like axial skeleton abnormalities, not normally observed in controls, were present in the F1 and F2 generations descended from the treated F0 founders. Egg release and fertilization success were reduced in the TCDD lineage F1 and F2 generations. This reduction in fertility in the TCDD lineage F2 generation could be attributed to alterations in the F2 males. Using zebrafish as a model allowed the simultaneous maintenance of different generations with relatively small space and costs. The zebrafish showed clear signs of transgenerational responses persisting into generations never directly exposed to TCDD. PMID:24470537

  11. Tanshinone IIA Exhibits Anticonvulsant Activity in Zebrafish and Mouse Seizure Models

    PubMed Central

    2013-01-01

    Danshen or Chinese red sage (Salvia miltiorrhiza, Bunge) is used by traditional Chinese medicine (TCM) practitioners to treat neurological, cardiovascular, and cerebrovascular disorders and is included in some TCM formulations to control epileptic seizures. In this study, acetonic crude extracts of danshen inhibited pentylenetetrazol (PTZ)-induced seizure activity in zebrafish larvae. Subsequent zebrafish bioassay-guided fractionation of the extract resulted in the isolation of four major tanshinones, which suppressed PTZ-induced activity to varying degrees. One of the active tanshinones, tanshinone IIA, also reduced c-fos expression in the brains of PTZ-exposed zebrafish larvae. In rodent seizure models, tanshinone IIA showed anticonvulsive activity in the mouse 6-Hz psychomotor seizure test in a biphasic manner and modified seizure thresholds in a complex manner for the mouse i.v. PTZ seizure assay. Interestingly, tanshinone IIA is used as a prescription drug in China to address cerebral ischemia in patients. Here, we provide the first in vivo evidence demonstrating that tanshinone IIA has anticonvulsant properties as well. PMID:23937066

  12. Tanshinone IIA exhibits anticonvulsant activity in zebrafish and mouse seizure models.

    PubMed

    Buenafe, Olivia Erin; Orellana-Paucar, Adriana; Maes, Jan; Huang, Hao; Ying, Xuhui; De Borggraeve, Wim; Crawford, Alexander D; Luyten, Walter; Esguerra, Camila V; de Witte, Peter

    2013-11-20

    Danshen or Chinese red sage (Salvia miltiorrhiza, Bunge) is used by traditional Chinese medicine (TCM) practitioners to treat neurological, cardiovascular, and cerebrovascular disorders and is included in some TCM formulations to control epileptic seizures. In this study, acetonic crude extracts of danshen inhibited pentylenetetrazol (PTZ)-induced seizure activity in zebrafish larvae. Subsequent zebrafish bioassay-guided fractionation of the extract resulted in the isolation of four major tanshinones, which suppressed PTZ-induced activity to varying degrees. One of the active tanshinones, tanshinone IIA, also reduced c-fos expression in the brains of PTZ-exposed zebrafish larvae. In rodent seizure models, tanshinone IIA showed anticonvulsive activity in the mouse 6-Hz psychomotor seizure test in a biphasic manner and modified seizure thresholds in a complex manner for the mouse i.v. PTZ seizure assay. Interestingly, tanshinone IIA is used as a prescription drug in China to address cerebral ischemia in patients. Here, we provide the first in vivo evidence demonstrating that tanshinone IIA has anticonvulsant properties as well. PMID:23937066

  13. Activin type IB receptor signaling in prostate cancer cells promotes lymph node metastasis in a xenograft model

    SciTech Connect

    Nomura, Masatoshi, E-mail: nomura@med.kyushu-u.ac.jp [Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)] [Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Tanaka, Kimitaka; Wang, Lixiang; Goto, Yutaka; Mukasa, Chizu; Ashida, Kenji; Takayanagi, Ryoichi [Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)] [Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer ActRIB signaling induces Snail and S100A4 expressions in prostate cancer cells. Black-Right-Pointing-Pointer The prostate cancer cell lines expressing an active form of ActRIB were established. Black-Right-Pointing-Pointer ActRIB signaling promotes EMT and lymph node metastasis in xenograft model. -- Abstract: Activin, a member of the transforming growth factor-{beta} family, has been known to be a growth and differentiating factor. Despite its pluripotent effects, the roles of activin signaling in prostate cancer pathogenesis are still unclear. In this study, we established several cell lines that express a constitutive active form of activin type IB receptor (ActRIBCA) in human prostate cancer cells, ALVA41 (ALVA-ActRIBCA). There was no apparent change in the proliferation of ALVA-ActRIBCA cells in vitro; however, their migratory ability was significantly enhanced. In a xenograft model, histological analysis revealed that the expression of Snail, a cell-adhesion-suppressing transcription factor, was dramatically increased in ALVA-ActRIBCA tumors, indicating epithelial mesenchymal transition (EMT). Finally, mice bearing ALVA-ActRIBCA cells developed multiple lymph node metastases. In this study, we demonstrated that ActRIBCA signaling can promote cell migration in prostate cancer cells via a network of signaling molecules that work together to trigger the process of EMT, and thereby aid in the aggressiveness and progression of prostate cancers.

  14. Relationship between differentially expressed mRNA and mRNA-protein correlations in a xenograft model system

    PubMed Central

    Koussounadis, Antonis; Langdon, Simon P.; Um, In Hwa; Harrison, David J.; Smith, V. Anne

    2015-01-01

    Differential mRNA expression studies implicitly assume that changes in mRNA expression have biological meaning, most likely mediated by corresponding changes in protein levels. Yet studies into mRNA-protein correspondence have shown notoriously poor correlation between mRNA and protein expression levels, creating concern for inferences from only mRNA expression data. However, none of these studies have examined in particular differentially expressed mRNA. Here, we examined this question in an ovarian cancer xenograft model. We measured protein and mRNA expression for twenty-nine genes in four drug-treatment conditions and in untreated controls. We identified mRNAs differentially expressed between drug-treated xenografts and controls, then analysed mRNA-protein expression correlation across a five-point time-course within each of the four experimental conditions. We evaluated correlations between mRNAs and their protein products for mRNAs differentially expressed within an experimental condition compared to those that are not. We found that differentially expressed mRNAs correlate significantly better with their protein product than non-differentially expressed mRNAs. This result increases confidence for the use of differential mRNA expression for biological discovery in this system, as well as providing optimism for the usefulness of inferences from mRNA expression in general. PMID:26053859

  15. Relationship between differentially expressed mRNA and mRNA-protein correlations in a xenograft model system.

    PubMed

    Koussounadis, Antonis; Langdon, Simon P; Um, In Hwa; Harrison, David J; Smith, V Anne

    2015-01-01

    Differential mRNA expression studies implicitly assume that changes in mRNA expression have biological meaning, most likely mediated by corresponding changes in protein levels. Yet studies into mRNA-protein correspondence have shown notoriously poor correlation between mRNA and protein expression levels, creating concern for inferences from only mRNA expression data. However, none of these studies have examined in particular differentially expressed mRNA. Here, we examined this question in an ovarian cancer xenograft model. We measured protein and mRNA expression for twenty-nine genes in four drug-treatment conditions and in untreated controls. We identified mRNAs differentially expressed between drug-treated xenografts and controls, then analysed mRNA-protein expression correlation across a five-point time-course within each of the four experimental conditions. We evaluated correlations between mRNAs and their protein products for mRNAs differentially expressed within an experimental condition compared to those that are not. We found that differentially expressed mRNAs correlate significantly better with their protein product than non-differentially expressed mRNAs. This result increases confidence for the use of differential mRNA expression for biological discovery in this system, as well as providing optimism for the usefulness of inferences from mRNA expression in general. PMID:26053859

  16. Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection.

    PubMed

    Harvie, Elizabeth A; Huttenlocher, Anna

    2015-01-01

    The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection. The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection. PMID:25938624

  17. Assessment of a Novel VEGF Targeted Agent Using Patient-Derived Tumor Tissue Xenograft Models of Colon Carcinoma with Lymphatic and Hepatic Metastases

    Microsoft Academic Search

    Ketao Jin; Guangliang Li; Binbin Cui; Jing Zhang; Huanrong Lan; Na Han; Bojian Xie; Feilin Cao; Kuifeng He; Haohao Wang; Zhenzhen Xu; Lisong Teng; Tieming Zhu

    2011-01-01

    The lack of appropriate tumor models of primary tumors and corresponding metastases that can reliably predict for response to anticancer agents remains a major deficiency in the clinical practice of cancer therapy. It was the aim of our study to establish patient-derived tumor tissue (PDTT) xenograft models of colon carcinoma with lymphatic and hepatic metastases useful for testing of novel

  18. Developing zebrafish models relevant to PTSD and other trauma- and stressor-related disorders.

    PubMed

    Stewart, Adam Michael; Yang, Ester; Nguyen, Michael; Kalueff, Allan V

    2014-12-01

    While post-traumatic stress disorder (PTSD) and other trauma- and stress-related disorders (TSRDs) represent a serious societal and public health concern, their pathogenesis is largely unknown. Given the clinical complexity of TSRD development and susceptibility, greater investigation into candidate biomarkers and specific genetic pathways implicated in both risk and resilience to trauma becomes critical. In line with this, numerous animal models have been extensively used to better understand the pathogenic mechanisms of PTSD and related TSRD. Here, we discuss the rapidly increasing potential of zebrafish as models of these disorders, and how their use may aid researchers in uncovering novel treatments and therapies in this field. PMID:25138994

  19. Switching to zebrafish neurobehavioral models: The obsessive-compulsive disorder paradigm.

    PubMed

    D'Amico, Davide; Estivill, Xavier; Terriente, Javier

    2015-07-15

    Obsessive-compulsive disorder (OCD) is the tenth most disabling illness of any kind. OCD stands as a paradigm for complex neurobehavioral disorders due to its polygenic origin. It presents heterogenic clinical presentation, variable disease onset, progression and treatment responses, what makes its understanding a major neuropsychiatric challenge. Like with other neurobehavioral disorders, animal models are essential tools for decoding OCD genetic complexity, understanding its biological base and discovering novel treatments and diagnostic methods. 20 years of rodent OCD modeling have helped to understand the disease better, but multiple questions remain regarding OCD. Innovative whole genome sequencing (WGS) approaches might provide important answers on OCD risk associated genes. However, exploiting those large data sets through the use of traditional animal models is costly and time consuming. Zebrafish might be an appropriate animal model to streamline the pipeline of gene functional validation. This animal model shows several advantages versus rodent models, such as faster and cheaper genetic manipulation, strong impact on the 3Rs implementation, behavioral phenotypic reproducibility of OCD-like behaviors (obsessions and compulsions) and feasibility to develop high-throughput assays for novel OCD drug therapies discovery. In conclusion, zebrafish could be an innovative and relevant model for understanding OCD. PMID:25814246

  20. A privileged intraphagocyte niche is responsible for disseminated infection of Staphylococcus aureus in a zebrafish model

    PubMed Central

    Prajsnar, Tomasz K; Hamilton, Ruth; Garcia-Lara, Jorge; McVicker, Gareth; Williams, Alexander; Boots, Michael; Foster, Simon J; Renshaw, Stephen A

    2012-01-01

    The innate immune system is the primary defence against the versatile pathogen, Staphylococcus aureus. How this organism is able to avoid immune killing and cause infections is poorly understood. Using an established larval zebrafish infection model, we have shown that overwhelming infection is due to subversion of phagocytes by staphylococci, allowing bacteria to evade killing and found foci of disease. Larval zebrafish coinfected with two S. aureus strains carrying different fluorescent reporter gene fusions (but otherwise isogenic) had bacterial lesions, at the time of host death, containing predominantly one strain. Quantitative data using two marked strains revealed that the strain ratios, during overwhelming infection, were often skewed towards the extremes, with one strain predominating. Infection with passaged bacterial clones revealed the phenomenon not to bedue to adventitious mutations acquired by the pathogen. After infection of the host, all bacteria are internalized by phagocytes and the skewing of population ratios is absolutely dependent on the presence of phagocytes. Mathematical modelling of pathogen population dynamics revealed the data patterns are consistent with the hypothesis that a small number of infected phagocytes serve as an intracellular reservoir for S. aureus, which upon release leads to disseminated infection. Strategies to specifically alter neutrophil/macrophage numbers were used to map the potential subpopulation of phagocytes acting as a pathogen reservoir, revealing neutrophils as the likely ‘niche’. Subsequently in a murine sepsis model, S. aureus abscesses in kidneys were also found to be predominantly clonal, therefore likely founded by an individual cell, suggesting a potential mechanism analogous to the zebrafish model with few protected niches. These findings add credence to the argument that S. aureus control regimes should recognize both the intracellular as well as extracellular facets of the S. aureus life cycle. PMID:22694745

  1. A human choriocarcinoma xenograft in nude mice; a model for the study of antibody localization.

    PubMed Central

    Searle, F.; Boden, J.; Lewis, J. C.; Bagshawe, K. D.

    1981-01-01

    The successful development of the concept of linking cell-killing agents to tumour-specific antibodies will be largely determined by the extent to which the antibodies are preferentially localized in the malignant tissue. A xenograft of human choriocarcinoma (CC3) has been established in nude mice, and the relative distribution of affinity-purified specific antibodies to human chorionic gonadotrophin has been compared with that of nonspecific antibodies from the same species. Treatment of the nonspecific antibodies with ammonium thiocyanate appeared to be important to ensure that the distributions in normal nude mice were equivalent. Specificity indices, derived from the comparative distributions of isotope activity in the tumour and lung of labelled specific and nonspecific antibodies, ranged between 1.3 and 2.0. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6168276

  2. The Zebrafish Brain in Research and Teaching: A Simple in Vivo and in Vitro Model for the Study of Spontaneous Neural Activity

    ERIC Educational Resources Information Center

    Vargas, R.; Johannesdottir, I. P.; Sigurgeirsson, B.; Porsteinsson, H.; Karlsson, K. AE.

    2011-01-01

    Recently, the zebrafish ("Danio rerio") has been established as a key animal model in neuroscience. Behavioral, genetic, and immunohistochemical techniques have been used to describe the connectivity of diverse neural circuits. However, few studies have used zebrafish to understand the function of cerebral structures or to study neural circuits.…

  3. The first mecp2-null zebrafish model shows altered motor behaviors

    PubMed Central

    Pietri, Thomas; Roman, Angel-Carlos; Guyon, Nicolas; Romano, Sebastián A.; Washbourne, Philip; Moens, Cecilia B.; de Polavieja, Gonzalo G.; Sumbre, Germán

    2013-01-01

    Rett syndrome (RTT) is an X-linked neurodevelopmental disorder and one of the most common causes of mental retardation in affected girls. Other symptoms include a rapid regression of motor and cognitive skills after an apparently early normal development. Sporadic mutations in the transcription factor MECP2 has been shown to be present in more than 90% of the patients and several models of MeCP2-deficient mice have been created to understand the role of this gene. These models have pointed toward alterations in the maintenance of the central nervous system rather than its development, in line with the late onset of the disease in humans. However, the exact functions of MeCP2 remain difficult to delineate and the animal models have yielded contradictory results. Here, we present the first mecp2-null allele mutation zebrafish model. Surprisingly and in contrast to MeCP2-null mouse models, mecp2-null zebrafish are viable and fertile. They present nonetheless clear behavioral alterations during their early development, including spontaneous and sensory-evoked motor anomalies, as well as defective thigmotaxis. PMID:23874272

  4. In vivo antimetastatic effects of uPAR retargeted measles virus in syngeneic and xenograft models of mammary cancer

    PubMed Central

    Jing, Yuqi; Bejarano, Marcela Toro; Zaias, Julia; Merchan, Jaime R.

    2014-01-01

    Purpose The urokinase receptor (uPAR) plays a critical role in breast cancer (BC) progression and metastases, and is a validated target for novel therapies. The current study investigates the effects of MV-uPA, an oncolytic measles virus fully retargeted against uPAR in syngeneic and xenograft BC metastases models. Methods In vitro replication and cytotoxicity of MVs retargeted against human (MV-h-uPA) or mouse (MV-m-uPA) uPAR were assessed in human and murine cancer and non-cancer mammary epithelial cells. The in vivo effects of species-specific uPAR retargeted MVs were assessed in syngeneic and xenograft models of experimental metastases, established by intravenous administration of luciferase expressing 4T1 or MDA-MD-231 cells. Metastases progression was assessed by in vivo bioluminescence imaging. Tumor targeting was evaluated by qRT-PCR of MV-N, rescue of viable viral particles and immunostaining of MV particles in lungs from tumor bearing mice. Results In vitro, MV-h-uPA and MV-m-uPA selectively infected, replicated and induced cytotoxicity in cancer compared to non-cancer cells in a species-specific manner. In vivo, MV-m-uPA delayed 4T1 lung metastases progression and prolonged survival. These effects were associated with identification of viable viral particles, viral RNA and detection of MV-N by immunostaining from lung tissues in treated mice. In the human MDA-MB-231 metastases model, intravenous administration of MV-h-uPA markedly inhibited metastases progression and significantly improved survival, compared to controls. No significant treatment related toxicity was observed in treated mice. Conclusions The above preclinical findings strongly suggest that uPAR retargeted measles virotherapy is a novel and feasible systemic therapy strategy against metastatic breast cancer. PMID:25519042

  5. Early Interneuron Dysfunction in ALS: Insights from a Mutant sod1 Zebrafish Model

    PubMed Central

    McGown, Alexander; McDearmid, Jonathan R; Panagiotaki, Niki; Tong, Huaxia; Al Mashhadi, Sufana; Redhead, Natasha; Lyon, Alison N; Beattie, Christine E; Shaw, Pamela J; Ramesh, Tennore M

    2013-01-01

    Objective To determine, when, how, and which neurons initiate the onset of pathophysiology in amyotrophic lateral sclerosis (ALS) using a transgenic mutant sod1 zebrafish model and identify neuroprotective drugs. Methods Proteinopathies such as ALS involve mutant proteins that misfold and activate the heat shock stress response (HSR). The HSR is indicative of neuronal stress, and we used a fluorescent hsp70-DsRed reporter in our transgenic zebrafish to track neuronal stress and to measure functional changes in neurons and muscle over the course of the disease. Results We show that mutant sod1 fish first exhibited the HSR in glycinergic interneurons at 24 hours postfertilization (hpf). By 96 hpf, we observed a significant reduction in spontaneous glycinergic currents induced in spinal motor neurons. The loss of inhibition was followed by increased stress in the motor neurons of symptomatic adults and concurrent morphological changes at the neuromuscular junction (NMJ) indicative of denervation. Riluzole, the only approved ALS drug and apomorphine, an NRF2 activator, reduced the observed early neuronal stress response. Interpretation The earliest event in the pathophysiology of ALS in the mutant sod1 zebrafish model involves neuronal stress in inhibitory interneurons, resulting from mutant Sod1 expression. This is followed by a reduction in inhibitory input to motor neurons. The loss of inhibitory input may contribute to the later development of neuronal stress in motor neurons and concurrent inability to maintain the NMJ. Riluzole, the approved drug for use in ALS, modulates neuronal stress in interneurons, indicating a novel mechanism of riluzole action. ANN NEUROL 2013;73:246–258 PMID:23281025

  6. A zebrafish model of manganism reveals reversible and treatable symptoms that are independent of neurotoxicity

    PubMed Central

    Bakthavatsalam, Subha; Das Sharma, Shreya; Sonawane, Mahendra; Thirumalai, Vatsala; Datta, Ankona

    2014-01-01

    Manganese (manganese ion; referred to as Mn) is essential for neuronal function, yet it is toxic at high concentrations. Environmental and occupational exposure to high concentrations of Mn causes manganism, a well-defined movement disorder in humans, with symptoms resembling Parkinson’s disease (PD). However, manganism is distinct from PD and the neural basis of its pathology is poorly understood. To address this issue, we generated a zebrafish model of manganism by incubating larvae in rearing medium containing Mn. We find that Mn-treated zebrafish larvae exhibit specific postural and locomotor defects. Larvae begin to float on their sides, show a curved spine and swim in circles. We discovered that treatment with Mn causes postural defects by interfering with mechanotransduction at the neuromasts. Furthermore, we find that the circling locomotion could be caused by long-duration bursting in the motor neurons, which can lead to long-duration tail bends in the Mn-treated larvae. Mn-treated larvae also exhibited fewer startle movements. Additionally, we show that the intensity of tyrosine hydroxylase immunoreactivity is reversibly reduced after Mn-treatment. This led us to propose that reduced dopamine neuromodulation drives the changes in startle movements. To test this, when we supplied an external source of dopamine to Mn-treated larvae, the larvae exhibited a normal number of startle swims. Taken together, these results indicate that Mn interferes with neuronal function at the sensory, motor and modulatory levels, and open avenues for therapeutically targeted studies on the zebrafish model of manganism. PMID:25261567

  7. Unique and potent effects of acute ibogaine on zebrafish: the developing utility of novel aquatic models for hallucinogenic drug research.

    PubMed

    Cachat, Jonathan; Kyzar, Evan J; Collins, Christopher; Gaikwad, Siddharth; Green, Jeremy; Roth, Andrew; El-Ounsi, Mohamed; Davis, Ari; Pham, Mimi; Landsman, Samuel; Stewart, Adam Michael; Kalueff, Allan V

    2013-01-01

    An indole alkaloid, ibogaine is the principal psychoactive component of the iboga plant, used by indigenous peoples in West Africa for centuries. Modulating multiple neurotransmitter systems, the drug is a potent hallucinogen in humans, although its psychotropic effects remain poorly understood. Expanding the range of model species is an important strategy for translational neuroscience research. Here we exposed adult zebrafish (Danio rerio) to 10 and 20mg/L of ibogaine, testing them in the novel tank, light-dark box, open field, mirror stimulation, social preference and shoaling tests. In the novel tank test, the zebrafish natural diving response (geotaxis) was reversed by ibogaine, inducing initial top swimming followed by bottom dwelling. Ibogaine also attenuated the innate preference for dark environments (scototaxis) in the light-dark box test. While it did not exert overt locomotor or thigmotaxic responses in the open field test, the drug altered spatiotemporal exploration of novel environment, inducing clear preference of some areas over others. Ibogaine also promoted 'mirror' exploration in the mirror stimulation test, disrupted group cohesion in the shoaling test, and evoked strong coloration responses due to melanophore aggregation, but did not alter brain c-fos expression or whole-body cortisol levels. Overall, our results support the complex pharmacological profile of ibogaine and its high sensitivity in zebrafish models, dose-dependently affecting multiple behavioral domains. While future investigations in zebrafish may help elucidate the mechanisms underlying these unique behavioral effects, our study strongly supports the developing utility of aquatic models in hallucinogenic drug research. High sensitivity of three-dimensional phenotyping approaches applied here to behavioral effects of ibogaine in zebrafish provides further evidence of how 3D reconstructions of zebrafish swimming paths may be useful for high-throughput pharmacological screening. PMID:22974549

  8. Hypoxia-Induced Retinal Neovascularization in Zebrafish Embryos: A Potential Model of Retinopathy of Prematurity

    PubMed Central

    Kao, Alex; Hsi, Brian; Lee, Shwu-Huey; Chen, Yau-Hung; Wang, I-Jong

    2015-01-01

    Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2–4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression. PMID:25978439

  9. Transcriptional changes and developmental abnormalities in a zebrafish model of myotonic dystrophy type 1

    PubMed Central

    Todd, Peter K.; Ackall, Feras Y.; Hur, Junguk; Sharma, Kush; Paulson, Henry L.; Dowling, James J.

    2014-01-01

    Myotonic dystrophy type I (DM1) is a multi-system, autosomal dominant disorder caused by expansion of a CTG repeat sequence in the 3?UTR of the DMPK gene. The size of the repeat sequence correlates with age at onset and disease severity, with large repeats leading to congenital forms of DM1 associated with hypotonia and intellectual disability. In models of adult DM1, expanded CUG repeats lead to an RNA toxic gain of function, mediated at least in part by sequestering specific RNA splicing proteins, most notably muscleblind-related (MBNL) proteins. However, the impact of CUG RNA repeat expression on early developmental processes is not well understood. To better understand early developmental processes in DM1, we utilized the zebrafish, Danio rerio, as a model system. Direct injection of (CUG)91 repeat-containing mRNA into single-cell embryos induces toxicity in the nervous system and muscle during early development. These effects manifest as abnormal morphology, behavioral abnormalities and broad transcriptional changes, as shown by cDNA microarray analysis. Co-injection of zebrafish mbnl2 RNA suppresses (CUG)91 RNA toxicity and reverses the associated behavioral and transcriptional abnormalities. Taken together, these findings suggest that early expression of exogenously transcribed CUG repeat RNA can disrupt normal muscle and nervous system development and provides a new model for DM1 research that is amenable to small-molecule therapeutic development. PMID:24092878

  10. Use of the Zebrafish Larvae as a Model to Study Cigarette Smoke Condensate Toxicity

    PubMed Central

    Ellis, Lee D.; Soo, Evelyn C.; Achenbach, John C.; Morash, Michael G.; Soanes, Kelly H.

    2014-01-01

    The smoking of tobacco continues to be the leading cause of premature death worldwide and is linked to the development of a number of serious illnesses including heart disease, respiratory diseases, stroke and cancer. Currently, cell line based toxicity assays are typically used to gain information on the general toxicity of cigarettes and other tobacco products. However, they provide little information regarding the complex disease-related changes that have been linked to smoking. The ethical concerns and high cost associated with mammalian studies have limited their widespread use for in vivo toxicological studies of tobacco. The zebrafish has emerged as a low-cost, high-throughput, in vivo model in the study of toxicology. In this study, smoke condensates from 2 reference cigarettes and 6 Canadian brands of cigarettes with different design features were assessed for acute, developmental, cardiac, and behavioural toxicity (neurotoxicity) in zebrafish larvae. By making use of this multifaceted approach we have developed an in vivo model with which to compare the toxicity profiles of smoke condensates from cigarettes with different design features. This model system may provide insights into the development of smoking related disease and could provide a cost-effective, high-throughput platform for the future evaluation of tobacco products. PMID:25526262

  11. INDUCED AND SPONTANEOUS NEOPLASIA IN ZEBRAFISH.

    EPA Science Inventory

    To address the potential of zebrafish as a cancer model, it is important to determine the susceptibility of zebrafish to tumors, and to compare zebrafish tumors with human tumors. To determine whether the commonly-used germ line mutagen, ethylnitrosourea (ENU) induces tumors, we ...

  12. The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models

    PubMed Central

    2013-01-01

    Background Melanoma is the most deadly form of skin cancer. Expression of oncogenic BRAF or NRAS, which are frequently mutated in human melanomas, promote the formation of nevi but are not sufficient for tumorigenesis. Even with germline mutated p53, these engineered melanomas present with variable onset and pathology, implicating additional somatic mutations in a multi-hit tumorigenic process. Results To decipher the genetics of these melanomas, we sequence the protein coding exons of 53 primary melanomas generated from several BRAFV600E or NRASQ61K driven transgenic zebrafish lines. We find that engineered zebrafish melanomas show an overall low mutation burden, which has a strong, inverse association with the number of initiating germline drivers. Although tumors reveal distinct mutation spectrums, they show mostly C?>?T transitions without UV light exposure, and enrichment of mutations in melanogenesis, p53 and MAPK signaling. Importantly, a recurrent amplification occurring with pre-configured drivers BRAFV600E and p53-/- suggests a novel path of BRAF cooperativity through the protein kinase A pathway. Conclusion This is the first analysis of a melanoma mutational landscape in the absence of UV light, where tumors manifest with remarkably low mutation burden and high heterogeneity. Genotype specific amplification of protein kinase A in cooperation with BRAF and p53 mutation suggests the involvement of melanogenesis in these tumors. This work is important for defining the spectrum of events in BRAF or NRAS driven melanoma in the absence of UV light, and for informed exploitation of models such as transgenic zebrafish to better understand mechanisms leading to human melanoma formation. PMID:24148783

  13. Assessment of extravascular extracellular space fraction in human melanoma xenografts by DCE-MRI and kinetic modeling.

    PubMed

    Benjaminsen, Ilana C; Brurberg, Kjetil G; Ruud, Else-Beate M; Rofstad, Einar K

    2008-02-01

    Tumor aggressiveness and response to therapy are influenced by the extravascular extracellular space fraction (EESF) of the malignant tissue. The EESF may, therefore, be an important prognostic parameter for cancer patients. The aim of this study was to investigate whether gadopentetate dimeglumine (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can be used to assess the EESF of tumors. Amelanotic human melanoma xenografts (A-07, R-18) were used as preclinical models of human cancer. Images of E.F (E is the initial extraction fraction of Gd-DTPA and F is perfusion) and lambda (the partition coefficient of Gd-DTPA) were obtained by Kety analysis of DCE-MRI data. Our study was based on the hypothesis that lambda is governed by the EESF and is not influenced significantly by microvascular density (MVD) or blood perfusion. To test this hypothesis, we searched for correlations between lambda and E.F, MVD or EESF by comparing lambda images with E.F images, histological preparations from the imaged tissue and the radial heterogeneity in EESF obtained by invasive imaging. Positive correlations were found between lambda and EESF. Thus, median lambda was larger in A-07 tumors than in R-18 tumors by a factor of 4.2 (P<.00001), consistent with the histological observation that EESF is approximately fourfold larger in A-07 tumors than in R-18 tumors. The radial heterogeneity in lambda in A-07 and R-18 tumors was almost identical to the radial heterogeneity in EESF. Moreover, lambda was larger in tissue regions with high EESF than in tissue regions with low EESF in A-07 tumors (P=.048). On the other hand, significant correlations between lambda and MVD or E.F could not be detected. Consequently, Kety analysis of Gd-DTPA-based DCE-MRI series of xenografted tumors provides lambda images that primarily reflect the EESF of the tissue. PMID:17692490

  14. Targeting Tumor Vasculature Endothelial Cells and Tumor Cells for Immunotherapy of Human Melanoma in a Mouse Xenograft Model

    NASA Astrophysics Data System (ADS)

    Hu, Zhiwei; Sun, Ying; Garen, Alan

    1999-07-01

    An immunotherapy treatment for cancer that targets both the tumor vasculature and tumor cells has shown promising results in a severe combined immunodeficient mouse xenograft model of human melanoma. The treatment involves systemic delivery of an immunoconjugate molecule composed of a tumor-targeting domain conjugated to the Fc effector domain of human IgG1. The effector domain induces a cytolytic immune response against the targeted cells by natural killer cells and complement. Two types of targeting domains were used. One targeting domain is a human single-chain Fv molecule that binds to a chondroitin sulfate proteoglycan expressed on the surface of most human melanoma cells. Another targeting domain is factor VII (fVII), a zymogen that binds with high specificity and affinity to the transmembrane receptor tissue factor (TF) to initiate the blood coagulation cascade. TF is expressed by endothelial cells lining the tumor vasculature but not the normal vasculature, and also by many types of tumor cells including melanoma. Because the binding of a fVII immunoconjugate to TF might cause disseminated intravascular coagulation, the active site of fVII was mutated to inhibit coagulation without affecting the affinity for TF. The immunoconjugates were encoded as secreted molecules in a replication-defective adenovirus vector, which was injected into the tail vein of severe combined immunodeficient mice. The results demonstrate that a mutated fVII immunoconjugate, administered separately or together with a single-chain Fv immunoconjugate that binds to the tumor cells, can inhibit the growth or cause regression of an established human tumor xenograft. This procedure could be effective in treating a broad spectrum of human solid tumors that express TF on vascular endothelial cells and tumor cells.

  15. WaveCNV: allele-specific copy number alterations in primary tumors and xenograft models from next-generation sequencing

    PubMed Central

    Holt, Carson; Losic, Bojan; Pai, Deepa; Zhao, Zhen; Trinh, Quang; Syam, Sujata; Arshadi, Niloofar; Jang, Gun Ho; Ali, Johar; Beck, Tim; McPherson, John; Muthuswamy, Lakshmi B.

    2014-01-01

    Motivation: Copy number variations (CNVs) are a major source of genomic variability and are especially significant in cancer. Until recently microarray technologies have been used to characterize CNVs in genomes. However, advances in next-generation sequencing technology offer significant opportunities to deduce copy number directly from genome sequencing data. Unfortunately cancer genomes differ from normal genomes in several aspects that make them far less amenable to copy number detection. For example, cancer genomes are often aneuploid and an admixture of diploid/non-tumor cell fractions. Also patient-derived xenograft models can be laden with mouse contamination that strongly affects accurate assignment of copy number. Hence, there is a need to develop analytical tools that can take into account cancer-specific parameters for detecting CNVs directly from genome sequencing data. Results: We have developed WaveCNV, a software package to identify copy number alterations by detecting breakpoints of CNVs using translation-invariant discrete wavelet transforms and assign digitized copy numbers to each event using next-generation sequencing data. We also assign alleles specifying the chromosomal ratio following duplication/loss. We verified copy number calls using both microarray (correlation coefficient 0.97) and quantitative polymerase chain reaction (correlation coefficient 0.94) and found them to be highly concordant. We demonstrate its utility in pancreatic primary and xenograft sequencing data. Availability and implementation: Source code and executables are available at https://github.com/WaveCNV. The segmentation algorithm is implemented in MATLAB, and copy number assignment is implemented Perl. Contact: lakshmi.muthuswamy@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24192544

  16. Ginsenoside Rh2 induces Bcl-2 family proteins-mediated apoptosis in vitro and in xenografts in vivo models.

    PubMed

    Choi, Sunga; Oh, Jun-Young; Kim, Soo-Jin

    2011-01-01

    The cancer chemoprevention effects of ginseng saponins have been demonstrated against a variety of experimental tumors; however, their molecular mechanisms in vitro and in in vivo models are not well studied. This study was undertaken to gain insights into the molecular mechanisms of ginsenoside Rh2 (Rh2)-induced cell death in human breast cancer cell lines as well as in in vivo xenografts. Rh2 treatment significantly inhibited viability of both MCF-7 and MDA-MB-231 human breast cells in a concentration-dependent manner, which correlated with mitochondria-mediated apoptosis. Rh2-induced apoptosis was accompanied by the down-regulation of antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1. It also caused induction of the proapoptotic members Bak, Bax, and Bim leading to mitochondrial translocation of Bax and activation of caspases. Moreover, Rh2-induced apoptosis was partially, yet significantly protected by transient transfection of MCF-7 cells with Bax- and Bak-targeted siRNAs. Oral gavage of 5?mg Rh2/kg of mouse (three times a week) significantly caused apoptosis of MDA-MB-231 xenografts. An increase in Bax and Bak and a decrease in Bcl-2 and Bcl-xL transcript levels, in accordance with their protein expression, were observed in tumor tissue. Tumors from Rh2-treated mice exhibited a markedly higher count of apoptotic bodies and reduced proliferation index compared with control tumors. Our data suggest that Rh2 used in traditional oriental medicine for the treatment of various ailments, may be an attractive agent for the treatment and/or prevention of human breast cancers. PMID:21080338

  17. Vascular Development in the Zebrafish

    PubMed Central

    Gore, Aniket V.; Monzo, Kathryn; Cha, Young R.; Pan, Weijun; Weinstein, Brant M.

    2012-01-01

    The zebrafish has emerged as an excellent vertebrate model system for studying blood and lymphatic vascular development. The small size, external and rapid development, and optical transparency of zebrafish embryos are some of the advantages the zebrafish model system offers. Multiple well-established techniques have been developed for imaging and functionally manipulating vascular tissues in zebrafish embryos, expanding on and amplifying these basic advantages and accelerating use of this model system for studying vascular development. In the past decade, studies performed using zebrafish as a model system have provided many novel insights into vascular development. In this article we discuss the amenability of this model system for studying blood vessel development and review contributions made by this system to our understanding of vascular development. PMID:22553495

  18. A Novel Synthetic Smoothened Antagonist Transiently Inhibits Pancreatic Adenocarcinoma Xenografts in a Mouse Model

    PubMed Central

    Strand, Martin F.; Wilson, Steven R.; Dembinski, Jennifer L.; Holsworth, Daniel D.; Khvat, Alexander; Okun, Ilya; Petersen, Dirk; Krauss, Stefan

    2011-01-01

    Background Hedgehog (Hh) signaling is over-activated in several solid tumors where it plays a central role in cell growth, stroma recruitment and tumor progression. In the Hh signaling pathway, the Smoothened (SMO) receptor comprises a primary drug target with experimental small molecule SMO antagonists currently being evaluated in clinical trials. Principal Findings Using Shh-Light II (Shh-L2) and alkaline phosphatase (AP) based screening formats on a “focused diversity” library we identified a novel small molecule inhibitor of the Hh pathway, MS-0022 (2-bromo-N-(4-(8-methylimidazo[1,2-a]pyridin-2-yl)phenyl)benzamide). MS-0022 showed effective Hh signaling pathway inhibition at the level of SMO in the low nM range, and Hh pathway inhibition downstream of Suppressor of fused (SUFU) in the low µM range. MS-0022 reduced growth in the tumor cell lines PANC-1, SUIT-2, PC-3 and FEMX in vitro. MS-0022 treatment led to a transient delay of tumor growth that correlated with a reduction of stromal Gli1 levels in SUIT-2 xenografts in vivo. Significance We document the in vitro and in vivo efficacy and bioavailability of a novel small molecule SMO antagonist, MS-0022. Although MS-0022 primarily interferes with Hh signaling at the level of SMO, it also has a downstream inhibitory effect and leads to a stronger reduction of growth in several tumor cell lines when compared to related SMO antagonists. PMID:21698280

  19. Modulation of eicosanoid metabolism in endothelial cells in a xenograft model. Role of cyclooxygenase-2.

    PubMed Central

    Bustos, M; Coffman, T M; Saadi, S; Platt, J L

    1997-01-01

    Lipid inflammatory mediators are thought to play a critical role in the pathogenesis of vascular injury. Among the events which might cause the synthesis of eicosanoids in blood vessels is activation of the complement. To evaluate how complement might influence eicosanoid metabolism, we investigated endothelial cells exposed to xenoreactive antibodies and complement, as might occur in rejecting xenografts where severe vascular injury is a typical feature. While resting porcine aortic endothelial cells released only prostaglandin (PG) I2, endothelial cells stimulated with xenoreactive antibodies and complement released PGE2 and thromboxane A2 (TXA2), in addition to increased amounts of PGI2. This alteration in eicosanoid metabolism was associated with induction of cyclooxygenase (Cox)-2 and thromboxane synthase, but not Cox-1. Unlike results seen in other systems, the upregulation of Cox-2 and the subsequent release of eicosanoids by endothelial cells was not directly induced by complement but rather required production of IL-1alpha, which acted on endothelial cells as an autocrine factor. Since eicosanoids have a potent effect on inflammation, vascular tone and platelet aggregation, we postulated that the abnormalities in eicosanoid release induced by xenoreactive antibodies and complement might provide one explanation for the vascular injury, focal ischemia, and thrombosis observed in acute vascular rejection and other vasculitides mediated by complement. PMID:9276732

  20. Novel mechanism of macrophage-mediated metastasis revealed in a zebrafish model of tumor development.

    PubMed

    Wang, Jian; Cao, Ziquan; Zhang, Xing-Mei; Nakamura, Masaki; Sun, Meili; Hartman, Johan; Harris, Robert A; Sun, Yuping; Cao, Yihai

    2015-01-15

    Cancer metastasis can occur at early stages of tumor development due to facilitative alterations in the tumor microenvironment. Although imaging techniques have considerably improved our understanding of metastasis, early events remain challenging to study due to the small numbers of malignant cells involved that are often undetectable. Using a novel zebrafish model to investigate this process, we discovered that tumor-associated macrophages (TAM) acted to facilitate metastasis by binding tumor cells and mediating their intravasation. Mechanistic investigations revealed that IL6 and TNF? promoted the ability of macrophages to mediate this step. M2 macrophages were particularly potent when induced by IL4, IL10, and TGF?. In contrast, IFN?-lipopolysaccharide-induced M1 macrophages lacked the capability to function in the same way in the model. Confirming these observations, we found that human TAM isolated from primary breast, lung, colorectal, and endometrial cancers exhibited a similar capability in invasion and metastasis. Taken together, our work shows how zebrafish can be used to study how host contributions can facilitate metastasis at its earliest stages, and they reveal a new macrophage-dependent mechanism of metastasis with possible prognostic implications. PMID:25492861

  1. Identification of a New Modulator of the Intercalated Disc in a Zebrafish Model of Arrhythmogenic Cardiomyopathy

    PubMed Central

    Asimaki, Angeliki; Kapoor, Sudhir; Plovie, Eva; Arndt, Anne Karin; Adams, Edward; Liu, ZhenZhen; James, Cynthia A.; Judge, Daniel P.; Calkins, Hugh; Churko, Jared; Wu, Joseph C.; MacRae, Calum A.; Kléber, André G.; Saffitz, Jeffrey E.

    2015-01-01

    Arrhythmogenic cardiomyopathy (ACM) is characterized by frequent cardiac arrhythmias. To elucidate the underlying mechanisms and discover potential chemical modifiers, we created a zebrafish model of ACM with cardiac myocyte–specific expression of the human 2057del2 mutation in the gene encoding plakoglobin. A high-throughput screen identified SB216763 as a suppressor of the disease phenotype. Early SB216763 therapy prevented heart failure and reduced mortality in the fish model. Zebrafish ventricular myocytes that expressed 2057del2 plakoglobin exhibited 70 to 80% reductions in INa and IK1 current densities, which were normalized by SB216763. Neonatal rat ventricular myocytes that expressed 2057del2 plakoglobin recapitulated pathobiological features seen in patients with ACM, all of which were reversed or prevented by SB216763. The reverse remodeling observed with SB216763 involved marked subcellular redistribution of plakoglobin, connexin 43, and Nav1.5, but without changes in their total cellular content, implicating a defect in protein trafficking to intercalated discs. In further support of this mechanism, we observed SB216763-reversible, abnormal subcellular distribution of SAP97 (a protein known to mediate forward trafficking of Nav1.5 and Kir2.1) in rat cardiac myocytes expressing 2057del2 plakoglobin and in cardiac myocytes derived from induced pluripotent stem cells from two ACM probands with plakophilin-2 mutations. These observations pinpoint aberrant trafficking of intercalated disc proteins as a central mechanism in ACM myocyte injury and electrical abnormalities. PMID:24920660

  2. Modeling nociception in zebrafish: a way forward for unbiased analgesic discovery.

    PubMed

    Curtright, Andrew; Rosser, Micaela; Goh, Shamii; Keown, Bailey; Wagner, Erinn; Sharifi, Jasmine; Raible, David W; Dhaka, Ajay

    2015-01-01

    Acute and chronic pain conditions are often debilitating, inflicting severe physiological, emotional and economic costs and affect a large percentage of the global population. However, the development of therapeutic analgesic agents based primarily on targeted drug development has been largely ineffective. An alternative approach to analgesic development would be to develop low cost, high throughput, untargeted animal based behavioral screens that model complex nociceptive behaviors in which to screen for analgesic compounds. Here we describe the development of a behavioral based assay in zebrafish larvae that is effective in identifying small molecule compounds with analgesic properties. In a place aversion assay, which likely utilizes supraspinal neuronal circuitry, individually arrayed zebrafish larvae show temperature-dependent aversion to increasing and decreasing temperatures deviating from rearing temperature. Modeling thermal hyperalgesia, the addition of the noxious inflammatory compound and TRPA1 agonist allyl isothiocyanate sensitized heat aversion and reversed cool aversion leading larvae to avoid rearing temperature in favor of otherwise acutely aversive cooler temperatures. We show that small molecules with known analgesic properties are able to inhibit acute and/or sensitized temperature aversion. PMID:25587718

  3. Both sunitinib and sorafenib are effective treatments for pheochromocytoma in a xenograft model.

    PubMed

    Denorme, M; Yon, L; Roux, C; Gonzalez, B J; Baudin, E; Anouar, Y; Dubessy, C

    2014-10-01

    Pheochromocytomas and paragangliomas are rare neuroendocrine tumors which develop from chromaffin cells of the adrenal medulla and extra-adrenal sites, leading to excess catecholamine release and hypertension. Many of the tumors are characterized by a high vascularity, suggesting the possible implementation of anti-angiogenic therapies for patients. Here, the efficacy of the tyrosine kinase inhibitors sunitinib and sorafenib was investigated in vivo and in vitro. Oral treatment with either sunitinib or sorafenib (40mg/kg/day) for 14days induced a marked reduction in the volume and weight of PC12 pheochromocytoma cell tumor xenografts in mice. Assessment of tumoral neo-angiogenesis, assessed by morphometric analysis of the vascular network after CD31 immunolabeling, showed that both sunitinib and sorafenib reduced the microvessel area (-85% and -80%, respectively) and length (-80% and -78%, respectively) in treated compared to control tumors. In addition, the number of vessel nodes was significantly lower in treated tumors (-95% and -84%, respectively). Furthermore, cleaved caspase 3 immunolabeling revealed a marked increase in the number of apoptotic cells in tumors from treated animals. Sunitinib and sorafenib could exert a direct effect on PC12 cell viability in vitro. While sunitinib induced a rapid (4h) and pronounced (5-fold) increase in caspase-3/7-dependent apoptosis, sorafenib seems to exert its cytotoxic activity through a different mechanism. Altogether, our data demonstrate that sunitinib and sorafenib have the ability to impair pheochromocytoma development by inhibiting angiogenesis and reducing tumor cell viability. These results strongly suggest that both sunitinib and sorafenib could represent valuable therapeutic tools for pheochromocytoma. PMID:25016061

  4. Effect of Melatonin on Tumor Growth and Angiogenesis in Xenograft Model of Breast Cancer

    PubMed Central

    Jardim-Perassi, Bruna Victorasso; Arbab, Ali S.; Ferreira, Lívia Carvalho; Borin, Thaiz Ferraz; Varma, Nadimpalli R. S.; Iskander, A. S. M.; Shankar, Adarsh; Ali, Meser M.; de Campos Zuccari, Debora Aparecida Pires

    2014-01-01

    As neovascularization is essential for tumor growth and metastasis, controlling angiogenesis is a promising tactic in limiting cancer progression. Melatonin has been studied for their inhibitory properties on angiogenesis in cancer. We performed an in vivo study to evaluate the effects of melatonin treatment on angiogenesis in breast cancer. Cell viability was measured by MTT assay after melatonin treatment in triple-negative breast cancer cells (MDA-MB-231). After, cells were implanted in athymic nude mice and treated with melatonin or vehicle daily, administered intraperitoneally 1 hour before turning the room light off. Volume of the tumors was measured weekly with a digital caliper and at the end of treatments animals underwent single photon emission computed tomography (SPECT) with Technetium-99m tagged vascular endothelial growth factor (VEGF) C to detect in vivo angiogenesis. In addition, expression of pro-angiogenic/growth factors in the tumor extracts was evaluated by membrane antibody array and collected tumor tissues were analyzed with histochemical staining. Melatonin in vitro treatment (1 mM) decreased cell viability (p<0.05). The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size and cell proliferation (Ki-67) compared to control animals after 21 days of treatment (p<0.05). Expression of VEGF receptor 2 decreased significantly in the treated animals compared to that of control when determined by immunohistochemistry (p<0.05) but the changes were not significant on SPECT (p>0.05) images. In addition, there was a decrease of micro-vessel density (Von Willebrand Factor) in melatonin treated mice (p<0.05). However, semiquantitative densitometry analysis of membrane array indicated increased expression of epidermal growth factor receptor and insulin-like growth factor 1 in treated tumors compared to vehicle treated tumors (p<0.05). In conclusion, melatonin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis. PMID:24416386

  5. Validation of visualized transgenic zebrafish as a high throughput model to assay bradycardia related cardio toxicity risk candidates.

    PubMed

    Wen, Dingsheng; Liu, Aiming; Chen, Feng; Yang, Julin; Dai, Renke

    2012-10-01

    Drug-induced QT prolongation usually leads to torsade de pointes (TdP), thus for drugs in the early phase of development this risk should be evaluated. In the present study, we demonstrated a visualized transgenic zebrafish as an in vivo high-throughput model to assay the risk of drug-induced QT prolongation. Zebrafish larvae 48 h post-fertilization expressing green fluorescent protein in myocardium were incubated with compounds reported to induce QT prolongation or block the human ether-a-go-go-related gene (hERG) K? current. The compounds sotalol, indapaminde, erythromycin, ofoxacin, levofloxacin, sparfloxacin and roxithromycin were additionally administrated by microinjection into the larvae yolk sac. The ventricle heart rate was recorded using the automatic monitoring system after incubation or microinjection. As a result, 14 out of 16 compounds inducing dog QT prolongation caused bradycardia in zebrafish. A similar result was observed with 21 out of 26 compounds which block hERG current. Among the 30 compounds which induced human QT prolongation, 25 caused bradycardia in this model. Thus, the risk of compounds causing bradycardia in this transgenic zebrafish correlated with that causing QT prolongation and hERG K? current blockage in established models. The tendency that high logP values lead to high risk of QT prolongation in this model was indicated, and non-sensitivity of this model to antibacterial agents was revealed. These data suggest application of this transgenic zebrafish as a high-throughput model to screen QT prolongation-related cardio toxicity of the drug candidates. PMID:22744888

  6. Prey capture in zebrafish larvae serves as a model to study cognitive functions

    PubMed Central

    Muto, Akira; Kawakami, Koichi

    2013-01-01

    Prey capture in zebrafish larvae is an innate behavior which can be observed as early as 4~days postfertilization, the day when they start to swim. This simple behavior apparently involves several neural processes including visual perception, recognition, decision-making, and motor control, and, therefore, serves as a good model system to study cognitive functions underlying natural behaviors in vertebrates. Recent progresses in imaging techniques provided us with a unique opportunity to image neuronal activity in the brain of an intact fish in real-time while the fish perceives a natural prey, paramecium. By expanding this approach, it would be possible to image entire brain areas at a single-cell resolution in real-time during prey capture, and identify neuronal circuits important for cognitive functions. Further, activation or inhibition of those neuronal circuits with recently developed optogenetic tools or neurotoxins should shed light on their roles. Thus, we will be able to explore the prey capture in zebrafish larvae more thoroughly at cellular levels, which should establish a basis of understanding of the cognitive function in vertebrates. PMID:23781176

  7. Vitamin K reduces hypermineralisation in zebrafish models of PXE and GACI.

    PubMed

    Mackay, Eirinn W; Apschner, Alexander; Schulte-Merker, Stefan

    2015-03-15

    The mineralisation disorder pseudoxanthoma elasticum (PXE) is associated with mutations in the transporter protein ABCC6. Patients with PXE suffer from calcified lesions in the skin, eyes and vasculature, and PXE is related to a more severe vascular calcification syndrome called generalised arterial calcification of infancy (GACI). Mutations in ABCC6 are linked to reduced levels of circulating vitamin K. Here, we describe a mutation in the zebrafish (Danio rerio) orthologue abcc6a, which results in extensive hypermineralisation of the axial skeleton. Administration of vitamin K to embryos was sufficient to restore normal levels of mineralisation. Vitamin K also reduced ectopic mineralisation in a zebrafish model of GACI, and warfarin exacerbated the mineralisation phenotype in both mutant lines. These data suggest that vitamin K could be a beneficial treatment for human patients with PXE or GACI. Additionally, we found that abcc6a is strongly expressed at the site of mineralisation rather than the liver, as it is in mammals, which has significant implications for our understanding of the function of ABCC6. PMID:25758222

  8. From Omics to Drug Metabolism and High Content Screen of Natural Product in Zebrafish: A New Model for Discovery of Neuroactive Compound

    PubMed Central

    Hung, Ming Wai; Zhang, Zai Jun; Li, Shang; Lei, Benson; Yuan, Shuai; Cui, Guo Zhen; Man Hoi, Pui; Chan, Kelvin; Lee, Simon Ming Yuen

    2012-01-01

    The zebrafish (Danio rerio) has recently become a common model in the fields of genetics, environmental science, toxicology, and especially drug screening. Zebrafish has emerged as a biomedically relevant model for in vivo high content drug screening and the simultaneous determination of multiple efficacy parameters, including behaviour, selectivity, and toxicity in the content of the whole organism. A zebrafish behavioural assay has been demonstrated as a novel, rapid, and high-throughput approach to the discovery of neuroactive, psychoactive, and memory-modulating compounds. Recent studies found a functional similarity of drug metabolism systems in zebrafish and mammals, providing a clue with why some compounds are active in zebrafish in vivo but not in vitro, as well as providing grounds for the rationales supporting the use of a zebrafish screen to identify prodrugs. Here, we discuss the advantages of the zebrafish model for evaluating drug metabolism and the mode of pharmacological action with the emerging omics approaches. Why this model is suitable for identifying lead compounds from natural products for therapy of disorders with multifactorial etiopathogenesis and imbalance of angiogenesis, such as Parkinson's disease, epilepsy, cardiotoxicity, cerebral hemorrhage, dyslipidemia, and hyperlipidemia, is addressed. PMID:22919414

  9. Identification of anti-prion compounds as efficient inhibitors of polyglutamine protein aggregation in a zebrafish model.

    PubMed

    Schiffer, Niclas W; Broadley, Sarah A; Hirschberger, Thomas; Tavan, Paul; Kretzschmar, Hans A; Giese, Armin; Haass, Christian; Hartl, F Ulrich; Schmid, Bettina

    2007-03-23

    Several neurodegenerative diseases, including Huntington disease (HD), are associated with aberrant folding and aggregation of polyglutamine (polyQ) expansion proteins. Here we established the zebrafish, Danio rerio, as a vertebrate HD model permitting the screening for chemical suppressors of polyQ aggregation and toxicity. Upon expression in zebrafish embryos, polyQ-expanded fragments of huntingtin (htt) accumulated in large SDS-insoluble inclusions, reproducing a key feature of HD pathology. Real time monitoring of inclusion formation in the living zebrafish indicated that inclusions grow by rapid incorporation of soluble htt species. Expression of mutant htt increased the frequency of embryos with abnormal morphology and the occurrence of apoptosis. Strikingly, apoptotic cells were largely devoid of visible aggregates, suggesting that soluble oligomeric precursors may instead be responsible for toxicity. As in nonvertebrate polyQ disease models, the molecular chaperones, Hsp40 and Hsp70, suppressed both polyQ aggregation and toxicity. Using the newly established zebrafish model, two compounds of the N'-benzylidene-benzohydrazide class directed against mammalian prion proved to be potent inhibitors of polyQ aggregation, consistent with a common structural mechanism of aggregation for prion and polyQ disease proteins. PMID:17170113

  10. A Novel Zebrafish Model to Provide Mechanistic Insights into the Inflammatory Events in Carrageenan-Induced Abdominal Edema

    PubMed Central

    Huang, Shi-Ying; Feng, Chien-Wei; Hung, Han-Chun; Chakraborty, Chiranjib; Chen, Chun-Hong; Chen, Wu-Fu; Jean, Yen-Hsuan; Wang, Hui-Min David; Sung, Chun-Sung; Sun, Yu-Min; Wu, Chang-Yi; Liu, Wangta; Hsiao, Chung-Der; Wen, Zhi-Hong

    2014-01-01

    A suitable small animal model may help in the screening and evaluation of new drugs, especially those from natural products, which can be administered at lower dosages, fulfilling an urgent worldwide need. In this study, we explore whether zebrafish could be a model organism for carrageenan-induced abdominal edema. The research results showed that intraperitoneal (i.p.) administration of 1.5% ?-carrageenan in a volume of 20 µL significantly increased abdominal edema in adult zebrafish. Levels of the proinflammatory proteins tumor necrosis factor-? (TNF-?) and inducible nitric oxide synthase (iNOS) were increased in carrageenan-injected adult zebrafish during the development of abdominal edema. An associated enhancement was also observed in the leukocyte marker, myeloperoxidase (MPO). To support these results, we further observed that i.p. methylprednisolone (MP; 1 µg), a positive control, significantly inhibited carrageenan-induced inflammation 24 h after carrageenan administration. Furthermore, i.p. pretreatment with either an anti-TNF-? antibody (1?5 dilution in a volume of 20 µL) or the iNOS-selective inhibitor aminoguanidine (AG; 1 µg) inhibited carrageenan-induced abdominal edema in adult zebrafish. This new animal model is uncomplicated, easy to develop, and involves a straightforward inducement of inflammatory edema for the evaluation of small volumes of drugs or test compounds. PMID:25141004

  11. Delayed xenograft rejection

    Microsoft Academic Search

    Fritz H. Bach; Hans Winkler; Christiane Ferran; Wayne W. Hancock; Simon C. Robson

    1996-01-01

    Despite considerable progress in understanding the mechanisms of discordant xenograft rejection, and overcoming hyperacute rejection through targeting of complement or antibody, vascularized xenografts are typically rejected within days. Here, Fritz Bach and colleagues discuss the importance of endothelial cell activation, platelet aggregation and other aspects of thrombosis, as well as the contribution of host natural killer cell and monocyte activation

  12. Hydrogel-Based 3D Model of Patient-Derived Prostate Xenograft Tumors Suitable for Drug Screening

    PubMed Central

    2015-01-01

    The lack of effective therapies for bone metastatic prostate cancer (PCa) underscores the need for accurate models of the disease to enable the discovery of new therapeutic targets and to test drug sensitivities of individual tumors. To this end, the patient-derived xenograft (PDX) PCa model using immunocompromised mice was established to model the disease with greater fidelity than is possible with currently employed cell lines grown on tissue culture plastic. However, poorly adherent PDX tumor cells exhibit low viability in standard culture, making it difficult to manipulate these cells for subsequent controlled mechanistic studies. To overcome this challenge, we encapsulated PDX tumor cells within a three-dimensional hyaluronan-based hydrogel and demonstrated that the hydrogel maintains PDX cell viability with continued native androgen receptor expression. Furthermore, a differential sensitivity to docetaxel, a chemotherapeutic drug, was observed as compared to a traditional PCa cell line. These findings underscore the potential impact of this novel 3D PDX PCa model as a diagnostic platform for rapid drug evaluation and ultimately push personalized medicine toward clinical reality. PMID:24779589

  13. Modeling Stress and Anxiety in Zebrafish Jonathan M. Cachat, Peter R. Canavello, Marco F. Elegante,

    E-print Network

    Kalueff, Allan V.

    and chronic anxiety. Key words: Novel environment, video-aided analysis, stress, anxiety, fear, affective, zebrafish possess all of the "classical" neurotransmitters found in vertebrates (11), suggesting

  14. Novel Mouse Xenograft Models Reveal a Critical Role of CD4+ T Cells in the Proliferation of EBV-Infected T and NK Cells

    Microsoft Academic Search

    Ken-Ichi Imadome; Misako Yajima; Ayako Arai; Atsuko Nakazawa; Fuyuko Kawano; Sayumi Ichikawa; Norio Shimizu; Naoki Yamamoto; Tomohiro Morio; Shouichi Ohga; Hiroyuki Nakamura; Mamoru Ito; Osamu Miura; Jun Komano; Shigeyoshi Fujiwara

    2011-01-01

    Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic herpesvirus, ectopically infects T or NK cells to cause severe diseases of unknown pathogenesis, including chronic active EBV infection (CAEBV) and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). We developed xenograft models of CAEBV and EBV-HLH by transplanting patients' PBMC to immunodeficient mice of the NOD\\/Shi-scid\\/IL-2R?null strain. In these models, EBV-infected T, NK, or B cells proliferated

  15. Comprehensive characterization of chemotherapeutic efficacy on metastases in the established gastric neuroendocrine cancer patient derived xenograft model.

    PubMed

    Jiang, Jiahua; Wang, Daisy Dandan; Yang, Mengmeng; Chen, Dawei; Pang, Liang; Guo, Sheng; Cai, Jie; Wery, Jean-Pierre; Li, Linda; Li, Henry Qixiang; Lin, Peter Ping

    2015-06-20

    The HuPrime® human gastric neuroendocrine carcinoma derived xenograft model GA0087 was established in this study. GA0087 PDX model showed high gene expression of vascular endothelial growth factors (VEGF)-A and B, and high potential of lung metastasis. Circulating tumor cells (CTCs) with either large or small size, circulating tumor microemboli (CTM) and lung metastatic lesions were detected in GA0087 PDX mice. The number of CTC correlated to the number of metastatic nodules in lung. Both primary tumor growth and metastasis in terms of the number of dynamically monitored CTCs and metastatic nodules were effectively suppressed by Cisplatin. Diverse subtypes of CTCs in the context of sensitivity to Cisplatin were specifically identified by subtraction enrichment (SE) integrated with in situ Phenotyping of cytokeratin 18 (CK18) and Karyotyping of chromosome 8 (in situ PK CTC by CK-iFISH). All the CK18-/diploid and majority of CK18+/diploid CTC subtypes were chemosensitive, whereas a higher percentage of CK18+/multiploid subtype of CTC were Cisplatin-insensitive. Combined histopathological examination of metastatic lesion and in situ PK CTC in a metastatic PDX (mPDX) tumor model are of particular significance, and may provide an unique and robust platform for cancer research as well as pre-clinical evaluation of therapeutic efficacy of new anti-cancer drugs. PMID:25909226

  16. Emerging applications of zebrafish as a model organism to study oxidative mechanisms and their role in inflammation and vascular accumulation of oxidized lipids

    PubMed Central

    Fang, Longhou; Miller, Yury I.

    2012-01-01

    With the advent of genetic engineering, zebrafish (Danio rerio) were recognized as an attractive model organism to study many biological processes. Remarkably, the small size and optical transparency of zebrafish larvae enable high-resolution imaging of live animals. Zebrafish respond to various environmental and pathological factors with robust oxidative stress. In this article, we provide an overview of molecular mechanisms involved in oxidative stress and antioxidant response in zebrafish. Existing applications of generically-encoded fluorescent sensors allow imaging, in real time, production of H2O2 and studying its involvement in inflammatory responses, as well as activation of oxidation-sensitive transcription factors HIF and NRF2. Oxidative stress, combined with hypelipidemia, leads to oxidation of lipoproteins, the process that contributes significantly to development of human atherosclerosis. Recent work found that feeding zebrafish a high-cholesterol diet results in hypercholesterolemia, vascular lipid accumulation and extreme lipoprotein oxidation. Generation of a transgenic zebrafish expressing a GFP-tagged human antibody to malondialdehyde (MDA)-modified LDL makes possible in vivo visualization of MDA epitopes in the vascular wall and testing the efficacy of antioxidants and dietary interventions. Thus, using zebrafish as a model organism provides important advantages in studying the role of ROS and lipid oxidation in basic biologic and pathologic processes. PMID:22906686

  17. Metallofullerene-based Nanoplatform for Brain Tumor Brachytherapy and Longitudinal Imaging in a Murine Orthotopic Xenograft Model

    PubMed Central

    Shultz, Michael D.; Wilson, John D.; Fuller, Christine E.; Zhang, Jianyuan; Dorn, Harry C.

    2011-01-01

    Purpose: To demonstrate in an orthotopic xenograft brain tumor model that a functionalized metallofullerene (f-Gd3N@C80) can enable longitudinal tumor imaging and, when radiolabeled with lutetium 177 (177Lu) and tetraazacyclododecane tetraacetic acid (DOTA) (177Lu-DOTA-f-Gd3N@C80), provide an anchor to deliver effective brachytherapy. Materials and Methods: All experiments involving the use of mice were carried out in accordance with protocols approved by the institutional animal care and use committee. Human glioblastoma U87MG cells were implanted by using stereotactic procedures into the brains of 37 female athymic nude-Foxn1nu mice and allowed to develop into a tumor for 8 days. T1- and T2-weighted magnetic resonance (MR) imaging was performed in five mice. Biodistribution studies were performed in 12 mice at four time points over 7 days to evaluate gadolinium content. Survival studies involved 20 mice that received infusion of a nanoplatform by means of convection-enhanced delivery (CED) 8 days after tumor implantation. Mice in survival studies were divided into two groups: one comprised untreated mice that received f-Gd3N@C80 alone and the other comprised mice treated with brachytherapy that received 1.11 MBq of 177Lu-DOTA-f-Gd3N@C80. Survival data were evaluated by using Kaplan-Meier statistical methods. Results: MR imaging showed extended tumor retention (25.6% ± 1.2 of the infused dose at 52 days, confirmed with biodistribution studies) of the f-Gd3N@C80 nanoplatform, which enabled longitudinal imaging. Successful coupling of 177Lu to the f-Gd3N@C80 surface was achieved by using a bifunctional macrocyclic chelator. The extended tumor retention allowed for effective brachytherapy, as indicated by extended survival time (>2.5 times that of the untreated group) and histologic signs of radiation-induced tumor damage. Conclusion: The authors have developed a multimodal nanoplatform and have demonstrated longitudinal tumor imaging, prolonged intratumoral probe retention, biodistribution, and extended survival in an orthotopic xenograft brain tumor model. © RSNA, 2011 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.11102569/-/DC1 PMID:21813738

  18. Quantitative Phenotyping-Based In Vivo Chemical Screening in a Zebrafish Model of Leukemia Stem Cell Xenotransplantation

    PubMed Central

    Zhang, Beibei; Shimada, Yasuhito; Kuroyanagi, Junya; Umemoto, Noriko; Nishimura, Yuhei; Tanaka, Toshio

    2014-01-01

    Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs) is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo) and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro), the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+) cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange) and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine). Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs. PMID:24454867

  19. A novel AMPK activator reduces glucose uptake and inhibits tumor progression in a mouse xenograft model of colorectal cancer.

    PubMed

    Valtorta, Silvia; Nicolini, Gabriella; Tripodi, Farida; Meregalli, Cristina; Cavaletti, Guido; Avezza, Federica; Crippa, Luca; Bertoli, Gloria; Sanvito, Francesca; Fusi, Paola; Pagliarin, Roberto; Orsini, Fulvia; Moresco, Rosa Maria; Coccetti, Paola

    2014-12-01

    The anticancer activity of a novel pure 1,4-Diaryl-2-azetidinone (1), endowed with a higher solubility than the well known Combretastatin A4, is tested in mice. We previously reported that Compound (1) showed specific antiproliferative activity against duodenal and colon cancer cells, inducing activation of AMP-activated protein kinase and apoptosis. Here we estimate that the maximum tolerated dose in a mouse model is 40 mg/kg; the drug is well tolerated both in single dose and in repeated administration schedules. The drug displays a significant antitumor activity and a tumor growth delay when administered at the MTD both in single and fractionated i.v. administration in a mouse xenograft model of colorectal cancer. Arrest of tumor growth and relapse after drug suspension are parallel to modification in glucose demand as shown by PET studies with [(18)F] FDG. These data strongly support Compound (1) as a promising molecule for in vivo treatment of colorectal cancer. PMID:25134489

  20. The isothiocyanate erucin abrogates telomerase in hepatocellular carcinoma cells in vitro and in an orthotopic xenograft tumour model of HCC

    PubMed Central

    Herz, Corinna; Hertrampf, Anke; Zimmermann, Stefan; Stetter, Nadine; Wagner, Meike; Kleinhans, Claudia; Erlacher, Miriam; Schüler, Julia; Platz, Stefanie; Rohn, Sascha; Mersch-Sundermann, Volker; Lamy, Evelyn

    2014-01-01

    In contrast to cancer cells, most normal human cells have no or low telomerase levels which makes it an attractive target for anti-cancer drugs. The small molecule sulforaphane from broccoli is known for its cancer therapeutic potential in vitro and in vivo. In animals and humans it was found to be quickly metabolized into 4-methylthiobutyl isothiocyanate (MTBITC, erucin) which we recently identified as strong selective apoptosis inducer in hepatocellular carcinoma (HCC) cells. Here, we investigated the relevance of telomerase abrogation for cytotoxic efficacy of MTBITC against HCC. The drug was effective against telomerase, independent from TP53 and MTBITC also blocked telomerase in chemoresistant subpopulations. By using an orthotopic human liver cancer xenograft model, we give first evidence that MTBITC at 50 mg/KG b.w./d significantly decreased telomerase activity in vivo without affecting enzyme activity of adjacent normal tissue. Upon drug exposure, telomerase decrease was consistent with a dose-dependent switch to anti-survival, cell arrest and apoptosis in our in vitro HCC models. Blocking telomerase by the specific inhibitor TMPyP4 further sensitized cancer cells to MTBITC-mediated cytotoxicity. Overexpression of hTERT, but not enzyme activity deficient DNhTERT, protected against apoptosis; neither DNA damage nor cytostasis induction by MTBITC was prevented by hTERT overexpression. These findings imply that telomerase enzyme activity does not protect against MTBITC-induced DNA damage but impacts signalling processes upstream of apoptosis execution level. PMID:25256442

  1. A novel cytidine analog, RX-3117, shows potent efficacy in xenograft models, even in tumors that are resistant to gemcitabine.

    PubMed

    Yang, Mi Young; Lee, Young Bok; Ahn, Chang-Ho; Kaye, Joel; Fine, Tania; Kashi, Rina; Ohne, Osnat; Smid, Kees; Peters, Godefridus J; Kim, Deog Joong

    2014-12-01

    RX-3117 (fluorocyclopentenylcytosine) is a cytidine analog and this class of drugs, including gemcitabine, has been widely used for the treatment of various types of cancers. However, there is no oral formulation of gemcitabine and drug resistance to gemcitabine is common. In this study, the efficacy of orally-administered RX-3117 was examined in 9 different human tumor xenograft models (colon, non-small cell lung, small cell lung, pancreatic, renal and cervical), grown subcutaneously in athymic nude mice. In the Colo 205, H460, H69 and CaSki models, gemcitabine treatment resulted in 28%, 30%, 25% and 0% tumor growth inhibition (TGI), respectively, whereas oral treatment with RX-3117 induced 100%, 78%, 62% and 66% TGI, respectively. This indicates that RX-3117 may have the potential to be used for the treatment of tumors that do not respond to gemcitabine. RX-3117 was also evaluated in a single primary low-passage human pancreatic Tumorgraft™CTG-0298 (TGI 76%), which is relatively resistant to gemcitabine (TGI 38%) and has a favorable RX-3117-activating enzyme profile. These studies demonstrated the therapeutic potential and anticancer efficacy of RX-3117. PMID:25503121

  2. Using Zebrafish to Study Renal Regeneration

    PubMed Central

    McCampbell, Kristen K.; Wingert, Rebecca A.

    2013-01-01

    Over the past several decades, the zebrafish has become one of the major vertebrate model organisms used in biomedical research. In this arena, the zebrafish has emerged as an applicable system for the study of kidney diseases and renal regeneration. The relevance of the zebrafish model for nephrology research has been increasingly appreciated as the understanding of zebrafish kidney structure, ontogeny, and the response to damage has steadily expanded. Recent studies have documented the amazing regenerative characteristics of the zebrafish kidney, which include the ability to replace epithelial populations after acute injury and to grow new renal functional units, termed nephrons. Here we discuss how nephron composition is conserved between zebrafish and mammals, and highlight how recent findings from zebrafish studies utilizing transgenic technologies and chemical genetics can complement traditional murine approaches in the effort to dissect how the kidney responds to acute damage and identify therapeutics that enhance human renal regeneration. PMID:24183931

  3. Mapping of Zebrafish Research: A Global Outlook

    PubMed Central

    Mahesh, Gopalakrishnan; Panwar, Yatish

    2013-01-01

    Abstract On the basis of analysis of 17,151 records on zebrafish identified from Zebrafish Information Network: the zebrafish model organism database and Web of Science, the research performance on this model organism has been evaluated. The earliest research work on zebrafish as reflected in the databases goes back to 1951. After a rather slow growth till the 1980s, research on zebrafish gained momentum in the 1990s. Analysis shows a rapid and consistent increase in the publication output with 226 publications in the year 1996, to 1929 publications in the year 2012. The prominent areas of zebrafish research, journals, and leading authors as reflected from the research output have been identified. USA is the most productive country with 8196 articles. The most frequently used keywords were also determined to gain insights about the research trends and some of the commonly used keywords other than zebrafish and Danio rerio are development, retina, and gene expression. PMID:24131434

  4. Identification, Modeling and Ligand Affinity of Early Deuterostome CYP51s, and Functional Characterization of Recombinant Zebrafish Sterol 14?-Demethylase

    PubMed Central

    Morrison, Ann Michelle Stanley; Goldstone, Jared V.; Lamb, David C.; Kubota, Akira; Lemaire, Benjamin; Stegeman, John. J.

    2014-01-01

    Background Sterol 14?-demethylase (cytochrome P450 51, CYP51, P45014DM) is a microsomal enzyme that in eukaryotes catalyzes formation of sterols essential for cell membrane function and as precursors in biosynthesis of steroid hormones. Functional properties of CYP51s are unknown in non-mammalian deuterostomes. Methods PCR-cloning and sequencing and computational analyses (homology modeling and docking) addressed CYP51 in zebrafish Danio rerio, the reef fish sergeant major Abudefduf saxatilis, and the sea urchin Strongylocentrotus purpuratus. Following N-terminal amino acid modification, zebrafish CYP51 was expressed in Escherichia coli, and lanosterol 14?-demethylase activity and azole inhibition of CYP51 activity were characterized using GC/MS. Results Molecular phylogeny positioned S. purpuratus CYP51 at the base of the deuterostome clade. In zebrafish, CYP51 is expressed in all organs examined, most strongly in intestine. The recombinant protein bound lanosterol and catalyzed 14?-demethylase activity, at 3.2 nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave KS (dissociation constant) values of 0.26 ?M for ketoconazole and 0.64 ?M for propiconazole. Displacement of carbon monoxide also indicated zebrafish CYP51 has greater affinity for ketoconazole. Docking to homology models showed that lanosterol docks in fish and sea urchin CYP51s with an orientation essentially the same as in mammalian CYP51. Docking of ketoconazole indicates it would inhibit fish and sea urchin CYP51s. Conclusions Biochemical and computational analyses are consistent with lanosterol being a substrate for early deuterostome CYP51s. General Significance The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications. PMID:24361620

  5. The search for evolutionary developmental origins of aging in zebrafish: a novel intersection of developmental and senescence biology in the zebrafish model system.

    PubMed

    Kishi, Shuji

    2011-09-01

    Senescence may be considered the antithesis of early development, but yet there may be factors and mechanisms in common between these two phenomena during the process of aging. We investigated whether any relationship exists between the regulatory mechanisms that function in early development and in senescence using the zebrafish (Danio rerio), a small freshwater fish and a useful model animal for genetic studies. We conducted experiments to isolate zebrafish mutants expressing an apparent senescence phenotype during embryogenesis (embryonic senescence). Some of the genes we thereby identified had already been associated with cellular senescence and chronological aging in other organisms, but many had not yet been linked to these processes. Complete loss-of-function of developmentally essential genes induce embryonic (or larval) lethality, whereas it seems like their partial loss-of-function (i.e., decrease-of-function by heterozygote or hypomorphic mutations) still remains sufficient to go through the early developmental process because of its adaptive plasticity or rather heterozygote advantage. However, in some cases, such partial loss-of-function of genes compromise normal homeostasis due to haploinsufficiency later in adult life having many environmental stress challenges. By contrast, any heterozygote-advantageous genes might gain a certain benefit(s) (much more fitness) by such partial loss-of-function later in life. Physiological senescence may evolutionarily arise from both genetic and epigenetic drifts as well as from losing adaptive developmental plasticity in face of stress signals from the external environment that interacts with functions of multiple genes rather than effects of only a single gene mutation or defect. Previously uncharacterized developmental genes may thus mediate the aging process and play a pivotal role in senescence. Moreover, unexpected senescence-related genes might also be involved in the early developmental process and regulation. We wish to ascertain whether we can identify such genes promptly in a comprehensive manner. The ease of manipulation using the zebrafish system allows us to conduct an exhaustive exploration of novel genes and small molecular compounds that can be linked to the senescence phenotype and thereby facilitates searching for the evolutionary and developmental origins of aging in vertebrates. PMID:21932432

  6. Transforming Growth Factor-B Can Suppress Tumorigenesis through Effects on the Putative Cancer Stem or Early Progenitor Cell and Committed Progeny in a Breast Cancer Xenograft Model

    Microsoft Academic Search

    Binwu Tang; Naomi Yoo; Mary Vu; Mizuko Mamura; Jeong-Seok Nam; Akira Ooshima; Zhijun Du; Pierre-Yves Desprez; Miriam R. Anver; Aleksandra M. Michalowska; Joanna Shih; W. Tony Parks; Lalage M. Wakefield

    The transforming growth factor-B (TGF-B) pathway has tumor-suppressor activity in many epithelial tissues. Because TGF-B is a potent inhibitor of epithelial cell proliferation, it has been widely assumed that this property underlies the tumor-suppressor effect. Here, we have used a xenograft model of breast cancer to show that endogenous TGF-B has the potential to suppress tumorigenesis through a novel mechanism,

  7. Therapeutic potential of chimeric anti-(ganglioside GD3) antibody KM871: antitumor activity in xenograft model of melanoma and effector function analysis

    Microsoft Academic Search

    Junji Kanazawa; So Ohta; Fumiko Fujita; Masahide Fujita; Nobuo Hanai; Shiro Akinaga; Masami Okabe

    2000-01-01

    KM871 is a chimeric antibody recognizing ganglioside GD3, which is one of the major gangliosides expressed on the cell surface\\u000a of human tumors of neuroectodermal origin. This study demonstrates the antitumor activity of KM871 against human melanoma\\u000a xenografts in nude mice, and analyzes the effector function operating in mice. In a well-established tumor model, KM871 showed\\u000a antitumor activity against H-15

  8. Three-dimensional MR mapping of angiogenesis with  5 1(   3)-targeted theranostic nanoparticles in the MDA-MB-435 xenograft mouse model

    Microsoft Academic Search

    Anne H. Schmieder; Shelton D. Caruthers; Huiying Zhang; Todd A. Williams; J. David Robertson; Samuel A. Wickline; Gregory M. Lanza

    2008-01-01

    Our objectives were 1) to characterize angiogenesis in the MDA-MB-435 xenograft mouse model with three-dimensional (3D) MR molecular im- aging using 51(RGD)- or irrelevant RGS-targeted paramagnetic nanoparticles and 2) to use MR molecu- lar imaging to assess the antiangiogenic effectiveness of 51(3)- vs. 3-targeted fumagillin (50 g\\/kg) nanoparticles. Tumor-bearing mice were imaged with MR before and after administration of either

  9. Survivin-targeting Artificial MicroRNAs Mediated by Adenovirus Suppress Tumor Activity in Cancer Cells and Xenograft Models

    PubMed Central

    Chi, Yudan; Wang, Xiang; Yang, Yong; Zhang, Chao; Ertl, Hildegund C J; Zhou, Dongming

    2014-01-01

    Survivin is highly expressed in most human tumors and fetal tissue, and absent in terminally differentiated cells. It promotes tumor cell proliferation by negatively regulating cell apoptosis and facilitating cell division. Survivin's selective expression pattern suggests that it might be a suitable target for cancer therapy, which would promote death of transformed but not normal cells. This was tested using artificial microRNAs (amiRNAs) targeting survivin. After screening, two effective amiRNAs, which knocked down survivin expression, were identified and cloned into a replication-defective adenoviral vector. Tumor cells infected with the recombinant vector downregulated expression of survivin and underwent apoptotic cell death. Further studies showed that apoptosis was associated with increases in caspase 3 and cleaved Poly (ADP-ribose) polymerase, and activation of the p53 signaling pathway. Furthermore, amiRNA treatment caused blockade of mitosis and cell cycle arrest at the G2/M phase. In vivo, survivin-targeting amiRNAs expressed by adenoviral vectors effectively delayed growth of hepatocellular and cervical carcinomas in mouse xenograft models. These results indicate that silencing of survivin by amiRNA has potential for treatment of cancer. PMID:25368912

  10. Triptolide and its prodrug minnelide suppress Hsp70 and inhibit in vivo growth in a xenograft model of mesothelioma.

    PubMed

    Jacobson, Blake A; Chen, Esther Z; Tang, Shaogeng; Belgum, Holly Sedgwick; McCauley, Joel A; Evenson, Kristen A; Etchison, Ryan G; Jay-Dixon, Joe; Patel, Manish R; Raza, Ahmad; Saluja, Ashok K; D'Cunha, Jonathan; Kratzke, Robert A

    2015-03-01

    Malignant mesothelioma is a devastating disease with a poor prognosis for which there is a clear need for more successful therapeutic approaches. Triptolide, a diterpenoid triepoxide, is a highly effective agent against several cancer types in animal models. Owing to triptolide's poor solubility in water, a water-soluble analog, minnelide, was synthesized. Minnelide is a prodrug of triptolide and is activated by exposure to phosphatases that are found in all body tissues, including blood. Mesothelioma cells were treated in vitro with minnelide or its parent compound, triptolide. Minnelide and triptolide were both found to significantly reduce mesothelioma cell viability and induce apoptosis. The level of Hsp70, a protein that promotes cancer cell survival, was measured in mesothelioma cells before and after treatment with triptolide. Hsp70 levels were decreased in a dose-dependent manner. In addition, triptolide sensitized cells to gemcitabine and pemetrexed as measured by cell viability. Mice bearing mesothelioma flank tumors were treated with daily injections (28 d) of minnelide or saline solution and xenograft tumor growth recorded. Mice displayed significantly reduced tumor burden. These findings support the clinical evaluation of minnelide therapy for mesothelioma. PMID:26000097

  11. Zoledronic acid reduces bone loss and tumor growth in an orthotopic xenograft model of osteolytic oral squamous cell carcinoma.

    PubMed

    Martin, Chelsea K; Werbeck, Jillian L; Thudi, Nanda K; Lanigan, Lisa G; Wolfe, Tobie D; Toribio, Ramiro E; Rosol, Thomas J

    2010-11-01

    Squamous cell carcinoma (SCC) is the most common form of oral cancer. Destruction and invasion of mandibular and maxillary bone frequently occurs and contributes to morbidity and mortality. We hypothesized that the bisphosphonate drug zoledronic acid (ZOL) would inhibit tumor-induced osteolysis and reduce tumor growth and invasion in a murine xenograft model of bone-invasive oral SCC (OSCC) derived from an osteolytic feline OSCC. Luciferase-expressing OSCC cells (SCCF2Luc) were injected into the perimaxillary subgingiva of nude mice, which were then treated with 100 ?g/kg ZOL or vehicle. ZOL treatment reduced tumor growth and prevented loss of bone volume and surface area but had no effect on tumor invasion. Effects on bone were associated with reduced osteolysis and increased periosteal new bone formation. ZOL-mediated inhibition of tumor-induced osteolysis was characterized by reduced numbers of tartrate-resistant acid phosphatase-positive osteoclasts at the tumor-bone interface, where it was associated with osteoclast vacuolar degeneration. The ratio of eroded to total bone surface was not affected by treatment, arguing that ZOL-mediated inhibition of osteolysis was independent of effects on osteoclast activation or initiation of bone resorption. In summary, our results establish that ZOL can reduce OSCC-induced osteolysis and may be valuable as an adjuvant therapy in OSCC to preserve mandibular and maxillary bone volume and function. PMID:20959474

  12. Anti-Cancer Effect of Ginsenoside F2 against Glioblastoma Multiforme in Xenograft Model in SD Rats.

    PubMed

    Shin, Ji Yon; Lee, Jung Min; Shin, Heon Sub; Park, Sang Yong; Yang, Jung Eun; Cho, Somi Kim; Yi, Tae-Hoo

    2012-01-01

    The glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. Despite combination treatments of radiation and chemotherapy, the survival periods are very short. Therefore, this study was conducted to assess the potential of ginsenoside F2 (F2) to treat GBM. In in vitro experiments with glioblastoma cells U373MG, F2 showed the cytotoxic effect with IC50 of 50 ?g/mL through apoptosis, confirmed by DNA condensation and fragmentation. The cell population of cell cycle sub-G1 as indicative of apoptosis was also increased. In xenograft model in SD rats, F2 at dosage of 35 mg/kg weight was intravenously injected every two days. This reduced the tumor growth in magnetic resonance imaging images. The immunohistochemistry revealed that the anticancer activity might be mediated through inhibition of proliferation judged by Ki67 and apoptosis induced by activation of caspase-3 and -8. And the lowered expression of CD31 showed the reduction in blood vessel densities. The expression of matrix metalloproteinase-9 for invasion of cancer was also inhibited. The cell populations with cancer stem cell markers of CD133 and nestin were reduced. The results of this study suggested that F2 could be a new potential chemotherapeutic drug for GBM treatment by inhibiting the growth and invasion of cancer. PMID:23717108

  13. Anti-Cancer Effect of Ginsenoside F2 against Glioblastoma Multiforme in Xenograft Model in SD Rats

    PubMed Central

    Shin, Ji Yon; Lee, Jung Min; Shin, Heon Sub; Park, Sang Yong; Yang, Jung Eun; Cho, Somi Kim; Yi, Tae-Hoo

    2012-01-01

    The glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. Despite combination treatments of radiation and chemotherapy, the survival periods are very short. Therefore, this study was conducted to assess the potential of ginsenoside F2 (F2) to treat GBM. In in vitro experiments with glioblastoma cells U373MG, F2 showed the cytotoxic effect with IC50 of 50 ?g/mL through apoptosis, confirmed by DNA condensation and fragmentation. The cell population of cell cycle sub-G1 as indicative of apoptosis was also increased. In xenograft model in SD rats, F2 at dosage of 35 mg/kg weight was intravenously injected every two days. This reduced the tumor growth in magnetic resonance imaging images. The immunohistochemistry revealed that the anticancer activity might be mediated through inhibition of proliferation judged by Ki67 and apoptosis induced by activation of caspase-3 and -8. And the lowered expression of CD31 showed the reduction in blood vessel densities. The expression of matrix metalloproteinase-9 for invasion of cancer was also inhibited. The cell populations with cancer stem cell markers of CD133 and nestin were reduced. The results of this study suggested that F2 could be a new potential chemotherapeutic drug for GBM treatment by inhibiting the growth and invasion of cancer. PMID:23717108

  14. [18F]-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography of LAPC4-CR Castration-Resistant Prostate Cancer Xenograft Model in Soft Tissue Compartments1

    PubMed Central

    McCall, Keisha C.; Cheng, Su-Chun; Huang, Ying; Kohl, Nancy E.; Tupper, Tanya; Van den Abbeele, Annick D.; Zukotynski, Katherine A.; Sweeney, Christopher J.

    2015-01-01

    Preclinical xenograft models have contributed to advancing our understanding of the molecular basis of prostate cancer and to the development of targeted therapy. However, traditional preclinical in vivo techniques using caliper measurements and survival analysis evaluate the macroscopic tumor behavior, whereas tissue sampling disrupts the microenvironment and cannot be used for longitudinal studies in the same animal. Herein, we present an in vivo study of [18F]-fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) designed to evaluate the metabolism within the microenvironment of LAPC4-CR, a unique murine model of castration-resistant prostate cancer. Mice bearing LAPC4-CR subcutaneous tumors were administered [18F]-FDG via intravenous injection. After a 60-minute distribution phase, the mice were imaged on a PET/CT scanner with submillimeter resolution; and the fused PET/CT images were analyzed to evaluate tumor size, location, and metabolism across the cohort of mice. The xenograft tumors showed [18F]-FDG uptake that was independent of tumor size and was significantly greater than uptake in skeletal muscle and liver in mice (Wilcoxon signed-rank P values of .0002 and .0002, respectively). [18F]-FDG metabolism of the LAPC4-CR tumors was 2.1 ± 0.8 ID/cm3*wt, with tumor to muscle ratio of 7.4 ± 4.7 and tumor to liver background ratio of 6.7 ± 2.3. Noninvasive molecular imaging techniques such as PET/CT can be used to probe the microenvironment of tumors in vivo. This study showed that [18F]-FDG-PET/CT could be used to image and assess glucose metabolism of LAPC4-CR xenografts in vivo. Further work can investigate the use of PET/CT to quantify the metabolic response of LAPC4-CR to novel agents and combination therapies using soft tissue and possibly bone compartment xenograft models. PMID:26055171

  15. Ovarian Tumor Attachment, Invasion, and Vascularization Reflect Unique Microenvironments in the Peritoneum: Insights from Xenograft and Mathematical Models

    PubMed Central

    Steinkamp, Mara P.; Winner, Kimberly Kanigel; Davies, Suzy; Muller, Carolyn; Zhang, Yong; Hoffman, Robert M.; Shirinifard, Abbas; Moses, Melanie; Jiang, Yi; Wilson, Bridget S.

    2013-01-01

    Ovarian cancer relapse is often characterized by metastatic spread throughout the peritoneal cavity with tumors attached to multiple organs. In this study, interaction of ovarian cancer cells with the peritoneal tumor microenvironment was evaluated in a xenograft model based on intraperitoneal injection of fluorescent SKOV3.ip1 ovarian cancer cells. Intra-vital microscopy of mixed GFP-red fluorescent protein (RFP) cell populations injected into the peritoneum demonstrated that cancer cells aggregate and attach as mixed spheroids, emphasizing the importance of homotypic adhesion in tumor formation. Electron microscopy provided high resolution structural information about local attachment sites. Experimental measurements from the mouse model were used to build a three-dimensional cellular Potts ovarian tumor model (OvTM) that examines ovarian cancer cell attachment, chemotaxis, growth, and vascularization. OvTM simulations provide insight into the relative influence of cancer cell–cell adhesion, oxygen availability, and local architecture on tumor growth and morphology. Notably, tumors on the mesentery, omentum, or spleen readily invade the “open” architecture, while tumors attached to the gut encounter barriers that restrict invasion and instead rapidly expand into the peritoneal space. Simulations suggest that rapid neovascularization of SKOV3.ip1 tumors is triggered by constitutive release of angiogenic factors in the absence of hypoxia. This research highlights the importance of cellular adhesion and tumor microenvironment in the seeding of secondary ovarian tumors on diverse organs within the peritoneal cavity. Results of the OvTM simulations indicate that invasion is strongly influenced by features underlying the mesothelial lining at different sites, but is also affected by local production of chemotactic factors. The integrated in vivo mouse model and computer simulations provide a unique platform for evaluating targeted therapies for ovarian cancer relapse. PMID:23730620

  16. Pdgfra protects against ethanol-induced craniofacial defects in a zebrafish model of FASD

    PubMed Central

    McCarthy, Neil; Wetherill, Leah; Lovely, C. Ben; Swartz, Mary E.; Foroud, Tatiana M.; Eberhart, Johann K.

    2013-01-01

    Human birth defects are highly variable and this phenotypic variability can be influenced by both the environment and genetics. However, the synergistic interactions between these two variables are not well understood. Fetal alcohol spectrum disorders (FASD) is the umbrella term used to describe the wide range of deleterious outcomes following prenatal alcohol exposure. Although FASD are caused by prenatal ethanol exposure, FASD are thought to be genetically modulated, although the genes regulating sensitivity to ethanol teratogenesis are largely unknown. To identify potential ethanol-sensitive genes, we tested five known craniofacial mutants for ethanol sensitivity: cyp26b1, gata3, pdgfra, smad5 and smoothened. We found that only platelet-derived growth factor receptor alpha (pdgfra) interacted with ethanol during zebrafish craniofacial development. Analysis of the PDGF family in a human FASD genome-wide dataset links PDGFRA to craniofacial phenotypes in FASD, prompting a mechanistic understanding of this interaction. In zebrafish, untreated pdgfra mutants have cleft palate due to defective neural crest cell migration, whereas pdgfra heterozygotes develop normally. Ethanol-exposed pdgfra mutants have profound craniofacial defects that include the loss of the palatal skeleton and hypoplasia of the pharyngeal skeleton. Furthermore, ethanol treatment revealed latent haploinsufficiency, causing palatal defects in ?62% of pdgfra heterozygotes. Neural crest apoptosis partially underlies these ethanol-induced defects in pdgfra mutants, demonstrating a protective role for Pdgfra. This protective role is mediated by the PI3K/mTOR pathway. Collectively, our results suggest a model where combined genetic and environmental inhibition of PI3K/mTOR signaling leads to variability within FASD. PMID:23861062

  17. Microgavage of zebrafish larvae.

    PubMed

    Cocchiaro, Jordan L; Rawls, John F

    2013-01-01

    The zebrafish has emerged as a powerful model organism for studying intestinal development(1-5), physiology(6-11), disease(12-16), and host-microbe interactions(17-25). Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae(26). Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system. PMID:23463135

  18. Rapamycin targeting mTOR and hedgehog signaling pathways blocks human rhabdomyosarcoma growth in xenograft murine model.

    PubMed

    Kaylani, Samer Z; Xu, Jianmin; Srivastava, Ritesh K; Kopelovich, Levy; Pressey, Joseph G; Athar, Mohammad

    2013-06-14

    Rhabdomyosarcomas (RMS) represent the most common childhood soft-tissue sarcoma. Over the past few decades outcomes for low and intermediate risk RMS patients have slowly improved while patients with metastatic or relapsed RMS still face a grim prognosis. New chemotherapeutic agents or combinations of chemotherapies have largely failed to improve the outcome. Based on the identification of novel molecular targets, potential therapeutic approaches in RMS may offer a decreased reliance on conventional chemotherapy. Thus, identification of effective therapeutic agents that specifically target relevant pathways may be particularly beneficial for patients with metastatic and refractory RMS. The PI3K/AKT/mTOR pathway has been found to be a potentially attractive target in RMS therapy. In this study, we provide evidence that rapamycin (sirolimus) abrogates growth of RMS development in a RMS xenograft mouse model. As compared to a vehicle-treated control group, more than 95% inhibition in tumor growth was observed in mice receiving parenteral administration of rapamycin. The residual tumors in rapamycin-treated group showed significant reduction in the expression of biomarkers indicative of proliferation and tumor invasiveness. These tumors also showed enhanced apoptosis. Interestingly, the mechanism by which rapamycin diminished RMS tumor growth involved simultaneous inhibition of mTOR and hedgehog (Hh) pathways. Diminution in these pathways in this model of RMS also inhibited epithelial mesenchymal transition (EMT) which then dampened the invasiveness of these tumors. Our data provide bases for using rapamycin either alone or in combination with traditional chemotherapeutic drugs to block the pathogenesis of high risk RMS. PMID:23665330

  19. Carbon Quantum Dots for Zebrafish Fluorescence Imaging

    PubMed Central

    Kang, Yan-Fei; Li, Yu-Hao; Fang, Yang-Wu; Xu, Yang; Wei, Xiao-Mi; Yin, Xue-Bo

    2015-01-01

    Carbon quantum dots (C-QDs) are becoming a desirable alternative to metal-based QDs and dye probes owing to their high biocompatibility, low toxicity, ease of preparation, and unique photophysical properties. Herein, we describe fluorescence bioimaging of zebrafish using C-QDs as probe in terms of the preparation of C-QDs, zebrafish husbandry, embryo harvesting, and introduction of C-QDs into embryos and larvae by soaking and microinjection. The multicolor of C-QDs was validated with their imaging for zebrafish embryo. The distribution of C-QDs in zebrafish embryos and larvae were successfully observed from their fluorescence emission. the bio-toxicity of C-QDs was tested with zebrafish as model and C-QDs do not interfere to the development of zebrafish embryo. All of the results confirmed the high biocompatibility and low toxicity of C-QDs as imaging probe. The absorption, distribution, metabolism and excretion route (ADME) of C-QDs in zebrafish was revealed by their distribution. Our work provides the useful information for the researchers interested in studying with zebrafish as a model and the applications of C-QDs. The operations related zebrafish are suitable for the study of the toxicity, adverse effects, transport, and biocompatibility of nanomaterials as well as for drug screening with zebrafish as model. PMID:26135470

  20. Pharmacological Characterization of an Antisense Knockdown Zebrafish Model of Dravet Syndrome: Inhibition of Epileptic Seizures by the Serotonin Agonist Fenfluramine

    PubMed Central

    Copmans, Daniëlle; Langlois, Mélanie; Crawford, Alexander D.; Ceulemans, Berten; Lagae, Lieven; de Witte, Peter A. M.; Esguerra, Camila V.

    2015-01-01

    Dravet syndrome (DS) is one of the most pharmacoresistant and devastating forms of childhood epilepsy syndromes. Distinct de novo mutations in the SCN1A gene are responsible for over 80% of DS cases. While DS is largely resistant to treatment with existing anti-epileptic drugs, promising results have been obtained in clinical trials with human patients treated with the serotonin agonist fenfluramine as an add-on therapeutic. We developed a zebrafish model of DS using morpholino antisense oligomers (MOs) targeting scn1Lab, the zebrafish ortholog of SCN1A. Zebrafish larvae with an antisense knockdown of scn1Lab (scn1Lab morphants) were characterized by automated behavioral tracking and high-resolution video imaging, in addition to measuring brain activity through local field potential recordings. Our findings reveal that scn1Lab morphants display hyperactivity, convulsive seizure-like behavior, loss of posture, repetitive jerking and a myoclonic seizure-like pattern. The occurrence of spontaneous seizures was confirmed by local field potential recordings of the forebrain, measuring epileptiform discharges. Furthermore, we show that these larvae are remarkably sensitive to hyperthermia, similar to what has been described for mouse models of DS, as well as for human DS patients. Pharmacological evaluation revealed that sodium valproate and fenfluramine significantly reduce epileptiform discharges in scn1Lab morphants. Our findings for this zebrafish model of DS are in accordance with clinical data for human DS patients. To our knowledge, this is the first study demonstrating effective seizure inhibition of fenfluramine in an animal model of Dravet syndrome. Moreover, these results provide a basis for identifying novel analogs with improved activity and significantly milder or no side effects. PMID:25965391

  1. Additional effects of engineered stem cells expressing a therapeutic gene and interferon-? in a xenograft mouse model of endometrial cancer.

    PubMed

    Yi, Bo-Rim; Kim, Seung U; Choi, Kyung-Chul

    2015-07-01

    Endometrial cancer is the most common gynecologic malignancy in women worldwide. In the present study, we evaluated the effects of neural stem cell-directed enzyme/prodrug therapy (NDEPT) designed to more selectively target endometrial cancer. For this, we employed two different types of neural stem cells (NSCs), HB1.F3.CD and HB1.F3.CD.IFN-? cells. Cytosine deaminase (CD) can convert the non-toxic prodrug, 5-fluorocytosine (5-FC), into a toxic agent, 5-fluorouracil (5-FU), which inhibits DNA synthesis. IFN-? is a powerful cytotoxic cytokine that is released by activated immune cells or lymphocytes. In an animal model xenografted with endometrial Ishikawa cancer cells, the stem cells stained with CM-DiI were injected into nearby tumor masses and 5-FC was delivered by intraperitoneal injection. Co-expression of CD and IFN-? significantly inhibited the growth of cancer (~50-60%) in the presence of 5-FC. Among migration-induced factors, VEGF gene was highly expressed in endometrial cancer cells. Histological analysis showed that the aggressive nature of cancer was inhibited by 5-FC in the mice treated with the therapeutic stem cells. Furthermore, PCNA expression was more decreased in HB1.F3.CD.IFN-? treated mice rather than HB1.F3.CD treated mice. To confirm the in vitro combined effects of 5-FU and IFN-?, 5-FU was treated in Ishikawa cells. 5-FU increased the IFN-?/receptor 2 (IFNAR2) and BXA levels, indicating that 5-FU increased sensitivity of endometrial cancer cells to IFN-?, leading to apoptosis of cancer cells. Taken together, these results provide evidence for the efficacy of therapeutic stem cell-based immune therapy involving the targeted expression of CD and IFN-? genes at endometrial cancer sites. PMID:25963746

  2. Anti-tumor activity of oridonin on SNU-5 subcutaneous xenograft model via regulation of c-Met pathway.

    PubMed

    Liu, Hua; Qian, Changlin; Shen, Zhiyong

    2014-09-01

    Gastric cancer is the leading cause of cancer death worldwide. Oridonin, a diterpenoid isolated from Rabdosia rubescens, has attracted considerable attention as a potential treatment for gastric cancer based on its anti-tumor effects in many tumor cell lines. However, detailed anti-tumor mechanisms of oridonin remain a matter of speculation. In the present study, a gastric carcinoma cell line harboring c-Met gene amplification SNU-5 was used to investigate the underlying mechanisms. The results showed that in vitro, oridonin potently inhibited c-Met phosphorylation and c-Met-dependent cell proliferation (IC50 value, 36.8 ?M), meanwhile down-regulated the expression of the downstream signaling molecules including phospho-c-Raf, phospho-Erk, and phospho-Akt. In vivo, oridonin showed efficacy at well-tolerated doses, including marked cytoreductive anti-tumor activity in SNU-5 subcutaneous xenograft model. The anti-tumor efficacy of oridonin was dose-dependent and showed strong inhibition of c-Met phosphorylation. Additional mechanism of action studies showed dose-dependent inhibition of c-Met-dependent signal transduction, tumor cell proliferation (Ki67), and reduction of microvessel density (CD31). These results suggested that the anti-tumor activity of oridonin may be mediated by direct effects on tumor cell growth or survival as well as anti-angiogenic mechanisms. In summary, the results indicated that oridonin exerted anti-tumor growth on human gastric cancer SNU-5 in vitro and in vivo by direct regulation of c-Met signaling pathway and the anti-tumor effects was mainly based on its anti-proliferation and anti-angiogenesis. PMID:24916572

  3. Tumor-Targeted Hyaluronan Nanoliposomes Increase the Antitumor Activity of Liposomal Doxorubicin in Syngeneic and Human Xenograft Mouse Tumor Models

    PubMed Central

    Peer, Dan; Margalit, Rimona

    2004-01-01

    Abstract Naturally occurring high-Mr hyaluronan, bound to the surface of nanoliposomes (denoted targeted hyaluronan liposomes, or tHA-LIP), is a candidate for active targeting to tumors, many of which overexpress the hyaluronan receptors CD44 and RHAMM. The surfacebound hyaluronan also provides a hydrophilic coat that, similar to polyethylene glycol, may promote longterm circulation. We recently reported the successful targeting of mitomycin C, mediated by tHA-LIP, in tumor-bearing syngeneic mice. Hypothesizing that this targeting is carrier-specific, rather than drug-specific, we report here studies with doxorubicin (DXR)-loaded tHA-LIP, in syngeneic and human xenograft models. Saline, free DXR, DXR-loaded nontargeted liposomes (nt-LIP), and Doxil served as controls. The tHA-LIP were long-circulating, more than all controls, in healthy and tumor-bearing (C57BL/6/B16F10.9; BALB/c/C-26) mice. Mediated by tHA-LIP, DXR accumulation in tumor-bearing lungs was 30-, 6.7-, and 3.5-fold higher than free DXR, nt-LIP, and Doxil, respectively. Key indicators of therapeutic responses—tumor progression, metastatic burden, and survival—were superior (P < .001) in animals receiving DXR-loaded tHA-LIP compared with controls, in tumor-bearing syngeneic mice (BDF1/P388/ADR ascites, C57BL/6/B16F10.9 lung metastasis, and BALB/c/C-26 solid tumors), and in nude mice bearing PANC-1 solid tumors. In conclusion, tHA-LIP, performing as tumor-targeted carriers, have the potential to join the arsenal of carrier-formulated anticancer drugs. PMID:15256056

  4. Naltrindole Inhibits Human Multiple Myeloma Cell Proliferation In Vitro and in a Murine Xenograft Model In Vivo

    PubMed Central

    Mundra, Jyoti Joshi; Terskiy, Alexandra

    2012-01-01

    It has been demonstrated previously that immune cell activation and proliferation were sensitive to the effects of naltrindole, a nonpeptidic ?-opioid receptor-selective antagonist; therefore, we hypothesized that human multiple myeloma (MM) would be a valuable model for studying potential antineoplastic properties of naltrindole. [3H]naltrindole exhibited saturable, low-affinity binding to intact human MM cells; however, the pharmacological profile of the binding site differed considerably from the properties of ?-, ?-, and ?-opioid receptors, and opioid receptor mRNA was not detected in MM cells by reverse transcriptase-polymerase chain reaction. Naltrindole inhibited the proliferation of cultured human U266 MM cells in a time- and dose-dependent manner with an EC50 of 16 ?M. The naltrindole-induced inhibition of U266 cell proliferation was not blocked by a 10-fold molar excess of naltrexone, a nonselective opioid antagonist. Additive inhibition of MM cell proliferation was observed when using a combination of naltrindole with the histone deacetylase inhibitor sodium valproate, the proteasome inhibitor bortezomib, the glucocorticoid receptor agonist dexamethasone, and the HMG CoA reductase inhibitor simvastatin. Treatment of U266 cells with naltrindole significantly decreased the level of the active, phosphorylated form of the kinases, extracellular signal-regulated kinase and Akt, which may be related to its antiproliferative activity. The antiproliferative activity of naltrindole toward MM cells was maintained in cocultures of MM and bone marrow-derived stromal cells, mimicking the bone marrow microenvironment. In vivo, naltrindole significantly decreased tumor cell volumes in human MM cell xenografts in severe combined immunodeficient mice. We hypothesize that naltrindole inhibits the proliferation of MM cells through a nonopioid receptor-dependent mechanism. PMID:22537770

  5. Zebrafish Models for Ectopic Mineralization Disorders: Practical Issues from Morpholino Design to Post-Injection Observations

    PubMed Central

    Hosen, Mohammad Jakir; Vanakker, Olivier M.; Willaert, Andy; Huysseune, Ann; Coucke, Paul; De Paepe, Anne

    2013-01-01

    Zebrafish (ZF, Danio rerio) has emerged as an important and popular model species to study different human diseases. Key regulators of skeletal development and calcium metabolism are highly conserved between mammals and ZF. The corresponding orthologs share significant sequence similarities and an overlap in expression patterns when compared to mammals, making ZF a potential model for the study of mineralization-related disorders and soft tissue mineralization. To characterize the function of early mineralization-related genes in ZF, these genes can be knocked down by injecting morpholinos into early stage embryos. Validation of the morpholino needs to be performed and the concern of aspecific effects can be addressed by applying one or more independent techniques to knock down the gene of interest. Post-injection assessment of early mineralization defects can be done using general light microscopy, calcein staining, Alizarin red staining, Alizarin red-Alcian blue double staining, and by the use of transgenic lines. Examination of general molecular defects can be done by performing protein and gene expression analysis, and more specific processes can be explored by investigating ectopic mineralization-related mechanisms such as apoptosis and mitochondrial dysfunction. In this paper, we will discuss all details about the aforementioned techniques; shared knowledge will be very useful for the future investigation of ZF models for ectopic mineralization disorders and to understand the underlying pathways involved in soft tissue calcification. PMID:23760765

  6. Effect of Citrus bergamia juice on human neuroblastoma cells in vitro and in metastatic xenograft models.

    PubMed

    Navarra, M; Ursino, M R; Ferlazzo, N; Russo, M; Schumacher, U; Valentiner, U

    2014-06-01

    Neuroblastoma is the most common extracranial pediatric solid tumor with poor prognosis in children with disseminated stage of disease. A number of studies show that molecules largely distributed in commonly consumed fruits and vegetables may have anti-tumor activity. In this study we evaluate the effect of Citrus bergamia (bergamot) juice (BJ) in vitro and in a spontaneous metastatic neuroblastoma SCID mouse model. Qualitative and quantitative characterizations of BJ flavonoid fractions were performed by RP-HPLC/PDA/MS. We show that BJ significantly affects SK-N-SH and LAN-1 cell proliferation in vitro, but fails to reduce primary tumor weight in vivo. Moreover, BJ reduced cell adhesiveness and invasion of LAN-1 and SK-N-SH cells in vitro and the number of pulmonary metastases under consideration of the number of tumor cells in the blood in mice inoculated with LAN-1 cells in vivo. These effects without any apparent sign of systemic toxicity confirm the potential clinical interest of BJ and lay the basis for further investigation in cancer. PMID:24594241

  7. A rapid in vivo zebrafish model to elucidate oxidative stress-mediated PCB126-induced apoptosis and developmental toxicity.

    PubMed

    Liu, Han; Gooneratne, Ravi; Huang, Xin; Lai, Ruifang; Wei, Jin; Wang, Weimin

    2015-07-01

    Dioxin-like 3,3',4,4',5-pentachlorobiphenyl (PCB126) is one of the most potent and widespread environmental pollutants. Although PCB126-induced toxicity is related to the aryl hydrocarbon receptor pathway, there is still no study that has constructed an in vivo visual model to clarify the role of the Nrf2/ARE signaling pathway in the oxidative stress mechanism of PCB126-induced toxicity. In the present study, an in vivo zebrafish model of nrf2a fused to enhanced green fluorescent protein (nrf2a-eGFP) was constructed. The zebrafish embryos microinjected with nrf2a-eGFP (72h postfertilization) were exposed to various concentrations of PCB126 (0, 25, 50, 100, 200?g/L) or 30mMN-acetylcysteine (NAC)+200?g/L PCB126. After 72h exposure, PCB126 significantly increased the malformation rates and induced eGFP expression in a dose-dependent manner in several zebrafish tissue types. The distribution of eGFP fluorescence coincided with developmental deformity sites. NAC pretreatment effectively counteracted PCB126-induced developmental toxicity including heart rate, pericardial edema, and body length. The highest PCB126 dose, 200?g/L, produced marked apoptosis in the eye, gill, and trunk detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. At 48 and 72h exposure, 200?g/L PCB126 affected glutathione metabolism as evidenced by decreased glutathione and increased glutathione disulfide concentrations, indicative of oxidative stress. These effects were also counteracted by NAC pretreatment. Furthermore, the Nrf2-regulated genes gclc, gpx, gstp1, and hmox1 were significantly induced at 24, 48, and 72h at the highest PCB126 exposures but not in the NAC-pretreated group. In addition, a significant increase in ROS generation was detected in zebrafish larvae at 72h PCB126 exposure, which might offer a link for future mechanistic studies. Collectively, these data suggest that PCB126-induced developmental toxicity and apoptosis in the nrf2a-eGFP-injected zebrafish model are due to oxidative stress mediated by disruption to glutathione metabolism and changes in Nrf2-regulated gene expression. PMID:25770664

  8. Rapamycin targeting mTOR and hedgehog signaling pathways blocks human rhabdomyosarcoma growth in xenograft murine model

    SciTech Connect

    Kaylani, Samer Z. [Division of Hematology and Oncology, Department of Pediatrics, University of Alabama at Birmingham, 1600 7th Avenue South, ACC 414, Birmingham, AL 35233 (United States)] [Division of Hematology and Oncology, Department of Pediatrics, University of Alabama at Birmingham, 1600 7th Avenue South, ACC 414, Birmingham, AL 35233 (United States); Xu, Jianmin; Srivastava, Ritesh K. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States)] [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States); Kopelovich, Levy [Division of Cancer Prevention, National Cancer Institute, Bethesda (United States)] [Division of Cancer Prevention, National Cancer Institute, Bethesda (United States); Pressey, Joseph G. [Division of Hematology and Oncology, Department of Pediatrics, University of Alabama at Birmingham, 1600 7th Avenue South, ACC 414, Birmingham, AL 35233 (United States)] [Division of Hematology and Oncology, Department of Pediatrics, University of Alabama at Birmingham, 1600 7th Avenue South, ACC 414, Birmingham, AL 35233 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States)] [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States)

    2013-06-14

    Graphical abstract: Intervention of poorly differentiated RMS by rapamycin: In poorly differentiated RMS, rapamycin blocks mTOR and Hh signaling pathways concomitantly. This leads to dampening in cell cycle regulation and induction of apoptosis. This study provides a rationale for the therapeutic intervention of poorly differentiated RMS by treating patients with rapamycin alone or in combination with other chemotherapeutic agents. -- Highlights: •Rapamycin abrogates RMS tumor growth by modulating proliferation and apoptosis. •Co-targeting mTOR/Hh pathways underlie the molecular basis of effectiveness. •Reduction in mTOR/Hh pathways diminish EMT leading to reduced invasiveness. -- Abstract: Rhabdomyosarcomas (RMS) represent the most common childhood soft-tissue sarcoma. Over the past few decades outcomes for low and intermediate risk RMS patients have slowly improved while patients with metastatic or relapsed RMS still face a grim prognosis. New chemotherapeutic agents or combinations of chemotherapies have largely failed to improve the outcome. Based on the identification of novel molecular targets, potential therapeutic approaches in RMS may offer a decreased reliance on conventional chemotherapy. Thus, identification of effective therapeutic agents that specifically target relevant pathways may be particularly beneficial for patients with metastatic and refractory RMS. The PI3K/AKT/mTOR pathway has been found to be a potentially attractive target in RMS therapy. In this study, we provide evidence that rapamycin (sirolimus) abrogates growth of RMS development in a RMS xenograft mouse model. As compared to a vehicle-treated control group, more than 95% inhibition in tumor growth was observed in mice receiving parenteral administration of rapamycin. The residual tumors in rapamycin-treated group showed significant reduction in the expression of biomarkers indicative of proliferation and tumor invasiveness. These tumors also showed enhanced apoptosis. Interestingly, the mechanism by which rapamycin diminished RMS tumor growth involved simultaneous inhibition of mTOR and hedgehog (Hh) pathways. Diminution in these pathways in this model of RMS also inhibited epithelial mesenchymal transition (EMT) which then dampened the invasiveness of these tumors. Our data provide bases for using rapamycin either alone or in combination with traditional chemotherapeutic drugs to block the pathogenesis of high risk RMS.

  9. Photofrin-mediated Photodynamic Therapy Induces Vascular Occlusion and Apoptosis in a Human Sarcoma Xenograft Model1

    Microsoft Academic Search

    Brett W. Engbrecht; Chandrakala Menon; Alexander V. Kachur; Stephen M. Hahn; Douglas L. Fraker

    Photodynamic therapy (PDT) involves light activation of a photosensi- tizer, resulting in oxygen-dependent, free radical-mediated cell death. Little is known about the efficacy of PDT in treating human sarcomas, despite an ongoing clinical trial treating i.p. sarcomatosis. The present study evaluates PDT treatment of a human sarcoma xenograft in nude mice and explores the mechanism of PDT-mediated antitumor effect. Athymic

  10. Rev. Neurosci., Vol. 22(1): 95105, 2011 Copyright by Walter de Gruyter Berlin New York. DOI 10.1515/RNS.2011.011 Zebrafish models to study drug abuse-related phenotypes

    E-print Network

    Kalueff, Allan V.

    . DOI 10.1515/RNS.2011.011 Zebrafish models to study drug abuse-related phenotypes Adam Stewarta , Keith and addiction research. Modeling drug abuse-related behavior in both adult and lar- val zebrafish produced a wealth of clinically translatable data, also demonstrating their sensitivity to various drugs of abuse

  11. Establishment of a Patient-Derived Orthotopic Xenograft (PDOX) Model of HER-2-Positive Cervical Cancer Expressing the Clinical Metastatic Pattern

    PubMed Central

    Hiroshima, Yukihiko; Zhang, Yong; Zhang, Nan; Maawy, Ali; Mii, Sumiyuki; Yamamoto, Mako; Uehara, Fuminari; Miwa, Shinji; Yano, Shuya; Murakami, Takashi; Momiyama, Masashi; Chishima, Takashi; Tanaka, Kuniya; Ichikawa, Yasushi; Bouvet, Michael; Murata, Takuya; Endo, Itaru; Hoffman, Robert M.

    2015-01-01

    Squamous cell carcinoma of the cervix, highly prevalent in the developing world, is often metastatic and treatment resistant with no standard treatment protocol. Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) nude mouse model with the technique of surgical orthotopic implantation (SOI). Unlike subcutaneous transplant patient-derived xenograft (PDX) models, PDOX models metastasize. Most importantly, the metastasis pattern correlates to the patient. In the present report, we describe the development of a PDOX model of HER-2-positive cervical cancer. Metastasis after SOI in nude mice included peritoneal dissemination, liver metastasis, lung metastasis as well as lymph node metastasis reflecting the metastatic pattern in the donor patient. Metastasis was detected in 4 of 6 nude mice with primary tumors. Primary tumors and metastases in the nude mice had histological structures similar to the original tumor and were stained by an anti-HER-2 antibody in the same pattern as the patient’s cancer. The metastatic pattern, histology and HER-2 tumor expression of the patient were thus preserved in the PDOX model. In contrast, subcutaneous transplantation of the patient’s cervical tumors resulted in primary growth but not metastasis. PMID:25689852

  12. Indole Alkaloids from Fischerella Inhibit Vertebrate Development in the Zebrafish (Danio rerio) Embryo Model

    PubMed Central

    Walton, Katherine; Gantar, Miroslav; Gibbs, Patrick D. L.; Schmale, Michael C.; Berry, John P.

    2014-01-01

    Cyanobacteria are recognized producers of toxic or otherwise bioactive metabolite associated, in particular, with so-called “harmful algal blooms” (HABs) and eutrophication of freshwater systems. In the present study, two apparently teratogenic indole alkaloids from a freshwater strain of the widespread cyanobacterial genus, Fischerella (Stigonemataceae), were isolated by bioassay-guided fractionation, specifically using the zebrafish (Danio rerio) embryo, as a model of vertebrate development. The two alkaloids include the previously known 12-epi-hapalindole H isonitrile (1), and a new nitrile-containing variant, 12-epi-ambiguine B nitrile (2). Although both compounds were toxic to developing embryos, the former compound was shown to be relatively more potent, and to correlate best with the observed embryo toxicity. Related indole alkaloids from Fischerella, and other genera in the Stigonemataceae, have been widely reported as antimicrobial compounds, specifically in association with apparent allelopathy. However, this is the first report of their vertebrate toxicity, and the observed teratogenicity of these alkaloids supports a possible contribution to the toxicity of this widespread cyanobacterial family, particularly in relation to freshwater HABs and eutrophication. PMID:25533520

  13. Targeted tumor theranostics using folate-conjugated and camptothecin-loaded acoustic nanodroplets in a mouse xenograft model.

    PubMed

    Chen, Wei-Tsung; Kang, Shih-Tsung; Lin, Jian-Liang; Wang, Chung-Hsin; Chen, Ran-Chou; Yeh, Chih-Kuang

    2015-06-01

    In this study, we aimed to validate the feasibility of receptor-targeted tumor theranostics with folate-conjugated (FA) and camptothecin-loaded (CPT) acoustic nanodroplets (NDs) (collectively termed FA-CPT-NDs). The ND formulation was based on lipid-stabilized low-boiling perfluorocarbon that can undergo acoustic droplet vaporization (ADV) under ultrasound (US) exposure. Conjugation of folate enhanced the selective delivery to tumors expressing high levels of folate receptor (FR) under mediation by the enhanced permeability and retention effect. In vitro and in vivo studies were performed using FR-positive KB and FR-negative HT-1080 cell lines and mouse xenograft tumor models. Simultaneous therapy and imaging were conducted with a clinical US imaging system at mechanical indices of up to 1.4 at a center frequency of 10 MHz. The results demonstrated that FA-CPT-NDs selectively attached to KB cells, but not HT-1080 cells. The targeted ADV caused instant and delayed damage via mechanical disruption and chemical toxicity to decrease the viability of KB cells by up to 45%, a much higher decrease than that achieved by the NDs without folate conjugation. The in vivo experiments showed that FR-mediated targeting successfully enhanced the EPR of FA-CPT-NDs in KB tumors mainly on the tumor periphery as indicated by immunofluorescence microscopy and US B-mode imaging. Treatments with FA-CPT-NDs at a CPT dose of 50 ?g/kg inhibited the growth of KB tumors for up to six weeks, whereas treatment with NDs lacking folate produced a 4.6-fold increase in tumor volume. For HT-1080 tumors, neither the treatments with FA-CPT-NDs nor those with the NDs lacking folate presented tumor growth inhibition. In summary, FR-targeted tumor theranostics has been successfully implemented with FA-CPT-NDs and a clinical US unit. The ligand-directed and EPR-mediated accumulation provides active and passive targeting capabilities, permitting the antitumor effects of FA-CPT-NDs to be exerted selectively to FR-positive tumors and simultaneously providing targeted US imaging capabilities. PMID:25890765

  14. Viral Diseases in Zebrafish: What Is Known and Unknown

    PubMed Central

    Crim, Marcus J.; Riley, Lela K.

    2013-01-01

    Naturally occurring viral infections have the potential to introduce confounding variability that leads to invalid and misinterpreted data. Whereas the viral diseases of research rodents are well characterized and closely monitored, no naturally occurring viral infections have been characterized for the laboratory zebrafish (Danio rerio), an increasingly important biomedical research model. Despite the ignorance about naturally occurring zebrafish viruses, zebrafish models are rapidly expanding in areas of biomedical research where the confounding effects of unknown infectious agents present a serious concern. In addition, many zebrafish research colonies remain linked to the ornamental (pet) zebrafish trade, which can contribute to the introduction of new pathogens into research colonies, whereas mice used for research are purpose bred, with no introduction of new mice from the pet industry. Identification, characterization, and monitoring of naturally occurring viruses in zebrafish are crucial to the improvement of zebrafish health, the reduction of unwanted variability, and the continued development of the zebrafish as a model organism. This article addresses the importance of identifying and characterizing the viral diseases of zebrafish as the scope of zebrafish models expands into new research areas and also briefly addresses zebrafish susceptibility to experimental viral infection and the utility of the zebrafish as an infection and immunology model. PMID:23382345

  15. Novel Vitamin K analogues suppress seizures in zebrafish and mouse models of epilepsy

    PubMed Central

    Rahn, Jennifer J.; Bestman, Jennifer E.; Josey, Benjamin J.; Inks, Elizabeth S.; Stackley, Krista D.; Rogers, Carolyn E.; Chou, C. James; Chan, Sherine S. L.

    2014-01-01

    Epilepsy is a debilitating disease affecting 1-2% of the world’s population. Despite this high prevalence, 30% of patients suffering from epilepsy are not successfully managed by current medication suggesting a critical need for new anti-epileptic drugs (AEDs). In an effort to discover new therapeutics for the management of epilepsy, we began our study by screening drugs that, like some currently used AEDs, inhibit HDACs using a well-established larval zebrafish model. In this model, 7-day post fertilization (dpf) larvae are treated with the widely used seizure-inducing compound pentylenetetrazol (PTZ) which stimulates a rapid increase in swimming behavior previously determined to be a measurable manifestation of seizures. In our first screen, we tested a number of different HDAC inhibitors and found that one, NQN1, significantly decreased swim activity to levels equal to that of VPA. We continued to screen structurally related compounds including Vitamin K3 (VK3) and a number of novel Vitamin K (VK) analogues. We found that VK3 was a robust inhibitor of the PTZ-induced swim activity, as were several of our novel compounds. Three of these compounds were subsequently tested on mouse seizure models at the National Institute of Neurological Disorders and Stroke (NINDS) Anticonvulsant Screening Program. Compound 2h reduced seizures particularly well in the minimal clonic seizure (6 Hz) and corneal kindled mouse models of epilepsy, with no observable toxicity. As VK3 affects mitochondrial function, we tested the effects of our compounds on mitochondrial respiration and ATP production in a mouse hippocampal cell line. We demonstrate that these compounds affect ATP metabolism and increase total cellular ATP. Our data indicate the potential utility of these and other VK analogues for prevention of seizures and suggest the potential mechanism for this protection may lie in the ability of these compounds to affect energy production. PMID:24291671

  16. Enhanced anticancer activity of a combination of docetaxel and Aneustat (OMN54) in a patient-derived, advanced prostate cancer tissue xenograft model.

    PubMed

    Qu, Sifeng; Wang, Kendric; Xue, Hui; Wang, Yuwei; Wu, Rebecca; Liu, Chengfei; Gao, Allen C; Gout, Peter W; Collins, Colin C; Wang, Yuzhuo

    2014-03-01

    The current first-line treatment for advanced metastatic prostate cancer, i.e. docetaxel-based therapy, is only marginally effective. The aim of the present study was to determine whether such therapy can be improved by combining docetaxel with Aneustat (OMN54), a multivalent botanical drug candidate shown to have anti-prostate cancer activity in preliminary in vitro experiments, which is currently undergoing a Phase-I Clinical Trial. Human metastatic, androgen-independent C4-2 prostate cancer cells and NOD-SCID mice bearing PTEN-deficient, metastatic and PSA-secreting, patient-derived subrenal capsule LTL-313H prostate cancer tissue xenografts were treated with docetaxel and Aneustat, alone and in combination. In vitro, Aneustat markedly inhibited C4-2 cell replication in a dose-dependent manner. When Aneustat was combined with docetaxel, the growth inhibitions of the drugs were essentially additive. In vivo, however, the combination of docetaxel and Aneustat enhanced anti-tumor activity synergistically and very markedly, without inducing major host toxicity. Complete growth inhibition and shrinkage of the xenografts could be obtained with the combined drugs as distinct from the drugs on their own. Analysis of the gene expression of the xenografts using microarray indicated that docetaxel + Aneustat led to expanded anticancer activity, in particular to targeting of cancer hallmarks that were not affected by the single drugs. Our findings, obtained with a highly clinically relevant prostate cancer model, suggest, for the first time, that docetaxel-based therapy of advanced human prostate cancer may be improved by combining docetaxel with Aneustat. PMID:24388358

  17. Host immune response and acute disease in a zebrafish model of francisella pathogenesis

    USGS Publications Warehouse

    Vojtech, L.N.; Sanders, G.E.; Conway, C.; Ostland, V.; Hansen, J.D.

    2009-01-01

    Members of the bacterial genus Francisella are highly virulent and infectious pathogens. New models to study Francisella pathogenesis in evolutionarily distinct species are needed to provide comparative insight, as the mechanisms of host resistance and pathogen virulence are not well understood. We took advantage of the recent discovery of a novel species of Francisella to establish a zebrafish/Francisella comparative model of pathogenesis and host immune response. Adult zebraflsh were susceptible to acute Francisella-induced disease and suffered mortality in a dose-dependent manner. Using immunohistochemical analysis, we localized bacterial antigens primarily to lymphoid tissues and livers of zebraflsh following infection by intraperitoneal injection, which corresponded to regions of local cellular necrosis. Francisella sp. bacteria replicated rapidly in these tissues beginning 12 h postinfection, and bacterial titers rose steadily, leveled off, and then decreased by 7 days postinfection. Zebraflsh mounted a significant tissue-specific proinflammatory response to infection as measured by the upregulation of interleukin-l?? (IL-1??), gamma interferon, and tumor necrosis factor alpha mRNA beginning by 6 h postinfection and persisting for up to 7 days postinfection. In addition, exposure of zebraflsh to heat-killed bacteria demonstrated that the significant induction of IL-?? was highly specific to live bacteria. Taken together, the pathology and immune response to acute Francisella infection in zebraflsh share many features with those in mammals, highlighting the usefulness of this new model system for addressing both general and specific questions about Francisella host-pathogen interactions via an evolutionary approach. Copyright ?? 2009, American Society for Microbiology. All Rights Reserved.

  18. Anti-CD45 pretargeted radioimmunotherapy using bismuth-213: high rates of complete remission and long-term survival in a mouse myeloid leukemia xenograft model

    PubMed Central

    Kenoyer, Aimee L.; Bäck, Tom; Hamlin, Donald K.; Wilbur, D. Scott; Fisher, Darrell R.; Park, Steven I.; Frayo, Shani; Axtman, Amanda; Orgun, Nural; Orozco, Johnnie; Shenoi, Jaideep; Lin, Yukang; Gopal, Ajay K.; Green, Damian J.; Appelbaum, Frederick R.; Press, Oliver W.

    2011-01-01

    Pretargeted radioimmunotherapy (PRIT) using an anti-CD45 antibody (Ab)–streptavidin (SA) conjugate and DOTA-biotin labeled with ?-emitting radionuclides has been explored as a strategy to decrease relapse and toxicity. ?-emitting radionuclides exhibit high cytotoxicity coupled with a short path length, potentially increasing the therapeutic index and making them an attractive alternative to ?-emitting radionuclides for patients with acute myeloid leukemia. Accordingly, we have used 213Bi in mice with human leukemia xenografts. Results demonstrated excellent localization of 213Bi-DOTA-biotin to tumors with minimal uptake into normal organs. After 10 minutes, 4.5% ± 1.1% of the injected dose of 213Bi was delivered per gram of tumor. ?-imaging demonstrated uniform radionuclide distribution within tumor tissue 45 minutes after 213Bi-DOTA-biotin injection. Radiation absorbed doses were similar to those observed using a ?-emitting radionuclide (90Y) in the same model. We conducted therapy experiments in a xenograft model using a single-dose of 213Bi-DOTA-biotin given 24 hours after anti-CD45 Ab-SA conjugate. Among mice treated with anti-CD45 Ab-SA conjugate followed by 800 ?Ci of 213Bi- or 90Y-DOTA-biotin, 80% and 20%, respectively, survived leukemia-free for more than 100 days with minimal toxicity. These data suggest that anti-CD45 PRIT using an ?-emitting radionuclide may be highly effective and minimally toxic for treatment of acute myeloid leukemia. PMID:21613259

  19. Antiproliferative efficacies but minor drug transporter inducing effects of paclitaxel, cisplatin, or 5-fluorouracil in a murine xenograft model for head and neck squamous cell carcinoma.

    PubMed

    Theile, Dirk; Gal, Zoltan; Warta, Rolf; Rigalli, Juan Pablo; Lahrmann, Bernd; Grabe, Niels; Herold-Mende, Christel; Dyckhoff, Gerhard; Weiss, Johanna

    2014-04-01

    Drug-induced multidrug resistance (MDR) has been linked to overexpression of drug transporting proteins in head and neck squamous cell carcinoma (HNSCC) in vitro. The aim of this work was to reassess these findings in a murine xenograft model. NOD-SCID mice xenotransplanted with 10 (6) HNO97 cells were treated for four consecutive weeks with weekly paclitaxel, biweekly cisplatin (both intraperitoneal), or 5-fluorouracil (5-FU, administered by osmotic pump). Tumor volume and body weight were weekly documented. Expression of drug transporters and Ki-67 marker were examined using quantitative real-time polymerase chain reaction and/or immunohistochemistry. Both paclitaxel and cisplatin significantly reduced tumor volumes after 2-3 weeks. 5-FU-treated animals had significantly lower body weights after 2 or 4 weeks of chemotherapy. None of the drugs affected expression of drug transporters at the mRNA level. However, P-glycoprotein (Pgp) protein expression was increased by paclitaxel (P<0.01). Ki-67 expression did not change during treatment irrespective of the drug applied. Paclitaxel and cisplatin are effectively tumor volume reducing drugs in a murine xenograft model of HNSCC. Paclitaxel enhanced Pgp expression at the protein level, but not at the mRNA level suggesting transcriptional induction to be of minor relevance. In contrast, posttranscriptional mechanisms or Darwinian selection of intrinsically drug transporter overexpressing MDR cells might lead to iatrogenic chemotherapy resistance in HNSCC. PMID:24448417

  20. Efficacy of JAK/STAT pathway inhibition in murine xenograft models of early T-cell precursor (ETP) acute lymphoblastic leukemia.

    PubMed

    Maude, Shannon L; Dolai, Sibasish; Delgado-Martin, Cristina; Vincent, Tiffaney; Robbins, Alissa; Selvanathan, Arthavan; Ryan, Theresa; Hall, Junior; Wood, Andrew C; Tasian, Sarah K; Hunger, Stephen P; Loh, Mignon L; Mullighan, Charles G; Wood, Brent L; Hermiston, Michelle L; Grupp, Stephan A; Lock, Richard B; Teachey, David T

    2015-03-12

    Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a recently described subtype of T-ALL characterized by a unique immunophenotype and genomic profile, as well as a high rate of induction failure. Frequent mutations in cytokine receptor and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways led us to hypothesize that ETP-ALL is dependent on JAK/STAT signaling. Here we demonstrate aberrant activation of the JAK/STAT pathway in ETP-ALL blasts relative to non-ETP T-ALL. Moreover, ETP-ALL showed hyperactivation of STAT5 in response to interleukin-7, an effect that was abrogated by the JAK1/2 inhibitor ruxolitinib. In vivo, ruxolitinib displayed activity in 6 of 6 patient-derived murine xenograft models of ETP-ALL, with profound single-agent efficacy in 5 models. Ruxolitinib treatment decreased peripheral blast counts relative to pretreatment levels and compared with control (P < .01) in 5 of 6 ETP-ALL xenografts, with marked reduction in mean splenic blast counts (P < .01) in 6 of 6 samples. Surprisingly, both JAK/STAT pathway activation and ruxolitinib efficacy were independent of the presence of JAK/STAT pathway mutations, raising the possibility that the therapeutic potential of ruxolitinib in ETP-ALL extends beyond those cases with JAK mutations. These findings establish the preclinical in vivo efficacy of ruxolitinib in ETP-ALL, a biologically distinct subtype for which novel therapies are needed. PMID:25645356

  1. Axon degeneration and PGC-1?-mediated protection in a zebrafish model of ?-synuclein toxicity

    PubMed Central

    O’Donnell, Kelley C.; Lulla, Aaron; Stahl, Mark C.; Wheat, Nickolas D.; Bronstein, Jeff M.; Sagasti, Alvaro

    2014-01-01

    ?-synuclein (aSyn) expression is implicated in neurodegenerative processes, including Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). In animal models of these diseases, axon pathology often precedes cell death, raising the question of whether aSyn has compartment-specific toxic effects that could require early and/or independent therapeutic intervention. The relevance of axonal pathology to degeneration can only be addressed through longitudinal, in vivo monitoring of different neuronal compartments. With current imaging methods, dopaminergic neurons do not readily lend themselves to such a task in any vertebrate system. We therefore expressed human wild-type aSyn in zebrafish peripheral sensory neurons, which project elaborate superficial axons that can be continuously imaged in vivo. Axonal outgrowth was normal in these neurons but, by 2 days post-fertilization (dpf), many aSyn-expressing axons became dystrophic, with focal varicosities or diffuse beading. Approximately 20% of aSyn-expressing cells died by 3 dpf. Time-lapse imaging revealed that focal axonal swelling, but not overt fragmentation, usually preceded cell death. Co-expressing aSyn with a mitochondrial reporter revealed deficits in mitochondrial transport and morphology even when axons appeared overtly normal. The axon-protective protein Wallerian degeneration slow (WldS) delayed axon degeneration but not cell death caused by aSyn. By contrast, the transcriptional coactivator PGC-1?, which has roles in the regulation of mitochondrial biogenesis and reactive-oxygen-species detoxification, abrogated aSyn toxicity in both the axon and the cell body. The rapid onset of axonal pathology in this system, and the relatively moderate degree of cell death, provide a new model for the study of aSyn toxicity and protection. Moreover, the accessibility of peripheral sensory axons will allow effects of aSyn to be studied in different neuronal compartments and might have utility in screening for novel disease-modifying compounds. PMID:24626988

  2. Axon degeneration and PGC-1?-mediated protection in a zebrafish model of ?-synuclein toxicity.

    PubMed

    O'Donnell, Kelley C; Lulla, Aaron; Stahl, Mark C; Wheat, Nickolas D; Bronstein, Jeff M; Sagasti, Alvaro

    2014-05-01

    ?-synuclein (aSyn) expression is implicated in neurodegenerative processes, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In animal models of these diseases, axon pathology often precedes cell death, raising the question of whether aSyn has compartment-specific toxic effects that could require early and/or independent therapeutic intervention. The relevance of axonal pathology to degeneration can only be addressed through longitudinal, in vivo monitoring of different neuronal compartments. With current imaging methods, dopaminergic neurons do not readily lend themselves to such a task in any vertebrate system. We therefore expressed human wild-type aSyn in zebrafish peripheral sensory neurons, which project elaborate superficial axons that can be continuously imaged in vivo. Axonal outgrowth was normal in these neurons but, by 2 days post-fertilization (dpf), many aSyn-expressing axons became dystrophic, with focal varicosities or diffuse beading. Approximately 20% of aSyn-expressing cells died by 3 dpf. Time-lapse imaging revealed that focal axonal swelling, but not overt fragmentation, usually preceded cell death. Co-expressing aSyn with a mitochondrial reporter revealed deficits in mitochondrial transport and morphology even when axons appeared overtly normal. The axon-protective protein Wallerian degeneration slow (WldS) delayed axon degeneration but not cell death caused by aSyn. By contrast, the transcriptional coactivator PGC-1?, which has roles in the regulation of mitochondrial biogenesis and reactive-oxygen-species detoxification, abrogated aSyn toxicity in both the axon and the cell body. The rapid onset of axonal pathology in this system, and the relatively moderate degree of cell death, provide a new model for the study of aSyn toxicity and protection. Moreover, the accessibility of peripheral sensory axons will allow effects of aSyn to be studied in different neuronal compartments and might have utility in screening for novel disease-modifying compounds. PMID:24626988

  3. l-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome

    PubMed Central

    Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E.; Gerton, Jennifer L.

    2015-01-01

    Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (?-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite ?-KIC can partially rescue development in zebrafish models for CdLS. PMID:25378554

  4. L-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome.

    PubMed

    Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E; Gerton, Jennifer L

    2015-03-15

    Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (?-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite ?-KIC can partially rescue development in zebrafish models for CdLS. PMID:25378554

  5. Relative developmental toxicities of pentachloroanisole and pentachlorophenol in a zebrafish model (Danio rerio).

    PubMed

    Cheng, Yan; Ekker, Marc; Chan, Hing Man

    2015-02-01

    Pentachloroanisole (PCA) and pentachlorophenol (PCP) are chlorinated aromatic compounds that have been found in the environment and in human populations. The objective of this study is to characterize the effects of PCA in comparison to those of PCP on development at environmental relevant levels using a fish model. Zebrafish embryos were exposed to 0.1, 1, 10, 100, 500, 1000 ?g/L PCA and PCP respectively for 96 h. Malformation observation, LC50 testing for survival rate at 96 hours post fertilization (hpf) and EC50 testing for hatching rate at 72 hpf indicated that the developmental toxicity of PCP was about 15 times higher than that of PCA. PCP exposure at 10 ?g/L resulted in elevated 3, 3', 5-triiodothyronine (T3) levels and decreased thyroxine (T4) levels, whereas PCA had no effects on T3 or T4 levels. PCP and PCA exposure at 1 and 10 ?g/L showed possible hyperthyroid effects similar to that of T3, due to increased relative mRNA expression of synapsin I (SYN), iodothyronine deiodinase type III (Dio3), thyroid hormone receptor alpha a (THR?a) and thyroid hormone receptor beta (THR?), and decreased expression of iodothyronine deiodinase type II (Dio2). The results indicate that both PCA and PCP exposure can cause morphological deformities, possibly affect the timing and coordination of development in the central nervous system, and alter thyroid hormone levels by disrupting thyroid hormone regulating pathways. However, the developmental toxicity of PCA is at least ten times lower than that of PCP. Our results on the relative developmental toxicities of PCA and PCP and the possible underlying mechanisms will be useful to support interpretation of envrionmental concentrations and body burden levels observed in human populations. PMID:25463847

  6. Correlative light and electron microscopy imaging of autophagy in a zebrafish infection model.

    PubMed

    Hosseini, Rohola; Lamers, Gerda Em; Hodzic, Zlatan; Meijer, Annemarie H; Schaaf, Marcel Jm; Spaink, Herman P

    2014-10-01

    High-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we present a versatile method for high-resolution imaging of microbial infection in zebrafish larvae by injecting pathogens into the tail fin. This allows visualization of autophagic compartments by light and electron microscopy, which makes it possible to correlate images acquired by the 2 techniques. Using this method we have studied the autophagy response against Mycobacterium marinum infection. We show that mycobacteria during the progress of infection are frequently associated with GFP-Lc3-positive vesicles, and that 2 types of GFP-Lc3-positive vesicles were observed. The majority of these vesicles were approximately 1 ?m in size and in close vicinity of bacteria, and a smaller number of GFP-Lc3-positive vesicles was larger in size and were observed to contain bacteria. Quantitative data showed that these larger vesicles occurred significantly more in leukocytes than in other cell types, and that approximately 70% of these vesicles were positive for a lysosomal marker. Using electron microscopy, it was found that approximately 5% of intracellular bacteria were present in autophagic vacuoles and that the remaining intracellular bacteria were present in phagosomes, lysosomes, free inside the cytoplasm or occurred as large aggregates. Based on correlation of light and electron microscopy images, it was shown that GFP-Lc3-positive vesicles displayed autophagic morphology. This study provides a new approach for injection of pathogens into the tail fin, which allows combined light and electron microscopy imaging in vivo and opens new research directions for studying autophagy process related to infectious diseases. PMID:25126731

  7. Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-to-primate kidney transplant model.

    PubMed

    Higginbotham, Laura; Mathews, Dave; Breeden, Cynthia A; Song, Mingqing; Farris, Alton Brad; Larsen, Christian P; Ford, Mandy L; Lutz, Andrew J; Tector, Matthew; Newell, Kenneth A; Tector, A Joseph; Adams, Andrew B

    2015-05-01

    Xenotransplantation has the potential to alleviate the organ shortage that prevents many patients with end-stage renal disease from enjoying the benefits of kidney transplantation. Despite significant advances in other models, pig-to-primate kidney xenotransplantation has met limited success. Preformed anti-pig antibodies are an important component of the xenogeneic immune response. To address this, we screened a cohort of 34 rhesus macaques for anti-pig antibody levels. We then selected animals with both low and high titers of anti-pig antibodies to proceed with kidney transplant from galactose-?1,3-galactose knockout/CD55 transgenic pig donors. All animals received T-cell depletion followed by maintenance therapy with costimulation blockade (either anti-CD154 mAb or belatacept), mycophenolate mofetil, and steroid. The animal with the high titer of anti-pig antibody rejected the kidney xenograft within the first week. Low-titer animals treated with anti-CD154 antibody, but not belatacept exhibited prolonged kidney xenograft survival (>133 and >126 vs. 14 and 21 days, respectively). Long-term surviving animals treated with the anti-CD154-based regimen continue to have normal kidney function and preserved renal architecture without evidence of rejection on biopsies sampled at day 100. This description of the longest reported survival of pig-to-non-human primate kidney xenotransplantation, now >125 days, provides promise for further study and potential clinical translation. PMID:25847130

  8. Metformin impairs Rho GTPase signaling to induce apoptosis in neuroblastoma cells and inhibits growth of tumors in the xenograft mouse model of neuroblastoma

    PubMed Central

    Kumar, Ambrish; Al-Sammarraie, Nadia; DiPette, Donald J.; Singh, Ugra S.

    2014-01-01

    Metformin has been shown to inhibit tumor growth in xenograft rodent models of adult cancers, and various human clinical trials are in progress. However, the precise molecular mechanisms of metformin action are largely unknown. In the present study we examined the anti-tumor activity of metformin against neuroblastoma, and determined the underlying signaling mechanisms. Using human neuroblastoma xenograft mice, we demonstrated that oral administration of metformin (100 and 250 mg/kg body weight) significantly inhibited the growth of tumors. The interference of metformin in spheroid formation further confirmed the anti-tumor activity of metformin. In tumors, the activation of Rac1 (GTP-Rac1) and Cdc42 (GTP-Cdc42) was increased while RhoA activation (GTP-RhoA) was decreased by metformin. It also induced phosphorylation of JNK and inhibited the phosphorylation of ERK1/2 without affecting p38 MAP Kinase. Infection of cells by adenoviruses expressing dominant negative Rac1 (Rac1-N17), Cdc42 (Cdc42-N17) or constitutively active RhoA (RhoA-V14), or incubation of cells with pharmacological inhibitors of Rac1 (NSC23766) or Cdc42 (ML141) significantly protected neuroblastoma cells from metformin-induced apoptosis. Additionally, inhibition of JNK activity along with Rac1 or Cdc42 attenuated cytotoxic effects of metformin. These studies demonstrated that metformin impairs Rho GTPases signaling to induce apoptosis via JNK pathway. PMID:25365944

  9. Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, shows broad antitumor activity in human tumor xenograft models associated with microvessel density and pericyte coverage

    PubMed Central

    2014-01-01

    Background Lenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor ? (PDGFR ?), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study. Method We evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors. Result Lenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100?mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100?mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p?

  10. Enhanced anti-tumor activity of the glycoengineered type II CD20 antibody obinutuzumab (GA101) in combination with chemotherapy in xenograft models of human lymphoma

    PubMed Central

    Herting, Frank; Friess, Thomas; Bader, Sabine; Muth, Gunter; Hölzlwimmer, Gabriele; Rieder, Natascha; Umana, Pablo

    2014-01-01

    Obinutuzumab (GA101) is a novel glycoengineered type II CD20 antibody in development for non-Hodgkin lymphoma. We compared the anti-tumor activity of obinutuzumab and rituximab in preclinical studies using subcutaneous Z138 and WSU-DLCL2 xenograft mouse models. Obinutuzumab and rituximab were assessed alone and in combination with bendamustine, fludarabine, chlorambucil, doxorubicin and cyclophosphamide/vincristine. Owing to strong single-agent efficacy in these models, suboptimal doses of obinutuzumab were applied to demonstrate a combination effect. Obinutuzumab plus bendamustine achieved superior tumor growth inhibition versus rituximab plus bendamustine and showed a statistically significant effect versus the respective single treatments. Combinations of obinutuzumab with fludarabine, chlorambucil or cyclophosphamide/vincristine demonstrated significantly superior activity to rituximab-based treatment. Obinutuzumab monotherapy was at least as effective as rituximab plus chemotherapy in vivo, and obinutuzumab plus chemotherapy was superior to the respective monotherapies. These data support further clinical investigation of obinutuzumab plus chemotherapy. PMID:24304419

  11. Enhanced anti-tumor activity of the glycoengineered type II CD20 antibody obinutuzumab (GA101) in combination with chemotherapy in xenograft models of human lymphoma.

    PubMed

    Herting, Frank; Friess, Thomas; Bader, Sabine; Muth, Gunter; Hölzlwimmer, Gabriele; Rieder, Natascha; Umana, Pablo; Klein, Christian

    2014-09-01

    Obinutuzumab (GA101) is a novel glycoengineered type II CD20 antibody in development for non-Hodgkin lymphoma. We compared the anti-tumor activity of obinutuzumab and rituximab in preclinical studies using subcutaneous Z138 and WSU-DLCL2 xenograft mouse models. Obinutuzumab and rituximab were assessed alone and in combination with bendamustine, fludarabine, chlorambucil, doxorubicin and cyclophosphamide/vincristine. Owing to strong single-agent efficacy in these models, suboptimal doses of obinutuzumab were applied to demonstrate a combination effect. Obinutuzumab plus bendamustine achieved superior tumor growth inhibition versus rituximab plus bendamustine and showed a statistically significant effect versus the respective single treatments. Combinations of obinutuzumab with fludarabine, chlorambucil or cyclophosphamide/vincristine demonstrated significantly superior activity to rituximab-based treatment. Obinutuzumab monotherapy was at least as effective as rituximab plus chemotherapy in vivo, and obinutuzumab plus chemotherapy was superior to the respective monotherapies. These data support further clinical investigation of obinutuzumab plus chemotherapy. PMID:24304419

  12. Enhanced antitumor effect of anti-tissue factor antibody-conjugated epirubicin-incorporating micelles in xenograft models.

    PubMed

    Yamamoto, Yoshiyuki; Hyodo, Ichinosuke; Koga, Yoshikatsu; Tsumura, Ryo; Sato, Ryuta; Obonai, Toshihumi; Fuchigami, Hirobumi; Furuya, Fumiaki; Yasunaga, Masahiro; Harada, Mitsunori; Kato, Yasuki; Ohtsu, Atsushi; Matsumura, Yasuhiro

    2015-05-01

    For the creation of a successful antibody-drug conjugate (ADC), both scientific and clinical evidence has indicated that highly toxic anticancer agents (ACA) should be conjugated to a monoclonal antibody (mAb) to administer a reasonable amount of ADC to patients without compromising the affinity of the mAb. For ordinary ACA, the conjugation of a mAb to ACA-loaded micellar nanoparticles is clinically applicable. Tissue factor (TF) is often overexpressed in various cancer cells and tumor vascular endothelium. Accordingly, anti-TF-NC-6300, consisting of epirubicin-incorporating micelles (NC-6300) conjugated with the F(ab')2 of anti-TF mAb was developed. The in vitro and in vivo efficacy and pharmacokinetics of anti-TF-NC-6300 were compared to NC-6300 using two human pancreatic cancer cell lines, BxPC3 (high TF expression) and SUIT2 (low TF expression), and a gastric cancer cell line, 44As3 (high TF expression). The intracellular uptake of epirubicin was faster and greater in BxPC3 cells treated with anti-TF-NC-6300, compared with NC-6300. Anti-TF-NC-6300 showed a superior antitumor activity in BxPC3 and 44As3 xenografts, compared with NC-6300, while the activities of both micelles were similar in the SUIT2 xenograft. A higher tumor accumulation of anti-TF-NC-6300 compared to NC-6300 was seen, regardless of the TF expression levels. However, anti-TF-NC-6300 appeared to be localized to the tumor cells with high TF expression. These results indicated that the enhanced antitumor effect of anti-TF-NC6300 may be independent of the tumor accumulation but may depend on the selective intratumor localization and the preferential internalization of anti-TF-NC-6300 into high TF tumor cells. PMID:25711681

  13. GH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model

    Microsoft Academic Search

    Rafael Y. Kuradomi; Márcio A. Figueiredo; Carlos F. C. Lanes; Carlos E. da Rosa; Daniela V. Almeida; Rodrigo Maggioni; Maeli D. P. Silva; Luis F. Marins

    2011-01-01

    The aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic\\u000a zebrafish (Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis\\u000a demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic\\u000a females being more hypertrophic

  14. A novel orally available inhibitor of focal adhesion signaling increases survival in a xenograft model of diffuse large B-cell lymphoma with central nervous system involvement

    PubMed Central

    Bosch, Rosa; Moreno, María José; Dieguez-Gonzalez, Rebeca; Céspedes, María Virtudes; Gallardo, Alberto; Trias, Manuel; Grañena, Albert; Sierra, Jorge; Casanova, Isolda; Mangues, Ramon

    2013-01-01

    Central nervous system dissemination is a relatively uncommon but almost always fatal complication in diffuse large B-cell lymphoma patients. Optimal therapy for central nervous involvement in this malignancy has not been established. In this paper, we aimed to evaluate the therapeutic effect of E7123, a celecoxib derivative that inhibits focal adhesion signaling, in a novel xenograft model of diffuse large B-cell lymphoma with central nervous system involvement. Cells obtained after disaggregation of HT subcutaneous tumors (HT-SC cells) were intravenously injected in NOD/SCID mice. These mice received oral vehicle or 75 mg/kg of E7123 daily until they were euthanized for weight loss or signs of sickness. The antitumor effect of E7123 was validated in an independent experiment using a bioluminescent mouse model. Intravenously injected HT-SC cells showed higher take rate and higher central nervous system tropism (associated with increased expression of ?1-integrin and p130Cas proteins) than HT cells. The oral administration of E7123 significantly increased survival time in 2 independent experiments using mice injected with unmodified or bioluminescent HT-SC cells. We have developed a new xenograft model of diffuse large B-cell lymphoma with central nervous system involvement that can be used in the pre-clinical evaluation of new drugs for this malignancy. E7123 is a new, well-tolerated and orally available therapeutic agent that merits further investigation since it may improve current management of diffuse large B-cell lymphoma patients with central nervous system involvement. PMID:23716554

  15. In Silico cancer cell versus stroma cellularity index computed from species-specific human and mouse transcriptome of xenograft models: towards accurate stroma targeting therapy assessment

    PubMed Central

    2014-01-01

    Background The current state of the art for measuring stromal response to targeted therapy requires burdensome and rate limiting quantitative histology. Transcriptome measures are increasingly affordable and provide an opportunity for developing a stromal versus cancer ratio in xenograft models. In these models, human cancer cells are transplanted into mouse host tissues (stroma) and together coevolve into a tumour microenvironment. However, profiling the mouse or human component separately remains problematic. Indeed, laser capture microdissection is labour intensive. Moreover, gene expression using commercial microarrays introduces significant and underreported cross-species hybridization errors that are commonly overlooked by biologists. Method We developed a customized dual-species array, H&M array, and performed cross-species and species-specific hybridization measurements. We validated a new methodology for establishing the stroma vs cancer ratio using transcriptomic data. Results In the biological validation of the H&M array, cross-species hybridization of human and mouse probes was significantly reduced (4.5 and 9.4 fold reduction, respectively; p < 2x10-16 for both, Mann-Whitney test). We confirmed the capability of the H&M array to determine the stromal to cancer cells ratio based on the estimation of cellularity index of mouse/human mRNA content in vitro. This new metrics enable to investigate more efficiently the stroma-cancer cell interactions (e.g. cellularity) bypassing labour intensive requirement and biases of laser capture microdissection. Conclusion These results provide the initial evidence of improved and cost-efficient analytics for the investigation of cancer cell microenvironment, using species-specificity arrays specifically designed for xenografts models. PMID:25079962

  16. GH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model.

    PubMed

    Kuradomi, Rafael Y; Figueiredo, Márcio A; Lanes, Carlos F C; da Rosa, Carlos E; Almeida, Daniela V; Maggioni, Rodrigo; Silva, Maeli D P; Marins, Luis F

    2011-06-01

    The aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic zebrafish (Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic females being more hypertrophic than transgenic males. The expression of genes related to muscle growth revealed that transgenic hypertrophy is independent from local induction of insulin-like growth factor 1 gene (igf1). In addition, transgenic males exhibited significant induction of myogenin gene (myog) expression, indicating that myog may mediate hypertrophic growth in zebrafish males overexpressing GH. Induction of the ?-actin gene (acta1) in males, independently from transgenesis, also was observed. There were no significant differences in total protein content from the muscle. Our results show that muscle hypertrophy is independent from muscle igf1, and is likely to be a direct effect of excess circulating GH and/or IGF1 in this transgenic zebrafish lineage. PMID:20640508

  17. Ahsa1 and Hsp90 activity confers more severe craniofacial phenotypes in a zebrafish model of hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR)

    PubMed Central

    Sheehan-Rooney, Kelly; Swartz, Mary E.; Zhao, Feng; Liu, Dong; Eberhart, Johann K.

    2013-01-01

    SUMMARY The severity of most human birth defects is highly variable. Our ability to diagnose, treat and prevent defects relies on our understanding of this variability. Mutation of the transcription factor GATA3 in humans causes the highly variable hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR) syndrome. Although named for a triad of defects, individuals with HDR can also exhibit craniofacial defects. Through a forward genetic screen for craniofacial mutants, we isolated a zebrafish mutant in which the first cysteine of the second zinc finger of Gata3 is mutated. Because mutation of the homologous cysteine causes HDR in humans, these zebrafish mutants could be a quick and effective animal model for understanding the role of gata3 in the HDR disease spectrum. We demonstrate that, unexpectedly, the chaperone proteins Ahsa1 and Hsp90 promote severe craniofacial phenotypes in our zebrafish model of HDR syndrome. The strengths of the zebrafish system, including rapid development, genetic tractability and live imaging, make this an important model for variability. PMID:23720234

  18. Functional Ginger Extracts from Supercritical Fluid Carbon Dioxide Extraction via In Vitro and In Vivo Assays: Antioxidation, Antimicroorganism, and Mice Xenografts Models

    PubMed Central

    Lee, Chih-Chen; Chiou, Li-Yu; Wang, Jheng-Yang; Chou, Sin-You; Lan, John Chi-Wei; Huang, Tsi-Shu; Huang, Kuo-Chuan

    2013-01-01

    Supercritical fluid carbon dioxide extraction technology was developed to gain the active components from a Taiwan native plant, Zingiber officinale (ginger). We studied the biological effects of ginger extracts via multiple assays and demonstrated the biofunctions in each platform. Investigations of ginger extracts indicated antioxidative properties in dose-dependant manners on radical scavenging activities, reducing powers and metal chelating powers. We found that ginger extracts processed moderate scavenging values, middle metal chelating levels, and slight ferric reducing powers. The antibacterial susceptibility of ginger extracts on Staphylococcus aureus, Streptococcus sobrinus, S. mutans, and Escherichia coli was determined with the broth microdilution method technique. The ginger extracts had operative antimicroorganism potentials against both Gram-positive and Gram-negative bacteria. We further discovered the strong inhibitions of ginger extracts on lethal carcinogenic melanoma through in vivo xenograft model. To sum up, the data confirmed the possible applications as medical cosmetology agents, pharmaceutical antibiotics, and food supplements. PMID:23983624

  19. A Small Molecule Inhibitor of ETV1, YK-4-279, Prevents Prostate Cancer Growth and Metastasis in a Mouse Xenograft Model

    PubMed Central

    Rahim, Said; Minas, Tsion; Hong, Sung-Hyeok; Justvig, Sarah; Çelik, Haydar; Kont, Yasemin Saygideger; Han, Jenny; Kallarakal, Abraham T.; Kong, Yali; Rudek, Michelle A.; Brown, Milton L.; Kallakury, Bhaskar; Toretsky, Jeffrey A.; Üren, Aykut

    2014-01-01

    Background The erythroblastosis virus E26 transforming sequences (ETS) family of transcription factors consists of a highly conserved group of genes that play important roles in cellular proliferation, differentiation, migration and invasion. Chromosomal translocations fusing ETS factors to promoters of androgen responsive genes have been found in prostate cancers, including the most clinically aggressive forms. ERG and ETV1 are the most commonly translocated ETS proteins. Over-expression of these proteins in prostate cancer cells results in a more invasive phenotype. Inhibition of ETS activity by small molecule inhibitors may provide a novel method for the treatment of prostate cancer. Methods and Findings We recently demonstrated that the small molecule YK-4-279 inhibits biological activity of ETV1 in fusion-positive prostate cancer cells leading to decreased motility and invasion in-vitro. Here, we present data from an in-vivo mouse xenograft model. SCID-beige mice were subcutaneously implanted with fusion-positive LNCaP-luc-M6 and fusion-negative PC-3M-luc-C6 tumors. Animals were treated with YK-4-279, and its effects on primary tumor growth and lung metastasis were evaluated. YK-4-279 treatment resulted in decreased growth of the primary tumor only in LNCaP-luc-M6 cohort. When primary tumors were grown to comparable sizes, YK-4-279 inhibited tumor metastasis to the lungs. Expression of ETV1 target genes MMP7, FKBP10 and GLYATL2 were reduced in YK-4-279 treated animals. ETS fusion-negative PC-3M-luc-C6 xenografts were unresponsive to the compound. Furthermore, YK-4-279 is a chiral molecule that exists as a racemic mixture of R and S enantiomers. We established that (S)-YK-4-279 is the active enantiomer in prostate cancer cells. Conclusion Our results demonstrate that YK-4-279 is a potent inhibitor of ETV1 and inhibits both the primary tumor growth and metastasis of fusion positive prostate cancer xenografts. Therefore, YK-4-279 or similar compounds may be evaluated as a potential therapeutic tool for treatment of human prostate cancer at different stages. PMID:25479232

  20. Cisplatin and 4-hexylresorcinol synergise to decrease metastasis and increase survival rate in an oral mucosal melanoma xenograft model: a preliminary study.

    PubMed

    Lee, Sang-Woon; Kim, Seong-Gon; Park, Young-Wook; Kweon, Haeyong; Kim, Jwa-Young; Rotaru, Horatiu

    2013-06-01

    The present study was undertaken to examine the effects of cisplatin plus 4-hexylresorcinol (4-HR) combination therapy on oral mucosal melanoma (OMM) using cultured primary OMM cells in a tumour xenograft model. Cultured primary OMM cells were used for the MTT assay and DNA microarray. OMM cells were implanted into the submandibular glands of nude mice. The mice were then treated with cisplatin only or cisplatin plus 4-HR. Tumour size changes, survival rate and tumour metastasis were compared between the two groups by observation, micro-positron emission tomography (PET) and histological examination. In the MTT assay, the cisplatin plus 4-HR group showed significantly higher inhibition of OMM cell growth compared to the other groups (p<0.05). DNA microarray results showed significant inhibition of matrix metalloproteinase (MMP)-2 gene expression upon 4-HR application. The necropsy and micro-PET results showed that the mice from the cisplatin-only group had more distant metastases than the mice from the cisplatin plus 4-HR combination group (p=0.002). MMP-2 expression was lower in the primary tumours in the cisplatin plus 4-HR combination group than in the cisplatin-only group (p<0.001). Overall survival was longer in mice from the cisplatin plus 4-HR combination group than in the cisplatin-only group (p=0.049). In conclusion, the combined effect of cisplatin and 4-HR resulted in fewer metastases and longer survival than cisplatin-only treatment in the OMM xenograft model. PMID:23412976

  1. Poly(ethylene glycol)-block-poly(?-caprolactone) micelles for combination drug delivery: evaluation of paclitaxel, cyclopamine and gossypol in intraperitoneal xenograft models of ovarian cancer.

    PubMed

    Cho, Hyunah; Lai, Tsz Chung; Kwon, Glen S

    2013-02-28

    Ovarian cancer is the most lethal gynecological malignancy, characterized by a high rate of chemoresistance. Current treatment strategies for ovarian cancer focus on novel drug combinations of cytotoxic agents and molecular targeted agents or novel drug delivery strategies that often involve intraperitoneal (IP) injection. Poly(ethylene glycol)-block-poly(?-caprolactone) (PEG-b-PCL) micelles were loaded with paclitaxel (cytotoxic agent), cyclopamine (hedgehog inhibitor), and gossypol (Bcl-2 inhibitor). After physicochemical studies focusing on combination drug solubilization, 3-drug PEG-b-PCL micelles were evaluated in vitro in 2-D and 3-D cell culture and in vivo in xenograft models of ovarian cancer, tracking bioluminescence signals from ES-2 and SKOV3 human ovarian cancer cell lines after IP injection. 3-Drug PEG-b-PCL micelles were not significantly more potent in 2-D cell culture in comparison to paclitaxel; however, they disaggregated ES-2 tumor spheroids, whereas single drugs or 2-drug combinations only slowed growth of ES-2 tumor spheroids or had no noticeable effects. In ES-2 and SKOV3 xenograft models, 3-drug PEG-b-PCL micelles had significantly less tumor burden than paclitaxel based on bioluminescence imaging, 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET imaging, and overall survival. (18)F-FLT-PET images clearly showed that 3-drug PEG-b-PCL micelles dramatically reduce tumor volumes over paclitaxel and vehicle controls. In summary, PEG-b-PCL micelles enable the IP combination drug delivery of paclitaxel, cyclopamine and gossypol, resulting in tumor growth inhibition and prolonged survival over paclitaxel alone. These results validate a novel treatment strategy for ovarian cancer based on drug combinations of cytotoxic agents and molecular targeted agents, delivered concurrently by a nanoscale drug delivery system, e.g. PEG-b-PCL micelles. PMID:23246471

  2. T Cells Derived From Human Melanoma Draining Lymph Nodes Mediate Melanoma-specific Antitumor Responses In Vitro and In Vivo in Human Melanoma Xenograft Model.

    PubMed

    Zhang, Mei; Graor, Hallie; Visioni, Anthony; Strohl, Madeleine; Yan, Lu; Caja, Kevin; Kim, Julian A

    2015-01-01

    It has been established in murine models that lymph nodes draining a progressively growing tumor contain antigen-specific T cells capable of mediating protective immune responses upon adoptive transfer. However, naturally occurring human tumor-draining lymph nodes (TDLNs) have yet to be fully investigated. In this study, we analyzed TDLNs from patients with stage III melanoma who were undergoing routine lymph node dissection. Following short-term (14 d) culture activation with anti-CD3/anti-CD28 microbeads and expansion in low concentrations of IL-2, the melanoma-draining lymph node (MDLN) cells were ?60% CD4-activated and ?40% CD8-activated T cells. The activated MDLN cells demonstrated reactivity in response to overlapping peptides spanning the sequence of 4 different known melanoma antigens MAGEA1, Melan-A/MART-1, NY-ESO-1, and Prame/OIP4, suggesting the presence of melanoma-specific T cells. Coculture of activated MDLN T cells with cancer cells in vitro resulted in preferential apoptosis of human cancer cell lines that were cocultured with T cells with high degree of MHC matching. Adoptive transfer of MDLN T cells with high degree of MHC matching to A375 to mice-bearing human A375 melanoma xenografts resulted in dose-dependent improvement in survival. Although prior human studies have demonstrated the immune responses within melanoma vaccine-draining lymph nodes, this study presents evidence for the first time that naturally occurring human MDLN samples contain melanoma-experienced CD4 and CD8 T cells that can be readily cultured and expanded to mediate protective immune responses both in vitro and in vivo in a human melanoma xenograft model. PMID:26049546

  3. Anti-CD45 Pretargeted Radioimmunotherapy using Bismuth-213: High Rates of Complete Remission and Long-Term Survival in a Mouse Myeloid Leukemia Xenograft Model

    SciTech Connect

    Pagel, John M.; Kenoyer, Aimee L.; Back, Tom; Hamlin, Donald K.; Wilbur, D. Scott; Fisher, Darrell R.; Park, Steven I.; Frayo, Shani; Axtman, Amanda; Orgun, Nural; Orozoco, Johnnie; Shenoi, Jaideep; Lin, Yukang; Gopal, Ajay K.; Green, Damian J.; Appelbaum, Frederick R.; Press, Oliver W.

    2011-07-21

    Pretargeted radioimmunotherapy (PRIT) using an anti-CD45 antibody (Ab)-streptavidin (SA) conjugate and DOTA-biotin labeled with beta-emitting radionuclides has been explored as a strategy to decrease relapse and toxicity. Alpha-emitting radionuclides exhibit high cytotoxicity coupled with a short path-length, potentially increasing the therapeutic index and making them an attractive alternative to beta-emitting radionuclides for patients with Acute Myeloid Leukemia (AML). Accordingly, we have used 213Bi in mice with human leukemia xenografts. Results demonstrated excellent localization of 213Bi-DOTA-biotin to tumors with minimal uptake into normal organs. After 10 minutes, 4.5 ± 1.1% of the injected dose of 213Bi was delivered per gram of tumor. Alpha imaging demonstrated uniform radionuclide distribution within tumor tissue 45 minutes after 213Bi-DOTA-biotin injection. Radiation absorbed doses were similar to those observed using a beta-emitting radionuclide (90Y) in the same model. We conducted therapy experiments in a xenograft model using a single-dose of 213Bi-DOTA-biotin given 24 hours after anti-CD45 Ab-SA conjugate. Among mice treated with anti-CD45 Ab-SA conjugate followed by 800 ?Ci of 213Bi- or 90Y-DOTA-biotin, 80% and 20%, respectively, survived leukemia-free for >100 days with minimal toxicity. These data suggest that anti-CD45 PRIT using an alpha-emitting radionuclide may be highly effective and minimally toxic for treatment of AML.

  4. Abundance of the Quorum-Sensing Factor Ax21 in Four Strains of Stenotrophomonas maltophilia Correlates with Mortality Rate in a New Zebrafish Model of Infection

    PubMed Central

    Yero, Daniel; Mongiardini, Elías; Torrent, Gerard; Huedo, Pol; Martínez, Paula; Roher, Nerea; Mackenzie, Simon; Gibert, Isidre; Daura, Xavier

    2013-01-01

    Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence. Little is known about its pathogenesis and the genomic diversity exhibited by clinical isolates complicates the study of pathogenicity and virulence factors. Here, we present a strategy to identify such factors in new clinical isolates of S. maltophilia, incorporating an adult-zebrafish model of S. maltophilia infection to evaluate relative virulence coupled to 2D difference gel electrophoresis to explore underlying differences in protein expression. In this study we report upon three recent clinical isolates and use the collection strain ATCC13637 as a reference. The adult-zebrafish model shows discrimination capacity, i.e. from very low to very high mortality rates, with clinical symptoms very similar to those observed in natural S. maltophilia infections in fish. Strain virulence correlates with resistance to human serum, in agreement with previous studies in mouse and rat and therefore supporting zebrafish as a replacement model. Despite its clinical origin, the collection strain ATCC13637 showed obvious signs of attenuation in zebrafish, with null mortality. Multilocus-sequence-typing analysis revealed that the most virulent strains, UV74 and M30, exhibit the strongest genetic similitude. Differential proteomic analysis led to the identification of 38 proteins with significantly different abundance in the three clinical strains relative to the reference strain. Orthologs of several of these proteins have been already reported to have a role in pathogenesis, virulence or resistance mechanisms thus supporting our strategy. Proof of concept is further provided by protein Ax21, whose abundance is shown here to be directly proportional to mortality in the zebrafish infection model. Indeed, recent studies have demonstrated that this protein is a quorum-sensing-related virulence factor. PMID:23840626

  5. Silencing of APE1 enhances sensitivity of human hepatocellular carcinoma cells to radiotherapy in vitro and in a xenograft model.

    PubMed

    Cun, Yanping; Dai, Nan; Xiong, Chengjie; Li, Mengxia; Sui, Jiangdong; Qian, Chengyuan; Li, Zheng; Wang, Dong

    2013-01-01

    Resistance to radiotherapy is a key limitation for the treatment of human hepatocellular carcinoma (HCC). To overcome this problem, we investigated the correlation between radioresistance and the human apurinic/apyrimidinic endonuclease (APE1), a bifunctional protein, which plays an important role in DNA repair and redox regulation activity of transcription factors. In the present study, we examined the radiosensitivity profiles of three human HCC cell lines, HepG2, Hep3B, and MHCC97L, using the adenoviral vector Ad5/F35-mediated APE1 siRNA (Ad5/F35-siAPE1). The p53 mutant cell lines MHCC97L showed radioresistance, compared with HepG2 and Hep3B cells. APE1 was strongly expressed in MHCC97L cells and was induced by irradiation in a dose-dependent manner, and Ad5/F35-siAPE1 effectively inhibited irradiation-induced APE1 and p53 expression. Moreover, silencing of APE1 significantly potentiated the growth inhibition and apoptosis induction by irradiation in all tested human HCC cell lines. In addition, Ad5/F35-siAPE1 significantly enhanced inhibition of tumor growth and potentiated cell apoptosis by irradiation both in HepG2 and MHCC97L xenografts. In conclusion, down regulation of APE1 could enhance sensitivity of human HCC cells to radiotherapy in vitro and in vivo. PMID:23418439

  6. Combination of angiogenesis inhibitors increases the anti-tumor efficacy of photodynamic therapy in a human bladder tumor xenograft model

    NASA Astrophysics Data System (ADS)

    Bhuvaneswari, Ramaswamy; Gan, Yik Yuen; Thong, Patricia S. P.; Chin, William Wei L.; Soo, Khee Chee; Olivo, Malini

    2009-06-01

    Photodynamic therapy (PDT) is a standard treatment for various malignant and non-malignant conditions. Though therapeutic responses are encouraging, recurrences have been noted, as one of the limitations of PDT is treatment-induced hypoxia that triggers angiogenesis. The present study evaluates the use of angiogenic inhibitors Avastin, that targets vascular endothelial growth factor (VEGF) and Erbitux that targets epidermal growth factor receptor (EGFR) with PDT in an in vivo bladder carcinoma xenograft. Tumor bearing mice were assigned to 6 different categories: control, PDT only, Avastin + Erbitux, PDT + Avastin, PDT + Erbitux and PDT + Avastin and Erbitux. Treated and control tumors were monitored for recurrence for up to 90 days. VEGF and EGFR expression was detected in the tumor tissue. Migratory assay was performed to establish the inhibitory effect of the angiogenesis agents. Using confocal laser endomicroscopy, the tumor microvasculature was assessed. Tumors treated with the combination therapy of PDT + inhibitors showed significantly greater response compared to control and PDT only treated group. Combination therapy treated tumors also showed the most post-treatment damage with reduced tumor vasculature. These results demonstrate that the combination of PDT with inhibitors that target different angiogenesis pathways can improve tumor control.

  7. Meta-[{sup 211}At]astatobenzylguanidine (MABG): In vivo evaluation in an athymic mouse human neuroblastoma xenograft model

    SciTech Connect

    Vaidyanathan, G.; Friedman, H.S.; Keir, S.T. [Duke Univ. Medical Center, Durhamk, NC (United States)] [and others

    1996-05-01

    Because of the short range and high linear energy transfer of {sup 211}At {alpha}-particles, the MIBG analogue MABG might be useful for the therapy of micrometastatic neuroblastoma and previous in vitro studies have demonstrated that under single-cell conditions, the cytotoxicity of MABG is > 1000 times higher than [{sup 131}I]MIBG. A paired label protocol was used to compare the tissue distribution of MABG and [{sup 131}I]MIBG in athymic mice bearing subcutaneous SK-N-SH human neuroblastoma xenografts from 1-24 hr after injection. In tumor, significantly higher (p < 0.05) uptake was observed for MABG (3.8 {plus_minus} 0.8%ID/g vs 3.1 {plus_minus} 0.7%ID/g at 8 hr). Pretreatment with desipramine reduced tumor uptake of MABG by 43%, suggesting that accumulation was related to the uptake-1 mechanism. Significantly higher uptake of MABG also was observed in normal tissue targets. For example, at 8 hr, heart uptake of MABG was 6.0 {plus_minus} 0.9 % ID/g compared with 4.5 {plus_minus} 0.8%ID/g for [{sup 131}I]MIBG. Two strategies were investigated to increase the tumor-to-hear uptake ratio. Pretreatment of mice with unlabeled MIBG (4 mg/kg) increased MABG tumor uptake by 1.5-fold while reducing uptake in several normal tissues including heart. The vesicular uptake blocker tetrabenazine (TBZ; 20 mg/kg), reduced MABG hear uptake by 30% of control values with not significant decrease in tumor levels. We conclude that MABG deserves further evaluation as a potential agent for the treatment of neuroblastoma, particularly in combination with strategies to minimize radiation dose to normal target tissues.

  8. Characterization and assessment of the sensitivity and resistance of a newly established human gastrointestinal stromal tumour xenograft model to treatment with tyrosine kinase inhibitors

    PubMed Central

    2014-01-01

    Background Acquired resistance to tyrosine kinase inhibitors (TKIs) in gastrointestinal stromal tumours (GISTs) is most commonly caused by secondary KIT or PDGFRA mutations. In this study we characterize a newly established GIST xenograft model, UZLX-GIST9, and evaluate the in vivo response of the model to standard TKIs (imatinib, sunitinib, and regorafenib). Methods Tumour fragments from a metastatic lesion of a GIST patient clinically progressing after treatment with imatinib, sunitinib and regorafenib were engrafted in a nude, immunodeficient mouse. Upon sequential passaging from mouse to mouse, tumour fragments were collected for histopathological and molecular characterization. The sensitivity of the model to treatment with TKIs was evaluated in 28 mice [passage 2 (n?=?8), passage 4 (n?=?20), 41 tumours]. Mice were grouped as follows: control (untreated), imatinib (50 mg/kg/BID), imatinib (100 mg/kg/BID), sunitinib (40 mg/kg/QD), and regorafenib (30 mg/kg/QD). After three weeks of oral treatment, tumours were collected for subsequent analysis. The efficacy of treatment was assessed by tumour volume, histopathology and Western immunoblotting. Results UZLX-GIST9 maintains the same typical morphological features and immunohistochemical characteristics as the original patient biopsy and expresses CD117 and DOG1. The KIT mutational profile (p.P577del + W557LfsX5+ D820G) remains the same as the original tissue sample originating from an intraspinal metastatic site. Three week treatment with different TKIs showed that the model is resistant to imatinib. Sunitinib induces tumour growth delay and regorafenib reduces the tumour burden by 30% as compared to control animals. While none of the TKIs had a significant effect on cell proliferation or cell survival, a remarkable increase of necrosis and significant reduction of microvessel density was observed under sunitinib and regorafenib. Western immunoblotting showed a mild reduction in KIT and AKT activation only in regorafenib treated tumours. Conclusions We established a novel human GIST xenograft, UZLX-GIST9, harbouring KIT exon 11 and 17 mutations and maintaining the pheno-and genotype of the original tumour. UZLX-GIST9 shows different levels of response to standard TKIs. This model will help to study TKI resistance and to explore novel treatment approaches for patients with TKI-resistant GIST. PMID:25132955

  9. A zebrafish model of Roberts syndrome reveals that Esco2 depletion interferes with development by disrupting the cell cycle.

    PubMed

    Mönnich, Maren; Kuriger, Zoë; Print, Cristin G; Horsfield, Julia A

    2011-01-01

    The human developmental diseases Cornelia de Lange Syndrome (CdLS) and Roberts Syndrome (RBS) are both caused by mutations in proteins responsible for sister chromatid cohesion. Cohesion is mediated by a multi-subunit complex called cohesin, which is loaded onto chromosomes by NIPBL. Once on chromosomes, cohesin binding is stabilized in S phase upon acetylation by ESCO2. CdLS is caused by heterozygous mutations in NIPBL or cohesin subunits SMC1A and SMC3, and RBS is caused by homozygous mutations in ESCO2. The genetic cause of both CdLS and RBS reside within the chromosome cohesion apparatus, and therefore they are collectively known as "cohesinopathies". However, the two syndromes have distinct phenotypes, with differences not explained by their shared ontology. In this study, we have used the zebrafish model to distinguish between developmental pathways downstream of cohesin itself, or its acetylase ESCO2. Esco2 depleted zebrafish embryos exhibit features that resemble RBS, including mitotic defects, craniofacial abnormalities and limb truncations. A microarray analysis of Esco2-depleted embryos revealed that different subsets of genes are regulated downstream of Esco2 when compared with cohesin subunit Rad21. Genes downstream of Rad21 showed significant enrichment for transcriptional regulators, while Esco2-regulated genes were more likely to be involved the cell cycle or apoptosis. RNA in situ hybridization showed that runx1, which is spatiotemporally regulated by cohesin, is expressed normally in Esco2-depleted embryos. Furthermore, myca, which is downregulated in rad21 mutants, is upregulated in Esco2-depleted embryos. High levels of cell death contributed to the morphology of Esco2-depleted embryos without affecting specific developmental pathways. We propose that cell proliferation defects and apoptosis could be the primary cause of the features of RBS. Our results show that mutations in different elements of the cohesion apparatus have distinct developmental outcomes, and provide insight into why CdLS and RBS are distinct diseases. PMID:21637801

  10. Toxicity to embryo and adult zebrafish of copper complexes with two malonic acids as models for dissolved organic matter

    SciTech Connect

    Palmer, F.B.; Evans, C.W. [Univ. of Auckland (New Zealand); Butler, C.A. [Woodward Clyde NZ Ltd., Auckland (New Zealand); Timperley, M.H. [National Inst. of Water and Atmospheric Research, Auckland (New Zealand)

    1998-08-01

    The toxicity to embryo and adult zebrafish (Danio rerio) of Cu complexes with two substituted malonic acids, benzyl- and n-hexadecyl-, chosen as models for low-molecular-weight natural dissolved organic matter, were investigated. Toxicity test solutions at pH 6.5 {+-} 0.1 with the required Cu ion-specific electrode. In the absence of malonic acids, concentrations of Cu{sup 2+} up to 1.13 {mu}mol/L increased the embryo hatching time from approx. 2 d in control solutions (no Cu or malonic acid) and solutions containing malonic acids without Cu to approx. 8 d. The Cu-benzylmalonic acid complex in the presence of inorganic Cu species did not delay hatching beyond that attributable to Cu{sup 2+}. In contrast, 0.60 {mu}mol/L Cu-n-hexadecylmalonic complexes delayed hatching by 5.5 d in excess of that attributable to 1.13 {mu}mol/L Cu{sup 2+}, assuming that the hatching delays caused by the different Cu species were additive, possibly because of Cu entry into the embryo as the lipophilic Cu-n-hexadecylmalonic complex. None of the Cu-malonic acid complexes was acutely toxic to adult zebrafish at concentrations up to 1.4 {mu}mol/L, possibly because Cu was removed from the Cu-malonic acid complexes by stronger chelating groups at the gill surface. Substituted malonic acids with similar proton and Cu association constants can be readily prepared with a variety of simple substituents, radiolabeled if required. Their results show that these acids could be useful ligands for investigating intracellular transport and metabolism of metal-organic complexes.

  11. Zebrafish as a model for apolipoprotein biology: comprehensive expression analysis and a role for ApoA-IV in regulating food intake.

    PubMed

    Otis, Jessica P; Zeituni, Erin M; Thierer, James H; Anderson, Jennifer L; Brown, Alexandria C; Boehm, Erica D; Cerchione, Derek M; Ceasrine, Alexis M; Avraham-Davidi, Inbal; Tempelhof, Hanoch; Yaniv, Karina; Farber, Steven A

    2015-03-01

    Improved understanding of lipoproteins, particles that transport lipids throughout the circulation, is vital to developing new treatments for the dyslipidemias associated with metabolic syndrome. Apolipoproteins are a key component of lipoproteins. Apolipoproteins are proteins that structure lipoproteins and regulate lipid metabolism through control of cellular lipid exchange. Constraints of cell culture and mouse models mean that there is a need for a complementary model that can replicate the complex in vivo milieu that regulates apolipoprotein and lipoprotein biology. Here, we further establish the utility of the genetically tractable and optically clear larval zebrafish as a model of apolipoprotein biology. Gene ancestry analyses were implemented to determine the closest human orthologs of the zebrafish apolipoprotein A-I (apoA-I), apoB, apoE and apoA-IV genes and therefore ensure that they have been correctly named. Their expression patterns throughout development were also analyzed, by whole-mount mRNA in situ hybridization (ISH). The ISH results emphasized the importance of apolipoproteins in transporting yolk and dietary lipids: mRNA expression of all apolipoproteins was observed in the yolk syncytial layer, and intestinal and liver expression was observed from 4-6 days post-fertilization (dpf). Furthermore, real-time PCR confirmed that transcription of three of the four zebrafish apoA-IV genes was increased 4 hours after the onset of a 1-hour high-fat feed. Therefore, we tested the hypothesis that zebrafish ApoA-IV performs a conserved role to that in rat in the regulation of food intake by transiently overexpressing ApoA-IVb.1 in transgenic larvae and quantifying ingestion of co-fed fluorescently labeled fatty acid during a high-fat meal as an indicator of food intake. Indeed, ApoA-IVb.1 overexpression decreased food intake by approximately one-third. This study comprehensively describes the expression and function of eleven zebrafish apolipoproteins and serves as a springboard for future investigations to elucidate their roles in development and disease in the larval zebrafish model. PMID:25633982

  12. Rapamycin enhances docetaxel-induced cytotoxicity in a androgen-independent prostate cancer xenograft model by survivin downregulation

    SciTech Connect

    Morikawa, Yasuyuki, E-mail: yasu-m@med.gunma-u.ac.jp [Department of Urology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maeabshi, Gunma 3718511 (Japan)] [Department of Urology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maeabshi, Gunma 3718511 (Japan); Koike, Hidekazu; Sekine, Yoshitaka; Matsui, Hiroshi; Shibata, Yasuhiro; Ito, Kazuto; Suzuki, Kazuhiro [Department of Urology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maeabshi, Gunma 3718511 (Japan)] [Department of Urology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maeabshi, Gunma 3718511 (Japan)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer Rapamycin (RPM) enhances the susceptibility of PC3 cells to docetaxel. Black-Right-Pointing-Pointer Low-dosage of docetaxel (DTX) did not reduce survivin expression levels in PC3 cells. Black-Right-Pointing-Pointer Combination treatment of RPM with DTX suppressed the expression of surviving. Black-Right-Pointing-Pointer SiRNA against survivin enhanced the susceptibility of PC3 cells to DTX. Black-Right-Pointing-Pointer RPM and DTX cotreatment inhibited PC3 cell growth and decreased surviving in vivo. -- Abstract: Background: Docetaxel is a first-line treatment choice in castration-resistant prostate cancer (CRPC). However, the management of CRPC remains an important challenge in oncology. There have been many reports on the effects of rapamycin, which is an inhibitor of the mammalian target of rapamycin (mTOR), in the treatment of carcinogenesis. We assessed the cytotoxic effects of the combination treatment of docetaxel and rapamycin in prostate cancer cells. Furthermore, we examined the relationship between these treatments and survivin, which is a member of the inhibitory apoptosis family. Methods: Prostate cancer cells were cultured and treated with docetaxel and rapamycin. The effects on proliferation were evaluated with the MTS assay. In addition, we evaluated the effect on proliferation of the combination treatment induced knockdown of survivin expression by small interfering RNA transfection and docetaxel. Protein expression levels were assayed using western blotting. PC3 cells and xenograft growth in nude mice were used to evaluate the in vivo efficacy of docetaxel and its combination with rapamycin. Results: In vitro and in vivo, the combination of rapamycin with docetaxel resulted in a greater inhibition of proliferation than treatment with rapamycin or docetaxel alone. In addition, in vitro and in vivo, rapamycin decreased basal surviving levels, and cotreatment with docetaxel further decreased these levels. Transfection siRNA against survivin enhanced the cytotoxicity of docetaxel in PC3 cells. Conclusion: The rapamycin-dependent enhancement of the cytotoxic effects of docetaxel was associated with the downregulation of survivin expression. Our results suggest that the combination of docetaxel and rapamycin is a candidate for the improved treatment of advanced prostate cancer.

  13. The use of mass spectrometry imaging to predict treatment response of patient-derived xenograft models of triple-negative breast cancer.

    PubMed

    Mascini, Nadine E; Eijkel, Gert B; ter Brugge, Petra; Jonkers, Jos; Wesseling, Jelle; Heeren, Ron M A

    2015-02-01

    In recent years, mass spectrometry imaging (MSI) has been shown to be a promising technique in oncology. The effective application of MSI, however, is hampered by the complexity of the generated data. Bioinformatic approaches that reduce the complexity of these data are needed for the effective use in a (bio)medical setting. This holds especially for the analysis of tissue microarrays (TMA), which consist of hundreds of small tissue cores. Here we present an approach that combines MSI on tissue microarrays with principal component linear discriminant analysis (PCA-LDA) to predict treatment response. The feasibility of such an approach was evaluated on a set of patient-derived xenograft models of triple-negative breast cancer (TNBC). PCA-LDA was used to classify TNBC tumor tissues based on the proteomic information obtained with matrix-assisted laser desorption ionization (MALDI) MSI from the TMA surface. Classifiers based on two different tissue microarrays from the same tumor models showed overall classification accuracies between 59 and 77%, as determined by cross-validation. Reproducibility tests revealed that the two models were similar. A clear effect of intratumor heterogeneity of the classification scores was observed. These results demonstrate that the analysis of MALDI-MSI data by PCA-LDA is a valuable approach for the classification of treatment response and tumor heterogeneity in breast cancer. PMID:25553735

  14. Molecular control of neurogenesis in the regenerating central nervous system of the adult zebrafish 

    E-print Network

    Dias, Tatyana Beverly

    2012-11-30

    In contrast to mammals, adult zebrafish display cellular regeneration of lost motor neurons and achieve functional recovery following a complete spinal cord transection. Using adult zebrafish as a model to study how key ...

  15. Navigation by environmental geometry: the use of zebrafish as a model

    PubMed Central

    Lee, Sang Ah; Vallortigara, Giorgio; Flore, Michele; Spelke, Elizabeth S.; Sovrano, Valeria A.

    2013-01-01

    SUMMARY Sensitivity to environmental shape in spatial navigation has been found, at both behavioural and neural levels, in virtually every species tested, starting early in development. Moreover, evidence that genetic deletions can cause selective deficits in such navigation behaviours suggests a genetic basis to navigation by environmental geometry. Nevertheless, the geometric computations underlying navigation have not been specified in any species. The present study teases apart the geometric components within the traditionally used rectangular enclosure and finds that zebrafish selectively represent distance and directional relationships between extended boundary surfaces. Similar behavioural results in geometric navigation tasks with human children provide prima facie evidence for similar underlying cognitive computations and open new doors for probing the genetic foundations that give rise to these computations. PMID:23788708

  16. A zebrafish model of glucocorticoid resistance shows serotonergic modulation of the stress response

    PubMed Central

    Griffiths, Brian B.; Schoonheim, Peter J.; Ziv, Limor; Voelker, Lisa; Baier, Herwig; Gahtan, Ethan

    2012-01-01

    One function of glucocorticoids is to restore homeostasis after an acute stress response by providing negative feedback to stress circuits in the brain. Loss of this negative feedback leads to elevated physiological stress and may contribute to depression, anxiety, and post-traumatic stress disorder. We investigated the early, developmental effects of glucocorticoid signaling deficits on stress physiology and related behaviors using a mutant zebrafish, grs357, with non-functional glucocorticoid receptors (GRs). These mutants are morphologically inconspicuous and adult-viable. A previous study of adult grs357 mutants showed loss of glucocorticoid-mediated negative feedback and elevated physiological and behavioral stress markers. Already at 5 days post-fertilization, mutant larvae had elevated whole body cortisol, increased expression of pro-opiomelanocortin (POMC), the precursor of adrenocorticotropic hormone (ACTH), and failed to show normal suppression of stress markers after dexamethasone treatment. Mutant larvae had larger auditory-evoked startle responses compared to wildtype sibling controls (grwt), despite having lower spontaneous activity levels. Fluoxetine (Prozac) treatment in mutants decreased startle responding and increased spontaneous activity, making them behaviorally similar to wildtype. This result mirrors known effects of selective serotonin reuptake inhibitors (SSRIs) in modifying glucocorticoid signaling and alleviating stress disorders in human patients. Our results suggest that larval grs357 zebrafish can be used to study behavioral, physiological, and molecular aspects of stress disorders. Most importantly, interactions between glucocorticoid and serotonin signaling appear to be highly conserved among vertebrates, suggesting deep homologies at the neural circuit level and opening up new avenues for research into psychiatric conditions. PMID:23087630

  17. A xenograft mouse model coupled with in-depth plasma proteome analysis facilitates identification of novel serum biomarkers for human ovarian cancer

    PubMed Central

    Tang, Hsin-Yao; Beer, Lynn A.; Chang-Wong, Tony; Hammond, Rachel; Gimotty, Phyllis; Coukos, George; Speicher, David W.

    2011-01-01

    Proteomics discovery of novel cancer serum biomarkers is hindered by the great complexity of serum, patient-to-patient variability, and triggering by the tumor of an acute-phase inflammatory reaction. This host response alters many serum protein levels in cancer patients, but these changes have low specificity as they can be triggered by diverse causes. We addressed these hurdles by utilizing a xenograft mouse model coupled with an in-depth 4-D protein profiling method to identify human proteins in the mouse serum. This strategy ensures identified putative biomarkers are shed by the tumor, and detection of low-abundance proteins shed by the tumor is enhanced because the mouse blood volume is more than a thousand times smaller than that of a human. Using TOV-112D ovarian tumors, more than 200 human proteins were identified in the mouse serum, including novel candidate biomarkers and proteins previously reported to be elevated in either ovarian tumors or the blood of ovarian cancer patients. Subsequent quantitation of selected putative biomarkers in human sera using label-free multiple reaction monitoring (MRM) mass spectrometry (MS) showed that chloride intracellular channel 1, the mature form of cathepsin D, and peroxiredoxin 6 were elevated significantly in sera from ovarian carcinoma patients. PMID:22032327

  18. Systemic siRNA Delivery via Peptide-Tagged Polymeric Nanoparticles, Targeting PLK1 Gene in a Mouse Xenograft Model of Colorectal Cancer

    PubMed Central

    Malhotra, Meenakshi; Tomaro-Duchesneau, Catherine; Saha, Shyamali; Prakash, Satya

    2013-01-01

    Polymeric nanoparticles were developed from a series of chemical reactions using chitosan, polyethylene glycol, and a cell-targeting peptide (CP15). The nanoparticles were complexed with PLK1-siRNA. The optimal siRNA loading was achieved at an N?:?P ratio of 129.2 yielding a nanoparticle size of >200?nm. These nanoparticles were delivered intraperitoneally and tested for efficient delivery, cytotoxicity, and biodistribution in a mouse xenograft model of colorectal cancer. Both unmodified and modified chitosan nanoparticles showed enhanced accumulation at the tumor site. However, the modified chitosan nanoparticles showed considerably, less distribution in other organs. The relative gene expression as evaluated showed efficient delivery of PLK1-siRNA (0.5?mg/kg) with 50.7 ± 19.5% knockdown (P = 0.031) of PLK1 gene. The in vivo data reveals no systemic toxicity in the animals, when tested for systemic inflammation and liver toxicity. These results indicate a potential of using peptide-tagged nanoparticles for systemic delivery of siRNA at the targeted tumor site. PMID:24159333

  19. Transforming growth factor-beta can suppress tumorigenesis through effects on the putative cancer stem or early progenitor cell and committed progeny in a breast cancer xenograft model.

    PubMed

    Tang, Binwu; Yoo, Naomi; Vu, Mary; Mamura, Mizuko; Nam, Jeong-Seok; Ooshima, Akira; Du, Zhijun; Desprez, Pierre-Yves; Anver, Miriam R; Michalowska, Aleksandra M; Shih, Joanna; Parks, W Tony; Wakefield, Lalage M

    2007-09-15

    The transforming growth factor-beta (TGF-beta) pathway has tumor-suppressor activity in many epithelial tissues. Because TGF-beta is a potent inhibitor of epithelial cell proliferation, it has been widely assumed that this property underlies the tumor-suppressor effect. Here, we have used a xenograft model of breast cancer to show that endogenous TGF-beta has the potential to suppress tumorigenesis through a novel mechanism, involving effects at two distinct levels in the hierarchy of cellular progeny that make up the epithelial component of the tumor. First, TGF-beta reduces the size of the putative cancer stem or early progenitor cell population, and second it promotes differentiation of a more committed, but highly proliferative, progenitor cell population to an intrinsically less proliferative state. We further show that reduced expression of the type II TGF-beta receptor correlates with loss of luminal differentiation in a clinical breast cancer cohort, suggesting that this mechanism may be clinically relevant. At a molecular level, the induction of differentiation by TGF-beta involves down-regulation of Id1, and forced overexpression of Id1 can promote tumorigenesis despite persistence of the antiproliferative effect of TGF-beta. These data suggest new roles for the TGF-beta pathway in regulating tumor cell dynamics that are independent of direct effects on proliferation. PMID:17875704

  20. Alkylpurine–DNA–N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients

    PubMed Central

    Agnihotri, Sameer; Gajadhar, Aaron S.; Ternamian, Christian; Gorlia, Thierry; Diefes, Kristin L.; Mischel, Paul S.; Kelly, Joanna; McGown, Gail; Thorncroft, Mary; Carlson, Brett L.; Sarkaria, Jann N.; Margison, Geoffrey P.; Aldape, Kenneth; Hawkins, Cynthia; Hegi, Monika; Guha, Abhijit

    2011-01-01

    Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). O6-methylguanine–DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O6-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine–DNA–N-glycosylase (APNG), which repairs the cytotoxic lesions N3-methyladenine and N7-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease. PMID:22156195

  1. TGF-? can suppress tumorigenesis through effects on the putative cancer stem or early progenitor cell and committed progeny in a breast cancer xenograft model

    PubMed Central

    Tang, Binwu; Yoo, Naomi; Vu, Mary; Mamura, Mizuko; Nam, Jeong-Seok; Ooshima, Akira; Du, Zhijun; Desprez, Pierre-Yves; Anver, Miriam R.; Michalowska, Aleksandra M.; Shih, Joanna; Parks, W. Tony; Wakefield, Lalage M.

    2008-01-01

    The TGF-? pathway has tumor suppressor activity in many epithelial tissues. Since TGF-? is a potent inhibitor of epithelial cell proliferation, it has been widely assumed that this property underlies the tumor suppressor effect. Here we have used a xenograft model of breast cancer to show that endogenous TGF-? has the potential to suppress tumorigenesis through a novel mechanism, involving effects at two distinct levels in the hierarchy of cellular progeny that make up the epithelial component of the tumor. Firstly TGF-? reduces the size of the putative cancer stem or early progenitor cell population, and secondly it promotes differentiation of a more committed, but highly proliferative, progenitor cell population to an intrinsically less proliferative state. We further show that reduced expression of the type II TGF-? receptor correlates with loss of luminal differentiation in a clinical breast cancer cohort, suggesting that this mechanism may be clinically relevant. At a molecular level, the induction of differentiation by TGF-? involves down-regulation of Id1, and forced overexpression of Id1 can promote tumorigenesis despite persistence of the anti-proliferative effect of TGF-?. These data suggest new roles for the TGF-? pathway in regulating tumor cell dynamics that are independent of direct effects on proliferation. PMID:17875704

  2. Autophagy induction by leptin contributes to suppression of apoptosis in cancer cells and xenograft model: Involvement of p53/FoxO3A axis

    PubMed Central

    Nepal, Saroj; Kim, Mi Jin; Hong, Jin Tae; Kim, Sang Hyun; Sohn, Dong-Hwan; Lee, Sung Hee; Song, Kyung; Choi, Dong Young; Lee, Eung Seok; Park, Pil-Hoon

    2015-01-01

    Leptin, a hormone mainly produced from adipose tissue, has been shown to induce proliferation of cancer cells. However, the molecular mechanisms underlying leptin-induced tumor progression have not been clearly elucidated. In the present study, we investigated the role of autophagy in leptin-induced cancer cell proliferation using human hepatoma (HepG2) and breast cancer cells (MCF-7), and tumor growth in a xenograft model. Herein, we showed that leptin treatment caused autophagy induction as assessed by increase in expression of autophagy-related genes, including beclin-1, Atg5 and LC3 II, further induction of autophagosome formation and autophagic flux. Interestingly, inhibition of autophagic process by treatment with inhibitors and LC3B gene silencing blocked leptin-induced increase in cell number and suppression of apoptosis, indicating a crucial role of autophagy in leptin-induced tumor progression. Moreover, gene silencing of p53 or FoxO3A prevented leptin-induced LC3 II protein expression, suggesting an involvement of p53/FoxO3A axis in leptin-induced autophagy activation. Leptin administration also accelerated tumor growth in BALB/c nude mice, which was found to be autophagy dependent. Taken together, our results demonstrate that leptin-induced tumor growth is mediated by autophagy induction and autophagic process would be a promising target to regulate development of cancer caused by leptin production. PMID:25704884

  3. Advances in zebrafish chemical screening technologies

    PubMed Central

    Mathias, Jonathan R; Saxena, Meera T; Mumm, Jeff S

    2013-01-01

    Due to several inherent advantages, zebrafish are being utilized in increasingly sophisticated screens to assess the physiological effects of chemical compounds directly in living vertebrate organisms. Diverse screening platforms showcase these advantages. Morphological assays encompassing basic qualitative observations to automated imaging, manipulation, and data-processing systems provide whole organism to subcellular levels of detail. Behavioral screens extend chemical screening to the level of complex systems. In addition, zebrafish-based disease models provide a means of identifying new potential therapeutic strategies. Automated systems for handling/sorting, high-resolution imaging and quantitative data collection have significantly increased throughput in recent years. These advances will make it easier to capture multiple streams of information from a given sample and facilitate integration of zebrafish at the earliest stages of the drug-discovery process, providing potential solutions to current drug-development bottlenecks. Here we outline advances that have been made within the growing field of zebrafish chemical screening. PMID:23043478

  4. Zebrafish screen identifies novel compound with selective toxicity against leukemia.

    PubMed

    Ridges, Suzanne; Heaton, Will L; Joshi, Deepa; Choi, Henry; Eiring, Anna; Batchelor, Lance; Choudhry, Priya; Manos, Elizabeth J; Sofla, Hossein; Sanati, Ali; Welborn, Seth; Agarwal, Archana; Spangrude, Gerald J; Miles, Rodney R; Cox, James E; Frazer, J Kimble; Deininger, Michael; Balan, Kaveri; Sigman, Matthew; Müschen, Markus; Perova, Tatiana; Johnson, Radia; Montpellier, Bertrand; Guidos, Cynthia J; Jones, David A; Trede, Nikolaus S

    2012-06-14

    To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces long-term remission in adult zebrafish with cMYC-induced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells. PMID:22490804

  5. Zebrafish screen identifies novel compound with selective toxicity against leukemia

    PubMed Central

    Ridges, Suzanne; Heaton, Will L.; Joshi, Deepa; Choi, Henry; Eiring, Anna; Batchelor, Lance; Choudhry, Priya; Manos, Elizabeth J.; Sofla, Hossein; Sanati, Ali; Welborn, Seth; Agarwal, Archana; Spangrude, Gerald J.; Miles, Rodney R.; Cox, James E.; Frazer, J. Kimble; Deininger, Michael; Balan, Kaveri; Sigman, Matthew; Müschen, Markus; Perova, Tatiana; Johnson, Radia; Montpellier, Bertrand; Guidos, Cynthia J.; Jones, David A.

    2012-01-01

    To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces long-term remission in adult zebrafish with cMYC-induced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells. PMID:22490804

  6. Pharmacologic Inhibition of MLK3 Kinase Activity Blocks the In Vitro Migratory Capacity of Breast Cancer Cells but Has No Effect on Breast Cancer Brain Metastasis in a Mouse Xenograft Model

    PubMed Central

    Rhoo, Kun Hyoe; Granger, Megan; Sur, Joynita; Feng, Changyong; Gelbard, Harris A.; Dewhurst, Stephen; Polesskaya, Oksana

    2014-01-01

    Brain metastasis of breast cancer is an important clinical problem, with few therapeutic options and a poor prognosis. Recent data have implicated mixed lineage kinase 3 (MLK3) in controlling the in vitro migratory capacity of breast cancer cells, as well as the metastasis of MDA-MB-231 breast cancer cells from the mammary fat pad to distant lymph nodes in a mouse xenograft model. We therefore set out to test whether MLK3 plays a role in brain metastasis of breast cancer cells. To address this question, we used a novel, brain penetrant, MLK3 inhibitor, URMC099. URMC099 efficiently inhibited the migration of breast cancer cells in an in vitro cell monolayer wounding assay, and an in vitro transwell migration assay, but had no effect on in vitro cell growth. We also tested the effect of URMC099 on tumor formation in a mouse xenograft model of breast cancer brain metastasis. This analysis showed that URMC099 had no effect on the either the frequency or size of breast cancer brain metastases. We conclude that pharmacologic inhibition of MLK3 by URMC099 can reduce the in vitro migratory capacity of breast cancer cells, but that it has no effect on either the frequency or size of breast cancer brain metastases, in a mouse xenograft model. PMID:25264786

  7. Radioimmunoscintigraphy and radioimmunotherapy of renal cell carcinoma xenografts

    SciTech Connect

    Vessella, R.L.; Alvarez, V.; Chiou, R.K.; Rodwell, J.; Elson, M.; Palme, D.; Shafer, R.; Lange, P.

    1987-01-01

    From a panel of monoclonal antibodies (MAb) reactive with human renal cell carcinoma generated by this laboratory, three (designated A6H, C5H, and D5D) were selected for in vivo studies with a nude mouse xenograft model. These studies included /sup 131/I- and /sup 111/In-labeled MAb radioimmunoscintigraphy and /sup 131/I-labeled MAb radioimmunotherapy. In the imaging studies, these radiolabeled MAb allowed visualization of subcutaneous xenografts larger than 40 mg and subrenal capsule xenografts smaller than 20 mg. Comparisons of tumor to non-tumor tissue radiolabel distribution yielded unusually high ratios and depended on the MAb-xenograft combination. The /sup 111/In-radiolabeled A6H showed increased accumulation in the liver compared with /sup 131/I-A6H, but this still did not necessitate background subtraction for good visualization of small, subrenal capsule renal cell carcinoma xenografts. Radioimmunotherapy studies with /sup 131/I-A6H in BALB/c nu/nu mice bearing established renal cell carcinoma xenografts showed a prolonged (greater than 90 days) regression in tumor burden and possible cures, whereas three sets of control mice showed progressive and rapid increases in tumor size. These studies indicated that MAb, which show good tissue biodistribution and high imaging sensitivity, could also be capable of delivering effective radiotherapy to the tumor when human equivalent radiolabeled-MAb doses are used.

  8. Developmental defects and neuromuscular alterations due to mitofusin 2 gene (MFN2) silencing in zebrafish: a new model for Charcot-Marie-Tooth type 2A neuropathy.

    PubMed

    Vettori, Andrea; Bergamin, Giorgia; Moro, Enrico; Vazza, Giovanni; Polo, Giulia; Tiso, Natascia; Argenton, Francesco; Mostacciuolo, Maria Luisa

    2011-01-01

    The development of new animal models is a crucial step in determining the pathological mechanism underlying neurodegenerative diseases and is essential for the development of effective therapies. We have investigated the zebrafish (Danio rerio) as a new model to study CMT2A, a peripheral neuropathy characterized by the selective loss of motor neurons, caused by mutations of mitofusin 2 gene. Using a knock-down approach, we provide evidence that during embryonic development, mitofusin 2 loss of function is responsible of several morphological defects and motility impairment. Immunohistochemical investigations, revealing the presence of severe alterations in both motor neurons and muscles fibres, indicated the central role played by MFN2 in axonal and neuromuscular development. Finally, we demonstrated the ability of human MFN2 to balance the downregulation of endogenous mfn2 in zebrafish, further supporting the conserved function of the MFN2 gene. These results highlight the essential role of mitofusin 2 in the motor axon development and demonstrate the potential of zebrafish as a suitable and complementary platform for dissecting pathogenetic mechanisms of MFN2 mutations in vivo. PMID:20951042

  9. Understanding the transformation, speciation, and hazard potential of copper particles in a model septic tank system using zebrafish to monitor the effluent.

    PubMed

    Lin, Sijie; Taylor, Alicia A; Ji, Zhaoxia; Chang, Chong Hyun; Kinsinger, Nichola M; Ueng, William; Walker, Sharon L; Nel, André E

    2015-02-24

    Although copper-containing nanoparticles are used in commercial products such as fungicides and bactericides, we presently do not understand the environmental impact on other organisms that may be inadvertently exposed. In this study, we used the zebrafish embryo as a screening tool to study the potential impact of two nano Cu-based materials, CuPRO and Kocide, in comparison to nanosized and micron-sized Cu and CuO particles in their pristine form (0-10 ppm) as well as following their transformation in an experimental wastewater treatment system. This was accomplished by construction of a modeled domestic septic tank system from which effluents could be retrieved at different stages following particle introduction (10 ppm). The Cu speciation in the effluent was identified as nondissolvable inorganic Cu(H2PO2)2 and nondiffusible organic Cu by X-ray diffraction, inductively coupled plasma mass spectrometry (ICP-MS), diffusive gradients in thin-films (DGT), and Visual MINTEQ software. While the nanoscale materials, including the commercial particles, were clearly more potent (showing 50% hatching interference above 0.5 ppm) than the micron-scale particulates with no effect on hatching up to 10 ppm, the Cu released from the particles in the septic tank underwent transformation into nonbioavailable species that failed to interfere with the function of the zebrafish embryo hatching enzyme. Moreover, we demonstrate that the addition of humic acid, as an organic carbon component, could lead to a dose-dependent decrease in Cu toxicity in our high content zebrafish embryo screening assay. Thus, the use of zebrafish embryo screening, in combination with the effluents obtained from a modeled exposure environment, enables a bioassay approach to follow the change in the speciation and hazard potential of Cu particles instead of difficult-to-perform direct particle tracking. PMID:25625504

  10. Novel Mouse Xenograft Models Reveal a Critical Role of CD4+ T Cells in the Proliferation of EBV-Infected T and NK Cells

    PubMed Central

    Arai, Ayako; Nakazawa, Atsuko; Kawano, Fuyuko; Ichikawa, Sayumi; Shimizu, Norio; Yamamoto, Naoki; Morio, Tomohiro; Ohga, Shouichi; Nakamura, Hiroyuki; Ito, Mamoru; Miura, Osamu; Komano, Jun; Fujiwara, Shigeyoshi

    2011-01-01

    Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic herpesvirus, ectopically infects T or NK cells to cause severe diseases of unknown pathogenesis, including chronic active EBV infection (CAEBV) and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). We developed xenograft models of CAEBV and EBV-HLH by transplanting patients' PBMC to immunodeficient mice of the NOD/Shi-scid/IL-2R?null strain. In these models, EBV-infected T, NK, or B cells proliferated systemically and reproduced histological characteristics of the two diseases. Analysis of the TCR repertoire expression revealed that identical predominant EBV-infected T-cell clones proliferated in patients and corresponding mice transplanted with their PBMC. Expression of the EBV nuclear antigen 1 (EBNA1), the latent membrane protein 1 (LMP1), and LMP2, but not EBNA2, in the engrafted cells is consistent with the latency II program of EBV gene expression known in CAEBV. High levels of human cytokines, including IL-8, IFN-?, and RANTES, were detected in the peripheral blood of the model mice, mirroring hypercytokinemia characteristic to both CAEBV and EBV-HLH. Transplantation of individual immunophenotypic subsets isolated from patients' PBMC as well as that of various combinations of these subsets revealed a critical role of CD4+ T cells in the engraftment of EBV-infected T and NK cells. In accordance with this finding, in vivo depletion of CD4+ T cells by the administration of the OKT4 antibody following transplantation of PBMC prevented the engraftment of EBV-infected T and NK cells. This is the first report of animal models of CAEBV and EBV-HLH that are expected to be useful tools in the development of novel therapeutic strategies for the treatment of the diseases. PMID:22028658

  11. Alarm substance induced behavioral responses in zebrafish ( Danio rerio)

    Microsoft Academic Search

    Natasha Speedie; Robert Gerlai

    2008-01-01

    Zebrafish (zebra danio) are becoming increasingly popular in behavioral neuroscience and behavior genetics. This small vertebrate may be utilized in modeling human brain disorders. One of the major neuropsychiatric conditions still not well understood is abnormally increased fear and anxiety. Zebrafish may be an appropriate organism with which these human diseases can be modeled and their biological mechanisms investigated. Predator

  12. The Zebrafish Models to Explore Genetic and Epigenetic Impacts on Evolutionary Developmental Origins of Aging

    PubMed Central

    Kishi, Shuji

    2014-01-01

    Can we reset, reprogram, rejuvenate or reverse the organismal aging process? Certain genetic manipulations could at least reset and reprogram epigenetic dynamics beyond phenotypic plasticity and elasticity in cells, which can be further manipulated into organisms. However, in a whole complex aging organism, how can we rejuvenate intrinsic resources and infrastructures in an intact/noninvasive manner? The incidence of diseases increases exponentially with age, accompanied by progressive deteriorations of physiological functions in organisms. Aging-associated diseases are sporadic but essentially inevitable complications arising from senescence. Senescence is often considered the antithesis of early development, but yet there may be factors and mechanisms in common between these two phenomena to rejuvenate over the dynamic process of aging. The association between early development and late-onset disease with advancing age is thought to come from a consequence of developmental plasticity, the phenomenon by which one genotype can give rise to a range of physiologically and/or morphologically adaptive states based on diverse epigenotypes, in response to intrinsic or extrinsic environmental cues and genetic perturbations. We hypothesized that the future aging process can be predictive based on adaptivity during the early developmental period. Modulating the thresholds and windows of plasticity and its robustness by molecular genetic and chemical epigenetic approaches, we have successfully conducted experiments to isolate zebrafish mutants expressing apparently altered senescence phenotypes during their embryonic and/or larval stages (“embryonic/larval senescence”). Subsequently, at least some of these mutant animals were found to show shortened lifespan, while some others would be expected to live longer in adulthoods. We anticipate that previously uncharacterized developmental genes may mediate the aging process and play a pivotal role in senescence. On the other hand, unexpected senescence-related genes might also be involved in the early developmental process and its regulation. The ease of manipulation using the zebrafish system allows us to conduct an exhaustive exploration of novel genes/genotypes and epigenotype that can be linked to the senescence phenotype, and thereby facilitates searching for the evolutionary and developmental origins of aging in vertebrates. PMID:24239812

  13. Using zebrafish models to explore genetic and epigenetic impacts on evolutionary developmental origins of aging.

    PubMed

    Kishi, Shuji

    2014-02-01

    Can we reset, reprogram, rejuvenate, or reverse the organismal aging process? Certain genetic manipulations could at least reset and reprogram epigenetic dynamics beyond phenotypic plasticity and elasticity in cells, which can be manipulated further into organisms. However, in a whole complex aging organism, how can we rejuvenate intrinsic resources and infrastructures in an intact and noninvasive manner? The incidence of diseases increases exponentially with age, accompanied by progressive deteriorations of physiological functions in organisms. Aging-associated diseases are sporadic but essentially inevitable complications arising from senescence. Senescence is often considered the antithesis of early development, but yet there may be factors and mechanisms in common between these 2 phenomena to rejuvenate over the dynamic process of aging. The association between early development and late-onset disease with advancing age is thought to come from a consequence of developmental plasticity, the phenomenon by which one genotype can give rise to a range of physiologically and/or morphologically adaptive states based on diverse epigenotypes in response to intrinsic or extrinsic environmental cues and genetic perturbations. We hypothesized that the future aging process can be predictive based on adaptivity during the early developmental period. Modulating the thresholds and windows of plasticity and its robustness by molecular genetic and chemical epigenetic approaches, we have successfully conducted experiments to isolate zebrafish mutants expressing apparently altered senescence phenotypes during their embryonic and/or larval stages ("embryonic/larval senescence"). Subsequently, at least some of these mutant animals were found to show a shortened life span, whereas others would be expected to live longer into adulthood. We anticipate that previously uncharacterized developmental genes may mediate the aging process and play a pivotal role in senescence. On the other hand, unexpected senescence-related genes might also be involved in the early developmental process and its regulation. The ease of manipulation using the zebrafish system allows us to conduct an exhaustive exploration of novel genes, genotypes, and epigenotypes that can be linked to the senescence phenotype, which facilitates searching for the evolutionary and developmental origins of aging in vertebrates. PMID:24239812

  14. Anti-CCR7 therapy exerts a potent anti-tumor activity in a xenograft model of human mantle cell lymphoma

    PubMed Central

    2013-01-01

    Background The chemokine receptor CCR7 mediates lymphoid dissemination of many cancers, including lymphomas and epithelial carcinomas, thus representing an attractive therapeutic target. Previous results have highlighted the potential of the anti-CCR7 monoclonal antibodies to inhibit migration in transwell assays. The present study aimed to evaluate the in vivo therapeutic efficacy of an anti-CCR7 antibody in a xenografted human mantle cell lymphoma model. Methods NOD/SCID mice were either subcutaneously or intravenously inoculated with Granta-519 cells, a human cell line derived from a leukemic mantle cell lymphoma. The anti-CCR7 mAb treatment (3 × 200 ?g) was started on day 2 or 7 to target lymphoma cells in either a peri-implantation or a post-implantation stage, respectively. Results The anti-CCR7 therapy significantly delayed the tumor appearance and also reduced the volumes of tumors in the subcutaneous model. Moreover, an increased number of apoptotic tumor cells was detected in mice treated with the anti-CCR7 mAb compared to the untreated animals. In addition, significantly reduced number of Granta-519 cells migrated from subcutaneous tumors to distant lymphoid organs, such as bone marrow and spleen in the anti-CCR7 treated mice. In the intravenous models, the anti-CCR7 mAb drastically increased survival of the mice. Accordingly, dissemination and infiltration of tumor cells in lymphoid and non-lymphoid organs, including lungs and central nervous system, was almost abrogated. Conclusions The anti-CCR7 mAb exerts a potent anti-tumor activity and might represent an interesting therapeutic alternative to conventional therapies. PMID:24305507

  15. From DNA to Protein: a study of genomic instability candidate genes during zebrafish development

    Microsoft Academic Search

    Kristine Griffett

    2011-01-01

    The zebrafish, Danio rerio, is a type of freshwater minnow often used to model human diseases including cancer, anxiety and aging diseases. The overall biology of zebrafish is strikingly similar to that of humans, allowing these fish to be used for drug discovery and toxicology studies for preclinical trials. In this study, zebrafish embryos were used to identify and characterize

  16. Mycobacteria Counteract a TLR-Mediated Nitrosative Defense Mechanism in a Zebrafish Infection Model

    PubMed Central

    van Hensbergen, Vincent; Schutz, Esther; Redd, Michael J.; Murayama, Emi; Spaink, Herman P.; Meijer, Annemarie H.

    2014-01-01

    Pulmonary tuberculosis (TB), caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb), is a major world health problem. The production of reactive nitrogen species (RNS) is a potent cytostatic and cytotoxic defense mechanism against intracellular pathogens. Nevertheless, the protective role of RNS during Mtb infection remains controversial. Here we use an anti-nitrotyrosine antibody as a readout to study nitration output by the zebrafish host during early mycobacterial pathogenesis. We found that recognition of Mycobacterium marinum, a close relative of Mtb, was sufficient to induce a nitrosative defense mechanism in a manner dependent on MyD88, the central adaptor protein in Toll like receptor (TLR) mediated pathogen recognition. However, this host response was attenuated by mycobacteria via a virulence mechanism independent of the well-characterized RD1 virulence locus. Our results indicate a mechanism of pathogenic mycobacteria to circumvent host defense in vivo. Shifting the balance of host-pathogen interactions in favor of the host by targeting this virulence mechanism may help to alleviate the problem of infection with Mtb strains that are resistant to multiple drug treatments. PMID:24967596

  17. TET2 Plays an Essential Role in Erythropoiesis by Regulating Lineage-Specific Genes via DNA Oxidative Demethylation in a Zebrafish Model

    PubMed Central

    Ge, Liang; Zhang, Rui-peng; Wan, Feng; Guo, Dong-yang; Wang, Ping

    2014-01-01

    Although epigenetic modulation is critical for a variety of cellular activities, its role in erythropoiesis remains poorly understood. Ten-eleven translocation (TET) molecules participate in methylcytosine (5mC) hydroxylation, which results in DNA demethylation in several biological processes. In this research, the role of TETs in erythropoiesis was investigated by using the zebrafish model, where three TET homologs were identified. These homologs share conserved structural domains with their mammalian counterparts. Zebrafish TETs mediate the conversion of 5mC to hydroxymethylcytosine (5hmC) in zebrafish embryos, and the deletion of TET2 inhibits erythropoiesis by suppressing the expression of the scl, gata-1, and cmyb genes. TET2-upregulated lineage-specific genes and erythropoiesis are closely associated with the occurrence of 5hmC and demethylation in the intermediate CpG promoters (ICPs) of scl, gata-1, cmyb, which frequently occur at specific regions or CpG sites of these ICPs. Moreover, TET2 regulates the formation and differentiation of erythroid progenitors, and deletion of TET2 leads to erythrocyte dysplasia and anemia. Here, we preliminarily proved that TET2 plays an essential role in erythrocyte development by regulating lineage-specific genes via DNA oxidative demethylation. This report is anticipated to broaden current information on hematopoiesis and pathogenesis of hematopoiesis-related diseases. PMID:24396069

  18. Biodegradable polymeric micelle-encapsulated quercetin suppresses tumor growth and metastasis in both transgenic zebrafish and mouse models

    NASA Astrophysics Data System (ADS)

    Wu, Qinjie; Deng, Senyi; Li, Ling; Sun, Lu; Yang, Xi; Liu, Xinyu; Liu, Lei; Qian, Zhiyong; Wei, Yuquan; Gong, Changyang

    2013-11-01

    Quercetin (Que) loaded polymeric micelles were prepared to obtain an aqueous formulation of Que with enhanced anti-tumor and anti-metastasis activities. A simple solid dispersion method was used, and the obtained Que micelles had a small particle size (about 31 nm), high drug loading, and high encapsulation efficiency. Que micelles showed improved cellular uptake, an enhanced apoptosis induction effect, and stronger inhibitory effects on proliferation, migration, and invasion of 4T1 cells than free Que. The enhanced in vitro antiangiogenesis effects of Que micelles were proved by the results that Que micelles significantly suppressed proliferation, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs). Subsequently, transgenic zebrafish models were employed to investigate anti-tumor and anti-metastasis effects of Que micelles, in which stronger inhibitory effects of Que micelles were observed on embryonic angiogenesis, tumor-induced angiogenesis, tumor growth, and tumor metastasis. Furthermore, in a subcutaneous 4T1 tumor model, Que micelles were more effective in suppressing tumor growth and spontaneous pulmonary metastasis, and prolonging the survival of tumor-bearing mice. Besides, immunohistochemical and immunofluorescent assays suggested that tumors in the Que micelle-treated group showed more apoptosis, fewer microvessels, and fewer proliferation-positive cells. In conclusion, Que micelles, which are synthesized as an aqueous formulation of Que, possess enhanced anti-tumor and anti-metastasis activity, which can serve as potential candidates for cancer therapy.

  19. Effect of a platelet-activating factor (PAF) receptor antagonist on hyperacute xenograft rejection; evaluation in a pig kidney–human blood xenoperfusion model

    PubMed Central

    Cruzado, J M; Torras, J; Riera, M; Lloberas, N; Herrero, I; Condom, E; Martorell, J; Alsina, J; Grinyó, J M

    1998-01-01

    In pig to human discordant xenotransplantation, PAF may contribute to the pathogenesis of hyperacute xenograft rejection (HXR). We examined the release of PAF and the effect of a PAF receptor antagonist (BN 52021) on HXR in a pig kidney–human blood xenoperfusion model. Pig kidneys were perfused with porcine blood (AUTO group, n = 5), human blood (HETER group, n = 6) or human blood plus BN 52021 (BN group, n = 4), respectively. In contrast to HETER kidneys that never produced urine and were rejected in 15–30 min, the administration of BN 52021 induced a partial recovery of glomerular filtration rate and allowed kidneys to function until the end of the study. The release of PAF and soluble P-selectin, as well as endothelial P-selectin expression and tissue myeloperoxidase (MPO), were much higher in the HETER than in the AUTO group. HETER and BN kidneys displayed similar natural xenoantibody titres, CH50, PAF, soluble P-selectin as well as renal immunoglobulin (IgM, IgG, IgA) and complement (C3, C1q) deposition. However, HETER kidneys displayed a full histologic picture of HXR (mainly interstitial haemorrhage and vascular microthrombi) and BN kidneys had only endothelial cell swelling. Also, BN 52021 administration attenuated glomerular and vascular P-selectin expression and renal tissue MPO activity. We conclude that in the pig kidney–human blood xenoperfusion model, PAF is produced in higher amounts than in the pig kidney–pig blood autologous combination. The administration of BN 52021 exerts a protective effect by means of attenuating the acute inflammatory response and blocking vascular microthrombi formation. PMID:9697996

  20. Preclinical activity of the type II CD20 antibody GA101 (obinutuzumab) compared with rituximab and ofatumumab in vitro and in xenograft models.

    PubMed

    Herter, Sylvia; Herting, Frank; Mundigl, Olaf; Waldhauer, Inja; Weinzierl, Tina; Fauti, Tanja; Muth, Gunter; Ziegler-Landesberger, Doris; Van Puijenbroek, Erwin; Lang, Sabine; Duong, Minh Ngoc; Reslan, Lina; Gerdes, Christian A; Friess, Thomas; Baer, Ute; Burtscher, Helmut; Weidner, Michael; Dumontet, Charles; Umana, Pablo; Niederfellner, Gerhard; Bacac, Marina; Klein, Christian

    2013-10-01

    We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation. PMID:23873847

  1. EGFRvIII-specific chimeric antigen receptor T cells migrate to and kill tumor deposits infiltrating the brain parenchyma in an invasive xenograft model of glioblastoma.

    PubMed

    Miao, Hongsheng; Choi, Bryan D; Suryadevara, Carter M; Sanchez-Perez, Luis; Yang, Shicheng; De Leon, Gabriel; Sayour, Elias J; McLendon, Roger; Herndon, James E; Healy, Patrick; Archer, Gary E; Bigner, Darell D; Johnson, Laura A; Sampson, John H

    2014-01-01

    Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and is uniformly lethal. T-cell-based immunotherapy offers a promising platform for treatment given its potential to specifically target tumor tissue while sparing the normal brain. However, the diffuse and infiltrative nature of these tumors in the brain parenchyma may pose an exceptional hurdle to successful immunotherapy in patients. Areas of invasive tumor are thought to reside behind an intact blood brain barrier, isolating them from effective immunosurveillance and thereby predisposing the development of "immunologically silent" tumor peninsulas. Therefore, it remains unclear if adoptively transferred T cells can migrate to and mediate regression in areas of invasive GBM. One barrier has been the lack of a preclinical mouse model that accurately recapitulates the growth patterns of human GBM in vivo. Here, we demonstrate that D-270 MG xenografts exhibit the classical features of GBM and produce the diffuse and invasive tumors seen in patients. Using this model, we designed experiments to assess whether T cells expressing third-generation chimeric antigen receptors (CARs) targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, would localize to and treat invasive intracerebral GBM. EGFRvIII-targeted CAR (EGFRvIII+ CAR) T cells demonstrated in vitro EGFRvIII antigen-specific recognition and reactivity to the D-270 MG cell line, which naturally expresses EGFRvIII. Moreover, when administered systemically, EGFRvIII+ CAR T cells localized to areas of invasive tumor, suppressed tumor growth, and enhanced survival of mice with established intracranial D-270 MG tumors. Together, these data demonstrate that systemically administered T cells are capable of migrating to the invasive edges of GBM to mediate antitumor efficacy and tumor regression. PMID:24722266

  2. Magnetic resonance tracking of endothelial progenitor cells labeled with alkyl-polyethylenimine 2 kDa/superparamagnetic iron oxide in a mouse lung carcinoma xenograft model.

    PubMed

    Chen, Cong; Yu, Hong; Xia, Rui; Wang, Lei; Ai, Hua; Liu, Shiyuan; Xu, Zhiming; Xiao, Xiangsheng; Gao, Fabao

    2014-01-01

    The potential of using endothelial progenitor cells (EPCs) in novel anticancer therapy and the repair of vascular injury has been increasingly recognized. In the present study, EPCs were labeled with N-alkyl-polyethylenimine 2 kDa (PEI2k)-stabilized superparamagnetic iron oxide (SPIO) to facilitate magnetic resonance imaging (MRI) of EPCs in a mouse lung carcinoma xenograft model. EPCs derived from human peripheral blood were labeled with alkyl-PEI2k/SPIO. The viability and activity of labeled cells were evaluated using proliferation, migration, and tubulogenesis assays. Alkyl-PEI2k/SPIO-labeled EPCs were injected intravenously (group 1) or mixed and injected together with A549 cells subcutaneously (group 2) into groups of six mice with severe combined immunodeficiency. The labeling efficiency with alkyl-PEI2k/SPIO at 7 ?g Fe/mL concentration was approximately 100%. Quantitative analysis of cellular iron was 6.062 ± 0.050 pg/cell. No significant effects on EPC proliferation, migration, or tubulogenesis were seen after labeling. Seven-tesla micro-MRI showed the presence of schistic or linear hypointense regions at the tumor margins starting from days 7 to 8 after EPC administration. This gradually extended into the inner tumor layers in group 1. In group 2, tumor growth was accompanied by dispersion of low-signal intensity regions inside the tumor. Iron-positive cells identified by Prussian blue dye were seen at the sites identified using MRI. Human CD31-positive cells and mouse CD31-positive cells were present in both groups. Labeling EPCs with alkyl-PEI2k/SPIO allows noninvasive magnetic resonance investigation of EPC involvement in tumor neovasculature and is associated with excellent biocompatibility and MRI sensitivity. PMID:25248646

  3. FXR controls the tumor suppressor NDRG2 and FXR agonists reduce liver tumor growth and metastasis in an orthotopic mouse xenograft model.

    PubMed

    Deuschle, Ulrich; Schüler, Julia; Schulz, Andreas; Schlüter, Thomas; Kinzel, Olaf; Abel, Ulrich; Kremoser, Claus

    2012-01-01

    The farnesoid X receptor (FXR) is expressed predominantly in tissues exposed to high levels of bile acids and controls bile acid and lipid homeostasis. FXR(-/-) mice develop hepatocellular carcinoma (HCC) and show an increased prevalence for intestinal malignancies, suggesting a role of FXR as a tumor suppressor in enterohepatic tissues. The N-myc downstream-regulated gene 2 (NDRG2) has been recognized as a tumor suppressor gene, which is downregulated in human hepatocellular carcinoma, colorectal carcinoma and many other malignancies.We show reduced NDRG2 mRNA in livers of FXR(-/-) mice compared to wild type mice and both, FXR and NDRG2 mRNAs, are reduced in human HCC compared to normal liver. Gene reporter assays and Chromatin Immunoprecipitation data support that FXR directly controls NDRG2 transcription via IR1-type element(s) identified in the first introns of the human, mouse and rat NDRG2 genes. NDRG2 mRNA was induced by non-steroidal FXR agonists in livers of mice and the magnitude of induction of NDRG2 mRNA in three different human hepatoma cell lines was increased when ectopically expressing human FXR. Growth and metastasis of SK-Hep-1 cells was strongly reduced by non-steroidal FXR agonists in an orthotopic liver xenograft tumor model. Ectopic expression of FXR in SK-Hep1 cells reduced tumor growth and metastasis potential of corresponding cells and increased the anti-tumor efficacy of FXR agonists, which may be partly mediated via increased NDRG2 expression. FXR agonists may show a potential in the prevention and/or treatment of human hepatocellular carcinoma, a devastating malignancy with increasing prevalence and limited therapeutic options. PMID:23056173

  4. In-vivo monitoring of angiogenesis-inhibitory treatment effects by contrast-enhanced dynamic CT in a xenograft tumor model

    PubMed Central

    Raatschen, Hans-Juergen; Fu, Yanjun; Brasch, Robert C.; Pietsch, Hubertus; Shames, David M.; Yeh, Benjamin M.

    2014-01-01

    Rationale and Objectives To evaluate the potential of dynamic CT enhanced by Iohexol or a novel macromolecular contrast agent, PEG12000-Gen4-triiodo, to monitor microvascular changes in tumors treated with the angiogenesis inhibitor bevacizumab. Materials and Methods Ten female nude rats with MDA-MB 435 xenograft tumors were treated with 1 mg intraperitoneal bevacizumab when tumors reached 1 cm diameter and, for 4 rats, treated again 7 days later. Just prior to and 24 hours after the first injection of anti-VEGF antibody, the tumors were imaged by dynamic CT scans enhanced with PEG12000-Gen4-triiodo [n=3 rats] or Iohexol [n=3 rats]. The other 4 rats underwent dynamic CT scans enhanced with PEG12000-Gen4-triiodo just prior to and 24 hours after the second injection of anti-VEGF antibody. Microvascular leakiness (KPS) was calculated for the tumors using a two-compartment tissue model. Results PEG12000-Gen4-triiodo-enhanced CT scans showed progressive reductions in KPS from Day 1 to 2 to 9 (from 2.55 to 1.27 to 0.69 ?l min?1 cm?3, respectively, p < 0.005 for each comparison of Day 1 to 2, and Day 2 to 9). No significant difference was seen in the KPS estimates derived from Iohexol-enhanced CT scans obtained before or after treatment (276 versus 223.8 ?l min?1cm?3, respectively, p = 0.54). The microvascular leak (KPS) was significantly larger for Iohexol than for PEG12000-Gen4-triiodo-enhanced CT, p<0.05. Conclusion Dynamic macromolecular contrast-enhanced CT can be used to monitor serial decreases in tumor microvessel leakiness induced by repeated doses of an angiogenesis inhibitor drug PMID:19346961

  5. Oral administration of naturally occurring chitosan-based nanoformulated green tea polyphenol EGCG effectively inhibits prostate cancer cell growth in a xenograft model.

    PubMed

    Khan, Naghma; Bharali, Dhruba J; Adhami, Vaqar M; Siddiqui, Imtiaz A; Cui, Huadong; Shabana, Sameh M; Mousa, Shaker A; Mukhtar, Hasan

    2014-02-01

    In preclinical animal models, several phytochemicals have shown excellent potential to be used as effective agents in preventing and treating many cancers. However, the limited bioavailability of active agents could be one reason for their restricted usefulness for human consumption. To overcome this limitation, we recently introduced the concept of nanochemoprevention by encapsulating useful bioactive food components for their slow and sustained release. Here, we report the synthesis, characterization and efficacy assessment of a nanotechnology-based oral formulation of chitosan nanoparticles encapsulating epigallocatechin-3-gallate (Chit-nanoEGCG) for the treatment of prostate cancer (PCa) in a preclinical setting. Chit-nanoEGCG with a size of <200nm diameter and encapsulating EGCG as determined by dynamic light scattering and transmission electron microscope showed slow release of EGCG in simulated gastric juice acidic pH and faster release in simulated intestinal fluid. The antitumor efficacy of Chit-nanoEGCG was assessed in subcutaneously implanted 22R?1 tumor xenografts in athymic nude mice. Treatment with Chit-nanoEGCG resulted in significant inhibition of tumor growth and secreted prostate-specific antigen levels compared with EGCG and control groups. In tumor tissues of mice treated with Chit-nanoEGCG, compared with groups treated with EGCG and controls, there was significant (i) induction of poly (ADP-ribose) polymerases cleavage, (ii) increase in the protein expression of Bax with concomitant decrease in Bcl-2, (iii) activation of caspases and (iv) reduction in Ki-67 and proliferating cell nuclear antigen. Through this study, we propose a novel preventive and therapeutic modality for PCa using EGCG that addresses issues related to bioavailability. PMID:24072771

  6. Oral administration of naturally occurring chitosan-based nanoformulated green tea polyphenol EGCG effectively inhibits prostate cancer cell growth in a xenograft model

    PubMed Central

    Mukhtar, Hasan

    2014-01-01

    In preclinical animal models, several phytochemicals have shown excellent potential to be used as effective agents in preventing and treating many cancers. However, the limited bioavailability of active agents could be one reason for their restricted usefulness for human consumption. To overcome this limitation, we recently introduced the concept of nanochemoprevention by encapsulating useful bioactive food components for their slow and sustained release. Here, we report the synthesis, characterization and efficacy assessment of a nanotechnology-based oral formulation of chitosan nanoparticles encapsulating epigallocatechin-3-gallate (Chit-nanoEGCG) for the treatment of prostate cancer (PCa) in a preclinical setting. Chit-nanoEGCG with a size of <200nm diameter and encapsulating EGCG as determined by dynamic light scattering and transmission electron microscope showed slow release of EGCG in simulated gastric juice acidic pH and faster release in simulated intestinal fluid. The antitumor efficacy of Chit-nanoEGCG was assessed in subcutaneously implanted 22R?1 tumor xenografts in athymic nude mice. Treatment with Chit-nanoEGCG resulted in significant inhibition of tumor growth and secreted prostate-specific antigen levels compared with EGCG and control groups. In tumor tissues of mice treated with Chit-nanoEGCG, compared with groups treated with EGCG and controls, there was significant (i) induction of poly (ADP-ribose) polymerases cleavage, (ii) increase in the protein expression of Bax with concomitant decrease in Bcl-2, (iii) activation of caspases and (iv) reduction in Ki-67 and proliferating cell nuclear antigen. Through this study, we propose a novel preventive and therapeutic modality for PCa using EGCG that addresses issues related to bioavailability. PMID:24072771

  7. Function of the Blood-Brain Barrier and Restriction of Drug Delivery to Invasive Glioma Cells: Findings in an Orthotopic Rat Xenograft Model of Glioma

    PubMed Central

    Agarwal, Sagar; Manchanda, Pooja; Vogelbaum, Michael A.; Ohlfest, John R.

    2013-01-01

    Despite aggressive treatment with radiation and chemotherapy, recurrence of glioblastoma multiforme (GBM) is inevitable. The objective of this study was to show that the blood-brain barrier (BBB), through a combination of tight junctions and active efflux transporters in the brain microvasculature, can significantly restrict delivery of molecularly targeted agents to invasive glioma cells. Transgenic mice lacking P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) were used to study efflux of erlotinib at the BBB. A U87 rat xenograft model of GBM was used to investigate the regional distribution of erlotinib to the tumor, and brain regions surrounding the tumor. The effect of concurrent administration of elacridar on regional tumor distribution of erlotinib was evaluated. We show that erlotinib transport across an intact BBB is significantly restricted due to P-gp- and Bcrp-mediated efflux transport. We then show that the BBB is sufficiently intact in areas of brain adjacent to the tumor core to significantly restrict erlotinib delivery. Inhibition of P-gp and Bcrp by the dual inhibitor elacridar dramatically increased erlotinib delivery to the tumor core, rim, and normal brain. These results provide conclusive evidence of the impact that active efflux at the BBB has on the delivery of molecularly targeted therapy to different tumor regions in glioma. These data also support the possibility that the repeated failure of clinical trials of new drugs for gliomas may be in part due to a failure to achieve effective concentrations in invasive tumor cells that reside behind an intact BBB. PMID:23014761

  8. Blockade of the ERK pathway enhances the therapeutic efficacy of the histone deacetylase inhibitor MS-275 in human tumor xenograft models

    SciTech Connect

    Sakamoto, Toshiaki; Ozaki, Kei-ichi; Fujio, Kohsuke; Kajikawa, Shu-hei [Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521 (Japan)] [Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521 (Japan); Uesato, Shin-ichi [Department of Biotechnology, Faculty of Engineering, Kansai University, Osaka 564-8680 (Japan)] [Department of Biotechnology, Faculty of Engineering, Kansai University, Osaka 564-8680 (Japan); Watanabe, Kazushi [Proubase Technology Inc., Kanagawa 211-0063 (Japan)] [Proubase Technology Inc., Kanagawa 211-0063 (Japan); Tanimura, Susumu [Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521 (Japan)] [Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521 (Japan); Koji, Takehiko [Department of Histology and Cell Biology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523 (Japan)] [Department of Histology and Cell Biology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523 (Japan); Kohno, Michiaki, E-mail: kohnom@nagasaki-u.ac.jp [Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521 (Japan) [Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521 (Japan); Proubase Technology Inc., Kanagawa 211-0063 (Japan); Kyoto University Graduate School of Pharmaceutical Sciences, Kyoto 606-8501 (Japan)

    2013-04-19

    Highlights: •Blockade of the ERK pathway enhances the anticancer efficacy of HDAC inhibitors. •MEK inhibitors sensitize human tumor xenografts to HDAC inhibitor cytotoxicity. •Such the enhanced efficacy is achieved by a transient blockade of the ERK pathway. •This drug combination provides a promising therapeutic strategy for cancer patients. -- Abstract: The ERK pathway is up-regulated in various human cancers and represents a prime target for mechanism-based approaches to cancer treatment. Specific blockade of the ERK pathway alone induces mostly cytostatic rather than pro-apoptotic effects, however, resulting in a limited therapeutic efficacy of the ERK kinase (MEK) inhibitors. We previously showed that MEK inhibitors markedly enhance the ability of histone deacetylase (HDAC) inhibitors to induce apoptosis in tumor cells with constitutive ERK pathway activation in vitro. To evaluate the therapeutic efficacy of such drug combinations, we administered the MEK inhibitor PD184352 or AZD6244 together with the HDAC inhibitor MS-275 in nude mice harboring HT-29 or H1650 xenografts. Co-administration of the MEK inhibitor markedly sensitized the human xenografts to MS-275 cytotoxicity. A dose of MS-275 that alone showed only moderate cytotoxicity thus suppressed the growth of tumor xenografts almost completely as well as induced a marked reduction in tumor cellularity when administered with PD184352 or AZD6244. The combination of the two types of inhibitor also induced marked oxidative stress, which appeared to result in DNA damage and massive cell death, specifically in the tumor xenografts. The enhanced therapeutic efficacy of the drug combination was achieved by a relatively transient blockade of the ERK pathway. Administration of both MEK and HDAC inhibitors represents a promising chemotherapeutic strategy with improved safety for cancer patients.

  9. Identifying Structural Alerts Based on Zebrafish Developmental Morphological Toxicity (TDS)

    EPA Science Inventory

    Zebrafish constitute a powerful alternative animal model for chemical hazard evaluation. To provide an in vivo complement to high-throughput screening data from the ToxCast program, zebrafish developmental toxicity screens were conducted on the ToxCast Phase I (Padilla et al., 20...

  10. A new zebrafish model produced by TILLING of SOD1-related amyotrophic lateral sclerosis replicates key features of the disease and represents a tool for in vivo therapeutic screening

    PubMed Central

    Da Costa, Marc M. J.; Allen, Claire E.; Higginbottom, Adrian; Ramesh, Tennore; Shaw, Pamela J.; McDermott, Christopher J.

    2014-01-01

    Mutations in the superoxide dismutase gene (SOD1) are one cause of familial amyotrophic lateral sclerosis [ALS; also known as motor neuron disease (MND)] in humans. ALS is a relentlessly progressive neurodegenerative disease and, to date, there are no neuroprotective therapies with significant impact on the disease course. Current transgenic murine models of the disease, which overexpress mutant SOD1, have so far been ineffective in the identification of new therapies beneficial in the human disease. Because the human and the zebrafish (Danio rerio) SOD1 protein share 76% identity, TILLING (‘targeting induced local lesions in genomes’) was carried out in collaboration with the Sanger Institute in order to identify mutations in the zebrafish sod1 gene. A T70I mutant zebrafish line was characterised using oxidative stress assays, neuromuscular junction (NMJ) analysis and motor function studies. The T70I sod1 zebrafish model offers the advantage over current murine models of expressing the mutant Sod1 protein at a physiological level, as occurs in humans with ALS. The T70I sod1 zebrafish demonstrates key features of ALS: an early NMJ phenotype, susceptibility to oxidative stress and an adult-onset motor neuron disease phenotype. We have demonstrated that the susceptibility of T70I sod1 embryos to oxidative stress can be used in a drug screening assay, to identify compounds that merit further investigation as potential therapies for ALS. PMID:24092880

  11. A DN-mda5 transgenic zebrafish model demonstrates that Mda5 plays an important role in snakehead rhabdovirus resistance.

    PubMed

    Gabor, K A; Charette, J R; Pietraszewski, M J; Wingfield, D J; Shim, J S; Millard, P J; Kim, C H

    2015-08-01

    Melanoma Differentiation-Associated protein 5 (MDA5) is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family, which is a cytosolic pattern recognition receptor that detects viral nucleic acids. Here we show an Mda5-dependent response to rhabdovirus infection in vivo using a dominant-negative mda5 transgenic zebrafish. Dominant-negative mda5 zebrafish embryos displayed an impaired antiviral immune response compared to wild-type counterparts that can be rescued by recombinant full-length Mda5. To our knowledge, we have generated the first dominant-negative mda5 transgenic zebrafish and demonstrated a critical role for Mda5 in the antiviral response to rhabdovirus. PMID:25634485

  12. Prevention of EBV lymphoma development by oncolytic myxoma virus in a murine xenograft model of post-transplant lymphoproliferative disease.

    PubMed

    Kim, Manbok; Rahman, Masmudur M; Cogle, Christopher R; McFadden, Grant

    2015-07-10

    Epstein-Barr virus (EBV) has been associated with a variety of epithelial and hematologic malignancies, including B-, T- and NK cell-lymphomas, Hodgkin's disease (HD), post-transplant lymphoproliferative diseases (LPDs), nasopharyngeal and gastric carcinomas, smooth muscle tumors, and HIV-associated lymphomas. Currently, treatment options for EBV-associated malignancies are limited. We have previously shown that myxoma virus specifically targets various human solid tumors and leukemia cells in a variety of animal models, while sparing normal human or murine tissues. Since transplant recipients of bone marrow or solid organs often develop EBV-associated post-transplant LPDs and lymphoma, myxoma virus may be of utility to prevent EBV-associated malignancies in immunocompromised transplant patients where treatment options are frequently limited. In this report, we demonstrate the safety and efficacy of myxoma virus purging as a prophylactic strategy for preventing post-transplant EBV-transformed human lymphomas, using a highly immunosuppressed mouse xenotransplantation model. This provides support for developing myxoma virus as a potential oncolytic therapy for preventing EBV-associated LPDs following transplantation of bone marrow or solid organ allografts. PMID:25843801

  13. Forebrain electrophysiological recording in larval zebrafish.

    PubMed

    Baraban, Scott C

    2013-01-01

    Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 ?l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish. PMID:23380808

  14. Stressing Zebrafish for Behavioral Genetics

    PubMed Central

    Clark, Karl J.; Boczek, Nicole J.; Ekker, Stephen C.

    2012-01-01

    Synopsis The stress response is a normal reaction to a real or perceived threat. However, stress response systems that are overwhelmed or out of balance can increase both the incidence and severity of diseases including addiction and mood and anxiety disorders. Using an animal model with both genetic diversity and large family size can help discover the specific genetic and environmental contributions to these behavioral diseases. The stress response has been studied extensively in teleosts because of their importance in food production. The zebrafish (Danio rerio) is a major model organism with a strong record for use in developmental biology, genetic screening, and genomic studies. More recently, the stress response of larval and adult zebrafish has been documented. High-throughput automated tracking systems make possible behavioral readouts of the stress response in zebrafish. This non-invasive measure of the stress response can be combined with mutagenesis methods to dissect the genes involved in complex stress response behaviors in vertebrates. Understanding the genetic and epigenetic basis for the stress response in vertebrates will help to develop advanced screening and therapies for stress-aggravated diseases like addiction and mood and anxiety disorders. PMID:21615261

  15. Establishment of a human multiple myeloma xenograft model in the chicken to study tumor growth, invasion and angiogenesis.

    PubMed

    Martowicz, Agnieszka; Kern, Johann; Gunsilius, Eberhard; Untergasser, Gerold

    2015-01-01

    Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA). In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells. PMID:25993267

  16. Behavioral effects of MDMA (`ecstasy') on adult zebrafish Adam Stewart, Russell Riehl, Keith Wong, Jeremy Green, Jessica Cosgrove,

    E-print Network

    Kalueff, Allan V.

    of zebrafish models for screening of hallucinogenic compounds. Behavioural Pharmacology 22:275­280 c 2011 hallucinogenic drugs, zebrafish Department of Pharmacology and Neuroscience Program, Tulane Neurophenotyping pharmacological manipulations, includ- ing the hallucinogens lysergic acid diethylamide (LSD) and salvinorin

  17. Co-factors of LIM-HD transcription factors in neural development and axon pathfinding in zebrafish 

    E-print Network

    Zhong, Zhen

    2012-06-22

    The zebrafish neuromuscular system is an elegant model to study neural development. To reveal a specific programme for zebrafish motor axon pathfinding I established a method to selectively block motor axon pathfinding ...

  18. Anti-tumor activity of fenretinide complexed with human serum albumin in lung cancer xenograft mouse model

    PubMed Central

    Teti, Gabriella; Salvatore, Viviana; Focaroli, Stefano; Tesei, Anna; Pignatta, Sara; Falconi, Mirella

    2014-01-01

    Sufficient knowledge regarding cellular and molecular basis of lung cancer progression and metastasis would help in the development of novel and effective strategies for the treatment of lung cancer. 4HPR is a synthetic retinoid with potential anti-tumor activity but is still limited because of its poor bioavailability. The use of albumin as a complexing agent for a hydrophobic drug is expected to improve the water solubility and consequently their bioavailability.This study investigated the antitumor activity of a novel complex between albumin and 4-HPR in a mouse model of human lung cancer and focuses on role and mechanism of Cav-1 mainly involved in regulating cancer and Acsvl3 mainly connected with tumor growth. Their expressions were assayed by immunohistochemistry and qRT-PCR, to demonstrate the reduction of the tumor growth following the drug treatment. Our results showed a high antitumor activity of 4HPR-HSA by reduction of the volume of tumor mass and the presence of a high level of apoptotic cell by TUNEL assay. The downregulation of Cav-1 and Acsvl3 suggested a reduction of tumor growth. In conclusion, we demonstrated the great potential of 4HPR-HSA in the treatment of lung cancer. More data about the mechanism of drug delivery the 4HPR-HSA are necessary. PMID:25015569

  19. Correlation of ultrasound contrast agent derived blood flow parameters with immunohistochemical angiogenesis markers in murine xenograft tumor models

    PubMed Central

    Eisenbrey, John R.; Wilson, Christian C.; Ro, Raymond J.; Fox, Traci B; Liu, Ji-Bin; Chiou, See-Ying; Forsberg, Flemming

    2013-01-01

    Purpose In this study we used temporal analysis of ultrasound contrast agent (UCA) wash-in to estimate blood flow dynamics and demonstrate their improved correlation to angiogenesis markers relative to previously reported, non-temporal fractional vascularity estimates. Materials and Methods Breast tumor (NMU) or glioma (C6) cells were implanted in either the abdomen or thigh of 144 rats. After 6, 8 or 10 days, rats received a bolus UCA injection of Optison (GE Healthcare, Princeton, NJ; 0.4ml/kg) during power Doppler imaging (PDI), harmonic imaging (HI), and microflow imaging (MFI) using an Aplio ultrasound scanner with 7.5 MHz linear array (Toshiba America Medical Systems, Tustin, CA). Time-intensity curves of contrast wash-in were constructed on a pixel-by-pixel basis and averaged to calculate maximum intensity, time to peak, perfusion, and time integrated intensity (TII). Tumors were then stained for 4 immunohistochemical markers (bFGF, CD31, COX-2, and VEGF). Correlations between temporal parameters and the angiogenesis markers were investigated for each imaging mode. Effects of tumor model and implant location on these correlations were also investigated. Results Significant correlation over the entire dataset was only observed between TII and VEGF for all three imaging modes (R=?0.35,?0.54,?0.32 for PDI, HI and MFI, respectively; p<0.0001). Tumor type and location affected these correlations, with the strongest correlation of TII to VEGF found to be with implanted C6 cells (R=?0.43,?0.54,?0.52 for PDI, HI and MFI, respectively; p<0.0002). Conclusions While UCA-derived temporal blood flow parameters were found to correlate strongly with VEGF expression, these correlations were also found to be influenced by both tumor type and implant location. PMID:23659876

  20. Mechanical vessel injury in zebrafish embryos.

    PubMed

    Clay, Hilary; Coughlin, Shaun R

    2015-01-01

    Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model. PMID:25742284

  1. [ 186Re]Liposomal doxorubicin (Doxil): in vitro stability, pharmacokinetics, imaging and biodistribution in a head and neck squamous cell carcinoma xenograft model

    Microsoft Academic Search

    Anuradha Soundararajan; Ande Bao; William T. Phillips; Ricardo Perez III; Beth A. Goins

    2009-01-01

    The purpose of this study was to determine the feasibility of radiolabeling liposomal doxorubicin (Doxil) for cancer chemoradionuclide therapy by directly loading the therapeutic radionuclide rhenium-186 (186Re) into the liposome interior. The pharmacokinetics, imaging and biodistribution of [186Re]Doxil (555 MBq\\/kg) and control [186Re]polyethylene glycol (PEG) liposomes (555 MBq\\/kg) were determined after intravenous administration in a head and neck cancer xenograft

  2. Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

    PubMed Central

    Martin-Iverson, Mathew T.; Mondal, Alinda; Ong, Daniel; Rainey-Smith, Stephanie; Taddei, Kevin; Lardelli, Michael; Groth, David M.; Verdile, Giuseppe; Martins, Ralph N.

    2012-01-01

    This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model. PMID:23183629

  3. Efficacy of a Non-Hypercalcemic Vitamin-D2 Derived Anti-Cancer Agent (MT19c) and Inhibition of Fatty Acid Synthesis in an Ovarian Cancer Xenograft Model

    PubMed Central

    Moore, Richard G.; Lange, Thilo S.; Robinson, Katina; Kim, Kyu K.; Uzun, Alper; Horan, Timothy C.; Kawar, Nada; Yano, Naohiro; Chu, Sharon R.; Mao, Quanfu; Brard, Laurent; DePaepe, Monique E.; Padbury, James F.; Arnold, Leggy A.; Brodsky, Alexander; Shen, Tun-Li; Singh, Rakesh K.

    2012-01-01

    Background Numerous vitamin-D analogs exhibited poor response rates, high systemic toxicities and hypercalcemia in human trials to treat cancer. We identified the first non-hypercalcemic anti-cancer vitamin D analog MT19c by altering the A-ring of ergocalciferol. This study describes the therapeutic efficacy and mechanism of action of MT19c in both in vitro and in vivo models. Methodology/Principal Finding Antitumor efficacy of MT19c was evaluated in ovarian cancer cell (SKOV-3) xenografts in nude mice and a syngenic rat ovarian cancer model. Serum calcium levels of MT19c or calcitriol treated animals were measured. In-silico molecular docking simulation and a cell based VDR reporter assay revealed MT19c–VDR interaction. Genomewide mRNA analysis of MT19c treated tumors identified drug targets which were verified by immunoblotting and microscopy. Quantification of cellular malonyl CoA was carried out by HPLC-MS. A binding study with PPAR-Y receptor was performed. MT19c reduced ovarian cancer growth in xenograft and syngeneic animal models without causing hypercalcemia or acute toxicity. MT19c is a weak vitamin-D receptor (VDR) antagonist that disrupted the interaction between VDR and coactivator SRC2-3. Genome-wide mRNA analysis and western blot and microscopy of MT19c treated xenograft tumors showed inhibition of fatty acid synthase (FASN) activity. MT19c reduced cellular levels of malonyl CoA in SKOV-3 cells and inhibited EGFR/phosphoinositol-3kinase (PI-3K) activity independently of PPAR-gamma protein. Significance Antitumor effects of non-hypercalcemic agent MT19c provide a new approach to the design of vitamin-D based anticancer molecules and a rationale for developing MT19c as a therapeutic agent for malignant ovarian tumors by targeting oncogenic de novo lipogenesis. PMID:22509304

  4. Anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue in lipopolysaccharide-stimulated RAW 264.7 macrophages and in vivo zebrafish model

    PubMed Central

    Ko, Seok-Chun

    2015-01-01

    BACKGROUND/OBJECTIVES In this study, potential anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue was assessed via nitric oxide (NO) production in lipopolysaccahride (LPS) induced RAW 264.7 macrophages and in vivo zebrafish model. MATERIALS/METHODS We investigated the ability of enzymatic hydrolysates from Styela clava flesh tissue to inhibit LPS-induced expression of pro-inflammatory mediators in RAW 264.7 macrophages, and the molecular mechanism through which this inhibition occurred. In addition, we evaluated anti-inflammatory effect of enzymatic hydrolysates against a LPS-exposed in in vivo zebrafish model. RESULTS Among the enzymatic hydrolysates, Protamex-proteolytic hydrolysate exhibited the highest NO inhibitory effect and was fractionated into three ranges of molecular weight by using ultrafiltration (UF) membranes (MWCO 5 kDa and 10 kDa). The above 10 kDa fraction down-regulated LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), thereby reducing production of NO and prostaglandin E2 (PGE2) in LPS-activated RAW 264.7 macrophages. The above 10 kDa fraction suppressed LPS-induced production of pro-inflammatory cytokines, including interleukin (IL)-1?, IL-6, and tumor necrosis factor (TNF)-?. In addition, the above 10 kDa fraction inhibited LPS-induced phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38. Furthermore, NO production in live zebrafish induced by LPS was reduced by addition of the above 10 kDa fraction from S. clava enzymatic hydrolysate. CONCLUSION The results of this study suggested that hydrolysates derived from S. clava flesh tissue would be new anti-inflammation materials in functional resources.

  5. A zebrafish model of congenital disorders of glycosylation with phosphomannose isomerase deficiency reveals an early opportunity for corrective mannose supplementation.

    PubMed

    Chu, Jaime; Mir, Alexander; Gao, Ningguo; Rosa, Sabrina; Monson, Christopher; Sharma, Vandana; Steet, Richard; Freeze, Hudson H; Lehrman, Mark A; Sadler, Kirsten C

    2013-01-01

    Individuals with congenital disorders of glycosylation (CDG) have recessive mutations in genes required for protein N-glycosylation, resulting in multi-systemic disease. Despite the well-characterized biochemical consequences in these individuals, the underlying cellular defects that contribute to CDG are not well understood. Synthesis of the lipid-linked oligosaccharide (LLO), which serves as the sugar donor for the N-glycosylation of secretory proteins, requires conversion of fructose-6-phosphate to mannose-6-phosphate via the phosphomannose isomerase (MPI) enzyme. Individuals who are deficient in MPI present with bleeding, diarrhea, edema, gastrointestinal bleeding and liver fibrosis. MPI-CDG patients can be treated with oral mannose supplements, which is converted to mannose-6-phosphate through a minor complementary metabolic pathway, restoring protein glycosylation and ameliorating most symptoms, although liver disease continues to progress. Because Mpi deletion in mice causes early embryonic lethality and thus is difficult to study, we used zebrafish to establish a model of MPI-CDG. We used a morpholino to block mpi mRNA translation and established a concentration that consistently yielded 13% residual Mpi enzyme activity at 4 days post-fertilization (dpf), which is within the range of MPI activity detected in fibroblasts from MPI-CDG patients. Fluorophore-assisted carbohydrate electrophoresis detected decreased LLO and N-glycans in mpi morphants. These deficiencies resulted in 50% embryonic lethality by 4 dpf. Multi-systemic abnormalities, including small eyes, dysmorphic jaws, pericardial edema, a small liver and curled tails, occurred in 82% of the surviving larvae. Importantly, these phenotypes could be rescued with mannose supplementation. Thus, parallel processes in fish and humans contribute to the phenotypes caused by Mpi depletion. Interestingly, mannose was only effective if provided prior to 24 hpf. These data provide insight into treatment efficacy and the broader molecular and developmental abnormalities that contribute to disorders associated with defective protein glycosylation. PMID:22899857

  6. Neurogenesis in zebrafish – from embryo to adult

    PubMed Central

    2013-01-01

    Neurogenesis in the developing central nervous system consists of the induction and proliferation of neural progenitor cells and their subsequent differentiation into mature neurons. External as well as internal cues orchestrate neurogenesis in a precise temporal and spatial way. In the last 20 years, the zebrafish has proven to be an excellent model organism to study neurogenesis in the embryo. Recently, this vertebrate has also become a model for the investigation of adult neurogenesis and neural regeneration. Here, we summarize the contributions of zebrafish in neural development and adult neurogenesis. PMID:23433260

  7. Methods for generating and colonizing gnotobiotic zebrafish

    PubMed Central

    Pham, Linh N.; Kanther, Michelle; Semova, Ivana; Rawls, John F.

    2008-01-01

    Vertebrates are colonized at birth by complex and dynamic communities of microorganisms that can contribute significantly to host health and disease. The ability to raise animals in the absence of microorganisms has been a powerful tool for elucidating the relationships between animal hosts and their microbial residents. The optical transparency of the developing zebrafish and relative ease of generating germ-free zebrafish makes it an attractive model organism for gnotobiotic research. Here we provide a protocol for: generating zebrafish embryos; deriving and rearing germ-free zebrafish; and colonizing zebrafish with microorganisms. Using these methods, we typically obtain 80–90% sterility rates in our germ-free derivations with 90% survival in germ-free animals and 50–90% survival in colonized animals through larval stages. Obtaining embryos for derivation requires approximately 1–2 hours with a 3–8 hour incubation period prior to derivation. Derivation of germ-free animals takes 1–1.5 hours, and daily maintenance requires 1–2 hours. PMID:19008873

  8. The zebrafish organizer

    Microsoft Academic Search

    Alexander F Schier; William S Talbot

    1998-01-01

    Signals from the organizer play a crucial role in patterning the vertebrate embryo. Recent molecular analysis of zebrafish mutations has established an essential role for BMP2 and chordin in organizer function and has identified one-eyed pinhead as a novel EGF-like gene involved in prechordal plate and endoderm formation. In addition, embryological studies have suggested that the zebrafish organizer is induced

  9. Ultrasound Bio-Microscopic Image Segmentation for Evaluation of Zebrafish Cardiac Function

    PubMed Central

    Zhou, Xiaowei; Sun, Lei; Yu, Yanyan; Qiu, Weibao; Lien, Ching-Ling; Shung, K. Kirk; Yu, Weichuan

    2013-01-01

    Zebrafish can fully regenerate their myocardium after ventricular resection without evidence of scars. This extraordinary regenerative ability provides an excellent model system to study the activation of the regenerative potential for human heart tissue. In addition to the morphology, it is vital to understand the cardiac function of zebrafish. To characterize adult zebrafish cardiac function, an ultrasound biomicroscope (UBM) was customized for real-time imaging of the zebrafish heart (about 1 mm in diameter) at a resolution of around 37 µm. Moreover, we developed an image segmentation algorithm to track the cardiac boundary and measure the dynamic size of the zebrafish heart for further quantification of zebrafish cardiac function. The effectiveness and accuracy of the proposed segmentation algorithm were verified on a tissue-mimicking phantom and in vivo zebrafish echocardiography. The quantitative evaluation demonstrated that the accuracy of the proposed algorithm is comparable to the manual delineation by experts. PMID:23549532

  10. Conditional Gene-Trap Mutagenesis in Zebrafish

    PubMed Central

    Maddison, Lisette A; Li, Mingyu; Chen, Wenbiao

    2014-01-01

    Summary Zebrafish has become a widely used model for analysis of gene function. Several methods have been used to create mutations in this organism and thousands of mutant lines are available. However, all the conventional zebrafish mutations affect the gene in all cells at all time, making it difficult to determine tissue-specific functions. We have adopted a FlEx Trap approach to generate conditional mutations in zebrafish by gene trap mutagenesis. Combined with appropriate Cre or Flp lines, the insertional mutants not only allow spatial and temporal specific gene inactivation, but also permit spatial and temporal specific rescue of the disrupted gene. We provide experimental details on how to generate and use such mutations. PMID:24233792

  11. Sun light mediated synthesis of gold nanoparticles as carrier for 6-mercaptopurine: Preparation, characterization and toxicity studies in zebrafish embryo model

    SciTech Connect

    Ganeshkumar, Moorthy [Department of Biochemistry, Central Leather Research Institute, Council of Scientific and Industrial Research, Chennai 600020 (India)] [Department of Biochemistry, Central Leather Research Institute, Council of Scientific and Industrial Research, Chennai 600020 (India); Sastry, Thotapalli Parvathaleswara [Bioproducts Laboratory, Central Leather Research Institute, Chennai 600020 (India)] [Bioproducts Laboratory, Central Leather Research Institute, Chennai 600020 (India); Sathish Kumar, Muniram [Department of Pharmaceutics, Anna University, Trichy, Tamilnadu (India)] [Department of Pharmaceutics, Anna University, Trichy, Tamilnadu (India); Dinesh, Murugan Girija [Thanthai Hansroever College, Perambalur, Tamilnadu (India)] [Thanthai Hansroever College, Perambalur, Tamilnadu (India); Kannappan, Sudalyandi [Central Institute of Brackish Water Aquaculture, Chennai 600028 (India)] [Central Institute of Brackish Water Aquaculture, Chennai 600028 (India); Suguna, Lonchin, E-mail: slonchin@yahoo.co.uk [Department of Biochemistry, Central Leather Research Institute, Council of Scientific and Industrial Research, Chennai 600020 (India)] [Department of Biochemistry, Central Leather Research Institute, Council of Scientific and Industrial Research, Chennai 600020 (India)

    2012-09-15

    Highlights: ? Gold nanoparticles prepared using eco-friendly method with good in vitro stability. ? Can be used as drug delivery system. ? Did not show any toxicity in zebrafish embryo. ? More toxic to cancer cells when compared to N-Au-Mp and Mp. -- Abstract: The objective of this study is to synthesize green chemistry based gold nanoparticles by sun light irradiation method. The prepared gold nanoparticles (AuNPs) were modified using folic acid and then coupled with 6-mercaptopurine. These modified nanoparticles were used as a tool for targeted drug delivery to treat laryngeal cancer. In the present study, novel bionanocomposites containing nutrient agar coated gold nano particles (N-AuNPs) coupled with 6-mercaptopurine (drug) (N-AuNPs-Mp), folic acid (ligand) (N-AuNPs-Mp-Fa) and rhodamine (dye) (N-AuNPs-Rd), a fluorescent agent, were prepared and characterized by IR, UV, TEM, Particle size analysis and in vitro stability. The toxicity and fluorescence of N-Au was studied using zebrafish embryo model. The in vitro cytotoxicity of free Mp, N-Au-Mp and N-Au-Mp-Fa against HEp-2 cells was compared and found that the amount of Mp required to achieve 50% of growth of inhibition (IC{sub 50}) was much lower in N-Au-Mp-Fa than in free Mp and N-Au-Mp.

  12. Two knockdown models of the autism genes SYNGAP1 and SHANK3 in zebrafish produce similar behavioral phenotypes associated with embryonic disruptions of brain morphogenesis.

    PubMed

    Kozol, Robert A; Cukier, Holly N; Zou, Bing; Mayo, Vera; De Rubeis, Silvia; Cai, Guiqing; Griswold, Anthony J; Whitehead, Patrice L; Haines, Jonathan L; Gilbert, John R; Cuccaro, Michael L; Martin, Eden R; Baker, James D; Buxbaum, Joseph D; Pericak-Vance, Margaret A; Dallman, Julia E

    2015-07-15

    Despite significant progress in the genetics of autism spectrum disorder (ASD), how genetic mutations translate to the behavioral changes characteristic of ASD remains largely unknown. ASD affects 1-2% of children and adults, and is characterized by deficits in verbal and non-verbal communication, and social interactions, as well as the presence of repetitive behaviors and/or stereotyped interests. ASD is clinically and etiologically heterogeneous, with a strong genetic component. Here, we present functional data from syngap1 and shank3 zebrafish loss-of-function models of ASD. SYNGAP1, a synaptic Ras GTPase activating protein, and SHANK3, a synaptic scaffolding protein, were chosen because of mounting evidence that haploinsufficiency in these genes is highly penetrant for ASD and intellectual disability (ID). Orthologs of both SYNGAP1 and SHANK3 are duplicated in the zebrafish genome and we find that all four transcripts (syngap1a, syngap1b, shank3a and shank3b) are expressed at the earliest stages of nervous system development with pronounced expression in the larval brain. Consistent with early expression of these genes, knockdown of syngap1b or shank3a cause common embryonic phenotypes including delayed mid- and hindbrain development, disruptions in motor behaviors that manifest as unproductive swim attempts, and spontaneous, seizure-like behaviors. Our findings indicate that both syngap1b and shank3a play novel roles in morphogenesis resulting in common brain and behavioral phenotypes. PMID:25882707

  13. Human Immunodeficiency Virus Type 1 Infection of Neural Xenografts

    NASA Astrophysics Data System (ADS)

    Cvetkovich, Therese A.; Lazar, Eliot; Blumberg, Benjamin M.; Saito, Yoshihiro; Eskin, Thomas A.; Reichman, Richard; Baram, David A.; del Cerro, Coca; Gendelman, Howard E.; del Cerro, Manuel; Epstein, Leon G.

    1992-06-01

    Human immunodeficiency virus type 1 (HIV-1) infection is highly specific for its human host. To study HIV-1 infection of the human nervous system, we have established a small animal model in which second-trimester (11 to 17.5 weeks) human fetal brain or neural retina is transplanted to the anterior chamber of the eye of immunosuppressed adult rats. The human xenografts vascularized, formed a blood-brain barrier, and differentiated, forming neurons and glia. The xenografts were infected with cell-free HIV-1 or with HIV-1-infected human monocytes. Analysis by polymerase chain reaction revealed HIV-1 sequences in DNA from xenograft tissue exposed to HIV-1 virions, and in situ hybridization demonstrated HIV-1 mRNA localized in macrophages and multinucleated giant cells. Pathological damage was observed only in neural xenografts containing HIV-1-infected human monocytes, supporting the hypothesis that these cells mediate neurotoxicity. This small animal model allows the study of direct and indirect effects of HIV-1 infection on developing human fetal neural tissues, and it should prove useful in evaluating antiviral therapies, which must ultimately target HIV-1 infection of the brain.

  14. Culturable Gut Microbiota Diversity in Zebrafish

    PubMed Central

    Sørby, Jan Roger Torp; Aleström, Peter; Sørum, Henning

    2012-01-01

    Abstract The zebrafish (Danio rerio) is an increasingly used laboratory animal model in basic biology and biomedicine, novel drug development, and toxicology. The wide use has increased the demand for optimized husbandry protocols to ensure animal health care and welfare. The knowledge about the correlation between culturable zebrafish intestinal microbiota and health in relation to environmental factors and management procedures is very limited. A semi-quantitative level of growth of individual types of bacteria was determined and associated with sampling points. A total of 72 TAB line zebrafish from four laboratories (Labs A–D) in the Zebrafish Network Norway were used. Diagnostic was based on traditional bacterial culture methods and biochemical characterization using commercial kits, followed by 16S rDNA gene sequencing from pure subcultures. Also selected Gram-negative isolates were analyzed for antibiotic susceptibility to 8 different antibiotics. A total of 13 morphologically different bacterial species were the most prevalent: Aeromonas hydrophila, Aeromonas sobria, Vibrio parahaemolyticus, Photobacterium damselae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas luteola, Comamonas testosteroni, Ochrobactrum anthropi, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus warneri. Only Lab B had significantly higher levels of total bacterial growth (OR=2.03), whereas numbers from Lab C (OR=1.01) and Lab D (OR=1.12) were found to be similar to the baseline Lab A. Sexually immature individuals had a significantly higher level of harvested total bacterial growth than mature fish (OR=0.82), no statistically significant differences were found between male and female fish (OR=1.01), and the posterior intestinal segment demonstrated a higher degree of culturable bacteria than the anterior segment (OR=4.1). Multiple antibiotic (>3) resistance was observed in 17% of the strains. We propose that a rapid conventional diagnostic bacteriological assay on the culturable microbiota profiles can be designed and used as quality measure of the husbandry routines of a zebrafish facility to ensure a bacterial standard safeguarding the zebrafish health and welfare. PMID:22428747

  15. The Zebrafish Insertion Collection (ZInC): a web based, searchable collection of zebrafish mutations generated by DNA insertion

    PubMed Central

    Varshney, Gaurav K.; Huang, Haigen; Zhang, Suiyuan; Lu, Jing; Gildea, Derek E.; Yang, Zhongan; Wolfsberg, Tyra G.; Lin, Shuo; Burgess, Shawn M.

    2013-01-01

    ZInC (Zebrafish Insertional Collection, http://research.nhgri.nih.gov/ZInC/) is a web-searchable interface of insertional mutants in zebrafish. Over the last two decades, the zebrafish has become a popular model organism for studying vertebrate development as well as for modeling human diseases. To facilitate such studies, we are generating a genome-wide knockout resource that targets every zebrafish protein-coding gene. All mutant fish are freely available to the scientific community through the Zebrafish International Resource Center (ZIRC). To assist researchers in finding mutant and insertion information, we developed a comprehensive database with a web front-end, the ZInC. It can be queried using multiple types of input such as ZFIN (Zebrafish Information Network) IDs, UniGene accession numbers and gene symbols from zebrafish, human and mouse. In the future, ZInC may include data from other insertional mutation projects as well. ZInC cross-references all integration data with the ZFIN (http://zfin.org/). PMID:23180778

  16. Segmentation of zebrafish embryonic images using a geometric atlas deformation.

    PubMed

    Zacharia, Eleni; Bondesson, Maria; Gustafsson, Jan-Åke; Kakadiaris, Ioannis A

    2012-01-01

    Transgenic zebrafish expressing fluorescent proteins in specific tissues or organs are promising models for studies of normal developmental processes, or perturbations of these. However, for widespread use, reliable quantification of the observed effects is necessary. Therefore, accurate and automatic analysis of images obtained by fluorescent microscopy and depicting zebrafish embryos becomes crucial. In this paper, a segmentation approach for such images is proposed. The segmentation is achieved by fitting a species-specific 2D atlas to the zebrafish data depicted in the images. Experiments performed in a set of 50 images have provided promising results. PMID:23366804

  17. Evaluation of zebrafish kidney function using a fluorescent clearance assay.

    PubMed

    Christou-Savina, Sonia; Beales, Philip L; Osborn, Daniel P S

    2015-01-01

    The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria 'dipstick', which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function. PMID:25742415

  18. Of fish and men: using zebrafish to fight human diseases.

    PubMed

    Ablain, Julien; Zon, Leonard I

    2013-12-01

    Long restricted to the field of developmental biology, the use of the zebrafish (Danio rerio) has extended to the study of human pathogenesis. Fostered by the rapid adaptation of new technologies, the design and analysis of fish models of human diseases have contributed important findings that are now making their way from aquariums to clinics. Here we outline the clinical relevance of the zebrafish as a model organism. PMID:24275383

  19. Computerized image analysis for quantitative neuronal phenotyping in zebrafish.

    PubMed

    Liu, Tianming; Lu, Jianfeng; Wang, Ye; Campbell, William A; Huang, Ling; Zhu, Jinmin; Xia, Weiming; Wong, Stephen T C

    2006-06-15

    An integrated microscope image analysis pipeline is developed for automatic analysis and quantification of phenotypes in zebrafish with altered expression of Alzheimer's disease (AD)-linked genes. We hypothesize that a slight impairment of neuronal integrity in a large number of zebrafish carrying the mutant genotype can be detected through the computerized image analysis method. Key functionalities of our zebrafish image processing pipeline include quantification of neuron loss in zebrafish embryos due to knockdown of AD-linked genes, automatic detection of defective somites, and quantitative measurement of gene expression levels in zebrafish with altered expression of AD-linked genes or treatment with a chemical compound. These quantitative measurements enable the archival of analyzed results and relevant meta-data. The structured database is organized for statistical analysis and data modeling to better understand neuronal integrity and phenotypic changes of zebrafish under different perturbations. Our results show that the computerized analysis is comparable to manual counting with equivalent accuracy and improved efficacy and consistency. Development of such an automated data analysis pipeline represents a significant step forward to achieve accurate and reproducible quantification of neuronal phenotypes in large scale or high-throughput zebrafish imaging studies. PMID:16364449

  20. Effectiveness of Recommended Euthanasia Methods in Larval Zebrafish (Danio rerio)

    PubMed Central

    Strykowski, Jennifer L; Schech, Joseph M

    2015-01-01

    The popularity of zebrafish and its use as a model organism in biomedical research including genetics, development, and toxicology, has increased over the past 20 y and continues to grow. However, guidelines for euthanasia remain vague, and the responsibility of creating appropriate euthanasia protocols essentially falls on individual facilities. To reduce variation in experimental results among labs, a standard method of euthanasia for zebrafish would be useful. Although various euthanasia methods have been compared, few studies focus on the effectiveness of euthanasia methods for larval zebrafish. In this study, we exposed larval zebrafish to each of 3 euthanasia agents (MS222, eugenol, and hypothermic shock) and assessed the recovery rate. Hypothermic shock appeared to be the most effective method for euthanizing zebrafish at 14 d after fertilization; however, this method may not be considered an efficient method for large numbers of larval zebrafish. Exposure to chemicals, such as MS222 and eugenol, were ineffective methods for euthanasia at this stage of development. When these agents are used, secondary measures should be taken to ensure death. Choosing a euthanasia method that is effective, efficient, and humane can be challenging. Determining a method of euthanasia that is suitable for fish of all stages will bring the zebrafish community closer to meeting this challenge. PMID:25651095

  1. The zebrafish world of colors and shapes: preference and discrimination.

    PubMed

    Oliveira, Jessica; Silveira, Mayara; Chacon, Diana; Luchiari, Ana

    2015-04-01

    Natural environment imposes many challenges to animals, which have to use cognitive abilities to cope with and exploit it to enhance their fitness. Since zebrafish is a well-established model for cognitive studies and high-throughput screening for drugs and diseases that affect cognition, we tested their ability for ambient color preference and 3D objects discrimination to establish a protocol for memory evaluation. For the color preference test, zebrafish were observed in a multiple-chamber tank with different environmental color options. Zebrafish showed preference for blue and green, and avoided yellow and red. For the 3D objects discrimination, zebrafish were allowed to explore two equal objects and then observed in a one-trial test in which a new color, size, or shape of the object was presented. Zebrafish showed discrimination for color, shape, and color+shape combined, but not size. These results imply that zebrafish seem to use some categorical system to discriminate items, and distracters affect their ability for discrimination. The type of variables available (color and shape) may favor zebrafish objects perception and facilitate discrimination processing. We suggest that this easy and simple memory test could serve as a useful screening tool for cognitive dysfunction and neurotoxicological studies. PMID:25674976

  2. Imaging escape and avoidance behavior in zebrafish larvae.

    PubMed

    Colwill, Ruth M; Creton, Robbert

    2011-01-01

    This review provides an overview of the assays that are used for measuring escape and avoidance behavior in zebrafish, with a specific focus on zebrafish larvae during the first week of development. Zebrafish larvae display a startle response when exposed to tactile, acoustic, or visual stimuli and will avoid dark areas, moving objects, conspecifics, and open spaces. Emotional states such as fear and anxiety might be induced when larvae are exposed to stimuli that they would normally escape from or avoid. Although these emotional states probably differ between species and change during development, much can be learned about human fear and anxiety using zebrafish as a model system. The molecular mechanisms of fear and anxiety are highly conserved in vertebrates and are present during early zebrafish development. Larvae during the first week of development display elevated cortisol levels in response to stress and are sensitive to the same anxiolytics that are used for the management of anxiety in humans. Zebrafish larvae are well suited for high-throughput analyses of behavior, and automated systems have been developed for imaging and analyzing the behavior of zebrafish larvae in multiwell plates. These high-throughput analyses will not only provide a wealth of information on the genes and environmental factors that influence escape and avoidance behaviors and the emotional states that might accompany them but will also facilitate the discovery of novel pharmaceuticals that could be used in the management of anxiety disorders in humans. PMID:21572576

  3. Imaging escape and avoidance behavior in zebrafish larvae

    PubMed Central

    Colwill, Ruth M.; Creton, Robbert

    2011-01-01

    Synopsis This review provides an overview of the assays that are used for measuring escape and avoidance behavior in zebrafish, with a specific focus on zebrafish larvae during the first week of development. Zebrafish larvae display a startle response when exposed to tactile, acoustic, or visual stimuli and will avoid dark areas, moving objects, conspecifics, and open spaces. Emotional states such as fear and anxiety may be induced when larvae are exposed to stimuli that they would normally escape from or avoid. While these emotional states may differ between species and change during development, much can be learned about human fear and anxiety using zebrafish as a model system. The molecular mechanisms of fear and anxiety are highly conserved in vertebrates and are present during early zebrafish development. Larvae during the first week of development display elevated cortisol levels in response to stress and are sensitive to the same anxiolytics that are used for the management of anxiety in humans. Zebrafish larvae are well suited for high-throughput analyses of behavior, and automated systems have been developed for imaging and analyzing the behavior of zebrafish larvae in multiwell plates. These high-throughput analyses will not only provide a wealth of information on the genes and environmental factors that influence escape and avoidance behaviors and the emotional states that may accompany them, but will also facilitate the discovery of novel pharmaceuticals that may be used in the management of anxiety disorders in humans. PMID:21572576

  4. Farnesol inhibits tumor growth and enhances the anticancer effects of bortezomib in multiple myeloma xenograft mouse model through the modulation of STAT3 signaling pathway.

    PubMed

    Lee, Jong Hyun; Kim, Chulwon; Kim, Sung-Hoon; Sethi, Gautam; Ahn, Kwang Seok

    2015-05-01

    Aberrant activation of signal transducer and activator of transcription 3 (STAT3) is frequently observed in multiple myeloma (MM) cancer and can upregulate the expression of several genes involved in proliferation, survival, metastasis, and angiogenesis. The effect of farnesol (FOH) on STAT3 activation, associated protein kinases, its regulated gene products, cellular proliferation, and apoptosis was examined. The in vivo effect of FOH on the growth of human MM xenograft tumors alone and in combination with bortezomib (Bor) in athymic nu/nu female mice was also investigated. We found that FOH suppressed both constitutive and inducible STAT3 activation at Tyr705 in MM cells. The suppression of STAT3 was mediated through the inhibition of activation of upstream JAK1, JAK2, and c-Src kinases. Also, treatment with the protein tyrosine phosphatase (PTP) inhibitor, pervanadate treatment reversed the FOH-induced down-regulation of STAT3, possibly indicating the involvement of a PTP. Indeed, we found that FOH treatment induces the increased expression of SHP-2 protein and knockdown of the SHP-2 gene by small interfering RNA suppressed the ability of FOH to inhibit STAT3 activation. FOH inhibited proliferation and significantly potentiated the apoptotic effects of bortezomib (Bor) in U266 cells. When administered intraperitoneally, FOH enhanced Bor-induced growth suppression of human MM xenograft tumors in athymic nu/nu female mice. Our results suggest that FOH is a novel blocker of STAT3 signaling pathway and exerts both anti-proliferative and apoptotic activities in MM in vitro and in vivo. PMID:25697480

  5. Characterization of zebrafish caspase-3 and induction of apoptosis through ceramide generation in fish fathead minnow tailbud cells and zebrafish embryo.

    PubMed Central

    Yabu, T; Kishi, S; Okazaki, T; Yamashita, M

    2001-01-01

    Caspase-3 was cloned from zebrafish embryos and its properties were characterized to identify the biological implications of caspase in embryogenesis and apoptosis in zebrafish, which is a model organism in vertebrate developmental biology and genetics. The predicted amino acid sequence, totalling 282 amino acid residues, consisted of the prodomain and large and small subunits. Phylogenetic analysis showed that the cloned zebrafish caspase was a member of the caspase-3 subfamily with approx. 60% identity with caspase-3 from Xenopus, chicken and mammals. In addition, recombinant zebrafish caspase hydrolysed acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide, and exhibited similar substrate specificity to the mammalian caspase-3 subfamily. Therefore this caspase was designated zebrafish caspase-3. Overexpression of zebrafish caspase-3 induced apoptosis and increased ceramide levels in fish fathead minnow tailbud cells and zebrafish embryos. Both ceramide generation and apoptosis induction were inhibited by treatment with a caspase inhibitor, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone. Moreover, zebrafish caspase-3 mRNA was present in early embryos up to the 1000-cell stage as a maternal factor, and was then expressed throughout the body after the gastrula stage by zygotic expression. These findings indicate that the isolated caspase-3 plays an important role in the induction of ceramide generation as well as apoptosis in fish cells and the zebrafish embryo, and suggest that caspase-3 functions as a modulator of the pro-apoptotic signal in development. PMID:11695990

  6. Modeling Stress and Anxiety in Zebrafish Jonathan M. Cachat, Peter R. Canavello, Marco F. Elegante, Brett K. Bartels, Salem I. Elkhayat,

    E-print Network

    Kalueff, Allan V.

    , zebrafish possess all of the "classical" neurotransmitters found in vertebrates [31], suggesting to human error and incorrect data interpretation. In contrast, automated video-tracking technologies can influence [14]. Another advantage of using the video-tracking approach in zebrafish research is the ability

  7. Fundamental Approaches to the Study of Zebrafish Nutrition

    PubMed Central

    Watts, Stephen A.; Powell, Mickie; D’Abramo, Louis R.

    2014-01-01

    The value of the zebrafish model has been well established. However, culture variability within and among laboratories remains a concern, particularly as it relates to nutrition. Investigators using rodent models addressed this concern several decades ago and have developed strict nutritional regimes to which their models adhere. These investigators decreased the variability associated with nutrition in most studies by developing standardized reference and open formulation diets. Zebrafish investigators have not embraced this approach. In this article, we address the problems associated with the lack of nutritional information and standardization in the zebrafish research community. Based on the knowledge gained from studies of other animals, including traditional research models, other fish species, domesticated and companion animals, and humans, we have proposed an approach that seeks to standardize nutrition research in zebrafish. We have identified a number of factors for consideration in zebrafish nutrition studies and have suggested a number of proposed outcomes. The long term-goal of nutrition research will be to identify the daily nutritional requirements of the zebrafish and to develop appropriate standardized reference and open formulation diets. PMID:23382346

  8. Zebrafish: A possible tool to evaluate bioactive ions.

    PubMed

    Montazerolghaem, M; Nyström, L; Engqvist, H; Karlsson Ott, M

    2015-06-01

    Zebrafish is a well-established model organism with a skeletal structure that highly resembles mammalian bone. Yet its use in the research field of biomaterials has been limited. One area that could benefit from this model system is the evaluation of ionic dissolution products from different materials. As a proof of concept we have evaluated the effect of silicate ions on the zebrafish larvae and compared it to a well-known osteblastic cell line, MC3T3-E1 subclone 14. We have shown that sodium metasilicate (125?M and 625?M) induces more mineralisation in a dose-dependent manner in zebrafish larvae, 9days post fertilisation as compared to the non-treated group. Moreover the same trends were seen when adding sodium metasilicate to MC3T3-E1 cultures, with more mineralisation and higher ALP levels with higher doses of silicate (25, 125 and 625?M). These results indicate the feasibility of zebrafish larvae for ionic dissolution studies. The zebrafish model is superior to isolated cell cultures in the aspect that it includes the whole bone remodelling system, with osteoblasts, osteoclasts and osteocytes. Zebrafish could thus provide a powerful in vivo tool and be a bridge between cell culture systems and mammalian models. PMID:25770927

  9. A shifted repertoire of endocannabinoid genes in the zebrafish ( Danio rerio )

    Microsoft Academic Search

    J. M. McPartland; Michelle Glass; Isabel Matias; Ryan W. Norris; C. William Kilpatrick

    2007-01-01

    The zebrafish has served as a model organism for developmental biology. Sequencing its genome has expanded zebrafish research\\u000a into physiology and drug-development testing. Several cannabinoid pharmaceuticals are in development, but expression of endocannabinoid\\u000a receptors and enzymes remains unknown in this species. We conducted a bioinformatics analysis of the zebrafish genome using\\u000a 17 human endocannabinoid genes as a reference set. Putative

  10. Peroxisomes in zebrafish: distribution pattern and knockdown studies

    Microsoft Academic Search

    Olga Krysko; Mieke Stevens; Tobias Langenberg; Marc Fransen; Marc Espeel; Myriam Baes

    2010-01-01

    Peroxisomes are organelles that are essential for normal development in men and mice. In order to explore whether zebrafish\\u000a could be used as a model system to study the role of peroxisomes, we examined their distribution pattern in developing and\\u000a adult zebrafish and we tested different approaches to eliminate them during the first days after fertilization. In 4-day-old\\u000a embryos, catalase-containing

  11. Quantification of birefringence readily measures the level of muscle damage in zebrafish

    SciTech Connect

    Berger, Joachim, E-mail: Joachim.Berger@Monash.edu [Australian Regenerative Medicine Institute, EMBL Australia, Monash University, Clayton (Australia)] [Australian Regenerative Medicine Institute, EMBL Australia, Monash University, Clayton (Australia); Sztal, Tamar; Currie, Peter D. [Australian Regenerative Medicine Institute, EMBL Australia, Monash University, Clayton (Australia)] [Australian Regenerative Medicine Institute, EMBL Australia, Monash University, Clayton (Australia)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer Report of an unbiased quantification of the birefringence of muscle of fish larvae. Black-Right-Pointing-Pointer Quantification method readily identifies level of overall muscle damage. Black-Right-Pointing-Pointer Compare zebrafish muscle mutants for level of phenotype severity. Black-Right-Pointing-Pointer Proposed tool to survey treatments that aim to ameliorate muscular dystrophy. -- Abstract: Muscular dystrophies are a group of genetic disorders that progressively weaken and degenerate muscle. Many zebrafish models for human muscular dystrophies have been generated and analysed, including dystrophin-deficient zebrafish mutants dmd that model Duchenne Muscular Dystrophy. Under polarised light the zebrafish muscle can be detected as a bright area in an otherwise dark background. This light effect, called birefringence, results from the diffraction of polarised light through the pseudo-crystalline array of the muscle sarcomeres. Muscle damage, as seen in zebrafish models for muscular dystrophies, can readily be detected by a reduction in the birefringence. Therefore, birefringence is a very sensitive indicator of overall muscle integrity within larval zebrafish. Unbiased documentation of the birefringence followed by densitometric measurement enables the quantification of the birefringence of zebrafish larvae. Thereby, the overall level of muscle integrity can be detected, allowing the identification and categorisation of zebrafish muscle mutants. In addition, we propose that the establish protocol can be used to analyse treatments aimed at ameliorating dystrophic zebrafish models.

  12. Nicotine response genetics in the zebrafish.

    PubMed

    Petzold, Andrew M; Balciunas, Darius; Sivasubbu, Sridhar; Clark, Karl J; Bedell, Victoria M; Westcot, Stephanie E; Myers, Shelly R; Moulder, Gary L; Thomas, Mark J; Ekker, Stephen C

    2009-11-01

    Tobacco use is predicted to result in over 1 billion deaths worldwide by the end of the 21(st) century. How genetic variation contributes to the observed differential predisposition in the human population to drug dependence is unknown. The zebrafish (Danio rerio) is an emerging vertebrate model system for understanding the genetics of behavior. We developed a nicotine behavioral assay in zebrafish and applied it in a forward genetic screen using gene-breaking transposon mutagenesis. We used this method to molecularly characterize bdav/cct8 and hbog/gabbr1.2 as mutations with altered nicotine response. Each have a single human ortholog, identifying two points for potential scientific, diagnostic, and drug development for nicotine biology and cessation therapeutics. We show this insertional method generates mutant alleles that are reversible through Cre-mediated recombination, representing a conditional mutation system for the zebrafish. The combination of this reporter-tagged insertional mutagen approach and zebrafish provides a powerful platform for a rich array of questions amenable to genetic-based scientific inquiry, including the basis of behavior, epigenetics, plasticity, stress, memory, and learning. PMID:19858493

  13. Melanoma patient derived xenografts acquire distinct Vemurafenib resistance mechanisms

    PubMed Central

    Monsma, David J; Cherba, David M; Eugster, Emily E; Dylewski, Dawna L; Davidson, Paula T; Peterson, Chelsea A; Borgman, Andrew S; Winn, Mary E; Dykema, Karl J; Webb, Craig P; MacKeigan, Jeffrey P; Duesbery, Nicholas S; Nickoloff, Brian J; Monks, Noel R

    2015-01-01

    Variable clinical responses, tumor heterogeneity, and drug resistance reduce long-term survival outcomes for metastatic melanoma patients. To guide and accelerate drug development, we characterized tumor responses for five melanoma patient derived xenograft models treated with Vemurafenib. Three BRAFV600E models showed acquired drug resistance, one BRAFV600E model had a complete and durable response, and a BRAFV600V model was expectedly unresponsive. In progressing tumors, a variety of resistance mechanisms to BRAF inhibition were uncovered, including mutant BRAF alternative splicing, NRAS mutation, COT (MAP3K8) overexpression, and increased mutant BRAF gene amplification and copy number. The resistance mechanisms among the patient derived xenograft models were similar to the resistance pathways identified in clinical specimens from patients progressing on BRAF inhibitor therapy. In addition, there was both inter- and intra-patient heterogeneity in resistance mechanisms, accompanied by heterogeneous pERK expression immunostaining profiles. MEK monotherapy of Vemurafenib-resistant tumors caused toxicity and acquired drug resistance. However, tumors were eradicated when Vemurafenib was combined the MEK inhibitor. The diversity of drug responses among the xenograft models; the distinct mechanisms of resistance; and the ability to overcome resistance by the addition of a MEK inhibitor provide a scheduling rationale for clinical trials of next-generation drug combinations.

  14. A Zebrafish Model of Axenfeld-Rieger Syndrome Reveals That pitx2 Regulation by Retinoic Acid Is Essential for Ocular and Craniofacial Development

    PubMed Central

    Bohnsack, Brenda L.; Kasprick, Daniel S.; Kish, Phillip E.; Goldman, Daniel

    2012-01-01

    Purpose. The homeobox transcription factor PITX2 is a known regulator of mammalian ocular development, and human PITX2 mutations are associated with Axenfeld-Rieger syndrome (ARS). However, the treatment of patients with ARS remains mostly supportive and palliative. Methods. The authors used molecular genetic, pharmacologic, and embryologic techniques to study the biology of ARS in a zebrafish model that uses transgenes to mark neural crest and muscle cells in the head. Results. The authors demonstrated in vivo that pitx2 is a key downstream target of retinoic acid (RA) in craniofacial development, and this pathway is required for coordinating neural crest, mesoderm, and ocular development. pitx2a knockdown using morpholino oligonucleotides disrupts jaw and pharyngeal arch formation and recapitulates ocular characteristics of ARS, including corneal and iris stroma maldevelopment. These phenotypes could be rescued with human PITX2A mRNA, demonstrating the specificity of the knockdown and evolutionary conservation of pitx2a function. Expression of the ARS dominant negative human PITX2A K50E allele also caused ARS-like phenotypes. Similarly, inhibition of RA synthesis in the developing eye (genetic or pharmacologic) disrupted craniofacial and ocular development, and human PITX2A mRNA partially rescued these defects. Conclusions. RA regulation of pitx2 is essential for coordinating interactions among neural crest, mesoderm, and developing eye. The marked evolutionary conservation of Pitx2 function in eye and craniofacial development makes zebrafish a potentially powerful model of ARS, amenable to in vivo experimentation and development of potential therapies. PMID:22125274

  15. Abca12-mediated lipid transport and Snap29-dependent trafficking of lamellar granules are crucial for epidermal morphogenesis in a zebrafish model of ichthyosis.

    PubMed

    Li, Qiaoli; Frank, Michael; Akiyama, Masashi; Shimizu, Hiroshi; Ho, Shiu-Ying; Thisse, Christine; Thisse, Bernard; Sprecher, Eli; Uitto, Jouni

    2011-11-01

    Zebrafish (Danio rerio) can serve as a model system to study heritable skin diseases. The skin is rapidly developed during the first 5-6 days of embryonic growth, accompanied by expression of skin-specific genes. Transmission electron microscopy (TEM) of wild-type zebrafish at day 5 reveals a two-cell-layer epidermis separated from the underlying collagenous stroma by a basement membrane with fully developed hemidesmosomes. Scanning electron microscopy (SEM) reveals an ordered surface contour of keratinocytes with discrete microridges. To gain insight into epidermal morphogenesis, we have employed morpholino-mediated knockdown of the abca12 and snap29 genes, which are crucial for secretion of lipids and intracellular trafficking of lamellar granules, respectively. Morpholinos, when placed on exon-intron junctions, were >90% effective in preventing the corresponding gene expression when injected into one- to four-cell-stage embryos. By day 3, TEM of abca12 morphants showed accumulation of lipid-containing electron-dense lamellar granules, whereas snap29 morphants showed the presence of apparently empty vesicles in the epidermis. Evaluation of epidermal morphogenesis by SEM revealed similar perturbations in both cases in the microridge architecture and the development of spicule-like protrusions on the surface of keratinocytes. These morphological findings are akin to epidermal changes in harlequin ichthyosis and CEDNIK syndrome, autosomal recessive keratinization disorders due to mutations in the ABCA12 and SNAP29 genes, respectively. The results indicate that interference of independent pathways involving lipid transport in the epidermis can result in phenotypically similar perturbations in epidermal morphogenesis, and that these fish mutants can serve as a model to study the pathomechanisms of these keratinization disorders. PMID:21816950

  16. Abca12-mediated lipid transport and Snap29-dependent trafficking of lamellar granules are crucial for epidermal morphogenesis in a zebrafish model of ichthyosis

    PubMed Central

    Li, Qiaoli; Frank, Michael; Akiyama, Masashi; Shimizu, Hiroshi; Ho, Shiu-Ying; Thisse, Christine; Thisse, Bernard; Sprecher, Eli; Uitto, Jouni

    2011-01-01

    SUMMARY Zebrafish (Danio rerio) can serve as a model system to study heritable skin diseases. The skin is rapidly developed during the first 5–6 days of embryonic growth, accompanied by expression of skin-specific genes. Transmission electron microscopy (TEM) of wild-type zebrafish at day 5 reveals a two-cell-layer epidermis separated from the underlying collagenous stroma by a basement membrane with fully developed hemidesmosomes. Scanning electron microscopy (SEM) reveals an ordered surface contour of keratinocytes with discrete microridges. To gain insight into epidermal morphogenesis, we have employed morpholino-mediated knockdown of the abca12 and snap29 genes, which are crucial for secretion of lipids and intracellular trafficking of lamellar granules, respectively. Morpholinos, when placed on exon-intron junctions, were >90% effective in preventing the corresponding gene expression when injected into one- to four-cell-stage embryos. By day 3, TEM of abca12 morphants showed accumulation of lipid-containing electron-dense lamellar granules, whereas snap29 morphants showed the presence of apparently empty vesicles in the epidermis. Evaluation of epidermal morphogenesis by SEM revealed similar perturbations in both cases in the microridge architecture and the development of spicule-like protrusions on the surface of keratinocytes. These morphological findings are akin to epidermal changes in harlequin ichthyosis and CEDNIK syndrome, autosomal recessive keratinization disorders due to mutations in the ABCA12 and SNAP29 genes, respectively. The results indicate that interference of independent pathways involving lipid transport in the epidermis can result in phenotypically similar perturbations in epidermal morphogenesis, and that these fish mutants can serve as a model to study the pathomechanisms of these keratinization disorders. PMID:21816950

  17. Zebrafish embryo development

    NSDL National Science Digital Library

    Mildred Hoover (Salem State College; Biology Department)

    2008-07-19

    Early development of Danio eggs ? note the internal structure and the cluster of eggs (note the large yolk attached to the two-cell egg). The cells all divide at the same time. Within two days this mass of dividing cells will take on the shape of a zebrafish larvae.

  18. Non-associative learning in larval zebrafish.

    PubMed

    Best, Jonathan D; Berghmans, Stéphane; Hunt, Julia J F G; Clarke, Samantha C; Fleming, Angeleen; Goldsmith, Paul; Roach, Alan G

    2008-04-01

    Habituation, where a response is reduced when exposed to a continuous stimulus is one of the simplest forms of non-associative learning and has been shown in a number of organisms from sea slugs to rodents. However, very little has been reported in the zebrafish, a model that is gaining popularity for high-throughput compound screens. Furthermore, since most of the studies involving learning and memory in zebrafish have been conducted in adults, we sought to determine if zebrafish larvae could display non-associative learning and whether it could be modulated by compounds identified in previous rodent studies. We demonstrated that zebrafish larvae (7 days post fertilization) exhibit iterative reduction in a startle response to a series of acoustic stimuli. Furthermore, this reduction satisfied criteria for habituation: spontaneous recovery, more rapid reductions in startle to shorter intertrial intervals and dishabituation. We then investigated the pathways mediating this behavior using established compounds in learning and memory. Administration of rolipram (PDE4 inhibitor), donepezil (acetylcholinesterase inhibitor), and memantine (N-methyl-D-aspartic acid (NMDA) receptor antagonist) all increased the acoustic startle response and decreased habituation in the larvae, similar to previous rodent studies. Further studies demonstrated that NMDA blocked the memantine response and the effect of donepezil was blocked by mecamylamine but not atropine suggesting that the donepezil response was mediated by nicotinic rather than muscarinic receptors. Zebrafish larvae possess numerous advantages for medium to high-throughput screening; the model described herein therefore offers the potential to screen for additional compounds for further study on cognition function. PMID:17581529

  19. In vitro neutralization of viral hemorrhagic septicemia virus by plasma from immunized zebrafish.

    PubMed

    Chinchilla, Blanca; Gomez-Casado, Eduardo; Encinas, Paloma; Falco, Alberto; Estepa, Amparo; Coll, Julio

    2013-03-01

    We studied humoral long-term adaptive viral neutralization responses in zebrafish (Danio rerio), an increasingly useful vertebrate model for viral diseases actually limited by the absence of standardized anti-zebrafish immunoglobulin M (IgM) antibodies. We established an alternative method, similar to those used in other fish, to achieve a first estimation of zebrafish anti-viral antibody-like responses. We used the viral hemorrhagic septicemia virus (VHSV) model because, although protection after this non-natural infection was demonstrated in cold-acclimatized zebrafish, little is known about their induced anti-VHSV antibody-like responses. Therefore, we first optimized a micro-neutralization method based on immunostaining VHSV-infected fish cell monolayers to detect zebrafish neutralizing activity in plasma samples in one day. We then used the method to measure the specific anti-VHSV neutralization in plasma obtained from individual zebrafish under various VHSV challenges or immunization protocols. The neutralizing activity was inhibited by protein A-sepharose and rabbit anti-zebrafish IgM antibodies, suggesting the implication of IgM zebrafish antibodies in such responses. To our knowledge, this is the first report to demonstrate detectable and significant VHSV neutralization titers in zebrafish surviving VHSV infections. This micro-method might be useful, not only for the follow-up of infection/vaccine development in the zebrafish/VHSV model in particular, but also for similar work involving other in vitro neutralizable zebrafish pathogens. This technique might also further the development of alternative ELISA assay methods to measure specific immunoglobulins in zebrafish. PMID:23445428

  20. Therapeutic Silencing of Bcl-2 by Systemically Administered siRNA Nanotherapeutics Inhibits Tumor Growth by Autophagy and Apoptosis and Enhances the Efficacy of Chemotherapy in Orthotopic Xenograft Models of ER (?) and ER (+) Breast Cancer

    PubMed Central

    Tekedereli, Ibrahim; Alpay, S Neslihan; Akar, Ugur; Yuca, Erkan; Ayugo-Rodriguez, Cristian; Han, He-Dong; Sood, Anil K; Lopez-Berestein, Gabriel; Ozpolat, Bulent

    2013-01-01

    Bcl-2 is overexpressed in about a half of human cancers and 50–70% of breast cancer patients, thereby conferring resistance to conventional therapies and making it an excellent therapeutic target. Small interfering RNA (siRNA) offers novel and powerful tools for specific gene silencing and molecularly targeted therapy. Here, we show that therapeutic silencing of Bcl-2 by systemically administered nanoliposomal (NL)-Bcl-2 siRNA (0.15?mg siRNA/kg, intravenous) twice a week leads to significant antitumor activity and suppression of growth in both estrogen receptor-negative (ER(?)) MDA-MB-231 and ER-positive (+) MCF7 breast tumors in orthotopic xenograft models (P < 0.05). A single intravenous injection of NL-Bcl-2-siRNA provided robust and persistent silencing of the target gene expression in xenograft tumors. NL-Bcl-2-siRNA treatment significantly increased the efficacy of chemotherapy when combined with doxorubicin in both MDA-MB-231 and MCF-7 animal models (P < 0.05). NL-Bcl-2-siRNA treatment-induced apoptosis and autophagic cell death, and inhibited cyclin D1, HIF1? and Src/Fak signaling in tumors. In conclusion, our data provide the first evidence that in vivo therapeutic targeting Bcl-2 by systemically administered nanoliposomal-siRNA significantly inhibits growth of both ER(?) and ER(+) breast tumors and enhances the efficacy of chemotherapy, suggesting that therapeutic silencing of Bcl-2 by siRNA is a viable approach in breast cancers. PMID:24022053

  1. Functional inhibition of UQCRB suppresses angiogenesis in zebrafish

    SciTech Connect

    Cho, Yoon Sun; Jung, Hye Jin [Chemical Genomics National Research Laboratory, Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)] [Chemical Genomics National Research Laboratory, Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Seok, Seung Hyeok [Department of Microbiology and Immunology, Institute for Experimental Animals, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)] [Department of Microbiology and Immunology, Institute for Experimental Animals, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Payumo, Alexander Y.; Chen, James K. [Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305 (United States)] [Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305 (United States); Kwon, Ho Jeong, E-mail: kwonhj@yonsei.ac.kr [Chemical Genomics National Research Laboratory, Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2013-04-19

    Highlights: ? This is the first functional characterization of UQCRB in vivo model. ? Angiogenesis is inhibited with UQCRB loss of function in zebrafish. ? UQCRB is introduced as a prognostic marker for mitochondria- and angiogenesis-related diseases. -- Abstract: As a subunit of mitochondrial complex III, UQCRB plays an important role in complex III stability, electron transport, and cellular oxygen sensing. Herein, we report UQCRB function regarding angiogenesis in vivo with the zebrafish (Danio rerio). UQCRB knockdown inhibited angiogenesis in zebrafish leading to the suppression of VEGF expression. Moreover, the UQCRB-targeting small molecule terpestacin also inhibited angiogenesis and VEGF levels in zebrafish, supporting the role of UQCRB in angiogenesis. Collectively, UQCRB loss of function by either genetic and pharmacological means inhibited angiogenesis, indicating that UQCRB plays a key role in this process and can be a prognostic marker of angiogenesis- and mitochondria-related diseases.

  2. Modeling the Zebrafish Segmentation Clock’s Gene Regulatory Network Constrained by Expression Data Suggests Evolutionary Transitions Between Oscillating and Nonoscillating Transcription

    PubMed Central

    Schwendinger-Schreck, Jamie; Kang, Yuan; Holley, Scott A.

    2014-01-01

    During segmentation of vertebrate embryos, somites form in accordance with a periodic pattern established by the segmentation clock. In the zebrafish (Danio rerio), the segmentation clock includes six hairy/enhancer of split-related (her/hes) genes, five of which oscillate due to negative autofeedback. The nonoscillating gene hes6 forms the hub of a network of 10 Her/Hes protein dimers, which includes 7 DNA-binding dimers and 4 weak or non-DNA-binding dimers. The balance of dimer species is critical for segmentation clock function, and loss-of-function studies suggest that the her genes have both unique and redundant functions within the clock. However, the precise regulatory interactions underlying the negative feedback loop are unknown. Here, we combine quantitative experimental data, in silico modeling, and a global optimization algorithm to identify a gene regulatory network (GRN) designed to fit measured transcriptional responses to gene knockdown. Surprisingly, we find that hes6, the clock gene that does not oscillate, responds to negative feedback. Consistent with prior in silico analyses, we find that variation in transcription, translation, and degradation rates can mediate the gain and loss of oscillatory behavior for genes regulated by negative feedback. Extending our study, we found that transcription of the nonoscillating Fgf pathway gene sef responds to her/hes perturbation similarly to oscillating her genes. These observations suggest a more extensive underlying regulatory similarity between the zebrafish segmentation clock and the mouse and chick segmentation clocks, which exhibit oscillations of her/hes genes as well as numerous other Notch, Fgf, and Wnt pathway genes. PMID:24663100

  3. Ionic channels underlying the ventricular action potential in zebrafish embryo.

    PubMed

    Alday, Aintzane; Alonso, Hiart; Gallego, Monica; Urrutia, Janire; Letamendia, Ainhoa; Callol, Carles; Casis, Oscar

    2014-06-01

    Over the last years zebrafish has become a popular model in the study of cardiac physiology, pathology and pharmacology. Recently, the application of the 3Rs regulation and the characteristics of the embryo have reduced the use of adult zebrafish use in many studies. However, the zebrafish embryo cardiac physiology is poorly characterized since most works have used indirect techniques and direct recordings of cardiac action potential and ionic currents are scarce. In order to optimize the zebrafish embryo model, we used electrophysiological, pharmacological and immunofluorescence tools to identify the characteristics and the ionic channels involved in the ventricular action potentials of zebrafish embryos. The application of Na(+) or T-type Ca(+2) channel blockers eliminated the cardiac electrical activity, indicating that the action potential upstroke depends on Na(+) and T-type Ca(+2) currents. The plateau phase depends on L-type Ca(+2) channels since it is abolished by specific blockade. The direct channel blockade indicates that the action potential repolarization and diastolic potential depends on ERG K(+) channels. The presence in the embryonic heart of the Nav1.5, Cav1.2, Cav3.2 and ERG channels was also confirmed by immunofluorescence, while the absence of effect of specific blockers and immunostaining indicate that two K(+) repolarizing currents present in human heart, Ito and IKs, are absent in the embryonic zebrafish heart. Our results describe the ionic channels present and its role in the zebrafish embryo heart and support the use of zebrafish embryos to study human diseases and their use for drug testing. PMID:24747832

  4. Minimally invasive establishment of murine orthotopic bladder xenografts.

    PubMed

    Jäger, Wolfgang; Moskalev, Igor; Janssen, Claudia; Hayashi, Tetsutaro; Gust, Killian M; Awrey, Shannon; Black, Peter C

    2014-01-01

    Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse. With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period. In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model. PMID:24561487

  5. Ezetimibe and Simvastatin Reduce Cholesterol Levels in Zebrafish Larvae Fed a High-Cholesterol Diet

    PubMed Central

    Baek, Ji Sun; Fang, Longhou; Li, Andrew C.; Miller, Yury I.

    2012-01-01

    Cholesterol-fed zebrafish is an emerging animal model to study metabolic, oxidative, and inflammatory vascular processes relevant to pathogenesis of human atherosclerosis. Zebrafish fed a high-cholesterol diet (HCD) develop hypercholesterolemia and are characterized by profound lipoprotein oxidation and vascular lipid accumulation. Using optically translucent zebrafish larvae has the advantage of monitoring vascular pathology and assessing the efficacy of drug candidates in live animals. Thus, we investigated whether simvastatin and ezetimibe, the principal drugs used in management of hypercholesterolemia in humans, would also reduce cholesterol levels in HCD-fed zebrafish larvae. We found that ezetimibe was well tolerated by zebrafish and effectively reduced cholesterol levels in HCD-fed larvae. In contrast, simvastatin added to water was poorly tolerated by zebrafish larvae and, when added to food, had little effect on cholesterol levels in HCD-fed larvae. Combination of low doses of ezetimibe and simvastatin had an additive effect in reducing cholesterol levels in zebrafish. These results suggest that ezetimibe exerts in zebrafish a therapeutic effect similar to that in humans and that the hypercholesterolemic zebrafish can be used as a low-cost and informative model for testing new drug candidates and for investigating mechanisms of action for existing drugs targeting dyslipidemia. PMID:22693663

  6. Systematic comparison of tissue fixation with alternative fixatives to conventional tissue fixation with buffered formalin in a xenograft-based model.

    PubMed

    Nietner, Thorben; Jarutat, Tiantom; Mertens, Alfred

    2012-09-01

    In our study we systematically compared the alternative fixatives acidified formal alcohol (AFA), PAXgene®, HOPE®, and combinations of AFA or formalin with ultrasound treatment to standard (buffered) formalin fixation. We examined general morphology and detectability of protein structures by immunohistochemistry of the membrane receptors epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R), and phosphorylated human epidermal growth factor receptor 2 (phospho-HER2). In order to allow for stringent comparability of different fixation techniques, we used matched mouse xenograft tumor samples from three different human cancer cell lines (colon, ovarian, and non-small cell lung cancer), either fixed conventionally with formalin or an alternative fixative. Tissue morphology after fixation with AFA and PAXgene® was comparable to formalin-fixed paraffin-embedded tissue (FFPET) morphology. Ultrasound fixations resulted in slightly inferior morphology and HOPE® fixation preserved morphology only poorly compared to FFPET in this system. None of the tested alternative fixatives enabled immunohistochemical detectability of all three targets in the same manner as FFPET. Pronounced staining was possible for EGFR and IGF-1R with all alternative fixatives but HOPE®, and phospho-HER2 staining was only noteworthy with formalin-ultrasound-fixed tissue. Therefore, the use of alternative fixatives comes with the need for careful validation of obtained IHC results individually for each target. PMID:22814649

  7. Evaluation of heterogeneous metabolic profile in an orthotopic human glioblastoma xenograft model using compressed sensing hyperpolarized 3D 13C magnetic resonance spectroscopic imaging.

    PubMed

    Park, Ilwoo; Hu, Simon; Bok, Robert; Ozawa, Tomoko; Ito, Motokazu; Mukherjee, Joydeep; Phillips, Joanna J; James, C David; Pieper, Russell O; Ronen, Sabrina M; Vigneron, Daniel B; Nelson, Sarah J

    2013-07-01

    High resolution compressed sensing hyperpolarized (13)C magnetic resonance spectroscopic imaging was applied in orthotopic human glioblastoma xenografts for quantitative assessment of spatial variations in (13)C metabolic profiles and comparison with histopathology. A new compressed sensing sampling design with a factor of 3.72 acceleration was implemented to enable a factor of 4 increase in spatial resolution. Compressed sensing 3D (13)C magnetic resonance spectroscopic imaging data were acquired from a phantom and 10 tumor-bearing rats following injection of hyperpolarized [1-(13)C]-pyruvate using a 3T scanner. The (13)C metabolic profiles were compared with hematoxylin and eosin staining and carbonic anhydrase 9 staining. The high-resolution compressed sensing (13)C magnetic resonance spectroscopic imaging data enabled the differentiation of distinct (13)C metabolite patterns within abnormal tissues with high specificity in similar scan times compared to the fully sampled method. The results from pathology confirmed the different characteristics of (13)C metabolic profiles between viable, non-necrotic, nonhypoxic tumor, and necrotic, hypoxic tissue. PMID:22851374

  8. Morphologic and molecular characterization of ATRT xenografts adapted for orthotopic therapeutic testing.

    PubMed

    Hashizume, Rintaro; Gupta, Nalin; Berger, Mitchel S; Banerjee, Anu; Prados, Michael D; Ayers-Ringler, Jennifer; James, C David; VandenBerg, Scott R

    2010-04-01

    Atypical teratoid rhabdoid tumor (ATRT) is a malignant tumor of the central nervous system that most commonly arises in young children. The aggressive growth and propensity for early dissemination throughout the neuraxis confers a dismal prognosis. Large clinical trials that could test new therapeutic agents are difficult to conduct due to the low incidence of this cancer. For this reason, high throughput preclinical testing with suitable animal models for ATRT would serve a critical need for identifying the most efficacious treatments. In response to this need, we have adapted ATRT cell lines for bioluminescence imaging (BLI) of intracranial (orthotopic) xenografts established in athymic mice. Our results indicate that following supratentorial or infratentorial injection in athymic mice, ATRT cells produce rapidly growing tumors, often with intraventricular spread or neuraxis dissemination. When established as orthotopic xenografts, the tumors predominantly display cells with a rhabdoid-like cellular morphology that show a spectrum of immunophenotypes similar to primary ATRT tumors. To demonstrate the feasibility of this orthotopic ATRT xenograft model for therapeutic testing with correlation to biomarker analysis, we examined the responses of luciferase-modified ATRT cells to temozolomide (TMZ). These xenografts, which highly express MGMT, are resistant to TMZ treatment when compared with an orthotopic glioblastoma xenograft that is MGMT deficient and responsive to TMZ. These data suggest that an orthotopic ATRT xenograft model, in which BLI is used for monitoring tumor growth and response to therapy, should contribute to the identification of effective therapeutics and regimens for treating this highly aggressive pediatric brain tumor. PMID:20308314

  9. Constructing the habituome for phenotype-driven zebrafish research.

    PubMed

    Stewart, Adam Michael; Cachat, Jonathan; Green, Jeremy; Gaikwad, Siddharth; Kyzar, Evan; Roth, Andrew; Davis, Ari; Collins, Christopher; El-Ounsi, Mohamed; Pham, Mimi; Kalueff, Allan V

    2013-01-01

    Intra-session habituation to novelty reflects spatial working memory (related to exploration and cognition), and is observed in various species, including zebrafish (Danio rerio). With the growing understanding of complex zebrafish behaviors, the extent to which they habituate remains unclear. Here we perform a large-scale characterization of zebrafish novelty-evoked (novel tank and open field) behaviors, to establish their grouping based on intra-session habituation and sensitivity to anxiolytic or anxiogenic manipulations. We also assess multiple behaviors in high- and low-anxiety sub-cohorts of a large heterogeneous zebrafish population, comparing their habituation profiles. Overall, our analyses demonstrate that anxiety responsivity and the ability to habituate show little correlation for multiple zebrafish behaviors, suggesting that they most likely represent distinct behavioral phenomena in novel environments. Using these data, we also present the habituome--a new conceptual approach to study affective and cognitive responses in zebrafish by examining a big set of their habituation phenotypes. Given marked similarity in animal novelty exploration, this approach may also be used to construct habituomes in other model organisms, including rodents and humans. PMID:22944516

  10. Individual differences in activity levels in zebrafish (Danio rerio)

    PubMed Central

    Tran, Steven; Gerlai, Robert

    2013-01-01

    Individual differences and variation in behavioural responses have been identified in many animal species. These differences may be the result of genetic or environmental factors or the interaction between them. Analysis of individual differences in behaviour may be important for many reasons. The zebrafish is a powerful model organism that is rapidly gaining popularity in behavioural brain research. However, individual differences have rarely been explored in zebrafish although significant variation in their performance has been reported. In the current study we identified individual differences in activity levels of zebrafish using a genetically heterogeneous population. Groups of zebrafish classified as high, medium, or low activity performers demonstrated consistent activity levels over a period of 7 days, and also in a subsequent open field task, suggesting stable individual differences as opposed to stochastic variation among subjects. We also uncovered a sex dependent relationship between behavioural measures. Female zebrafish in the high activity group preferred the top portion of the tank, whereas low activity females preferred the lower portion but males did not show such a relationship. The relationship between these two behaviours in females implies the potential existence of a behavioural syndrome persisting between contexts. Furthermore, females demonstrated a higher level of consistency in their behaviour as compared to males, and the behavioural differences were found to be independent of both body size and weight of the tested subjects. The identification of individual differences in activity levels in zebrafish will allow the investigation of underlying genetic and/or environmental underpinnings. PMID:24084583

  11. Production of Haploid Zebrafish Embryos by In Vitro Fertilization

    PubMed Central

    Kroeger, Paul T.; Poureetezadi, Shahram Jevin; McKee, Robert; Jou, Jonathan; Miceli, Rachel; Wingert, Rebecca A.

    2014-01-01

    The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity – a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages. PMID:25046024

  12. Inbred strains of zebrafish exhibit variation in growth performance and myostatin expression following fasting

    PubMed Central

    Meyer, Ben M.; Froehlich, Jacob M.; Galt, Nicholas J.; Biga, Peggy R.

    2012-01-01

    Although the zebrafish (Danio rerio) has been widely utilized as a model organism for several decades, there is little information available on physiological variation underlying genetic variation among the most commonly used inbred strains. This study evaluated growth performance using physiological and molecular markers of growth in response to fasting in six commonly used zebrafish strains [AB, TU, TL, SJA, WIK, and petstore (PET) zebrafish]. Fasting resulted in a standard decrease in whole blood glucose levels, typical vertebrate glucose metabolism pattern, in AB, PET, TL, and TU zebrafish strains. Alternatively, fasting did not affect glucose levels in SJA and WIK zebrafish strains. Similarly, fasting had no effect on myostatin mRNA levels in AB, PET, TU, and WIK zebrafish strains, but decreased myostatin-1 and -2 mRNA levels in SJA zebrafish. Consistent with previous work, fasting increased myostatin-2 mRNA levels in TL zebrafish. These data demonstrate that variation is present in growth performance between commonly used inbred strains of zebrafish. These data can help future research endeavors by highlighting the attributes of each strain with regard to growth performance so that the most fitting strain may be utilized. PMID:23047051

  13. A proteome map of the zebrafish (Danio rerio) lens reveals similarities between zebrafish and mammalian crystallin expression

    PubMed Central

    Hawke, Molly; LaCava, Carrie; Prince, Courtney J.; Bellanco, Nicholas R.; Corbin, Rebecca W.

    2008-01-01

    Purpose To characterize the crystallin content of the zebrafish lens using two-dimensional gel electrophoresis (2-DE). These data will facilitate future investigations of vertebrate lens development, function, and disease. Methods Adult zebrafish lens proteins were separated by 2-DE, and the resulting spots were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The relative proportion of each crystallin was quantified by image analysis, and phosphospecific staining was used to identify phosphorylated ?-crystallins. The proportion of each crystallin in the soluble and insoluble fraction of the lens was also determined by resolving these lens fractions separately by 2-DE. Results ?-, ?-, and ?-crystallins comprised 7.8, 36.0, and 47.2% of the zebrafish lens, respectively. While the ?-crystallin content of the zebrafish lens is less than the amounts found in the human lens, the ratio of ?A:?B crystallin is very similar. The phosphorylation pattern of zebrafish ?A-crystallins was also similar to that of humans. The most abundant ?-crystallins were the diverse ?Ms, comprising 30.5% of the lens. Intact zebrafish crystallins were generally more common in the soluble fraction with truncated versions more common in the insoluble fraction. Conclusions While the total ?- and ?-crystallin content of the zebrafish lens differs from that of humans, similarities in ?-crystallin ratios and modifications and a link between crystallin truncation and insolubility suggest that the zebrafish is a suitable model for the vertebrate lens. The proteome map provided here will be of value to future studies of lens development, function, and disease. PMID:18449354

  14. Evaluation of color preference in zebrafish for learning and memory.

    PubMed

    Avdesh, Avdesh; Martin-Iverson, Mathew T; Mondal, Alinda; Chen, Mengqi; Askraba, Sreten; Morgan, Newman; Lardelli, Michael; Groth, David M; Verdile, Giuseppe; Martins, Ralph N

    2012-01-01

    There is growing interest in using zebrafish (Danio rerio) as a model of neurodegenerative disorders such as Alzheimer's disease. A zebrafish model of tauopathies has recently been developed and characterized in terms of presence of the pathological hallmarks (i.e., neurofibrillary tangles and cell death). However, it is also necessary to validate these models for function by assessing learning and memory. The majority of tools to assess memory and learning in animal models involve visual stimuli, including color preference. The color preference of zebrafish has received little attention. To validate zebrafish as a model for color-associated-learning and memory, it is necessary to evaluate its natural preferences or any pre-existing biases towards specific colors. In the present study, we have used four different colors (red, yellow, green, and blue) to test natural color preferences of the zebrafish using two procedures: Place preference and T-maze. Results from both experiments indicate a strong aversion toward blue color relative to all other colors (red, yellow, and green) when tested in combinations. No preferences or biases were found among reds, yellows, and greens in the place preference procedure. However, red and green were equally preferred and both were preferred over yellow by zebrafish in the T-maze procedure. The results from the present study show a strong aversion towards blue color compared to red, green, and yellow, with yellow being less preferred relative to red and green. The findings from this study may underpin any further designing of color-based learning and memory paradigms or experiments involving aversion, anxiety, or fear in the zebrafish. PMID:22008261

  15. Zebrafish brain ventricle injection.

    PubMed

    Gutzman, Jennifer H; Sive, Hazel

    2009-01-01

    Proper brain ventricle formation during embryonic brain development is required for normal brain function. Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid. In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation. In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue. However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space. Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy. The brightfield and the fluorescent images are then processed and superimposed in Photoshop. This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image. Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages. We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3). PMID:19352312

  16. Absence of rapid eye movements during sleep in adult zebrafish.

    PubMed

    Árnason, B B; Þorsteinsson, H; Karlsson, K Æ

    2015-09-15

    Sleep is not a uniform phenomenon, but is organized in alternating, fundamentally different states, rapid eye movement sleep and non-rapid eye movement sleep. Zebrafish (Danio rerio) have recently emerged as an excellent model for sleep research. Zebrafish are well characterized in terms of development, neurobiology and genetics. Moreover, there are many experimental tools not easily applied in mammalian models that can be readily applied to zebrafish, making them a valuable additional animal model for sleep research. Sleep in zebrafish is defined behaviorally and exhibits the hallmarks of mammalian sleep (e.g. sleep homeostasis and pressure). To our knowledge no attempts have been made to discern if sleep in zebrafish entails alternations of REM-NREM sleep cycles which are critical for further development of the model. In the current experiment we quantify two key REM sleep components, rapid eye movements and respiratory rates, across sleep-wake cycles. We find no sleep-related rapid eye movements. During sleep respiratory rates, however, are reduced and become less regular, further establishing that the behavioral definition used truly captures a change in the fish's physiology. We thus fail to find evidence for REM-NREM sleep cycles in zebrafish but demonstrate a physiological change that occurs concomitantly with the previously defined behavioral state of sleep. We do not rule out that other phasic REM components (e.g. atonia, cardiac arrhythmias, myoclonic twitches or desynchronized EEG) are coherently expressed during sleep but we conclude that adult zebrafish do not have REM-sleep-related rapid eye movements. PMID:26003945

  17. Zebrafish Developmental Screening of the ToxCast? Phase I Chemical Library

    EPA Science Inventory

    Zebrafish (Danio rerio) is an emerging toxicity screening model for both human health and ecology. As part of the Computational Toxicology Research Program of the U.S. EPA, the toxicity of the 309 ToxCast? Phase I chemicals was assessed using a zebrafish screen for developmental ...

  18. Cell cultures derived from early zebrafish embryos differentiate in vitro into neurons and astrocytes

    Microsoft Academic Search

    Chandramallika Ghosh; Yi Liu; Chunguang Ma; Paul Collodi

    1997-01-01

    The zebrafish is a polular nonmammalian model for studies of neural development. We have derived cell cultures, initiated from blastula-stage zebrafish embryos, that differentiate in vitro into neurons and astrocytes. Cultures were initiated in basal nutrient medium supplemented with bovine insulin, trout serum, trout embryo extract and fetal bovine serum. After two weeks in culture the cells exhibited extensive neurite

  19. Movement disorder and neuromuscular change in zebrafish embryos after exposure to caffeine

    Microsoft Academic Search

    Yau-Hung Chen; Yi-Hui Huang; Chi-Chung Wen; Yun-Hsin Wang; Wei-Li Chen; Li-Chao Chen; Huey-Jen Tsay

    2008-01-01

    Though caffeine is broadly distributed in many plants and foods, little is known about the teratogenic effects of caffeine during early embryonic development. Here, we used zebrafish as a model to test toxicity and teratogenicity since they have transparent eggs, making the organogenesis of zebrafish embryos easier to observe. When the exposure doses of caffeine were less than 150 ppm (17.5,

  20. Wnt Signaling in Zebrafish Fin Regeneration: Chemical Biology Using GSK3b Inhibitors Courtney Curtis1

    E-print Network

    Zhou, Yaoqi

    Wnt Signaling in Zebrafish Fin Regeneration: Chemical Biology Using GSK3b Inhibitors Courtney) protein inhibitors activate Wnt signaling, including in bone growth models. Our study utilized a zebrafish regeneration experiments were conducted using various concentrations of GSK3b inhibitor compound for different

  1. Growth and Survivorship of Larval Zebrafish Danio rerio on Processed Diets

    Microsoft Academic Search

    Edward M. Goolish; Ken Okutake; Sheffield Lesure

    1999-01-01

    Within the past decade the zebrafish or zebra danio Danio rerio has become a major vertebrate model for the study of developmental biology, neurobiology, and molecular genetics. Most research universities now include large zebrafish colonies where considerable resources are expended for their feeding and maintenance. Larvae are particularly labor intensive because they are commonly fed a live diet such as

  2. Identification of Estrogen Target Genes during Zebrafish Embryonic Development through Transcriptomic Analysis

    EPA Science Inventory

    Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17ß-estradiol (E2) or vehicle from 3 hours to 4 days post...

  3. Analysis of steroid hormone effects on xenografted human NF1 tumor Schwann cells

    PubMed Central

    Li, Hua; Zhang, Xuelian; Fishbein, Lauren; Kweh, Frederick; Thompson, Martha Campbell; Perrin, George Q; Muir, David

    2010-01-01

    The neurofibroma, a common feature of neurofibromatosis type 1 (NF1), is a benign peripheral nerve sheath tumor that contains predominantly Schwann cells (SC). There are reports that neurofibroma growth may be affected by hormonal changes, particularly in puberty and pregnancy, suggesting an influence by steroid hormones. This study examined the effects of estrogen and progesterone on proliferation and apoptosis in a panel of NF1 tumor xenografts. SC-enriched cultures derived from three human NF1 tumor types [dermal neurofibroma, plexiform neurofibroma and malignant peripheral nerve sheath tumor (MPNST)] were xenografted in sciatic nerves of ovariectomized scid/Nf1?/+ mice. At the same time, mice were implanted with time-release pellets for systemic delivery of progesterone, estrogen or placebo. Proliferation and apoptosis by the xenografted SC were examined two months after implantation, by Ki67 immunolabeling and TUNEL. Estrogen was found to increase the growth of all three MPNST xenografts. Progesterone was associated with increased growth in two of the three MPNSTs, yet decreased growth of the other. Of the four dermal neurofibroma xenografts tested, estrogen caused a statistically significant growth increase in one and progesterone did in another. Of the four plexiform neurofibroma SC xenografts, estrogen and progesterone significantly decreased growth in one of the xenografts, but not the other three. No relationship of patient age or gender to steroid response was observed. These findings indicate that human NF1 Schwann cells derived from some tumors show increased proliferation or decreased apoptosis in response to particular steroid hormones in a mouse xenograft model. This suggests that antiestrogen or anti-progesterone therapies may be worth considering for specific NF1 neurofibromas and MPNSTs. PMID:20699653

  4. Improving In Vivo High-Resolution CT Imaging of the Tumour Vasculature in Xenograft Mouse Models through Reduction of Motion and Bone-Streak Artefacts

    PubMed Central

    Kersemans, Veerle; Kannan, Pavitra; Beech, John S.; Bates, Russell; Irving, Benjamin; Gilchrist, Stuart; Allen, Philip D.; Thompson, James; Kinchesh, Paul; Casteleyn, Christophe; Schnabel, Julia; Partridge, Mike; Muschel, Ruth J.; Smart, Sean C.

    2015-01-01

    Introduction Preclinical in vivo CT is commonly used to visualise vessels at a macroscopic scale. However, it is prone to many artefacts which can degrade the quality of CT images significantly. Although some artefacts can be partially corrected for during image processing, they are best avoided during acquisition. Here, a novel imaging cradle and tumour holder was designed to maximise CT resolution. This approach was used to improve preclinical in vivo imaging of the tumour vasculature. Procedures A custom built cradle containing a tumour holder was developed and fix-mounted to the CT system gantry to avoid artefacts arising from scanner vibrations and out-of-field sample positioning. The tumour holder separated the tumour from bones along the axis of rotation of the CT scanner to avoid bone-streaking. It also kept the tumour stationary and insensitive to respiratory motion. System performance was evaluated in terms of tumour immobilisation and reduction of motion and bone artefacts. Pre- and post-contrast CT followed by sequential DCE-MRI of the tumour vasculature in xenograft transplanted mice was performed to confirm vessel patency and demonstrate the multimodal capacity of the new cradle. Vessel characteristics such as diameter, and branching were quantified. Results Image artefacts originating from bones and out-of-field sample positioning were avoided whilst those resulting from motions were reduced significantly, thereby maximising the resolution that can be achieved with CT imaging in vivo. Tumour vessels ? 77 ?m could be resolved and blood flow to the tumour remained functional. The diameter of each tumour vessel was determined and plotted as histograms and vessel branching maps were created. Multimodal imaging using this cradle assembly was preserved and demonstrated. Conclusions The presented imaging workflow minimised image artefacts arising from scanner induced vibrations, respiratory motion and radiopaque structures and enabled in vivo CT imaging and quantitative analysis of the tumour vasculature at higher resolution than was possible before. Moreover, it can be applied in a multimodal setting, therefore combining anatomical and dynamic information. PMID:26046526

  5. Global protein expression profiling of zebrafish organs based on in vivo incorporation of stable isotopes.

    PubMed

    Nolte, Hendrik; Konzer, Anne; Ruhs, Aaron; Jungblut, Benno; Braun, Thomas; Krüger, Marcus

    2014-04-01

    The zebrafish has become a widely used model organism employed for developmental studies, live cell imaging, and genetic screens. High-resolution transcriptional profiles of different developmental and adult stages of the fish and of its various organs were generated, which are readily accessible via the ZFIN database. In contrast, quantitative proteomic studies of zebrafish organs are still in their infancy. Here, we used the SILAC (stable isotope labeling by amino acids in cell culture) zebrafish as a "spike-in" reference to generate a protein atlas of nine organs including gills, brain, heart, muscle, liver, spleen, skin, swim bladder, and testis. Single-shot 4 h LC gradients coupled to a Quadrupole-Orbitrap (QExactive) instrument allowed identification of over 5000 proteins in less than 5 days, of which more than 70% were quantified in triplicate. Identified proteins were subjected to BLAST searches and Gene Ontology classification to improve annotation of zebrafish proteins and obtain insights into potential functions. Comparison to mouse tissue proteome data sets revealed differences and similarities in the protein composition of zebrafish versus mouse organs. We reason that the data set will be helpful for the proteomic characterization of zebrafish organs and identification of tissue-specific proteins that might serve as biomarkers. Our approach provides a complementary view into the biochemistry of zebrafish models and will assist large-scale protein quantification in zebrafish disease models. PMID:24611545

  6. Autophagy Induction Is a Tor- and Tp53-Independent Cell Survival Response in a Zebrafish Model of Disrupted Ribosome Biogenesis

    PubMed Central

    Boglev, Yeliz; Badrock, Andrew P.; Trotter, Andrew J.; Du, Qian; Richardson, Elsbeth J.; Parslow, Adam C.; Markmiller, Sebastian J.; Hall, Nathan E.; de Jong-Curtain, Tanya A.; Ng, Annie Y.; Verkade, Heather; Ober, Elke A.; Field, Holly A.; Shin, Donghun; Shin, Chong H.; Hannan, Katherine M.; Hannan, Ross D.; Pearson, Richard B.; Kim, Seok-Hyung; Ess, Kevin C.; Lieschke, Graham J.; Stainier, Didier Y. R.; Heath, Joan K.

    2013-01-01

    Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (ttis450), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In ttis450, the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in ttis450 larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in ttis450 larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death. PMID:23408911

  7. Mutations in zebrafish lrp2 result in adult-onset ocular pathogenesis that models myopia and other risk factors for glaucoma.

    PubMed

    Veth, Kerry N; Willer, Jason R; Collery, Ross F; Gray, Matthew P; Willer, Gregory B; Wagner, Daniel S; Mullins, Mary C; Udvadia, Ava J; Smith, Richard S; John, Simon W M; Gregg, Ronald G; Link, Brian A

    2011-02-01

    The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease. PMID:21379331

  8. Automatic zebrafish heartbeat detection and analysis for zebrafish embryos.

    PubMed

    Pylatiuk, Christian; Sanchez, Daniela; Mikut, Ralf; Alshut, Rüdiger; Reischl, Markus; Hirth, Sofia; Rottbauer, Wolfgang; Just, Steffen

    2014-08-01

    A fully automatic detection and analysis method of heartbeats in videos of nonfixed and nonanesthetized zebrafish embryos is presented. This method reduces the manual workload and time needed for preparation and imaging of the zebrafish embryos, as well as for evaluating heartbeat parameters such as frequency, beat-to-beat intervals, and arrhythmicity. The method is validated by a comparison of the results from automatic and manual detection of the heart rates of wild-type zebrafish embryos 36-120?h postfertilization and of embryonic hearts with bradycardia and pauses in the cardiac contraction. PMID:25003305

  9. High Cholesterol Diet Induces IL-1? Expression in Adult but Not Larval Zebrafish

    PubMed Central

    Jang, Man-Young; Na, Yirang; Ko, Youngho; Choi, Jae-Hoon; Seok, Seung Hyeok

    2013-01-01

    Recently, it has been demonstrated that high cholesterol diet induced hypercholesterolemia and vascular lipid oxidation and accumulation in zebrafish larvae, suggesting that zebrafish is a new promising atherosclerosis model in addition to mouse models. However, up to date, there was no report regarding inflammatory cytokine expression during the lipid accumulation in zebrafish larva and adult fish. In this study, we first demonstrated the expression levels of IL-1? and TNF-? in high cholesterol diet (HCD)-fed zebrafish larvae, and found that although HCD induced vascular lipid accumulation, the cytokine expressions in the larvae were not changed by HCD. Furthermore, there was no significant difference in leukocyte accumulation in vessels between control and HCD fed group. But prolonged HCD induced IL-1? expression in spleen and liver compared to those of control zebrafish, and produced very early stage of fatty streak lesion in dorsal aorta of 19 week HCD-fed zebrafish. These results indicate that HCD induced hypercholesterolemia and atherosclerotic changes in zebrafish are very early stage, and suggest the necessity of the generation of mutant zebrafish having a disruption in a lipid metabolism-related gene leading to severe hypercholesterolemia and advanced atherosclerosis. PMID:23825600

  10. Novel C-17-heteroaryl steroidal CYP17 inhibitors/antiandrogens: synthesis, in vitro biological activity, pharmacokinetics, and antitumor activity in the LAPC4 human prostate cancer xenograft model.

    PubMed

    Handratta, Venkatesh D; Vasaitis, Tadas S; Njar, Vincent C O; Gediya, Lalji K; Kataria, Ritesh; Chopra, Pankaj; Newman, Donnell; Farquhar, Rena; Guo, Zhiyong; Qiu, Yun; Brodie, Angela M H

    2005-04-21

    New chemical entities, steroidal C-17 benzoazoles (5, 6, 9 and 10) and pyrazines (14 and 15) were rationally designed and synthesized. The key reaction for synthesis of the benzoazoles involved the nucleophilic vinylic "addition-elimination" substitution reaction of 3beta-acetoxy-17-chloro-16-formylandrosta-5,16-diene (2) and benzoazole nucleophiles, while that for synthesis of pyrazines involved palladium-catalyzed cross-coupling reaction of 17-iodoandrosta-5,16-dien-3beta-ol (13) with tributylstannyl diazines. Some of the compounds were shown to be potent inhibitors of human CYP17 enzyme as well as potent antagonist of both wild type and mutant androgen receptors (AR). The most potent CYP17 inhibitors were 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (5, code named VN/124-1), 3beta-hydroxy-17-(5(1)-pyrimidyl)androsta-5,16-diene (15) and 17-(1H-benzimidazole-1-yl)androsta-4,16-dien-3-one (6), with IC(50) values of 300, 500 and 915 nM, respectively. Compounds 5, 6, 14 and 15 were effective at preventing binding of (3)H-R1881 (methyltrienolone, a stable synthetic androgen) to both the mutant LNCaP AR and the wild-type AR, but with a 2.2- to 5-fold higher binding efficiency to the latter. Compounds 5 and 6 were also shown to be potent pure AR antagonists. The cell growth studies showed that 5 and 6 inhibit the growth of DHT-stimulated LNCaP and LAPC4 prostate cancer cells with IC(50) values in the low micromolar range (i.e., <10 microM). Their inhibitory potencies were comparable to that of casodex but remarkably superior to that of flutamide. The pharmacokinetics of compounds 5 and 6 in mice were investigated. Following s.c. administration of 50 mg/kg of 5 and 6, peak plasma levels of 16.82 and 5.15 ng/mL, respectively, occurred after 30 to 60 min, both compounds were cleared rapidly from plasma (terminal half-lives of 44.17 and 39.93 min, respectively), and neither was detectable at 8 h. Remarkably, compound 5 was rapidly converted into a metabolite tentatively identified as 17-(1H-benzimidazol-1-yl)androsta-3-one. When tested in vivo, 5 proved to be very effective at inhibiting the growth of androgen-dependent LAPC4 human prostate tumor xenograft, while 6 was ineffective. Compound 5 (50 mg/kg/twice daily) resulted in a 93.8% reduction (P = 0.00065) in the mean final tumor volume compared with controls, and it was also significantly more effective than castration. To our knowledge, this is the first example of an antihormonal agent (an inhibitor of androgen synthesis (CYP17 inhibitor)/antiandrogen) that is significantly more effective than castration in suppression of androgen-dependent prostate tumor growth. In view of these impressive anticancer properties, compound 5 is a strong candidate for development for the treatment of human prostate cancer. PMID:15828836

  11. Plumbagin, a medicinal plant (Plumbago zeylanica) - derived 1,4-naphthoquinone, inhibits growth and metastasis of human prostate cancer PC-3M-luciferase cells in an orthotopic xenograft mouse model

    PubMed Central

    Hafeez, Bilal Bin; Zhong, Weixiong; Fischer, Joseph W.; Mustafa, Ala; Shi, Xudong Daniel; Meske, Louise; Hong, Hao; Cai, Weibo; Havighurst, Thomas; Kim, KyungMann; Verma, Ajit. K

    2012-01-01

    We present here first time that Plumbagin (PL), a medicinal plant-derived 1,4-naphthoquinone, inhibits the growth and metastasis of prostate cancer (PCa) in an orthotopic xenograft mouse model. In this study, human PCa PC-3M-luciferase cells (2X106) were injected into the prostate of athymic nude mice. Three days post cell implantation, mice were treated with PL (2 mg/kg body wt. i.p five days in a week) for 8 weeks. Growth and metastasis of PC-3M-luciferase cells was examined weekly by bioluminescence imaging of live mice. PL-treatment significantly (p=0.0008) inhibited the growth of orthotopic xenograft tumors. PCa metastasis into the liver, lungs and lymph nodes was determined by bioluminescence imaging and histopathology. Results demonstrated a significant inhibition of metastasis into liver (p=0.037), but inhibition of metastasis into the lungs (p=0.60) and liver (p=0.27) was not observed to be significant. These results were further confirmed by histopathology of these organs. Results of histopathology demonstrated a significant inhibition of metastasis into lymph nodes (p=0.034) and lungs (p=0.028), and a trend to significance in liver (p=0.075). None of the mice in the PL-treatment group showed PCa metastasis into the liver, but these mice had small metastasis foci into the lymph nodes and lungs. However, control mice had large metastatic foci into the lymph nodes, lungs, and liver. PL-caused inhibition of the growth and metastasis of PC-3M cells accompanies inhibition of the expression of: 1) PKC?, pStat3Tyr705, and pStat3Ser727, 2) Stat3 downstream target genes (survivin and BclxL), 3) proliferative markers Ki-67 and PCNA, 4) metastatic marker MMP9, MMP2, and uPA, and 5) angiogenesis markers CD31 and VEGF. Taken together, these results suggest that PL inhibits tumor growth and metastasis of human PCa PC3-M-luciferase cells, which could be used as a therapeutic agent for the prevention and treatment of human PCa. PL: Plumbagin, PCa: Prostate cancer. PMID:23273564

  12. Interplay of choline metabolites and genes in patient-derived breast cancer xenografts

    PubMed Central

    2014-01-01

    Introduction Dysregulated choline metabolism is a well-known feature of breast cancer, but the underlying mechanisms are not fully understood. In this study, the metabolomic and transcriptomic characteristics of a large panel of human breast cancer xenograft models were mapped, with focus on choline metabolism. Methods Tumor specimens from 34 patient-derived xenograft models were collected and divided in two. One part was examined using high-resolution magic angle spinning (HR-MAS) MR spectroscopy while another part was analyzed using gene expression microarrays. Expression data of genes encoding proteins in the choline metabolism pathway were analyzed and correlated to the levels of choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) using Pearson’s correlation analysis. For comparison purposes, metabolic and gene expression data were collected from human breast tumors belonging to corresponding molecular subgroups. Results Most of the xenograft models were classified as basal-like (N?=?19) or luminal B (N?=?7). These two subgroups showed significantly different choline metabolic and gene expression profiles. The luminal B xenografts were characterized by a high PCho/GPC ratio while the basal-like xenografts were characterized by highly variable PCho/GPC ratio. Also, Cho, PCho and GPC levels were correlated to expression of several genes encoding proteins in the choline metabolism pathway, including choline kinase alpha (CHKA) and glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). These characteristics were similar to those found in human tumor samples. Conclusion The higher PCho/GPC ratio found in luminal B compared with most basal-like breast cancer xenograft models and human tissue samples do not correspond to results observed from in vitro studies. It is likely that microenvironmental factors play a role in the in vivo regulation of choline metabolism. Cho, PCho and GPC were correlated to different choline pathway-encoding genes in luminal B compared with basal-like xenografts, suggesting that regulation of choline metabolism may vary between different breast cancer subgroups. The concordance between the metabolic and gene expression profiles from xenograft models with breast cancer tissue samples from patients indicates that these xenografts are representative models of human breast cancer and represent relevant models to study tumor metabolism in vivo. PMID:24447408

  13. Mechanisms of Ca2+ Handling in Zebrafish Ventricular Myocytes

    PubMed Central

    Bovo, Elisa; Dvornikov, Alexey V.; Mazurek, Stefan R.; de Tombe, Pieter P.; Zima, Aleksey V.

    2014-01-01

    Zebrafish serves as a promising transgenic animal model that can be used to study cardiac Ca2+ regulation. However, mechanisms of sarcoplasmic reticulum (SR) Ca2+ handling in zebrafish heart have not been systematically explored. We found that in zebrafish ventricular myocytes the action potential (AP)-induced Ca2+ transient is mainly (80%) mediated by Ca2+ influx via L-type Ca2+ channels (LTCC) and only 20% by Ca2+ released from the SR. This small contribution of the SR to the Ca2+ transient was not the result of depleted SR Ca2+ load. We found that the ryanodine receptor (RyR) expression level in zebrafish myocytes was 78% lower compared to rabbit myocytes. In permeabilized myocytes, increasing cytosolic [Ca2+] from 100 to 350 nM did not trigger SR Ca2+ release. However, an application of a low dose of caffeine activated Ca2+ sparks. These results show that the zebrafish cardiac RyR is not sensitive to the mechanism of Ca2+-induced Ca2+ release. Activation of protein kinase A by forskolin increased phosphorylation of the RyR in zebrafish myocardium. In half of the studied cells, an increased Ca2+ transient by forskolin was entirely mediated by augmentation of LTCC current. In the remaining myocytes, the forskolin action was associated with an increase of both LTCC and SR Ca2+ release. These results indicate that the mechanism of excitation-contraction coupling in zebrafish myocytes differs from the mammalian one mainly because of the small contribution of SR Ca2+ release to the Ca2+ transient. This difference is due to a low sensitivity of RyRs to cytosolic [Ca2+]. PMID:23821298

  14. High fidelity patient-derived xenografts for accelerating prostate cancer discovery and drug development.

    PubMed

    Lin, Dong; Wyatt, Alexander W; Xue, Hui; Wang, Yuwei; Dong, Xin; Haegert, Anne; Wu, Rebecca; Brahmbhatt, Sonal; Mo, Fan; Jong, Lina; Bell, Robert H; Anderson, Shawn; Hurtado-Coll, Antonio; Fazli, Ladan; Sharma, Manju; Beltran, Himisha; Rubin, Mark; Cox, Michael; Gout, Peter W; Morris, James; Goldenberg, Larry; Volik, Stanislav V; Gleave, Martin E; Collins, Colin C; Wang, Yuzhuo

    2014-02-15

    Standardized and reproducible preclinical models that recapitulate the dynamics of prostate cancer are urgently needed. We established a bank of transplantable patient-derived prostate cancer xenografts that capture the biologic and molecular heterogeneity currently confounding prognostication and therapy development. Xenografts preserved the histopathology, genome architecture, and global gene expression of donor tumors. Moreover, their aggressiveness matched patient observations, and their response to androgen withdrawal correlated with tumor subtype. The panel includes the first xenografts generated from needle biopsy tissue obtained at diagnosis. This advance was exploited to generate independent xenografts from different sites of a primary site, enabling functional dissection of tumor heterogeneity. Prolonged exposure of adenocarcinoma xenografts to androgen withdrawal led to castration-resistant prostate cancer, including the first-in-field model of complete transdifferentiation into lethal neuroendocrine prostate cancer. Further analysis of this model supports the hypothesis that neuroendocrine prostate cancer can evolve directly from adenocarcinoma via an adaptive response and yielded a set of genes potentially involved in neuroendocrine transdifferentiation. We predict that these next-generation models will be transformative for advancing mechanistic understanding of disease progression, response to therapy, and personalized oncology. PMID:24356420

  15. Metabolism of ibuprofen in zebrafish larvae.

    PubMed

    Jones, Huw S; Trollope, Henry T; Hutchinson, Thomas H; Panter, Grace H; Chipman, J Kevin

    2012-11-01

    The application of zebrafish (Danio rerio) larvae to drug discovery assays and toxicity testing, and the occurrence of pharmaceuticals in the environment, has resulted in a need to understand the extent of the metabolic capabilities in the early life stages of this species. The aims of this study were to determine if zebrafish larvae absorbed, metabolized and excreted the model pharmaceutical, ibuprofen. Zebrafish larvae (72 h post fertilization) were exposed to ibuprofen (100 µg/L), (14)C-ibuprofen (100 µg/L) or a solvent control (ethanol) for ? 24 h. Water samples and larval extracts were assessed for metabolites of ibuprofen using liquid chromatography mass spectrometry (LC-MS-MS). Fractions from the separation of the samples treated with (14)C-ibuprofen were collected after chromatography and analysed for (14)C content by scintillation counting. Assessment of larval extracts and water samples by LC-MS-MS at 24 h resulted in the identification of hydroxy-ibuprofen in both water samples and larval extracts (8.2 and 0.08% of the total detected (14)C, respectively). A second putative hydroxy-ibuprofen moiety was also observed in water samples at trace levels, and a third minor unknown metabolite was detected in larval extracts only by scintillation counting (0.02% of the total (14)C detected). This study provides evidence that zebrafish larvae can metabolize and excrete ibuprofen in a manner known to be cytochrome P450-dependent in mammals, and the similarity to the mammalian pathway supports the use of this system as a surrogate in toxicity and efficacy screening. PMID:22594345

  16. Functional effects of spinocerebellar ataxia type 13 mutations are conserved in zebrafish Kv3.3 channels

    Microsoft Academic Search

    Allan F. Mock; Jessica L. Richardson; Jui-Yi Hsieh; Gina Rinetti; Diane M. Papazian

    2010-01-01

    BACKGROUND: The zebrafish has been suggested as a model system for studying human diseases that affect nervous system function and motor output. However, few of the ion channels that control neuronal activity in zebrafish have been characterized. Here, we have identified zebrafish orthologs of voltage-dependent Kv3 (KCNC) K+ channels. Kv3 channels have specialized gating properties that facilitate high-frequency, repetitive firing

  17. In vivo imaging of zebrafish embryogenesis

    PubMed Central

    Keller, Philipp J.

    2013-01-01

    The zebrafish Danio rerio has emerged as a powerful vertebrate model system that lends itself particularly well to quantitative investigations with live imaging approaches, owing to its exceptionally high optical clarity in embryonic and larval stages. Recent advances in light microscopy technology enable comprehensive analyses of cellular dynamics during zebrafish embryonic development, systematic mapping of gene expression dynamics, quantitative reconstruction of mutant phenotypes and the system-level biophysical study of morphogenesis. Despite these technical breakthroughs, it remains challenging to design and implement experiments for in vivo long-term imaging at high spatio-temporal resolution. This article discusses the fundamental challenges in zebrafish long-term live imaging, provides experimental protocols and highlights key prop1erties and capabilities of advanced fluorescence microscopes. The article focuses in particular on experimental assays based on light sheet-based fluorescence microscopy, an emerging imaging technology that achieves exceptionally high imaging speeds and excellent signal-to-noise ratios, while minimizing light-induced damage to the specimen. This unique combination of capabilities makes light sheet microscopy an indispensable tool for the in vivo long-term imaging of large developing organisms. PMID:23523701

  18. Comparative studies of Toll-like receptor signalling using zebrafish.

    PubMed

    Kanwal, Zakia; Wiegertjes, Geert F; Veneman, Wouter J; Meijer, Annemarie H; Spaink, Herman P

    2014-09-01

    Zebrafish model systems for infectious disease are increasingly used for the functional analysis of molecular pattern recognition processes. These studies benefit from the high conservation level of all innate immune factors in vertebrates. Zebrafish studies are strategically well positioned for this because of the ease of comparisons with studies in other fish species of which the immune system also has been intensively studied, but that are currently still less amendable to detailed genetic or microscopic studies. In this paper we focus on Toll-like receptor (TLR) signalling factors, which currently are the best characterized in mammalian systems. We review the knowledge on TLR signalling in the context of recent advances in zebrafish studies and discuss possibilities for future approaches that can complement studies in cell cultures and rodent models. A focus in these comparisons is the role of negative control mechanisms in immune responses that appear very important in a whole organism to keep adverse systemic responses in check. We also pay much attention to comparisons with studies in common carp that is highly related to zebrafish and that because of its large body mass can complement immune studies in zebrafish. PMID:24560981

  19. In vivo alternative testing with zebrafish in ecotoxicology

    PubMed Central

    Seok, Seung-Hyeok; Baek, Min-Won; Lee, Hui-Young; Kim, Dong-Jae; Na, Yi-Rang; Noh, Kyoung-Jin; Park, Sung-Hoon; Lee, Hyun-Kyoung; Lee, Byoung-Hee

    2008-01-01

    Although rodents have previously been used in ecotoxicological studies, they are expensive, time-consuming, and are limited by strict legal restrictions. The present study used a zebrafish (Danio rerio) model and generated data that was useful for extrapolating toxicant effects in this system to that of humans. Here we treated embryos of the naive-type as well as a transiently transfected zebrafish liver cell line carrying a plasmid (phAhRE-EGFP), for comparing toxicity levels with the well-known aryl hydrocarbon receptor (AhR)-binding toxicants: 3,3',4,4',5-pentachlorobiphenyl (PCB126), 2,3,7,8-tetrachlorodibenzo-p-dioxin, and 3-methylcholanthrene. These toxicants induced a concentration-dependent increase in morphological disruption, indicating toxicity at early life-stages. The transient transgenic zebrafish liver cell line was sensitive enough to these toxicants to express the CYP1A1 regulated enhanced green fluorescent protein. The findings of this study demonstrated that the zebrafish in vivo model might allow for extremely rapid and reproducible toxicological profiling of early life-stage embryo development. We have also shown that the transient transgenic zebrafish liver cell line can be used for research on AhR mechanism studies. PMID:19043309

  20. Discovery of Highly Potent Tyrosinase Inhibitor, T1, with Significant Anti-Melanogenesis Ability by zebrafish in vivo Assay and Computational Molecular Modeling

    NASA Astrophysics Data System (ADS)

    Chen, Wang-Chuan; Tseng, Tien-Sheng; Hsiao, Nai-Wan; Lin, Yun-Lian; Wen, Zhi-Hong; Tsai, Chin-Chuan; Lee, Yu-Ching; Lin, Hui-Hsiung; Tsai, Keng-Chang

    2015-01-01

    Tyrosinase is involved in melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders that can be treated with depigmenting agents. A natural product T1, bis(4-hydroxybenzyl)sulfide, isolated from the Chinese herbal plant, Gastrodia elata, is a strong competitive inhibitor against mushroom tyrosinase (IC50 = 0.53 ?M, Ki = 58 +/- 6 nM), outperforms than kojic acid. The cell viability and melanin quantification assay demonstrate that 50 ?M of T1 apparently attenuates 20% melanin content of human normal melanocytes without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that T1 effectively reduces melanogenesis with no adverse side effects. The acute oral toxicity study evidently confirms that T1 molecule is free of discernable cytotoxicity in mice. Furthermore, the molecular modeling demonstrates that the sulfur atom of T1 coordinating with the copper ions in the active site of tyrosinase is essential for mushroom tyrosinase inhibition and the ability of diminishing the human melanin synthesis. These results evident that T1 isolated from Gastrodia elata is a promising candidate in developing pharmacological and cosmetic agents of great potency in skin-whitening.

  1. Discovery of highly potent tyrosinase inhibitor, T1, with significant anti-melanogenesis ability by zebrafish in vivo assay and computational molecular modeling.

    PubMed

    Chen, Wang-Chuan; Tseng, Tien-Sheng; Hsiao, Nai-Wan; Lin, Yun-Lian; Wen, Zhi-Hong; Tsai, Chin-Chuan; Lee, Yu-Ching; Lin, Hui-Hsiung; Tsai, Keng-Chang

    2015-01-01

    Tyrosinase is involved in melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders that can be treated with depigmenting agents. A natural product T1, bis(4-hydroxybenzyl)sulfide, isolated from the Chinese herbal plant, Gastrodia elata, is a strong competitive inhibitor against mushroom tyrosinase (IC50 = 0.53??M, Ki = 58 ± 6?nM), outperforms than kojic acid. The cell viability and melanin quantification assay demonstrate that 50??M of T1 apparently attenuates 20% melanin content of human normal melanocytes without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that T1 effectively reduces melanogenesis with no adverse side effects. The acute oral toxicity study evidently confirms that T1 molecule is free of discernable cytotoxicity in mice. Furthermore, the molecular modeling demonstrates that the sulfur atom of T1 coordinating with the copper ions in the active site of tyrosinase is essential for mushroom tyrosinase inhibition and the ability of diminishing the human melanin synthesis. These results evident that T1 isolated from Gastrodia elata is a promising candidate in developing pharmacological and cosmetic agents of great potency in skin-whitening. PMID:25613357

  2. The zebrafish as a tool to identify novel therapies for human cardiovascular disease

    PubMed Central

    Asnani, Aarti; Peterson, Randall T.

    2014-01-01

    Over the past decade, the zebrafish has become an increasingly popular animal model for the study of human cardiovascular disease. Because zebrafish embryos are transparent and their genetic manipulation is straightforward, the zebrafish has been used to recapitulate a number of cardiovascular disease processes ranging from congenital heart defects to arrhythmia to cardiomyopathy. The use of fluorescent reporters has been essential to identify two discrete phases of cardiomyocyte differentiation necessary for normal cardiac development in the zebrafish. These phases are analogous to the differentiation of the two progenitor heart cell populations in mammals, termed the first and second heart fields. The small size of zebrafish embryos has enabled high-throughput chemical screening to identify small-molecule suppressors of fundamental pathways in vasculogenesis, such as the BMP axis, as well as of common vascular defects, such as aortic coarctation. The optical clarity of zebrafish has facilitated studies of valvulogenesis as well as detailed electrophysiological mapping to characterize the early cardiac conduction system. One unique aspect of zebrafish larvae is their ability to oxygenate through diffusion alone, permitting the study of mutations that cause severe cardiomyopathy phenotypes such as silent heart and pickwickm171, which mimic titin mutations observed in human dilated cardiomyopathy. Above all, the regenerative capacity of zebrafish presents a particularly exciting opportunity to discover new therapies for cardiac injury, including scar formation following myocardial infarction. This Review will summarize the current state of the field and describe future directions to advance our understanding of human cardiovascular disease. PMID:24973746

  3. Characterization of the Z lineage Major histocompatability complex class I genes in zebrafish

    PubMed Central

    Dirscherl, Hayley

    2015-01-01

    Zebrafish (Danio rerio) are a valuable model for studying immunity, infection, and hematopoietic disease and have recently been employed for transplantation assays. However, the lack of syngeneic zebrafish creates challenges with identifying immune-matched individuals. The MHC class I genes, which mediate allogeneic recognition in mammals, have been grouped into three broad lineages in zebrafish: the classical U genes on chromosome 19, the Z genes which have been reported to map to chromosome 1, and the L genes that map to multiple loci. Transplantations between individual zebrafish that are matched at the U locus fail to consistently engraft suggesting that additional loci contribute to allogeneic recognition. Although two full-length zebrafish Z transcripts have been described, the genomic organization and diversity of these genes have not been reported. Herein we define ten Z genes on chromosomes 1 and 3 and on an unplaced genomic scaffold. We report that neither of the Z transcripts previously described match the current genome assembly and classify these transcripts as additional gene loci. We characterize full-length transcripts for 9 of these 12 genes. We demonstrate a high level of expression variation of the Z genes between individual zebrafish suggestive of haplotypic variation. We report low level sequence variation for individual Z genes between individual zebrafish reflecting a possible nonclassical function, although these molecules may still contribute to allogeneic recognition. Finally, we present a gene nomenclature system for the Z genes consistent with MHC nomenclature in other species and with the zebrafish gene nomenclature guidelines. PMID:24287892

  4. Technical brief: Constant intense light exposure to lesion and initiate regeneration in normally pigmented zebrafish

    PubMed Central

    Rajaram, Kamya; Summerbell, Emily R.

    2014-01-01

    Zebrafish are capable of robust and spontaneous regeneration of injured retina. Constant intense light exposure to adult albino zebrafish specifically causes apoptosis of rod and cone photoreceptor cells and is an excellent model to study the molecular mechanisms underlying photoreceptor regeneration. However, this paradigm has only been applied to lesion zebrafish of the nonpigmented albino genetic background, which precludes the use of numerous transgenic reporter lines that are widely used to study regeneration. Here, we explored the effectiveness of constant intense light exposure in causing photoreceptor apoptosis and stimulating regeneration in normally pigmented zebrafish retinas. We show that constant intense light exposure causes widespread photoreceptor damage in the dorsal-central retinas of pigmented zebrafish. Photoreceptor loss triggers dedifferentiation and proliferation of Müller glia as well as progenitor cell proliferation. We also demonstrate that the timeline of regeneration response is comparable between the albino and the pigmented retinas. PMID:25324680

  5. Anthocyanin Induces Apoptosis of DU-145 Cells In Vitro and Inhibits Xenograft Growth of Prostate Cancer

    PubMed Central

    Ha, U-Syn; Bae, Woong Jin; Kim, Su Jin; Yoon, Byung Il; Hong, Sung Hoo; Lee, Ji Youl; Hwang, Tae-Kon; Hwang, Sung Yeoun; Wang, Zhiping

    2015-01-01

    Purpose To investigate the effects of anthocyanins extracted from black soybean, which have antioxidant activity, on apoptosis in vitro (in hormone refractory prostate cancer cells) and on tumor growth in vivo (in athymic nude mouse xenograft model). Materials and Methods The growth and viability of DU-145 cells treated with anthocyanins were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis was assessed by DNA laddering. Immunoblotting was conducted to evaluate differences in the expressions of p53, Bax, Bcl, androgen receptor (AR), and prostate specific antigen (PSA). To study the inhibitory effects of anthocyanins on tumor growth in vivo, DU-145 tumor xenografts were established in athymic nude mice. The anthocyanin group was treated with daily oral anthocyanin (8 mg/kg) for 14 weeks. After 2 weeks of treatment, DU-145 cells (2×106) were inoculated subcutaneously into the right flank to establish tumor xenografts. Tumor dimensions were measured twice a week using calipers and volumes were calculated. Results Anthocyanin treatment of DU-145 cells resulted in 1) significant increase in apoptosis in a dose-dependent manner, 2) significant decrease in p53 and Bcl-2 expressions (with increased Bax expression), and 3) significant decrease in PSA and AR expressions. In the xenograft model, anthocyanin treatment significantly inhibit tumor growth. Conclusion This study suggests that anthocyanins from black soybean inhibit the progression of prostate cancer in vitro and in a xenograft model. PMID:25510742

  6. Atlas of Cellular Dynamics during Zebrafish Adult Kidney Regeneration.

    PubMed

    McCampbell, Kristen K; Springer, Kristin N; Wingert, Rebecca A

    2015-01-01

    The zebrafish is a useful animal model to study the signaling pathways that orchestrate kidney regeneration, as its renal nephrons are simple, yet they maintain the biological complexity inherent to that of higher vertebrate organisms including mammals. Recent studies have suggested that administration of the aminoglycoside antibiotic gentamicin in zebrafish mimics human acute kidney injury (AKI) through the induction of nephron damage, but the timing and details of critical phenotypic events associated with the regeneration process, particularly in existing nephrons, have not been characterized. Here, we mapped the temporal progression of cellular and molecular changes that occur during renal epithelial regeneration of the proximal tubule in the adult zebrafish using a platform of histological and expression analysis techniques. This work establishes the timing of renal cell death after gentamicin injury, identifies proliferative compartments within the kidney, and documents gene expression changes associated with the regenerative response of proliferating cells. These data provide an important descriptive atlas that documents the series of events that ensue after damage in the zebrafish kidney, thus availing a valuable resource for the scientific community that can facilitate the implementation of zebrafish research to delineate the mechanisms that control renal regeneration. PMID:26089919

  7. Atlas of Cellular Dynamics during Zebrafish Adult Kidney Regeneration

    PubMed Central

    McCampbell, Kristen K.; Springer, Kristin N.; Wingert, Rebecca A.

    2015-01-01

    The zebrafish is a useful animal model to study the signaling pathways that orchestrate kidney regeneration, as its renal nephrons are simple, yet they maintain the biological complexity inherent to that of higher vertebrate organisms including mammals. Recent studies have suggested that administration of the aminoglycoside antibiotic gentamicin in zebrafish mimics human acute kidney injury (AKI) through the induction of nephron damage, but the timing and details of critical phenotypic events associated with the regeneration process, particularly in existing nephrons, have not been characterized. Here, we mapped the temporal progression of cellular and molecular changes that occur during renal epithelial regeneration of the proximal tubule in the adult zebrafish using a platform of histological and expression analysis techniques. This work establishes the timing of renal cell death after gentamicin injury, identifies proliferative compartments within the kidney, and documents gene expression changes associated with the regenerative response of proliferating cells. These data provide an important descriptive atlas that documents the series of events that ensue after damage in the zebrafish kidney, thus availing a valuable resource for the scientific community that can facilitate the implementation of zebrafish research to delineate the mechanisms that control renal regeneration. PMID:26089919

  8. Analyzing craniofacial morphogenesis in zebrafish using 4D confocal microscopy.

    PubMed

    McGurk, Patrick D; Lovely, C Ben; Eberhart, Johann K

    2014-01-01

    Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos. PMID:24514435

  9. Assessing epilepsy-related behavioral phenotypes in adult zebrafish Daniel Desmond, Evan Kyzar, Siddharth Gaikwad, Jeremy Green,

    E-print Network

    Kalueff, Allan V.

    1 Assessing epilepsy-related behavioral phenotypes in adult zebrafish Daniel Desmond, Evan Kyzar, zebrafish have been presented as a novel and valuable tool for modeling complex human diseases. Epilepsy pathogenesis requires novel paradigms and model organism for translational experimental epilepsy research

  10. Altered Behavioral Performance and Live Imaging of Circuit-Specific Neural Deficiencies in a Zebrafish Model for Psychomotor Retardation

    PubMed Central

    Lerer-Goldshtein, Tali; Vatine, Gad David; Appelbaum, Lior

    2014-01-01

    The mechanisms and treatment of psychomotor retardation, which includes motor and cognitive impairment, are indefinite. The Allan-Herndon-Dudley syndrome (AHDS) is an X-linked psychomotor retardation characterized by delayed development, severe intellectual disability, muscle hypotonia, and spastic paraplegia, in combination with disturbed thyroid hormone (TH) parameters. AHDS has been associated with mutations in the monocarboxylate transporter 8 (mct8/slc16a2) gene, which is a TH transporter. In order to determine the pathophysiological mechanisms of AHDS, MCT8 knockout mice were intensively studied. Although these mice faithfully replicated the abnormal serum TH levels, they failed to exhibit the neurological and behavioral symptoms of AHDS patients. Here, we generated an mct8 mutant (mct8?/?) zebrafish using zinc-finger nuclease (ZFN)-mediated targeted gene editing system. The elimination of MCT8 decreased the expression levels of TH receptors; however, it did not affect the expression of other TH-related genes. Similar to human patients, mct8?/? larvae exhibited neurological and behavioral deficiencies. High-throughput behavioral assays demonstrated that mct8?/? larvae exhibited reduced locomotor activity, altered response to external light and dark transitions and an increase in sleep time. These deficiencies in behavioral performance were associated with altered expression of myelin-related genes and neuron-specific deficiencies in circuit formation. Time-lapse imaging of single-axon arbors and synapses in live mct8?/? larvae revealed a reduction in filopodia dynamics and axon branching in sensory neurons and decreased synaptic density in motor neurons. These phenotypes enable assessment of the therapeutic potential of three TH analogs that can enter the cells in the absence of MCT8. The TH analogs restored the myelin and axon outgrowth deficiencies in mct8?/? larvae. These findings suggest a mechanism by which MCT8 regulates neural circuit assembly, ultimately mediating sensory and motor control of behavioral performance. We also propose that the administration of TH analogs early during embryo development can specifically reduce neurological damage in AHDS patients. PMID:25255244

  11. Cocktails of human anti-cancer antibodies show a synergistic effect in nude mouse tumor xenografts.

    PubMed

    Glassy, Mark C; McKnight, Michael E; Kotlan, Beatrix; Glassy, Eric F; Koda, Keiji

    2007-01-01

    A panel of four natural human monoclonal IgG antibodies derived from B lymphocytes isolated from regional draining lymph nodes of cancer patients has been developed and characterized. The four human antibodies are termed, RM1, RM2, RM3, and RM4. The immunoreactivity of this panel of four human antibodies is restricted to tumor cells. Individually, these human MAbs show tumor targeting and are effective in inhibiting tumor growth in nude mouse xenograft models. When used in combination the antibodies show an additive effect in slowing down the progression of tumors in xenograft models suggesting that cocktails of antibodies may be useful in the clinic. PMID:18334744

  12. Assessment of immunotoxicity of dibutyl phthalate using live zebrafish embryos.

    PubMed

    Xu, Hai; Dong, Xing; Zhang, Zhen; Yang, Ming; Wu, Xiangyang; Liu, Hongcui; Lao, Qiaocong; Li, Chunqi

    2015-08-01

    This study set out to understand the immune-toxic effects of dibutyl phthalate (DBP) using transgenic, albino or AB line zebrafish. Zebrafish embryos were exposed to different concentrations of DBP, and the immune cells formation, phagocytosis ability were measured after a short-term exposure to DBP for 6 h post-fertilization (hpf) to 72 or 96 hpf. Exposure to DBP was found to inhibit the neutrophils and macrophage formation in a concentration-dependent manner. The ability of macrophage phagocytosis was all decreased after exposure to DBP, indicating the occurrence of immunotoxicity. The respiratory burst was induced, and the transcription levels of T/B cell-related genes rag1/2 were up-regulated. The overall results indicate that DBP in aquatic environment greatly influence the immune system in fish, and zebrafish embryos can serve as a reliable model for the developmental immunotoxicity of toxic-chemicals. PMID:25956719

  13. [Osteostimulating effect of bone xenograft on bone tissue regeneration].

    PubMed

    Balin, V N; Balin, D V; Iordanishvili, A K; Musikin, M I

    2015-01-01

    The aim of experimental case-control study performed in 28 dogs divided in 2 groups was to assess local tissue reactions on bone xenograft transplantation; dynamics of bone remodeling and formation at the site of bone defect wall contacting with bone xenograft; dynamics and mechanisms of xenograft remodeling. Transplantation of xenograft in conventional bone defects did not cause inflammatory of destructive reactions because of high biocompatibility of the material. At transplantation site active fibrous bone trabeculae formation filling the spaces between xenograft participles was observed. On the 90th day newly formed bone showed lammelar structure. Simultaneously from the 42d day the invasion of cell elements from recipient bed into the material was seen leading to xenograft resorption. The observed dynamics may be assessed as gradual substitution of xenograft with newly formed host bone structures. PMID:26145467

  14. Fishing for drugs from nature: zebrafish as a technology platform for natural product discovery.

    PubMed

    Crawford, Alexander D; Esguerra, Camila V; de Witte, Peter A M

    2008-05-01

    Emerging challenges within the current drug discovery paradigm are prompting renewed interest in natural products as a source of novel, bioactive small molecules. With the recent validation of zebrafish as a biomedically relevant model for functional genomics and in vivo drug discovery, the zebrafish bioassay-guided identification of natural products may be an attractive strategy to generate new lead compounds in a number of indication areas. Here, we review recent natural product research using zebrafish and evaluate the potential of this vertebrate model as a discovery platform for the systematic identification of bioactive natural products. PMID:18584811

  15. Zebrafish Behavioral Response to Chronic Drug

    E-print Network

    Kalueff, Allan V.

    study the effects of withdrawal from addictive substances to produce anxiogenic-like responses in zebrafish. Key words: Zebrafish, Withdrawal, Cortisol, Anxiety, Stress, Novel Tank test, Addiction #12;3 | P are being utilized in research for human brain disorders (Speedie & Gerlai, (2007). Zebrafish organs

  16. Kidney organogenesis in the zebrafish: insights into vertebrate nephrogenesis and regeneration

    PubMed Central

    Gerlach, Gary F.; Wingert, Rebecca A.

    2012-01-01

    Vertebrates form a progressive series of up to three kidney organs during development—the pronephros, mesonephros, and metanephros. Each kidney derives from the intermediate mesoderm and is comprised of conserved excretory units called nephrons. The zebrafish is a powerful model for vertebrate developmental genetics, and recent studies have illustrated that zebrafish and mammals share numerous similarities in nephron composition and physiology. The zebrafish embryo forms an architecturally simple pronephros that has two nephrons, and these eventually become a scaffold onto which a mesonephros of several hundred nephrons is constructed during larval stages. In adult zebrafish, the mesonephros exhibits ongoing nephrogenesis, generating new nephrons from a local pool of renal progenitors during periods of growth or following kidney injury. The characteristics of the zebrafish pronephros and mesonephros make them genetically tractable kidney systems in which to study the functions of renal genes and address outstanding questions about the mechanisms of nephrogenesis. Here, we provide an overview of the formation and composition of these zebrafish kidney organs, and discuss how various zebrafish mutants, gene knockdowns, and transgenic models have created frameworks in which to further delineate nephrogenesis pathways. PMID:24014448

  17. Assessment of Acute Antivascular Effects of Vandetanib with High-Resolution Dynamic Contrast-Enhanced Computed Tomographic Imaging in a Human Colon Tumor Xenograft Model in the Nude Rat1

    PubMed Central

    Tai, Joo Ho; Tessier, Jean; Ryan, Anderson J; Hoffman, Lisa; Chen, Xiaogang; Lee, Ting-Yim

    2010-01-01

    Tumor size is not a reliable marker for the assessment of early antivascular effects of antiangiogenics. In the present study, we used 200-µm in-plane high-resolution dynamic contrast-enhanced computed tomography (DCE-CT) to noninvasively assess the immediate antivascular effects of vandetanib in a subcutaneous human colon cancer (LoVo) xenograft model in nude rats and to investigate correlation between changes in CT perfusion parameters and tumor volume or immunohistochemical end points. At 3 to 4 weeks after LoVo cell implantation, the animal was gavaged with either vandetanib (50 mg/kg) or vehicle twice (22 hours apart) and scanned with a preclinical DCE-CT scanner before (0 hour) and after treatment (24 hours). Quantitative maps of blood flow (BF) and volume (BV) of the tumor were calculated from the acquired DCE-CT images. The rats were divided into nonhypovascular, hypovascular, and combined (regardless of vascularity) groups. In the nonhypovascular group, significant decreases in both tumor BF and BV were observed in the vandetanib-treated rats compared with increases in the vehicle-treated rats. A significant decrease in BV was detected in the vandetanib-treated rats in the combined group as well. No differences in tumor growth, vascular endothelial growth factor expression, microvessel density, or apoptosis were observed between vandetanib- and vehicle-treated rats in all three groups. These results demonstrate that BF and BV imaging biomarkers from DCE-CT imaging can be used for rapid monitoring of immediate (24 hours after) antimicrovascular effects of vandetanib on tumors, even in the absence of significant changes of tumor volume or clinically relevant immunohistochemical end points. PMID:20824046

  18. Optimization of Whole-body Zebrafish Sectioning Methods for Mass Spectrometry Imaging (2013)

    EPA Science Inventory

    Mass spectrometry imaging methods and protocols have become widely adapted to a variety of tissues and species. However, the mass spectrometry imaging literature contains minimal information on whole-body cryosection preparation for the zebrafish (Danio rerio), a model organism ...

  19. Frequency Response of the Swim Bladder and Weberian Ossicles of Zebrafish (Danio Rerio) 

    E-print Network

    Wisniowiecki, Anna Marie

    2014-09-18

    of zebrafish “hearing” is limited compared to other current models and human physiology. Examining the frequency response of the swim bladder and Weberian ossicles would allow the development of a mechanical understanding of the ear and comparison to the human...

  20. Ganglioside mapping of a human medulloblastoma xenograft

    Microsoft Academic Search

    J. Gottfries; J.-E. Mansson; P. Fredman; C. J. Wikstrand; H. S. Friedman; D. D. Bigner; L. Svennerholm

    1989-01-01

    The ganglioside patterns of medulloblastomas have never been established; in this study we report the ganglioside profile of the human medulloblastoma cell line TE-671 grown as a xenograft in nude mice. Gangliosides were isolated and structurally analyzed by fast atom bombardment mass spectometry following permethylation. Identification of individual gangliosides was also performed by immunostaining of high-performance thin-layer chromatography-separated bands. Total

  1. Endosulfan I and endosulfan sulfate disrupts zebrafish embryonic development

    Microsoft Academic Search

    Kerri A. Stanley; Lawrence R. Curtis; Staci L. Massey Simonich; Robert L. Tanguay

    2009-01-01

    Fish in agricultural and remote areas may be exposed to endosulfan and its degradation products as a result of direct runoff, atmospheric transport and deposition. The following study used the zebrafish developmental model to investigate the responses to endosulfan I and endosulfan sulfate, the major degradation product of endosulfan I and II. Embryos were dechorionated and waterborne exposed to the

  2. Neoplasia and Neoplasm Associated Lesions in Laboratory Colonies of Zebrafish Emphasizing Key Influences of Diet and Aquaculture System Design

    PubMed Central

    Spitsbergen, Jan M.; Buhler, Donald R.; Peterson, Tracy S.

    2014-01-01

    During the past decade the zebrafish has emerged as a leading model for mechanistic cancer research due to its sophisticated genetic and genomic resources, its tractability for tissue targeting of transgene expression, its efficiency for forward genetic approaches to cancer model development, and its cost-effectiveness for enhancer and suppressor screens once a cancer model is established. However, in contrast to other laboratory animal species widely used as cancer models, much basic cancer biology information is lacking in zebrafish. As yet data are not published regarding dietary influences on neoplasm incidences in zebrafish. Little information is available regarding spontaneous tumor incidences or histologic types in wild-type (wt) lines of zebrafish. So far a comprehensive database documenting the full spectrum of neoplasia in various organ systems and tissues in not available for zebrafish as it is for other intensely studied laboratory animal species. This manuscript confirms that as in other species diet and husbandry can profoundly influence tumor incidences and histologic spectra in zebrafish. We show that in many laboratory colonies wt lines of zebrafish exhibit elevated neoplasm incidences and neoplasm associated lesions such as heptocyte megalocytosis. We present experimental evidence showing that certain diet and water management regimens can result in high incidences of neoplasia and neoplasm associated lesions. We document the wide array of benign and malignant neoplasms affecting nearly every organ, tissue and cell type in zebrafish, in some cases as a spontaneous aging change, and in other cases due to carcinogen treatment or genetic manipulation. PMID:23382343

  3. Brain on the stage – Spotlight on nervous system development in zebrafish: EMBO practical course, KIT, Sept. 2013

    PubMed Central

    2013-01-01

    During the EMBO course ‘Imaging of Neural Development in Zebrafish’, held on September 9–15th 2013, researchers from different backgrounds shared their latest results, ideas and practical expertise on zebrafish as a model to address open questions regarding nervous system development. PMID:24350623

  4. HCV IRES-Mediated Core Expression in Zebrafish

    PubMed Central

    Zhang, Jing-Pu; Hu, Zhan-Ying; Tong, Jun-Wei; Ding, Cun-Bao; Peng, Zong-Gen; Zhao, Li-Xun; Song, Dan-Qing; Jiang, Jian-Dong

    2013-01-01

    The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-?, PDGF-?, etc. The model was used to evaluate three clinical drugs, ribavirin, IFN?-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFN?-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism. PMID:23469178

  5. Developing highER-throughput zebrafish screens for in-vivo CNS drug discovery.

    PubMed

    Stewart, Adam Michael; Gerlai, Robert; Kalueff, Allan V

    2015-01-01

    The high prevalence of brain disorders and the lack of their efficient treatments necessitate improved in-vivo pre-clinical models and tests. The zebrafish (Danio rerio), a vertebrate species with high genetic and physiological homology to humans, is an excellent organism for innovative central nervous system (CNS) drug discovery and small molecule screening. Here, we outline new strategies for developing higher-throughput zebrafish screens to test neuroactive drugs and predict their pharmacological mechanisms. With the growing application of automated 3D phenotyping, machine learning algorithms, movement pattern- and behavior recognition, and multi-animal video-tracking, zebrafish screens are expected to markedly improve CNS drug discovery. PMID:25729356

  6. Immunostaining of dissected zebrafish embryonic heart.

    PubMed

    Yang, Jingchun; Xu, Xiaolei

    2012-01-01

    Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects. The expression of any gene can be manipulated via morpholino technology or RNA injection. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis. Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals. PMID:22258109

  7. Antitumor activity of selective MEK1/2 inhibitor AZD6244 in combination with PI3K/mTOR inhibitor BEZ235 in gefitinib-resistant NSCLC xenograft models

    PubMed Central

    2014-01-01

    Purpose Although the EGF receptor tyrosine kinase inhibitors (EGFR-TKI) gefitinib have shown dramatic effects against EGFR mutant lung cancer, patients become resistant by various mechanisms, including gatekeeper EGFR-T790M mutation, MET amplification, and KRAS mutation, thereafter relapsing. AZD6244 is a potent, selective, and orally available MEK1/2 inhibitor. In this study, we evaluated the therapeutic efficacy of AZD6244 alone or with BEZ235, an orally available potent inhibitor of phosphatidylinositol 3–kinase (PI3K) and mammalian target of rapamycin (mTOR), in gefitinib-resistant non-small cell lung carcinoma (NSCLC) models. Experimental design NCI-H1975 with EGFR-T790M mutation, NCI-H1993 with MET amplification and NCI-H460 with KRAS/PIK3CA mutation human NSCLC cells were subcutaneous injected into the athymic nude mice respectively. Mice were randomly assigned to treatment with AZD6244, BEZ235, AZD6244 plus BEZ235, or control for 3 weeks, then all mice were sacrificed and tumor tissues were subjected to western blot analyses and immunohistochemical staining. Results AZD6244 could inhibit the tumor growth of NCI-H1993, but slightly inhibit the tumor growth of NCI-1975 and NCI-H460. Combining AZD6244 with BEZ235 markedly enhanced their antitumor effects and without any marked adverse events. Western blot analysis and immunohistochemical staining revealed that AZD6244 alone reduced ERK1/2 phosphorylation, angiogenesis, and tumor cell proliferation. Moreover, MEK1/2 inhibition resulted in decreased AKT phosphorylation in NCI-H1993 tumor model. BEZ235 also inhibited AKT phosphorylation as well as their downstream molecules in all three tumor models. The antiangiogenic effects were substantially enhanced when the agents were combined, which may due to the reduced expression of matrix metallopeptidase-9 in tumor tissues (MMP-9). Conclusions In this study, we evaluated therapy directed against MEK and PI3K/mTOR in distinct gefitinib-resistant NSCLC xenograft models. Combining AZD6244 with BEZ235 enhanced their antitumor and antiangiogenic effects. We concluded that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in suppressing the growth of gefitinib-resistant tumors caused by EGFR T790M mutation, MET amplification, and KRAS/PIK3CA mutation. This new therapeutic strategy may be a practical approach in the treatment of these patients. PMID:24939055

  8. A Student Team in a University of Michigan Biomedical Engineering Design Course Constructs a Microfluidic Bioreactor for Studies of Zebrafish Development

    PubMed Central

    Shen, Yu-chi; Li, David; Al-Shoaibi, Ali; Bersano-Begey, Tom; Chen, Hao; Ali, Shahid; Flak, Betsy; Perrin, Catherine; Winslow, Max; Shah, Harsh; Ramamurthy, Poornapriya; Schmedlen, Rachael H.; Takayama, Shuichi

    2009-01-01

    Abstract The zebrafish is a valuable model for teaching developmental, molecular, and cell biology; aquatic sciences; comparative anatomy; physiology; and genetics. Here we demonstrate that zebrafish provide an excellent model system to teach engineering principles. A seven-member undergraduate team in a biomedical engineering class designed, built, and tested a zebrafish microfluidic bioreactor applying microfluidics, an emerging engineering technology, to study zebrafish development. During the semester, students learned engineering and biology experimental design, chip microfabrication, mathematical modeling, zebrafish husbandry, principles of developmental biology, fluid dynamics, microscopy, and basic molecular biology theory and techniques. The team worked to maximize each person's contribution and presented weekly written and oral reports. Two postdoctoral fellows, a graduate student, and three faculty instructors coordinated and directed the team in an optimal blending of engineering, molecular, and developmental biology skill sets. The students presented two posters, including one at the Zebrafish meetings in Madison, Wisconsin (June 2008). PMID:19292670

  9. 2?-(2-bromohexadecanoyl)-paclitaxel conjugate nanoparticles for the treatment of non-small cell lung cancer in an orthotopic xenograft mouse model

    PubMed Central

    Peng, Lei; Schorzman, Allison N; Ma, Ping; Madden, Andrew J; Zamboni, William C; Benhabbour, Soumya Rahima; Mumper, Russell J

    2014-01-01

    A nanoparticle (NP) formulation with 2?-(2-bromohexadecanoyl)-paclitaxel (Br-16-PX) conjugate was developed in these studies for the treatment of non-small cell lung cancer (NSCLC). The lipophilic paclitaxel conjugate Br-C16-PX was synthesized and incorporated into lipid NPs where the 16-carbon chain enhanced drug entrapment in the drug delivery system and improved in vivo pharmacokinetics. The electron-withdrawing bromine group was used to facilitate the conversion of Br-C16-PX to paclitaxel at the tumor site. The developed system was evaluated in luciferase-expressing A549 cells in vitro and in an orthotopic NSCLC mouse model. The results demonstrated that the Br-C16-PX NPs had a higher maximum tolerated dose (75 mg/kg) than Taxol® (19 mg/kg) and provided significantly longer median survival (88 days versus 70 days, P<0.05) in the orthotopic NSCLC model. An improved pharmacokinetic profile was observed for the Br-C16-PX NPs at 75 mg/kg compared to Taxol at 19 mg/kg. The area under the concentration versus time curve (AUC)0–96 h of Br-C16-PX from the NPs was 91.7-fold and 49.6-fold greater than Taxol in plasma and tumor-bearing lungs, respectively, which provided sustained drug exposure and higher antitumor efficacy in the NP-treated group. PMID:25114529

  10. Association of Diet-Induced Hyperinsulinemia With Accelerated Growth of Prostate Cancer (LNCaP) Xenografts

    Microsoft Academic Search

    Vasundara Venkateswaran; Ahmed Q. Haddad; Neil E. Fleshner; Rong Fan; Linda M. Sugar; Rob Nam; Laurence H. Klotz; Michael Pollak

    2007-01-01

    Background Prior research suggested that energy balance and fat intake influence prostate cancer progression, but the influence of dietary carbohydrate on prostate cancer progression has not been well characterized. We hypothesized that hyperinsulinemia resulting from high intake of refined carbohydrates would lead to more rapid growth of tumors in the murine LNCaP xenograft model of prostate cancer. Methods Athymic mice

  11. Testicular Xenografts: A Novel Approach to Study Cytotoxic Damage in Juvenile Primate Testis

    Microsoft Academic Search

    Kirsi Jahnukainen; Jens Ehmcke; Stefan Schlatt

    2006-01-01

    The underlying primary damage to the testis caused by chemotherapeutic regimens during childhood is largely unknown. Xenografting of monkey testes was successfully applied in maturation of juvenile testis to the point of complete spermatogenesis. This allows us to manipulate developing primate testis without direct treatment of patients. This new model is validated establishing the effects of cyto- toxic treatment in

  12. In vivo Efficacy of Marimastat and Chemoradiation in Head and Neck Cancer Xenografts

    Microsoft Academic Search

    Joni B. Skipper; Lacey R. McNally; Eben L. Rosenthal; Wenquan Wang; Donald J. Buchsbaum

    2009-01-01

    Objective: To assess the effect of combining a synthetic matrix metalloprotease inhibitor and chemoradiation therapy on tumor growth in a murine model of head and neck squamous-cell carcinoma (SCC). Methods: Athymic, nude mice bearing SCC-1 xenografts were used to comprise 4 treatment groups: (1) control receiving vehicle alone, (2) marimastat alone, (3) cisplatin + radiation in combination and (4) marimastat

  13. Convection-enhanced delivery of liposomal doxorubicin in intracranial brain tumor xenografts

    Microsoft Academic Search

    Yoji Yamashita; Ryuta Saito; Michal T. Krauze; Tomohiro Kawaguchi; Charles Noble; Daryl C. Drummond; Dmitri B. Kirpotin; John W. Park; Mitchel S. Berger; Krystof S. Bankiewicz

    2006-01-01

    We previously reported that convection-enhanced delivery (CED) of liposomes into brain tissue and intracranial brain tumor xenografts produced robust tissue distribution that can be detected by magnetic resonance imaging. Considering image-guided CED of therapeutic liposomes as a promising strategy for the treatment of brain tumors, we evaluated the efficacy of pegylated liposomal doxorubicin delivered by CED in an animal model.

  14. Imaging biomarkers to monitor response to the hypoxia-activated prodrug TH-302 in the MiaPaCa2 flank xenograft model

    PubMed Central

    Cárdenas-Rodríguez, Julio; Li, Yuguo; Galons, Jean-Philippe; Cornnell, Heather; Gillies, Robert J.; Pagel, Mark D.; Baker, Amanda F.

    2012-01-01

    TH-302, a hypoxia-activated anticancer prodrug was evaluated for anti-tumor activity and changes in dynamic contrast enhanced (DCE)- and diffusion weighted (DW)-magnetic resonance imaging (MRI) in a mouse model of pancreatic cancer. TH-302 monotherapy resulted in a significant delay in tumor growth compared to vehicle treated controls. TH-302 treatment was also associated with a significant decrease in the volume transfer constant (Ktrans) compared to vehicle treated controls one day following the first dose measured using DCE-MRI. This early decrease in Ktrans following the first dose as measured is consistent with selective killing of the hypoxic fraction of cells which are associated with enhanced expression of HIF-1? that regulates expression of permeability & perfusion factors including VEGF-A. No changes were observed in DW-MRI following treatment with TH-302, which may indicate this technique is not sensitive enough to detect changes in small hypoxic fractions of the tumor targeted by TH-302. These results suggest that changes in tumor permeability and/or perfusion may be an early imaging biomarker for response to TH-302 therapy. PMID:22554971

  15. Pharmaceutical inhibition of glycogen synthetase kinase-3? reduces multiple myeloma–induced bone disease in a novel murine plasmacytoma xenograft model

    PubMed Central

    Gunn, W. Grady; Krause, Ulf; Lee, Narae

    2011-01-01

    Multiple myeloma (MM) is a malignancy of plasma cells that accumulate in the bone marrow. MM is incurable with approximately 100 000 patients currently in the United States and 20 000 new cases diagnosed yearly. The malignancy causes displacement of hematopoiesis and formation of osteolytic bone lesions also known as myeloma bone disease (MBD). At diagnosis, 79% of patients suffer from MBD associated with severe pain and increased mortality. Wnt inhibitors secreted by MM cells inhibit osteogenesis and promote osteoclastogenesis, therefore rapid targeting of Wnt inhibitors is necessary to prevent potentially irreversible effects on the stroma, which could lead to incurable MBD. Inhibition of glycogen synthetase kinase-3? (GSK3?) causes accelerated Wnt signaling and enhanced osteogenesis in mesenchymal stem/progenitor cells, irrespective of the extracellular concentration of Wnt inhibitors. Our primary goal of this study was to evaluate a GSK3? inhibitor (6-bromoindirubin-3?-oxime BIO) for amelioration of bone destruction in a murine model of MBD. When measured using histomorphometry, peritumoral BIO administration improved bone quality at the bone-tumor interface and, surprisingly, increased histologically apparent tumor necrosis. Furthermore, in vitro assays demonstrated a proapoptotic effect on numerous MM cell lines. These preliminary data suggest that pharmaceutical GSK3? inhibition may improve bone quality in myeloma and other malignant bone diseases. PMID:21123822

  16. Two Zebrafish hsd3b Genes Are Distinct in Function, Expression, and Evolution.

    PubMed

    Lin, Jen-Chieh; Hu, Shing; Ho, Pei-Hung; Hsu, Hwei-Jan; Postlethwait, John H; Chung, Bon-Chu

    2015-08-01

    HSD3B catalyzes the synthesis of ?4 steroids such as progesterone in the adrenals and gonads. Individuals lacking HSD3B2 activity experience congenital adrenal hyperplasia with imbalanced steroid synthesis. To develop a zebrafish model of HSD3B deficiency, we characterized 2 zebrafish hsd3b genes. Our phylogenetic and conserved synteny analyses showed that the tandemly duplicated human HSD3B1 and HSD3B2 genes are coorthologs of zebrafish hsd3b1 on chromosome 9 (Dre9), whereas the gene called hsd3b2 resides on Dre20 in an ancestral chromosome segment, from which its ortholog was lost in the tetrapod lineage. Zebrafish hsd3b1(Dre 9) was expressed in adult gonads and headkidney, which contains interrenal glands, the zebrafish counterpart of the tetrapod adrenal. Knockdown of hsd3b1(Dre 9) caused the interrenal and anterior pituitary to expand and pigmentation to increase, resembling human HSD3B2 deficiency. The zebrafish hsd3b2(Dre 20) gene was expressed in zebrafish early embryos as maternal transcripts that disappeared 1 day after fertilization. Morpholino inactivation of hsd3b2(Dre 20) led to embryo elongation, which was rescued by the injection of hsd3b2 mRNA. Thus, zebrafish hsd3b2(Dre 20) evolved independently of hsd3b1(Dre 9) with a morphogenetic function during early embryogenesis. Zebrafish hsd3b1(Dre 9), on the contrary, functions like mammalian HSD3B2, whose deficiency leads to congenital adrenal hyperplasia. PMID:25974401

  17. Genetically designing a more potent anti-pancreatic cancer agent by simultaneously cotargeting human IL-13 and EGF receptors in a mouse xenograft model

    PubMed Central

    Vallera, Daniel A; Stish, Brad J.; Shu, Yanqun; Chen, Hua; Saluja, Ashok; Buchsbaum, Donald J.; Vickers, Selwyn M.

    2009-01-01

    Objective Investigators are currently interested in the EGFR and IL-13R as potential targets in the development of new biologicals for pancreatic cancer. Attempts to develop successful agents have met with difficulty. Our novel approach was to simultaneously target these receptors with EGF and IL-13 cloned on the same bispecific single chain molecule with truncated diphtheria toxin (DT390) to determine if co-targeting with DTEGF13 had any advantages. Design Proliferation experiments were performed to measure the potency and selectivity of bispecific DTEGF13 and its monospecific counterparts against pancreatic cancer cell lines Panc-1 and MiaPaCa-2 in vitro. DTEGF13 was then administered intratumorally to nude mice with MiaPaCa-2 flank tumors to measure efficacy and toxicity (weight loss). Results In vitro, bispecific DTEGF13 was 2,800-fold more toxic than monospecific DTEGF or DTIL13 against Panc-1. A similar enhancement was observed in vitro when MiaPaCa-2 pancreatic cancer cells or H2981-T3 lung adenocarcinoma cells were studied. DTEGF13 activity was blockable with recombinant EGF13. DTEGF13 was potent (IC50 = 0.00017 nM) against MiaPaCa-2, receptor specific, and significantly inhibited MiaPaCa-2 tumor in nude mice (p<0.008). Conclusions In vitro studies show that the presence of both ligands on the same bispecific molecule is responsible for the superior activity of DTEGF13. Intratumoral administration showed that DTEGF13 was highly effective in checking aggressive tumor progression in mice. Lack of weight loss in these mice indicated that the drug was tolerated and a therapeutic index exists in an “on target” model in which DTEGF13 is cross-reactive with native mouse receptors. PMID:18222985

  18. Dual-Modality Micro-Positron Emission Tomography/Computed Tomography and Near-Infrared Fluorescence Imaging of EphB4 in Orthotopic Glioblastoma Xenograft Models

    PubMed Central

    Huang, Miao; Xiong, Chiyi; Lu, Wei; Zhang, Rui; Zhou, Min; Huang, Qian; Weinberg, Jeffrey; Li, Chun

    2013-01-01

    Purpose In glioblastoma, EphB4 receptors, a member of the largest family of receptor tyrosine kinases, are overexpressed in both tumor cells and angiogenic blood vessels. The purpose of this study was to examine whether the EphB4-binding peptide TNYL-RAW labeled with both 64Cu and near-infrared fluorescence dye Cy5.5 could be used as a molecular imaging agent for dual-modality positron emission tomography/computed tomography [PET/CT] and optical imaging of human glioblastoma in orthotopic brain tumor models. Materials and Methods TNYL-RAW was conjugated to Cy5.5 and the radiometal chelator 1,4,7,10-tetraazadodecane-N,N?,N?,N? -tetraacetic acid. The conjugate was then labeled with 64Cu for in vitro binding and in vivo dual ?PET/CT and optical imaging studies in nude mice implanted with EphB4-expressing U251 and EphB4-negative U87 human glioblastoma cells. Tumors and brains were removed at the end of the imaging sessions for immunohistochemical staining and fluorescence microscopic examinations. Results ?PET/CT and near-infrared optical imaging clearly showed specific uptake of the dual-labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of the nude mice after intravenous injection of the peptide. In U251 tumors, the Cy5.5-labeled peptide colocalized with both tumor blood vessels and tumor cells; in U87 tumors, the tracer colocalized only with tumor blood vessels, not with tumor cells. Conclusions Dual-labeled EphB4-specific peptide could be used as a noninvasive molecular imaging agent for PET/CT and optical imaging of glioblastoma owing to its ability to bind to both EphB4-expressing angiogenic blood vessels and EphB4-expressing tumor cells. PMID:23918654

  19. [Effects of compound Zhe-Bei granule (CZBG) combined with doxorubicin on expression of membrane transport proteins in K562/A02 cell xenografts].

    PubMed

    Li, Dong-Yun; Zheng, Zhi; Hou, Li; Jiang, Miao; Dong, Qing; Tian, Shao-Dan; Ma, Wei; Chen, Ju; Wang, Jing; Chen, Xin-Yi

    2010-02-01

    This study was purposed to investigate the effects of compound Zhe-Bei granule (CZBG) combined with doxorubicin on expressions of P-gp, MRP, LRP in K562/A02 cell xenografts. Tumor xenograft model were established by injecting the multidrug resistant cell line K562/A02 in the axillary flank of BALB/c-nu-nu mice. CZBG-intragastric administration and doxorubicin-intraperitoneal injection in combination were given to the BALB/c-nu nude mice. The tumor xenografts were made into slice after the dissection, and the expression of P-gp, MRP, LRP in K562/A02 tumor xenografts in mice were investigated by immunohistochemical technique. The integral optical density (IOD) of P-gp, MRP, LRP in K562/A02 tumor xenografts were measured by Image Pro Plus 6.0. The results showed that as compared with the doxorubicin alone, the combination of the doxorubicin and CZBG with high, middle and low dosage could decrease IOD of P-gp, MRP in K562/A02 tumor xenografts with statistical significance (p < 0.05). The LRP expression in K562/A02 tumor xenografts was not observed in five groups. It is concluded that the combination of CZBG with doxorubicin decreases the expressions of P-gp, MRP in K562/A02 tumor xenografts of mice. PMID:20137116

  20. PI3K-mTOR inhibitor PF-04691502 anti-tumor activity is enhanced with induction of wild-type TP53 in human xenograft and murine knockout models of head and neck cancer

    PubMed Central

    Herzog, Amanda; Bian, Yansong; Broek, Robert Vander; Hall, Bradford; Coupar, Jamie; Cheng, Hui; Sowers, Anastasia L.; Cook, John D.; Mitchell, James B.; Chen, Zhong; Kulkarni, Ashok B.; VanWaes, Carter

    2013-01-01

    Purpose PI3K-mTOR pathway activation is often associated with altered expression or mutations of PIK3CA, TP53/p73, PTEN and TGF?R in head and neck squamous cell carcinomas (HNSCC). However, little is known about how these alterations affect response to PI3K-mTOR targeted agents. Experimental Design In this preclinical study, PI3K-Akt-mTOR signaling was characterized in 9 HNSCC (UM-SCC) cell lines and Human Oral Keratinocytes (HOK). We investigated the molecular and anti-cancer effects of dual PI3K/mTOR inhibitor PF-04691502(PF-502) in UM-SCC expressing PIK3CA with decreased wtTP53, mtTP53-/+mtTGF?R2, and in HNSCC of a conditional Pten/Tgfbr1 double knockout (2cKO) mouse model, displaying PI3K-Akt-mTOR activation. Results UM-SCC showed increased PIK3CA expression and Akt/mTOR activation, and PF-502 inhibited PI3K/mTORC1/2 targets. In human HNSCC expressing PIK3CA and decreased wtTP53 and p73, PF-502 reciprocally enhanced TP53/p73 expression and growth inhibition, which was partially reversible by p53 inhibitor pifithrin-?. Most UM-SCC with wtTP53 exhibited a lower IC50 than those with mtTP53 status. PF-502 blocked growth in G0/G1 and increased apoptotic subG0 DNA. PF502 suppressed tumorigenesis and showed combinatorial activity with radiation in a wtTP53 UMSCC xenograft model. PF-502 also significantly delayed HNSCC tumorigenesis and prolonged survival of Pten/Tgfbr1 deficient mice. Significant inhibition of p-Akt, p-4EBP1, p-S6, Ki67, as well as increased p53 and TUNEL were observed in tumor specimens. Conclusions PI3K-mTOR inhibition can enhance TP53/p73 expression and significantly inhibit tumor growth alone or when combined with radiation in HNSCC with wtTP53. PIK3CA, TP53/p73, PTEN and TGF? alterations are potential modifiers of response and merit investigation in future clinical trials with PI3K-mTOR inhibitors. PMID:23640975

  1. Growth of LAPC4 prostate cancer xenograft tumor is insensitive to 5?-reductase inhibitor dutasteride

    PubMed Central

    Garcia, Raquel Ramos; Masoodi, Khalid Z; Pascal, Laura E; Nelson, Joel B; Wang, Zhou

    2014-01-01

    Intermittent androgen deprivation therapy (IADT) allows prostate cancer patients a break from the side-effects of continuous androgen deprivation therapy (ADT). Although clinical studies suggest that IADT can significantly improve patient quality of life over ADT, it has not been demonstrated to improve patient survival. Recently, increased survival has been demonstrated when 5?-reductase inhibitors have been used during the off-cycle of IADT in animal xenograft tumor models LNCaP and LuCaP35. In the current study, the sensitivity of LAPC4 xenograft tumor regrowth to the 5ARI dutasteride was determined. Tumor regrowth and gene expression changes in LAPC4 tumors were compared to the previously determined response of LNCaP and LuCaP35 xenograft tumors to 5ARI treatment during the off-cycle of IADT, LAPC4, LNCaP and LuCaP35 tumors were sensitive to androgen manipulation. However, in contrast to LNCaP and LuCaP35, dutasteride treatment during testosterone-stimulated prostate regrowth did not affect tumor regrowth or the expression of androgen responsive genes. Tumor response to dutasteride during the off-cycle of IADT is variable in xenograft prostate tumor models. Future studies will be required to elucidate the mechanisms contributing to the dutasteride resistance observed in the LAPC4 model during the off-cycle. PMID:25374909

  2. Growth of LAPC4 prostate cancer xenograft tumor is insensitive to 5?-reductase inhibitor dutasteride.

    PubMed

    Garcia, Raquel Ramos; Masoodi, Khalid Z; Pascal, Laura E; Nelson, Joel B; Wang, Zhou

    2014-01-01

    Intermittent androgen deprivation therapy (IADT) allows prostate cancer patients a break from the side-effects of continuous androgen deprivation therapy (ADT). Although clinical studies suggest that IADT can significantly improve patient quality of life over ADT, it has not been demonstrated to improve patient survival. Recently, increased survival has been demonstrated when 5?-reductase inhibitors have been used during the off-cycle of IADT in animal xenograft tumor models LNCaP and LuCaP35. In the current study, the sensitivity of LAPC4 xenograft tumor regrowth to the 5ARI dutasteride was determined. Tumor regrowth and gene expression changes in LAPC4 tumors were compared to the previously determined response of LNCaP and LuCaP35 xenograft tumors to 5ARI treatment during the off-cycle of IADT, LAPC4, LNCaP and LuCaP35 tumors were sensitive to androgen manipulation. However, in contrast to LNCaP and LuCaP35, dutasteride treatment during testosterone-stimulated prostate regrowth did not affect tumor regrowth or the expression of androgen responsive genes. Tumor response to dutasteride during the off-cycle of IADT is variable in xenograft prostate tumor models. Future studies will be required to elucidate the mechanisms contributing to the dutasteride resistance observed in the LAPC4 model during the off-cycle. PMID:25374909

  3. Teratogenic and toxic effects of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt.:Fr.) P. Karst. (higher Basidiomycetes), on zebrafish embryo as model.

    PubMed

    Dulay, Rich Milton R; Kalaw, Sofronio P; Reyes, Renato G; Alfonso, Noel F; Eguchi, Fumio

    2012-01-01

    This paper highlights the teratogenic and toxic effects of Ganoderma lucidum (Lingzhi or Reishi mushroom) extract on zebrafish embryos. Hatchability, malformations, and lethality rate of zebrafish embryos were assessed to provide valuable information regarding the potential teratogenic activity of G. lucidum. Hatching was completed 48 h post treatment application (hpta) at 1% or lower concentrations of extract and embryo water. The hatching rate of embryos treated with 5% or higher concentrations was significantly lower (p> 0.05) than the control. Tail malformation was the most marked morphological abnormality in embryos at 72 hpta, which was obviously caused by 1% extract (55.56% tail malformation) and was observed in all embryos exposed to 5% of extract. Growth retardation was evident in embryos exposed to 5%, 10%, and 20%. However, lethal effect of extract of G. lucidum was dependent on dose and time of exposure. Mortality rates of embryos treated with 5% (44.44%) or higher concentrations of the extract was significantly higher (p > 0.05) than that of the control embryos at 72 hpta. These results suggest that G. lucidum extract has lethal and sub-lethal effects on zebrafish embryos. PMID:23510220

  4. Assessment of anti-inflammatory effect of 5?-hydroxypalisadin B isolated from red seaweed Laurencia snackeyi in zebrafish embryo in vivo model.

    PubMed

    Wijesinghe, W A J P; Kim, Eun-A; Kang, Min-Cheol; Lee, Won-Woo; Lee, Hyi-Seung; Vairappan, Charles S; Jeon, You-Jin

    2014-01-01

    5?-Hydroxypalisadin B, a halogenated secondary metabolite isolated from red seaweed Laurencia snackeyi was evaluated for its anti-inflammatory activity in lipopolysaccharide (LPS)-induced zebrafish embryo. Preliminary studies suggested the effective concentrations of the compound as 0.25, 0.5, 1 ?g/mL for further in vivo experiments. 5?-Hydroxypalisadin B, exhibited profound protective effect in the zebrafish embryo as confirmed by survival rate, heart beat rate, and yolk sac edema size. The compound acts as an effective agent against reactive oxygen species (ROS) formation induced by LPS and tail cut. Moreover, 5?-hydroxypalisadin B effectively inhibited the LPS-induced nitric oxide (NO) production in zebrafish embryo. All the tested protective effects of 5?-hydroxypalisadin B were comparable to the well-known anti-inflammatory agent dexamethasone. According to the results obtained, 5?-hydroxypalisadin B isolated from red seaweed L. snackeyi could be considered as an effective anti-inflammatory agent which might be further developed as a functional ingredient. PMID:24317194

  5. Chevron formation of the zebrafish muscle segments

    PubMed Central

    Rost, Fabian; Eugster, Christina; Schröter, Christian; Oates, Andrew C.; Brusch, Lutz

    2014-01-01

    The muscle segments of fish have a folded shape, termed a chevron, which is thought to be optimal for the undulating body movements of swimming. However, the mechanism shaping the chevron during embryogenesis is not understood. Here, we used time-lapse microscopy of developing zebrafish embryos spanning the entire somitogenesis period to quantify the dynamics of chevron shape development. By comparing such time courses with the start of movements in wildtype zebrafish and analysing immobile mutants, we show that the previously implicated body movements do not play a role in chevron formation. Further, the monotonic increase of chevron angle along the anteroposterior axis revealed by our data constrains or rules out possible contributions by previously proposed mechanisms. In particular, we found that muscle pioneers are not required for chevron formation. We put forward a tension-and-resistance mechanism involving interactions between intra-segmental tension and segment boundaries. To evaluate this mechanism, we derived and analysed a mechanical model of a chain of contractile and resisting elements. The predictions of this model were verified by comparison with experimental data. Altogether, our results support the notion that a simple physical mechanism suffices to self-organize the observed spatiotemporal pattern in chevron formation. PMID:25267843

  6. Neurotransmitter map of the asymmetric dorsal habenular nuclei of zebrafish

    PubMed Central

    deCarvalho, Tagide N.; Subedi, Abhignya; Rock, Jason; Harfe, Brian D.; Thisse, Christine; Thisse, Bernard; Halpern, Marnie E.; Hong, Elim

    2014-01-01

    The role of the habenular nuclei in modulating fear and reward pathways has sparked a renewed interest in this conserved forebrain region. The bilaterally paired habenular nuclei, each consisting of a medial/dorsal and lateral/ventral nucleus, can be further divided into discrete subdomains whose neuronal populations, precise connectivity and specific functions are not well understood. An added complexity is that the left and right habenulae show pronounced morphological differences in many non-mammalian species. Notably, the dorsal habenulae of larval zebrafish provide a vertebrate genetic model to probe the development and functional significance of brain asymmetry. Previous reports have described a number of genes that are expressed in the zebrafish habenulae, either in bilaterally symmetric patterns or more extensively on one side of the brain than the other. The goal of our study was to generate a comprehensive map of the zebrafish dorsal habenular nuclei, by delineating the relationship between gene expression domains, comparing the extent of left-right asymmetry at larval and adult stages, and identifying potentially functional subnuclear regions as defined by neurotransmitter phenotype. While many aspects of habenular organization appear conserved with rodents, the zebrafish habenulae also possess unique properties that may underlie lateralization of their functions. PMID:24753112

  7. Shoaling develops with age in Zebrafish (Danio rerio)

    PubMed Central

    Buske, Christine; Gerlai, Robert

    2010-01-01

    The biological mechanisms of human social behavior are complex. Animal models may facilitate the understanding of these mechanisms and may help one to develop treatment strategies for abnormal human social behavior, a core symptom in numerous clinical conditions. The zebrafish is perhaps the most social vertebrate among commonly used laboratory species. Given its practical features and the numerous genetic tools developed for it, it should be a promising tool. Zebrafish shoal, i.e. form tight multimember groups, but the ontogenesis of this behavior has not been described. Analyzing the development of shoaling is a step towards discovering the mechanisms of this behavior. Here we study age-dependent changes of shoaling in zebrafish from day 7 post fertilization to over 5 months of age by measuring the distance between all pairs of fish in freely swimming groups of ten subjects. Our longitudinal (repeated measure within subject) and cross sectional (non-repeated measure between subject) analyses both demonstrated a significant increase of shoaling with age (decreased distance between shoal members). Given the sophisticated genetic and developmental biology methods already available for zebrafish, we argue that our behavioral results open a new avenue towards the understanding of the development of vertebrate social behavior and of its mechanisms and abnormalities. PMID:20837077

  8. The identification and characterization of zebrafish hematopoietic stem cells

    PubMed Central

    Ma, Dongdong; Zhang, Jing; Lin, Hui-feng; Italiano, Joseph

    2011-01-01

    HSCs are defined by their ability to self-renew and maintain hematopoiesis throughout the lifespan of an organism. The optical clarity of their embryos and the ease of genetic manipulation make the zebrafish (Danio rerio) an excellent model for studying hematopoiesis. Using flow cytometry, we identified 2 populations of CD41-GFP+ cells (GFPhi and GFPlo) in the whole kidney marrow of Tg(CD41:GFP) zebrafish. Past studies in humans and mice have shown that CD41 is transiently expressed in the earliest hematopoietic progenitors and is then silenced, reappearing in the platelet/thrombocyte lineage. We have transplanted flow-sorted GFPhi and GFPlo cells into irradiated adult zebrafish and assessed long-term hematopoietic engraftment. Transplantation of GFPhi cells did not reconstitute hematopoiesis. In contrast, we observed multilineage hematopoiesis up to 68 weeks after primary and secondary transplantation of GFPlo cells. We detected the CD41-GFP transgene in all major hematopoietic lineages and CD41-GFP+ cells in histologic sections of kidneys from transplant recipients. These studies show that CD41-GFPlo cells fulfill generally accepted criteria for HSCs. The identification of fluorescent zebrafish HSCs, coupled with our ability to transplant them into irradiated adult recipients, provide a valuable new tool to track HSC homing, proliferation, and differentiation into hematopoietic cells. PMID:21586750

  9. Automated processing of zebrafish imaging data: a survey.

    PubMed

    Mikut, Ralf; Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A; Kausler, Bernhard X; Ledesma-Carbayo, María J; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

    2013-09-01

    Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines. PMID:23758125

  10. [A brief protocol for sperm cryopreservation and revival in zebrafish].

    PubMed

    Zheng, Nai-Zhong; Zhang, Bo

    2012-09-01

    Zebrafish is an important vertebrate model organism for the study of embryonic development and the underlying genetic mechanism. Numerous mutants and transgenics have been generated in recent years, long-term and safe storage of these fish lines is of crucial importance for every zebrafish community/lab. Sperm cryopreservation and revival has become a preferred method for this purpose, which provides extra and reliable security as a backup for the cost-effective maintenance of genetic stocks in addition to reducing space demanding for housing large amount of live fish. This is especially critical for invaluable fish lines against accidental loss. Generally, the sperm are obtained by either squeezing the male fish or dissecting out and homogenizing the testes, then they are mixed with the freezing medium before gradually frozen as aliquots in liquid nitrogen. They can be easily revived through in vitro fertilization whenever necessary. This technique was introduced into zebrafish research three decades ago and has gradually become mature and more reliable following the im-provement of many critical factors and steps, including cryoprotectants and conditions for freezing and revival. Base on pioneers' work, our lab has established and improved a simple method for sperm cryopreservation and revival which shows high recovery rate after relatively long storage time. Here we briefly summarize the history and development of the methods for sperm cryopreservation and revival in zebrafish and present a brief protocol for the practice of sperm cryopreservation and revival, which is routinely used in our lab. PMID:23017463

  11. Proteomics profiling of single organs from individual adult zebrafish.

    PubMed

    Abramsson, Alexandra; Westman-Brinkmalm, Ann; Pannee, Josef; Gustavsson, Mikael; von Otter, Malin; Blennow, Kaj; Brinkmalm, Gunnar; Kettunen, Petronella; Zetterberg, Henrik

    2010-06-01

    The model organism zebrafish (Danio rerio) is extensively utilized in studies of developmental biology but is also being investigated in the context of a growing list of human age-related diseases. To facilitate such studies, we here present protein expression patterns of adult zebrafish organs, including blood, brain, fin, heart, intestine, liver, and skeletal muscle. Protein extracts were prepared from the different organs of two zebrafish and analyzed using liquid chromatography coupled to high-resolution tandem mass spectrometry. Zebrafish tissue was digested directly after minimal fractionation and cleaned up (the shotgun approach). Proteins were identified using Mascot software. In total, 1394 proteins were identified of which 644 were nonredundant. Of these, 373 demonstrated an organ-specific expression pattern and 57 had not been shown on protein level before. These data emphasize the need for increased research at the protein level to facilitate the selection of candidate proteins for targeted quantification and to refine systematic genetic network analysis in vertebrate development, biology, and disease. PMID:20392139

  12. Differential reinforcement of an approach response in zebrafish (Danio rerio).

    PubMed

    Manabe, Kazuchika; Dooling, R J; Takaku, Shinichi

    2013-09-01

    Five zebrafish were trained to approach a target using a fully automated training procedure. During a training session, if the distance between the fish and the target was closer than an arbitrarily set distance, the approach response was reinforced by food. The fish continued to respond under this reinforcement contingency and the distance criterion could be shortened up to eighty times within a 1h session. The initial distance limit was then shortened for the next test training session. Once the initial distance criterion was reduced to a final minimum distance, the distance criterion was fixed at this value for the next nine successive sessions. In a second experiment using different fish, we manipulated approach distances in three conditions. The first condition was identical to the changing criterion training as in Experiment 1. In the second condition, only response distances under a distance criterion were reinforced. And in the last condition, only response distances over the distance criterion were reinforced. Results show that zebrafish can control the distance between themselves and a target. In other words, zebrafish are sensitive to the spatial consequences of their behavior. The present results show that a differential reinforcement paradigm can be successfully applied to zebrafish which therefore enhances their value as a vertebrate model for studies of complex behavior including visuomotor learning. PMID:23727035

  13. Zebrafish sex: a complicated affair

    PubMed Central

    Liew, Woei Chang

    2014-01-01

    In this review, we provide a detailed overview of studies on the elusive sex determination (SD) and gonad differentiation mechanisms of zebrafish (Danio rerio). We show that the data obtained from most studies are compatible with polygenic sex determination (PSD), where the decision is made by the allelic combinations of several loci. These loci are typically dispersed throughout the genome, but in some teleost species a few of them might be located on a preferential pair of (sex) chromosomes. The PSD system has a much higher level of variation of SD genotypes both at the level of gametes and the sexual genotype of individuals, than that of the chromosomal sex determination systems. The early sexual development of zebrafish males is a complicated process, as they first develop a ‘juvenile ovary’, that later undergoes a transformation to give way to a testis. To date, three major developmental pathways were shown to be involved with gonad differentiation through the modulation of programmed cell death. In our opinion, there are more pathways participating in the regulation of zebrafish gonad differentiation/transformation. Introduction of additional powerful large-scale genomic approaches into the analysis of zebrafish reproduction will result in further deepening of our knowledge as well as identification of additional pathways and genes associated with these processes in the near future. PMID:24148942

  14. Advanced Echocardiography in Adult Zebrafish Reveals Delayed Recovery of Heart Function after Myocardial Cryoinjury

    PubMed Central

    Kossack, Mandy; Juergensen, Lonny; Fuchs, Dieter; Katus, Hugo A.; Hassel, David

    2015-01-01

    Translucent zebrafish larvae represent an established model to analyze genetics of cardiac development and human cardiac disease. More recently adult zebrafish are utilized to evaluate mechanisms of cardiac regeneration and by benefiting from recent genome editing technologies, including TALEN and CRISPR, adult zebrafish are emerging as a valuable in vivo model to evaluate novel disease genes and specifically validate disease causing mutations and their underlying pathomechanisms. However, methods to sensitively and non-invasively assess cardiac morphology and performance in adult zebrafish are still limited. We here present a standardized examination protocol to broadly assess cardiac performance in adult zebrafish by advancing conventional echocardiography with modern speckle-tracking analyses. This allows accurate detection of changes in cardiac performance and further enables highly sensitive assessment of regional myocardial motion and deformation in high spatio-temporal resolution. Combining conventional echocardiography measurements with radial and longitudinal velocity, displacement, strain, strain rate and myocardial wall delay rates after myocardial cryoinjury permitted to non-invasively determine injury dimensions and to longitudinally follow functional recovery during cardiac regeneration. We show that functional recovery of cryoinjured hearts occurs in three distinct phases. Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi). The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage. PMID:25853735

  15. Embryos of the zebrafish Danio rerio in studies of non-targeted effects of ionizing radiation.

    PubMed

    Choi, V W Y; Yu, K N

    2015-01-01

    The use of embryos of the zebrafish Danio rerio as an in vivo tumor model for studying non-targeted effects of ionizing radiation was reviewed. The zebrafish embryo is an animal model, which enables convenient studies on non-targeted effects of both high-linear-energy-transfer (LET) and low-LET radiation by making use of both broad-beam and microbeam radiation. Zebrafish is also a convenient embryo model for studying radiobiological effects of ionizing radiation on tumors. The embryonic origin of tumors has been gaining ground in the past decades, and efforts to fight cancer from the perspective of developmental biology are underway. Evidence for the involvement of radiation-induced genomic instability (RIGI) and the radiation-induced bystander effect (RIBE) in zebrafish embryos were subsequently given. The results of RIGI were obtained for the irradiation of all two-cell stage cells, as well as 1.5 hpf zebrafish embryos by microbeam protons and broad-beam alpha particles, respectively. In contrast, the RIBE was observed through the radioadaptive response (RAR), which was developed against a subsequent challenging dose that was applied at 10 hpf when <0.2% and <0.3% of the cells of 5 hpf zebrafish embryos were exposed to a priming dose, which was provided by microbeam protons and broad-beam alpha particles, respectively. Finally, a perspective on the field, the need for future studies and the significance of such studies were discussed. PMID:24176822

  16. The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish

    PubMed Central

    Cameron, Donald Joshua; Rassamdana, Faydim; Tam, Peony; Dang, Kathleen; Yanez, Carolina; Ghaemmaghami, Saman; Dehkordi, Mahsa Iranpour

    2013-01-01

    Zebrafish are a proven model for vision research, however many of the earlier methods generally focused on larval fish or demonstrated a simple response. More recently adult visual behavior in zebrafish has become of interest, but methods to measure specific responses are new coming. To address this gap, we set out to develop a methodology to repeatedly and accurately utilize the optokinetic response (OKR) to measure visual acuity in adult zebrafish. Here we show that the adult zebrafish's visual acuity can be measured, including both binocular and monocular acuities. Because the fish is not harmed during the procedure, the visual acuity can be measured and compared over short or long periods of time. The visual acuity measurements described here can also be done quickly allowing for high throughput and for additional visual procedures if desired. This type of analysis is conducive to drug intervention studies or investigations of disease progression. PMID:24145465

  17. Effective heritable gene knockdown in zebrafish using synthetic microRNAs

    PubMed Central

    Giacomotto, Jean; Rinkwitz, Silke; Becker, Thomas S.

    2015-01-01

    Although zebrafish is used to model human diseases through mutational and morpholino-based knockdown approaches, there are currently no robust transgenic knockdown tools. Here we investigate the knockdown efficiency of three synthetic miRNA-expressing backbones and show that these constructs can downregulate a sensor transgene with different degrees of potency. Using this approach, we reproduce spinal muscular atrophy (SMA) in zebrafish by targeting the smn1 gene. We also generate different transgenic lines, with severity and age of onset correlated to the level of smn1 inhibition, recapitulating for the first time the different forms of SMA in zebrafish. These lines are proof-of-concept that miRNA-based approaches can be used to generate potent heritable gene knockdown in zebrafish. PMID:26051838

  18. The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental research.

    PubMed

    Sprague, J; Doerry, E; Douglas, S; Westerfield, M

    2001-01-01

    The Zebrafish Information Network, ZFIN, is a WWW community resource of zebrafish genetic, genomic and developmental research information (http://zfin.org). ZFIN provides an anatomical atlas and dictionary, developmental staging criteria, research methods, pathology information and a link to the ZFIN relational database (http://zfin. org/ZFIN/). The database, built on a relational, object-oriented model, provides integrated information about mutants, genes, genetic markers, mapping panels, publications and contact information for the zebrafish research community. The database is populated with curated published data, user submitted data and large dataset uploads. A broad range of data types including text, images, graphical representations and genetic maps supports the data. ZFIN incorporates links to other genomic resources that provide sequence and ortholog data. Zebrafish nomenclature guidelines and an automated registration mechanism for new names are provided. Extensive usability testing has resulted in an easy to learn and use forms interface with complex searching capabilities. PMID:11125057

  19. Effective heritable gene knockdown in zebrafish using synthetic microRNAs.

    PubMed

    Giacomotto, Jean; Rinkwitz, Silke; Becker, Thomas S

    2015-01-01

    Although zebrafish is used to model human diseases through mutational and morpholino-based knockdown approaches, there are currently no robust transgenic knockdown tools. Here we investigate the knockdown efficiency of three synthetic miRNA-expressing backbones and show that these constructs can downregulate a sensor transgene with different degrees of potency. Using this approach, we reproduce spinal muscular atrophy (SMA) in zebrafish by targeting the smn1 gene. We also generate different transgenic lines, with severity and age of onset correlated to the level of smn1 inhibition, recapitulating for the first time the different forms of SMA in zebrafish. These lines are proof-of-concept that miRNA-based approaches can be used to generate potent heritable gene knockdown in zebrafish. PMID:26051838

  20. Metabolic profile analysis of zebrafish embryos.

    PubMed

    Gibert, Yann; McGee, Sean L; Ward, Alister C

    2013-01-01

    A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model. PMID:23353983

  1. Brain and gonadal aromatase as potential targets of endocrine disrupting chemicals in a model species, the zebrafish (Danio rerio).

    E-print Network

    Boyer, Edmond

    ). However, the endocrine system may also be disrupted by environmental substances through pathways1 Brain and gonadal aromatase as potential targets of endocrine disrupting chemicals in a model effects of endocrine disrupting chemicals (EDCs) on aromatase function, we first developed methods

  2. Shuttle box learning in zebrafish (Danio rerio)

    PubMed Central

    Pather, Shalini; Gerlai, Robert

    2009-01-01

    Zebrafish is used in forward genetic and drug screening and is gaining popularity in behavioral brain research but high throughput learning paradigms are lacking. The sight of conspecifics has been shown to be rewarding in zebrafish. Here, in a novel paradigm, subjects learn to respond to alternating presentation of computer animated zebrafish images. The simplicity and computerization of the paradigm will make it useful for high throughput screening. PMID:18926855

  3. Behavioral and Molecular Analysis of Nicotine-Conditioned Place Preference in Zebrafish

    PubMed Central

    Kedikian, Ximena; Faillace, Maria Paula; Bernabeu, Ramón

    2013-01-01

    Studies using mice and rats have demonstrated that nicotine induces a conditioned place preference (CPP), with more effective results obtained by using biased procedures. Zebrafish have also been used as a model system to identify factors influencing nicotine-associated reward by using an unbiased design. Here, we report that zebrafish exhibited putative nicotine biased CPP to an initially aversive compartment (nicotine-paired group). A counterbalanced nicotine-exposed control group did not show a significant preference shift, providing evidence that the preference shift in the nicotine-paired group was not due to a reduction of aversion for this compartment. Zebrafish preference was corroborated by behavioral analysis of several indicators of drug preference, such as time spent in the drug-paired side, number of entries to the drug-paired side, and distance traveled. These results provided strong evidence that zebrafish may actually develop a preference for nicotine, although the drug was administrated in an aversive place for the fish, which was further supported by molecular studies. Reverse transcription-quantitative real-time PCR analysis depicted a significant increase in the expression of