Dataset Information

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study.

ABSTRACT:

Background

Cryopreservation of peripheral blood mononuclear cells has been used to preserve and standardize immunologic measurements for multicenter studies, however, effects of cryopreservation on cytokine responses are incompletely understood. In designing immunologic studies for a new multicenter birth cohort study of childhood asthma, we performed a series of experiments to determine the effects of two different methods of cryopreservation on the cytokine responses of cord and peripheral blood mononuclear cells.

Results

Paired samples of PBMC were processed freshly, or after cryopreservation in a Nalgene container (NC) or a controlled-rate freezer (CRF). Although there were some differences between the methods, cryopreservation inhibited PHA-induced IL-10 secretion and Der f 1-induced IL-2 secretion, and augmented PHA-induced IL-2 secretion and spontaneous secretion of TNF-alpha. In separate experiments, NC cryopreservation inhibited secretion of several cytokines (IL-13, IL-10, IFN-gamma, TNF-alpha) by PHA-stimulated cord blood mononuclear cells. With the exception of PHA-induced IL-13, results from fresh and cryopreserved cord blood samples were not significantly correlated. Finally, in reproducibility studies involving processing of identical cell samples in up to 4 separate laboratories, variances in cytokine responses of fresh cells stimulated at separate sites did not exceed those in cryopreserved cells stimulated at a central site.

Conclusion

Collectively, these studies indicate that cryopreservation can affect mononuclear cell cytokine response profiles, and that IL-10 secretion and antigen-induced responses may be especially vulnerable. These studies also demonstrate that mononuclear cell responses can be standardized for performance in a small number of laboratories for multicenter studies, and underscore the importance of measuring reproducibility and of testing whether cryopreservation techniques alter specific immunologic outcomes.

SUBMITTER: Shreffler WG

PROVIDER: S-EPMC1762025 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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Publications

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study.

Shreffler Wayne G WG Visness Cynthia M CM Burger Melissa M Cruikshank William W WW Lederman Howard M HM de la Morena Maite M Grindle Kristine K Calatroni Agustin A Sampson Hugh A HA Gern James E JE

BMC immunology 20061212

<h4>Background</h4>Cryopreservation of peripheral blood mononuclear cells has been used to preserve and standardize immunologic measurements for multicenter studies, however, effects of cryopreservation on cytokine responses are incompletely understood. In designing immunologic studies for a new multicenter birth cohort study of childhood asthma, we performed a series of experiments to determine the effects of two different methods of cryopreservation on the cytokine responses of cord and periph ...[more]

PMID: 17156490

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Project description:Study questionWhat is the effect of saturated fat ingestion on mononuclear cell (MNC) TNFα, IL-6 and IL-1β secretion and circulating IL-6 levels in women with polycystic ovary syndrome (PCOS)?Summary answerWomen with PCOS exhibit increases in MNC-derived TNFα, IL-6 and IL-1β secretion and circulating IL-6 following saturated fat ingestion even in the absence of obesity, and these increases are linked to metabolic aberration and androgen excess.What is known alreadyCytokine excess and metabolic aberration is often present in PCOS.Study design, size, durationA cross-sectional design was used in this study of 38 reproductive-age women.Participants/materials, setting, methodsGroups of 19 reproductive-age women with PCOS (10 lean, 9 obese) and 19 ovulatory controls (10 lean, 9 obese) participated in this study that was performed at a tertiary academic medical centre. TNFα, IL-6 and IL-1β secretion was measured from cultured MNC, and IL-6 was measured in plasma from blood sampling while fasting and 2, 3 and 5 h after saturated fat ingestion. Insulin sensitivity was determined using the Matsuda index following an oral glucose tolerance test. Androgen secretion was evaluated with blood sampling while fasting and 24, 48 and 72 h after an HCG injection.Main results and the role of chanceLean and obese women with PCOS exhibited lipid-induced incremental AUC increases in MNC-derived TNFα (489-611%), IL-6 (333-398%) and IL-1β (560-695%) secretion and in plasma IL-6 levels (426-474%), in contrast with lean control subjects. In both PCOS groups, insulin sensitivity was lower (42-49%) and androgen secretion after HCG injection was greater (63-110%) compared with control subjects. The MNC-derived TNFα, IL-6 and IL-1β and circulating IL-6 responses were inversely associated with insulin sensitivity and directly associated with fasting lipids and androgen secretion after HCG injection.Limitations, reasons for cautionThe sample size of each of the four study groups was modest following group assignment of subjects by body mass.Wider implications of the findingsThis study showcases the unique pro-inflammatory contribution of circulating MNC in the development of metabolic aberration and androgen excess in PCOS.Study funding/competing interest(s)This research was supported by grant R01 DK107605 to F.G. from the National Institutes of Health, the Indiana Clinical and Translational Sciences Institute Clinical Research Center which is funded in part by grant UL1TR002529 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award, and the Indiana University Center for Diabetes and Metabolic Diseases funded by grant P30 DK097512 from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. No conflicts of interest, financial or otherwise, are declared by the authors.Trial registration numberClinicalTrials.gov NCT01489319.

Project description:Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current definition of a probiotic.

Dataset Information

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study.

Background

Results

Conclusion

Publications

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study.

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