Infectivity was detected in blood components of mice infected with a human-derived strain of vCJD during both the preclinical and clinical phases of disease in a similarly low range of concentrations as in mice infected with a human-derived nonvariant strain (GSS, Fukuoka-1). Other measures of virulence, including brain infectivity titers, incubation periods, and the accumulation of PrPres in spleens and brains, were also comparable in both experimental models.

Background: Prions can be transmitted by blood transfusion, but their origin and distribution in blood are unknown. Results: Prions were detected in plasma extracellular vesicles from preclinical and clinically sick mice. Conclusion: Prions associate with blood-circulating extracellular vesicles. Significance: These findings provide information about prion distribution in blood and set the groundwork for novel prion removal and disease diagnosis technologies.

The emergence of a new environmentally caused variant of Creutzfeldt-Jakob disease (vCJD), the result of food-born infection by the causative agent of bovine spongiform encephalopathy (BSE), has stimulated research on a practical diagnostic screening test. The immunocompetitive capillary electrophoresis (ICCE) assay has been reported to detect disease-specific, proteinase-resistant prion protein (PrPres) in the blood of scrapie-infected sheep. We have applied this method to blood from CJD-infected chimpanzees and humans. The threshold of detection achieved with our ICCE was 0.6 nM of synthetic peptide corresponding to the prion protein (PrP) C-terminus, and 2 nM of recombinant human PrP at the optimized conditions. However, the test was unable to distinguish between extracts of leucocytes from healthy and CJD-infected chimpanzees, and from healthy human donors and patients affected with various forms of CJD. Thus, the ICCE assay as presently performed is not suitable for use as a screening test in human transmissible spongiform encephalopathies (TSEs).

Infectivity in plasma of vCJD-infected mice showed a trend toward reduction following enzymatic treatment with increasing doses of PK, possibly because of activity against proteolysis-sensitive isoforms of abnormal prion protein. It is concluded that the use of PK in protocols for the detection of PrPres may decrease the sensitivity of blood-based assays.

Squirrel monkeys were experimentally infected with the classical bovine spongiform encephalopathy (BSE) agent. Two to four years later, six of the monkeys developed alterations in interactive behavior, cognition and other neurological signs typical of transmissible spongiform encephalopathy (TSE). At necropsy, all brains showed pathological changes similar to those described in variant Creutzfeldt-Jakob disease (vCJD) of humans, except that the squirrel monkey brains contained no PrP amyloid plaques typical of that disease. Constant neuropathological features included spongiform degeneration, gliosis, deposition of abnormal prion protein (PrPTSE) and many deposits of abnormally phosphorylated tau protein (p-Tau) in several areas of the cerebrum and cerebellum. Western blots showed large amounts of proteinase-K-resistant prion protein in the central nervous system. The striking absence of PrP plaques (prominent in brains of cynomolgus macaques with both experimental BSE and vCJD and in patients with vCJD) reinforces the conclusion that the host plays a major role in determining the neuropathology of TSEs. Results of this study suggest that p-Tau, found in the brains of all BSE-infected monkeys, might play a role in the pathogenesis of TSEs. Whether p-Tau contributes to development of disease or appears as a secondary change late in the course of illness remains to be determined.

BACKGROUND The possible risk of iatrogenic transmissible spongiform encephalopathies (TSEs, prion diseases) from transplantation of bone marrow-derived mesenchymal stem cells (MSCs) is uncertain. While most cell lines resist infection, a few propagate TSE agents. STUDY DESIGN AND METHODS We generated MSC-like (MSC-L) cell cultures from bone marrow (BM) of mice inoculated with the human-derived Fukuoka-1 (Fu) strain of TSE agent. Cultured cells were characterized for various markers and cellular prion protein (PrPC) by FACS and for PrPC and its pathologic TSE-associated form (PrPTSE) by Western blotting (WB). Cell cultures were tested for their susceptibility to infection with Fu in vitro. Infectivity of one Fu-infected cell culture was assayed in mice. RESULTS BM cells from Fu-infected mice expressed neither PrPC nor PrPTSE after three days in culture as demonstrated by WB. Cells adherent to plastic and maintained under two different culture conditions became spontaneously immortalized and began to express PrPC at about the same time. One culture became transformed shortly after exposure to Fu in vitro and remained persistently infected, continuously generating PrPTSE through multiple passages; infectivity of cultured cells was confirmed by intracerebral inoculation of lysates into mice. Both persistently TSE-infected and uninfected cells expressed a number of typical MSC markers. CONCLUSION BM-derived MSC-L cells of mice became persistently infected with the Fu agent under certain conditions in culture—conditions that differ substantially from those currently used to develop investigational human stem cell therapies.

The transmission of variant Creutzfeldt-Jakob disease (vCJD) through blood transfusions has created new concerns about the iatrogenic spread of transmissible spongiform encephalopathies (TSEs)/prion diseases through blood and plasma-derived products and has increased the need to develop efficient methods for detection of the agent in biologics. Here, we report the first successful generation of spleen-derived murine stromal cell cultures that persistently propagate two mouse-adapted isolates of human TSE agents, mouseadapted vCJD, and Fukuoka 1. These new cell cultures can be used efficiently for studies of the pathogenesis of the disease, for development of diagnostics and therapeutics, and as a rapid ex vivo assay for TSE inactivation/removal procedures.

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