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Pubmed Article
Blockade of the programmed death-1 (PD1) pathway undermines potent genetic protection from type 1 diabetes.
PLoS ONE
PUBLISHED: 01-01-2014
Inhibition of PD1-PDL1 signaling in NOD mice accelerates onset of type 1 diabetes implicating this pathway in suppressing the emergence of pancreatic beta cell reactive T-cells. However, the molecular mechanism by which PD1 signaling protects from type 1 diabetes is not clear. We hypothesized that differential susceptibility of Idd mouse strains to type 1 diabetes when challenged with anti PDL1 will identify genomic loci that collaborate with PD1 signaling in suppressing type 1 diabetes.
ABSTRACT
The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.
24 Related JoVE Articles!
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Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets
Authors: Patrick T. Fueger, Angelina M. Hernandez, Yi-Chun Chen, E. Scott Colvin.
Institutions: Indiana University School of Medicine, Indiana University School of Medicine.
Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes 1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes. Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa. By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass 4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets 4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function 5,6,8,9,16,17.
Medicine, Issue 64, Physiology, beta cell, gene expression, islet, diabetes, insulin secretion, proliferation, adenovirus, rat
4080
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Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Authors: James Smadbeck, Meghan B. Peterson, George A. Khoury, Martin S. Taylor, Christodoulos A. Floudas.
Institutions: Princeton University.
The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity. To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
50476
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Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue
Authors: Elaine Bigelow, Katherine M. Bever, Haiying Xu, Allison Yager, Annie Wu, Janis Taube, Lieping Chen, Elizabeth M. Jaffee, Robert A. Anders, Lei Zheng.
Institutions: The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, Yale School of Medicine, The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine.
B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8. Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12. In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.
Cancer Biology, Issue 71, Medicine, Immunology, Biochemistry, Molecular Biology, Cellular Biology, Chemistry, Oncology, immunohistochemistry, B7-H1 (PD-L1), pancreatic adenocarcinoma, pancreatic cancer, pancreas, tumor, T-cell immunity, cancer
4059
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Analysis of Oxidative Stress in Zebrafish Embryos
Authors: Vera Mugoni, Annalisa Camporeale, Massimo M. Santoro.
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
51328
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Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease
Authors: Vivian P. Chou, Novie Ko, Theodore R. Holman, Amy B. Manning-Boğ.
Institutions: SRI International, University of California-Santa Cruz.
Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer's disease, but its effects in Parkinson's disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone. To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder. Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD. The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates. This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.
Medicine, Issue 83, MPTP, dopamine, Iba1, TH, GFAP, lipoxygenase, transgenic, gene-environment interactions, mouse, Parkinson's disease, neurodegeneration, neuroinflammation
50960
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Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen
Authors: Dennis Ma, Jonathan Collins, Tomas Hudlicky, Siyaram Pandey.
Institutions: University of Windsor, Brock University.
Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.
Cancer Biology, Issue 63, Medicine, Biochemistry, Breast adenocarcinoma, neuroblastoma, tamoxifen, combination therapy, apoptosis, autophagy
3586
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Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells
Authors: Jennifer L. Fogel, Thu Zan Tun Thein, Francesca V. Mariani.
Institutions: University of Southern California.
Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells. Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death. To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase. In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.
Developmental Biology, Issue 68, Molecular Biology, Stem Cell Biology, Cellular Biology, mouse embryo, embryonic stem cells, lysosome, programmed cell death, imaging, sonic hedgehog
4254
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Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3
Authors: Shelly Sorrells, Cristhian Toruno, Rodney A. Stewart, Cicely Jette.
Institutions: University of Utah.
Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.
Developmental Biology, Issue 82, zebrafish, embryo, apoptosis, Caspase 3, Immunofluorescence, whole-mount, cell death
51060
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In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function
Authors: Filippos Porichis, Meghan G. Hart, Jennifer Zupkosky, Lucie Barblu, Daniel E. Kaufmann.
Institutions: The Ragon Institute of MGH, MIT and Harvard, Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM).
T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects. Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10Rα and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 °C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.
Immunology, Issue 80, Virus Diseases, Immune System Diseases, HIV, CD4 T cell, CD8 T cell, antigen-presenting cell, Cytokines, immunoregulatory networks, PD-1: IL-10, exhaustion, monocytes
50821
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Creating Objects and Object Categories for Studying Perception and Perceptual Learning
Authors: Karin Hauffen, Eugene Bart, Mark Brady, Daniel Kersten, Jay Hegdé.
Institutions: Georgia Health Sciences University, Georgia Health Sciences University, Georgia Health Sciences University, Palo Alto Research Center, Palo Alto Research Center, University of Minnesota .
In order to quantitatively study object perception, be it perception by biological systems or by machines, one needs to create objects and object categories with precisely definable, preferably naturalistic, properties1. Furthermore, for studies on perceptual learning, it is useful to create novel objects and object categories (or object classes) with such properties2. Many innovative and useful methods currently exist for creating novel objects and object categories3-6 (also see refs. 7,8). However, generally speaking, the existing methods have three broad types of shortcomings. First, shape variations are generally imposed by the experimenter5,9,10, and may therefore be different from the variability in natural categories, and optimized for a particular recognition algorithm. It would be desirable to have the variations arise independently of the externally imposed constraints. Second, the existing methods have difficulty capturing the shape complexity of natural objects11-13. If the goal is to study natural object perception, it is desirable for objects and object categories to be naturalistic, so as to avoid possible confounds and special cases. Third, it is generally hard to quantitatively measure the available information in the stimuli created by conventional methods. It would be desirable to create objects and object categories where the available information can be precisely measured and, where necessary, systematically manipulated (or 'tuned'). This allows one to formulate the underlying object recognition tasks in quantitative terms. Here we describe a set of algorithms, or methods, that meet all three of the above criteria. Virtual morphogenesis (VM) creates novel, naturalistic virtual 3-D objects called 'digital embryos' by simulating the biological process of embryogenesis14. Virtual phylogenesis (VP) creates novel, naturalistic object categories by simulating the evolutionary process of natural selection9,12,13. Objects and object categories created by these simulations can be further manipulated by various morphing methods to generate systematic variations of shape characteristics15,16. The VP and morphing methods can also be applied, in principle, to novel virtual objects other than digital embryos, or to virtual versions of real-world objects9,13. Virtual objects created in this fashion can be rendered as visual images using a conventional graphical toolkit, with desired manipulations of surface texture, illumination, size, viewpoint and background. The virtual objects can also be 'printed' as haptic objects using a conventional 3-D prototyper. We also describe some implementations of these computational algorithms to help illustrate the potential utility of the algorithms. It is important to distinguish the algorithms from their implementations. The implementations are demonstrations offered solely as a 'proof of principle' of the underlying algorithms. It is important to note that, in general, an implementation of a computational algorithm often has limitations that the algorithm itself does not have. Together, these methods represent a set of powerful and flexible tools for studying object recognition and perceptual learning by biological and computational systems alike. With appropriate extensions, these methods may also prove useful in the study of morphogenesis and phylogenesis.
Neuroscience, Issue 69, machine learning, brain, classification, category learning, cross-modal perception, 3-D prototyping, inference
3358
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A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation
Authors: Erik J. Zmuda, Catherine A. Powell, Tsonwin Hai.
Institutions: The Ohio State University, The Ohio State University, The Ohio State University.
Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation. Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.
Medicine, Issue 50, islet isolation, islet transplantation, diabetes, murine, pancreas
2096
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Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes
Authors: Jing Chen, Scott Grieshaber, Clayton E. Mathews.
Institutions: University of Florida.
Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr. As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37°C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes. Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.
Immunology, Issue 52, cell, Type 1 Diabetes, Autoimmunity, Cytotoxic T Lymphocyte
2724
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Staining Protocols for Human Pancreatic Islets
Authors: Martha L. Campbell-Thompson, Tiffany Heiple, Emily Montgomery, Li Zhang, Lynda Schneider.
Institutions: University of Florida .
Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region. The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&E and IHC stains. The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.
Medicine, Issue 63, Physiology, type 1 diabetes, histology, H&E, immunohistochemistry, insulin, beta-cells, glucagon, alpha-cells, pancreatic polypeptide, islet, pancreas, spleen, organ donor
4068
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Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells
Authors: Charles Zhang, Arthur T. Suckow, Steven D. Chessler.
Institutions: University of California, San Diego, Janssen Research & Development, University of California, San Diego.
Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function. Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions. Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.
Medicine, Issue 76, Cellular Biology, Molecular Biology, Biomedical Engineering, Immunology, Hepatology, Islets of Langerhans, islet, Insulin, Coculture, pancreatic beta cells, INS-1 cells, extracellular contact, transmembrane protein, transcellular interactions, insulin secretion, diabetes, cell culture
50365
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Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)
Authors: Sandra Deliard, Jianhua Zhao, Qianghua Xia, Struan F.A. Grant.
Institutions: Children's Hospital of Philadelphia Research Institute, University of Pennsylvania .
ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions. For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers. Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.
Molecular Biology, Issue 74, Genetics, Biochemistry, Microbiology, Medicine, Proteins, DNA-Binding Proteins, Transcription Factors, Chromatin Immunoprecipitation, Genes, chromatin, immunoprecipitation, ChIP, DNA, PCR, sequencing, antibody, cross-link, cell culture, assay
50286
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Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research
Authors: Anna U. Eriksson, Christoffer Svensson, Andreas Hörnblad, Abbas Cheddad, Elena Kostromina, Maria Eriksson, Nils Norlin, Antonello Pileggi, James Sharpe, Fredrik Georgsson, Tomas Alanentalo, Ulf Ahlgren.
Institutions: Umeå University, University of Miami,, Catalan Institute of Research and Advanced Studies, Umeå University.
By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.
Medicine, Issue 71, Biomedical Engineering, Cellular Biology, Molecular Biology, Biophysics, Pancreas, Islets of Langerhans, Diabetes Mellitus, Imaging, Three-Dimensional, Optical Projection Tomography, Beta-cell Mass, Near Infrared, Computational Processing
50238
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Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters
Authors: Ana D. Lopez, Ayse G. Kayali, Alberto Hayek, Charles C. King.
Institutions: University of California, San Diego.
For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.
Medicine, Issue 87, human fetal pancreas, islet cell cluster (ICC), transplantation, immunofluorescence, endocrine cell proliferation, differentiation, C-peptide
50796
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A Zebrafish Model of Diabetes Mellitus and Metabolic Memory
Authors: Robert V. Intine, Ansgar S. Olsen, Michael P. Sarras Jr..
Institutions: Rosalind Franklin University of Medicine and Science, Rosalind Franklin University of Medicine and Science.
Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined. We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.
Medicine, Issue 72, Genetics, Genomics, Physiology, Anatomy, Biomedical Engineering, Metabolomics, Zebrafish, diabetes, metabolic memory, tissue regeneration, streptozocin, epigenetics, Danio rerio, animal model, diabetes mellitus, diabetes, drug discovery, hyperglycemia
50232
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A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
Authors: Joshua C. Neuman, Nathan A. Truchan, Jamie W. Joseph, Michelle E. Kimple.
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison, University of Waterloo.
Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.
Physiology, Issue 88, islet, isolation, insulin secretion, β-cell, diabetes, cAMP production, mouse
50374
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Murine Pancreatic Islet Isolation
Authors: Gregory L. Szot, Pavel Koudria, Jeffrey A. Bluestone.
Institutions: University of California, San Francisco - UCSF.
Issue 7, Immunology, Pancreatic Islets, Cell Culture, Diabetes, Ficoll Gradient
255
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Extraction of Tissue Antigens for Functional Assays
Authors: Andra Necula, Rochna Chand, Batool Albatat, Stuart I. Mannering.
Institutions: St. Vincent's Institute of Medical Research, University of Melbourne.
Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.
Immunology, Issue 67, Medicine, Physiology, Biomedical Engineering, Tissue Engineering, tissue lysate, functional assay, extraction, autoimmune disease, T cells, spleen
4230
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Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice
Authors: Gregory L. Szot, Pavel Koudria, Jeffrey A. Bluestone.
Institutions: University of California, San Francisco - UCSF.
Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse. Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped. Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.
Immunology, Issue 9, Mouse, Pancreas, Kidney, Diabetes, Transplantation, Islets, Translational Research
404
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Regulatory T cells: Therapeutic Potential for Treating Transplant Rejection and Type I Diabetes
Authors: Jeffry A. Bluestone.
Institutions: University of California, San Francisco - UCSF.
Issue 7, Immunology, Pancreatic Islets, Cell Culture, Diabetes, Ficoll Gradient, Translational Research
257
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Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection
Authors: Sang Mo Kang.
Institutions: University of California, San Francisco - UCSF.
Issue 7, Immunology, Heterotopic Heart Transplant, Small Bowel Transplant, Transplant Rejection, T regs, Diabetes, Autoimmune Disease, Translational Research
256
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