SEARCH VEGSOURCE:

 

 

Follow Ups | Post Followup | Back to Discussion Board | VegSource
See spam or
inappropriate posts?
Please let us know.
  




From: TSS ()
Subject: FAILURE TO DETECT PRION PROTEIN BY IHC IN STRIATED MUSCLE TISSUES OF ANIMALS EXPERIMENTALLY INOCULATED WITH TSE QUESTIONED
Date: March 27, 2005 at 9:29 am PST

FAILURE TO DETECT PRION PROTEIN (PRPRES) BY IMMUNOHISTOCHEMISTRY IN STRIATED MUSCLE TISSUES OF ANIMALS EXPERIMENTALLY INOCULATED WITH AGENTS OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY Authors

Submitted to: Veterinary Pathology
Publication Acceptance Date: July 28, 2003
Publication Date: January 1, 2004
Citation: Hamir, A.N., Miller, J.M., Cutlip, R.C. 2004. Failure To Detect Prion Protein (Prpres) By Immunohistochemistry In Striated Muscle Tissues Of Animals Experimentally Inoculated With Agents Of Transmissible Spongiform Encephalopathy. Veterinary Pathology. 41(1):78-81. Interpretive Summary: Transmissible spongiform encephalopathies (TSEs) are fatal, neurologic diseases. Infection by the causative agent, a prion, induces accumulations of an abnormal form of prion protein (PrPres) in tissues of affected animals. Presence of characteristic lesions and detection of protease-resistant PrPres in tissues are the basis of currently available methods for diagnosis of TSEs. In this study, samples of muscle tissues (tongue, heart, diaphragm and masseter muscle) from 20 animals (cattle, sheep, elk, and raccoons) were examined for PrPres. All of the animals had developed TSE after experimental inoculation with scrapie, chronic wasting disease, or transmissible mink encephalopathy agents. PrPres was found in the brains, but not in muscle tissues of all examined animals. These results are contradictory to recently published data in laboratory animals with TSEs, which may indicate that laboratory-adapted TSE strains have acquired the capacity to amplify in muscles during propagation in laboratory animals. Although further testing of muscle tissues is needed to confirm findings of the present study, meat consumers may be relieved to know that at present no PrPres can be detected in muscle tissues of experimental animals with TSEs. Technical Abstract: Transmissible spongiform encephalopathies (TSEs) are fatal, neurologic diseases. Infection by the causative agent, a prion, induces accumulations of an abnormal form of prion protein (PrPres) in tissues of nervous and lymphoid systems. Presence of characteristic histopathological changes (spongiform encephalopathy) and detection of protease-resistant PrPres in neural and lymphoid tissues are the basis of currently available methods for diagnosis of TSEs. In this study, samples of striated muscle tissues (tongue, heart, diaphragm and masseter muscle) from 20 animals (cattle, sheep, elk, and raccoons) were examined for PrPres by immunohistochemistry. All of the animals had developed TSE after experimental inoculation with scrapie, chronic wasting disease, or transmissible mink encephalopathy. PrPres was found in the brains, but not in muscle tissues of all examined animals. These results are contradictory to recently published data in laboratory animals with TSEs, which may indicate that laboratory-adapted TSE strains have acquired the capacity to amplify in muscles during propagation in laboratory animals. Further testing of muscle tissues is needed to confirm findings of the present study. ...

http://www.ars.usda.gov/

107
Vet Pathol 42:107–108 (2005)
Letters to the Editor
Editor:
Absence of evidence is not always evidence of absence.
In the article ‘‘Failure to detect prion protein (PrPres) by
immunohistochemistry in striated muscle tissues of animals
experimentally inoculated with agents of transmissible spongiform
encephalopathy,’’ recently published in Veterinary
Pathology (41:78–81, 2004), PrPres was not detected in striated
muscle of experimentally infected elk, cattle, sheep, and
raccoons by immunohistochemistry (IHC). Negative IHC,
however, does not exclude the presence of PrPSc. For example,
PrPres was detected in skeletal muscle in 8 of 32
humans with the prion disease, sporadic Creutzfeldt-Jakob
disease (CJD), using sodium phosphotungstic acid (NaPTA)
precipitation and western blot.1 The NaPTA precipitation,
described by Wadsworth et al.,3 concentrates the abnormal
isoform of the prion, PrPres, from a large tissue homogenate
volume before western blotting. This technique has increased
the sensitivity of the western blot up to three orders
of magnitude and could be included in assays to detect
PrPres. Extremely conspicuous deposits of PrPres in muscle
were detected by IHC in a recent case report of an individual
with inclusion body myositis and CJD.2 Here, PrPres was
detected in the muscle by immunoblotting, IHC, and paraf-
fin-embedded tissue blot. We would therefore caution that,
in addition to IHC, highly sensitive biochemical assays and
bioassays of muscle are needed to assess the presence or
absence of prions from muscle in experimental and natural
TSE cases.
Christina Sigurdson, Markus Glatzel, and Adriano Aguzzi
Institute of Neuropathology
University Hospital of Zurich
Zurich, Switzerland
References
1 Glatzel M, Abela E, et al: Extraneural pathologic prion
protein in sporadic Creutzfeldt-Jakob disease. N Engl J
Med 349(19):1812–1820, 2003
2 Kovacs GG, Lindeck-Pozza E, et al: Creutzfeldt-Jakob
disease and inclusion body myositis: abundant diseaseassociated
prion protein in muscle. Ann Neurol 55(1):
121–125, 2004
3 Wadsworth JDF, Joiner S, et al: Tissue distribution of protease
resistant prion protein in variant CJD using a highly
sensitive immuno-blotting assay. Lancet 358:171–180,
2001


====================================


Diagnosis of human prion disease


*Institute for Neurodegenerative Diseases, §Memory and Aging Center, and Departments of {dagger}Neurology, ¶Pathology, and {dagger}{dagger}Biochemistry and Biophysics, University of California, San Francisco, CA 94143; ||ZLB Behring, 35041 Marburg, Germany; and **ZLB Behring, 75601 Paris, France

Contributed by Stanley B. Prusiner, December 22, 2004


Abstract


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, {approx} 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.

snip...

Discussion


The clinical diagnosis of human prion disease is often difficult until the patient shows profound signs of neurologic dysfunction. It is widely accepted that the clinical diagnosis must be provisional until a tissue diagnosis either confirms or rules out the clinical assessment. Before the availability of Abs to PrP, a tissue diagnosis was generally made by histologic evaluation of neuropil vacuolation. IHC with anti-glial-fibrillary-acidic-protein Abs in combination with H&E staining preceded the use of anti-PrP Ab staining.

Recently, the role of IHC in the diagnosis of scrapie in the brains of eight clinically affected goats inoculated with the SSBP1 prion isolate has been challenged (14). Thalamic samples taken from seven of eight goats with scrapie were positive for PrPSc by Western blotting but negative by IHC. The eighth goat was negative by Western blotting and IHC. Consistent with these findings in goats are the data reported here, in which IHC of formalin-fixed, paraffin-embedded human brain samples was substantially less sensitive than the CDI.

The CDI was developed to quantify PrPSc in tissue samples from mammals producing prions. Concerned that limited PK digestion was hydrolyzing some or even most of the PrPSc, we developed a CDI that does not require PK digestion. The CDI revealed that as much as 90% of PrPSc is sPrPSc; thus, it was being destroyed during limited proteolytic digestion used to hydrolyze PrPC. sPrPSc comprises {approx}80% of PrPSc in the frontal lobe and in the white matter (Fig. 4).

The CDI detected HuPrPSc with a sensitivity comparable to the bioassay for prion infectivity in Tg(MHu2M) mice (Fig. 1). The high sensitivity achieved by the CDI is due to several factors (8, 10, 11, 15). First, both sPrPSc and rPrPSc conformers are specifically precipitated by PTA (Table 5) (8, 9). PTA has also been used to increase the sensitivity of Western blots enabling the detection of rPrPSc in human muscle and other peripheral tissues (16, 17). Second, a sandwich protocol was used with the high-affinity MAR1 mAb (11) to capture HuPrPSc and Eu-labeled 3F4 mAb to detect HuPrPSc (12). Third, the CDI detects PrPSc by Ab-binding to native and denatured forms of the protein and, therefore, does not depend on proteolytic degradation of PrPC. We chose not to perform Western blots on most of the samples used in this study because such immunoblots require denaturation of the sample, which eliminates measurement of the native signal corresponding to PrPC (Table 5). Moreover, a comparison between the CDI and Western blotting on brain samples from sCJD and variant CJD patients showed that the CDI was 50- to 100-fold more sensitive (15). Additionally, Western blots combined with densitometry are linear over a 10- to 100-fold range of concentrations, whereas the CDI is linear over a >104-fold range. The CDI has been automated, which not only improves accuracy and reproducibility (10) but also allows numerous samples to be analyzed, as reported here. Western blots are difficult to automate and are labor intensive.

Our studies show that only the CDI detected PrPSc in all regions examined in 24 sCJD and 3 fCJD(E200K) brains (Figs. 2 and 6). Comparative analyses demonstrated that the CDI was vastly superior to histology and IHC. When 18 regions of 8 sCJD and 2 fCJD(E200K) brains were compared, we discovered that histology and IHC were unreliable diagnostic tools except for samples from a few brain regions. In contrast, the CDI was a superb diagnostic procedure because it detected PrPSc in all 18 regions in 8 of 8 sCJD and 2 of 2 fCJD(E200K) cases (Tables 1 and 2).

Histologic changes in prion disease have been shown to follow the accumulation of prions as measured by bioassay of infectivity and by PrPSc accumulation (18–22). Because low levels of PrPSc are not associated with neuropathologic changes, some discrepancy between vacuolation and PrPSc was expected. In contrast to histology, IHC measures PrP immunostaining after autoclaving tissue sections exposed to formic acid. Because IHC measures PrP, we expected the sensitivity of this procedure might be similar to the CDI, but that proved not to be the case. Whether exposure of formic acid-treated tissue sections to elevated temperature destroys not only PrPC but also sPrPSc and only denatures rPrPSc remains to be determined. Such a scenario could account for the lower sensitivity of IHC compared with CDI or bioassay (Tables 1 and 2).

Studies of the white matter in CJD brains were particularly informative with respect to the sensitivity of the CDI, where PrPSc levels were low but readily detectable, 10- to 100-fold above the threshold value (Fig. 4). Because animal studies have shown that PrPSc and infectivity are transported anterogradely from one brain region to another along neuroanatomical pathways (23–25), we expected to find PrPSc in white matter as demonstrated by the CDI but not IHC. Axonal transport of PrPSc is also suggested by diffusion-weighted MRI scans of CJD cases, which show high-intensity signals in analogous neocortical regions of the right and left cerebral hemispheres (26). This symmetry of neuroradiological abnormalities is consistent with spread of PrPSc to the contralateral cortex by means of callosal commissural pathways.

Most immunoassays that detect HuPrPSc do so only after subjecting the sample to limited proteolysis to form PrP 27–30, followed by denaturation. Because the CDI measures the immunoreactivity before and after denaturation to an epitope that is exposed in native PrPC but buried in PrPSc, limited proteolysis to eliminate PrPC is unnecessary. Assays based on limited proteolysis underestimate the level of PrPSc because they digest sPrPSc, which represents 80–90% of PrPSc in CJD and scrapie brains (Fig. 4 and Table 5).

Gerstmann–Sträussler–Scheinker, an inherited human prion disease, is caused by the P102L mutation in the PRNP gene. In mice expressing the Gerstmann–Sträussler–Scheinker mutant PrP transgene, the CDI detected high levels of sPrPSc(P101L) as well as low levels of rPrPSc(P101L) long before neurodegeneration and clinical symptoms occurred (9). sPrPSc(P101L) as well as low concentrations of rPrPSc(P101L) previously escaped detection (27). Whether a similar situation applies in other genetic forms of prion disease, sCJD, or variant CJD remains to be determined. Because most of the PrPSc in the brains of sCJD patients is protease-sensitive (Fig. 4), it is likely that the lower sensitivity of IHC is due to its inability to detect sPrPSc. Presently, we have no information about the kinetics of either sPrPSc or rPrPSc accumulation in human brain. Limited information on the kinetics of PrPSc accumulation in livestock comes from studies of cattle, sheep, and goats inoculated orally, but most of the bioassays were performed in non-Tg mice (28–30) in which prion titers were underestimated by as much as a factor of 104 (10).

The studies reported here are likely to change profoundly the approach to the diagnosis of prion disease in both humans and livestock (31–33). The superior performance of the CDI in diagnosing prion disease compared to routine neuropathologic examination and IHC demands that the CDI be used in future diagnostic evaluations of prion disease. Prion disease can no longer be ruled out by routine histology or IHC. Moreover, the use of IHC to confirm cases of bovine spongiform encephalopathy after detection of bovine PrPSc by the CDI (10) seems an untenable approach in the future. Clearly, the CDI for HuPrPSc is as sensitive or more sensitive than bioassays in Tg(MHu2M) mice (Fig. 1).

Our results suggest that using the CDI to test large numbers of samples for human prions might alter the epidemiology of prion diseases. At present, there is limited data on the frequency of subclinical variant CJD infections in the U.K. population (34). Because appendixes and tonsils were evaluated only by IHC, many cases might have escaped detection (Tables 1 and 2). Equally important may be the use of CDI-like tests to diagnose other neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, and the frontotemporal dementias. Whether IHC underestimates the incidence of one or more of these common degenerative diseases is unknown. Moreover, CDI-like tests may help determine the frequency with which these disorders and the prion diseases occurs concomitantly in a single patient (35, 36).


snip...

http://www.pnas.org/cgi/content/abstract/102/9/3501

ORAL 8

Bovine spongiform encephalopathy (BSE) in Japan

Takashi Yokoyama, Kumiko M. Kimura, Morikazu Shinagawa
Prion Disease Research Center, National Institute of Animal Health, Japan

Bovine spongiform encephalopathy (BSE) has become an important problem not only for animal industry, but
also for public health. In Japan, BSE was first recognized in September 2001 by fallen stock surveillance.
Since October 2001, BSE examination for all cattle slaughtered at abattoirs has started. In April 2004, all dead
cattle examination (over 24 months) has been conducted at livestock hygiene service center. Samples positive
in enzyme linked immunosorbent assay (ELISA) are further subjected to western blot (WB) and
immunohistochemistry (IHC). Thirteen BSE cases have been reported by September 2004. Twelve cases
were classified as typical BSE, and the remained one was an atypical BSE. Variant forms of BSE with atypical
histopathological and/or biochemical phenotype were reported in Italy and France. Further study is required
for BSE prion characteristics.
To characterize BSE prion properties, brain homogenates of Japanese BSE cases were intracerebrally
inoculated into wild-type mice. The first case (BSE/Chiba) was successfully transmitted to rodents. The mean
incubation periods (409.0 days) in this experiment was preferably longer than that of previously reported.
PrPSc distribution, prion titer, mice susceptibility and/or storage condition of sample might be influenced the
result. Recently, we introduced transgenic mice that overexpress a bovine PrP gene to overcome the species
barrier problem. These mice are expected to accelerate the transmission experiment of BSE prion.
Transmission of atypical BSE case is undergoing by using these transgenic mice.


http://www.knt.co.jp/ec/2004/prion/E2.htm


Japan Consumer Press online
Nippon shouhisha shinbun
http://www.jc-press.com/eg.htm
http://www.jc-press.com/En/Latest%20News/200411/20041109BSE%20death%20cow%27s%20anomalous%20prion.htm
Last modified, 11/09/2004 13:42:49

BSE death cow's anomalous prion detected from peripheral nerve tissue,
suprarenal gland

First time from non-Specified Risk Material, or SRM

By JCPRESS

National Institute of Animal Health Animal announced on November 1 that it had detected the anomalous prion protein that was the etiologic agent of the mad cow disease, or BSE, or bovine spongiform encephaalopathy, from the peripheral nerve tissue and the suprarenal gland of the cow of the age in the mad cow disease for the dying infection 94 months on March 9 this year.
Japan is obligating the removal of the Specified Risk Material, or SRM such as the head, the spinal cord, the vertebral columns, and the small
intestines that accumulate the anomalous prion protein easily as a BSE
(bovine spongiform encephaalopathy) measures.
Because the mad cow disease etiologic agent was detected from a tissue
different from the Specified Risk Material, or SRM, the review of the
Specified Risk Material, or SRM might be urged on the Japanese Government.
International Symposium of PRION DISEASES for food and drug safety
http://www.knt.co.jp/ec/2004/prion/
national institute of animal health(only in Japanese)
http://niah.naro.affrc.go.jp/index-j.html
The statement of the Ministry of Health, Labour and Welfare
(only in Japanese)
http://www.maff.go.jp/www/press/cont2/20041101press_7.htm
Yomiuri on line (only in Japanese)
http://www.yomiuri.co.jp/science/news/20041102i503.htm
Asahi on line(only in Japanese)
http://www.asahi.com/special/bse/TKY200411010291.html
Mainichi on line(only in Japanese)
http://www.mainichi-msn.co.jp/shakai/jiken/disease/news/20041102ddm041040128000c.html

TSS




Follow Ups:



Post a Followup

Name:
E-mail: (optional)
Subject:

Comments:

Optional Link URL:
Link Title:
Optional Image URL: