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Feline cell-mediated immune response against viral pathogens using 5-bromo-2’-deoxyuridine (BrdU) labeling

Ben Vermeulen UGent, Leslie Bosseler UGent, Hannah Dewerchin UGent and Hans Nauwynck UGent (2010) Molecular dialogue in host-parasite interaction. p.86-86
abstract
The feline immune response against viruses consists of a humoral and cellular immune response. At present, four immune parameters can be monitored by flowcytometry in order to evaluate the CMI: activation-proliferation, cytokine-production, cytotoxicity and receptor-detection. In this study, the lymphocytic proliferative response to infectious and inactivated virus (feline calicivirus (FCV), feline parvovirus (FPV) and feline herpesvirus 1 (FHV)) was investigated in vitro by using 5-bromo-2’-deoxyuridine (BrdU) labeling in combination with a double staining (CD3 - CD8) and subsequent flow cytometrical analysis to specifically identify the subset of proliferating T cells. Macrophages (MΦ) and monocyte derived dendritic cells (mDC) were used as antigen presenting cells (APC). Additionally, the production of interferon-γ, the signature molecule of Th1 cells, was evaluated in cell culture supernatant using an enzyme-linked immune sorbent assay (ELISA). Briefly, feline MΦ and mDC from FCV/FPV/FHV-vaccinated cats were incubated with several infectious viruses (FCV, FHV and FPV) and with equivalent amounts of inactivated virus. After washing, the cells were further incubated with 1.105 ml-1 autologous lymphocytes for 4 days at 37°C, 5% CO2. Cells were then stained for proliferation with BrdU and against CD3 and CD8 with specific monoclonal antibodies to identify the proliferating cell population. With mDC as APC, significant proliferation signals (comparable to mitogen treated lymphocytes) were seen when adding inactivated FCV, FHV and FPV. When adding infectious virus, severe proliferation suppression was seen with FPV and FHV in comparison with inactivated virus. Infectious FCV induced a proliferation equivalent to that of inactivated virus. The distribution of all signals was comparable between the CD8+ and CD8- lymphocyte population (Figure 1a). When MΦ were used as APC, both CD8+ and CD8- lymphocyte populations did not show any significant proliferation when incubated with infectious and inactivated virus, with the exception of inactivated calicivirus which gave a 12.2% proliferation signal in the CD8- lymphocyte population (Figure 1b). Additionally, the IFN-γ concentrations found in the culture supernatant concurred well with the amount of proliferation of the DC-stimulated lymphocytes (Figure 1c), validating this technique as a tool for studying the feline CMI against viruses. Figure 1: Percentage proliferating lymphocytes (CD3+) in response to antigen stimulated dendritic cells (DC) (a) and macrophages (MΦ) (b) as APC. (c) IFN-γ concentration in supernatant of lymphocytes in (a). To the author’s knowledge this is the first time that the BrdU technique was used in combination with MΦ and mDC as APC and both infectious and inactivated virus in order to evaluate the CMI of individual cats upon virus vaccination and subsequent viral infection in vitro.
Please use this url to cite or link to this publication:
author
organization
year
type
conference
publication status
published
subject
in
Molecular dialogue in host-parasite interaction
pages
86 - 86
publisher
Belgian Society for Microbiology ; Belgian Society for Biochemistry and Molecular Biology ; Royal Academies of Science and the Arts of Belgium. National Committee for Microbiology
conference name
Molecular Dialogue in Host-Parasite Interaction
conference location
Brussels, Belgium
conference start
2010-11-18
conference end
2010-11-19
language
English
UGent publication?
yes
classification
C3
copyright statement
I have transferred the copyright for this publication to the publisher
id
3051932
handle
http://hdl.handle.net/1854/LU-3051932
date created
2012-11-13 10:38:55
date last changed
2012-11-13 15:19:36
@inproceedings{3051932,
  abstract     = {The feline immune response against viruses consists of a humoral and cellular immune response. At present, four immune parameters can be monitored by flowcytometry in order to evaluate the CMI: activation-proliferation, cytokine-production, cytotoxicity and receptor-detection. 
In this study, the lymphocytic proliferative response to infectious and inactivated virus (feline calicivirus (FCV), feline parvovirus (FPV) and feline herpesvirus 1 (FHV)) was investigated in vitro by using 5-bromo-2{\textquoteright}-deoxyuridine (BrdU) labeling in combination with a double staining (CD3 - CD8) and subsequent flow cytometrical analysis to specifically identify the subset of proliferating T cells. Macrophages (M\ensuremath{\Phi}) and monocyte derived dendritic cells (mDC) were used as antigen presenting cells (APC). Additionally, the production of interferon-\ensuremath{\gamma}, the signature molecule of Th1 cells, was evaluated in cell culture supernatant using an enzyme-linked immune sorbent assay (ELISA). Briefly, feline M\ensuremath{\Phi} and mDC from FCV/FPV/FHV-vaccinated cats were incubated with several infectious viruses (FCV, FHV and FPV) and with equivalent amounts of inactivated virus. After washing, the cells were further incubated with 1.105 ml-1 autologous lymphocytes for 4 days at 37{\textdegree}C, 5\% CO2. Cells were then stained for proliferation with BrdU and against CD3 and CD8 with specific monoclonal antibodies to identify the proliferating cell population. With mDC as APC, significant proliferation signals (comparable to mitogen treated lymphocytes) were seen when adding inactivated FCV, FHV and FPV. When adding infectious virus, severe proliferation suppression was seen with FPV and FHV in comparison with inactivated virus. Infectious FCV induced a proliferation equivalent to that of inactivated virus. The distribution of all signals was comparable between the CD8+ and CD8- lymphocyte population (Figure 1a). When M\ensuremath{\Phi} were used as APC, both CD8+ and CD8- lymphocyte populations did not show any significant proliferation when incubated with infectious and inactivated virus, with the exception of inactivated calicivirus which gave a 12.2\% proliferation signal in the CD8- lymphocyte population (Figure 1b). Additionally, the IFN-\ensuremath{\gamma} concentrations found in the culture supernatant concurred well with the amount of proliferation of the DC-stimulated lymphocytes (Figure 1c), validating this technique as a tool for studying the feline CMI against viruses.
Figure 1: Percentage proliferating lymphocytes (CD3+) in response to antigen stimulated dendritic cells (DC) (a) and macrophages (M\ensuremath{\Phi}) (b) as APC. (c) IFN-\ensuremath{\gamma} concentration in supernatant of lymphocytes in (a).
To the author{\textquoteright}s knowledge this is the first time that the BrdU technique was used in combination with M\ensuremath{\Phi} and mDC as APC and both infectious and inactivated virus in order to evaluate the CMI of individual cats upon virus vaccination and subsequent viral infection in vitro.},
  author       = {Vermeulen, Ben and Bosseler, Leslie and Dewerchin, Hannah and Nauwynck, Hans},
  booktitle    = {Molecular dialogue in host-parasite interaction},
  language     = {eng},
  location     = {Brussels, Belgium},
  pages        = {86--86},
  publisher    = {Belgian Society for Microbiology ; Belgian Society for Biochemistry and Molecular Biology ; Royal Academies of Science and the Arts of Belgium. National Committee for Microbiology},
  title        = {Feline cell-mediated immune response against viral pathogens using 5-bromo-2{\textquoteright}-deoxyuridine (BrdU) labeling},
  year         = {2010},
}

Chicago
Vermeulen, Ben, Leslie Bosseler, Hannah Dewerchin, and Hans Nauwynck. 2010. “Feline Cell-mediated Immune Response Against Viral Pathogens Using 5-bromo-2’-deoxyuridine (BrdU) Labeling.” In Molecular Dialogue in Host-parasite Interaction, 86–86. Belgian Society for Microbiology ; Belgian Society for Biochemistry and Molecular Biology ; Royal Academies of Science and the Arts of Belgium. National Committee for Microbiology.
APA
Vermeulen, Ben, Bosseler, L., Dewerchin, H., & Nauwynck, H. (2010). Feline cell-mediated immune response against viral pathogens using 5-bromo-2’-deoxyuridine (BrdU) labeling. Molecular dialogue in host-parasite interaction (pp. 86–86). Presented at the Molecular Dialogue in Host-Parasite Interaction, Belgian Society for Microbiology ; Belgian Society for Biochemistry and Molecular Biology ; Royal Academies of Science and the Arts of Belgium. National Committee for Microbiology.
Vancouver
1.
Vermeulen B, Bosseler L, Dewerchin H, Nauwynck H. Feline cell-mediated immune response against viral pathogens using 5-bromo-2’-deoxyuridine (BrdU) labeling. Molecular dialogue in host-parasite interaction. Belgian Society for Microbiology ; Belgian Society for Biochemistry and Molecular Biology ; Royal Academies of Science and the Arts of Belgium. National Committee for Microbiology; 2010. p. 86–86.
MLA
Vermeulen, Ben, Leslie Bosseler, Hannah Dewerchin, et al. “Feline Cell-mediated Immune Response Against Viral Pathogens Using 5-bromo-2’-deoxyuridine (BrdU) Labeling.” Molecular Dialogue in Host-parasite Interaction. Belgian Society for Microbiology ; Belgian Society for Biochemistry and Molecular Biology ; Royal Academies of Science and the Arts of Belgium. National Committee for Microbiology, 2010. 86–86. Print.