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From: TSS (216-119-144-16.ipset24.wt.net)
Subject: Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient [FULL TEXT]
Date: August 7, 2004 at 9:28 am PST

-------- Original Message --------
Subject: Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient [FULL TEXT]
Date: Sat, 07 Aug 2004 11:21:46 -0500
From: "Terry S. Singeltary Sr."
To: Bovine Spongiform Encephalopathy
CC: cjdvoice@yahoogroups.com


Preclinical vCJD after blood transfusion in a PRNP codon 129
heterozygous patient

Alexander H Peden, Mark W Head, Diane L Ritchie, Jeanne E Bell, James W
Ironside
We report a case of preclinical variant Creutzfeldt-Jakob disease (vCJD)
in a patient who died from a nonneurological
disorder 5 years after receiving a blood transfusion from a donor who
subsequently developed vCJD.
Protease-resistant prion protein (PrPres) was detected by western blot,
paraffin-embedded tissue blot, and
immunohistochemistry in the spleen, but not in the brain.
Immunohistochemistry for prion protein was also
positive in a cervical lymph node. The patient was a heterozygote at
codon 129 of PRNP, suggesting that
susceptibility to vCJD infection is not confined to the methionine
homozygous PRNP genotype. These findings
have major implications for future estimates and surveillance of vCJD in
the UK.

In 2003, an elderly patient in the UK was diagnosed
with variant Creutzfeldt-Jakob disease (vCJD) that
seemed to have been transmitted by a transfusion of
non-leucodepleted red cells from a patient who
developed vCJD after the donation.1 The same
investigation also reported 17 individuals alive in
December, 2003, who had received labile blood
components from donors who subsequently developed
vCJD.1 We report an autopsy detection of a preclinical
case of vCJD infection, which appears to have been
transmitted by blood transfusion in one of this cohort.

In 1999, an elderly patient received a unit of nonleucodepleted
red blood cells from a donor who
developed symptoms of vCJD 18 months after
donation. The donor died in 2001 and vCJD was
confirmed after autopsy. The recipient died 5 years
after receiving the transfusion, with no evidence of a
neurological disorder. Medicolegal instruction for
autopsy was issued. The immediate cause of death was
a ruptured abdominal aortic aneurysm. We are bound
by a medicolegal restriction regarding disclosure of the
patients age, sex, and geographical location.

We assessed samples of frozen brain, spinal cord,
dorsal root ganglion, lymphoid tissues, and muscle for
the presence of protease-resistant prion protein (PrPres)
by western blot with phosphotungstic acid precipitation
and the monoclonal antibody 3F4.2 Immunohistochemistry
and paraffin-embedded tissue blotting was done
on protease-treated tissue sections from a wide range of
tissues, with a panel of four antibodies raised against
different epitopes of prion protein (PrP).3 Restriction
fragment length polymorphism analysis of DNA
extracted from frozen brain material identified the
patient as being heterozygous (methionine/valine) at
codon 129 of the prion protein gene (PRNP). Consent
for full sequence analysis of PRNP had not been
obtained. Western blot analysis showed the presence of
PrPres in spleen (figure 1). The mobility and glycoform
ratio of the signals in spleen were similar to those seen
in spleen from patients with clinical vCJD and in vCJD
brain diluted in non-CJD spleen (figure 1), and were
distinct from those described in a subset of sporadic
CJD cases, usually with a relatively lengthy clinical
illness.2 We found that PrPres positivity by this method
was a consistent feature of four autopsy specimens of
spleen from patients who had vCJD, but was absent
from a series of nine spleens from controls without
CJD (data not shown).

The brain (1337 g) showed only age-related changes,
with no pathological features of vCJD. PrPres was
undetectable in the brain and spinal cord by western
blotting, paraffin embedded tissue blotting, and
immunohistochemistry. Immunoreactivity for PrP was
found in a few germinal centres in the spleen, in a
pattern consistent with staining of follicular dendritic
cells (figure 2, A). The number of positive follicles was
far lower than in clinical cases of vCJD, with a less
aggregated accumulation of immunoreactivity.3
Immunoreactivity for PrP was also found in a germinal
centre within a cervical lymph node, with similar
pattern of positivity to that noted in the spleen
(figure 2, B). PrPres was not detectable by western

Lancet 2004; 264: 52729
National Creutzfeldt-Jakob
Disease Surveillance Unit,
Division of Pathology, School of
Molecular and Clinical Medicine,
University of Edinburgh,
Western General Hospital,
Edinburgh EH4 2XU, UK
(A H Peden PhD, M W Head PhD,
D L Ritchie BSc,
Prof J E Bell FRCPath,
Prof J W Ironside FRCPath)
Correspondence to:
Prof James W Ironside
james.ironside@ed.ac.uk
www.thelancet.com Vol 364 August 7, 2004 527
vCJD
brain
spike
49
kDa  + Case sample 1
Neurological control
Case sample 2
vCJD
38
28
18
14
Figure 1: PrPres analysis of spleen by western blot
Two samples of the patients spleen were compared with spleen samples from a
control with non-CJD neurological disease and from a patient with vCJD, and
with vCJD brain homogenate (10g) diluted in non-CJD spleen, with (+) or
without () proteinase K digestion. Every lane represents the
phosphotungstate
precipitate from 50 mg wet weight of spleen. Horizontal lines indicate
positions
of molecular weight markers. Amounts of PrPres in eight samples of
spleen from
the patient were undetectable in two samples (not shown), intermediate
in five
(sample 1), and similar to those found in the spleen of a patient with
vCJD at
autopsy in one (sample 2).
Research Letters


blotting in samples of tonsil, another cervical lymph
node, dorsal root ganglion, and muscle; neither was it
detected in the lymphoid follicles within the tonsil,
appendix, and large intestine by immunohistochemistry.

This is the first recorded case in the UK of autopsy
detection of preclinical vCJD infection. We have
previously shown preclinical PrP immunoreactivity in
germinal centres within appendix tissue from two
patients who underwent appendectomy 8 months and
2 years before the onset of vCJD.4 The patterns of PrP
accumulation within the germinal centres in the spleen
and cervical lymph node in the present case were
similar to those seen in three surgically removed
appendices from a large anonymised retrospective
study, suggesting that these findings might also
represent preclinical vCJD infection.4

Our findings also show that vCJD infection can be
confirmed by western blot analysis of PrPres in an
individual who is a heterozygote at codon 129 of
PRNP.1,3 This finding has major implications for future
estimations of numbers of vCJD cases in the UK, since
individuals with this genotype constitute the largest
genetic subgroup in the population.4 This subgroup
might have a different incubation period after exposure
to either primary infection by the bovine spongiform
encephalopathy (BSE) agent or secondary infection by
blood transfusion. A very lengthy incubation period
might explain why no clinical cases of vCJD have yet
been observed in this subgroup. Such preclinical cases
might also represent a source of iatrogenic infection
themselves, either by blood donation or by
contamination of surgical instruments coming into
contact with lymphoid tissues, even in the absence of
infectivity in the brain.

This patient was a UK resident and might therefore
have had dietary exposure to the BSE agent. However,
the chance of observing vCJD transmission in the
absence of a transfusion infection in a second recipient
of blood from a donor with vCJD must be far less likely
than the 1 in 15 000 to 1 in 30 000 chance for the first
reported case.1 PrPres was not detected in the nine
patients without CJD used as negative controls in this
study, and in a previous study we and others did not
detect PrP accumulation in lymphoid tissues in 56
cases of other forms of human prion disease and in 85
non-CJD cases.5 The restriction of PrPres to the spleen
and cervical lymph node (but not the tonsil or gutassociated
lymphoid tissue) in this case is consistent
with an intravenous rather than oral route of exposure.
It is also possible that the PRNP codon 129 genotype
might affect the distribution of PrPres in tissues.

This case highlights the need for continuing
surveillance for CJD in the UK, and strongly reinforces
the role of the autopsy in the investigation and
diagnosis of both clinical and preclinical forms of
human prion disease.

Conflict of interest statement
None declared.
Contributors
A H Peden did biochemical analysis and photography, and contributed
to drafting of the manuscript. M W Head did biochemical analysis,
and contributed to the drafting of the manuscript. D L Ritchie did
histological analysis and photography, and contributed to the drafting
of the manuscript. J E Bell did the autopsy, provided the autopsy data,
and contributed to the drafting of the manuscript. J W Ironside did the
histological analysis and coordinated the preparation and drafting of
the manuscript.
Acknowledgments
We are grateful to the Transfusion Medicine Epidemiology Review
study (C A Llewelyn, P E Hewitt, J MacKenzie, R G Will) for
identifying the case in this report and for provision of data. We thank
Matthew Bishop and Kelly Connolly for genetic analysis, and Suzanne
Lowrie for expert technical assistance. The National Creutzfeldt-Jakob
Disease Surveillance Unit is funded by the UK Department of Health
and the Scottish Executive. A H Peden is funded by the EC TSELAB
Project (ref QLK2 -CT-2002-81523). This work is part of the EU
NeuroPrion project (FOOD-CT-2004-506579 (NOE) subproject:
PRIOGEN). The funding sources had no input into the design of this
study, the collection, analysis. and interpretation of data, the writing of
the report, or the decision to submit the paper for publication.
References
1 Llewelyn CA, Hewitt PE, Knight RS, et al. Possible transmission of
variant Creutzfeldt-Jakob disease by blood transfusion. Lancet 2004;
363: 41721.
528 www.thelancet.com Vol 364 August 7, 2004
Figure 2: PrP in germinal centres within the spleen and cervical lymph node
Germinal centres are labelled (brown) with the anti-PrP antibodies 3F4
in spleen
(A) and 12F10 in a cervical lymph node (B) in a pattern similar to that
noted in
the follicular dendritic cell network, with less aggregated positivity
than in cases
of clinical vCJD. Original magnifications (A) 20 and (B) 10.
Research Letters
2 Glatzel M, Abela E, Maissen M, Aguzzi A. Extraneural pathologic
prion protein in sporadic Creutzfeldt-Jakob disease. N Engl J Med
2003; 349: 181220.
3 Head MW, Ritchie D, Smith N, et al. Peripheral tissue involvement
in sporadic, iatrogenic, and variant Creutzfeldt-Jakob disease: an
immunohistochemical, quantitative, and biochemical study.
Am J Pathol 2004; 164: 14353.
4 Hilton DA, Ghani A, Conyers L, et al. Prevalence of
lymphoreticular prion protein accumulation in UK tissue samples.
J Pathol 2004; 203: 73339.
5 Hilton DA, Sutak J, Smith MEF, et al. Specificity of
lymphoreticular accumulation of prion protein for variant
Creutzfeldt-Jakob disease. J Clin Pathol 2004; 57: 30002.

http://www.thelancet.com/

TSS





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