Prion proteins in subpopulations of white blood cells from patients with sporadic Creutzfeldt-Jakob disease.

Recent cases of prion transmission in humans following transfusions using blood donated by patients with asymptomatic variant Creutzfeldt-Jakob disease (CJD) implicate the presence of prion infectivity in peripheral blood. In this study, we examined the levels of the normal, cellular prion protein (PrPC), and the disease-causing isoform (PrPSc) in subpopulations of circulating white blood cells (WBCs) from patients with sporadic (s) CJD, age-matched neurological controls and healthy donors. Though widely distributed, the highest levels of PrPC were found in a subpopulation of T lymphocytes: approximately 12,000 PrPC molecules were found per CD4+CD45RA-CD62L- effector memory T helper cell. Although platelets expressed low levels of PrPC on their surface, their high abundance in circulation resulted in the majority of PrPC being platelet associated. Using quantitative fluorescence-activated cell sorting analysis, we found that neither WBC composition nor the amount of cell-surface PrPC molecules was altered in patients with sCJD. Eight different WBC fraction types from the peripheral blood of patients with sCJD were assessed for PrPSc. We were unable to find any evidence for PrPSc in purified granulocytes, monocytes, B cells, CD4+ T cells, CD8+ T cells, natural killer cells, nonclassical gamma delta T cells, or platelets. If human WBCs harbor prion infectivity in patients with sCJD, then the levels are likely to be low.

Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice.

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.

The neurodegeneration sequence in prion diseases: evidence from functional, morphological and ultrastructural studies of the GABAergic system.

Loss of the GABAergic system of neurons has been reported to be the first detectable neuropathological change in prion diseases, which features the accumulation of an aberrant isoform of the prion protein (PrP(Sc)). To determine the timing of GABAergic system dysfunction and degeneration and its relationship to PrP(Sc) accumulation during the course of prion disease in Syrian hamsters, we applied 3 approaches: i) quantifying GABA-immunopositive neurons and their processes by light and electron microscopy to test for selective loss; ii) measuring evoked [3H]-GABA release from synaptosomes to test for functional abnormalities; and iii) determining the kinetics of PrP(Sc) accumulation in subcellular fractions to correlate it with GABAergic dysfunction. At the terminal stages of disease, we found a significant increase in the number of GABA-positive and -negative presynaptic boutons with abnormally aggregated synaptic vesicles. At the same stage, we also found an equal degree of GABA-immunopositive and -immunonegative presynaptic bouton loss. In contrast, GABA-positive neocortical cell bodies increased, based on stereologic estimates in the terminal stage of scrapie. In the context of these abnormalities, evoked release of [3H]-GABA from cortical and thalamic synaptosomes was significantly decreased, which correlated well with the accumulation of PrP(Sc) in synaptosomes and cell membrane fractions.

Transmission and detection of prions in feces.

In chronic wasting disease (CWD) in cervids and in scrapie in 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 of CWD and scrapie among herbivores susceptible to these prion diseases. To explore the transmission of prions through feces, uninoculated Syrian hamsters (SHas) were cohabitated with or exposed to the bedding of SHas orally infected with Sc237 prions. Incubation times of 140 days and a rate of prion infection of 80%-100% among exposed animals suggested transmission by feces, probably via coprophagy. We measured the disease-causing isoform of the prion protein (PrP(Sc)) in feces by use of the conformation-dependent immunoassay, and we titrated the irradiated feces intracerebrally in transgenic mice that overexpressed SHa prion protein (SHaPrP). Fecal samples collected from infected SHas in the first 7 days after oral challenge harbored 60 ng/g PrP(Sc) and prion titers of approximately 10(6.6) ID(50)/g. Excretion of infectious prions continued at lower levels throughout the asymptomatic phase of the incubation period, most likely by the shedding of prions from infected Peyer patches. Our findings suggest that horizontal transmission of disease among herbivores may occur through the consumption of feces or foodstuff tainted with prions from feces of CWD-infected cervids and scrapie-infected sheep.

Human prions and plasma lipoproteins.

Prions are composed solely of an alternatively folded isoform of the prion protein (PrP), designated PrP(Sc). The polyoxometalate phosphotungstic acid has been used to separate PrP(Sc) from its precursor PrP(C) by selective precipitation; notably, native PrP(Sc) has not been solubilized by using nondenaturing detergents. Because of the similarities between PrP(Sc) and lipoproteins with respect to hydrophobicity and formation of phosphotungstic acid complexes, we asked whether these molecules are bound to each other in blood. Here we report that prions from the brains of patients with sporadic Creutzfeldt-Jakob disease (CJD) bind to very low-density (VLDL) and low-density (LDL) lipoproteins but not to high-density lipoproteins (HDL) or other plasma components, as demonstrated both by affinity assay and electron microscopy. Immunoassays demonstrated that apolipoprotein B (apoB), which is the major protein component of VLDL and LDL, bound PrP(Sc) through a highly cooperative process. Approximately 50% of the PrP(Sc) bound to LDL particles was released after exposure to 4 M guanidine hydrochloride at 80 degrees C for 20 min. The apparent binding constants of native human (Hu) PrP(Sc) or denatured recombinant HuPrP(90-231) for apoB and LDL ranged from 28 to 212 pM. Whether detection of PrP(Sc) in VLDL and LDL particles can be adapted into an antemortem diagnostic test for prions in the blood of humans, livestock, and free-ranging cervids remains to be determined.

Prion clearance in bigenic mice.

The clearance of prions from the brain was investigated in bigenic mice designated Tg(tTA : PrP(+/0))3, in which expression of the cellular prion protein (PrP(C)) was regulated by oral doxycycline administration. With suppression of PrP(C) expression, the incubation time for RML prions was prolonged almost threefold from approximately 150 to approximately 430 days. To determine the clearance rate of disease-causing PrP(Sc), bigenic mice were given oral doxycycline beginning 98 days after inoculation with RML prions and sacrificed at various time points over the subsequent 56 days. The half-life (t1/2) for PrP(Sc) was approximately 1.5 days in mouse brain, in reasonable agreement with the apparent t1/2 of 30 h that was determined in a separate study for scrapie-infected mouse neuroblastoma (ScN2a) cells in culture. Both protease-sensitive and -resistant conformers of PrP(Sc) were cleared at the same rate. The t1/2 value for PrP(C) clearance from brain was approximately 18 h, which was considerably longer than the t1/2 of 5 h found in ScN2a cells. The capability of the brain to clear prions raises the possibility that PrP(Sc) is normally made at low levels and continually cleared, and that PrP(Sc) may have a function in cellular metabolism. Moreover, these bigenic mice make it possible to determine both components of PrP(Sc) accumulation, i.e. the rates of formation and clearance, for various strains of prions exhibiting different incubation times.

Diagnosis of human prion disease.

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 (PrPSc) used limited proteolysis to digest the precursor cellular PrP (PrPC). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrPSc, we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrPSc 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, approximately 90% of the total PrPSc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrPC. 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.