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From: TSS ()
Subject: Coincident Scrapie Infection and Nephritis Lead to Urinary Prion Excretion {FULL TEXT}
Date: October 14, 2005 at 7:20 am PST

Science, Vol 310, Issue 5746, 324-326 , 14 October 2005

Reports
Coincident Scrapie Infection and Nephritis Lead to Urinary Prion Excretion
Harald Seeger,1* Mathias Heikenwalder,1* Nicolas Zeller,1 Jan Kranich,1 Petra Schwarz,1 Ariana Gaspert,2 Burkhardt Seifert,3 Gino Miele,1 Adriano Aguzzi1

Prion infectivity is typically restricted to the central nervous and lymphatic systems of infected hosts, but chronic inflammation can expand the distribution of prions. We tested whether chronic inflammatory kidney disorders would trigger excretion of prion infectivity into urine. Urinary proteins from scrapie-infected mice with lymphocytic nephritis induced scrapie upon inoculation into noninfected indicator mice. Prionuria was found in presymptomatic scrapie-infected and in sick mice, whereas neither prionuria nor urinary PrPSc was detectable in prion-infected wild-type or PrPC-overexpressing mice, or in nephritic mice inoculated with noninfectious brain. Thus, urine may provide a vector for horizontal prion transmission, and inflammation of excretory organs may influence prion spread.

1 Institute of Neuropathology, University Hospital of Zrich, Schmelzbergstrasse 12, CH-8091 Zrich, Switzerland.
2 Institute of Clinical Pathology, University Hospital of Zrich, Schmelzbergstrasse 12, CH-8091 Zrich, Switzerland.
3 Institute of Biostatistics, University of Zrich, Sumatrastrasse 30, CH-8006 Zrich, Switzerland.

* These authors contributed equally to this work.

To whom correspondence should be addressed. E-mail: adriano@pathol.unizh.ch

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The prion, the infectious agent of transmissible spongiform encephalopathies (TSEs), is detectable at extraneural sites long before clinical symptoms appear (1). PrPSc, a protease-resistant isoform of the host protein PrPC, accumulates mostly in central nervous system and lymphoid organs of infected organisms and may represent the infectious principle (2, 3). In addition to PrPC (4), splenic prion replication requires follicular dendritic cells (FDCs), the maintenance of which depends on B cells expressing lymphotoxins (LT) and (5). By activating local LT/ signaling, which induces lymphoneogenesis, chronic inflammation enables ectopic prion replication (6). Inflammatory kidney conditions induced by bacteria, viruses, or autoimmunity are frequent in animals and humans, and urosepsis can occur in terminally demented patients (7). We therefore wondered whether renal inflammatory conditions might lead to urinary prion excretion.
To probe this possibility, we administered prions to RIPLT and NZB x NZW F1 mice (henceforth termed NZBW) suffering from lymphocytic nephritis (figs. S1 and S2 and table S1), as well as NZW mice and milk fat globuleepidermal growth factor 8 (MFG-E8)deficient mice, which develop glomerulonephritis but lack lymphofollicular inflammation (fig. S1).

After intraperitoneal (i.p.) prion inoculation [3 and 5 log LD50 (50% lethal dose) units of the Rocky Mountain Laboratory (RML) scrapie strain (passage 5, henceforth called RML5) (8)], brains and spleens of RIPLT, NZBW, MFG-E8/, and control mice displayed similar prion and PrPSc loads (fig. S3, A to C). Whereas RIPLT and NZBW kidneys progressively accumulated PrPSc and prion infectivity at 60 to 90 days postinoculation (dpi), presymptomatic (66 dpi) and terminally sick MFG-E8/ mice lacked renal PrPSc (fig. S3D). Histoblot and immunohistochemical analysis identified PrPSc in renal lymphofollicular infiltrates of RIPLT and NZBW mice (6).

RIPLT, AlbLT, C57BL/6 (4 to 6 months old), NZW, NZB, NZBW, MFG-E8/, tga20, and 129Sv x C57BL/6 mice (8 to 16 weeks old) were inoculated i.p. with 3 or 5 logLD50 scrapie prions. We dialyzed and purified urinary proteins from pools of three to six mice of each genotype at 30, 45, 60, 85, 110, 120, and 130 dpi (all presymptomatic) and from terminally scrapie-sick mice (Fig. 1). Each urine donor was confirmed to contain brain or spleen PrPSc and/or infectivity upon necropsy (fig. S3, A to C).

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Fig. 1. Transmission of prions through urine. Urine samples were collected from individual donors (horizontal lines) at time points after inoculation, denoted by vertical lines, and pooled (intersections between lines, arrows). Squares represent individual tga20 mice inoculated i.c. with urinary proteins. White squares: no scrapie symptoms; red squares: histopathologically confirmed scrapie; green squares: positive PrPSc immunoblot. Numbers within squares: days to terminal disease. Clinical disease: red line. Prion incubation time is expressed in days. Asterisk: intercurrent death without clinical scrapie signs. [View Larger Version of this Image (34K GIF file)]


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Next, we quantified the recovery of spiked PrPSc and infectivity from urinary proteins (fig. S4). Scrapie cell endpoint assay (9) revealed a higher prion titer in dialyzed samples (fig. S4, C and D), possibly because dialysis removed biocontaminants inhibiting infection of PK1 cells.

Urinary proteins were purified by ultrafiltration followed by dialysis (600 g pooled from groups of three to six mice), or by dialysis followed by ultracentrifugation, and inoculated intracerebrally (i.c.) into groups of three to eight tga20 mice that overexpress PrPC (10). We found prion infectivity within pools of presymptomatic (120 dpi, n = 3) and scrapie-sick RIPLT (n = 6) and NZBW mice (n = 16). However, we did not find infectivity in C57BL/6 (n = 18), MFG-E8/ (n = 8), 129Sv x C57BL/6 (n = 4), NZW (n = 12), or NZB (n = 4) urine at any time point after prion inoculation (Fig. 1). Urine from terminally scrapie-sick NZBW, NZW, and NZB mice could not be collected because the incubation time of scrapie exceeded the natural life span of these mice.

All clinically unaffected tga20 indicator mice were killed at 200 dpi. Histopathological and immunoblot analyses confirmed scrapie in all clinically diagnosed tga20 mice and excluded it from all others (Fig. 2, A to C, and fig. S5C). Phosphotungstate-mediated concentration of PrPSc from 1000 g of protein did not reveal PrPSc in brains of clinically healthy urine-inoculated tga20 mice (fig. S5B). Thus, two pathogenetically distinct chronic inflammatory conditions of the kidney, in concert with prion infection, result in prionuria well before the onset of clinically overt prion disease.

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Fig. 2. Scrapie pathology in mice exposed to urine of nephritic mice. (A and B) Brain sections of tga20 mice that succumbed to scrapie after i.c. inoculation with urinary proteins from RIPLT (terminal) (A) or NZBW mice (130 dpi) (B), showing gliosis (GFAP, glial fibrillary acidic protein) and PrP deposition (SAF84). Tga20 brains inoculated with urine from terminally sick C57BL/6 or presymptomatic NZW mice showed little or no astrogliosis and no PrP deposition. (C) (Upper panels) PrPSc in brains of tga20 mice inoculated i.c. with NZBW urinary proteins (130 dpi). Ten micrograms (left) or 20 g (right) of tga20 brain were digested with proteinase K and immunoblotted. (Lower left panel) PrPSc in brains of tga20 mice inoculated i.c. with NZBW or RIPLT urinary proteins. Lanes 4 to 7: Inoculation with NZBW urinary proteins at 60 dpi (lanes 4 and 5) and 110 dpi (lanes 6 and 7). Positive controls: scrapie-sick tga20 brain homogenate (left two lanes of each blot). Negative control: brain homogenate of a healthy tga20 mouse. (Lower right panel) Inoculation with RIPLT urinary proteins at 120 dpi. (D) Prions were detected in tga20 mice exposed to urine from mice with lymphocytic nephritis (18.2%), but not in mice without kidney pathology or with isolated glomerulonephritis. [View Larger Version of this Image (96K GIF file)]


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Whereas RIPLT and NZBW mice suffer from combined interstitial lymphofollicular inflammation and glomerulonephritis, MFG-E8/, NZW, and NZB mice display glomerulonephritis but lack lymphofollicular foci (figs. S1 and S2). Hence, prionuria necessitates intrarenal organized inflammatory foci (6) and is not elicited by isolated glomerulonephritis (Fisher's exact test, P = 0.031). Urinary proteins from presymptomatic and terminal RIPLT mice induced similar attack rates, suggesting similar urinary prion infectivity titers in presymptomatic and scrapie-sick mice. The consistent lack of infectivity in urine from noninoculated mice and prion-sick wild-type mice makes it unlikely that infectivity found in urine of nephritic mice represents a contaminant.

Scrapie-infected hamsters and Creutzfeldt-Jakob disease (CJD) patients were reported to excrete urinary PrPSc (UPrPSc) (11). However, these findings were not reproduced (12) and were deemed artifactual (13, 14). We attempted to detect UPrPSc in presymptomatic and terminally sick RIPLT, MFG-E8/, tga20, C57BL/6, and 129Sv x C57BL/6 mice, as well as in presymptomatic NZW, NZB, and NZBW mice. Overnight dialysis did not affect the quantitative recovery of spiked PrPSc from urine (fig. S4, A and B); the detection threshold was 100 ng of terminal brain homogenate per milliliter of urine (Fig. 3, B and D), equivalent to 103 median infectious dose (ID50) units/ml. Under these conditions, we failed to reveal any UPrPSc, even in prionuric mice (Fig. 3 A, C, and D). These negative findings are not unexpected, because urinary infectivity titers were typically 1 ID50 units per 2 ml of pooled urine (Fig. 1), which is below the detectability of PrPSc (Fig. 3B).

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Fig. 3. Failure to detect urinary PrPSc. (A) Immunoblot analysis of urinary proteins from terminally scrapie-sick C57BL/6 mice. No PrPSc was found after ultracentrifugation. For control, Prnpo/o urine was spiked with scrapie brain homogenate. (B) Threshold of PrPSc detection in urinary proteins purified by dialysis and ultracentrifugation. C57BL/6 urine was spiked with serial dilutions of brain homogenate. Assay sensitivity: 100 ng of terminal brain homogenate per milliliter of urine (103 ID50 units/ml). (C) Immunoblot analysis of urinary proteins after ultracentrifugation. Scrapie-sick tga20 mice lacked UPrPSc. PK, proteinase K digestion; ICSM-18, primary antibody to PrP. Omission of primary antibody (right) abolished all signals. (D) Immunoblot analysis of urinary proteins from presymptomatic [NZB, NZW, and NZBW (100 dpi)] and terminally scrapie-sick mice. No PrPSc was detected after ultracentrifugation (long exposure). Controls: scrapie brain homogenate used for spiking (lane 1); urine spiked with brain homogenate from scrapie-sick (lanes 2 to 5) or healthy mice (lane 6). [View Larger Version of this Image (58K GIF file)]


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We then tested whether inflammation of nonexcretory organs leads to prionuria. We administered prions to AlbLT mice, which lack nephritis but develop hepatitis (6). Urine from AlbLT and appropriate wild-type control mice (four pools of n = 4 mice, 120 dpi) lacked prion infectivity and UPrPSc (Figs. 1 and 3D; fig. S5, B and C). Thus, extrarenal inflammation, though enabling prion accumulation at the site of inflammation, does not induce prionuria.

Because PrPC is necessary for prion replication (4), its expression may be rate-limiting for urinary prion excretion. We assessed prionuria in tga20 mice, whose renal PrPC content is six to eight times that of wild-type mice (fig. S3F). Pooled urinary proteins (600 g each) from six terminally scrapie-sick tga20 mice were inoculated i.c. into tga20 mice (Fig. 1). None of the recipient tga20 mice developed scrapie. Upon necropsy (>200 dpi), no scrapie histopathology was detected (fig. S5C). Thus, PrPC overexpression does not induce prionuria. The PrPC content of RIPLT, NZBW, and MFG-E8/ kidneys was similar to those of wild-type controls (fig. S3, G and H). RIPLT and NZBW kidneys contain FDC-M1+ cells with high, focal levels of PrPC (6), which may facilitate local prion replication (5). Inoculation of urinary protein from noninfected mice did not elicit any abnormality in tga20 mice (fig. S5C).

How do prions enter the urine? Upon extrarenal replication, blood-borne prions may be excreted by a defective filtration apparatus. Alternatively, prions may be produced locally and excreted during leukocyturia. Although prionemia occurs in many paradigms of peripheral prion pathogenesis (15, 16), the latter hypothesis appears more likely, because prionuria was invariably associated with local prion replication within kidneys.

Urine from one CJD patient was reported to elicit prion disease in mice (17, 18), but not in primates (19). Perhaps unrecognized nephritic conditions may underlie these discrepant observations. Inflammation-associated prionuria may also contribute to horizontal transmission among sheep, deer, and elk, whose high efficiency of lateral transmission is not understood.


References and Notes

1. H. Fraser, A. G. Dickinson, Nature 226, 462 (1970).[CrossRef][ISI][Medline]
2. J. Castilla, P. Saa, C. Hetz, C. Soto, Cell 121, 195 (2005).[CrossRef][ISI][Medline]
3. S. B. Prusiner, Science 216, 136 (1982).[ISI][Medline]
4. H. Beler et al., Cell 73, 1339 (1993).[CrossRef][ISI][Medline]
5. A. Aguzzi, M. Heikenwalder, Immunity 22, 145 (2005).[CrossRef][ISI][Medline]
6. M. Heikenwalder et al., Science 307, 1107 (2005).[Abstract/Free Full Text]
7. S. R. Jones, Am. J. Med. 88, S30 (1990).
8. Materials and methods are available as supporting material on Science Online.
9. P. C. Klohn, L. Stoltze, E. Flechsig, M. Enari, C. Weissmann, Proc. Natl. Acad. Sci. U.S.A. 100, 11666 (2003).[Abstract/Free Full Text]
10. M. Fischer et al., EMBO J. 15, 1255 (1996).[Abstract]
11. G. M. Shaked et al., J. Biol. Chem. 276, 31479 (2001).[Abstract/Free Full Text]
12. M. W. Head, E. Kouverianou, L. Taylor, A. Green, R. Knight, Neurology 64, 1794 (2005).[Abstract/Free Full Text]
13. A. Serban, G. Legname, K. Hansen, N. Kovaleva, S. B. Prusiner, J. Biol. Chem. 279, 48817 (2004).[Abstract/Free Full Text]
14. H. Furukawa et al., J. Biol. Chem. 279, 23661 (2004).[Abstract/Free Full Text]
15. C. A. Llewelyn et al., Lancet 363, 417 (2004).[CrossRef][ISI][Medline]
16. F. Houston, J. D. Foster, A. Chong, N. Hunter, C. J. Bostock, Lancet 356, 999 (2000).[CrossRef][ISI][Medline]
17. Y. Shibayama et al., Acta Pathol. Jpn. 32, 695 (1982).[Medline]
18. J. Tateishi, Y. Sato, M. Koga, H. Doi, M. Ohta, Acta Neuropathol. (Berlin) 51, 127 (1980).[CrossRef][ISI][Medline]
19. D. C. Gajdusek, C. J. Gibbs Jr., M. Alpers, Science 155, 212 (1967).[ISI][Medline]
20. We thank H. Moch, C. Sigurdson, M. Kurrer, P. Klhn, M. Prinz, R. Moos, A. Marcel, J. Collinge, and B. Odermatt for technical help. N. Ruddle provided RIPLT mice, and S. Nagata provided MFG-E8/ mice. Supported by grants from the Bundesamt fr Bildung und Wissenschaft, the Swiss National Foundation, and the National Center of Competence in Research on neural plasticity and repair (to A.A.). M.H. is supported by a Career Development Award of the University of Zrich.


Supporting Online Material

www.sciencemag.org/TSS

Materials and Methods

Figs. S1 to S5

Table S1

References

15 August 2005; accepted 18 September 2005
10.1126/science.1118829
Include this information when citing this paper.

http://www.sciencemag.org/TSS

www.sciencemag.org/cgi/content/full/310/5746/324/DC1

Supporting Online Material for

Coincident Scrapie Infection and Nephritis Lead to Urinary Prion Excretion

Harald Seeger, Mathias Heikenwalder, Nicolas Zeller, Jan Kranich,

Petra Schwarz, Ariana Gaspert, Burkhardt Seifert, Gino Miele, Adriano Aguzzi*

*To whom correspondence should be addressed. E-mail: adriano@pathol.unizh.ch

Published 14 October 2005, Science 310, 324 (2005)

DOI: 10.1126/science.1118829

This PDF file includes:

Materials and Methods

Figs. S1 to S5

Tables S1

References

Supporting Online material: "Coincident Scrapie Infection and Nephritis Lead to Urinary

Prion Excretion", by Seeger et al.

Material and methods

Mice: All aspects of animal procedures, including criteria for termination at onset of terminal

disease, were approved by local authorities. AlbLTαβ, RIPLTα, MFG-E8-/-, tga20, C57BL/6,

129SvxC57BL/6, NZW, NZB and NZBW mice have been described previously (S1-6).

RIPLTα mice (fig. S1A and S2A) suffer from follicular nephritis (S2-4, S7), membrano/

mesangioproliferative glomerulonephritis, and mesangiolysis with mild proteinuria (fig.

S1A, S2 and table S1) indicative of a glomerular filtration barrier defect.

NZBW mice (S5) develop severe membrano-/mesangioproliferative glomerulonephritis with

segmental sclerosis, crescents, abundant proteinuria (fig. S1B, D, F, and table S1), conspicuous

mesangial complement deposits (fig. S2I), and lymphoid inflammatory foci (fig.

S1B, S2A) (S4,S7).

Parental NZW mice suffer from glomerulonephritis with immunoglobulin and complement

deposits (fig. S2B, E, G), but lack lymphocytic inflammatory foci (fig. S1B). Therefore,

RIPLTα and NZBW mice suffer from severe immunopathology of the filtration apparatus

combined with interstitial lymphofollicular inflammation.

Mice deficient for milk fat globule-epidermal growth factor 8 (MFG-E8) develop membrano/

mesangioproliferative glomerulonephritis with proteinuria and display mesangial immunoglobulin

and glomerular complement deposits (fig. S2F-J and table S1), but lack lymphocytic

inflammation (S6).

Prion inoculations: Transgenic and wild-type mice were inoculated i.p. with brain homogenate

diluted in 100 l sterile PBS/5% BSA, equivalent to 3 and 5 logLD50 units (primary inoculations

and time-course experiments, respectively) of the Rocky Mountain Laboratory

(RML) scrapie strain (passage 5, henceforth called RML5). The titer of RML5 was previously

assessed by i.c. inoculation of serial dilutions into tga20 mice, and was found to be

8.9 logLD50/g of brain tissue (S8). Mouse urine (3-4 ml) from presymptomatic RIPLTα, wild-

type, NZBW, NZW, NZB, MFG-E8-/-, 129Sv x C57BL/6 and tga20 animals (for each genotype

n=3-6) was collected overnight in metabolic cages at 30, 45, 60, 85, 110, 120, 125, 130 dpi,

or at terminal stage of disease by bladder puncture after euthanasia, and pooled for each

group. Mice were monitored clinically every second day, and scrapie was diagnosed according

to clinical criteria including ataxia, kyphosis, priapism, tail rigidity and hind leg paresis.

Intracerebral inoculation of urinary proteins: The possible pathological consequences of

intracerebral (i.c.) urinary protein inoculation are unknown. We therefore inoculated purified

urinary proteins from non-prion-inoculated NZBW (n=6), C57BL/6 (n=6), RIPLTα (n=7),

AlbLTαβ (n=8), MFG-E8-/-(n=8) mice into groups of 6-8 tga20 indicator mice. None of the

latter mice developed any abnormal clinical signs at .120 dpi; no abnormal histopathological

findings and no brain PrPSc were detected in indicator mice exposed to C57BL/6 (n=6) and

NZBW (n=8) urine (fig. S5C). These results argue against any nonspecific effects of urinary

protein inoculation into indicator mice.

Dialysis and precipitation of urinary proteins: Two ml of urine pooled from 4 healthy or 4

terminally scrapie-sick wild-type (C57BL/6) or tga20 mice were dialyzed against 5 liters of

PBS (changed after 8 and 16 hrs) in dialysis cassettes (slide-a-lyzer, molecular weight cutoff:

7kD, Pierce, Rockford, Il) at 4C. In order to determine the extent of UPrPSc recovery,

urine from healthy wild-type mice (2 ml) was spiked with 20 l of 1% (w/v) RML5 brain homogenate

and dialyzed as described. Samples were centrifuged at 100000g for 1h at 4C,

and pellets were resuspended in 40 l STE buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1

mM EDTA) containing 2% Sarkosyl. Aliquots of each sample were incubated in the presence

or absence of proteinase K (PK, 20 g/ml, 30 min at 37C), heated at 95C for 5 min in SDS

loading buffer containing 100 mM DTT, and loaded on 12% SDS-polyacrylamide gels. The

specificity of PrP detection was assessed by incubating urinary protein blots of healthy and

terminally scrapie-sick tga20 mice with secondary antibody only. To assess the detection

limit of this assay, a range of serial dilutions of RML5 brain homogenate (10-2-10-4) was prepared

from a stock of 10-1 RML5 (w/v) brain homogenate, and spiked into healthy murine

(strain CD1) brain homogenate (10% w/v). Twenty l from each of these dilution steps were

2

added to 2 ml of urine from healthy mice. Spiked urine samples were dialyzed and processed

for immunoblot analysis as described above. For the immunoblot analysis showing the dilution

series, half of the resuspended pellets were used.

Concentration of urinary protein: Urine was centrifuged at 500 g at 4C for 5 min in a microcentrifuge

to pellet cellular debris. The supernatant was cell-free as assessed by light microscopy.

Three ml of the supernatant were concentrated approximately 10-fold in an ultrafree-

4 spin device (10kD NMWL; Millipore, Bedford, MN) at 3000 g at 4C in a swinging

bucket rotor. Samples were then dialyzed in dialysis cassettes (slide-a-lyzer, molecular

weight cutoff: 10 kD) against 5 liters of PBS (changed twice) for 24 hrs at 4C. Aliquots of 30

l of the dialyzed concentrated urinary proteins were inoculated i.c. into tga20 indicator mice.

Additionally, urine was collected in metabolic cages from groups of 4-6 animals at given time

points (30-130 dpi as indicated) from scrapie-inoculated AlbLTαβ, RIPLTα, MFG-E8-/-, tga20,

C57BL/6, 129Sv x C57BL/6, NZW, NZB and NZBW mice, as well as non-inoculated C57BL/6

and NZBW mice, centrifuged at 500g at 4C for 5 min in a microcentrifuge to pellet debris,

and dialyzed (slide-a-lyzer, molecular weight cutoff: 10 kD, Pierce, Rockford, Il) against saline

(5 l, changed twice) for twenty-four hours at 4C. Dialyzed urine from each group (2.2 ml)

was ultracentrifuged at 100000g for 1h. Pellets were resuspended in 150 l PBS containing

5mg/ml bovine serum albumin (Sigma), and inoculated into groups of four tga20 indicator

mice.

Histology and immunohistochemistry: Paraffin sections (1-2 m) and frozen sections of

brain (10 m) and kidney (5 m) were stained with hematoxylin/eosin. For generating paraffin

sections, formaldehyde-fixed brain and renal tissues were treated with 98% formic acid for 60

min. Postfixation in formaldehyde was performed for at least 1 d, and tissues were embedded

in paraffin. Antibodies FDC-M1 (clone 4C11; 1:50; Becton Dickinson), FDC-M2 (S9)

(1:50; Immunokontakt 212-M/C-1FDCM2), B220/CD45R (RA3-6B2, Pharmingen 553084;

1:400 in PBS/0.15% BSA), CD35 (8C12, Pharmingen, San Diego, CA; 1:100), CD4 cells

(YTS 191; 1:200) and CD8 (YTS 169; 1:50), both rat anti-mouse kindly provided by Dr. Rolf

Zinkernagel (S10), NLDC-145 (BMA T-2013; 1:1000), F4/80 (Serotec; 1:50), GFAP (1:300;

3

DAKO, Carpinteris, CA), and PNA lectin (Vector L-1070; 1:100) were applied and visualized

using standard methods. IgA (Jackson Immuno Research Laboratories, Inc.; Lot No

M051778; 1:2000), IgM (Pharmingen; Becton Dickinson; Lot No M026103; 1:2000), IgG

(Pharmingen; Becton Dickinson; Lot No M016245; IgG1 1:5000; Pharmingen; Becton Dickinson;

Lot No M025639; IgG2a/b 1:1000; Pharmingen; Becton Dickinson; Lot No M016316;

IgG3 1:2000), C1qa (Dako, A0136/rabbit anti human; 1:100), C3 (Conex: 171403150/13/15

IgG1 mouse C3b, iC3b, C3dg; 1:300) and C4 (Immuno kontact 212-MK-1FDC-M2 anti

mouse; 1:200) were stained on consecutive cryosections and visualized by immunohistochemistry

and/or immunofluorescence. For PrP staining, after deparaffination and pretreatment

in concentrated formic acid for 6 min sections (1-2 m) were heated to 100C in a

steamer in citrate buffer (pH 6.0) for 3 min, and allowed to cool down to room temperature.

Sections were incubated in Ventana buffer, and stains were performed on a NEXEX immunohistochemistry

robot (Ventana instruments, Switzerland) using an IVIEW DAB Detection

Kit (Ventana). After incubation with protease 1 (Ventana) for 16 min, sections were incubated

with anti-PrP SAF-84 (SPI bio, A03208, 1:200) for 32 min. Sections were counterstained with

hematoxylin. Histoblots were performed as described (S11). Periodic acid Schiff (PAS) were

performed according to standard procedures.

Electron microscopy (EM): Cubes from RIPLTα, wild-type, NZBW, NZW, MFG-E8-/- and

129Sv x C57BL/6 kidneys (size: 1-5 mm3) were fixed (4 hrs) in 2.5% glutaraldehyde, 0.1 M

phosphate, pH 7.4 at 4C, osmified, dehydrated, and cut according to standard procedures.

Scrapie cell assay in endpoint format (SCEPA): Prion-susceptible neuroblastoma cells

(subclone N2aPK1) were exposed to prion samples in 96-well plates for three days. Cells

were then split three times 1:3 every two days, and three times 1:10 every three days. After

confluence is reached, 25000 cells from each well are filtered onto the membrane of an

ELISPOT plate, treated with PK, denatured and individual infected (PrPSc-positive) cells are

detected by an ELISA using a PrP antibody. The number of "infectious tissue culture units"

(TCI) per aliquot was calculated from the proportion of negative to total wells using the Poisson

equation.

4

To investigate whether prion infectivity was lost during dialysis (fig. S4C), precipitates of dialyzed

and non-dialyzed urine spiked with scrapie brain homogenate at a dilution of 10-4 were

subjected to SCEPA. Precipitates were resuspended in 10 l PBS containing 0.1% sarcosyl.

These samples (inoculum; dilution 10-1) were then diluted 100-fold in PBS (dilution 10-3);

subsequent serial ten-fold dilutions were performed in cell culture medium containing healthy

mouse brain homogenate at a dilution of 10-4 until a final dilution of 10-7. Scrapie-susceptible

PK1 cells were then exposed to dilutions of the experimental samples ranging from 10-4 to

10-7, or a 10-4 dilution of healthy mouse brain homogenate ("mock").

Immunoblot analysis: 10% (w/v) tissue homogenates were prepared in a Ribolyzer as described

and optionally treated with proteinase K (50g/ml, 30 min, 37oC). Proteins were electrophoresed

through 12% SDS-PA gels and transferred to nitrocellulose membranes (Schleicher-

Schuell, Germany) by wet blotting. Membranes were blocked with TBS-T containing 5%

Topblock (Juro, Switzerland), and incubated with monoclonal anti-mouse PrP antibody

POM1 (400 ng/ml, M. Polymenidou and AA, unpublished). Detection was performed with

horseradish-peroxidase coupled rabbit anti-mouse IgG1 antibody (Zymed).

Sodium phosphotungstate (PTA) precipitation assay: Brain and kidney homogenates

(10%) were prepared in 0.32 M sucrose or PBS as described above. For kidneys, gross cellular

debris was removed by centrifugation at 500 g for 5 min. 100 l of the resultant supernatant

was mixed (1:1) with 4% Sarkosyl in PBS. Samples were incubated for 15 min at 37C

under constant agitation. Benzonase and MgCl2 were added to a final concentration of 50

U/ml and 1 mM, respectively, and incubated for 30 min at 37C under continuous agitation.

Samples were digested with 50 g/ml proteinase K (PK) for 30 min at 37C under agitation.

Complete TM mini protease inhibitor mix (Roche) and pre-warmed PTA stock solution (pH

7.4) prepared in 170 mM MgCl2 were added (final concentration: 0.3%). Samples were incubated

at 37C for 30 min with constant agitation, and centrifuged at 37C for 30 min at maximum

speed in an Eppendorf microcentrifuge. Pellets were resuspended in 20 l 0.1% Sarkosyl

in PBS, and heated at 95C for 10 min in SDS-containing loading buffer before loading

onto 12% NuPAGE polyacrylamide gels (Invitrogen, USA).

5

Measurements of albumin in urine: Urinary albumin concentration was determined by

ELISA (Albuwell M kit, Exocell Inc, USA). Urinary creatinine was quantified spectrophotometrically

using a creatinine assay kit (Exocell, Inc). Urinary albumin was normalized to

creatinine excretion and presented as micrograms of albumin per milligram of creatinine as

described (S12). For all experiments, pools of 3-6 age-matched mice were used and measured

in triplicates.

Statistical analysis: We examined the probability of death of tga20 indicator mice after i.c.

inoculation of urinary proteins derived from mice with isolated glomerulonephritis (NZB, and

NZW), and mice with lymphofollicular nephritis (RIPLTα and NZBW). The different numbers

of tga20 mice (n=4 or n=8) that had been used in each bioassay (inflammation/

glomerulonephritis versus no inflammation, or isolated glomerulonephritis versus no

glomerulonephritis) were accounted for by translating the results into "standard experiments"

with the size of 4 indicator mice. Seventy such standard experiments can be drawn from one

transmission experiment to 8 indicator mice. If the attack rate was e.g. 2/8 (urinary proteins

of terminal RIPLTα mice), 15 of these standard experiments are negative, whereas scrapie is

observed in the remaining 55. For experiments with a null attack rate, all standard subexperiments

are identical, and no correction is necessary. To compare different groups of

experiments, Fisher's exact test was applied iteratively in order to accommodate the existence

of various standard experiments. Unconditional p-values were computed using Bayes'

formula. The following groups were included in the statistical analysis: RIPLTα (120 dpi),

RIPLTα (terminal disease); C57BL/6 (120 dpi); C57BL/6 (terminal disease); tga20 (terminal

disease) NZBW I (130 dpi); NZBW II (130 dpi); NZBW III (120 dpi); NZBW IV (120 dpi);

NZW I (130 dpi); NZB I (130 dpi); NZW II (120 dpi); and NZW III (120 dpi). For Fig. 4D all

tga20 mice (.200 dpi) inoculated i.c. with urinary proteins (as depicted in Fig. 1) were classified

according to their affiliation to the following groups: prion infected/mock infected, glomerulonephritic/

not glomerulonephritic, and renal lymphofollicular inflammation/no renal inflammation.

6

Figure legends

Supplemental Figure 1: Renal lymphocytic infiltrates in RIPLTα and NZBW, but not in

MFG-E8-/- kidneys. (A-B) H&E-stained paraffin sections of age-matched RIPLTα, C57BL/6,

NZBW, and NZW kidneys, showing capsular and subcapsular lymphoid follicles, as well as

focal interstitial lymphocytic infiltrates (arrowheads). All RIPLTα and NZBW mice developed

nephritis, defined as the presence of interstitial and capsular follicular lymphoid infiltrates. (C)

No follicular infiltrates were identified in MFG-E8-/- and 129Sv x C57BL/6 kidneys.

Supplemental Figure 2: Glomerular pathology in RIPLTα, NZBW and MFG-E8-/- mice.

(A-C) Consecutive cryosections identifying IgA, IgM and IgG deposits in RIPLTα, but not in

C57BL/6, glomeruli. NZBW glomeruli display IgM and IgG, and NZW glomeruli display IgM

deposits. IgG and IgM deposits were also found in MFG-E8-/-, but not in age-matched 129Sv

x C57BL/6 glomeruli. (D-E) Six-eight month-old RIPLTα and NZBW glomeruli showing mesangial

proliferation and mesangiolysis (arrowhead) (RIPLTα), segmental sclerosis, crescents

(arrowhead) and hyperplastic tubules (NZBW). (F) Glomeruli of 40 week-old MFG-E8-/-

mice displayed parietal epithelial proliferation of the Bowman capsule, glomerular basement

membrane thickening, and mesangial hypercellularity. In contrast, PAS-stained paraffin sections

did not reveal any differences between young (approx. 20 week-old) MFG-E8-/- and

129Sv x C57BL/6 mice (data not shown). (G) Electron microscopy of RIPLTα, C57BL/6,

NZBW, NZW, MFG-E8-/-, and 129Sv x C57BL/6 glomeruli (Scale bar: 1m). Left: C57BL/6

(wild-type) capillary loops and mesangium appear normal and do not display electron dense

deposits. In contrast, subendothelial electron dense deposits and abnormal basement membrane

are seen in RIPLTα capillary loops (arrowhead). Middle panels: large mesangial electron-

dense deposits in NZW glomeruli. NZBW glomeruli show mesangial proliferation, mesangial

electron dense deposits (Inset A), and subendothelial electron dense deposits (Inset

B). These findings underscore the profound pathology of the glomerular filtration apparatus in

RIPLTα and NZBW mice. Right: mesangial/segmental endocapillary proliferation and mesangiolysis

in glomeruli of 37 week-old MFG-E8-/-, but not 129Sv x C57BL/6 mice (EM; scale

7

bar: 10 m). Inset A: small mesangial and paramesangial electron dense deposits (scale bar:

2 m). Inset B: small subendothelial electron dense deposits (scale bar: 2 m). Wild-type

mice glomeruli display minimal mesangial expansion and no electron dense deposits (scale

bar: 2 m). Electron microscopical analyses reveal destruction of the glomerular filtration

apparatus in MFG-E8-/- mice. (H) Immunofluorescence (IF) revealed abnormal mesangial and

capillary IgA, IgG and IgM deposits in RIPLTα, but not C57BL/6 glomeruli. DIC: Differential

interference contrast. Scale bar: 50 m. (I and J) Complement components C1q, C3 and C4

in kidney cryosections of RIPLTα, MFG-E8-/- and NZBW mice. (I) Complement components

were detectable in renal lymphofollicular infiltrates (right column), and more rarely in RIPLTα

glomeruli (left column). C57BL/6 glomeruli displayed no or weak signals (middle column). (J)

Left and middle panel: immunostained cryosections reveal prominent positivity for complement

components C1q and C4 in MFG-E8-/- glomeruli (40 week-old), but only for C4 in 129Sv

x C57BL/6 controls. Right panel: Strong immunostain for C1q, C3 and C4 in NZBW

glomeruli.

Supplemental Figure 3: PrPSc in brain, spleen and kidney of prion-infected mice. Similar

levels of PrPSc were detected in brains (A) and spleens (B) of prion-infected NZBW, NZB

and NZW mice. (C) Comparable levels of PrPSc were found in spleens of MFG-E8-/- and

129Sv x C57BL/6 mice inoculated with 3 logLD50 (left panel) or 6 logLD50 (right panel) of

RML5 at 66 dpi. (D-E) PTA precipitation of PrPSc from MFG-E8-/- and control kidney homogenates

(1 mg/100 l). No PrPSc was detectable at 66 dpi. (F-G) Immunoblots of two-fold dilution

series of kidney homogenate, showing increased PrPC levels (6-8 fold) in kidneys of

tga20 mice compared to wild-type mice, but not in kidneys of NZBW compared to NZB and

NZW mice. Blots were re-probed with antibody against β-actin (lower panels). (H) Quantification

of PrPC content of kidneys of MFG-E8-/- and control 129Sv x C57BL/6 mice did not reveal

any significant difference.

8

Supplemental Figure 4: Quantitative, lossless recovery of PrPSc and of prion infectivity

upon dialysis and concentration of urinary proteins. To assess the extent to which PrPSc

and prion infectivity are recovered after dialysis, serial ten-fold dilutions of RML brain homogenate

(10-3 - 10-5) were prepared in mouse urine as described in Material and Methods.

PTA precipitation was then performed on aliquots of each dilution step prior to, or after, dialysis.

Optionally dialyzed precipitates (+ or -) were subjected to semiquantitative immunoblot

analysis after PK digest and to scrapie cell assay in endpoint format (SCEPA). (A) Semiquantitative

immunoblot analysis indicating that no PrPSc was lost during the dialysis procedure

(right panel: long exposure visualizing PrPSc at dilution 10-5). (B) Integrals of immunoblot

signal intensities are expressed in arbitrary units. (C) To investigate whether infectivity was

lost during dialysis, precipitates of dialyzed and non-dialyzed urine spiked with scrapie brain

homogenate at a dilution of 10-4 were subjected to SCEPA. Membranes of ELISPOT plates

of the SCEPA stained with antibody POM-1 against PrP are shown. Each dot represents the

progeny of one scrapie-infected cell. (D) Table indicating the number of wells containing infected

cells relative to the total number of wells exposed to a given dilution of inoculum.

Whereas no prion infectivity could be detected at a dilution of 10-7 in the non-dialyzed sample,

3 of 6 wells tested positive in the dialyzed sample. This corresponds to 5.7 log tissue

culture infectivity (TCI) units/ml in the non-dialyzed sample, and to 6.3 log TCI units/ml inoculum

in the dialyzed sample. Images in (E) represent higher magnifications of selected areas

of ELISPOT membranes, as labeled with "X" in panel C.

Supplemental Figure 5: Biochemical and histological analysis of tga20 indicator mice.

(A-B) PTA-enhanced Western blot analyses of brains from tga20 mice that had been exposed

to urine from prion-infected mice, yet did not develop clinical signs of scrapie at .200

dpi. PK: Proteinase K. Ct-: brain of a healthy tga20 mouse; ct+: brain of a terminally scrapiesick

tga20 mouse, without PTA precipitation. Alb: tga20 mouse was inoculated with AlbLTαβ

urine. "Scr+" or "scr-" indicates whether the urine donors had been inoculated with prions. (C)

Immunohistochemistry of consecutive brain sections for PrP (antibody SAF84) or GFAP.

9

Only brains of clinically scrapie-sick tga20 indicator mice exhibited severe astrogliosis and

PrP deposits. Healthy tga20 indicator mice euthanized .200 days after urinary protein inoculation

occasionally showed mild astrogliosis, but never PrP deposits.

10

Supplemental Table 1: Analysis of urinary proteins in various mouse models. 3-6 individual

age-matched mice of each genotype were analyzed.

Genotype g albumin/mg creatinine

C57BL/6 17.2 0.3

RIPLTα 79.3 6.8

AlbLTαβ 51.78 6.6

MFGE8-/- 617.7 38

C57BL/6 x Sv129 36.1 2.2

NZBW 47078.5 2965.4

NZW 78.1 12.8

NZB 622.4 114.5

tga20 39.0 1.6

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S2. D. E. Picarella, A. Kratz, C. B. Li, N. H. Ruddle, R. A. Flavell, Proc Natl Acad Sci U S

A 89, 10036 (1992).

S3. A. Kratz, A. Campos-Neto, M. S. Hanson, N. H. Ruddle, Journal of Experimental

Medicine 183, 1461 (1996).

S4. M. Heikenwalder et al., Science 307, 1107 (2005).

S5. N. Talal, Clin Rheum Dis 11, 633 (1985).

S6. R. Hanayama et al., Science 304, 1147 (2004).

S7. H. Seeger, M. Heikenwalder, A. Aguzzi, data not shown.

S8. P. S. Kaeser, M. A. Klein, P. Schwarz, A. Aguzzi, J Virol 75, 7097 (2001).

S9. P. R. Taylor et al., Eur J Immunol 32, 1888 (2002).

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11


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