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Impact of rituximab on immunoglobulin concentrations and B cell numbers after cyclophosphamide treatment in patients with ANCA-associated vasculitides.
To assess the impact of immunosuppressive therapy with cyclophosphamide (CYC) and rituximab (RTX) on serum immunoglobulin (Ig) concentrations and B lymphocyte counts in patients with ANCA-associated vasculitides (AAVs).
Authors: Hilary C. Rees, Voichita Ianas, Patricia McCracken, Shannon Smith, Anca Georgescu, Tirdad Zangeneh, Jane Mohler, Stephen A. Klotz.
Published: 07-24-2013
A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2. In HIV infection the syndrome occurs at a younger age. HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal. The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.
23 Related JoVE Articles!
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Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade
Authors: Jeff K. Davies, Christine M. Barbon, Annie R. Voskertchian, Lee M. Nadler, Eva C. Guinan.
Institutions: Dana Farber Cancer Institute, Brigham and Womens Hospital, Dana Farber Cancer Institute, Children’s Hospital Boston.
Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.
Immunology, Issue 49, Allogeneic stem cell transplantation, alloreactivity, Graft-versus-Host Disease, T cell costimulation, anergy, mixed lymphocyte reaction.
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The α-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva
Authors: Cynthia L. Bristow, Mariya A. Babayeva, Rozbeh Modarresi, Carole P. McArthur, Santosh Kumar, Charles Awasom, Leo Ayuk, Annette Njinda, Paul Achu, Ronald Winston.
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 α1proteinase inhibitor (α1PI, α1antitrypsin, SerpinA1)3. In HIV-1 disease, α1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active α1PI 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 α1PI (r2=0.93, p<0.0001, n=26) and inactive α1PI (r2=0.91, p<0.0001, n=26) 5. Administration of α1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting α1PI 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 α1PI 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 α1PI 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 α1PI 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 (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active α1PI in saliva. The resulting inhibition of PPE by active α1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/μl), the concentration of α1PI 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 α1PI in saliva is calculated as a ratio to saliva protein content and is termed the α1PI Index. Results presented herein demonstrate that the α1PI 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
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The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation
Authors: Benjamin J. C. Quah, Christopher R. Parish.
Institutions: John Curtin School of Medical Research, Australian National University.
Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.
Immunology, Issue 44, carboxyfluorescein diacetate succinimidyl ester (CFSE), labeling, lymphocytes, proliferation.
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Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions
Authors: Kevin Wilhelmsen, Katherine Farrar, Judith Hellman.
Institutions: University of California, San Francisco, University of California, San Francisco.
The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.
Immunology, Issue 78, Cellular Biology, Infection, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Endothelium, Vascular, Neutrophils, Inflammation, Inflammation Mediators, Neutrophil, Leukocyte Adhesion, Endothelial cells, assay
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The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism
Authors: Sara Tremblay, Vincent Beaulé, Sébastien Proulx, Louis-Philippe Lafleur, Julien Doyon, Małgorzata Marjańska, Hugo Théoret.
Institutions: University of Montréal, McGill University, University of Minnesota.
Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.
Neuroscience, Issue 93, proton magnetic resonance spectroscopy, transcranial direct current stimulation, primary motor cortex, GABA, glutamate, stroke
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Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications
Authors: Lori E. Lowes, Benjamin D. Hedley, Michael Keeney, Alison L. Allan.
Institutions: London Health Sciences Centre, Western University, London Health Sciences Centre, Lawson Health Research Institute, Western University.
The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.
Medicine, Issue 84, Metastasis, circulating tumor cells (CTCs), CellSearch system, user defined marker characterization, in vivo, preclinical mouse model, clinical research
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Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma
Authors: Ayman I. Omar.
Institutions: Southern Illinois University School of Medicine.
A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1. Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians’ choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10. There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.
Medicine, Issue 92, Tumor Treating Fields, TTF System, TTF Therapy, Recurrent Glioblastoma, Bevacizumab, Brain Tumor
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Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins
Authors: Ruben Magni, Benjamin H. Espina, Lance A. Liotta, Alessandra Luchini, Virginia Espina.
Institutions: George Mason University, Ceres Nanosciences.
Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.
Bioengineering, Issue 90, biomarker, hydrogel, low abundance, mass spectrometry, nanoparticle, plasma, protein, urine
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Methods to Assess Subcellular Compartments of Muscle in C. elegans
Authors: Christopher J. Gaffney, Joseph J. Bass, Thomas F. Barratt, Nathaniel J. Szewczyk.
Institutions: University of Nottingham.
Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.
Developmental Biology, Issue 93, Physiology, C. elegans, muscle, mitochondria, sarcomeres, ageing
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An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Authors: Justen Manasa, Siva Danaviah, Sureshnee Pillay, Prevashinee Padayachee, Hloniphile Mthiyane, Charity Mkhize, Richard John Lessells, Christopher Seebregts, Tobias F. Rinke de Wit, Johannes Viljoen, David Katzenstein, Tulio De Oliveira.
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
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HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL
Authors: Yen-Ling Chiu, Jonathan P. Schneck, Mathias Oelke.
Institutions: Johns Hopkins University, Far-Eastern Memorial Hospital, Johns Hopkins University, Johns Hopkins University.
CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNFα and IFNγ (Figure 3).
Immunology, Issue 50, immunotherapy, adoptive T cell therapy, CD8+ T cells, HLA-A2-Ig, CMV, aAPC, DC
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Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization
Authors: Julie Chaumeil, Mariann Micsinai, Jane A. Skok.
Institutions: New York University School of Medicine, New York University Center for Health Informatics and Bioinformatics, NYU Cancer Institute, Yale University School of Medicine .
Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.
Genetics, Issue 72, Molecular Biology, Bioinformatics, Cancer Biology, Pathology, Biomedical Engineering, Immunology, Intranuclear Space, Nuclear Matrix, Fluorescence in situ Hybridization, FISH, 3D DNA FISH, DNA, immunofluorescence, immuno-FISH, 3D microscopy, Nuclear organization, interphase nuclei, chromatin modifications
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Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR
Authors: Alessandra Sottini, Federico Serana, Diego Bertoli, Marco Chiarini, Monica Valotti, Marion Vaglio Tessitore, Luisa Imberti.
Institutions: Spedali Civili di Brescia.
T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.
Immunology, Issue 94, B lymphocytes, primary immunodeficiency, real-time PCR, immune recovery, T-cell homeostasis, T lymphocytes, thymic output, bone marrow output
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Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development
Authors: Daniel P. Vang, Gregory T. Wurz, Stephen M. Griffey, Chiao-Jung Kao, Audrey M. Gutierrez, Gregory K. Hanson, Michael Wolf, Michael W. DeGregorio.
Institutions: University of California, Davis, University of California, Davis, Merck KGaA, Darmstadt, Germany.
A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-γ)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.
Medicine, Issue 80, Urinary Bladder, Animals, Genetically Modified, Cancer Vaccines, Immunotherapy, Animal Experimentation, Models, Neoplasms Bladder Cancer, C57BL/6 Mouse, MUC1, Immunotherapy, Preclinical Model
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Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells
Authors: James E. East, Wenji Sun, Tonya J. Webb.
Institutions: University of Maryland .
Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCRα chain rearrangement: Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβ chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with α-Galactosylceramide (α-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.
Immunology, Issue 70, Medicine, Molecular Biology, Cellular Biology, Microbiology, Cancer Biology, Natural killer T cells, in vitro expansion, cancer immunology, artificial antigen presenting cells, adoptive transfer
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Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield
Authors: Andrea M. Collins, Jamie Rylance, Daniel G. Wootton, Angela D. Wright, Adam K. A. Wright, Duncan G. Fullerton, Stephen B. Gordon.
Institutions: National Institute for Health Research, Royal Liverpool and Broadgreen University Hospital Trust, Liverpool School of Tropical Medicine, University of Liverpool, Royal Liverpool and Broadgreen University Hospital Trust, University Hospital Aintree.
We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung. BAL is internationally accepted for research purposes and since 1999 the technique has been performed in > 1,000 subjects in the UK and Malawi by our group. Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage. Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria. This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.
Medicine, Issue 85, Research bronchoscopy, bronchoalveolar lavage (BAL), fiberoptic bronchoscopy, lymphocyte, macrophage
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Isolation of Precursor B-cell Subsets from Umbilical Cord Blood
Authors: Md Almamun, Jennifer L. Schnabel, Susan T. Gater, Jie Ning, Kristen H. Taylor.
Institutions: University of Missouri-Columbia, University of Missouri-Columbia.
Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation. This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.
Immunology, Issue 74, Cellular Biology, Molecular Biology, Genetics, Medicine, Biomedical Engineering, Anatomy, Physiology, Neoplasms, Precursor B-cells, B cells, Umbilical cord blood, Cell sorting, DNA methylation, Tissue specific expression, labeling, enrichment, isolation, blood, tissue, cells, flow cytometry
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Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells
Authors: Christian Schütz, Martin Fleck, Jonathan P. Schneck, Mathias Oelke.
Institutions: Johns Hopkins University, University of Regensburg, Asklepios Medical Center.
Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.
Immunology, Issue 90, Autoimmunity, Apoptosis, antigen-specific CD8+ T cells, HLA-A2-Ig, Fas/FasL, KaAPC
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High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum
Authors: Mazen Amatoury, Vera Merheb, Jessica Langer, Xin Maggie Wang, Russell Clive Dale, Fabienne Brilot.
Institutions: The University of Sydney, Westmead Millennium Institute for Medical Research.
Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.
Medicine, Issue 81, Flow cytometry, cell-based assay, autoantibody, high-throughput sampler, autoimmune CNS disease
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A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Authors: Lisa M. Weatherly, Rachel H. Kennedy, Juyoung Shim, Julie A. Gosse.
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here. Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
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Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting
Authors: Michelle L. Wunderlich, Megan E. Dodge, Rajeev K. Dhawan, William R. Shek.
Institutions: Charles River .
To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (αIg) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The αIg control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.
Basic Protocols, Issue 58, Multiplexed Fluorometric ImmunoAssay, MFIA, bead, serum, BAG, SPE, aggregate, microarray
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Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation
Authors: Mitchell Kronenberg.
Institutions: La Jolla Institute for Allergy and Immunology.
Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens. Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells. The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells. Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy. Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.
Immunology, Issue 10, Natural Killer T cells, NKT cells, CD1 Tetramers, antigen presentation, glycolipid antigens, CD1d, Mucosal Immunity, Translational Research
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Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture
Authors: Christine Beeton, Adriana Garcia, K. George Chandy.
Institutions: University of California, Irvine (UCI).
Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3. Here, we present two techniques for drawing blood from rats, each with its specific applications. Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6. Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.
Immunology, Issue 7, Blood Sampling Method, Rodent, Blood Draw, Heart, Pharmacokinetics, Serum, Plasma, Blood Collection, Bleeding, Hematology
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