Determination of Cytokine Responses Using a Multiplexed Fluorescent Microsphere Immunoassay

Martins, Thomas B.; Pasi, Brian M.; Pickering, Jerry W.; Jaskowski, Troy D.; Litwin, Christine M.; Hill, Harry R.

doi:10.1309/n0t6-c56b-gxb2-nvfb

Cited by 62 publications

(43 citation statements)

References 14 publications

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“…A multiplexed panel for the detection of 15 cytokines was developed, building upon our previous in-house developed cytokine panel (15), in the ARUP Institute for Experimental and Clinical Pathology, University of Utah, Salt Lake City, which was utilized to assay for TNF-α, IL-1β, IL-6, IL-8, IL-4, IL-5, IL-10, IL-13, soluble CD40 ligand, IL-2, IL-2R, IL-12, IFN-γ, TGF-β, and IL-17.…”

Section: Luminex Multianalyte Profilingmentioning

confidence: 99%

“…To our knowledge, Th17 cell populations in human neonates have not yet been fully described. Here, we employ phytohemagglutinin (PHA), heat-inactivated group B streptococci (GBS), and Escherichia coli as stimulants, and measure IL-17 responses in mixed mononuclear cells (MMCs) isolated from umbilical cord blood, using an in-house developed multiplex cytokine panel (15), and compare these responses to those of mononuclear cells from healthy adult controls. Additionally, we employ a flow cytometry assay adapted from Eyerich et al (16), to examine cord blood for CD3 + CD8 -T cells that express CCR6, a surface Th17 cell marker, as well as intracellular IL-17.…”

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confidence: 99%

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Severely depressed interleukin-17 production by human neonatal mononuclear cells

et al. 2014

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Background:The role of T-helper 17 cells (Th17) in neonatal host defense remains to be fully elucidated. Interleukin (IL)-17 plays an important role in the immune response to bacterial and fungal pathogens by promoting inflammation. Methods: We examined neonatal production of IL-17 in mixed mononuclear cells (MMCs) isolated from umbilical cord blood for comparison with adult peripheral blood mononuclear cell controls. results: IL-17 production was profoundly diminished in MMCs isolated from cord blood when compared with MMCs from adult blood. This was associated with a marked reduction in the population of CCR6 + IL-17 + T-cells in the neonatal cord blood. We also found diminished intracellular formation of IL-17, and diminished IL-17 responses to both group B streptococci (GBS) and Escherichia coli. Neonatal mononuclear cells were found to adequately phosphorylate signal transducer and activator of transcription 3, pY705, and pS727. We and others have reported markedly reduced interferon-γ production by neonate mononuclear cells exposed to GBS. Here, we correct that profound abnormality with added IL-17. conclusion: Our results suggest that profound deficiency of IL-17 production associated with a marked decrease in Th17 cells likely contributes significantly to the increased susceptibility of human neonates to invasive bacterial and fungal infections.

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Section: Luminex Multianalyte Profilingmentioning

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Severely depressed interleukin-17 production by human neonatal mononuclear cells

et al. 2014

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“…To our knowledge, multiplex technology has not been used to characterize TLR-stimulated cytokine production in neonates. Multiplex cytokine analysis is a useful alternative to ELISA that allows profile characterization of multiple cytokines in a single sample [21] . Here, we employed a TLR assay as described by Deering and Orange [22] to evaluate TLR function in cord blood compared to blood from adults, utilizing our own multianalyte 12 cytokine assay developed in-house [21] .…”

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Multiplex Analysis of Toll-Like Receptor-Stimulated Neonatal Cytokine Response

Caron

Pine²,

Augustine³

et al. 2009

Neonatology

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Background: The human neonate’s increased susceptibility to bacterial infections is not completely understood. Toll-like receptors (TLRs) have been recognized as pattern-recognition receptors critical to the innate immune response. TLR function in neonates, however, remains incompletely defined. Objective: To examine regulatory and proinflammatory cytokine responses to TLR-1–6 stimulation of cord blood compared to adult blood. Methods: We stimulated cord blood with ligands for each of TLRs 1–6 and compared these responses to adult controls. The following TLR ligands were utilized: Pam3CSK4 (TLR-1 and 2), zymosan (TLR-2 and 6), poly I:C (TLR-3), LPS (TLR-4), and flagellin (TLR-5). Cytokine production was measured with an assay developed in-house utilizing multi-analyte technology. Results: TLR-1–6 stimulation produced higher concentrations of proinflammatory cytokines (IL-1β, IL-6, and IL-8) in cord blood compared to adult blood, with the exception of TLR-4-stimulated TNF-α production, which was significantly lower in cord blood (319 pg/ml) compared to adult blood (645 pg/ml; p = 0.027). In contrast, TLR-1–6 stimulation resulted in decreased concentrations of Th1 and Th2 cytokines in cord blood compared to adult blood, with significantly diminished production of IL-12 (TLRs 1/2, 2/6, 3 and 4), IL-13 (TLR-1–6), and IL-10 (TLR-4). Conclusion: Cord blood production of regulatory Th1 and Th2 cytokines following TLR stimulation is decreased compared to that of adult blood. In contrast, TLR-stimulated proinflammatory cytokine production was markedly higher in neonates than in adults, with the exception of TLR-4-induced TNF-α production. The human neonate’s increased susceptibility to bacterial infections may be related to abnormal TLR responsiveness, with enhanced proinflammatory and decreased regulatory cytokine production.

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“…Recently, flow cytometric, bead-based technologies have been used for the development of assays for the simultaneous detection of multiple antibodies to ENA. The emerging multiplexed technology we have used is the Luminex 100 (Lab-MAP) system, which allows multiple analytes to be assessed simultaneously in a single sample (3,7,11,12,17,18,21). The technology uses 5.6-m polystyrene particles, called microspheres, that are internally labeled with two fluorescent dyes.…”

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Evaluation of Multiplexed Fluorescent Microsphere Immunoassay for Detection of Autoantibodies to Nuclear Antigens

Martins

Burlingame

Mühlen

et al. 2004

Clin Vaccine Immunol

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Antibodies to extractable nuclear antigens (ENA) are found in a variety of collagen vascular diseases. Determining the individual specificities of these antibodies is extremely useful in establishing the disease diagnosis and in some cases the prognosis. With a multiplexed fluorescent microsphere immunoassay, reactivity to five of the most diagnostically useful ENA was measured in 249 serum samples, including samples from 56 patients previously documented to have systemic lupus erythematosus (SLE). Results of the multiplexed assay were compared to results from established ENA enzyme-linked immunosorbent assays (ELISAs), and the agreement, sensitivity, and specificity, respectively, for the five ENA evaluated were as follows: SSA, 99.1, 100.0, and 98.8%; SSB, 98.6, 88.9, and 99.5%; Sm, 97.6, 95.8, and 97.9%; RNP, 97.2, 92.7, and 98.8%; Scl-70, 93.6, 50.0, and 99.0%. In the 56 confirmed SLE patients, the frequency of significant concentrations of autoantibodies with the multiplexed assay was 21.4% for SSA, 7.1% for SSB, 10.7% for Sm, 32.1% for RNP, and 0% for Scl-70. The new flow cytometric bead-based multiplexed assay showed excellent correlation with the well-established single-analyte ELISA methods for four of five the ENA markers investigated in this study. The most notable discrepancies between the two assays were for the Scl-70 antigen, which was most often resolved in favor of the multiplexed assay. Our studies show that the multiplexed microsphere-based immunoassay is a sensitive and specific method for the detection and semiquantitation of ENA antibodies in human sera.

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Determination of Cytokine Responses Using a Multiplexed Fluorescent Microsphere Immunoassay

Cited by 62 publications

References 14 publications

Severely depressed interleukin-17 production by human neonatal mononuclear cells

Severely depressed interleukin-17 production by human neonatal mononuclear cells

Multiplex Analysis of Toll-Like Receptor-Stimulated Neonatal Cytokine Response

Evaluation of Multiplexed Fluorescent Microsphere Immunoassay for Detection of Autoantibodies to Nuclear Antigens

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