Departments of
* Pediatrics/Neonatology, and
Environmental and Community Medicine, UMDNJ-Robert Wood Johnson Medical School, and
Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey
Correspondence: Barry Weinberger, M.D., Division of Neonatology, St. Peter’s University Hospital, 254 Easton Avenue, New Brunswick, NJ 08903. E-mail: barryw{at}pol.net
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B nuclear
binding activity induced by fMLP and DL1.2, as well as expression of
the p65 NF-B subunit and IB-
, was also significantly reduced
in neonatal cells, when compared with adult cells. In contrast,
although fMLP, but not DL1.2, activated p42/44 and p38
mitogen-activated protein (MAP) kinases in neutrophils, no differences
were observed between adults and neonates. Chemotaxis of adult and
neonatal neutrophils toward fMLP and DL1.2 was also blocked to a
similar extent by inhibitors of phosphatidylinositol 3-kinase, as well
as an inhibitor of NF-B. These findings indicate that reduced
chemotactic responsiveness in neonatal neutrophils is a result of, at
least in part, aberrations in chemoattractant-induced signaling.
However, the biochemical pathways mediating this defect appear to be
related to the specific chemoattractant.
Key Words: neonates • chemotaxis • NF-B
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The biochemical signaling pathways leading to chemotaxis in neonatal
cells have not been elucidated. In adult cells, fMLP binding is
associated with a rapid increase in intracellular calcium
[11
]. This is followed by activation of protein kinases,
including members of the mitogen-activated protein (MAP) kinase family
and nuclear translocation of nuclear factor B (NF-B)
[12
, 13
]. To analyze potential mechanisms
underlying impaired chemotactic responsiveness in neonates, we compared
the responses of adult and neonatal cells with fMLP and a unique
chemotactic antibody, DL1.2, which activates distinct signaling
pathways in neutrophils. We speculate that aberrations in signaling
mechanisms contribute to defects in chemotaxis in neonatal cells.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B subunits were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and mouse mAb
to the fMLP receptor was from BD PharMingen (San Diego, CA).
Horseradish peroxidase (HRP) and fluorescein-labeled goat anti-mouse or
sheep anti-rabbit immunoglobulin (Ig)G secondary antibodies were
obtained from BD Transduction Laboratories (Lexington, KY) and New
England Biolabs. Rabbit IgG and mouse IgG1 controls were
purchased from Santa Cruz Biotechnology and Coulter Immunotech (Miami,
FL). Pertussis toxin was from Gibco BRL Life Technologies (San Diego,
CA).
Cell isolation
Human neutrophils were isolated from heparinized umbilical cord
blood from healthy, full-term infants or from venous blood from healthy
adults. Cells were separated by dextran sedimentation and Ficoll
density gradient centrifugation as previously described
[14
]. These studies were approved by the Institutional
Review Board of St. Peter’s University Hospital (New Brunswick, NJ).
DL1.2 antibody
We have previously described a mouse mAb, L12.2, which is
chemotactic for human neutrophils [15
]. Using agarose
and checkerboard assays, we confirmed that the response to this
antibody involves directed migration (chemotaxis) of the cells
[15
]. The antibody used in the present studies was
derived from cells that were recloned three times by serial dilution
from this hybridoma and designated DL1.2. The hybridoma was cultured in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal
bovine serum. In dose-response experiments, we found that maximal
chemotactic activity was observed with a 1:3 dilution of concentrated
hybridoma culture supernatants, which corresponded to 100 ng/ml
protein, and this concentration was used in our studies. An irrelevant
mouse anti-human mAb, W6/32 [16
], prepared from
hybridoma culture supernatants containing 10% serum (as described for
DL1.2), was used as a control.
Immunofluorescence and flow cytometry
Neutrophils (1.5x106/ml) were incubated for 60 min
at room temperature with DL1.2, anti-fMLP receptor antibody (1:1000),
or isotypic IgG1 control antibody; washed (300
g, 5 min); and then incubated with FITC-labeled goat
anti-mouse IgG. After 30 min, the cells were analyzed on a Coulter
EPICS Profile II flow cytometer (Coulter Electronics, Hialeah, FL). For
analysis of p65 and IB- expression, neutrophils were fixed with
0.1% paraformaldehyde, permeabilized with lysophosphatidylcholine (4
µM), and incubated overnight with a 1:500 dilution of anti-p65,
anti-IB-, or IgG control. Cells were then washed and incubated
with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG
(1:500, 30 min), and equal numbers of cells (104) were
analyzed by flow cytometry. Fluorescence histograms were analyzed by
Overton’s cumulative subtraction routine of the Coulter Cytologic
Software program. We found that expression of the DL1.2 antigen, like
the L12.2 parent clone [15
], was limited to neutrophils
(unpublished results).
Measurement of neutrophil chemotaxis
The modified Boyden chamber technique [17
] was
used to assay chemotaxis of neutrophils through Millipore filters. For
our studies, we used a 48-well microchemotaxis chamber (Neuro Probe,
Pleasanton, CA). DL1.2 (100 ng/mL), fMLP
(5x10-8 M), HBSS, or antibody control was
placed in each well of the lower chamber. A 5 µm pore-size
polycarbonate filter was then placed over the wells, and the upper
chamber was set into place. The neutrophil suspension [50 µl;
1x105 cells in HBSS containing 0.5% bovine serum albumin
(BSA) and 2.4 mg/ml HEPES, pH 7.2] was added to each well. After
incubation for 45 min at 37°C, the filter containing adhered,
migrated neutrophils was removed and stained with Wright-Giemsa.
Chemotaxis was quantified as the number of cells that migrated through
the filter in 10 oil immersion fields. Data are presented as the
mean ± SE of four experiments. In some experiments,
cells were incubated at room temperature with BAY11-7085 (10 µM,
1 h), LY 294002 (50 µM, 5 min), wortmannin (100 nM, 10 min),
pertussis toxin (1 µg/ml, 30 min), cycloheximide (10 µM, 30 min),
or actinomycin D (5 µg/ml, 30 min) prior to analysis of chemotactic
responsiveness.
Western blotting
Neutrophils were suspended in phosphate-buffered saline
(1x106/ml) containing CaCl2 (1.8 µM) with
and without fMLP, DL1.2, 12-O-tetradecanoylphorbol
13-acetate (TPA; 170 nM), or an irrelevant antibody control. The cells
were incubated for 5 min at 37°C and then solubilized in buffer
containing 50 mM HEPES, pH 7.4, 10 mM KCl, 1 mM
ethylenediaminetetraacetate (EDTA), 1 mM dithiothreitol (DTT), 1
µg/ml pepstatin A, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin
inhibitor, 10 µg/ml aprotinin, and 1% Triton X-100. After 10 min on
ice, lysates were centrifuged (4000 g, 5 min), and protein
concentrations in the supernatants were quantified using a BCA Protein
Assay Kit (Pierce, Rockford, IL) with BSA as the standard. Aliquots of
supernatants containing 10 µg protein were fractionated on 7.5%
sodium dodecyl sulfate (SDS) polyacrylamide gels and electroblotted
onto 0.45 µm nitrocellulose paper (Bio-Rad, Hercules, CA) at 250 mA
for 4 h in a Mini Trans-Blot Electrophoretic Transfer Cell
(Bio-Rad) in 25 mM Tris, pH 8.3, 192 mM glycine, 20% (v/v) methanol
[18
, 19
]. Blots were then incubated with
DL1.2 (100 ng/ml), mouse mAb to p42/44 MAP kinase (1:500), or rabbit
polyclonal anti-phosphorylated human p38 MAP kinase (1:250). After
washing, the blots were incubated with goat anti-mouse IgG (1:2000) or
sheep anti-rabbit IgG (1:1000) HRP-conjugated antibodies (BD
Transduction Laboratories and New England Biolabs) for 1 h at room
temperature. Proteins were detected using a Renaissance Western Blot
Chemiluminescence Reagent Plus kit (NEN Life Sciences, Boston, MA).
Treatment of the lysates with 2-mercaptoethanol did not alter the
apparent molecular mass of the DL1.2 antigen in the gels.
Glycosidase treatment
Deglycosylation reactions were carried out using a GlycoPro kit
(ProZyme, San Leandro, CA). Briefly, 15 µg neutrophil proteins were
diluted to 35 µl with distilled water. Sodium phosphate buffer (10
µl of 0.25 M; pH 7.0) and 2.5 µl denaturing solution (2% SDS, 1 M
2-mercaptoethanol) were then added. After boiling for 5 min and cooling
to room temperature, 2.5 µl of 15% Triton X-100 and 0.5 µl
glycosidase(s) [2500 U/ml peptide-N-glycosidase F; 1 U/ml
endo-O-glycosidase; 2.5 U/ml sialidase A; or 2 µl proteinase K (10
mg/ml)] were added. The samples were then incubated for 6 h at
37°C, and one-half of each reaction was analyzed by Western blotting
as described above. Aliquots of fetuin (15 µg) were incubated under
the same conditions and served as enzyme controls. These products were
stained with Coomassie Brilliant Blue after electrophoresis.
Analysis of intracellular-free calcium
Neutrophils suspended in HBSS containing 0.5% BSA and 2.4 mg/ml
HEPES, pH 7.2 (1x106/ml), were incubated for 30 min
(37°C) with Fluo-4 (5 µg/ml). The cells were then stimulated with
fMLP, DL1.2, or control and analyzed by flow cytometry 0.5–5 min
later. Fluo-4 exhibits characteristic fluorescence, which is
proportional to the binding of the dye to intracellular calcium
[20
].
NF-B nuclear-binding activity
An electrophoretic mobility shift assay (EMSA) was used to
detect nuclear binding of activated NF-B. Neutrophils were suspended
in HBSS (1.5x106/ml) containing 0.1% BSA and 2.4 mg/ml
HEPES, pH 7.2. Following incubation with fMLP, DL1.2, or control for 45
min, nuclear extracts were prepared [21
]. Briefly,
neutrophils were centrifuged for 15 s in a microfuge and then
resuspended in 400 µl cold buffer [10 mM HEPES, pH 7.9, 10 mM KCl,
0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl
fluoride (PMSF)]. After 15 min on ice, 25 µl of a 10% solution of
Nonidet P-40 (NP-40) was added, and the tube was mixed on a vortex for
10 s. The lysate was then centrifuged for 30 s in a
microfuge, and the nuclear pellet was resuspended in 50 µl ice-cold
buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
DTT, 1 mM PMSF) and placed on a rocker platform at 4°C for 15 min.
The sample was then microcentrifuged for 5 min at 4°C, and the
resulting soluble nuclear extract was collected for analysis.
A double-stranded probe containing an NF-B binding site (5'-AGT TGA
GGG GAC CGC CCG CGG CCC GT-3'; Integrated DNA Technologies, Skokie, IL)
was labeled with (-32P)-dCTP. For binding reactions,
nuclear extracts (50 µg protein) were incubated for 15 min at room
temperature with labeled probe in buffer containing 10 mM Tris-HCl, pH
7.4, 40 mM NaCl, 10 mM EDTA, 1 mM 2-mercaptoethanol, 0.1% NP-40,
4% glycerol, and 0.1 µg/µl poly(dI-dC) · poly(dI-dC)
(Pharmacia Biotech, Piscataway, NJ). For competitive binding assays,
nuclear extracts were incubated with a 100-fold excess unlabeled probe
together with a 32P-labeled probe. Supershift assays were
performed by incubation of the samples with polyclonal antibodies to
p50 or p65 (1 µg/60 µl reaction volume) for 1 h prior to
addition of the labeled probe. Samples were electrophoresed on 4%
nondenaturing polyacrylamide gels in buffer (0.04 M Tris-acetate, 0.001
M EDTA, pH 8.0) at 15 V/cm. Gels were then dried and autoradiographed.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
 View larger version (14K): [in a new window] |
Figure 1. Comparison of chemotactic responsiveness of adult and neonatal
neutrophils. Chemotaxis of neutrophils toward fMLP, DL1.2, or an
irrelevant control antibody (W6/32) was assayed using microwell
chambers as described in Materials and Methods. Each bar is the
mean ± SE of four experiments. *,
Significantly different (P<0.05) from adult
neutrophils.
|
 View larger version (12K): [in a new window] |
Figure 2. Flow cytometric analysis of DL1.2 antigen and fMLP receptor expression
in adult and neonatal neutrophils. Cells were incubated for 60 min at
room temperature with DL1.2, anti-fMLP receptor antibody, or
IgG1 control. Neutrophils were then washed and incubated
with FITC-labeled goat anti-mouse IgG. After 30 min, the cells were
analyzed by flow cytometry. Each curve represents an individual donor
sample. Four to six representative donors are shown.
|
 View larger version (20K): [in a new window] |
Figure 3. Comparison of the DL1.2 antigen in adult and neonatal cells. Extracts
from neonatal (lanes 1, 3, 7, 9, 11, and 13) or adult (lanes 2, 4, 5,
6, 8, 10, and 12) neutrophils were treated with buffer only (lanes
1–4, 6, 7), proteinase K (lane 5), N-glycosidase F (lanes 8 and 9),
sialidase (lanes 10 and 11), or N-glycosidase F, endo-O-glycosidase,
and sialidase (lanes 12 and 13). The proteins (7.5 µg/lane) were then
analyzed by Western blotting as described in Materials and Methods. The
arrow indicates the 120 kDa band of the DL1.2 antigen. One
representative blot of three is shown.
|
 View larger version (23K): [in a new window] |
Figure 4. Chemoattractant-induced calcium mobilization in adult and neonatal
neutrophils. Cells from adults or neonates loaded with Fluo-4 were
stimulated with fMLP or DL1.2. Samples (104 neutrophils)
were analyzed by flow cytometry 30 s, 1 min, 3 min, or 5 min later. One
representative histogram for adults and neonatal cells is shown
(n=3–4).
|
|
View this table: [in a new window] |
Table 1. Effects of Inhibitors on Neutrophil Chemotaxis
|
 View larger version (32K): [in a new window] |
Figure 5. Chemoattractant-induced activation of p42/44 and p38 MAP kinases.
Neutrophils from adults were incubated for 5 min at 37°C with medium
control (lanes 1 and 6), DL1.2 (lanes 3 and 8), fMLP (lanes 4 and 9),
an irrelevant antibody control (lanes 5 and 10), or TPA (lanes 2 and
7), which was used as a positive control [51
]. Cellular
extracts (10 µg/lane) were analyzed by Western blotting. One
representative blot of three is shown.
|
B is a downstream signaling molecule
known to be activated by chemoattractants [12
,
13
]. We found that fMLP, as well as DL1.2, rapidly
induced nuclear translocation of NF-B proteins in adults and
neonates (Fig. 6
). Supershift assays revealed that the NF-B complex contained
p50 and p65 subunits. Preincubation of nuclear extracts with excess
unlabeled probe competitively reduced binding of the labeled probe,
demonstrating its specificity. In adult and neonatal neutrophils,
BAY11-7085, which blocks NF-B activation [28
],
significantly reduced chemotaxis in response to fMLP, as well as to
DL1.2 (Table 1)
. As observed with chemotaxis, activation of NF-B by
fMLP and DL1.2 was reduced in neonatal relative to adult neutrophils
(Fig. 6)
. This was correlated with reduced constitutive expression of
NF-B p65 and IB- protein in neonatal neutrophils. Moreover,
whereas treatment of adult cells with fMLP or DL1.2 resulted in
decreased expression of p65 and IB-, this response was diminished
in cells from neonates (Fig. 7
).
 View larger version (104K): [in a new window] |
Figure 6. Chemoattractant-induced NF-B nuclear-binding activity. Neonatal or
adult neutrophils were incubated with fMLP, DL1.2, TPA (170 nM), LPS
(100 ng/ml), or medium control (unstimulated) for 15 min. Nuclear
extracts (50 µg protein/lane) were analyzed by an electrophoretic
mobility shift assay. Supershift assays were performed in
DL1.2-stimulated cells from adults using control medium (-) or
polyclonal antibodies to p50 or p65 proteins. To demonstrate
specificity, nuclear extracts from DL1.2-stimulated neutrophils were
incubated with (Cold Comp.) or without (-) 100-fold excess of
unlabeled, double-stranded probe in addition to the
32P-labeled probe. One representative blot of two is
shown.
|
 View larger version (25K): [in a new window] |
Figure 7. Flow cytometric analysis of NF-B p65 and IB- expression
in adult and neonatal neutrophils. Freshly isolated neutrophils were
fixed and permeabilized (top panels) or incubated for 60 min with fMLP,
DL1.2, or an irrelevant antibody (control) prior to fixation and
permeabilization (bottom panels). Cells were then incubated overnight
with anti-p65 or anti-IB- antibodies, or IgG control, washed,
incubated with FITC-labeled goat anti-rabbit IgG (30 min), and analyzed
by flow cytometry. Studies were performed on cells from three adults
and three neonates. Data from one representative donor are shown.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
One of the earliest biochemical responses observed in neutrophils following fMLP binding is mobilization of intracellular calcium [36 , 37 ]. We found that adult and neonatal neutrophils differed with respect to fMLP-induced calcium mobilization. Whereas intracellular calcium levels increased rapidly after treatment of adult and neonatal neutrophils with fMLP, this response was attenuated in cells from neonates. Moreover, in neonatal cells, a subpopulation was identified that was unresponsive to fMLP. It is possible that decreased calcium mobilization contributes to impaired chemotaxis of neonatal cells to fMLP. In contrast to fMLP, DL1.2 did not induce calcium mobilization in adult or neonatal neutrophils, suggesting that calcium does not play a role in DL1.2-induced chemotaxis. These findings are consistent with recent studies of other inflammatory mediators that induce neutrophil chemotaxis without altering intracellular calcium, including transforming growth factor-ß1 and soluble Fas ligand [38 , 39 ]. Thus, it appears that the requirement for calcium in neutrophil chemotaxis is dependent on the chemoattractant and that impaired calcium mobilization, by itself, is not sufficient to account for decreased chemotaxis in neonatal cells.
In adult and neonatal neutrophils, chemotaxis induced by fMLP, as well as DL1.2, was blocked by wortmannin and LY 294002, two inhibitors of PI3K. This enzyme catalyzes the phosphorylation of PIP2 to PIP3, a step required for calcium-dependent neutrophil chemotaxis in response to fMLP, IL-8, and PAF [40 41 42 ]. The enzymatic products of PI3K induce some of their effects by mobilizing intracellular calcium stores [43 , 44 ]. Our data suggest that PI3K activation is important in DL1.2-induced chemotaxis but that this enzyme exerts its effects in neutrophils through a calcium-independent pathway. It is possible that phosphoinositides alter neutrophil polarization and motility by interacting with actin-binding proteins and inducing cytoskeletal reorganization [45 ]. Alternatively, PI3K-regulated phospholipid signaling may modulate neutrophil adhesion and motility by activating Akt/protein kinase B [46 ].
We also found that chemotaxis in response to DL1.2 and fMLP was inhibited by pertussis toxin in adult and neonatal cells, indicating that their activity is linked to G proteins. These are heterotrimeric molecules that function as intermediates in transmembrane signaling pathways initiated by various hormones, neurotransmitters, and chemoattractants [24 , 36 ]. Activation of G proteins increases guanosine 5'-triphosphate (GTP) binding, leading to phosphoinositide hydrolysis, calcium mobilization, and induction of protein kinase C. Our finding that DL1.2-induced chemotaxis is calcium-independent indicates that the signaling pathways initiated by G-protein activation are distinct for different chemoattractants.
Members of the MAP kinase family are also thought to be important in the signaling pathways leading to chemotaxis [25 26 27 ]. The p38 MAP kinase and the p42/44 extracellular signal-related kinase (ERK) are distinct proteins that have been identified in neutrophils [47 ]. We found that both enzymes were activated by fMLP in neonatal and adult cells. Similar effects of fMLP have been shown in adult neutrophils [25 26 27 ]. The fact that no major differences were observed between adults and neonates indicates that these MAP kinases are not involved in reduced chemotaxis of neonatal cells to fMLP. In contrast to fMLP, DL1.2 had no effect on p38 or p42/44 MAP kinase activation in adult or neonatal neutrophils. These data support the concept that fMLP and DL1.2-induced chemotaxis are regulated by distinct signaling pathways.
NF-B is a ubiquitous transcription factor activated by a number of
inflammatory mediators, including fMLP [12
,
13
]. DL1.2, like fMLP, was found to induce nuclear
translocation and DNA binding of NF-B in adult and neonatal
neutrophils. Moreover, inhibition of NF-B activity resulted in
decreased DL1.2 as well as fMLP-induced chemotaxis in both cell types.
These results suggest that the NF-B signaling pathway plays an
important role in neutrophil chemotaxis. This is consistent with the
observation that suppression of NF-B activation significantly
reduced endotoxin-induced neutrophil infiltration into the lung
[48
, 49
]. We also found that NF-B
nuclear binding activity in response to fMLP and DL1.2 was decreased in
neonatal neutrophils when compared with adult cells. Expression of the
NF-B p65 subunit and IB- proteins was also reduced in neonatal
cells. Following treatment with fMLP or DL1.2, levels of these proteins
were reduced to a greater extent in adult when compared with neonatal
neutrophils. Based on these findings, it appears that impaired
responsiveness of neonatal neutrophils to chemoattractants is, at least
in part, a result of altered NF-B signaling. This may be mediated by
diminished levels of p65 in neonatal cells and/or to reduced
regeneration of transcription factor proteins following
chemoattractant-induced activation. The findings that chemotaxis is
inhibited by actinomycin D and cycloheximide, which block RNA and
protein synthesis, respectively, provide support for this possibility.
The present studies suggest that deficiencies in neonatal neutrophil
chemotaxis are a result of aberrations in chemoattractant-induced
signaling pathways. Moreover, maturational changes underlying
developmental defects in neutrophil-functional activity are specific
for the chemoattractant. Thus, whereas reduced NF-B activity appears
to contribute to the diminished responsiveness of neonatal cells to
fMLP and DL1.2, defective calcium mobilization is important only in the
response to fMLP. The relative inability of neutrophils in newborns to
accumulate at sites of bacterial invasion is the most consistent defect
described in neonatal sepsis [50
, 51
]. A
more complete understanding of the pathways controlling neutrophil
migration and activation may provide insights into new therapeutic
approaches for treating infectious and inflammatory diseases in
neonates.
Received November 30, 2000; revised June 26, 2001; accepted June 28, 2001.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
triggered with formyl-methionyl-leucyl-phenylalanine Pediatr. Res. 34,243-248[Medline]B pathway by inflammatory stimuli in human neutrophils Blood 89,3421-3433B by phagocytic stimuli in human neutrophils FEBS Lett 412,583-586[Medline]B activation and cytokine release in human alveolar macrophages is PKC-independent and TK- and PC-PLC-dependent Am. J. Respir. Cell Mol. Biol. 18,384-391 or FMLP stimulation J. Immunol. 160,1982-1989B- phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo J. Biol. Chem. 272,21096-21103 mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils J. Clin. Invest. 103,851-858[Medline]B in endotoxin-tolerant rats is associated with down-regulation of chemokine gene expression and inhibition of neutrophilic lung infiltration J. Immunol. 158,5934-5940[Abstract]This article has been cited by other articles:
 |
|
 |
|
  S. Chandra, H. Haines, C. Michie, and A. Maheshwari Developmental Defects in Neutrophils from Preterm Infants NeoReviews, September 1, 2007; 8(9): e368 - e376. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Urlichs and C. P. Speer Neutrophil Function in Preterm and Term Infants NeoReviews, October 1, 2004; 5(10): e417 - e430. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
