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Journal of Immunological Methods

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https://www.readbyqxmd.com/read/29337047/imaging-flow-cytometry-a-method-for-examining-dynamic-native-foxo1-localization-in-human-lymphocytes
#1
Molly K Hritzo, Jean-Paul Courneya, Amit Golding
While flow cytometry can reliably assess surface and intracellular marker expression within small cell populations, it does not provide any information on protein localization. Several key transcription factors (TF) downstream of lymphocyte surface receptors are regulated by nuclear versus cytoplasmic localization, and one such TF is Forkhead box O1 (FOXO1). FOXO1 integrates antigen-binding, co-receptor activation and metabolic signals in lymphocytes, leading to proliferation and differentiation. Importantly, the nuclear or cytoplasmic localization of FOXO1 is key for gene expression leading to different lymphocyte phenotypes...
January 11, 2018: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29278684/isolation-of-human-lymphocytes-with-high-yield-and-viability-from-the-gastrointestinal-and-female-reproductive-tract-of-a-humanized-drag-mouse
#2
Atef Allam, Kristina K Peachman, Rodrigo Aguilera-Olvera, Sofia Casares, Mangala Rao
The mucosal tissues of the gut and female reproductive tract (FRT) are susceptible to pathogen infections including bacteria, viruses, and parasites, and are also the targets for immune disorders such as Crohn's disease, inflammatory bowel disease (IBD), and many types of cancers. However, the role of the mucosal immune cells to control these diseases is largely unknown. The limited availability of human mucosal biopsy tissue and the low number of cells that can be isolated from these tissues hampers the characterization of the phenotype and function of human mucosal immune cell subsets...
December 23, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29277486/development-of-a-high-throughput-bead-based-assay-system-to-measure-hiv-1-specific-immune-signatures-in-clinical-samples
#3
Thomas Liechti, Claus Kadelka, Hanna Ebner, Nikolas Friedrich, Roger D Kouyos, Huldrych F Günthard, Alexandra Trkola
The monitoring and assessment of a broadly neutralizing antibody (bnAb) based HIV-1 vaccine require detailed measurements of HIV-1 binding antibody responses to support the detection of correlates of protection. Here we describe the development of a flexible, high-throughput microsphere based multiplex assay system that allows monitoring complex binding antibody signatures. Studying a panel of 13 HIV-1 antigens in a parallel assessment of different IgG subclasses (IgG1, IgG2 and IgG3) we demonstrate the potential of our strategy...
December 22, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29258749/a-simple-method-for-measuring-immune-complex-mediated-fc-gamma-receptor-dependent-antigen-specific-activation-of-primary-human-t-cells
#4
Junker Fabian, Krishnarajah Sinduya, Qureshi Omar, Humphreys David, Fallah-Arani Farnaz
Immune complex (IC) deposition of IgG containing autologous antigens has been observed in autoimmunity. This can lead to IC-mediated antigen uptake and presentation by antigen presenting cells (APC) driving T cell dependent inflammation. IgG receptors (FcγRs) have been suggested to be involved in this process. Since ICs have been linked to autoimmune diseases, interfering with IC mediated effects on APCs and subsequent autoimmune T cell activation via FcγR blockade may be therapeutically beneficial. However, this is currently challenging due to a lack of translatable animal models and specific human in vitro assays to study IC-driven T cell responses...
December 16, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29253503/facs-isolation-of-live-mouse-eosinophils-at-high-purity-via-a-protocol-that-does-not-target-siglec-f
#5
Wendy E Geslewitz, Caroline M Percopo, Helene F Rosenberg
Flow cytometry protocols designed to identify mouse eosinophils typically target Siglec F, an α-2,3-sialic acid binding transmembrane protein expressed universally on cells of this lineage. While a convenient target, antibody-mediated ligation of Siglec F induces eosinophil apoptosis, which limits its usefulness for isolations that are to be followed by functional and/or gene expression studies. We present here a method for FACS isolation which does not target Siglec F and likewise utilizes no antibodies targeting IL5Rα (CD125) or CCR3...
December 15, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29175391/integrated-functional-and-mass-spectrometry-based-flow-cytometric-phenotyping-to-describe-the-immune-microenvironment-in-acute-myeloid-leukemia
#6
Adam J Lamble, Matthew Dietz, Ted Laderas, Shannon McWeeney, Evan F Lind
A hallmark of the development of cancer is its ability to avoid detection and elimination by the immune system. There are many identified mechanisms of this immune evasion that can be measured both phenotypically and functionally. Functional studies directly show the ability of the tumor microenvironment to suppress immune responses, typically measured as lymphocyte proliferation, cytokine production or killing ability. While a direct measurement of function is ideal, these assays require ex vivo activation which may not accurately mimic in vivo conditions...
November 23, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29174717/comparison-of-cytof-assays-across-sites-results-of-a-six-center-pilot-study
#7
Michael D Leipold, Gerlinde Obermoser, Craig Fenwick, Katja Kleinstuber, Narges Rashidi, John P McNevin, Allison N Nau, Lisa E Wagar, Virginie Rozot, Mark M Davis, Stephen DeRosa, Giuseppe Pantaleo, Thomas J Scriba, Bruce D Walker, Lars R Olsen, Holden T Maecker
For more than five years, high-dimensional mass cytometry has been employed to study immunology. However, these studies have typically been performed in one laboratory on one or few instruments. We present the results of a six-center study using healthy control human peripheral blood mononuclear cells (PBMCs) and commercially available reagents to test the intra-site and inter-site variation of mass cytometers and operators. We used prestained controls generated by the primary center as a reference to compare against samples stained at each individual center...
November 22, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29162425/usefulness-of-basophil-activation-test-for-the-diagnosis-of-ige-mediated-hypersensitivity-to-tetanus-toxoid-vaccine
#8
Blanca Herreros, Yesica Méndez, Francisco Feo-Brito, José Miguel Urra
A great number of vaccinated patients develop specific anti-tetanus toxoid IgE, but usually do not undergo any adverse effect. Most of the allergic reactions to tetanus toxoid vaccine usually present with unspecific symptoms of local inflammation. In the presence of severe reactions, and in a special way if the vaccine is provided together with other drugs, it is difficult to establish which is the harmful drug responsible for IgE-mediated adverse reaction. A patient with an anaphylactic reaction after the administration of Toxoid Tetanic (TT) along with several drugs is described...
November 18, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29154914/how-to-make-usage-of-the-standardized-euroflow-8-color-protocols-possible-for-instruments-of-different-manufacturers
#9
Michaela Nováková, Hana Glier, Naděžda Brdičková, Marcela Vlková, Ana Helena Santos, Margarida Lima, Mikael Roussel, Juan Flores-Montero, Tomasz Szczepanski, Sebastian Böttcher, Vincent H J van der Velden, Paula Fernandez, Ester Mejstříková, Leire Burgos, Bruno Paiva, Jacques J M van Dongen, Alberto Orfao, Tomáš Kalina
A critical component of the EuroFlow standardization of leukemia/lymphoma immunophenotyping is instrument setup. Initially, the EuroFlow consortium developed a step-by-step standard operating protocol for instrument setup of ≥8-color flow cytometers that were available in 2006, when the EuroFlow activities started. Currently, there are 14 instruments from 9 manufacturers capable of 3-laser excitation and ≥8 color measurements. The specific adaptations required in the instrument set-up to enable them to acquire the standardized 8-color EuroFlow protocols are described here...
November 15, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29154772/endogenous-peroxidases-in-sputum-interfere-with-horse-radish-peroxidase-based-elisas
#10
Lisa Ha-Yeon Kim, Karin Plaza, Sruthi R Thomas, Christina Draijer, Katherine Radford, Marc Peters-Golden, Manali Mukherjee, Parameswaran Nair
Peroxidase-based immunoassays are commonly used for detecting inflammatory mediators in biological samples. We suggest caution while interpreting assays particularly in sputum samples that have endogenous peroxidases like eosinophil peroxidase (EPX), which may interact with a horseradish peroxidase (HRP)-based ELISA. Using IL-8 as an example, we demonstrate that values generated with an HRP-ELISA (n=47) show significant positive correlation with the sputum EPX content (r=0.6, P=0.0004), which can be misconstrued to be affiliated with an eosinophilic event...
November 14, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29154771/in-vitro-blockade-of-the-cd4-receptor-co-signal-in-antigen-specific-t-cell-stimulation-cultures-induces-the-outgrowth-of-potent-cd4-independent-t-cell-effectors
#11
Sarah Vatter, Maximilian Schmid, Claudia Gebhard, Carina Mirbeth, Sebastian Klobuch, Michael Rehli, Wolfgang Herr, Simone Thomas
T-cell receptor (TCR) redirected T cells are promising tools for adoptive cancer immunotherapy. Since not only CD8 but also CD4 T cells are key players for efficient antitumor responses, the targeted redirection of both subsets with the same antigen-specific TCR comes more and more into focus. Although rapidly evolving technologies enable the reliable genetic re-programming of T cells, the limited availability of TCRs that induce T-cell activation in both T-cell subsets without CD4/CD8 co-receptor contribution hampers the broad application of this approach...
November 14, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29128425/using-sweat-to-measure-cytokines-in-older-adults-compared-to-younger-adults-a-pilot-study
#12
Melissa Hladek, Sarah L Szanton, Young-Eun Cho, Chen Lai, Caroline Sacko, Laken Roberts, Jessica Gill
BACKGROUND/OBJECTIVES: Current measures of cytokines involve urine, blood or saliva which have drawbacks including circadian rhythm variations and complicated collection methods. Sweat has been used to measure cytokines in young and middle-aged adults, but not older adults. We sought to determine the feasibility of using sweat to measure cytokines in older adults compared to younger adults. DESIGN: Two visit cross-sectional pilot study stratified by age group. SETTING: Independent living facility and Johns Hopkins University both in Maryland...
November 8, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29128424/the-use-of-synthetic-peptides-for-detection-of-anti-citrullinated-protein-antibodies-in-rheumatoid-arthritis
#13
Nicole Hartwig Trier, Bettina Eide Holm, Julie Heiden, Ole Slot, Henning Locht, Bente Jensen, Hanne Lindegaard, Anders Svendsen, Christoffer Tandrup Nielsen, Søren Jacobsen, Elke Theander, Gunnar Houen
Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. A characteristic feature of RA is the presence of anti-citrullinated protein antibodies (ACPA). Since ACPAs are highly specific for RA and are often present before the onset of RA symptoms, they have become valuable diagnostic and prognostic. As a result, several assays for detection of ACPAs exist, which vary in sensitivity and specificity. In this study, we analyzed the reactivity of RA sera to selected peptides by solid-phase immunoassays in order to develop an ACPA assay with improved sensitivity and specificity...
November 8, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29126964/simple-and-robust-two-step-ex-vivo-whole-blood-stimulation-assay-suitable-for-investigating-il-17-pathway-in-a-clinical-laboratory-setting
#14
Muir Russell, Abraham Mellkvist-Roos, John Mo, Rabia Hidi
We describe a simple and robust tube-based ex vivo whole blood stimulation procedure suitable for use in clinical laboratories by multiple operators on repeated occasions to study cytokine production in phase 1 human trials of investigational medicines, developed by the authors specifically to study IL-17A expression in man. The stimulation procedure is further proposed as a useful tool for biomarker assay development and validation, for example to prepare quality control samples without reliance on recombinant materials for spiking...
November 7, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29113954/an-improved-method-to-quantify-human-nk-cell-mediated-antibody-dependent-cell-mediated-cytotoxicity-adcc-per-igg-fcr-positive-nk-cell-without-purification-of-nk-cells
#15
Alexander P Sung, Jennifer J-J Tang, Michael J Guglielmo, Doug Redelman, Julie Smith-Gagen, Lucinda Bateman, Dorothy Hudig
Natural killer (NK) lymphocyte ADCC supports anti-viral protection and monoclonal antibody (mAb) anti-tumor therapies. To predict in vivo ADCC therapeutic responses of different individuals, measurement of both ADCC cellular lytic capacity and their NK cellular receptor recognition of antibodies on 'target' cells are needed, using clinically available amounts of blood. Twenty ml of blood provides sufficient peripheral blood mononuclear cells (PBMCs) for the new assay for lytic capacity described here and for an antibody EC50 assay for Fc-receptor recognition...
November 4, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28827189/prediction-of-antibody-structural-epitopes-via-random-peptide-library-screening-and-next-generation-sequencing
#16
Kelly N Ibsen, Patrick S Daugherty
Next generation sequencing (NGS) is widely applied in immunological research, but has yet to become common in antibody epitope mapping. A method utilizing a 12-mer random peptide library expressed in bacteria coupled with magnetic-based cell sorting and NGS correctly identified >75% of epitope residues on the antigens of two monoclonal antibodies (trastuzumab and bevacizumab). PepSurf, a web-based computational method designed for structural epitope mapping was utilized to compare peptides in libraries enriched for monoclonal antibody (mAb) binders to antigen surfaces (HER2 and VEGF-A)...
December 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28802832/retroviral-gene-transfer-into-primary-human-nk-cells-activated-by-il-2-and-k562-feeder-cells-expressing-membrane-bound-il-21
#17
Maria A Streltsova, Eugene Barsov, Sofia A Erokhina, Elena I Kovalenko
Natural killer (NK) cells are capable of rapidly recognizing and efficiently killing tumor cells. This makes them a potentially promising agent for cancer immunotherapy. Additional genetic modifications of NK cells may further improve their anti-tumor efficacy. Numerous technical challenges associated with gene delivery into NK cells have significantly tempered this approach. We achieved efficient retroviral vector transduction of primary human NK cells that were stimulated by a combination of IL-2 and engineered K562 cells expressing membrane-bound IL-21...
November 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28789924/constitutive-interaction-between-4-1bb-and-4-1bbl-on-murine-lps-activated-bone-marrow-dendritic-cells-masks-detection-of-4-1bbl-by-tks-1-but-not-19h3-antibody
#18
Achire N Mbanwi, Gloria H Y Lin, Kuan Chung Wang, Tania H Watts
4-1BB is a TNFR family member associated with NF-κB mediated survival signaling. 4-1BB is widely expressed on activated cells of the immune system, including activated T cells, NK cells and dendritic cells. Its ligand, 4-1BBL, is transiently expressed on activated antigen presenting cells and at low levels on activated T cells. Although 4-1BBL-deficient mice clearly demonstrate a role for 4-1BBL in CD8 T cell responses to viruses such as influenza, 4-1BBL can be difficult to detect following infection of mice...
November 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28782523/monitoring-native-hla-i-trimer-specific-antibodies-in-luminex-multiplex-single-antigen-bead-assay-evaluation-of-beadsets-from-different-manufacturers
#19
COMPARATIVE STUDY
Mepur H Ravindranath, Vadim Jucaud, Soldano Ferrone
Luminex single antigen bead (SAB) assay utilizes beadsets coated with a set of cloned and purified HLA molecules, for monitoring serum anti-HLA antibodies. Particularly, the level of serum IgG against native HLA-I trimers (heavy chain (HC) and β2-microglobulin (β2m) with a peptide), expressed in allograft tissues is correlated with graft failure. In addition to native trimeric HLAI, the beadsets may carry HC only or the dimeric variants, peptide-free HC with β2m and β2m-free HC with or without peptides...
November 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28780040/long-term-direct-visualization-of-passively-transferred-fluorophore-conjugated-antibodies
#20
Jeffrey R Schneider, Ann M Carias, Arangaserry R Bastian, Gianguido C Cianci, Patrick F Kiser, Ronald S Veazey, Thomas J Hope
The use of therapeutic antibodies, delivered by intravenous (IV) instillation, is a rapidly expanding area of biomedical treatment for a variety of conditions. However, little is known about how the antibodies are anatomically distributed following infusion and the underlying mechanism mediating therapeutic antibody distribution to specific anatomical sites remains to be elucidated. Current efforts utilize low resolution and sensitivity methods such as ELISA and indirect labeling imaging techniques, which often leads to high background and difficulty in assessing biodistribution...
November 2017: Journal of Immunological Methods
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