Friday, December 23, 2011

Assessing Prion Infectivity of Human Urine in Sporadic Creutzfeldt-Jakob Disease

Volume 18, Number 1—January 2012


Assessing Prion Infectivity of Human Urine in Sporadic Creutzfeldt-Jakob Disease

Silvio Notari1, Liuting Qing1, Maurizio Pocchiari, Ayuna Dagdanova, Kristin Hatcher, Arend Dogterom, Jose F. Groisman, Ib Bo Lumholtz, Maria Puopolo, Corinne Lasmezas, Shu G. Chen, Qingzhong Kong, and Pierluigi Gambetti Author affiliations: Case Western Reserve University, Cleveland, Ohio, USA (S. Notari, L. Qing, A. Dagdanova, K. Hatcher, S.G. Chen, Q. Kong, P. Gambetti); Istituto Superiore di Sanità, Rome, Italy (M. Pocchiari, M. Puopolo); Ferring Pharmaceuticals, Hvidore, Denmark (A. Dogterom); Instituto Massone, Buenos Aires, Argentina (J.F. Groisman); BL Consult ApS, Copenhagen, Denmark (I.B. Lumholtz); The Scripps Research Institute, Jupiter, Florida, USA (C. Lasmezas)

Abstract Prion diseases are neurodegenerative conditions associated with a misfolded and infectious protein, scrapie prion protein (PrPSc). PrPSc propagate prion diseases within and between species and thus pose risks to public health. Prion infectivity or PrPSc presence has been demonstrated in urine of experimentally infected animals, but there are no recent studies of urine from patients with Creutzfeldt-Jakob disease (CJD). We performed bioassays in transgenic mice expressing human PrP to assess prion infectivity in urine from patients affected by a common subtype of sporadic CJD, sCJDMM1. We tested raw urine and 100-fold concentrated and dialyzed urine and assessed the sensitivity of the bioassay along with the effect of concentration and dialysis on prion infectivity. Intracerebral inoculation of transgenic mice with urine from 3 sCJDMM1 patients failed to demonstrate prion disease transmission, indicating that prion infectivity in urine from sCJDMM1 patients is either not present or is <0.38 infectious units/mL.


Discussion The present study demonstrates that the urine from patients affected by advanced sCJDMM1, the most common sCJD subtype that alone accounts for ≈60% of all human prion diseases, contains either no prion infectivity or an infectivity titer that is below the detection limit of our bioassays. The bioassays were done in Tg mice expressing human PrP-129M (Tg40) following inoculation with urine obtained from patients with sCJDMM1 and a variety of positive and negative controls. In limit dilution experiments, Tg40 mice inoculated with MF preparations obtained from the brains of 3 urine donors with sCJDMM1 had prion disease develop at up to 105 or 104 dilutions of the brain tissue equivalent depending on whether the MF preparations were inoculated directly or after spiking into concentrated and dialyzed normal human urine.

To enhance the sensitivity of our system, urine samples were concentrated and dialyzed before inoculation. Similar procedures have been used in all the previous studies on prion infectivity of urine (13,22,23), except for the study by Gregori et al. (21). Our procedure is similar to that used by Seeger et al., who reported the detection of prion infectivity in urine from scrapie inoculated mice affected by nephritis (23). Although we demonstrated in the spiking experiment with MF from sCJDMM1 that the 100× concentration and dialysis procedure did not cause infectivity loss (Table 3), the infectivity of the prion-spiked preparation decreased 20-fold when 100× concentrated and dialyzed urine was used as carrier compared with PBS. On the basis of these findings, we estimated that if prion infectivity is present at all in sCJDMM1 urine, it is at most 0.38 IU/mL if the 20-fold infectivity loss is factored in. Because the nature of the potential PrPSc in urine from CJD patients is not known, this urine PrPSc species might show even higher loss of infectivity in the concentrated and dialyzed urine carrier than the brain PrPSc preparations used in the spiking experiments. To address this concern, we inoculated 33 Tg40 mice with raw urine from one of the 3 donors with sCJDMM1. No recipient mice showed evidence of prion disease suggesting an infectivity ranging from 0.0 and 0.11 IU/per mL (upper limit of 95% CI) as estimated by the Poisson distribution.

Although asymptomatic disease in recipient mice associated with NaPTA- undetectable minute amounts of PrPSc cannot be excluded, our inability to detect prion infectivity in human urine of patients with sCJDMM1 differs from several recent experimental studies on urine of prion-affected animals. Low prion infectivity has been demonstrated in urine from scrapie-infected hamsters (21,22), CWD-infected deer (13), and in scrapie-infected mice affected by lymphocytic nephritis. In the last study, however, no urine infectivity was found in non-nephritic mice (23). Three additional studies have demonstrated the presence of PrPSc in urine from scrapie-infected hamsters and CWD-infected deer using protein misfolding cyclic amplification (PMCA) (13,34,35). However, this highly sensitive procedure can detect prion concentrations below the level of detectability of bioassays.

The most likely explanation for the discrepancy between our negative results on human urine and the positive findings by bioassay in urine from animals resides in the different locale and mode of formation of the prion agents. In all the published animal experiments, including bioassays and PMCA, the prion disease was induced by intracerebral or oral administration of exogenous prions, whereas we examined urine infectivity in a naturally occurring sporadic human prion disease. In exogenously acquired prion diseases, PrPSc is much more likely to be widely present in nonneural peripheral organs, including blood, kidney, and bladder, which is likely the result of early exposure of peripheral organs to the inoculated prions. In sCJD, which is believed to occur spontaneously in the brain (rather than being acquired by infection from exogenous prions), only minute amounts of PrPSc have been detected in a few nonneural peripheral organs and tissues such as skeletal muscle and spleen (6–8,10). In contrast, in variant CJD (vCJD), the form of CJD acquired by consumption of BSE-infected beef, the spread of PrPSc to peripheral organs is much wider and typically involves lymph nodes, tonsil, spleen, portions of the intestinal tract, and the skeletal muscle (7,10,36,37), as well as kidney and other organs (9). These considerations indicate that vCJD (not sCJD) in principle is more similar to the exogenously acquired animal prion diseases that have been used to study prion infectivity in urine. Therefore, vCJD urine that is more likely to contain prion infectivity should be tested by PMCA or bioassay. However, the reported infectivity of animal urine might also result, at least in some instances, from contamination with feces.

Recent data have proved that feces from hamsters infected with scrapie by the oral route and to a lesser extent through intracerebral and intraperitoneal inoculation, contain a discrete amount of PrPSc and prion infectivity (38,39). Infectivity has been demonstrated also in feces from deer orally infected by CWD (40). In hamsters and mice, metabolic cages were used for urine collection, a method in which cross-contamination by feces may actually occur. However, prion infectivity was also demonstrated in urine from CWD-infected deer from which urine collection could easily be performed by catheterization (although this procedure was not mentioned by the authors) (13).

Although additional studies are still needed to determine whether minute amounts of prion infectivity or PrPSc are present in urine from patients with sCJD and to assess the presence of infectious prion in urine from patients with every other form and subtype of human prion diseases, our study shows that urine from patients with sCJDMM1, the most common subtype of sCJD, does not contain prion infectivity detectable by our bioassay and suggests that no significant prionuria occurs in this common subtype of human prion disease.

Dr Notari is an instructor at the Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA. His research interests are the prion diseases with particular focus on the characteristics of PrPSc in different CJD subtypes and their relationships with infectivity.

Friday, March 25, 2011

Detection of Prion Protein in Urine-Derived Injectable Fertility Products by a Targeted Proteomic Approach

Sunday, December 18, 2011

A blood test for variant Creutzfeldt‐Jakob disease: briefing note for patients, carers and health professionals

Friday, December 23, 2011

Oral Transmission of L-type Bovine Spongiform Encephalopathy in Primate Model

Volume 18, Number 1—January 2012 Dispatch



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