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
|
|