Mark D. Zabel

CCR2+Ly-6Chi monocytes are crucial for the effector phase of autoimmunity in the central nervous system

The chemokine receptor CCR2 plays a vital role for the induction of autoimmunity in the central nervous system. However, it remains unclear how the pathogenic response is mediated by CCR2-bearing cells. By combining bone marrow chimerism with gene targeting we detected a mild disease-modulating role of CCR2 during experimental autoimmune encephalomyelitis, a model for central nervous system autoimmunity, on radio-resistant cells that was independent from targeted CCR2 expression on endothelia. Interestingly, absence of CCR2 on lymphocytes did not influence autoimmune demyelination. In contrast, engagement of CCR2 on accessory cells was required for experimental autoimmune encephalomyelitis induction. CCR2+Ly-6Chi monocytes were rapidly recruited to the inflamed central nervous system and were crucial for the effector phase of disease. Selective depletion of this specific monocyte subpopulation through engagement of CCR2 strongly reduced central nervous system autoimmunity. Collectively, these data indicate a disease-promoting role of CCR2+Ly-6Chi monocytes during autoimmune inflammation of the central nervous system.

Detection of CWD Prions in Urine and Saliva of Deer by Transgenic Mouse Bioassay

Chronic wasting disease (CWD) is a prion disease affecting captive and free-ranging cervids (e.g. deer, elk, and moose). The mechanisms of CWD transmission are poorly understood, though bodily fluids are thought to play an important role. Here we report the presence of infectious prions in the urine and saliva of deer with chronic wasting disease (CWD). Prion infectivity was detected by bioassay of concentrated, dialyzed urine and saliva in transgenic mice expressing the cervid PrP gene (Tg[CerPrP] mice). In addition, PrPCWD was detected in pooled and concentrated urine by protein misfolding cyclic amplification (PMCA). The concentration of abnormal prion protein in bodily fluids was very low, as indicated by: undetectable PrPCWD levels by traditional assays (western blot, ELISA) and prolonged incubation periods and incomplete TSE attack rates in inoculated Tg(CerPrP) mice (373±3days in 2 of 9 urine-inoculated mice and 342±109 days in 8 of 9 saliva-inoculated mice). These findings help extend our understanding of CWD prion shedding and transmission and portend the detection of infectious prions in body fluids in other prion infections.

Complement Receptors CD21/35 Link Innate and Protective Immunity during Streptococcus pneumoniae Infection by Regulating IgG3 Antibody Responses

The CD21/35 receptor provides an important link between innate and adaptive immunity. Its importance during protective immune responses to encapsulated extracellular bacteria was assessed using a new line of mice completely deficient in CD21/35 expression (CD21/35(-/-)). CD21/35 expression was essential for the rapid trapping of C3dg-antigen complexes by B cells in vivo, especially in splenic marginal zones. Despite normal B cell development in CD21/35(-/-) mice, T cell-independent and -dependent antibody responses to low-dose antigens were significantly decreased, with a striking impairment in IgG3 responses. Accordingly, CD21/35(-/-) mice were more susceptible to acute lethal Streptococcus pneumoniae infection. Thus, CD21/35 expression is critical for early protective antibody responses to lethal pathogens that rapidly multiply and quickly overwhelm the immune system.

Detection of Sub-Clinical CWD Infection in Conventional Test-Negative Deer Long after Oral Exposure to Urine and Feces from CWD+ Deer

Background Chronic wasting disease (CWD) of cervids is a prion disease distinguished by high levels of transmissibility, wherein bodily fluids and excretions are thought to play an important role. Using cervid bioassay and established CWD detection methods, we have previously identified infectious prions in saliva and blood but not urine or feces of CWD+ donors. More recently, we identified very low concentrations of CWD prions in urine of deer by cervid PrP transgenic (Tg[CerPrP]) mouse bioassay and serial protein misfolding cyclic amplification (sPMCA). This finding led us to examine further our initial cervid bioassay experiments using sPMCA. Objectives We sought to investigate whether conventional test-negative deer, previously exposed orally to urine and feces from CWD+ sources, may be harboring low level CWD infection not evident in the 19 month observation period. We further attempted to determine the peripheral PrPCWD distribution in these animals. Methods Various neural and lymphoid tissues from conventional test-negative deer were reanalyzed for CWD prions by sPMCA and cervid transgenic mouse bioassay in parallel with appropriate tissue-matched positive and negative controls. Results PrPCWD was detected in the tissues of orally exposed deer by both sPMCA and Tg[CerPrP] mouse bioassay; each assay revealed very low levels of CWD prions previously undetectable by western blot, ELISA, or IHC. Serial PMCA analysis of individual tissues identified that obex alone was positive in 4 of 5 urine/feces exposed deer. PrPCWD was amplified from both lymphoid and neural tissues of positive control deer but not from identical tissues of negative control deer. Discussion Detection of subclinical infection in deer orally exposed to urine and feces (1) suggests that a prolonged subclinical state can exist, necessitating observation periods in excess of two years to detect CWD infection, and (2) illustrates the sensitive and specific application of sPMCA in the diagnosis of low-level prion infection. Based on these results, it is possible that low doses of prions, e.g. following oral exposure to urine and saliva of CWD-infected deer, bypass significant amplification in the LRS, perhaps utilizing a neural conduit between the alimentary tract and CNS, as has been demonstrated in some other prion diseases.

Liposome-siRNA-Peptide Complexes Cross the Blood-Brain Barrier and Significantly Decrease PrPC on Neuronal Cells and PrPRES in Infected Cell Cultures

Background Recent 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 Findings Here 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/Significance Liposome-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.

Efficient In Vitro Amplification of Chronic Wasting Disease PrPRES

ABSTRACT Chronic wasting disease (CWD) of cervids is associated with conversion of the normal cervid prion protein, PrPC, to a protease-resistant conformer, PrPCWD. Here we report the use of both nondenaturing amplification and protein-misfolding cyclic amplification (PMCA) to amplify PrPCWD in vitro. Normal brains from deer, transgenic mice expressing cervid PrPC [Tg(cerPrP)1536 mice], and ferrets supported amplification. PMCA using normal Tg(cerPrP)1536 brains as the PrPC substrate produced >6.5 × 109-fold amplification after six rounds. Highly efficient in vitro amplification of PrPCWD is a significant step toward detection of PrPCWD in the body fluids or excreta of CWD-susceptible species.

Detection of Chronic Wasting Disease Prions in Salivary, Urinary, and Intestinal Tissues of Deer: Potential Mechanisms of Prion Shedding and Transmission

ABSTRACT Efficient horizontal transmission is a signature trait of chronic wasting disease (CWD) in cervids. Infectious prions shed into excreta appear to play a key role in this facile transmission, as has been demonstrated by bioassays of cervid and transgenic species and serial protein misfolding cyclic amplification (sPMCA). However, the source(s) of infectious prions in these body fluids has yet to be identified. In the present study, we analyzed tissues proximate to saliva, urine, and fecal production by sPMCA in an attempt to elucidate this unique aspect of CWD pathogenesis. Oropharyngeal, urogenital, and gastrointestinal tissues along with blood and obex from CWD-exposed cervids (comprising 27 animals and >350 individual samples) were analyzed and scored based on the apparent relative CWD burden. PrPCWD-generating activity was detected in a range of tissues and was highest in the salivary gland, urinary bladder, and distal intestinal tract. In the same assays, blood from the same animals and unseeded normal brain homogenate controls (n = 116 of 117) remained negative. The PrP-converting activity in peripheral tissues varied from 10−11- to 100-fold of that found in brain of the same animal. Deer with highest levels of PrPCWD amplification in the brain had higher and more widely disseminated prion amplification in excretory tissues. Interestingly, PrPCWD was not demonstrable in these excretory tissues by conventional Western blotting, suggesting a low prion burden or the presence of protease-sensitive infectious prions destroyed by harsh proteolytic treatments. These findings offer unique insights into the transmission of CWD in particular and prion infection and trafficking overall.

Detection of protease-resistant cervid prion protein in water from a CWD-endemic area.

Chronic wasting disease (CWD) is the only known transmissible spongiform encephalopathy affecting free-ranging wildlife. Although the exact mode of natural transmission remains unknown, substantial evidence suggests that prions can persist in the environment, implicating components thereof as potential prion reservoirs and transmission vehicles. CWD-positive animals may contribute to environmental prion load via decomposing carcasses and biological materials including saliva, blood, urine and feces. Sensitivity limitations of conventional assays hamper evaluation of environmental prion loads in soil and water. Here we show the ability of serial protein misfolding cyclic amplification (sPMCA) to amplify a 1.3 x 10-7 dilution of CWD-infected brain homogenate spiked into water samples, equivalent to approximately 5 x 107 protease resistant cervid prion protein (PrPCWD) monomers. We also detected PrPCWD in one of two environmental water samples from a CWD endemic area collected at a time of increased water runoff from melting winter snow pack, as well as in water samples obtained concurrently from the flocculation stage of water processing by the municipal water treatment facility. Bioassays indicated that the PrPCWD detected was below infectious levels. These data demonstrate detection of very low levels of PrPCWD in the environment by sPMCA and suggest persistence and accumulation of prions in the environment that may promote CWD transmission.

Stromal Complement Receptor CD21/35 Facilitates Lymphoid Prion Colonization and Pathogenesis

We have studied the role of CD21/35, which bind derivatives of complement factors C3 and C4, in extraneural prion replication and neuroinvasion. Upon administration of small prion inocula, CD21/35−/− mice experienced lower attack rates and delayed disease over both wild-type (WT) mice and mice with combined C3 and C4 deficiencies. Early after inoculation, CD21/35−/− spleens were devoid of infectivity. Reciprocal adoptive bone marrow transfers between WT and CD21/35−/− mice revealed that protection from prion infection resulted from ablation of stromal, but not hemopoietic, CD21/35. Further adoptive transfer experiments between WT mice and mice devoid of both the cellular prion protein PrPC and CD21/35 showed that splenic retention of inoculum depended on stromal CD21/35 expression. Because both PrPC and CD21/35 are highly expressed on follicular dendritic cells, CD21/35 appears to be involved in targeting prions to follicular dendritic cells and expediting neuroinvasion following peripheral exposure to prions.

Trans-species amplification of PrPCWD and correlation with rigid loop 170N.

Chronic wasting disease (CWD) is an efficiently transmitted spongiform encephalopathy of cervids. Whether CWD could represent a threat to non-cervid species remains speculative. Here we show that brain homogenates from several CWD-susceptible non-cervid species, such as ferrets and hamsters, support amplification of PrP(CWD) by sPMCA, whereas brain homogenates from CWD-resistant species, such as laboratory mice and transgenic mice expressing human PrP(C) [Tg(HuPrP) mice], do not. We also investigated whether several North American species that share the environment with cervids would support amplification of PrP(CWD) by sPMCA. Three native rodent species, including voles and field mice, supported PrP(CWD) amplification, whereas other species (e.g. prairie dog, coyote) did not. Analysis of PrP sequences suggests that an ability to support amplification of PrP(CWD) in trans-species sPMCA is correlated with the presence of asparagine at position 170 of the substrate species PrP. Serial PMCA may offer insights into species barriers to transmission of CWD.