Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins (HDL). It has been recognized that high levels of plasma ApoCIII constitutea risk factor for cardiovascular diseases (CVD). Elevated plasma ApoCIII level often correlates with insulin resistance, obesity, and hypertriglyceridemia. Invaluable knowledge on the roles of ApoCIIIin lipid metabolisms and CVD has been obtained from transgenic mouse models including ApoCIII knockout (KO) mice; however, it is noted that the metabolism of lipoprotein in mice is different from that of humans in many aspects. It is not known until now whether elevated plasma ApoCIII is directly atherogenic. We worked to develop ApoCIII KO rabbits in the present study based on the hypothesis that rabbits can serve as a reasonablemodelfor studying human lipid metabolism and atherosclerosis. Zinc finger nuclease (ZFN) sets targeting rabbit ApoCIIIgene were subjected to in vitro validation prior to embryo microinjection. The mRNA was injected to the cytoplasm of 35 rabbit pronuclear stage embryos, and evaluated the mutation rates at the blastocyst state. Of sixteen blastocysts that were assayed, a satisfactory 50% mutation rate (8/16) at the targeting site was achieved, supporting the use of Set 1 for in vivo experiments. Next, we microinjected 145 embryos with Set 1 mRNA, and transferred these embryos to 7 recipient rabbits. After 30 days gestation, 21 kits were born, out of which five were confirmed as ApoCIII KO rabbits after PCR sequencing assays. The KO animal rate (#KO kits/total born) was 23.8%. The overall production efficiency is 3.4% (5 kits/145 embryos transferred). The present work demonstrated that ZFN is a highly efficient method to produce KO rabbits. These ApoCIII KO rabbits are novel resources to study the roles of ApoCIII in lipid metabolisms.
19 Related JoVE Articles!
Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Institutions: The Molecular Foundry.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2
, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3
. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5
. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6
. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
Environmental Sciences, Issue 90, small and asymmetric protein structure, electron microscopy, optimized negative staining
Cholesterol Efflux Assay
Institutions: Baker IDI Heart and Diabetes Institute.
Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1
. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2
. Excessive cholesterol is more common problem 3
. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I.
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.
Medicine, Issue 61, Lipids, lipoproteins, atherosclerosis, trafficking, cholesterol
Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy
Institutions: University Medical Center Utrecht.
We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus
on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+
. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.
To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSMα operon to GFP. When these reporter strains of S. aureus
are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.
Infection, Issue 77, Immunology, Cellular Biology, Infectious Diseases, Microbiology, Genetics, Medicine, Biomedical Engineering, Bioengineering, Neutrophils, Staphylococcus aureus, Bacterial Toxins, Microscopy, Fluorescence, Time-Lapse Imaging, Phagocytosis, phenol soluble modulins, PSMs, Polymorphonuclear Neutrophils, PMNs, intracellular expression, time-lapse microscopy, flow cytometry, cell, isolation, cell culture
On-Chip Endothelial Inflammatory Phenotyping
Institutions: University of California, Davis .
Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo
. We describe a lab-on-a-chip
based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC)
, which are bound directly to cultured EC monolayers.1
These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2
inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-α. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.
Biomedical Engineering, Issue 65, Bioengineering, Immunology, Molecular Biology, Genetics, endothelial cell, monocyte arrest, microfluidics, shear stress, cytokine, atherosclerosis, inflammation
Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER
Institutions: Washington University School of Medicine, Washington University School of Medicine, Washington University School of Medicine.
As DNA sequencing technology has markedly advanced in recent years2
, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3
. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5
. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8
. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9
and a novel software package1
for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (https://www.ibridgenetwork.org/wustl/splinter). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.
Genetics, Issue 64, Genomics, Cancer Biology, Bioinformatics, Pooled DNA sequencing, SPLINTER, rare genetic variants, genetic screening, phenotype, high throughput, computational analysis, DNA, PCR, primers
Assessment of Vascular Function in Patients With Chronic Kidney Disease
Institutions: University of Colorado, Denver, University of Colorado, Boulder.
Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA
) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA
, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA
, aPWV, and vascular endothelial cell protein expression.
Medicine, Issue 88, chronic kidney disease, endothelial cells, flow-mediated dilation, immunofluorescence, oxidative stress, pulse-wave velocity
Analysis of Oxidative Stress in Zebrafish Embryos
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
Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps
Institutions: The Ohio State University, College of Medicine, The Ohio State University.
Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2
. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3
, and ultimately to oxidative stress leading to cell injury or death4
Superoxide radical anion (O2
•-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2
•- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2
•- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2
•- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6
. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2
•- in a process called uncoupling, which is believed to be caused by oxidation of heme7
or the co-factor, tetrahydrobiopterin (BH4
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9
The cyclic nitrones, 5,5-dimethyl-pyrroline-N
, the phosphoryl-substituted DEPMPO11
, and the ester-substituted, EMPO12
, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2
•- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2
is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14
Molecular Biology, Issue 66, Cellular Biology, Physics, Biophysics, spin trap, eNOS, ROS, superoxide, NO, EPR
Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease
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
Blood Collection for Biochemical Analysis in Adult Zebrafish
Institutions: Centro de Pesquisa Experimental Laboratório de Hepatologia e Gastroenterologia Experimental, Universidade Federal do Rio Grande do Sul, UFRGS. Porto Alegre, RS, Brasil.
The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1
There are relatively few publications related to adult zebrafish physiology of organs and systems2
, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3
. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4
who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5
. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6
. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7
. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8
. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started.
Recently, Gupta et al.9
described how to dissect adult zebrafish organs, Kinkle et al.10
described how to perform intraperitoneal injections, and Pugach et al.11
described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.
Biochemistry, Issue 63, Developmental Biology, Zebrafish, Zebrafish blood, Hematologic, Biochemical analysis
In Vivo Modeling of the Morbid Human Genome using Danio rerio
Institutions: Duke University Medical Center, Duke University, Duke University Medical Center.
Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo
complementation in zebrafish. Zebrafish (Danio rerio
) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo,
and can be genetically manipulated.1
These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
Institutions: University of Maryland, University of Maryland.
Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200 nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125
I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125
I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.
Bioengineering, Issue 80, Antigens, Enzymes, Biological Therapy, bioengineering (general), Pharmaceutical Preparations, Macromolecular Substances, Therapeutics, Digestive System and Oral Physiological Phenomena, Biological Phenomena, Cell Physiological Phenomena, drug delivery systems, targeted nanocarriers, transcellular transport, epithelial cells, tight junctions, transepithelial electrical resistance, endocytosis, transcytosis, radioisotope tracing, immunostaining
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Assessing Murine Resistance Artery Function Using Pressure Myography
Institutions: Massachusetts General Hospital, Harvard Medical School.
Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo
behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd
order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the α1
subunit of soluble guanylate cyclase (sGCα1-/-
) are hypertensive when on a 129S6 (S6) background (sGCα1-/-S6
) but not when on a C57BL/6 (B6) background (sGCα1-/-B6
). Using pressure myography, we demonstrate that sGCα1
-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGCα1-/-S6
than in sGCα1-/-B6
mice, likely contributing to the higher blood pressure in sGCα1-/-S6
than in sGCα1-/-B6
Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.
Physiology, Issue 76, Biomedical Engineering, Medicine, Biophysics, Bioengineering, Anatomy, Cardiology, Hematology, Vascular Diseases, Cardiovascular System, mice, resistance arteries, pressure myography, myography, myograph, NO-cGMP signaling, signaling, animal model
Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research
Institutions: University of California, San Francisco, Veterans Affairs Medical Center, San Francisco, Veterans Affairs Medical Center, San Francisco.
The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo
method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.
Medicine, Issue 92, endothelial function, endothelial dysfunction, brachial artery, peripheral artery disease, ultrasound, vascular, endothelium, cardiovascular disease.
Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice
Institutions: Hunter College, CUNY.
Efficient expression of transgenes in vivo
is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).
Genetics, Issue 87, hydrodynamic gene delivery, hydrodynamics-based transfection, mouse, gene therapy, plasmid DNA, transient gene expression, tail vein injection
The α-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva
Institutions: Weill Cornell Medical College , University of Missouri-Kansas City-School of Dentistry, University of Missouri Kansas City- School of Pharmacy, Bamenda, NWP, Cameroon, Mezam Polyclinic HIV/AIDS Treatment Center, Cameroon, Institute for Human Genetics and Biochemistry.
There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1
. Thus, identification of cell-free correlates that directly regulate the number of CD4+
T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+
T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2
. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS
) and the HLECS
-reactive active α1
proteinase inhibitor (α1
. In HIV-1 disease, α1
PI is inactivated due to disease processes 4
. In the early asymptomatic categories of HIV-1 disease, active α1
PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 μM, and to achieve normal levels during the symptomatic categories4, 5
. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with >220 CD4 cells/μl, CD4 counts were correlated with serum levels of active α1
=0.93, p<0.0001, n=26) and inactive α1
=0.91, p<0.0001, n=26) 5
. Administration of α1
PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting α1
PI participates in regulating the number of CD4+
T cells in blood 3
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active α1
PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum α1
PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to α1
PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3
NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active α1
PI in saliva. The resulting inhibition of PPE by active α1
PI can be measured by adding the PPE substrate SA3
NA. (Figure 1)
. Although CD4 counts are measured in terms of blood volume (CD4 cells/μl), the concentration of α1
PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6
. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7
. Thus, active α1
PI in saliva is calculated as a ratio to saliva protein content and is termed the α1
PI Index. Results presented herein demonstrate that the α1
PI Index provides an accurate and precise physiologic method for calculating CD4 counts.
Medicine, Issue 63, CD4 count, saliva, antitrypsin, hematopoiesis, T cells, HIV/AIDS, clinical
A Zebrafish Model of Diabetes Mellitus and Metabolic Memory
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