Source: UNIVERSITY OF NEBRASKA submitted to
EFFECT OF VIRUS INFECTION ON CELLULAR GLUTATHIONE CONCENTRATION
Sponsoring Institution
State Agricultural Experiment Station
Project Status
TERMINATED
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
0188800
Grant No.
(N/A)
Project No.
NEB-13-152
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2001
Project End Date
Jun 30, 2004
Grant Year
(N/A)
Project Director
Brink, D. R.
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
ANIMAL SCIENCE
Non Technical Summary
Animal health is a leading concern in the cattle industry, with annual losses of $600-700 million due to non-specific respiratory disease. The purpose of this study is to determine if glutathione concentration is decreased after infection with bovine respiratory syncytial virus and if that decreases the efficacy of natural killer cell lysis.
Animal Health Component
100%
Research Effort Categories
Basic
75%
Applied
(N/A)
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3023310101020%
3023310109030%
3023310110110%
3113310101010%
3113310109010%
3113310110120%
Knowledge Area
311 - Animal Diseases; 302 - Nutrient Utilization in Animals;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1010 - Nutrition and metabolism; 1090 - Immunology; 1101 - Virology;
Goals / Objectives
Our objective is to determine if intracellular glutathione concentration is decreased in natural killer cells after infection with bovine respiratory syncytial virus (BRSV). The immune system has two arms, innate and acquired. The innate arm is the first line of defense in an infection and includes natural killer cells. Natural killer cells have anti-bacterial and anti-viral properties. A reduction in intracellular glutathione concentration of natural killer cells following BRSV infection could account for the reduced innate immunity that accompanies a viral infection. If our observations confirm a reduction in glutathione concentration in natural killer cells, we will determine the potential of enhancing cellular glutathione with dietary cysteine manipulation. We will also determine GGT expression in natural killer cells. At this time no one has measured natural killer cell glutathione concentration post-infection with BRSV. Our specific aims are to: 1) Determine whether intracellular glutathione concentration of NK cells is decreased post-infection with BRSV. 2) Determine the expression of gamma-glutamyl transpeptidase on NK cells before and after infection with BRSV. 3) Determine if specific nutrients can modulate the expression of gamma-glutamyl transpeptidase prior to and post-infection with BRSV.
Project Methods
Research Procedures: Specific Aim #1 We will obtain blood from donor animals, isolate cells and provide BRSV for infection of cells. The following techniques will be incorporated. Peripheral blood mononuclear cells (PBMC) will be obtained from healthy donor calves and centrifuged over Ficoll-Hypaque gradients. The PBMC will be collected, and cultured in RPMI 1640 culture medium. The PBMC cells will be infected with a dose of BRSV or treated with mock-infected culture supernatant for 2 h at 37C, washed with Hank's buffer, and then resuspended in 1 ml RPMI 1640 medium for 20 h at 37C. Natural killer cell populations will be isolated by depleting PBMC of other cell populations as described by Flamand et al. in 1996. Briefly, cell populations will be incubated with anti-CD3, anti-CD4, anti-CD8, anti-CD21, and anti-CD14 for 30 minutes followed by addition of complement for 1 hour at 37C. Intracellular glutathione concentration of the natural killer cell sub-population will be determined in the laboratory utilizing high-performance liquid chromatography with fluorometric detection. Specific Aim #2 Peripheral blood mononuclear cells will be isolated and infected with BRSV using the methods described above. The expression of gamma-glutamyl transpeptidase will be determined by flow cytometry using a monoclonal antibody to the enzyme. Flow cytometry will be conducted. Specific Aim #3 We will isolate PBMC and infect the cell population with BRSV as previously described. Then we will test several nutrients in the culture medium of the cells to determine any change in gamma-glutamyl transpeptidase expression on the natural killer cells. Expression of the enzyme will be determined as described above.

Progress 07/01/01 to 06/30/04

Outputs
The purpose of this study was to determine the effects of BRSV and BVDV infection on GSH concentration in peripheral blood mononuclear cells (PBMCs) and bovine natural killer (NK) cells. Mononuclear cells from calf peripheral blood were harvested by Ficoll-Paque density centrifugation. Cells were non-infected or infected with BRSV or BVDV, in the presence or absence of recombinant human IL-2. Glutathione concentrations in BRSV- or BVDV-infected PBMCs were less than that in the non-infected PBMCs. Given the importance of NK cells in halting a viral infection, NK cell GSH levels were also measured. Glutathione concentration was decreased in BRSV- and BVDV-infected NK cells compared to non-infected NK cells with maximum viral-induced reduction observed at 24 h (P < 0.001). Given cysteine is the precursor to biosynthesis of GSH; we measured the effect of supplementation with N-acetyl cysteine (NAC) after infection. Augmentation of cysteine with NAC resulted in restoration of GSH concentrations. These results indicate BRSV and BVDV infection are capable of modulating GSH status within the cell. A manuscipt has been submitted to Viral Immunology and is currently in review.

Impacts
The results of this study indicate that both BRSV and BVDV infection caused a decline in intracellular GSH in PBMCs and more specifically, NK cells. Given the importance of NK cells in halting a viral infection, alteration of the redox environment in the cell necessitates further studies to determine the effect of lower intracellular GSH on the immune response. Intracellular GSH levels may play a central role in tipping the balance toward and effective anti-viral immune response.

Publications

  • Matulka,L.A. 2004. Effects of Bovine Respiratory Syncytial Virus or Bovine Viral Diarrhea Virus Infection and N-Acetyl Cysteine Supplementation on Intracellular Glutathione Levels, Proliferation and Interferon Transcription by Bovine Peripheral Blood Mononuclear Cells and Natural Killer Cells.PhD.Diss.,University of Nebraska, Lincoln.


Progress 10/01/02 to 09/30/03

Outputs
In our first study intracellular glutathione(GSH)concentrations in bovine NK cells were determined using flow cytometric analysis.This method of fluorescence activated cell sorting coupled with multiparameter immunofluorescence sub setting allowed for GSH levels of a particular cell type (NK cells) to be determined. This method allows for a rapid means to sort viable cells by their GSH levels then assay functionality of the cell. A second study involved peripheral blood mononuclear cells infected with BRSV and BVDV to determine effect on intrcellular glutathione concentration.Intracellular GSH levels were decreased in the BRSV and BVDV infected NK cells compared to control (P<0.01).

Impacts
Measurement of intracellular glutathione concentration in bovine natural killer (NK) cells will play an important role in determining the basis for altered NK cell function during infection. Reduced glutathione levels in NK cells may contribute to development of respiratory disease.

Publications

  • Matulka, L. A., L. Wilkie, C. Kusznyski, D. R. Brink and C. L. Kelling. 2003. A flow cytometric method for intracellular analysis of glutathione concentration in bovine natural killer cells. ASAS:71.
  • Matulka, L. A., L. Wilkie, C. Kuszynski, S. Justice, D. Wylie, K. M. Eskridge, D. R. Brink and C. L. Kelling. 2003. Intracellular glutathione concentration in bovine natural killer cells after infection with bovine respiratory syncytial virsus or bovine viral diarrhea virus. J. Anim. Sci. 81, Suppl. 1/J. Dairy Sci. 86, Suppl 1: 14.


Progress 10/01/01 to 09/30/02

Outputs
We have developed a standard for relative intracellular glutathione within lymphocytes. Non-infected cells exhibit a mean glutathione level of 24 measured by log fluorescence. We soon will establish glutathione levels post-infection. To support our hypothesis that it is infection that results in reduction of glutathione within natural killer cells we conducted an infectivity study with peripheral blood mononuclear cells(PMBC). We infected PMBC with a strain of BRSV and incubated cells over a time course of 0-96 hours post infection. We then assayed for virus titers within the cells to determine permissiveness of cells to the virus.

Impacts
Measurement of intracellular glutathione concentration in bovine natural killer cells will play an important role in determining the basis for altered natural killer cell function during infection.

Publications

  • No publications reported this period


Progress 10/01/00 to 09/30/01

Outputs
Preliminary work is in progress. In the original proposal we were going to use HPLC for glutathione analysis of Natural Killer (NK) cells. Since then we have discovered research which utilizes flow cytometry to assess for glutathione in mononuclear cells. We are currently working out the details of this procedure in order to incorporate it into our study. At present we are titrating two antibodies to be used for flow cytometry to distinguish NK cells from the rest of the mononuclear cell population and glutathione level. Once the correct titrations have been discovered, we will begin infecting our cell populations with BRSV and conducting the next step of the research, utilizing flow cytometry.

Impacts
It is too early in the research to project specific impact beyond the general impact statement submitted with the AD-416 for this project.

Publications

  • No publications reported this period