Performing Department
Pathology, Microbiology & Immunology
Non Technical Summary
Some cells of the immune system become activated when there is an infection, but their response is not specific to the pathogen.Such cellsrespond to all pathogens the same whether it is the first or subsequent timethat particular infection has entered the body. These cells are part of what we call innateimmunity. Some work has been done by others that has shown that sometimes these cells can be trained to react more strongly to organisms that can cause infection in the host when they are exposed a second, third, or more times to that same organism or another organism.This so-called priming of the innate immune response is what we want to investigate. We will do this in the laboratory using cultures of the bovine monocyte cells and exposing them to anextract of a foreign organism. We will then measure products they produce (called cytokines). We can compare stimulated cells with those that are naive and not exposed to the extract of the foreign organism. If this priming works we may be able to used the results to test in a whole animal (a bovine calf) to see if treatment of the calf with the stimulant can make the calf better able to resist infection with a bacterial and/or viral pathogen.
Animal Health Component
100%
Research Effort Categories
Basic
70%
Applied
20%
Developmental
10%
Goals / Objectives
Goals / Objectives1) to expose peripheral blood monocytes and pulmonary macrophages from calves to various concentrations of Beta-glucan derived from Candida albicans or placebo in vitro and harvest cells to evaluate transcription of genes for IL-6, IL-8, and TNF alpha.2) to compare in vitro "trained" cells (described in objective 1) to naive cells for ability to respond to heterologous stimuli).3) to conduct an infection of 8 week old calves with in vivo trained innate immune cells versus placebo treated calves BRSV and H. somni co-infection, and to evaluate clinical signs, pathology and pro inflammatory cytokine production by innate cells before and after infection.?
Project Methods
Design:Experiment 1- Objective 1Blood will be harvested from 8 Holstein calves into citrate tubes and peripheral blood mononuclear cells (PBMCs) isolated by density gradient centrifugation. After resuspension of PBMC's in media monocytes will be isolated with magnetic activated cell sorting with monoclonal anti-CD14 (10). Anti mouse IgG1 labeled magnetic beads will then be used to obtain the monocytes by positive selection. Pulmonary macrophages will be harvested by pulmonary lavage. Cells will be harvested by centrifugation. Cytospin will verify purity of cell population. Both cell types will be plated in triplicate for each treatment in 96 well plates and treated for 24 hrs with the training stimulus (Beta-glucan) after which cells will be harvested for RNA to measure proinflammatory cytokine expression by qRT-PCR.Experiment 2 - Objective 2Cells will be obtained as described above and trained for 24 hours using the concentration of Beta-glucan that produced the greatest transcription response. Media will be changed and cells will rest in culture for 7 days. On day 8 secondary stimuli will be added: LPS from E. coli, Pam3Cys, killed H. somni and BRSV. Cells will be collected after 6 hours for RNA expression and after 72 hours for protein expression (using ELISA kits).