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Incunabular immunological events in prion trafficking.

Michel B, Meyerett-Reid C, Johnson T, Ferguson A, Wyckoff C, Pulford B, Bender H, Avery A, Telling G, Dow S, Zabel MD - Sci Rep (2012)

Bottom Line: Monocytes and dendritic cells (DCs) required Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking.B cells constituted the majority of prion-bearing cells in the mediastinal lymph node by six hours, indicating intranodal prion reception from resident DCs or subcapsulary sinus macrophages or directly from follicular conduits.These data reveal novel, cell autonomous prion lymphotropism, and a prominent role for B cells in intranodal prion movement.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine and Biomedical Sciences, Department of Microbiology, Immunology and Pathology, Prion Research Program at Colorado State University, Fort Collins, Colorado 80521, USA.

ABSTRACT
While prions probably interact with the innate immune system immediately following infection, little is known about this initial confrontation. Here we investigated incunabular events in lymphotropic and intranodal prion trafficking by following highly enriched, fluorescent prions from infection sites to draining lymph nodes. We detected biphasic lymphotropic transport of prions from the initial entry site upon peripheral prion inoculation. Prions arrived in draining lymph nodes cell autonomously within two hours of intraperitoneal administration. Monocytes and dendritic cells (DCs) required Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking. B cells constituted the majority of prion-bearing cells in the mediastinal lymph node by six hours, indicating intranodal prion reception from resident DCs or subcapsulary sinus macrophages or directly from follicular conduits. These data reveal novel, cell autonomous prion lymphotropism, and a prominent role for B cells in intranodal prion movement.

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Purification and conjugation of prion rods from infected brain homogenate and real-time whole-mouse in vivo imaging.(a) Western blot analysis depicting CWD infected elk brain homogenate used as starting material (5 µg in lane 1 and 100 µg in lane 2) from which we enriched prion rod aggregates (100 ng in lanes 3 and 4). Lanes 5 and 6 show 100 µg of control elk normal brain homogenate. (b) SDS page gel depicting fluorochrome tagged prion rods. Prions treated at 37°C without Proteinase K (PK, lane 1) or with PK (lane 2), and at 95°C with PK (lane 3). Molecular weight markers are shown in kilodaltons to the left of the blots. (c) In vivo detection of prion rods by epifluorescent whole-body optical imaging. Representative images of tg5037 mice injected orally or subcutaneously with PBS or fluorescent prion rods or 1 µm polystyrene microspheres (beads). Images were acquired at indicated time points after injection.
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f1: Purification and conjugation of prion rods from infected brain homogenate and real-time whole-mouse in vivo imaging.(a) Western blot analysis depicting CWD infected elk brain homogenate used as starting material (5 µg in lane 1 and 100 µg in lane 2) from which we enriched prion rod aggregates (100 ng in lanes 3 and 4). Lanes 5 and 6 show 100 µg of control elk normal brain homogenate. (b) SDS page gel depicting fluorochrome tagged prion rods. Prions treated at 37°C without Proteinase K (PK, lane 1) or with PK (lane 2), and at 95°C with PK (lane 3). Molecular weight markers are shown in kilodaltons to the left of the blots. (c) In vivo detection of prion rods by epifluorescent whole-body optical imaging. Representative images of tg5037 mice injected orally or subcutaneously with PBS or fluorescent prion rods or 1 µm polystyrene microspheres (beads). Images were acquired at indicated time points after injection.

Mentions: In order to monitor prion trafficking from inoculation sites to draining lymph nodes, we first enriched prion rods from a brain of an elk terminally sick with CWD from one liter of 10% crude brain homogenate, concentrating prion aggregate volume 104-fold to a final volume of 100 µl using detergent solubilization and ultracentrifugation through a sucrose cushion (figure 1A). We enriched aggregated prion rods approximately 103-fold (compare lanes 1 and 2 to 3 and 4). Like the crude brain homogenate, purified prion rods showed partial PK resistance (lanes 2 and 4). Normal brain homogenate contained no PK-resistant PrPC bands (lane 6). Intracranial injection of 1 µg of enriched, sonicated prion aggregates resulted in terminal disease in susceptible mice 122 ± 5 (n = 5) days post inoculation (DPI) compared to 157 ± 16 DPI for mice inoculated with 30 µg of 1% crude brain homogenate (n = 8, p = .0003). We then conjugated enriched prions to Dylight 649 fluorochrome (figure 1B). To evaluate the stability of the fluorochrome, we treated conjugated prions with PK (figure 1B, lanes 2 and 3) at physiological (lane 2) or supraphysiological temperatures (lane 3). DyLight 649 still fluoresced even after SDS and PK treatment followed by incubation at 95°C. In addition to demonstrating partial SDS and PK resistance (lanes 2 and 3), fluorescent prions could also aggregate into high molecular weight oligomers (lane 1).


Incunabular immunological events in prion trafficking.

Michel B, Meyerett-Reid C, Johnson T, Ferguson A, Wyckoff C, Pulford B, Bender H, Avery A, Telling G, Dow S, Zabel MD - Sci Rep (2012)

Purification and conjugation of prion rods from infected brain homogenate and real-time whole-mouse in vivo imaging.(a) Western blot analysis depicting CWD infected elk brain homogenate used as starting material (5 µg in lane 1 and 100 µg in lane 2) from which we enriched prion rod aggregates (100 ng in lanes 3 and 4). Lanes 5 and 6 show 100 µg of control elk normal brain homogenate. (b) SDS page gel depicting fluorochrome tagged prion rods. Prions treated at 37°C without Proteinase K (PK, lane 1) or with PK (lane 2), and at 95°C with PK (lane 3). Molecular weight markers are shown in kilodaltons to the left of the blots. (c) In vivo detection of prion rods by epifluorescent whole-body optical imaging. Representative images of tg5037 mice injected orally or subcutaneously with PBS or fluorescent prion rods or 1 µm polystyrene microspheres (beads). Images were acquired at indicated time points after injection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368226&req=5

f1: Purification and conjugation of prion rods from infected brain homogenate and real-time whole-mouse in vivo imaging.(a) Western blot analysis depicting CWD infected elk brain homogenate used as starting material (5 µg in lane 1 and 100 µg in lane 2) from which we enriched prion rod aggregates (100 ng in lanes 3 and 4). Lanes 5 and 6 show 100 µg of control elk normal brain homogenate. (b) SDS page gel depicting fluorochrome tagged prion rods. Prions treated at 37°C without Proteinase K (PK, lane 1) or with PK (lane 2), and at 95°C with PK (lane 3). Molecular weight markers are shown in kilodaltons to the left of the blots. (c) In vivo detection of prion rods by epifluorescent whole-body optical imaging. Representative images of tg5037 mice injected orally or subcutaneously with PBS or fluorescent prion rods or 1 µm polystyrene microspheres (beads). Images were acquired at indicated time points after injection.
Mentions: In order to monitor prion trafficking from inoculation sites to draining lymph nodes, we first enriched prion rods from a brain of an elk terminally sick with CWD from one liter of 10% crude brain homogenate, concentrating prion aggregate volume 104-fold to a final volume of 100 µl using detergent solubilization and ultracentrifugation through a sucrose cushion (figure 1A). We enriched aggregated prion rods approximately 103-fold (compare lanes 1 and 2 to 3 and 4). Like the crude brain homogenate, purified prion rods showed partial PK resistance (lanes 2 and 4). Normal brain homogenate contained no PK-resistant PrPC bands (lane 6). Intracranial injection of 1 µg of enriched, sonicated prion aggregates resulted in terminal disease in susceptible mice 122 ± 5 (n = 5) days post inoculation (DPI) compared to 157 ± 16 DPI for mice inoculated with 30 µg of 1% crude brain homogenate (n = 8, p = .0003). We then conjugated enriched prions to Dylight 649 fluorochrome (figure 1B). To evaluate the stability of the fluorochrome, we treated conjugated prions with PK (figure 1B, lanes 2 and 3) at physiological (lane 2) or supraphysiological temperatures (lane 3). DyLight 649 still fluoresced even after SDS and PK treatment followed by incubation at 95°C. In addition to demonstrating partial SDS and PK resistance (lanes 2 and 3), fluorescent prions could also aggregate into high molecular weight oligomers (lane 1).

Bottom Line: Monocytes and dendritic cells (DCs) required Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking.B cells constituted the majority of prion-bearing cells in the mediastinal lymph node by six hours, indicating intranodal prion reception from resident DCs or subcapsulary sinus macrophages or directly from follicular conduits.These data reveal novel, cell autonomous prion lymphotropism, and a prominent role for B cells in intranodal prion movement.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine and Biomedical Sciences, Department of Microbiology, Immunology and Pathology, Prion Research Program at Colorado State University, Fort Collins, Colorado 80521, USA.

ABSTRACT
While prions probably interact with the innate immune system immediately following infection, little is known about this initial confrontation. Here we investigated incunabular events in lymphotropic and intranodal prion trafficking by following highly enriched, fluorescent prions from infection sites to draining lymph nodes. We detected biphasic lymphotropic transport of prions from the initial entry site upon peripheral prion inoculation. Prions arrived in draining lymph nodes cell autonomously within two hours of intraperitoneal administration. Monocytes and dendritic cells (DCs) required Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking. B cells constituted the majority of prion-bearing cells in the mediastinal lymph node by six hours, indicating intranodal prion reception from resident DCs or subcapsulary sinus macrophages or directly from follicular conduits. These data reveal novel, cell autonomous prion lymphotropism, and a prominent role for B cells in intranodal prion movement.

Show MeSH
Related in: MedlinePlus