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Hematological shift in goat kids naturally devoid of prion protein.

Reiten MR, Bakkebø MK, Brun-Hansen H, Lewandowska-Sabat AM, Olsaker I, Tranulis MA, Espenes A, Boysen P - Front Cell Dev Biol (2015)

Bottom Line: Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities.Our data suggest that PrP(C) has a role in bone marrow physiology and warrant further studies of PrP(C) in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions.Altogether, this genetically unmanipulated PrP(C)-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrP(C).

View Article: PubMed Central - PubMed

Affiliation: Faculty of Veterinary Medicine and Biosciences, Norwegian University of Life Sciences Oslo, Norway.

ABSTRACT
The physiological role of the cellular prion protein (PrP(C)) is incompletely understood. The expression of PrP(C) in hematopoietic stem cells and immune cells suggests a role in the development of these cells, and in PrP(C) knockout animals altered immune cell proliferation and phagocytic function have been observed. Recently, a spontaneous nonsense mutation at codon 32 in the PRNP gene in goats of the Norwegian Dairy breed was discovered, rendering homozygous animals devoid of PrP(C). Here we report hematological and immunological analyses of homozygous goat kids lacking PrP(C) (PRNP(Ter/Ter) ) compared to heterozygous (PRNP (+/Ter)) and normal (PRNP (+/+)) kids. Levels of cell surface PrP(C) and PRNP mRNA in peripheral blood mononuclear cells (PBMCs) correlated well and were very low in PRNP (Ter/Ter), intermediate in PRNP (+/Ter) and high in PRNP (+/+) kids. The PRNP (Ter/Ter) animals had a shift in blood cell composition with an elevated number of red blood cells (RBCs) and a tendency toward a smaller mean RBC volume (P = 0.08) and an increased number of neutrophils (P = 0.068), all values within the reference ranges. Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities. Studies of relative composition of PBMCs, phagocytic ability of monocytes and T-cell proliferation revealed no significant differences between the genotypes. Our data suggest that PrP(C) has a role in bone marrow physiology and warrant further studies of PrP(C) in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions. Altogether, this genetically unmanipulated PrP(C)-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrP(C).

No MeSH data available.


Related in: MedlinePlus

Phagocytosis assays. Cytospots confirmed the cellular uptake of (A) latex beads (green fluorescence), (B)E. coli (red fluorescence), and (C) Zymosan (red fluorescence) in activated monocytes. Additional staining for nuclei (blue) and CD68 (A red, B and C green). (D) Gating of live cells based on FS and SS characteristics in flow cytometry. (E) Particle uptake in PRNPTer/Ter and PRNP+/+ cells based on the results from two representative animals. Medium only was used as control. Gates indicate particle-containing cells, and in the case of latex beads, also gates for cells that had engulfed 2 particles or more (F–H) Compiled results of all animals showing percentage of monocytes containing (F) latex beads, (G) Zymosan and (H)E. coli, as measured by flow cytometry. For each assay, n = 8, except PRNPTer/TerE. coli where n = 7.
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Figure 4: Phagocytosis assays. Cytospots confirmed the cellular uptake of (A) latex beads (green fluorescence), (B)E. coli (red fluorescence), and (C) Zymosan (red fluorescence) in activated monocytes. Additional staining for nuclei (blue) and CD68 (A red, B and C green). (D) Gating of live cells based on FS and SS characteristics in flow cytometry. (E) Particle uptake in PRNPTer/Ter and PRNP+/+ cells based on the results from two representative animals. Medium only was used as control. Gates indicate particle-containing cells, and in the case of latex beads, also gates for cells that had engulfed 2 particles or more (F–H) Compiled results of all animals showing percentage of monocytes containing (F) latex beads, (G) Zymosan and (H)E. coli, as measured by flow cytometry. For each assay, n = 8, except PRNPTer/TerE. coli where n = 7.

Mentions: To determine whether PrPC might have a functional impact on white blood cells, we performed phagocytosis and proliferation studies to assess two major functional properties of leukocytes. Positively selected CD14+ monocytes from peripheral blood were cultured for 24 h to stabilize cells following isolation, and supplemented with GM-CSF to activate the cells and prevent apoptosis (Bratton et al., 1995). The resulting short-term activated monocytes were incubated with latex beads, bacteria (Escherichia coli), or zymosan-covered yeast cells (Saccharomyces cerevisiae) for 30 min. Fluorescence and confocal microscopy of cytospots confirmed the cellular uptake of particles (Figures 4A–C and data not shown). A majority of the monocytes were CD68+, consistent with monocytes or macrophages as previously described (Fadini et al., 2013). These cells had numerous vacuoles in the cytoplasm and a round to bean-shaped nucleus. All particle types were efficiently phagocytized by activated monocytes (Figures 4D,E). When comparing activated monocytes from PRNP+/+ and PRNPTer/Ter goats by flow cytometry, we detected no significant differences between the genotypes in the proportions of cells that had taken up fluorescent particles, for none of the particle types (Figures 4 F–H). There was also no significant difference in the numbers of particles per cell measured as median fluorescent intensity of positive cells; or in the case of latex beads, the number of cells that had engulfed 2 beads or more (Figure 4E and data not shown).


Hematological shift in goat kids naturally devoid of prion protein.

Reiten MR, Bakkebø MK, Brun-Hansen H, Lewandowska-Sabat AM, Olsaker I, Tranulis MA, Espenes A, Boysen P - Front Cell Dev Biol (2015)

Phagocytosis assays. Cytospots confirmed the cellular uptake of (A) latex beads (green fluorescence), (B)E. coli (red fluorescence), and (C) Zymosan (red fluorescence) in activated monocytes. Additional staining for nuclei (blue) and CD68 (A red, B and C green). (D) Gating of live cells based on FS and SS characteristics in flow cytometry. (E) Particle uptake in PRNPTer/Ter and PRNP+/+ cells based on the results from two representative animals. Medium only was used as control. Gates indicate particle-containing cells, and in the case of latex beads, also gates for cells that had engulfed 2 particles or more (F–H) Compiled results of all animals showing percentage of monocytes containing (F) latex beads, (G) Zymosan and (H)E. coli, as measured by flow cytometry. For each assay, n = 8, except PRNPTer/TerE. coli where n = 7.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495340&req=5

Figure 4: Phagocytosis assays. Cytospots confirmed the cellular uptake of (A) latex beads (green fluorescence), (B)E. coli (red fluorescence), and (C) Zymosan (red fluorescence) in activated monocytes. Additional staining for nuclei (blue) and CD68 (A red, B and C green). (D) Gating of live cells based on FS and SS characteristics in flow cytometry. (E) Particle uptake in PRNPTer/Ter and PRNP+/+ cells based on the results from two representative animals. Medium only was used as control. Gates indicate particle-containing cells, and in the case of latex beads, also gates for cells that had engulfed 2 particles or more (F–H) Compiled results of all animals showing percentage of monocytes containing (F) latex beads, (G) Zymosan and (H)E. coli, as measured by flow cytometry. For each assay, n = 8, except PRNPTer/TerE. coli where n = 7.
Mentions: To determine whether PrPC might have a functional impact on white blood cells, we performed phagocytosis and proliferation studies to assess two major functional properties of leukocytes. Positively selected CD14+ monocytes from peripheral blood were cultured for 24 h to stabilize cells following isolation, and supplemented with GM-CSF to activate the cells and prevent apoptosis (Bratton et al., 1995). The resulting short-term activated monocytes were incubated with latex beads, bacteria (Escherichia coli), or zymosan-covered yeast cells (Saccharomyces cerevisiae) for 30 min. Fluorescence and confocal microscopy of cytospots confirmed the cellular uptake of particles (Figures 4A–C and data not shown). A majority of the monocytes were CD68+, consistent with monocytes or macrophages as previously described (Fadini et al., 2013). These cells had numerous vacuoles in the cytoplasm and a round to bean-shaped nucleus. All particle types were efficiently phagocytized by activated monocytes (Figures 4D,E). When comparing activated monocytes from PRNP+/+ and PRNPTer/Ter goats by flow cytometry, we detected no significant differences between the genotypes in the proportions of cells that had taken up fluorescent particles, for none of the particle types (Figures 4 F–H). There was also no significant difference in the numbers of particles per cell measured as median fluorescent intensity of positive cells; or in the case of latex beads, the number of cells that had engulfed 2 beads or more (Figure 4E and data not shown).

Bottom Line: Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities.Our data suggest that PrP(C) has a role in bone marrow physiology and warrant further studies of PrP(C) in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions.Altogether, this genetically unmanipulated PrP(C)-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrP(C).

View Article: PubMed Central - PubMed

Affiliation: Faculty of Veterinary Medicine and Biosciences, Norwegian University of Life Sciences Oslo, Norway.

ABSTRACT
The physiological role of the cellular prion protein (PrP(C)) is incompletely understood. The expression of PrP(C) in hematopoietic stem cells and immune cells suggests a role in the development of these cells, and in PrP(C) knockout animals altered immune cell proliferation and phagocytic function have been observed. Recently, a spontaneous nonsense mutation at codon 32 in the PRNP gene in goats of the Norwegian Dairy breed was discovered, rendering homozygous animals devoid of PrP(C). Here we report hematological and immunological analyses of homozygous goat kids lacking PrP(C) (PRNP(Ter/Ter) ) compared to heterozygous (PRNP (+/Ter)) and normal (PRNP (+/+)) kids. Levels of cell surface PrP(C) and PRNP mRNA in peripheral blood mononuclear cells (PBMCs) correlated well and were very low in PRNP (Ter/Ter), intermediate in PRNP (+/Ter) and high in PRNP (+/+) kids. The PRNP (Ter/Ter) animals had a shift in blood cell composition with an elevated number of red blood cells (RBCs) and a tendency toward a smaller mean RBC volume (P = 0.08) and an increased number of neutrophils (P = 0.068), all values within the reference ranges. Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities. Studies of relative composition of PBMCs, phagocytic ability of monocytes and T-cell proliferation revealed no significant differences between the genotypes. Our data suggest that PrP(C) has a role in bone marrow physiology and warrant further studies of PrP(C) in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions. Altogether, this genetically unmanipulated PrP(C)-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrP(C).

No MeSH data available.


Related in: MedlinePlus