Summary It is thought that monocytes rapidly differentiate to macrophages or dendritic cells (DCs) upon leaving blood. Here we have shown that Ly-6C+ monocytes constitutively trafficked into skin, lung, and lymph nodes (LNs). Entry was unaffected in gnotobiotic mice. Monocytes in resting lung and LN had similar gene expression profiles to blood monocytes, but elevated transcripts of a limited number of genes including cyclo-oxygenase-2 (COX-2) and major histocompatibility complex class II (MHC II), induced by monocyte interaction with endothelium. Parabiosis, bromodoxyuridine (BrdU) pulse-chase analysis, and intranasal instillation of tracers indicated that instead of contributing to resident macrophages in the lung, recruited endogenous monocytes acquired antigen for carriage to draining LNs, a function redundant with DCs though differentiation to DCs did not occur. Thus, monocytes can enter steady state non-lymphoid organs and recirculate to LNs without differentiation to macrophages or DCs, revising a long-held view that monocytes become tissue-resident macrophages by default.

We used serial protein misfolding cyclic amplification (sPMCA) to amplify the D10 strain of CWD prions in a linear relationship over two logs of D10 dilutions. The resultant PMCA-amplified D10 induced terminal TSE disease in CWD-susceptible Tg(cerPrP)1536 mice with a survival time approximately 80 days shorter than the original D10 inoculum, similar to that produced by in vivo sub-passage of D10 in Tg(cerPrP)1536 mice. Both in vitro-amplified and mouse-passaged D10 produced brain lesion profiles, glycoform ratios and conformational stabilities significantly different than those produced by the original D10 inoculum in Tg(cerPrP)1536 mice. These findings demonstrate that sPMCA can amplify and adapt prion strains in vitro as effectively and much more quickly than in vivo strain adaptation by mouse passage. Thus sPMCA may represent a powerful tool to assess prion strain adaptation and species barriers in vitro.

BackgroundRecent advances toward an effective therapy for prion diseases employ RNA interference to suppress PrPC expression and subsequent prion neuropathology, exploiting the phenomenon that disease severity and progression correlate with host PrPC expression levels. However, delivery of lentivirus encoding PrP shRNA has demonstrated only modest efficacy in vivo.Methodology/Principal FindingsHere we describe a new siRNA delivery system incorporating a small peptide that binds siRNA and acetylcholine receptors (AchRs), acting as a molecular messenger for delivery to neurons, and cationic liposomes that protect siRNA-peptide complexes from serum degradation.Conclusions/SignificanceLiposome-siRNA-peptide complexes (LSPCs) delivered PrP siRNA specifically to AchR-expressing cells, suppressed PrPC expression and eliminated PrPRES formation in vitro. LSPCs injected intravenously into mice resisted serum degradation and delivered PrP siRNA throughout the brain to AchR and PrPC-expressing neurons. These data promote LSPCs as effective vehicles for delivery of PrP and other siRNAs specifically to neurons to treat prion and other neuropathological diseases.

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.

The Complement System has been shown to facilitate peripheral prion pathogenesis. Mice lacking Complement receptors CD21/35 partially resist terminal prion disease when infected intraperitoneally with mouse-adapted scrapie prions. Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, like scrapie, has been shown to involve the immune system, which probably contributes to their relatively facile horizontal and environmental transmission. Here we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. CD21/35 deficient Tg5037 mice exhibit greatly impaired splenic prion accumulation and replication throughout disease, similar to CD21/35 deficient murine PrP mice infected with mouse scrapie. TgA5037;CD21/35-/- mice exhibited little or no neuropathology and deposition of misfolded, protease-resistant PrP associated with CWD. CD21/35 translocates to lipid rafts and mediates a strong germinal center response to prion infection that we propose provides the optimal environment for prion accumulation and replication. We further propose a potential role for CD21/35 in selecting prion quasi-species present in prion strains that may exhibit differential zoonotic potential compared to the parental strains.

Accumulating evidence shows a critical role of the complement system in facilitating attachment of prions to both B cells and follicular dendritic cells and assisting in prion replication. Complement activation intensifies disease in prion-infected animals, and elimination of complement components inhibits prion accumulation, replication and pathogenesis. Chronic wasting disease (CWD) is a highly infectious prion disease of captive and free-ranging cervid populations that utilizes the complement system for efficient peripheral prion replication and most likely efficient horizontal transmission. Here we show that complete genetic or transient pharmacological depletion of C3 prolongs incubation times and significantly delays splenic accumulation in a CWD transgenic mouse model. Using a semi-quantitative prion amplification scoring system we show that C3 impacts disease progression in the early stages of disease by slowing the rate of prion accumulation and/or replication. The delayed kinetics in prion replication correlate with delayed disease kinetics in mice deficient in C3. Taken together, these data support a critical role of C3 in peripheral CWD prion pathogenesis.

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie [1][2][3] . Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection [4][5][6] . In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10 , although their role in trafficking remains unclear [11][12][13][14][15][16] . Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17 . These prior studies also suffer from the problem of differentiating between endogenous PrP C and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.Protocol

Background Dogs with protein‐losing nephropathy (PLN) are treated with antiplatelet drugs for thromboprophylaxis but no standardized method exists to measure drug response. It is also unknown if clopidogrel metabolite concentrations [CM] differ between healthy and PLN dogs. Objectives Assess response to aspirin or clopidogrel in PLN dogs using platelet aggregometry (PA) and compare [CM] between healthy and PLN dogs. Animals Six healthy and 14 PLN dogs. Methods Platelet aggregometry using adenosine diphosphate (ADP), arachidonic acid (AA), and saline was performed in healthy dogs at baseline and 1‐week postclopidogrel administration to identify responders or nonresponders. A decrease of ≥60% for ADP or ≥30% for AA at 1 or 3 hours postpill was used to define a responder. At 1 and 3 hours postclopidogrel, [CM] and PA were measured in healthy and PLN dogs. Platelet aggregometry was performed in PLN dogs at baseline, 1, 6, and 12 weeks after clopidogrel or aspirin administration. Results In PLN dogs receiving clopidogrel, PA differed from baseline at all time points for ADP but not for AA at any time point. Most dogs responded at 1 or both time points except for 1 dog that showed no response. For PLN dogs receiving aspirin, no differences from baseline were observed at any time point for either ADP or AA. No differences in [CM] were found at either time point between healthy and PLN dogs. Conclusions and Clinical Importance Platelet aggregometry may represent an objective method to evaluate response to clopidogrel or aspirin treatment and PLN dogs appear to metabolize clopidogrel similarly to healthy dogs.