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Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4077-4083
Defective Proliferation of Primitive Myeloid Progenitor Cells in
Patients With Severe Congenital Neutropenia
By
Nakao Konishi,
Masao Kobayashi,
Shin-ichiro Miyagawa,
Takashi Sato,
Osamu Katoh, and
Kazuhiro Ueda
From The Department of Pediatrics, Hiroshima University School of
Medicine, Hiroshima; The Department of Child Health, Faculty of
Education, and Department of Environment and Mutation, Research
Institute for Radiation Biology and Medicine, Hiroshima University,
Hiroshima, Japan.
 |
ABSTRACT |
Although several mechanisms have been proposed to explain the
pathophysiology of severe congenital neutropenia (SCN), the precise
defect responsible for SCN remains unknown. We studied the
responsiveness of primitive myeloid progenitor cells to hematopoietic factors in 4 patients with SCN. The number of granulocyte-macrophage (GM) colonies formed in patients was decreased in response to granulocyte colony-stimulating factor (G-CSF) in both
serum-supplemented and serum-deprived culture. The polymerase chain
reaction-single-strand conformational polymorphism analysis of the
G-CSF receptor gene showed no variance in structure conformation
between the 4 patients and the normal subjects. In patients with SCN,
the nonadherent light density bone marrow cells and cells that were
purified on the basis of the expression of CD34 and Kit receptor
(CD34+/Kit+ cells) showed the reduced
response to the combination of steel factor (SF), the ligand for
flk2/flt3 (FL), and interleukin-3 (IL-3) with or without G-CSF in
serum-deprived culture. Furthermore, when individual
CD34+/Kit+ cells from patients were
cultured in the presence of SF, FL, and IL-3, with or without G-CSF for
10 days, the number of clones proliferated and the number of cells per
each proliferating clone was significantly less than those in normal
subjects. These results suggest that primitive myeloid progenitor cells
of patients with SCN have defective responsiveness to not only G-CSF,
but also the early- or intermediate-acting hematopoietic factors, SF,
FL, and IL-3.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
CONGENITAL NEUTROPENIAS are a
heterogeneous group of disorders. Severe congenital neutropenia (SCN),
also known as Kostmann-type neutropenia, is a severe form of
neutropenia that is characterized by early onset of chronic
life-threatening infections and severe neutropenia in the peripheral
blood.1-5 The bone marrow usually shows an arrest of
maturation of neutrophil precursors at the promyelocyte or myelocyte
stage of differentiation. In most patients with SCN, the treatment with
pharmacological doses of recombinant human granulocyte
colony-stimulating factor (G-CSF) leads to a significant increase of
absolute neutrophil count (ANC) and results in dramatic clinical
improvement.3,5-12
G-CSF and its receptor, granulocyte colony-stimulating factor receptor
(G-CSFR) are the major regulatory system for the production of
neutrophils.13-16 Mice lacking G-CSF have been
reported to develop congenital neutropenia.14 Mice
deficient in G-CSFR expression have chronic neutropenia and show
reduced numbers of marrow progenitors and impaired terminal
differentiation of neutrophils.15 However, although
numerous studies have been performed on the role of G-CSF and G-CSFR in
the pathogenesis of SCN, the underlying pathophysiology of SCN remains
unclear. The production of G-CSF by mononuclear leukocytes from SCN
patients appears to be normal, and the serum levels of G-CSF are often
increased.17-19 In addition, the G-CSFR has been found to
be present at normal or increased levels on myeloid
cells.19,20 Recently, mutations of the G-CSFR gene have
been reported in a subset of patients with SCN and have been determined
to contribute to neutropenia.21-26 However, the majority of
patients have not shown any mutations of the G-CSFR
gene.19,26,27
Bone marrow cells from patients with SCN frequently display a markedly
reduced or complete lack of responsiveness to G-CSF in vitro
culture.28-30 It has also been shown that this defect of
response to G-CSF can be partially restored by the addition of several
other hematopoietic factors.29,30 In this study, we
examined the responsiveness of purified myeloid progenitor cells to
hemtopoietic factors involved in myelopoiesis in 4 patients with SCN.
The results show the presence of qualitative and quantitative abnormalities in the proliferation of primitive myeloid progenitor cells from patients with SCN in response to hematopoietic factors including G-CSF.
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MATERIALS AND METHODS |
Subjects.
Laboratory and hematologic features of the patients with SCN enrolled
in this study are presented in Table 1. All
patients contracted bacterial infections at the age of less than 1 year and were referred to our hospital. None of the patients had a family
history of neutropenia. The complete blood cell count showed a normal
total white blood cell count with no neutrophils and normal red blood
cell and platelet counts. Bone marrow aspiration showed relative
myeloid hypocellularity with maturation arrest at the
promyelocyte/myelocyte stage. All patients have received the
administration of prophylactic sulfamethoxazole-trimethoprim or G-CSF
since the diagnosis was made. Patient 1 continued to have recurrent
skin abscesses and chronic gingivitis, and he has been maintained on
daily subcutaneous administration of G-CSF for the last 5 years. The 3 patients have received intermittent administration of G-CSF when
infections were observed.
Cytokines.
Recombinant human G-CSF, recombinant human interleukin-3 (IL-3) with a
specific activity of 1.0 × 108 U/mg and recombinant
human steel factor (SF) were supplied by Kirin Brewery Co Ltd (Tokyo,
Japan). The recombinant human ligand for flk2/flt3 (FL) was purchased
from PeproTech Inc (Rocky Hill, NJ). Unless otherwise specified, the
concentrations of factors used were as follows: G-CSF, 100 ng/mL; SF,
100 ng/mL; FL, 100 ng/mL; IL-3, 100 U/mL.
Bone marrow cell separation and purification.
Bone marrow samples were obtained with the informed consent of patients
and/or their guardians. Normal bone marrow cells for this study were
taken from healthy adult volunteers after obtaining informed consent.
Bone marrow samples were diluted with an equal volume of
 -modification of Eagle's medium (MEM; ICN Biomedicals, Inc,
Aurora, OH) and centrifuged over Lymphoprep (1.077 g/mL; Nycomed Pharma
AS, Oslo, Norway). The light density bone marrow cells (LDBMC) were
carefully harvested with a Pasteur pipette, washed 3 times with
phosphate-buffered saline (PBS) containing 2% human AB serum (Sigma
Chemical Co, St Louis, MO) and 0.1 mg/mL of DNase I (type II-S; Sigma
Chemical Co) and resuspended in MEM containing 10% fetal bovine
serum (FBS; ICN Biomedicals, Inc). Cells were incubated in plastic
culture flasks (Becton Dickinson Labware, Lincoln Park, NJ) at 37°C
for 1 hour to remove adherent cells. Nonadherent cells were used in
described purification or cryopreserved by a standard procedure using
10% dimethylsufoxide and stored in liquid nitrogen until use. Cells,
fresh or thawed, were washed and resuspended in PBS-human serum-DNase
solution containing 0.1% sodium azide for subsequent
immunofluorescence staining.
Cell purification was performed according to the methods reported
previously with modification.31 Cells (2 × 107/mL) were incubated with fluorescein isothiocyanate
(FITC)-labeled monoclonal anti-CD34 antibody (Beckman Coulter, Inc,
Fullerton, CA) at a concentration of 2 µg/106 cells for
30 minutes at 4°C. FITC-conjugated mouse IgG1a was used as an
isotype control. After the addition of propidium iodide (PI, Sigma
Chemical Co) at a concentration of 1 µg/mL, cells were washed twice
and resuspended in PBS-human serum-DNase-sodium azide solution. Initial
enrichment of CD34+ was performed by setting the FACS
Vantage (Becton Dickinson Immunocytometry Systems, San Jose, CA)
equipped with a 4-W argon laser to recognize only FITC-positive cells.
Low to medium forward scatter and low side scatter, as well as negative
PI fluorescence gates were also used. The resulting cell population
contained 30% to 50% CD34+ cells. Enriched
CD34+ cells were further stained with phycoerythrin
(PE)-conjugated anti-c-Kit (Beckman Coulter, Inc) for 30 to 40 minutes
at 4°C. After the addition of PI at a concentration of 1 µg/mL,
cells were washed twice and resuspended in PBS-human serum-DNase-sodium azide solution. The appropriate isotype controls were used to identify
background staining. Forward and orthogonal light scatter signals, as
well as specific fluorescence of FITC, PE, and PI excited at 488 nm and
633 nm, were used to establish sort windows. Cells were separated into
fractions expressing positive CD34 and c-Kit according to the methods
reported previously.31 Data acquisition and analysis was
performed with CellQuest software (Becton Dickinson Immunocytometry
Systems). Single cell sorting was performed using the Automated Cell
Deposition Unit (ACDU) with Clone-Cyt software (Becton Dickinson
Immunocytometry Systems).
Clonal cultures.
The clonal cell culture was performed in 35-mm Falcon
suspension culture dishes (Becton Dickinson Labware). In the
serum-deprived culture, 1 mL of the culture mixture contained purified
cells, 1% deionized crystallized bovine serum albumin (BSA, Sigma
Chemical Co), 300 µg/mL fully iron-saturated human transferrin (98%
pure; Sigma Chemical Co), 10 µg/mL soybean lecithin (Sigma Chemical Co), 6 µg/mL cholesterol (Sigma Chemical Co), 1 × 10 7 mol/L sodium selenite (Sigma Chemical Co), 10 µg/mL insulin (Sigma Chemical Co), 4.5 mmol/L L-glutamin (Sigma
Chemical Co), 1.5 mmol/L glycin (Sigma Chemical Co), 1.2%
1,500-centipoise methylcellulose (Shinetsu Chemical, Tokyo, Japan), and
designated cytokines.31,32 In the serum-supplemented
culture, the crystallized BSA transferrin, lecithin, cholesterol,
selenite, insulin, glutamin, and glycin were replaced by 30% FBS and
1% deionized fraction V BSA (Sigma Chemical Co). Cultures were
incubated at 37°C in a humidified atmosphere with 5%
CO2/95% air. On day 14 of incubation,
granulocyte-macrophage (GM) colonies were scored on an inverted
microscope using the criteria described previously.33 GM
colony contains pure granulocyte colonies consisting of mainly
neutrophils and their precursors, and mixed granulocyte-macrophage
colonies consisting of mainly neutrophils, macrophages/monocytes, and
their precursors. The numbers of colonies represent the mean of
triplicate cultures.
Single-cell suspension cultures.
Single cells were cultured in serum-deprived liquid suspension cultures
containing SF, FL, and IL-3 with or without G-CSF in 72-well round
bottom microtrays (Robbins Scientific, Sunnyvale, CA). Incubation was
performed at 37°C in a humidified atmosphere with 5%
CO2/95% air for 10 days. All wells were inspected
carefully on an inverted microscope 16 to 24 hours after the sorting.
We were able to detect 1 cell/well in more than 90% of the wells. We
then serially scored the number of cells in each well. In some experiments, some of the proliferated clones were individually picked,
centrifuged onto slides using Shandon's Cytospin 2 Centrifuge (Shandon
Inc, Pittsburgh, PA), and stained with Wright-Giemsa.
Reverse transcriptase-polymerase chain reaction (PCR) and
single-strand conformational polymorphism (SSCP).
Total cellular RNA was extracted from bone marrow mononuclear cells
using the guanidinium thiocyanate extraction method. RNA was converted
into cDNA by reverse transcriptase. PCR amplification of cDNA was
performed accord- ing to the method of Dong et al.21 The
primers used were as follows: FW3,
5'-CTGCTGTTGTTAACCTGCCTC-3' (nucleotides 2075 to 2095, forward); FW4, 5'-CCAAGAGCAGTTTCCACCCAGGCC-3' (nucleotides
2366 to 2389, forward); RV2,
5'-GTAGATCTTAGTCATGGGTTCATGG-3' (nucleotides 2750 to
2774, reverse); and RV6, 5'-TCTCAGGGGAGATAGTGGCCC-3' (nucleotides 2462 to 2481, reverse). The PCR products were applied to
electrophoresis in 12% polyaclylamide gel for 3 hours at 30 W at room
temperature. After electrophoresis, the bands were visualized by the
silver-staining method with a commercially available reagent kit
(Daiichi Pure Chemicals, Tokyo, Japan).34
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RESULTS |
Formation of GM colonies of nonadherent LDBMC in response to G-CSF.
First, we tested the formation of GM colonies of nonadherent LDBMC in
response to G-CSF in serum-supplemented and serum-deprived culture. As
shown in Fig 1, GM colony formation in
patients with SCN markedly decreased relative to that of controls at
all concentrations of G-CSF in both serum-supplemented and
serum-deprived culture. The difference between the number of GM
colonies formed in serum-supplemented and that formed in serum-deprived
culture indicates the presence of other factors that cooperate with
G-CSF in FBS. The decrease of G-CSF-dependent GM colony formation in
serum-deprived culture clearly suggests the defective responsiveness to
G-CSF alone in patients with SCN. These observations were consistent
with the data reported previously.6,23,28-30
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| Fig 1.
GM colony formation in response to various concentrations
of G-CSF. Nonadherent LDBMC (2 × 104 cells) were cultured
in a serum-supplemented (A) or serum-deprived (B) conditions containing
varying concentrations of G-CSF. Data represent the mean ± standard
deviation (SD) of 6 normal subjects ( ) and the mean of
triplicate cultures of 2 patients with SCN ( , patient 1;  ,
patient 2;  , patient 3).
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Analysis of structural conformation of G-CSFR.
Recently, nonsense mutations in the gene encoding the G-CSFR have been
described in some patients with SCN.21-26 However, the structure of G-CSFR in the majority of SCN patients is
normal.19,26,27 In this study, the structure of the G-CSFR
cDNA of patients was analyzed by PCR-SSCP. The cDNA was amplified with
primers FW4 and RV6 because the mutations were reported to be located
in the same cytoplasmic region of G-CSFR in a majority of
patients.26 As shown in Fig 2,
SSCP analysis showed that the patterns of morbidity of PCR products of
the 4 patients with SCN were indistinguishable from those of the normal
subjects. Patterns of morbidity of PCR products amplified with other
primers were similarly indistinguishable (data not shown), indicating
that there was no SSCP-detected abnormalities in the cytoplasmic domain
of G-CSFR in patients with SCN enrolled in this study.
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| Fig 2.
PCR-SSCP analysis of the G-CSFR cytosolic domain. The PCR
was performed with primers FW4 and RV6 as described in Materials and
Methods. The RT-PCR products from 4 patients (Patients 1 to 4) and
normal controls (control) underwent polyacrilamide gel electrophoresis
for 3 hours, and the bands were visualized by the silver-staining
method.
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GM colony formation of nonadherent LDBMC in response to various
hematopoietic factors.
Several hematopoietic factors, including SF, FL, IL-3, and G-CSF, have
been reported to be involved in myelopoiesis and classified into 3 different categories.35-38 We next examined the effects of
various hematopoietic factors on GM colony formation of nonadherent LDBMC from patients in response to such hematopoietic factors alone or
to various combinations of the factors in serum-deprived culture. As
presented in Table 2, single factors, such
as IL-3 and FL, gave rise to formation of a few GM colonies in both
patients with SCN and normal subjects. The various combinations of
G-CSF and 1 other factor induced the increase in the number of GM
colonies. However, the numbers of GM colonies in SCN patients were less than those in normal subjects. Furthermore, the numbers of GM colonies
of LDMNC from SCN patients were less than those from normal subjects in
response to SF, FL, or IL-3 with or without G-CSF.
GM colony formation of CD34+/Kit+ cells in
response to various hematopoietic factors.
To further examine the effects of various hematopoietic factors,
nonadherent LDBMC were enriched for primitive myeloid progenitors using
CD34 antibody and anti-c-Kit receptor antibody because colony-forming unit-GM (CFU-GM) was enriched in CD34+/Kit+
fraction. Figure 3 presents a
representative flow cytometric analysis of cells that had been enriched
for CD34. There was no difference between SCN patients and normal
subjects in the expression of CD34 or Kit of nonadherent LDBMC.
CD34+/Kit+ cells that were purified from
nonadherent LDBMC according to the gate indicated in Fig 3 were
cultured in serum-deprived conditions containing various hematopoietic
factors. As shown in Table 3, single
factors did not effectively support GM colony formation in patients
with SCN. Although 2-factor combinations that include G-CSF-induced
GM-colony formation in patients, the numbers of GM colonies were less
than those in normal subjects. Furthermore, GM colony formation of
patients with SCN showed a significant decrease in response to
combinations of SF, FL, and IL-3, both with or without G-CSF, compared
with that of normal subjects. These findings may suggest the
possibility that the primitive myeloid progenitor cells of patients
with SCN have defects in response not only to G-CSF, but also to other
hematopoietic factors.
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| Fig 3.
Flow cytometric analysis of CD34 and c-Kit expression on
bone marrow cells from normal subjects and the patients with SCN.
Nonadherent LDBMC enriched for CD34-FITC (see Materials and
Methods) were stained by Kit-PE. R indicates the gate for
CD34+/Kit+ cells. The figure shows a
representative flow cytometric analysis for 2 patients with SCN and 2 normal subjects.
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Proliferation of individual CD34+/Kit+ cells.
To confirm the direct evidence for proliferation of primitive myeloid
progenitors, we sorted the CD34+/Kit+ cells
using ACDU and analyzed the proliferation from single cells in response
to SF, FL, and IL-3 with or without G-CSF. All wells were inspected
carefully by an inverted microscope, and the number of proliferated
cells of clones was serially recorded. The number of clones
proliferated and the mean number of cells per each proliferated clone
after 10 days of culture from 2 patients with SCN and 5 normal subjects
are presented in Table 4. The number of
clones proliferated and the number of cells per each clone in patients with SCN were significantly less than those in normal subjects irrespective of the presence or absence of G-CSF. This result is
consistent with the data showing a decrease in the number of GM
colonies formed in methylcellulose. Although the number of the
proliferative clones was small in patients with SCN, several clones
showed normal proliferation. After 10 days culture, some of the
proliferating cells from single CD34+/Kit+ cell
were picked, centrifuged onto a slide, and stained.
Figure 4 shows the representative cytology
of cells from a single CD34+/Kit+ cell of
patients with SCN. Single CD34+/Kit+ cells in
the presence of SF, FL, and IL-3 developed to the myeloid precursor
level (Fig 4A), while the addition of G-CSF to SF, FL, and
IL-3 induced the development of mature segmented neutrophils (Fig
4B).
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| Fig 4.
Cytology of cells proliferated from a single
CD34+/Kit+ cell of patients with SCN.
Single CD34+/Kit+ cells were cultured in
the presence of SF, FL, and, IL-3 with (B) or without (A) G-CSF
according to the description in Table 4. Some of the proliferating
clones were picked, centrifuged onto a slide, and stained with
Wright-Giemsa.
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DISCUSSION |
Previously, we and others have shown that the bone marrow cells of
patients with SCN showed a reduced response to G-CSF and a partial
restoration of defective response to G-CSF by the addition of IL-3,
GM-CSF, or SF.29,30 These previous experiments used a
serum-supplemented culture with crude bone marrow cells. It is
well-known that G-CSF can synergize with early- and/or
intermediate-acting cytokines to support the proliferation of
neutrophils and their precursors in culture.36,39 The serum
in culture is a potential endogenous source of hematopoietic factors
that affect the responsiveness of progenitor cells in
culture.40,41 As shown in Fig 1, the number of GM colonies
supported with G-CSF in serum-supplemented culture was greater than
that in serum-deprived culture. In this study, we compared the effects
of the combination of the factors on supporting GM colony formation
between patients with SCN and normal subjects using a serum-deprived
culture system and purified myeloid progenitor cells. The results
presented here demonstrated that primitive myeloid progenitor cells
from patients with SCN had reduced response to hematopoietic factors
involved in myelopoiesis, including G-CSF. This observation was
confirmed by the single-cell proliferation studies of
CD34+/Kit+ cells in a serum-deprived suspension
culture. The number of clones proliferated and the mean number of cells
per each proliferated clone of patients in response to SF, FL, IL-3,
and G-CSF were significantly reduced when compared with those of normal
subjects. These findings directly suggest the presence of quantitative
and qualitative abnormalities in the proliferation of primitive myeloid progenitor cells in patients with SCN.
Somatic mutations in the gene for G-CSFR have been identified in some
patients with SCN and have been shown to result in a truncation from
the C-terminus of the receptor and in an inability of the receptor to
transduce the signal upon G-CSF stimulation. However, most patients
with SCN have not been found to have mutations in the G-CSFR gene.
Recently, Hermans et al42 and McLemore et al43
independently generated mice carrying a mutation of the G-CSFR that was
roughly identical to the mutation found in patients with SCN and
examined the effect of this mutation on granulopoiesis. Although the
G-CSFR mutation resulted in the reduced basal neutrophil levels, there
was no significant reduction in numbers of neutrophils and their
precursors in the bone marrow. Thus, the G-CSFR mutation found in
patients with SCN was not sufficient to induce an SCN phenotype in
mice. These results suggest that mutations of G-CSFR may not be
responsible for the impairment of granulopoiesis present in patients
with SCN. In this study, there is no SSCP-detected abnormalities in
cytoplasmic domain of G-CSFR. The majority of patients showed reduced
response to G-CSF irrespective of the presence or absence of G-CSFR
abnormalities. In addition, myeloid progenitor cells of the SCN
patients showed reduced response to the combination of SF, FL, and
IL-3, as well as to G-CSF alone. Thus, it is likely that abnormalities
of molecules involved in common hematopoietic growth factor signal
transduction downstream of the receptor might also play a role in the
pathogenesis of SCN.
Several hematopoietic factors, including SF, FL, IL-3,
GM-CSF, IL-6, and G-CSF, have been shown to be positive
regulators of granulopoiesis and to act at different stages of myeloid
cell development.13,16,36 Most of these factors support the
proliferation of early myeloid progenitor cells. However, G-CSF has the
ability to not only stimulate the proliferation, but also potentially induce the terminal maturation of myeloid progenitor cells to neutrophilic granulocytes. In the present single-cell proliferation studies, cytological examination of individual proliferated clones showed that single CD34+/Kit+ cells developed
into mature segmented neutrophils when G-CSF was added to SF, FL, and
IL-3 in culture. Although the number of responding clones and the
kinetic proliferation were less in patients than in normal subjects,
some enhancing effect of G-CSF on the proliferation and maturation of
progenitor cells in SCN patients was also observed. Therefore, in vitro
responsiveness of myeloid progenitor cells to hematopoietic factors in
patients with SCN was reduced, but not completely abolished. The
CD34+/Kit+ cells of patients had at least a
partial capacity to produce the mature neutrophils by addition of
G-CSF. These in vitro observations may indicate that most patients with
SCN respond favorably to in vivo administration of G-CSF, showing both
a significant increase in circulating neutropils and clinical
improvement. Furthermore, previous studies of both G-CSF-deficient and
G-CSFR-deficient mice suggest the existence of G-CSF or G-CSFR
independent of granulopoiesis.14,15
In conclusion, the present study may provide evidence that the bone
marrow cells of patients with SCN have abnormal responses not only to
G-CSF, but also to early- and intermediate-acting hematopoietic factors
involved in myelopoiesis. It appears that the cellular defect in
patients with SCN resides downstream of the receptors required for
hematopoietic factor-induced myeloid proliferation and maturation.
| |
ACKNOWLEDGMENT |
The authors are very grateful to Kirin Brewery Co Ltd (Tokyo, Japan)
for providing cytokines.
| |
FOOTNOTES |
Submitted March 5, 1999; accepted August 9, 1999.
Supported in part by Grant-in-Aid (to M.K.) for Scientific Research (C)
from the Ministry of Education, Science, Sports and Culture of Japan.
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 Masao Kobayashi, MD, Department of Child
Health, Faculty of Education, Hiroshima University, 1-1-2 Kagamiyama
Higashi-Hiroshima, Hiroshima, 739-8523 Japan; e-mail: masa{at}mcai.med.hiroshima-u.ac.jp.
| |
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