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Journal of Immunological Methods

M Kowalewicz-Kulbat, E Ograczyk, K Krawczyk, W Rudnicka, M Fol
Dendritic cells (DCs) are increasingly being used for multiple applications and are useful tools for many immunotherapeutic strategies. The understanding of the possible impact of the DCs-generation methods on the biological capacities of these cells is therefore essential. Although the immunomagnetic separation is regarded as a fast and accurate method yielding cells with the high purity and efficiency, still little is known about its impact on the properties of the generated DCs. The aim of this study was to compare the morphology of the monocyte derived dendritic cells (MoDCs), generated from monocytes selected with anti-CD14 mAbs (positive separation) and treated with anti-CD3, -CD7, -CD16, -CD19, -CD56, -CD123, glycophorin A (negative separation), using laser scanning microscopy...
October 13, 2016: Journal of Immunological Methods
Brice Nativel, Audrey Figuester, Jessica Andries, Cynthia Planesse, Joël Couprie, Philippe Gasque, Wildriss Viranaicken, Thomas Iwema
CD93 belongs to the group XIV C-type lectin like domain (CTLD) and is closely related to thrombomodulin (CD141). Although CD93 is known to be involved in the regulation of cell adhesion and phagocytosis, its role in innate immunity remains to be fully investigated. Critically, published data about CD141 suggest that CD93 CTLD could be involved in the control of inflammation. In order to address further functional and structural analyses, we expressed human CD93 CTLD with several disulfide bonds in an E. coli expression system...
October 12, 2016: Journal of Immunological Methods
Archana Prabahar, Jeyakumar Natarajan
Autoimmune diseases (AIDs) are incurable but suppressible diseases whose molecular mechanisms are yet to be elucidated. In this work, we selected five systemic autoimmune diseases such as Rheumatoid Arthritis (RA), Type 1 Diabetes (T1D), Inflammatory Bowel Disease (IBD), Autoimmune Thyroid Disease (ATD) and Systemic Lupus Erythematosus (SLE) and integrated their heterogeneous data such as miRNA, transcription factor (TF), target genes and protein-protein interactions involved in these AIDs to understand their roles at different functional levels of miRNA such as transcription initiation, gene regulatory network formation and post transcriptional regulation...
October 8, 2016: Journal of Immunological Methods
Thibaut Girardot, Julie Mouillaux, Estellie Idealisoa, Fanny Poujol, Christelle Rouget, Thomas Rimmelé, Guillaume Monneret, Julien Textoris, Fabienne Venet
In several clinical contexts, the measurement of ATP concentration in T lymphocytes has been proposed as a biomarker of immune status, predictive of secondary infections. However, the use of such biomarker in lymphopenic patients requires some adaptations in the ATP dosage protocol. We used blood from healthy volunteers to determine the optimal experimental settings. We investigated technical aspects such as the type of anticoagulant for blood sampling, the effect of freeze and thaw cycles, the reagent and sample mixing sequence, and the optimal dilution buffer...
October 6, 2016: Journal of Immunological Methods
Nerissa Lakhan, Natalie E Stevens, Kerrilyn R Diener, John D Hayball
Adjuvants are used to enhance the immune response against specific antigens for the production of antibodies, with the choice of adjuvant most critical for poorly immunogenic and self-antigens. This study quantitatively and qualitatively evaluated CoVaccine HT™ and Freund's adjuvants for eliciting therapeutic ovine polyclonal antibodies targeting the endogenous alarmin, high mobility group box-1 (HMGB1). Sheep were immunised with HMGB1 protein in CoVaccine HT™ or Freund's adjuvants, with injection site reactions and antibody titres periodically assessed...
September 30, 2016: Journal of Immunological Methods
M Jeraiby, K Sidi Yahya, Depince-Berger, C Lambert
: Microbicidal activity is related to the production of reactive oxygen species (ROS) that can be measured by Flow-Cytometry using Rhodamine 123 (R123). Few assays have been proposed to measure ROS production, usually on heparinized samples but none of them is standardized. Here we propose to improve the test by selecting polymorphonuclears (PMN) and monocytes, labelled and activated in one step to keep the test short, and to standardize the process even between different systems (i.e...
September 29, 2016: Journal of Immunological Methods
Shunsuke Sekiya, Makoto Murata, Satoshi Arai, Hiroshi Murayama, Atushi Kawasaki, Noriyuki Ashida, Kohki Okada, Masaki Ikemoto
Calprotectin, a heterodimer of S100A8 and S100A9, has been reported to be a useful biomarker in inflammatory bowel disease (IBD); however, the relationship between the fecal level of S100A9 and the extent of inflammation in IBD remains unclear. Our aim was to develop a new enzyme-linked immunosorbent assay (ELISA) for rat S100A9, and to investigate whether changes in fecal S100A9 levels reflect the inflammatory conditions in the intestinal tracts of rats with dextran sulfate sodium (DSS)-induced colitis. Anti-rat S100A9 monoclonal antibodies were raised in mice and used for the development of a novel ELISA for rat S100A9...
September 28, 2016: Journal of Immunological Methods
Jared L Spidel, Benjamin Vaessen, Yin Yin Chan, Luigi Grasso, J Bradford Kline
Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential...
September 24, 2016: Journal of Immunological Methods
Anastasia Zotova, Ivan Zotov, Alexander Filatov, Dmitriy Mazurov
An essential step in monoclonal antibody (mAb) development is the characterization and final identification of the specific target antigen and its epitope. Antibody validation is rather straightforward when immunization is carried out with peptide or purified protein, but is more difficult when whole cells or other complex antigens are used for the immunization. Determining antigen specificity of a mAb is further complicated, when reactivity of an antibody is not detected in Western blotting and/or immunoprecipitation assay...
September 21, 2016: Journal of Immunological Methods
Dejan Katanic, Atif Khan, Juilee Thakar
Pathogen specific immune response is a complex interplay between several innate and adaptive immune cell-types. Innate immune cells play a critical role in pathogen recognition and initiating the antigen specific adaptive immune response. Despite specific functional roles of the innate immune cells, they share several anti-viral pathways. The question then becomes, what is the overlap in the transcriptional changes induced upon viral infections across different cell-types? Here we investigate the extent to which gene signatures are conserved across innate immune cell-types by performing a comparative analysis of transcriptomic data...
September 19, 2016: Journal of Immunological Methods
Clarice Perry, Brianne Banasik, Summer R Gorder, Jingya Xia, Sarah Auclair, Nigel Bourne, Gregg N Milligan
Genital infections with herpes simplex virus type 2 (HSV-2) are a source of considerable morbidity and are a health concern for newborns exposed to virus during vaginal delivery. Additionally, HSV-2 infection diminishes the integrity of the vaginal epithelium resulting in increased susceptibility of individuals to infection with other sexually transmitted pathogens. Understanding immune protection against HSV-2 primary infection and immune modulation of virus shedding events following reactivation of the virus from latency is important for the development of effective prophylactic and therapeutic vaccines...
September 19, 2016: Journal of Immunological Methods
Luz M Cumba Garcia, April M Huseby Kelcher, Courtney S Malo, Aaron J Johnson
Effective recovery of activated brain infiltrating lymphocytes is critical for investigations involving murine neurological disease models. To optimize lymphocyte recovery, we compared two isolation methods using brains harvested from seven-day Theiler's murine encephalomyelitis virus (TMEV) and TMEV-OVA infected mice. Brains were processed using either a manual dounce based approach or enzymatic digestion using type IV collagenase. The resulting cell suspensions from these two techniques were transferred to a percoll gradient, centrifuged, and lymphocytes were recovered...
September 10, 2016: Journal of Immunological Methods
Margaret Beddall, Pratip K Chattopadhyay, Shing-Fen Kao, Kathy Foulds, Mario Roederer
Although cryopreserved cell specimens are used throughout biomedical research, the process for thawing samples is labor-intensive and prone to error. Here we describe a small laboratory device that couples an uncapped vial of frozen cells to a conical tube containing warm cell culture media. The entire complex is loaded directly into a centrifuge; within 5min, cells are thawed and diluted out of toxic cryopreservation medium. The recovery and viability of cells are slightly reduced compared to the common (traditional) method...
September 1, 2016: Journal of Immunological Methods
Maran L Sprouse, Gabriele Blahnik, Thomas Lee, Natalie Tully, Pinaki Banerjee, Eddie A James, Maria J Redondo, Matthew L Bettini, Maria Bettini
Single-cell paired TCR identification is a powerful tool, but has been limited in its previous incompatibility with further functional analysis. The current protocol describes a method to clone and functionally evaluate in vivo TCRs derived from single antigen-responsive human T cells and monoclonal T cell lines. We have improved upon current PCR-based TCR sequencing protocols by developing primers that allow amplification of human TCRα and TCRβ variable regions, while incorporating specific restriction cut sites for direct subcloning into the template retroviral vector...
August 31, 2016: Journal of Immunological Methods
Nicholas G Welch, Christopher D Easton, Judith A Scoble, Charlotte C Williams, Paul J Pigram, Benjamin W Muir
Enzyme linked immunosorbent assays (ELISAs) are employed for the detection and quantification of antigens from biological sources such as serum and cell culture media. A sandwich ELISA is dependent on the immobilization of a capture antibody, or antibody fragment, and the effective presentation of its antigen binding sites. Immobilization to common microtitre plates relies on non-specific interactions of the capture protein with a surface that may result in unfavourable orientation and conformation, compromising ELISA signal strength and performance...
November 2016: Journal of Immunological Methods
Irvin Y Ho, Jeffrey J Bunker, Steven A Erickson, Karlynn E Neu, Min Huang, Mario Cortese, Bali Pulendran, Patrick C Wilson
Generating monoclonal antibodies from single B cells is a valuable tool for characterizing the specificity and functional properties of humoral responses. We and others developed protocols that have facilitated major advances in our understanding of B cell development, tolerance, and effector responses to HIV and influenza. Here, we demonstrate various refinements and dramatically reduce the time required to produce recombinant antibodies. Further, we present new methods for cloning and isolating antibodies from cells with lower immunoglobulin mRNA levels that may be resistant to traditional techniques...
November 2016: Journal of Immunological Methods
Peter J Siska, Bumki Kim, Xiangming Ji, Megan D Hoeksema, Pierre P Massion, Kathryn E Beckermann, Jianli Wu, Jen-Tsan Chi, Jiyong Hong, Jeffrey C Rathmell
T and B lymphocytes undergo metabolic re-programming upon activation that is essential to allow bioenergetics, cell survival, and intermediates for cell proliferation and function. To support changes in the activity of signaling pathways and to provide sufficient and necessary intracellular metabolites, uptake of extracellular nutrients increases sharply with metabolic re-programming. One result of increased metabolic activity can be reactive oxygen species (ROS), which can be toxic when accumulated in excess...
November 2016: Journal of Immunological Methods
Paola Gastelum-Aviña, Fernando Lares-Villa, Clara Espitia, Olivia Valenzuela, Ramon Robles-Zepeda, Carlos Velazquez, Adriana Garibay-Escobar
T-cell hybridoma assays have been widely used for the in vitro study of antigen processing and presentation because they represent an unlimited source of cells and they bypass the difficulty of maintaining T-cell clones in culture. One of the most widely used methods to assess hybridoma activation is measurement of CTLL-2 cell proliferation, which is dependent on IL-2. However, continuous culture of this cell line results in a loss of sensitivity, and significant interassay variability can occur. Therefore, our goal was to develop a method to assess T-cell hybridoma activation that was fast and sensitive with low variability based on the IL-2 secretion assay...
November 2016: Journal of Immunological Methods
Shinji Matsuda, Alexandru Movila, Maiko Suzuki, Mikihito Kajiya, Wichaya Wisitrasameewong, Rayyan Kayal, Josefine Hirshfeld, Ayman Al-Dharrab, Irma J Savitri, Abdulghani Mira, Hidemi Kurihara, Martin A Taubman, Toshihisa Kawai
Using a mouse model of silk ligature-induced periodontal disease (PD), we report a novel method of sampling mouse gingival crevicular fluid (GCF) to evaluate the time-dependent secretion patterns of bone resorption-related cytokines. GCF is a serum transudate containing host-derived biomarkers which can represent cellular response in the periodontium. As such, human clinical evaluations of PD status rely on sampling this critical secretion. At the same time, a method of sampling GCF from mice is absent, hindering the translational value of mouse models of PD...
November 2016: Journal of Immunological Methods
Mykhailo Vladymyrov, Jun Abe, Federica Moalli, Jens V Stein, Akitaka Ariga
The development of multi-photon intravital microscopy, in particular two-photon microscopy (2PM), has been a breakthrough technique for deep-tissue imaging of dynamic cell behavior inside live organisms and has substantially advanced the field of immunology. However, intravital time-lapse imaging over prolonged time periods is complicated by slow tissue drifts caused by vital activity, leading to shifting fields of views and making the acquired image sequence partially or completely unanalyzable. To solve this issue, we have established a system that performs continuous drift offset correction in real time using fine pattern matching during 2PM acquisition...
November 2016: Journal of Immunological Methods
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