Remodeling of actin cytoskeleton in lupeol-induced B16 2F2 cell differentiation

Lupeol induces the formation of dendrites in B16 2F2 melanoma cells. The remodeling of cytoskeletal components contributes to the dendricity of melanoma cells. We studied the effects of lupeol on the remodeling of cytoplasmic filaments in B16 2F2 cells. Western blotting revealed no change in the levels of actin and tubulin. Lupeol attenuated stress fiber assembly, but did not promote the remodeling of microtubular networks. We examined the activation of cofilin, an actin-depolymerizing factor, in lupeol-treated B16 2F2 cells by western blotting. The level of phospho-cofilin was found to decrease in a time-dependent manner. Inhibition of p38 MAPK by SB203580 blocked tyrosinase induction by lupeol, but did not influence the disruption of stress fiber assembly or the dephosphorylation of cofilin. Furthermore, we studied the effects of lupeol on cell migration. At 10 microM, lupeol markedly inhibited the haptotaxis of B16 2F2 cells to fibronectin. Additionally, lupeol strongly inhibited the migration of human melanoma and neuroblastoma cells, and weakly suppressed the migration of lung adenocarcinoma cells. However, lupeol did not affect the motility of other cancer cells. The results suggest that lupeol suppresses the migration of malignant melanoma cells by disassembling the actin cytoskeleton.     

Treatment of human disease by adeno-associated viral gene transfer

During the past decade, in vivo administration of viral gene transfer vectors for treatment of numerous human diseases has been brought from bench to bedside in the form of clinical trials, mostly aimed at establishing the safety of the protocol. In preclinical studies in animal models of human disease, adeno-associated viral (AAV) vectors have emerged as a favored gene transfer system for this approach. These vectors are derived from a replication-deficient, non-pathogenic parvovirus with a single-stranded DNA genome. Efficient gene transfer to numerous target cells and tissues has been described. AAV is particularly efficient in transduction of non-dividing cells, and the vector genome persists predominantly in episomal forms. Substantial correction, and in some instances complete cure, of genetic disease has been obtained in animal models of hemophilia, lysosomal storage disorders, retinal diseases, disorders of the central nervous system, and other diseases. Therapeutic expression often lasted for months to years. Treatments of genetic disorders, cancer, and other acquired diseases are summarized in this review. Vector development, results in animals, early clinical experience, as well as potential hurdles and challenges are discussed.     

Organ-on-chip models: Implications in drug discovery and clinical applications

Before a lead compound goes through a clinical trial, preclinical studies utilize two-dimensional (2D) in vitro models and animal models to study the pharmacodynamics and pharmacokinetics of that lead compound. However, these current preclinical studies may not accurately represent the efficacy and safety of a lead compound in humans, as there has been a high failure rate of drugs that enter clinical trials. All of these failures and the associated costs demonstrate a need for more representative models of human organ systems for screening in the preclinical phase of drug development. In this study, we review the recent advances in in vitro modeling including three-dimensional (3D) organoids, 3D microfabrication, and 3D bioprinting for various organs including the heart, kidney, lung, gastrointestinal tract (intestine–gut–stomach), liver, placenta, adipose, retina, bone, and brain as well as multiorgan models. The availability of organ-on-chip models provides a wealth of opportunities to understand the pathogenesis of human diseases and provide a potentially better model to screen a drug, as these models utilize a dynamic 3D environment similar to the human body. Although there are limitations of organ-on-chip models, the emergence of new technologies have refined their capability for translational research as well as precision medicine.     

Green tea and its polyphenolic catechins: medicinal uses in cancer and noncancer applications

Can drinking several cups of green tea a day keep the doctor away? This certainly seems so, given the popularity of this practice in East Asian culture and the increased interest in green tea in the Western world. Several epidemiological studies have shown beneficial effects of green tea in cancer, cardiovascular, and neurological diseases. The health benefits associated with green tea consumption have also been corroborated in animal studies of cancer chemoprevention, hypercholesterolemia, artherosclerosis, Parkinson's disease, Alzheimer's disease, and other aging-related disorders. However, the use of green tea as a cancer chemopreventive or for other health benefits has been confounded by the low oral bioavailability of its active polyphenolic catechins, particularly epigallocatechin-3-gallate (EGCG), the most active catechin. This review summarizes the purported beneficial effects of green tea and EGCG in various animal models of human diseases. Dose-related differences in the effects of EGCG in cancer versus neurodegenerative and cardiovascular diseases, as well as discrepancies between doses used in in vitro studies and achievable plasma understanding of the in vivo effects of green tea catechins in humans, before the use of green tea is widely adopted as health-promoting measure.     

Lupeol reduces triglyceride and cholesterol synthesis in human hepatoma cells

Lupeol suppressed triglyceride and cholesterol secretion from HepG2-Lipo human hepatoma cells in a dose-dependent manner. Quantitative real-time RT-PCR analysis demonstrated that lupeol inhibited the expressions of sterol regulatory element-binding protein-1c and -2, fatty acid synthase, 3-hydroxy-3-methylglutaryl-Coenzyme A synthetase-1, and farnesyl-diphosphate farnesyl transferase-1, which are required for lipid synthesis in HepG2-Lipo cells. Furthermore, lupeol markedly inhibited apolipoproteinB-100 and microsomal triglyceride transfer protein in cells at the mRNA level. These results suggest that lupeol lowers lipid secretion from HepG2-Lipo cells by attenuating synthesis within the cells.     

Intermediate filaments of the lung

Intermediate filaments (IF), a subfamily of the cytoskeletal filaments, provide structural support to cells. Human diseases related to mutations in IF proteins in which their tissue-specific expression is reflected have been found in a broad range of patients. The properties of identified IF mutants are well-studied in vitro in cultured cells and in vivo using transgenic mice expressing IF mutants. However, the association of IF proteins with diseases of the lung is not fully studied yet. Epithelial cells in normal lung express vimentin and various keratins, and the patterns of their expression are altered depending on the progression of the lung diseases. A growing number of studies performed in alveolar epithelial cells demonstrated IF involvement in disease-related aspects including their usefulness as tumor marker, in epithelial-mesenchymal transition and cell migration. However, the lung disease-associated IF functions in animal models are poorly understood, and IF mutations associated with lung diseases in humans have not been reported. In this review, we summarize recent studies that show the significance of IF proteins in lung epithelial cells. Understanding these aspects is an important prerequisite for further investigations on the role of lung IF in animal models and human lung diseases.     

Hematopoietic cytokines--on the verge of conquering neurology

Two hematopoietic cytokines are currently gaining increasing attention within neurological research. Erythropoietin (EPO) and granulocyte-colony stimulating factor (G-CSF) have long been known for their ability to induce the proliferation of certain populations of hematopoietic lineage cells. However, it has recently been found that EPO, G-CSF, and their respective receptors are also expressed in the human central nervous system (CNS) and may be an important part of the brain's endogenous system of protection. Both hematopoietic cytokines have been shown to have neuroprotective potential in a variety of animal disease models both in vitro and in vivo, through the inhibition of apoptosis, induction of angiogenesis, exertion of anti-inflammatory and neurotrophic effects, as well as by the enhancement of neurogenesis. EPO and G-CSF have been extensively studied in the context of hematological disorders and have recently been successfully applied in the first clinical trials in stroke patients. Intravenous high-dose EPO therapy was associated with an improvement in the clinical outcome and preclinical studies with intravenous high-dose G-CSF therapy have clearly shown that it has considerable neuroprotective potential in the acute, as well as in the chronic phase of stroke. In this review, the current knowledge of the neuroprotective mechanisms of EPO and G-CSF is summarized with regard to in vitro and in vivo data. Focus is placed on the role of EPO in neurological disease models with an emphasis on its influence on functional outcome. New experimental results are assessed in detail and correlated with the findings of recent clinical studies.     

Mesenchymal stem cells in the treatment of inflammatory and autoimmune diseases in experimental animal models

Multipotent mesenchymal stromal cells [also known as mesenchymal stem cells(MSCs)] are currently being studied as a cell-based treatment for inflammatory disorders. Experimental animal models of human immune-mediated diseases have been instrumental in establishing their immunosuppressive properties. In this review, we summarize recent studies examining the effectiveness of MSCs as immunotherapy in several widely-studied animal models, including type 1 diabetes, experimental autoimmune arthritis, experimental autoimmune encephalomyelitis, inflammatory bowel disease, graft-vs-host disease, and systemic lupus erythematosus. In addition, we discuss mechanisms identified by which MSCs mediate immune suppression in specific disease models, and potential sources of functional variability of MSCs between studies.     

Genetics of interleukin 1 receptor-like 1 in immune and inflammatory diseases

Interleukin 1 receptor-like 1 (IL1RL1) is gaining in recognition due to its involvement in immune/inflammatory disorders. Well-designed animal studies have shown its critical role in experimental allergic inflammation and human in vitro studies have consistently demonstrated its up-regulation in several conditions such as asthma and rheumatoid arthritis. The ligand for IL1RL1 is IL33 which emerged as playing an important role in initiating eosinophilic inflammation and activating other immune cells resulting in an allergic phenotype.An IL1RL1 single nucleotide polymorphism (SNP) was among the most significant results of a genome-wide scan investigating eosinophil counts; in the same study, this SNP associated with asthma in 10 populations.The IL1RL1 gene resides in a region of high linkage disequilibrium containing interleukin 1 receptor genes as well as interleukin 18 receptor and accessory genes. This poses a challenge to researchers interested in deciphering genetic association signals in the region as all of the genes represent interesting candidates for asthma and allergic disease.The IL1RL1 gene and its resulting soluble and receptor proteins have emerged as key regulators of the inflammatory process implicated in a large variety of human pathologies We review the function and expression of the IL1RL1 gene. We also describe the role of IL1RL1 in asthma, allergy, cardiovascular disease, infections, liver disease and kidney disease.     

miRNAs芯片数据的聚类分析与羽扇豆醇抗癌途径预测

羽扇豆醇是一种植物天然提取物,具有高效低毒的抗癌活性,对于药物开发领域有很高价值。目前对其抗癌机制还不是十分明确。使用TaqMan MicroRNA Assay芯片技术,从羽扇豆醇处理的hela细胞中miRNA的差异表达入手,探索扇豆醇抗癌分子途径。运用聚类分析等统计学方法和targetscan,RNAfold等序列分析工具进行信号组分的预测筛选,再结合对NC-BI,Gene Ontology等大型生物信息数据库的检索,最终在生物信息学层面上,分析得到hela细胞中羽扇豆醇抗癌的分子调控途径,即以受体酪氨酸激酶ERBB4介导的,hsa-miR-23b,hsa-miR-200b等几种miRNA参与的分子通路。     

Overview of growth differentiation factor 15 in metabolic syndrome

Growth and differentiation factor 15 (GDF15) is a member of the transforming growth factor-β (TGF-β) superfamily. GDF15 has been linked with several metabolic syndrome pathologies such as obesity and cardiovascular diseases. GDF15 is considered to be a metabolic regulator, although its precise mechanisms of action remain to be determined. Glial cell-derived neurotrophic factor family receptor alpha-like (GRAL), located in the hindbrain, has been identified as the receptor for GDF15 and signals through the coreceptor receptor tyrosine kinase (RET). Administration of GDF15 analogues in preclinical studies using various animal models has consistently been shown to induce weight loss through a reduction in food intake. GDF15, therefore, represents an attractive target to combat the current global obesity epidemic. In this article, we review current knowledge on GDF15 and its involvement in metabolic syndrome.     

结核病动物模型研究进展

结核病是由结核分枝杆菌感染引起的传染病,是危害人类健康的主要传染病之一。动物模型已经成为研究人类传染病的标准化工具。虽然对于结核分枝杆菌而言并没有真正意义的动物资源,但由于不同种类的动物,对分枝杆菌的敏感性不一样,因此可以成为结核病研究的有利工具。结核病最常用的实验动物模型包括小鼠、兔和豚鼠。每种动物有其自身特点,但并不能完全模拟人类疾病。通过建立结核病的动物模型,可以大大增加我们对疾病的病因、毒力和发病机制的理解。除了这三种模型外,非人灵长类也常被用于结核病的研究。本文总结了这几种结核病模型的研究状况。     

Genetically modified pigs to model human diseases

Genetically modified mice are powerful tools to investigate the molecular basis of many human diseases. Mice are, however, of limited value for preclinical studies, because they differ significantly from humans in size, general physiology, anatomy and lifespan. Considerable efforts are, thus, being made to develop alternative animal models for a range of human diseases. These promise powerful new resources that will aid the development of new diagnostics, medicines and medical procedures. Here, we provide a comprehensive review of genetically modified porcine models described in the scientific literature: various cancers, cystic fibrosis, Duchenne muscular dystrophy, autosomal polycystic kidney disease, Huntington’s disease, spinal muscular atrophy, haemophilia A, X-linked severe combined immunodeficiency, retinitis pigmentosa, Stargardt disease, Alzheimer’s disease, various forms of diabetes mellitus and cardiovascular diseases.     

Tetracycline-inducible Expression Systems： New Strategies and Practices in the Transgenic Mouse Modeling

To accurately analyze the function of transgene(s)of interest in transgenic mice,and togenerate credible transgenic animal models for multifarious human diseases to precisely mimic human dis-ease states,it is critical to tightly regulate gene expression in the animals in a conditional manner.The abilityto turn gene expression on or off in the restricted cells or tissues at specific time permits unprecedentedflexibility in dissecting gene functions in health and disease.Pioneering studies in conditional transgene ex-pression have brought about the development of a wide variety of controlled gene expression systems,whichmeet this criterion.Among them,the tetracycline-controlled expression systems(e.g.Tet-off system andTet-on system)have been used extensively in vitro and in vivo.In recent years,some strategies derived fromtetracycline-inducible system alone,as well as the combined use of Tet-based systems and Cre/lox P switch-ing gene expression system,have been newly developed to allow more flexibility for exploring gene functionsin health and disease,and produce credible transgenic animal models for various human diseases.In thisreview these newly developed strategies are discussed.     

Promoting blood vessel growth in ischemic diseases: challenges in translating preclinical potential into clinical success

Angiogenic therapy, which involves the use of an exogenous stimulus to promote blood vessel growth, is an attractive approach for the treatment of ischemic diseases. It has been shown in animal models that the stimulation of blood vessel growth leads to the growth of the whole vascular tree, improvement of ischemic tissue perfusion and improved muscle aerobic energy metabolism. However, very few positive results have been gained from Phase 2 and 3 clinical angiogenesis trials. Many reasons have been given for the failures of clinical trials, including poor transgene expression (in gene-therapy trials) and instability of the vessels induced by therapy. In this Review, we discuss the selection of preclinical models as one of the main reasons why clinical translation has been unsuccessful thus far. This issue has received little attention, but could have had dramatic implications on the expectations of clinical trials. We highlight crucial differences between human patients and animal models with regards to blood flow and pressure, as well as issues concerning the chronic nature of ischemic diseases in humans. We use these as examples to demonstrate why the results from preclinical trials might have overestimated the efficacy of angiogenic therapies developed to date. We also suggest ways in which currently available animal models of ischemic disease could be improved to better mimic human disease conditions, and offer advice on how to work with existing models to avoid overestimating the efficacy of new angiogenic therapies.     

Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer

Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF) is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mice does not adequately replicate spontaneous bacterial infections observed in the human CF lung. Hence, several laboratories are pursuing alternative animal models of CF in larger species such as the pig, sheep, rabbits, and ferrets. Our laboratory has focused on developing the ferret as a CF animal model. Over the past few years, we have investigated several experimental parameters required for gene targeting and nuclear transfer (NT) cloning in the ferret using somatic cells. In this review, we will discuss our progress and the hurdles to NT cloning and gene-targeting that accompany efforts to generate animal models of genetic diseases in species such as the ferret.     

New insights on translational development of mesenchymal stromal cells for suppressor therapy

The discovery of the immunosuppressive properties of mesenchymal stromal cells (MSCs) has given new hope to patients suffering from autoimmune diseases as their lack of immunogenicity in addition to their immunosuppressive and regenerative properties makes them an ideal biological agent for the treatment of various disorders ranging from autoimmune diseases to tissue injury. The translational promises of a safer and more effective therapy has however suffered a setback with the recent release of the results from Phase III randomized clinical trial using MSCs for treatment of steroid refractory acute graft-versus-host disease (GvHD), which failed to meet its primary efficacy endpoint. In this review, we will address the current knowledge of the immunosuppressive mechanisms of MSCs from in vitro studies to animal models and then look upon the results obtained from human clinical trials in order to provide failure analysis of negative studies. We will conclude by proposing future directions which could help address this issue and allow rational development of MSCs as an effective and useful cell-based immunotherapeutic.     

Now and future of mouse mutagenesis for human disease models

One of the major objectives of the Human Genome Project is to understand the biological function of the gene and genome as well as to develop clinical applications for human diseases. For this purpose, the experimental validations and preclinical trails by using animal models are indispensable. The mouse (Mus musculus) is one of the best animal models because genetics is well established in the mouse and embryonic manipulation technologies are also well developed. Large-scale mouse mutagenesis projects have been conducted to de-velop various mouse models since 1997. Originally, the phenotype-driven mutagenesis with N-ethyl-N-nitrosourea (ENU) has been the major efforts internationally then knockout/conditional mouse projects and gene-driven mutagenesis have been following. At the beginning, simple monogenic traits in the experimental condition have been elucidated. Then, more complex traits with variety of environmental interactions and gene-to-gene interactions (epistasis) have been challenged with mutant mice. In addition, chromosomal substitution swains and collaborative cross strains are also available to elucidate the complex Waits in the mouse. Altogether, mouse models with mutagenesis and various laboratory strains will accelerate the studies of functional genomics in the mouse as well as in human.     

Synthesis of lupane-type saponins bearing mannosyl and 3,6-branched trimannosyl residues and their evaluation as anticancer agents

The saponins modified with mono- or trimannosyl residues can provide a convenient means of delivering drugs to certain human cells via interactions with mannose receptors. In the study reported therein, we developed a convenient approach for the synthesis of 3-O-mannoside and branched trimannoside derivatives of the saponin lupeol and of C-28 acyl esters of 3-O-acetyl-betulinic acid bearing the same mannosyl entities. Lupeol and 3-O-acetyl-betulinic acid were mannosylated with tetra-O-benzoyl- or tetra-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidates. De-esterification followed by regioselective dimannosylation of the unprotected monosaccharide derivatives with 2equiv of tetra-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate selectively yielded O-3,O-6-linked trimannosides. The cytotoxic activity of selected lupane-type saponins (derivatives of lupeol, betulinic acid, and betulin) toward normal human fibroblasts and various cancer cell lines was also compared.