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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1749-1756
Resistance of Paroxysmal Nocturnal Hemoglobinuria Cells to the
Glycosylphosphatidylinositol-Binding Toxin Aerolysin
By
Robert A. Brodsky,
Galina L. Mukhina,
Kim L. Nelson,
Tracy S. Lawrence,
Richard J. Jones, and
J. Thomas Buckley
From the Johns Hopkins Oncology Center, Baltimore, MD; and the
Department of Biochemistry and Microbiology, University of Victoria,
Victoria, British Columbia, Canada.
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ABSTRACT |
Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell
disorder caused by a somatic mutation of the PIGA gene. The product of
this gene is required for the biosynthesis of
glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic
hallmark of PNH cells is an absence or marked deficiency of all
GPI-anchored proteins. Aerolysin is a toxin secreted by the bacterial
pathogen Aeromonas hydrophila and is capable of killing target
cells by forming channels in their membranes after binding to
GPI-anchored receptors. We found that PNH blood cells (erythrocytes,
lymphocytes, and granulocytes), but not blood cells from normals or
other hematologic disorders, are resistant to the cytotoxic effects of
aerolysin. The percentage of lysis of PNH cells after aerolysin
exposure paralleled the percentage of CD59+ cells in the
samples measured by flow cytometry. The kinetics of red blood cell
lysis correlated with the type of PNH erythrocytes. PNH type III cells
were completely resistant to aerolysin, whereas PNH type II cells
displayed intermediate sensitivity. Importantly, the use of aerolysin
allowed us to detect PNH populations that could not be detected by
standard flow cytometry. Resistance of PNH cells to aerolysin allows
for a simple, inexpensive assay for PNH that is sensitive and
specific. Aerolysin should also be useful in studying PNH biology.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PAROXYSMAL NOCTURNAL hemoglobinuria (PNH)
is an acquired hematopoietic stem cell disorder manifested by abnormal
hematopoiesis, complement-mediated intravascular hemolysis, and a
propensity toward thrombosis.1 The disease usually results
from a somatic mutation in the X-linked gene, PIGA.2-5 The
product of this gene is necessary for the first step in the
biosynthesis of glycosylphosphatidylinositol (GPI) anchors; hence,
cells harboring PIGA mutations are characterized by a deficiency,
absolute or partial, of all proteins affixed to the cell membrane in
this way. GPI-anchored proteins have been shown to be involved in a
wide range of important cell functions, including signal
transduction6,7 and trafficking of apically expressed
epithelial proteins.8,9 They may also play a role in
regulating apoptosis.10 Two GPI-anchored proteins, CD55
(decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis), normally protect cells from the action of homologous
complement, and it is their absence that leads to the hemolytic anemia
associated with PNH.11,12
Aerolysin is a toxin secreted by the human bacterial pathogen
Aeromonas hydrophila and is perhaps the best characterized
member of a large group of water-soluble proteins that are capable of killing target cells by forming channels in their membranes (see Parker
et al13 for a recent review). Members of the group include a wide variety of virulence factors, such as Staphylococcus
aureus  toxin and aerolysin itself, as well as the T-cell
defense protein perforin. All of these proteins are able to undergo a
transformation from a water-soluble to an insertion-competent state by
oligomerizing to form amphipathic barrels.
Aerolysin is secreted as a completely inactive protoxin that is
converted to the active form by proteolytic removal of a C-terminal peptide.14 A number of mammalian proteases can activate
proaerolysin, including trypsin, chymotrypsin, and furin.15
Cells that are sensitive to aerolysin, such as T lymphocytes (which are
killed by 1 hour of exposure to as little as 10 11
mol/L toxin), contain specific high-affinity receptors that serve to
concentrate the toxin on the cell surface, promoting
insertion.16 Proaerolysin can also bind to the receptors,
but it is unable to oligomerize and therefore cannot form
channels.17
We have recently identified a number of proteins that can act as
aerolysin receptors.18-20 Among them are Thy-1 from brain and T lymphocytes, contactin from brain, the variant surface
glycoprotein (VSG) of trypanosomes, and a novel erythrocyte
glycoprotein (EAR). These proteins share one remarkable property: they
are all attached to the plasma membrane with GPI anchors. Importantly,
we demonstrated that the anchor itself is a primary determinant of
aerolysin binding.20 In addition, we showed that mutant
mouse T-cell lines that are unable to synthesize GPI-anchors are far
less sensitive to aerolysin than normal cells.19 In this
report, we show that human PNH cells are also resistant to the
cytotoxic effects of aerolysin. This provides a sensitive and specific
assay for PNH that is rapid and inexpensive.
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MATERIALS AND METHODS |
Cell lines.
The GPI-anchor deficient lymphoblastoid cell lines,
LD and JY5, harbor previously characterized PIGA
mutations.10,21 An expression vector containing the
full-length PIGA cDNA was stably transfected into the
LD and JY5 cell lines to establish the GPI-anchor
replete cell lines, LD(PIGA+) and
JY5(PIGA+), as previously described.10 All cell
lines were maintained in RPMI 1640 medium (GIBCO, Gaithersburg,
MD) with 10% heat-inactivated fetal calf serum. To
measure CD59 expression, cells were washed in RPMI with 0.2% fetal
calf serum, stained for CD59 with a fluorescein isothiocyanate-conjugated monoclonal antibody (MoAb; Research Diagnostics, Flanders, NJ), and analyzed by flow cytometry (FACscan; Becton Dickinson, San Jose, CA).
Preparation of cells for aerolysin assay.
Venous peripheral blood from patients with PNH, normal controls, or
disease controls was drawn into EDTA-containing tubes after informed
consent as approved by the Joint Committee on Clinical Investigation of
the Johns Hopkins Hospital. The blood was centrifuged at 400g
for 10 minutes and then the buffy coat was removed and the remaining
erythrocytes were washed two times in phosphate-buffered saline (PBS)
and resuspended in PBS to a concentration of 0.8%. Peripheral blood
granulocytes were isolated using Ficoll/Hypaque (density,
1.119 g/mL) as previously described.22
Titer assay of aerolysin-induced hemolysis.
Aerolysin (1.5 × 106 mol/L), produced by
trypsin activation of proaerolysin as described,15 was
diluted 1:16 in PBS to a final volume of 100 µL and added to the
first well of a 96-well plate. An equal volume of PBS was then added to
the first well (1:32 aerolysin) and 1:2 serial dilutions were made
across the plate. One hundred microliters of 0.8% red blood cells was
added to all wells, and the plate was incubated at 37°C. Absorbance at 620 nm was measured using a plate reader (Biotek Instruments, Inc,
Winooski, VT) at times 0, 5, 10, 15, and 20 minutes. Aerolysin and
proaerolysin are available from Protox Biotech (Victoria, British
Columbia, Canada).
Spectrophotometric assay of aerolysin-induced hemolysis.
Activated aerolysin was added to stirred cuvettes containing 1.5 mL of
0.8% vol/vol washed erythrocytes in PBS (10 mmol/L NaH2PO4, 150 mmol/L NaCl, pH 7.4) to a final
concentration of 8 nmol/L. The rate of hemolysis was determined by
measuring the change in optical density of the erythrocytes (which is
due to a decrease in light scattering as the cells lyse) at 600 nm and 37°C as a function of time. The instrument used was a Varian Cary 1 (Varian Instruments, Houston, TX) recording spectrophotometer.
Propidium iodide staining for viability.
Cells were suspended to 1 × 106/mL in 0.5 mL PBS. Fifty microliters of propidium iodide (10 µg/mL in PBS) was
added to each tube and the mixture was incubated for 5 minutes at
37°C. Approximately 10,000 data events per sample were collected
for analysis on a FACSCAN flow-cytometer (BDIS, Mansfield, MA).
Subcellular debris and remaining erythrocytes were excluded with a
forward-scatter (FS)/90°-scatter (SS) gate. Viable cells were those
exhibiting no fluorescence (propidium iodide excluding).
Detection of aerolysin-binding proteins by Western blotting.
Samples of cells dissolved in sample buffer were separated by sodium
dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) using the
method of Neville.23 Proteins were blotted onto
nitrocellulose and the blots were developed by sandwich Western
blotting as described by Nelson et al.19 This involved
incubation with 2 × 108 mol/L proaerolysin,
followed by polyclonal antiaerolysin and antirabbit horseradish
peroxidase. Blots were then developed by enhanced chemiluminescence
(ECL; Amersham Corp, Arlington Heights, IL).
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RESULTS |
Aerolysin-induced hemolysis of PNH erythrocytes.
In PNH, all hematopoietic lineages have a proportion of GPI-anchor
deficient cells, because the PIGA mutation initially occurs in a
pluripotent hematopoietic stem cell.1 The proportion for individual cell types can be estimated by measuring the fraction of
cells that lack CD59 by flow cytometry, although this is an expensive
procedure requiring complex instrumentation that is not widely
available.24,25
Based on our previous work, we expected the population of GPI-anchor
deficient cells in a PNH blood sample to be relatively resistant to
aerolysin compared with the normal, GPI-anchor replete cells.19 We initially performed a dose-response curve to
compare the sensitivity of normal erythrocytes and PNH erythrocytes to various concentrations of aerolysin. Normal erythrocytes, but not PNH
erythrocytes, were completely lysed after exposure to a 1:128 dilution
of aerolysin (1.5 × 10-6 mol/L stock solution) for 10 minutes at 37°C (Fig 1A). The
sensitivity of red blood cell lysis at a given dose of aerolysin varied
depending on whether the PNH patient had predominantly type II cells
( ) or type III cells ( ).
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| Fig 1.
Aerolysin assays for detection of PNH. (A) Dose-response
curve showing aerolysin sensitivity of two normal (solid lines, open
symbols) versus two PNH (dashed lines, solid symbols) erythrocytes.
() Cells that are primarily type II erythrocytes; () type III
cells. Serial dilutions of activated aerolysin (1.5 × 106 stock) were performed in a 96-well plate and mixed
with an equal volume of 0.8% erythrocytes. Absorbance at 620 nm was
measured after 10 minutes at 37°C using a plate reader. (B) Kinetic
analysis of aerolysin-induced hemolysis of normal erythrocytes (bottom
line) and erythrocytes from 5 different PNH patients. The rate of
hemolysis was determined by measuring the change in optical density of
erythrocytes at 600 nm and 37°C as a measure of time using a
spectrophotometer. The percentage of CD59 erythrocytes
determined by flow cytometry for each patient is shown to the right of
the figure. (C) Flow cytometric analysis for CD59 expression of PNH red
blood cells from the patient with 8% type III PNH erythrocytes (solid
line) and a normal control (dotted line). (D) Flow cytometric analysis
for CD59 expression of PNH red blood cells from the patient with 80%
PNH erythrocytes (solid line) and a normal control (dotted line). The
majority of cells represent type II PNH erythrocytes.
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We also performed a kinetic analysis of normal and PNH erythrocytes
after exposure to aerolysin. The spectrophotometric assay we used
depends on the decrease in light scattering that accompanies erythrocyte lysis. Aerolysin (8 nmol/L) resulted in the complete hemolysis of erythrocytes from normals within approximately 10 minutes
(Fig 1B). The time course was highly reproducible from one normal
sample to another. The proportion of unlysed cells, which should
correspond to those cells that did not display GPI-anchored proteins,
could easily be calculated. For every patient, the value that was
obtained corresponded very closely with the proportion of cells that
lacked CD59, as determined by flow cytometry (Fig 1B). Furthermore,
flow cytometric analysis showed that the cells remaining after exposure
to aerolysin were CD59 (data not shown).
Although all PNH samples showed patterns that were easily distinguished
from the controls, the kinetics of erythrocyte lysis was dependent on
the type of PNH erythrocytes tested. Patients with predominantly type
III PNH cells that completely lack GPI-anchors (eg, the patients with
85%, 20%, and 8% PNH cells; Fig 1C) exhibited a plateau of resistant
cells after 10 minutes in aerolysin that matched the frequency of PNH
cells in the patient (Fig 1B). In contrast, patients with a
predominance of PNH type II cells (eg, the patients with 80% and 32%
PNH cells; Fig 1D) showed slow ongoing lysis over time (Fig 1B).
To determine if the assay was specific for PNH, aerolysin sensitivity
of erythrocytes from patients with various hematologic disorders was
measured. In every disease state we tested, including myelodysplastic
syndromes, aplastic anemia, hemolytic anemias, myeloproliferative
disorders, and leukemias, we found that the cells were as sensitive as
normal erythrocytes to the lytic effects of aerolysin
(Fig 2). Absorbance greater than 0.1 10 minutes after exposure to a 1:128 dilution of aerolysin (1.5 × 10-6 mol/L stock) distinguished PNH from normals and from
other hematologic disorders (Fig 2).
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| Fig 2.
Aerolysin assay is specific for PNH. Mean absorbance of
erythrocytes from 7 normal controls (dots), 3 PNH patients (shaded),
and 20 disease controls (slanted lines) after 10 minutes of exposure to
a 1:128 dilution of aerolysin (1 × 108 mol/L). Disease
controls consisted of myelodysplastic syndromes (7), aplastic anemia
(5), polycythemia vera (2), myelofibrosis (2), sickle cell anemia (1),
autoimmune hemolytic anemia (1), iron deficiency anemia (1), and acute
myelogenous leukemia (1). Error bars represent the standard
deviation.
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Sensitivity of PNH leukocytes to aerolysin.
A major advantage of using flow cytometry over tests based on
complement sensitivity of erythrocytes (ie, sucrose hemolysis and Ham
test) is that immunophenotyping can detect abnormalities in multiple
hematopoietic lineages. To determine whether the aerolysin assay could
detect a GPI-anchor deficiency in nucleated cells as well as in
erythrocytes, the toxin was incubated with two PNH cell lines,
LD and JY5, that harbor a previously characterized
PIGA mutation and, hence, fail to express GPI-anchored proteins. The
absence of GPI anchors was confirmed using an aerolysin sandwich
Western blotting procedure (Fig 3) and flow
cytometric analysis for CD59 expression
(Fig 4A and C). The LD
and JY5 cell lines were essentially unaffected by 6 nmol/L aerolysin (Fig 4B and D). In contrast, the same dose of aerolysin produced rapid
death of the same cell lines stably transfected with the full-length
cDNA for PIGA (Fig 4B and D).
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| Fig 3.
PNH cells lack proaerolysin binding proteins.
Proaerolysin binding to proteins from LD cells (lane 1),
LD(PIGA+) cells (lane 2), and CEM cells
(lane 3). Equal numbers of cells (6 × 104/lane) were
applied to each lane. Lane 1 ( LD cells) is blank,
whereas lane 2 [LD(PIGA+)] has multiple
bands. For the sake of comparison, an immature human T-cell line (CEM)
was also run in this experiment. A band corresponding to Thy-1, which
we have detected previously in mouse T cells, is easily visible in the
figure.
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| Fig 4.
Restoration of GPI-anchor expression in PNH cell lines
overcomes resistance to aerolysin. Flow cytometric analysis for CD59
expression of LD and JY5 cells before (solid line) and
after (dotted line) stable transfection of the PIGA cDNA (A and C). (B)
Viability of LD (solid line) and
LDPIGA+ (dotted line) cells after exposure
to 1 nmol/L aerolysin at 37°C. (D) Viability of JY5 (solid line)
and JY5PIGA+ (dotted line) cells after exposure to 1 nmol/L aerolysin at 37°C. Cell viability was determined in
triplicate at 5-minute intervals. Error bars represent the standard
deviations.
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The percentage of PNH granulocytes in the circulation most accurately
reflects the size of the PNH clone, because the survival of PNH
granulocytes is normal or even increased,10,26,27 whereas erythrocyte survival is decreased.28 Therefore, we sought
to determine the sensitivity of PNH granulocytes to aerolysin.
Granulocytes from patients with PNH and from normal controls were
separated, treated with aerolysin, and analyzed using flow cytometry to
assess their ability to exclude propidium iodide. Forty minutes after exposure to 2.5 × 108 mol/L aerolysin, less
than 10% of normal granulocytes excluded propidium iodide
(Fig 5B). In contrast, more than 90% of
PNH granulocytes retained their ability to exclude propidium iodide
under identical conditions (Fig 5D). Granulocytes from disease controls
were as sensitive to the toxin as cells from normal controls (data not shown), demonstrating that the ability to exclude propidium iodide after exposure to aerolysin was specific for PNH cells. The percentage of PNH granulocytes that was resistant to aerolysin correlated with the
percentage of cells lacking CD59 expression (data not shown).
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| Fig 5.
PNH granulocytes are resistant to aerolysin.
Representative example of normal and PNH granulocytes stained with
propidium iodide before (A and C) and 40 minutes after (B and D)
incubation with 1 nmol/L activated aerolysin. Propidium iodide uptake
was assayed using flow cytometry.
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Sensitivity of aerolysin assay for PNH.
We next tested a known mixture of LD and
LD(PIGA+) to determine how accurately
the new assay could determine the percentage of GPI-anchor deficient
cells in a mixed population (10% PNH cells). Similar to the
above-mentioned experiments in erythrocytes, the assay accurately
determined the percentage of PNH cells in the population
(Fig 6). The lower limit of detection of a
PNH population using flow cytometry is 1% to 5%.24,25 To
determine whether aerolysin could be used to detect smaller PNH
populations than this, we mixed PNH cells (LD) with
increasing numbers of GPI anchor protein replete cells (CEM) and
assayed CD59 expression before and after exposure to aerolysin
(Fig 7). Before the addition of aerolysin,
PNH cells were undetectable when they comprised less than 1% of the
population; however, 30 minutes after exposure to aerolysin, PNH
populations as small as 0.1% were detected.
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| Fig 6.
Aerolysin sensitivity correlates CD59 expression in PNH
cell lines. Representative example showing correlation between
aerolysin sensitivity and flow cytometric detection of CD59 in PNH cell
lines. (A) Flow cytometric analysis for CD59 expression in a population
of cells consisting of 90% LDPIGA+ cells
and 10% LD cells (solid line). LD cells
are depicted by the dotted line. (B) The percentage of viability of the
LD (solid line) and mixed cell (dotted line) populations
after exposure to 1 nmol/L aerolysin at 37°C. Cell viability was
determined by trypan blue exclusion at 5-minute intervals.
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| Fig 7.
Use of aerolysin to detect small PNH populations.
LD cells (10,000) were mixed with CD59-expressing CEM
cells 1:1 (A), 1:10 (B), 1:100 (C), 1:500 (D), and 1:1,000 (E) and
analyzed for expression of CD59 before (solid line) and after (dotted
line) 30 minutes of exposure to 1 nmol/L aerolysin.
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PNH can arise de novo or evolve from aplastic anemia, suggesting a
pathophysiologic link between the two diseases.29,30 To
test whether the use of aerolysin in conjunction with flow cytometry
could detect small PNH populations, we used this assay to study the
peripheral blood from an aplastic anemia patient who did not respond to
immunosuppressive therapy. Before adding aerolysin, we were unable to
detect PNH erythrocytes using standard flow cytometry; however, 15 minutes after exposure to 5 × 10-9 mol/L aerolysin, a
small population of PNH cells was detected (Fig 8). Because aerolysin depleted the
erythrocyte population by 2.5 to 3 logs (determined by counting on a
hemacytometer), we calculated that this represents a PNH population of
less than 0.075%. A similar result was obtained in a patient with a
1-year history of moderate aplastic anemia; no PNH cells could be
detected in the peripheral blood of three normal control subjects
treated in an identical manner (data not shown).
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| Fig 8.
Use of aerolysin to detect a minor population of PNH
cells in patient with aplastic anemia. Two-color histogram of
peripheral blood cells stained with MoAb directed against CD59 antigen
(FITC) and MoAb directed against glycophorin-A (PE) before (A) and
after (B) exposure to aerolysin (5 × 109 mol/L for 15 minutes). Axis represent log red (PE) or log green (FITC) fluorescence
intensity.
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DISCUSSION |
We found that a simple, inexpensive assay that measures the absorbance
of cells after exposure to 1 × 108 mol/L
aerolysin for 10 minutes can reliably distinguish PNH cells from
normals and other disease states (Figs 1 and 2). The presence of
GPI-anchored proteins on cells sensitizes them to aerolysin because
these proteins act as toxin receptors and binding promotes oligomerization and channel formation. PNH cells are less sensitive to
the toxin because they have reduced (type II) or absent (type III)
expression of GPI-anchored proteins. Type II PNH cells show slow,
ongoing lysis to aerolysin over time because of their reduced but not
absent GPI-anchors; nevertheless, even type II PNH cells can be
unequivocally distinguished from normals and other disease states using
the above-mentioned conditions (1 × 108 mol/L
aerolysin for 10 minutes).
The sensitivity and specificity of the assay are not surprising given
that no other clinical syndrome is characterized by cells with
deficient GPI anchors. The assay represents an improvement over
traditional screening tests for PNH that measure the sensitivity of
erythrocytes to homologous complement, such as the sucrose hemolysis
and Ham tests. The Ham test is relatively specific for PNH, but lacks
sensitivity, whereas the sucrose hemolysis test is relatively
sensitive, but is not specific.25 Another advantage of the
aerolysin assay over these tests is that it accurately predicts the
size of the PNH clone and the type of PNH erythrocytes (Figs 1 and 4)
and can detect PNH populations in cells other than erythrocytes (Figs 3
and 7).
Flow cytometry is currently the gold standard for diagnosing PNH but
requires expensive equipment and significant technical expertise that
are not available in many laboratories. The simple aerolysin assay
offers most of the advantages of flow cytometry for the detection of
PNH and can be performed in any laboratory with access to a plate
reader or spectrophotometer. Moreover, it is currently not possible to
detect small (<1% to 2%) PNH populations.24,25 However,
by enriching for PNH cells, aerolysin can be used in conjunction with
flow cytometry to detect PNH populations that represent 0.075% or less
of the total cells (Figs 6 and 8). At this time it is unclear whether
these small PNH populations are clinically relevent; nevertheless, this
technology may be particularly helpful in studying the pathophysiology
of PNH and detecting small PNH populations in patients with aplastic
anemia (Fig 8).
Approximately one third of newly diagnosed PNH manifest as a secondary
clonal complication in patients with aplastic anemia.31 This is especially common after immunosuppressive therapy but is rare
after regimens that use immunoablative doses of
cyclophosphamide,32 with or without bone marrow
transplantation.33 The close relationship between aplastic
anemia and PNH suggests a pathophysiologic link between the two
disorders, but the precise mechanism remains
unclear.29,30,34,35 The fact that marrow failure in most
cases of aplastic anemia results from an autoimmune attack has led some
investigators to postulate that the immune attack in aplastic anemia is
directed toward a GPI-anchored protein.36,37 Thus, PNH
cells (with deficient GPI-anchored proteins) would be immune to attack
and could preferentially expand. Nevertheless, direct evidence for such
a mechanism in aplastic anemia or PNH is lacking and, with the
exception of CD59 and CD55, GPI-anchored proteins are not ubiquitously
expressed on both mature cells and progenitors. Our observations
demonstrate that aerolysin is the first natural toxin that selectively
kills normal cells while sparing PNH cells. This property may be
particularly useful not only in diagnosis, but also in studying the
biology of PNH by allowing the selection of PNH clones from the bone
marrow in patients with aplastic anemia or other disease states.
| |
ACKNOWLEDGMENT |
The authors thank Dr Jerry Spivak for patient samples and critical
reading of this manuscript and Marie C. Moineau for assistance in
manuscript preparation. Drs Dana Devine and Brian Berry supplied samples from PNH patients that were used in preliminary experiments.
| |
FOOTNOTES |
Submitted June 8, 1998; accepted October 21, 1998.
Supported in part by National Institutes of Health Grant No. CA74990 to
R.A.B. and a grant from the Natural Sciences and Engineering Research
Council of Canada (J.T.B.). R.A.B. is an American Society of Hematology Scholar.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Robert A. Brodsky, MD, Johns Hopkins
Oncology Center, Room 2-127, 600 N Wolfe St, Baltimore, MD 21287-8967.
| |
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Top
Abstract
Introduction