With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt-Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrP Sc ) used limited proteolysis to digest the precursor cellular PrP (PrP C ). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrP Sc , we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrP Sc in all 10 -24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, Ϸ90% of the total PrP Sc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrP C . Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.

There is increasing concern over the extent to which bovine spongiform encephalopathy (BSE) prions have been transmitted to humans, as a result of the rising number of variant Creutzfeldt-Jakob disease (vCJD) cases. Toward preventing new transmissions, diagnostic tests for prions in livestock have been developed using the conformation-dependent immunoassay (CDI), which simultaneously measures specific antibody binding to denatured and native forms of the prion protein (PrP). We employed high-affinity recombinant antibody fragments (recFab) reacting with residues 95-105 of bovine (Bo) PrP for detection and another recFab that recognizes residues 132-156 for capture in the CDI. We report that the CDI is capable of measuring the disease-causing PrP isoform (PrP(Sc)) in bovine brainstems with a sensitivity similar to that of end-point titrations in transgenic (Tg) mice expressing BoPrP. Prion titers were approximately 10(7) ID(50) units per gram of bovine brainstem when measured in Tg(BoPrP) mice, a figure approximately 10 times greater than that determined by bioassay in cattle and approximately 10,000x greater than in wild-type mice. We also report substantial differences in BoPrP(Sc) levels in different areas of the obex region, where neuropathology has been consistently observed in cattle with BSE. The CDI was able to discriminate between PrP(Sc) from BSE-infected cattle and Tg(BoPrP) mice as well as from chronic wasting disease (CWD)-infected deer and elk. Our findings argue that applying the CDI to livestock should considerably reduce human exposure to animal prions.

Gene transfer to airway epithelia is the most direct approach for treating the progressive lung disease associated with cystic fibrosis. However, the transduction efficiency is poor when viral vectors are applied to the mucosal surface. We reported previously that gene transfer via the apical surface of human airway epithelia in vitro was improved by formulating vectors with ethyleneglycol-bis-(2-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA) in a hypotonic buffer. First, we investigated the mechanism for this enhancement. When 100-nm fluorescent beads were applied to the apical surface in the presence of EGTA, paracellular deposition of the particles was noted. Transmission electron microscopy verified that the epithelial junction complex was disrupted under these conditions. The Ca(2+) chelators EGTA, 1,2-bis (2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ethylenediaminetetraacetic acid all caused a rapid, reversible drop in transepithelial resistance and facilitated gene transfer with retrovirus or adenovirus in vitro. When Ca(2+) chelators were applied to rabbit tracheal epithelia or human nasal epithelia in vivo, the transepithelial voltage decreased, and amiloride sensitivity was lost, suggesting that epithelial junctions opened. Importantly, this novel formulation enhanced both retroviral- and adenoviral-mediated gene transfer to rabbit tracheal epithelia in vivo. This technique may have applications for vector or drug delivery to airway epithelia and other polarized cells.

In chronic wasting disease (CWD) of cervids and scrapie of sheep, prions appear to be transmitted horizontally. Oral exposure to prion-tainted blood, urine, saliva and feces has been suggested as the mode of transmission for CWD and scrapie among herbivores susceptible to these prion diseases. To explore the transmission of prions through feces, uninoculated Syrian hamsters (SHa) were cohabitated with or exposed to the bedding of SHa orally infected with Sc237 prions. Incubation times of ~140 days and 80–100% prion infection rate in exposed animals suggested transmission by feces, probably via coprophagy. We measured the disease-causing isoform of the prion protein (PrPSc) in feces by the conformation-dependent immunoassay and titrated the irradiated feces intracerebrally in transgenic mice overexpressing SHaPrP. Feces collected from infected SHa in the first 7 days after oral challenge harbored ~60 ng/g of PrPSc and prion titers of ~106.6 ID50 units/g. The excretion of infectious prions continued at lower levels throughout the asymptomatic phase of the incubation period, most likely by shedding prions from infected Peyer’s patches. Our findings suggest that horizontal transmission among herbivores may occur through the consumption of feces or foodstuff tainted with prions from feces of CWD-infected cervids and scrapie-infected sheep.