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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 46-52
Concentrations of Thrombopoietin in Bone Marrow in Normal Subjects
and in Patients With Idiopathic Thrombocytopenic Purpura, Aplastic
Anemia, and Essential Thrombocythemia Correlate With Its mRNA
Expression of Bone Marrow Stromal Cells
By
Yasuo Hirayama,
Sumio Sakamaki,
Takuya Matsunaga,
Takashi Kuga,
Hiroyuki Kuroda,
Toshiro Kusakabe,
Katsunori Sasaki,
Koshi Fujikawa,
Junji Kato,
Katsuhisa Kogawa,
Ryuzo Koyama, and
Yoshiro Niitsu
From the 4th Department of Internal Medicine, Sapporo Medical
University School of Medicine, Sapporo, Japan; and Hokkaido Prefectural
Sapporo Kitano Hospital, Sapporo, Japan.
 |
ABSTRACT |
The function of bone marrow (BM) stromal thrombopoietin (TPO) in
megakaryopoiesis remains unknown. In the present study we attempted to
clarify the pathophysiological implications of stromal TPO in normal
subjects (NS) and in patients with idiopathic thrombocytopenic purpura
(ITP), aplastic anemia (AA), and essential thrombocythemia (ET) by
measuring TPO concentrations in BM and peripheral blood (PB) and by
estimating the levels of stromal TPO mRNA with TaqMan fluorescence-based post-reverse transcription-polymerase chain reaction product detection system. The results showed that TPO concentrations in PB were significantly elevated in patients with ITP
(34.9 ± 11.7 pg/mL) and AA (364.1 ± 153.5 pg/mL) but within normal
range in patients with ET (each 20.0 and 22.1; NS, 22.1 ± 8.2 pg/mL).
In all subjects, the TPO concentrations in BM correlated well with the
PB levels, and the former were consistently higher than the latter. The
concentrations of TPO in BM also correlated with the levels of TPO mRNA
in stromal cells. Furthermore, expression levels of TPO mRNA clearly
correlated with megakaryocyte counts in NS and patients with ITP,
indicating that stromal TPO actually enhances megakaryopoiesis. Thus,
our results in the present study indicate that TPO from BM stromal
cells is considered to play an essential role for megakaryopoiesis
under various patho-physiological conditions.
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INTRODUCTION |
SINCE THE INTRODUCTION of genetic cloning
of thrombopoietin (TPO), many studies have helped to clarify its
function in megakaryocyte-colony stimulation and
megakaryopotentiation.1-6 However, it is still not clear
which organs synthesize the TPO needed for megakaryopoiesis in bone
marrow (BM) and the mechanisms that regulate TPO production. Although
TPO is synthesized in liver, kidney, spleen, and lung,1 the
levels of TPO mRNA expression in these tissues do not vary in
accordance with circulating TPO concentrations or platelet numbers.7
Kuter and Rosenberg8 have proposed that the concentration
of TPO is passively regulated by its binding to TPO receptors on
platelets (sponge theory); this is based on the finding that, in
rabbits, circulating TPO increases with busulfan administration and
decreases after platelet transfusion.
Recently, BM has been implicated in the production of TPO for
megakaryopoiesis. McCarty et al9 have shown that giving
mice antibodies to platelets results in increased TPO mRNA expression in BM although they have not focused their interest particularly on
stromal cells. Although these are preliminary results, we have reported
that TPO from BM stromal cells play an important role for
megakaryopoiesis in normal subjects (NS) and patients with idiopathic
thrombocytopenic purpura (ITP).10 By using in
situ hybridization, Sungaran et al11 also showed an
increase in TPO mRNA expression in stromal cells from patients with
aplastic anemia (AA) and ITP, although, as they themselves pointed out,
more quantitative analysis is needed to conclusively signify their
finding. More recently, Guerriero et al12 confirmed
ubiquitous expression of TPO mRNA in stromal cultures initiated by
either CD34+ or CD34 BM cells in vitro.
In the present investigation we aimed to show that TPO in BM which
defines megakaryopoiesis under various pathophysiological conditions is
mainly derived from BM stromal cells by using enzyme-linked immunosorbent assay (ELISA) for measurement of TPO protein and TaqMan
reverse transcription-polymerase chain reaction (RT-PCR) for
semi-quantitation of TPO mRNA.13
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MATERIALS AND METHODS |
BM sample selection and preparation.
BM cells were obtained from 1-mL aliquots of BM obtained from 6 normal
volunteers, 6 patients with ITP, 6 patients with AA, and 2 patients
with essential thrombocythemia (ET) at the 4th Department of Internal
Medicine Sapporo Medical University, after informed consent had been
obtained. BM cells were then separated over Ficoll-Isopaque (Pharmacia
Biotech, Uppsala, Sweden) (1.077 g/mL, 400g; 20 minutes,
20°C), and the interface was harvested and washed
three times in RPMI 1640 medium. The mononuclear cells (5 × 105/mL) were plated in 25-cm2 tissue-culture
flasks (Corning Glass Works, Corning, NY) in 10 mL of  -minimum
essential medium (-MEM; Hazleton, Denver, CO) supplemented with
12.5% fetal calf serum (Whitakker, Walkersville, MD), 12.5% horse
serum (Whitakker), and 1.0 × 106 mol/L
hydrocortisone (Upjohn, Irving, TX). Cultures were maintained at
37°C in humidified chamber with 5% CO2 in air: the
medium was replaced weekly.14 After 4 weeks of culture, the
yield was usually 1 × 105 stromal cells/mL of BM.
Oligonucleotides for TaqMan PCR assay.
Table 1 shows the nucleotides sequences
from the oligonucleotide hybridization probes and primers used. These
were obtained from Applied Biosystems, A Division of Perkin Elmer
(Foster City, CA). The forward primer (P1) of TPO was designed to span
an exon 4/intron junction to avoid amplification of DNA sequences,
whereas the reverse TPO primer (P2) was complimentary to the exon 6. The TPO target probe (Pr A) was labeled at the 5 end with the
reporter dye molecule, FAM (6-carboxyfluorescein; emission l 538 nm).
The GAPDH target probe (Pr B) as the internal control was labeled with
JOE (6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein; emission l 546 nm). Both probes were labeled with the quencher fluor TAMRA (6-carboxytetramethylrodamine; emission l 582 nm) at the 3 end via a linker arm nucleotide (LAN) followed by the phosphorylation site
p.
TaqMan real-time quantitative RT-PCR assay.
RNA from an individual sample was applied for RT and amplification
using TaqMan EZ RT-PCR kit (Perkin Elmer, Foster City, CA) according to
the manufacture's protocol. In brief, a master mixture that contained
all reagents required for RT-PCR was prepared to give final
concentration of 1× TaqMan EZ buffer, 0.3 mmol/L dNTPs, 3 mmol/L
manganese acetate, 0.01 U/µL AmpErase UNG, and 0.1 U/µL rTth DNA
polymerase. Total RNA extract (containing unknown amount of target TPO
from BM stromal cells) was added to the master mixture. This mixture
was used to generate two sets of tubes, set I and set II. To detect the
amount of the TPO mRNA RT-PCR amplicon, target hybridization probe and
primers were added to set I, and internal control hybridization probe
and primers were added to set II, to give a final probe concentration
of 100 nmol/L-and 200 nmol/L-primers, and the total reaction volume was
increased to 50 µL. Each mixture was transferred to a set of
thermocycler tubes. Target and internal control were reverse
transcribed at 60°C for 30 minutes, followed by 50 cycles of
amplification at 94°C for 20 seconds and 62°C for 1 minute,
using ABI PRISM 7700 sequence detector (Applied Biosystems). During
each cycle of the PCR the 5  3 exonuclease
activity of rTth DNA polymerase cleaves the TaqMan probe, thereby
increasing the fluorescence of the reporter dye at the appropriate
wavelength. The increase in fluorescence ( Rn) was proportional to
the concentration of template in the PCR.
Threshold Rn is calculated by multiplying the standard deviation of
three Rn- values (no template controls) by 6.965 according to the
manufacturer's protocol for TaqMan RT-PCR Kit. PCR cycle number at
threshold line is represented as CT.
ELISA for TPO.
One milliliter of BM samples were diluted with normal saline
(threefold); peripheral blood (PB) samples were collected with heparin
(100 U/mL). Samples were centrifuged (400g, 15 minutes) and the supernatants were collected as plasma.
Concentrations of TPO in plasma of PB and BM were measured by ELISA
using the kit from Kirin Brewery Co Ltd (Tokyo, Japan).15 Each of the 96 wells of flat-bottomed microtiter plates (Maxisorp; Nunc, Roskilde, Denmark) was coated at 4°C overnight
with 100 µL of TNI (anti-human TPO mouse monoclonal antibody) at a
concentration of 10 µg/mL in 50 mmol/L carbonate buffer (pH 9.4).
After preincubation of the wells with 200 µL of a blocking reagent
(Super block in Tris-buffered saline [TBS]; Pierce, Rockford,
IL) for 30 minutes at room temperature (RT), 100 µL of
rhTPO standards (Kirin Brewery Co Ltd), serum sample, or
phosphate-buffered saline (PBS) were added to each well and reacted
with the coated TNI overnight at RT. After washing with 20 mmol/L
Tris-HCl containing 0.5 mol/L NaCl, 0.05% Tween 20, and 0.1%
NaN3, pH 7.5 (T-TBS), 100 µL of the biotinylated
anti-recombinant human (rh) TPO F(ab)2 antibody at
500 ng/mL in T-TBS containing 1% bovine serum albumin and 2% PEG 6000 (dilution buffer) was added to each well for 3 hours at RT. After
washing with T-TBS, 100 µL of streptavidin alkaline phosphatase
conjugate (1 mU/mL in dilution buffer; Boehringer Mannheim,
Indianapolis, IN) was added for 1 hour at RT. The color was developed
using an amplification system (GIBCO-BRL Life Technologies, Grand
Island, NY). After washing with T-TBS, 50 µL of substrate solution
was added for 40 minutes at RT. Subsequently, 50 µL of amplifier
solution was added for 30 minutes at RT. The reaction was stopped by
adding 50 µL of 0.3 mol/L H2SO4. The color
intensity was measured by a plate reader (Well Reader SK 601; Seikagaku Kogyo Co Ltd, Tokyo, Japan) with a measuring filter of 492 nm and
reference filter of 630 nm. The absorbance of each sample was
subtracted from that of the sample incubated with TNI.
Method for measurement of megakaryocyte counts.
After BM aspirates were 20-fold diluted with Turk medium, megakaryocyte
number was counted with Fuchs-Rosenthal calculation glass (Kayagaki,
Tokyo, Japan). Although counting megakaryocytes from aspirate may not
be as accurate as from biopsy samples, the latter technique was not
used in this particular investigation because it was difficult to
obtain informed consent for biopsy from normal volunteers.
Statistics.
Analysis of variance (ANOVA)-parallel line assay was used
to determine statistical significance between values for PB and BM. The
Mann-Whitney U-test was used to assess the standard error of difference
between two means; values were significant if P < .05.
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RESULTS |
TPO concentrations in BM and PB.
The mean TPO concentrations in BM and PB of NS and patients with ITP,
AA, and ET as measured by ELISA are given in
Table 2. Both BM and PB values in ITP were
higher (P < .05) than those in NS and returned to near-normal
value after steroid therapy. Patients with AA had extremely elevated
levels of TPO, ie, 20-fold above normal values. Two patients with ET
had TPO values within the normal range.
Relationship between TPO values in BM and PB.
We then analyzed the relationship between TPO values in BM and PB in
NS, ITP, AA, and ET. As shown in Fig 1,
there was a strong positive correlation between values in BM and those
in PB in these patients (r = .997); without exception, the
levels of TPO in BM were significantly higher than those in PB.
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| Fig 1.
Relationship between TPO concentrations in PB and BM. TPO
concentrations in NS ( ), patients with ITP before ( ) and after ( ) treatment, AA ( ), and ET ( ) were measured by ELISA. There was a significant positive correlation between TPO levels in BM and PB
in these patients (r = .997). In each subject, the TPO concentrations in BM were consistently higher than those in the PB.
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Verification of TaqMan RT-PCR for measurement of TPO mRNA in stromal
cells.
Before the application of TaqMan real-time quantitative RT-PCR for
assessing the stromal TPO mRNA levels in various hematological diseases, we verified the method by using the sample from a normal subject (Fig 2). Two-fold diluted samples
of total cell RNA (50 ng ~ 800 ng) from normal stromal
cells showed each distinct amplification curve in both TPO mRNA (Fig
2A) and GAPDH mRNA (internal control) assays (Fig 2B).
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| Fig 2.
Amplification plot of TPO mRNA (A) and GAPDH mRNA (B) of
BM stromal cells in real time by TaqMan RT-PCR method. Total cellular RNA (800 ng, ; 400 ng,  ; 200 ng, ; 100 ng, ; 50 ng, )
extracted from normal human stromal cells was subjected to TaqMan PCR
to detect a specific RT-PCR product of TPO mRNA and GAPDH mRNA in real
time. The arrows indicated the threshold Rn value obtained by
multiplying the standard deviation of Rn- (no template controls) by
6.965. In inserted figures of (A) and (B), mean CT (cycles at
threshold) of triplicate (bars represent one SD) was plotted against
applied RNA amount.
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For both mRNA, clear inverse correlations (inserted figures) were
observed between CT values (cycles at threshold line) and the amount of
applied cellular RNA.
Thus, this assay was confirmed to provide a high sensitivity with a
nanogram range of sample RNA and sufficient quantitativeness for
measuring TPO mRNA in stromal cells.
Expression levels of TPO mRNA of BM stromal cells in NS and in
patients with ITP, AA, and ET.
The levels of TPO mRNA and GAPDH mRNA expression in stromal cells from
6 NS, 5 patients with ITP, 6 patients with prednisolone treated ITP, 6 patients with AA, and 2 patients with ET were then examined by the
TaqMan RT-PCR assay (Fig 3). Two hundred
nanograms of total cellular RNA extracted from stromal cells of each
patient and normal subject was subjected to TaqMan RT-PCR method. In
each group, amplification curves of GAPDH mRNA from each individual overlapped to form almost a single line, and CT values were also almost
the same (Fig 3A). Contrarily, amplification curves of TPO mRNA
exhibited discrete patterns with different CT ranges indicated by
rectangles for each group of disease and NS (Fig 3B), and discrete
lines for each individual (Fig 3C).
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| Fig 3.
Amplification of BM stromal TPO mRNA by TaqMan RT-PCR
method in NS and in patients with ITP before and after treatment, AA, and ET. Two hundred nanograms of total cellular RNA extracted from
stromal cells of NS (n = 6) and patients with ITP before (n = 5)
and after (n = 6) treatment with steroid, AA (n = 6), and ET (n = 2) was subjected to TaqMan RT-PCR method. Threshold Rn value and CT
were obtained as described in Materials and Methods. In (A) and (B),
curves represent amplification plots of GAPDH mRNA and TPO mRNA,
respectively. In (C), magnifying view of rectangular area of (B) which
included minimum and maximum CT value for each disease group was shown
to illustrate the differences more apparently.
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Because TPO mRNA for standard was not available to quantitate its
absolute amount in stromal cells, the relative ratio of TPO mRNA/GAPDH
mRNA was used. In Fig 4, levels of stromal
TPO mRNA in these diseases and NS which were expressed as relative ratio of CT values for TPO mRNA and GAPDH mRNA were shown. In the
patients with ITP, the expression levels were significantly increased
compared to those in NS (P < .05) and were lowered by steroid
treatment. In patients with AA, the levels were as high as those of
patients with ITP. Two patients with ET showed normal expression of TPO mRNA.
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| Fig 4.
Levels of stromal TPO mRNA expression in NS and in
patients with ITP before and after treatment, AA, and ET.
Expression levels of stromal TPO mRNA in these diseases and NS
which were expressed as relative ratio of CT values for TPO mRNA and
GAPDH mRNA: (CT of TPO mRNA/CT of GAPDH
mRNA)1. The bars represent the mean ± SD.
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Relationship between TPO concentration in BM plasma and the TPO mRNA
expression of BM stromal cells in NS and in patients with ITP, AA, and
ET.
In all subjects there was a clear positive correlation between TPO mRNA
levels of stromal cells and TPO concentrations in BM
(Fig 5). However, the correlation
linearities found in patients with the three disorders (ITP, AA, and
ET) were all deviated from the expected lines based on those of normal
subjects (indicated as a dotted line). In nontreated ITP patients,
actual BM TPO concentrations were lower than one would predict from the
regression line of NS who had the same TPO mRNA expression level as
nontreated ITP. Conversely, in patients with AA, TPO concentrations
deviated toward far higher levels than those predicted from the
regression line of the NS.
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| Fig 5.
Relationship between TPO concentrations in BM and TPO
mRNA expression levels in BM stromal cells in NS and in patients with ITP before and after treatment, AA, and ET. BM TPO concentration of NS
(), the patients with ITP before () and after
prednisolone treatment (), AA (), and ET () were
plotted against BM stromal TPO mRNA expression.
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Relationship between megakaryocyte count and BM stromal TPO mRNA.
To investigate that stromal TPO indeed enhances megakaryopoiesis in
vivo, we analyzed a relationship between stromal TPO mRNA and
megakaryocyte counts in NS and in patients with ITP, AA, and ET. As
shown in Fig 6, except for the cases of AA
and ET whose megakaryopoiesis are not under the control of TPO because
of impaired responsiveness to TPO (AA) or autoproliferative property of
megakaryocyte (ET), megakaryocyte counts well correlated with the
levels of BM stromal TPO mRNA.
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| Fig 6.
Correlation between megakaryocyte counts and TPO mRNA
expression levels in BM stromal cells. Megakaryocyte counts of NS
(), the patients with ITP before () and after () prednisolone
treatment, and patients with AA () and ET () were plotted against
BM stromal TPO mRNA expression.
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DISCUSSION |
To explore the source(s) of TPO whose expression varies in response to
changes in platelet number, in this study we first measured the TPO
concentrations in BM and PB to compare them in NS and patients with
ITP, AA, and ET. The TPO concentrations in PB of our subjects agreed
with those NS reported previously.15,16 In all the subjects
examined, the TPO concentration in BM was positively correlated with
that in PB and always exceeded that of PB with significant difference.
This difference may be even more substantial in vivo because PB
inevitably contaminates marrow aspirate, resulting in dilution of TPO
in BM. These results indicated that TPO in BM was synthesized by some
BM constituent(s).
Because we10 and others11,12 have already
disclosed the fact that TPO mRNA is expressed in BM stromal cells, we
hypothesized that stromal TPO may be the key regulator of
megakaryopoiesis.
To evaluate the contribution of marrow stroma cell TPO to
megakaryopoiesis, we obtained RNA from stromal cells grown for 4 weeks
in Dexter cultures. Previous studies have suggested that such cells may
reflect the physiological state of the marrow stroma from which the
cultures were derived.17-20 This was true in our experiments, at least for the relative proportions of fibroblasts, endothelial cells, adipocytes, and macrophages.
Because RNAse protection assays for stromal TPO or mRNA were
insensitive, we turned to a TaqMan real-time RT-PCR assay to determine
stromal TPO mRNA levels. This assay has been recently applied for
measurement of trace substances in biological fluid and was proven to
be quite reliable in terms of reproducibility and
accuracy.21,22
By using this assay it was proven that TPO mRNA expression in stromal
cells of patients with ITP was significantly increased and returned to
normal with steroid treatment. This phenomenon may be explained by our
recent observation that transforming growth factor- (TGF-), which
is released from destructed platelets or megakaryocytes in this
disorder, stimulates stromal TPO mRNA, and the expression of stromal
TPO mRNA decreases along with TGF- with steroid
therapy.23 This notion apparently contradict the results by
Nagahisa et al,24,25 who found TPO mRNA levels in stromal
cell was not affected by acute thrombocytopenia or thrombocytosis in
mice. However, they have not examined the stromal TPO levels during
drastic change in platelet count and therefore most likely failed to
detect altered TPO expression.
Levels of TPO mRNA were correlated with TPO concentrations in BM in
ITP, suggesting that at least one of the factors that defines the
concentration of TPO in BM, ie, factors defining megakaryopoiesis, is
the production of TPO by stromal cells. However, the concentration of
TPO in the BM may not be solely determined by stromal TPO production because it was lower than the value predicted from the TPO mRNA level
of NS (Fig 1). This unexpectedly low level could be caused by the
adsorption of TPO on the surface of megakaryocytes that are increased
in the BM of this disease.15
In AA, stromal TPO mRNA was as high as that of ITP. Although merely
speculative, suppression of stromal TPO production by putative factors
from nonstromal hematopoietic cells may be relieved by aplasia of
hematopoietic cells because the expression of stromal TPO mRNA was
enhanced by abolishing hematopoietic cells with high doses of
chemotherapy (S. Sakamaki et al, unpublished data). TPO concentrations
in BM and stromal TPO mRNA in AA also showed a clear positive
correlation, again indicating that TPO in BM is derived from stromal
cells. However, patients with AA had extremely high TPO concentrations
in their BM (20-fold the normal value) and deviated significantly from
the predicted value of mRNA levels of NS. This may be explained in part
by the idea that in AA, megakaryocytes and platelets, on which TPO can
be adsorbed, are drastically suppressed, and also by the notion that
increased TPO itself may in turn suppress stromal TPO
mRNA.23
Two patients with ET had normal levels of both TPO mRNA and TPO
protein, despite the well-known abnormalities in megakaryopoiesis in
this disease. This apparent paradoxical observation may be explained by
a previous finding that megakaryocytes and platelets in this disease
have fewer TPO receptors than their normal counterparts26 and by the assumption that, in ET, both megakaryocytes and stromal cells may not be under regulation.
To verify that BM stromal TPO actually enhances megakaryopoiesis, the
relationship between the stromal TPO mRNA and megakaryocyte counts was
also analyzed. However, it is inappropriate to examine the effect of
stromal TPO on megakaryopoiesis in AA or ET whose hematopoietic cells,
including megakaryocytes, are not under the control of TPO. For this
reason, we analyzed the relationship in NS and in patients with ITP,
whose hematopoietic cells are supposed to be normal, and found a clear
positive correlation between stromal TPO mRNA and megakaryocyte counts.
The present notion that BM stromal cells is a major source for TPO
which locally regulates megakaryopoiesis appears to contradict to the
fact that TPO mRNA levels in organs such as liver and kidney are
substantially high compared with that in stromal cells. We believe that
high levels of TPO mRNA expression in liver or kidney do not necessary
mean that those organs are mainly involved in megakaryopoiesis because
TPO released from these organs may be diluted in the circulation so
that the concentration of TPO locally produced by stromal cells exceed
that of PB.
The physiological role of liver and kidney, although merely
speculative, may be to provide baseline TPO level in circulation preparing for stromal dysfunction.
In conclusion, the results of the present study strongly suggest that
TPO in BM is mainly derived from BM stromal cells and that the
concentration of TPO may be determined by its production rate from
stromal cells and possibly by its absorption rate on receptors of
platelets and megakaryocytes. Thus, in humans, TPO from BM stromal
cells is considered to play an essential role for megakaryopoiesis
under various pathophysiological conditions.
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FOOTNOTES |
Submitted July 28, 1997;
accepted April 20, 1998.
Supported in part by Grant-in-Aid for scientific research from the
Ministry of Education, Science and Culture of Japan.
Address reprint requests to Yoshiro Niitsu, MD, PhD, Chief and
Professor, 4th Department of Internal Medicine, Sapporo Medical University School of Medicine, South-1, West-16, Chuo-ku, Sapporo, 060-0061, Japan; e-mail: niitsu{at}sapmed.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Susan C. Feldman for editorial assistance. We also
thank Dr Irving Listowsky for helpful discussion and critical reading
of our manuscript.
| |
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Abstract
Introduction
Abstract