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Journal of Immunological Methods

Kohji Nozoe, Terukazu Sanui, Masaaki Takeshita, Takao Fukuda, Akira Haraguchi, Yoshitomi Aida, Fusanori Nishimura
Fimbriae are virulence factors of Porphyromonas gingivalis (P. gingivalis). In this study, the action of fimbriae on neutrophil respiratory burst and cytokine production by mononuclear cells (MNC) were investigated. Native or denatured form of purified P. gingivalis fimbriae contained endotoxin at an equivalence of 1-3μglipopolysaccharides(LPS)/mg protein. The endotoxin could be reduced to the equivalent of 1ng-LPS/mg protein by phase separation using Triton X-114. Unfractionated fimbriae caused serum-dependent priming of neutrophils for enhanced respiratory burst, but both native and denatured forms of Triton X-114-fractionated fimbriae were not active at 100μg/mL...
November 30, 2016: Journal of Immunological Methods
Karen Liao, Erik Meyer, Thomas N Lee, Amy Loercher, Daniel Sikkema
Mepolizumab, a humanized IgG1 monoclonal antibody that blocks native homodimeric interleukin-5 (IL-5) from binding to the IL-5 receptor, has recently been approved for treatment of severe eosinophilic asthma. Our initial immunogenicity assay method for phase I and II studies utilized a bridging electrochemiluminescence format with biotin and ruthenium-labelled mepolizumab linked by anti-drug antibodies (ADA). We discovered that IL-5 significantly increased in dosed subjects from a phase II study and that the increased IL-5 was in the form of a drug-bound complex...
November 24, 2016: Journal of Immunological Methods
Ankie Höglind, Irene Areström, Cecilia Ehrnfelt, Khosro Masjedi, Bartek Zuber, Luis Giavedoni, Niklas Ahlborg
Non-human primates (NHP) provide important animal models for studies on immune responses to infections and vaccines. When assessing cellular immunity in NHP, cytokines are almost exclusively analyzed utilizing cross-reactive anti-human antibodies. The functionality of antibodies has to be empirically established for each assay/application as well as NHP species. A rational approach was employed to identify monoclonal antibodies (mAb) cross-reactive with many NHP species. Panels of new and established mAbs against human Interferon (IFN)-γ and Interleukin (IL)-2 were assessed for reactivity with eukaryotically expressed recombinant IFN-γ and IL-2, respectively, from Old (rhesus, cynomolgus and pigtail macaques, African green monkey, sooty mangabey and baboon) and New World NHP (Ma's night monkey, squirrel monkey and common marmoset)...
November 23, 2016: Journal of Immunological Methods
C Pilat, K Krüger, T Frech, F C Mooren
Strenuous exercise is followed by an elevation of many cytokines with inflammation regulating properties. Since most cytokines act at pico- or nanomolar concentrations many investigations failed to detect their concentrations in vivo. Hence, the aim of this study was to evaluate the significance of cytokine measurements (IL-1β, TNF-α, IL-1ra, IL-6, CCL2 and CXCL8) in a stimulated whole-blood culture (sWBC) compared to serum with respect to their exercise-induced kinetics and detection rates. 40 male volunteers (age: 25,5 ± 4,3 years, BMI: 24,00 ± 2,24, VO2peak: 46,9 ± 4,1 ml/kg x min) performed 60 minutes of intensive bicycle exercise (80% VO2peak)...
November 19, 2016: Journal of Immunological Methods
Anne-Pauline Bellanger, Thibaud Lignon, Yann Godet, Bénédicte Rognon, Gabriel Reboux, Houssein Gbaguidi-Haore, Christophe Borg, Laurence Millon
PURPOSE: Hypersensitivity pneumonitis (HP) is an immunoallergic disease due to chronic exposure to high quantities of different microorganisms such as Mycobacterium immunogenum (Mi), a mycobacterium, and Lichtheimia corymbifera (Lc), a filamentous fungus. It has recently been demonstrated that the protein DLDH (dihydrolipoyl dehydrogenase), is common to these microorganisms. This study aimed to investigate the immune potential of overlapping peptide pools covering the MiDLDH and LcDLDH...
November 19, 2016: Journal of Immunological Methods
Peifang Sun, Brian J Morrison, Charmagne G Beckett, Zhaodong Liang, Nishith Nagabhushana, An Li, Kevin R Porter, Maya Williams
The study assessed antibody-dependent NK cell degranulation, a biomarker relevant to antibody-dependent cell cytotoxicity (ADCC), to analyze dengue immune sera. We first determined binding intensity of patient sera to the surface of DENV-infected cells and examined the types of antigens expressed on infected cells. Antigens from pre-membrane (PreM) and envelope (E), but not from NS proteins were detected on the surface of infected cells. After adding NK cells to infected target cells previously treated with patient sera, rapid NK cell degranulation was observed...
November 14, 2016: Journal of Immunological Methods
Ruth J Davis, Christopher Silvin, Clint T Allen
Myeloid-derived suppressor cells (MDSCs) have garnered much attention in recent years as a potential target for altering the immunosuppressive tumor microenvironment in a variety of solid tumor types. The ability to accurately assess the immunosuppressive capacity of MDSCs is fundamental to the development of therapeutic approaches aimed at disabling these immunosuppressive functions. In this article we provide evidence that the use of CD3/28 coated microbeads leads to artefactual T-lymphocyte suppression due to sequestration of beads by MDSCs isolated from the spleens of wild-type mice bearing subcutaneous syngeneic, carcinogen-induced oral cavity carcinomas...
November 14, 2016: Journal of Immunological Methods
Elizabeth Weingartner, Jean-Paul Courneya, Achsah Keegan, Amit Golding
We have established a highly reproducible and reliable protocol for testing human regulatory T cell function in suppressing IgM production from an immature human B cell line. The autoreactive Ramos B cell line provides a stable reporter of B cell effector function that can be tested by a straight-forward IgM ELISA. Tregs from healthy volunteers display a range of ability for suppressing baseline IgM production in a contact- and death-independent manner. Having established the normal range for human Treg direct suppression of B cell effector function, it will now be possible to efficiently test Tregs from various autoimmune conditions in which B cell hyperactivity and secretion of auto-antibodies are a hallmark of disease...
November 13, 2016: Journal of Immunological Methods
Lonneke M JanssenDuijghuijsen, Harry J Wichers, Klaske van Norren, Jaap Keijer, Joseph L Baumert, Govardus A H de Jong, Renger F Witkamp, Stef J Koppelman
Some studies have suggested that allergens may appear in the circulation after ingestion of allergenic food sources. The reported levels of allergen in serum, however, are low, and conclusions between studies differ. Here, we investigated factors that determine the detection of allergens in serum after consumption of peanuts. Ten healthy volunteers ingested 100g of light-roasted peanuts. Serum samples were taken at regular intervals for six hours. A double monoclonal sandwich ELISA was used to analyse the presence and quantity of the major peanut allergen Ara h 6 in serum...
November 11, 2016: Journal of Immunological Methods
Leroy Versteeg, Xavier Le Guezennec, Bin Zhan, Zhuyun Liu, Minilik Angagaw, Janice D Woodhouse, Subhabrata Biswas, Coreen M Beaumier
Luminex® technology provides a powerful methodology for multiplex cytokine detection but remains constrained by high costs and a minimum of 25-50μL sample volume requirement per assay-well often hindering analysis of limited biological samples. Here we compare the results of Luminex-based cytokine multiplexing assay performed using a conventional 96-well microtiter plates and using a particular 96-well wall-less plate based on Droparray® technology ("DA-Bead").The application of the DA-Bead plate allows 80% reduction of sample and reagent volume, thus an opportunity for significant Luminex reagents cost savings with no change to the workflow...
November 10, 2016: Journal of Immunological Methods
Claudio Fozza, Francesca Barraqueddu, Giovanna Corda, Salvatore Contini, Patrizia Virdis, Fausto Dore, Silvana Bonfigli, Maurizio Longinotti
The T-cell receptor (TCR) is the key player within the so called immunological synapse and the analysis of its repertoire offers a picture of both versatility and wideness of the whole immune T-cell compartment. Among the different approaches applied to its study the so-called spectratyping identifies the pattern of the third complementarity determining region (CDR3) length distribution in each one of the beta variable (TRBV) subfamilies encoded by the corresponding genes. This technique consists in a CDR3 fragment analysis through capillary electrophoresis, performed after cell separation, RNA extraction and reverse transcriptase PCR...
November 4, 2016: Journal of Immunological Methods
Andrew Melton, Russell Soon, Troy Tompkins, Brian Long, Becky Schweighardt, Yulan Qi, Catherine Vitelli, Anil Bagri, Celeste Decker, Charles A O'Neill, Stephen J Zoog, Lynne Jesaitis
Many enzyme replacement therapies (ERTs) for lysosomal storage disorders use the cell-surface cation-independent mannose-6 phosphate receptor (CI-M6PR) to deliver ERTs to the lysosome. However, neutralizing antibodies (NAb) may interfere with this process. We previously reported that most individuals with Morquio A who received elosulfase alfa in the phase 3 MOR-004 trial tested positive for NAbs capable of interfering with binding to CI-M6PR ectodomain in an ELISA-based assay. However, no correlation was detected between NAb occurrence and clinical efficacy or pharmacodynamics...
October 24, 2016: Journal of Immunological Methods
Songwang Hou, Paul E Harper, Nathalie Bardin, Ming Zhao
OBJECTIVE: The presence of circulating autoantibodies against phosphatidylethanolamine (PE) has been shown to be positively associated with symptoms of antiphospholipid syndromes (APS). However, the current ELISA-based tests for antiphosphatidylethanolamine (aPE) antibodies remain inconsistent and controversial. The term PE refers to a collection of phospholipids that have phosphorylethanolamine head group as a common structural feature, but can vary in fatty acids with diverse physicochemical properties...
October 23, 2016: 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, A E 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
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