Dataset Information

Generating Bovine Monocyte-Derived Dendritic Cells for Experimental and Clinical Applications Using Commercially Available Serum-Free Medium.

ABSTRACT: Advances in fundamental and applied immunology research often originate from pilot studies utilizing animal models. While cattle represent an ideal model for disease pathogenesis and vaccinology research for a number of human disease, optimized bovine culture models have yet to be fully established. Monocyte-derived dendritic cells (MoDC) are critical in activating adaptive immunity and are an attractive subset for experimental and clinical applications. The use of serum-supplemented culture medium in this ex vivo approach is undesirable as serum contains unknown quantities of immune-modulating components and may induce unwanted immune responses if not autologous. Here, we describe a standardized protocol for generating bovine MoDC in serum-free medium (AIM-V) and detail the MoDC phenotype, cytokine profile, and metabolic signature achieved using this culture methodology. MoDC generated from adult, barren cattle were used for a series of experiments that evaluated the following culture conditions: medium type, method of monocyte enrichment, culture duration, and concentration of differentiation additives. Viability and yield were assessed using flow cytometric propidium iodide staining and manual hemocytometer counting, respectively. MoDC phenotype and T cell activation and proliferation were assessed by flow cytometric analysis of surface markers (MHC class II, CD86, CD14, and CD205), and CD25 and CFSE respectively. Cytokine secretion was quantified using a multiplex bovine cytokine panel (IL-1?, IL-1?, IL-8, IL-10, IL-17A, IFN-?, MIP-1?, TNF-?, and IL-4). Changes in cell metabolism following stimulation were analyzed using an Extracellular Flux (XFe96) Seahorse Analyzer. Data were analyzed using paired t-tests and repeated measures ANOVA. Immature MoDC generated in serum-free medium using magnetic-activated cell sorting with plate adhesion to enrich monocytes and cultured for 4 days have the following phenotypic profile: MHC class II+++, CD86+, CD205++, and CD14-. These MoDC can be matured with PMA and ionomycin as noted by increased CD86 and CD40 expression, increased cytokine secretion (IL-1?, IL-10, MIP-1?, and IL-17A), a metabolic switch to aerobic glycolysis, and induction of T cell activation and proliferation following maturation. Cultivation of bovine MoDC utilizing our well-defined culture protocol offers a serum-free approach to mechanistically investigate mechanisms of diseases and the safety and efficacy of novel therapeutics for both humans and cattle alike.

SUBMITTER: Guinan J

PROVIDER: S-EPMC7596353 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Generating Bovine Monocyte-Derived Dendritic Cells for Experimental and Clinical Applications Using Commercially Available Serum-Free Medium.

Guinan Jack J Lopez Brina S BS

Frontiers in immunology 20201016

Advances in fundamental and applied immunology research often originate from pilot studies utilizing animal models. While cattle represent an ideal model for disease pathogenesis and vaccinology research for a number of human disease, optimized bovine culture models have yet to be fully established. Monocyte-derived dendritic cells (MoDC) are critical in activating adaptive immunity and are an attractive subset for experimental and clinical applications. The use of serum-supplemented culture med ...[more]

PMID: 33178224

Similar Datasets

Project description:BackgroundMobile applications or 'apps' intended to help people manage their health and chronic conditions are widespread and gaining in popularity. However, little is known about their acceptability and usability for low-income, racially/ethnically diverse populations who experience a disproportionate burden of chronic disease and its complications.ObjectiveThe objective of this study was to investigate the usability of existing mobile health applications ("apps") for diabetes, depression, and caregiving, in order to facilitate development and tailoring of patient-facing apps for diverse populations.DesignUsability testing, a mixed-methods approach that includes interviewing and direct observation of participant technology use, was conducted with participants (n = 9 caregivers; n = 10 patients with depression; and n = 10 patients with diabetes) on a total of 11 of the most popular health apps (four diabetes apps, four depression apps, and three caregiver apps) on both iPad and Android tablets.ParticipantsThe participants were diverse: 15 (58 %) African Americans, seven (27 %) Whites, two (8 %) Asians, two (8 %) Latinos with either diabetes, depression, or who were caregivers.Main measuresParticipants were given condition-specific tasks, such as entering a blood glucose value into a diabetes app. Participant interviews were video recorded and were coded using standard methods to evaluate attempts and completions of tasks. We performed inductive coding of participant comments to identify emergent themes.Key resultsParticipants completed 79 of 185 (43 %) tasks across 11 apps without assistance. Three themes emerged from participant comments: lack of confidence with technology, frustration with design features and navigation, and interest in having technology to support their self-management.ConclusionsApp developers should employ participatory design strategies in order to have an impact on chronic conditions such as diabetes and depression that disproportionately affect vulnerable populations. While patients express interest in using technologies for self-management, current tools are not consistently usable for diverse patients.

Project description:BackgroundColostrum, the milk produced during first few days after birth, is rich in immunoglobulins, antimicrobial peptides & growth factors. Multiple clinical trials using bovine colostrum are ongoing but with no assessment of test product bioactivity.ObjectivesTo examine variability of bioactivity between 20 commercial colostrum products, contribution of TGFβ and EGFR in mediating effects, heat sensitivity of bioactivity and changes in bioactivity of colostrum milkings in the days following calving.DesignIn vitro bioactivity used AGS, RIE-1 and Caco-2 cell proliferation (Alamar blue) and migration (wounded monolayers) assays. Changes in colostrum bioactivity determined following addition of TGFβ-neutralising antibody, EGFR blocker (Typhostin) and after heating (40-60°C, 60 min). In vivo bioassay assessed ability of colostrum gavage (2ml, 7mg/ml) to reduce gastric damage (NSAID + restraint) in rats. Milkings from 6 cows, days 0-3 post calving were assessed for bioactivity and growth factor concentrations.ResultSix-fold differences in pro-proliferative and migratory activity were seen comparing commercial products. Comparison of most- and least-active samples from in vitro studies showed two- to three-fold differences in ability to reduce gastric injury (86% reduction using most-active vs 48% using least-active, p<0.01). Tyrphostin reduced pro-migratory and proliferative activity by 23% and 55%. TGFβ neutralisation reduced migratory activity by 83% but did not affect proliferation Heating colostrum powder to 50°C did not affect immunoactivity of haptoglobin, EGF, TGFβ, IgG, IGF-1 or betacellulin but decreased bioactivity by >40%. Milking studies showed high bioactivity during first and second milkings on day 0 but 77% reduction by day 3. Changes in total protein, haptoglobin, EGF, TGFβ, IgG and IGF-1 paralleled falls in bioactivity.ConclusionCommercial colostrum products possess widely different bioactivity. Variation in heat exposure and/or proportion of day 0 colostrum content may contribute to this. Assessment of colostrum bioactivity has advantages to growth factor quantitation for quality control.

Dataset Information

Generating Bovine Monocyte-Derived Dendritic Cells for Experimental and Clinical Applications Using Commercially Available Serum-Free Medium.

Publications

Generating Bovine Monocyte-Derived Dendritic Cells for Experimental and Clinical Applications Using Commercially Available Serum-Free Medium.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets