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by Macrophages1
*
Department of Rheumatology, Hospital for Joint Diseases, New York University School of Medicine, New York, NY 10003;
Department of Chemical, Biochemical and Materials Engineering, Stevens Institute of Technology, Hoboken, NJ 07030; and
The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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from cocultured macrophages. In summary, apoptosis results
in surface accessibility of all SSA/Ro-SSB/La Ags for recognition by
circulating maternal Abs. It is speculated that in vivo such opsonized
apoptotic cardiocytes promote an inflammatory response by resident
macrophages with damage to surrounding conducting
tissue. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptors on the trophoblast into the fetal circulation
(1). This active placental transport is effective by 11 wk
of gestation (2). Fetal and neonatal disease appears to be
totally independent of maternal health status since the mothers can
have systemic lupus erythematosus, Sjögren’s syndrome, or in
fact be clinically asymptomatic (3, 4, 5). The two classic
manifestations of neonatal lupus are cardiac and cutaneous (1, 6), the former more serious and, with rare exception, permanent.
Cardiac injury characteristically involves the conduction system and
presents as congenital heart block
(CHB),3 which can be
first degree, but is most often second or third degree
(7). CHB is not associated with major structural heart
defects and is typically identified between 18 and 24 wk of gestation
(8). Despite the nearly universal association of CHB with
very specific maternal autoantibody responses, molecular mechanisms are
only recently being explored to explain how the targeted Ags, 48-kDa
SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro, which are normally
sequestered within the confines of the nucleus and cytoplasm, become
accessible to this immune response (9, 10). The traditional paradigm to account for tissue damage caused by the binding of autoantibodies to intracellular Ags is that immune complexes form in the intravascular space with subsequent deposition of the complexes, activation of complement, and influx of inflammatory cells (11). Alternatively, Abs could directly bind to the cell surface and/or penetrate across the membrane. It has recently been reported that anti-P Abs penetrate living cells and inhibit protein synthesis (12). In consideration of surface binding, one hypothesis is that apoptosis, a physiologic process of remodeling during embryogenesis (13, 14), might result in translocation of intracellular Ags to the external leaflet of the membrane. Rosen et al. (9) first demonstrated, by confocal microscopy, the presence of SSA/Ro and SSB/La in surface blebs of apoptotic keratinocytes. This observation was extended to the developing fetal heart in which we documented, by indirect immunofluorescence, that anti-SSA/Ro and anti-SSB/La Abs recognize the surface of nonpermeabilized cultured human fetal cardiocytes (10). Relevant to CHB, apoptosis occurs in scattered cells rather than tracts of contiguous cells in a tissue (15). Therefore, binding of autoantibodies to the surface of apoptotic cells could trigger an inflammatory response that results in damage to surrounding healthy tissue. It logically follows that organs with minimal regenerative capacities such as the heart, and particularly the specialized cells of the conduction system, would be most vulnerable.
The present study was initiated to determine whether Abs reactive to
each or all of the components of the SSA/Ro-SSB/La system truly bind to
the surface of human fetal cardiocytes following the induction of
apoptosis. Given the availability of a wide variety of human and murine
polyclonal Abs and mAbs, two initial approaches were exploited. The
methods included biotinylation of cell surface proteins and scanning
electron microscopy of immunogold-labeled cells. Subsequent experiments
addressed the consequences of surface binding by examining the release
of the inflammatory cytokine, TNF-, from macrophages cocultured with
apoptotic fetal cardiocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human fetal cardiocytes were cultured as described (16) with minor modifications. Briefly, human fetal hearts aged 16–24 wk were aseptically obtained after elective termination of normal pregnancy by dilatation and evacuation. This was done in accordance with the guidelines of the Institutional Review Board and after obtaining consent from the mothers. No cardiac toxic drugs were administered to the mothers during these procedures. The aorta was cannulated for continuous perfusion of the coronary arteries with calcium-free Tyrode’s solution (117 mM NaCl, 5.7 mM KCl, 11 mM glucose, 4.4 mM NaHCO3, 1.5 mM KH2PO4, 1.7 mM MgCl2, 20 mM HEPES, pH 7.4) containing 1 U/ml of sodium-heparin at 37°C, bubbled with 100% O2, as described for the Langendorff preparation (17). After 15 min of washing to clear the blood from the heart, fresh calcium-free Tyrode’s solution containing 1.5 mg/ml collagenase A (type III) was recirculated for approximately 20 min. The heart dissociated spontaneously, allowing cells to slowly drip and fall on a petri dish containing 0.25% trypsin, 1 mM EDTA in HBSS. Clumps of cells were dissociated and the resulting suspension poured over a cell strainer. Cells were centrifuged and the pellet resuspended in 20 ml of culture medium (DMEM supplemented with 10% FBS, 50 U/ml penicillin, 50 U/ml streptomycin, 100 mg/ml gentamicin, 1 mM nonessential amino acid (Life Technologies, Gaithersburg, MD), 0.1 mM essential medium vitamins (Life Technologies), 2 mM glutamine, and 0.1 mM sodium pyruvate). Cells were then plated at approximately 1.2 x 107 cells per 75-cm2 culture flask and grown in 5% CO2 at 37°C. After 4 days in culture, spontaneous contraction (30–40 beats per min) could be observed under phase-contrast microscopy.
Induction of apoptosis
Apoptosis was induced as previously described (10). Briefly, various concentrations of staurosporine (Sigma, St. Louis, MO) (0.5, 0.8, 1, 1.2 µM) were tested. Assessment of apoptosis was done by phase-contrast microscopy, fluorescence microscopy of propidium iodide-stained cells, and electron microscopy. Morphological signs of early apoptosis were observed in 40% of the cardiocytes after 3 h of incubation with 0.5 µM staurosporine; after 7 h, 97% of the cells showed signs of more advanced apoptosis. Trypan blue and propidium iodide exclusion indicated membrane integrity. Based on these observations, apoptosis was induced with 0.5 µM staurosporine for 6 h at 37°C in cardiocytes plated at a cell density of 1.2 x 107/75 cm2.
Biotinylation of cell surface proteins
Abs. To evaluate surface expression of SSA/Ro-SSB/La Ags, cell extracts from surface-biotinylated cardiocytes were immunoprecipitated with either human antisera or murine mAb. Three human antisera were used in these experiments: one contained anti-48-kDa SSB/La, 52- and 60-kDa SSA/Ro Abs (Met), and two sera were from healthy multigravidas with no known autoantibodies (Mo, Hai). Four murine mAb were used: mAb A2, IgG1 (recognizes 48-kDa SSB/La); mAb A9, IgG1 (recognizes 60-kDa SSA/Ro); W6/32, IgG2a (recognizes the HLA class I glycoproteins HLA-A, B, and C of all alloantigenic species, molecular mass 45 kDa); and MOPC-21, IgG1 (a nonspecific murine mAb).
Surface biotinylation. Surface proteins on apoptotic
and nonapoptotic human fetal cardiocytes, cultured in
75-cm2 flasks 4 days after isolation, were
labeled with 10 µg/ml D-biotinoyl-
-aminocaproic acid
hydroxysuccinimide ester (Boehringer Mannheim, Indianapolis, IN).
Specifically, apoptotic cells were processed 6–8 h after induction of
apoptosis with 0.5 µM staurosporine, when greater than 90% of the
cells were apoptotic. Cells in 75-cm2 culture
flasks were washed three times with ice-cold HBSS to remove traces of
serum proteins from the culture medium. Subsequently, 10 µg/ml
D-biotinoyl--aminocaproic acid in ice-cold saline
bicarbonate buffer (50 mM NaHCO3/0.9% NaCl)
was added and flasks incubated at 4°C for 20 min. After stopping the
biotinylation reaction with ice-cold 50 mM NH4Cl
in saline-bicarbonate buffer, flasks were washed three times with TBS
buffer (50 mM Tris, pH 7.5, 150 mM NaCl). Cells were then incubated for
20 min at 4°C in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1 mg/ml PMSF, and a protease
inhibitor cocktail (Complete Mini; Boehringer Mannheim), used as per
manufacturer’s instructions). Cells were then scraped off the flasks,
transferred to chilled tubes, and centrifuged at 3000 rpm for 10 min.
The supernatant was precleared twice with 50 µl/ml protein
A-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ). Before
immunoprecipitation, a 300-µl aliquot was taken from extracts of
apoptotic or control cells for subsequent SDS-PAGE. The cell extracts
were then divided into aliquots and incubated for 1 h at 4°C on
a rotating platform with 5 µl of one of the four Ab preparations, as
detailed above. Cell extracts corresponding to 3 x
107 cells were used per condition. The protein
concentration was equivalent for control and apoptotic cells (2.2
mg/ml), as determined by the BCA (bicinchoninic acid) Protein Assay
(Pierce, Rockford, IL). Protein A-Sepharose, 50 µl/ml, was then added
and immunoprecipitation was conducted overnight at 4°C on a rotating
platform. The beads were washed six times with chilled wash buffer (50
mM Tris, pH 7.4, 150 mM NaCl, 4 mM EDTA-Na2,
0.5% Nonidet P-40, 0.5% deoxycholic acid, sodium salt, 0.1% SDS),
eluted, and boiled with reducing SDS sample buffer, then run on 15%
high ratio monomer:cross-linker acrylamide gels overnight at 7.5 mAmps
until the dye front ran off. The gel was then transferred to
nitrocellulose at 40 V overnight. Biotinylated proteins were detected
using the bone marrow chemiluminescence blotting kit
(biotin/streptavidin; Boehringer Mannheim), according to the
manufacturer’s instructions, and filters wrapped in Saran wrap were
exposed to x-ray film (Fuji Medical Systems, Stamford, CT).
Scanning electron microscopy studies
To confirm surface expression of SSA/Ro-SSB/La Ags, nonpermeabilized apoptotic and nonapoptotic cardiocytes were incubated with human antisera or murine mAb at a dilution of 1/100 for 45 min in PBS-calcium. One human autoimmune serum contained anti-48-kDa SSB/La Abs alone, as evaluated by ELISA, immunoblot, and immunoprecipitation (Lew); one had 52- and 60-kDa SSA/Ro Abs alone (Dif); and one had 48-kDa SSB/La, and 52- and 60-kDa SSA/Ro Abs (Die). Sera from two healthy multigravidas (Mo, Hai) with no known autoantibodies served as negative controls. The murine mAb were as described above.
Preparation of cells for scanning electron microscopy. Cardiocytes were transferred on the fourth day after culture to 24-well plates with glass coverslips in the bottom, at a density of 1 x 105 cells/well. After 1 day, apoptosis was induced with 0.5 µM staurosporine, and 5 h later cells were incubated with human sera or murine mAb diluted 1/100 in PBS/0.1 mM CaCl2 (PBS-Ca2+) for 45 min at 37°C, 5% CO2. After washing three times with PBS-Ca2+, cells were fixed with 2% paraformaldehyde in PBS for 20 min at room temperature, washed again, and incubated for 2 h at room temperature with 18 nm goat anti-human (1/10 dilution, A520 = 2) or 12 nm goat anti-mouse (1/20 dilution, A520 = 2) colloidal gold affini-pure IgG (H+L), EM grade (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were washed again and fixed for 1 h at 4°C on a rocking platform with 2% glutaraldehyde in 100 mM cacodylate/100 mM sucrose buffer, pH 7.2, washed three times for 10 min with 100 mM cacodylate/100 mM sucrose buffer, pH 7.2, and dehydrated in increasing concentrations of ethanol. Cells were finally incubated for 1 h in 100% hexamethyldisilazane, air dried, and kept under vacuum until analysis.
Cell-coated coverslips were affixed to aluminum holders using double-sided carbon tape and silver paint, and subsequently coated with a thin layer of carbon to prevent electrical charging when scanning. Samples were examined in a LEO 982 field-emission digital SEM (LEO Electron Microscopy, Thornwood, NY) using 10 kV accelerating voltage and 8 mm working distance. Cell surface morphology was studied by secondary electron imaging with secondary electrons collected by a high-resolution in-lens secondary electron detector. Backscattered imaging was used in the immunogold studies. The electron backscattering coefficient is a strong function of atomic number; therefore, gold nanoparticles appear bright in a backscattered image. Backscattered electrons emitted from the samples were collected by a solid-state backscattered electron detector (KE Developments, Cambridge, U.K.). All images were recorded and stored digitally. After the images were filtered using DigitalMicrograph software, version 3.1 (Gatan, Pleasanton, CA), to remove the unflattened background, gold particles were counted by one of the investigators (T.-M.C.) without prior knowledge of the antisera being tested.
Cocultures of cardiocytes and macrophages
Preparation of affinity-purified Abs and IgG fractions. Abs against 48-kDa SSB/La, and 52- and 60-kDa SSA/Ro proteins were isolated from sera by affinity column chromatography using the respective recombinant proteins (18) coupled to CNBr-activated Sepharose 4B as Ags. Eluted Abs were neutralized with 1 M Tris and tested for specificity by ELISA, immunoblot, and immunoprecipitation of 35S-labeled proteins. Normal human IgG was obtained from normal human serum using the ImmunoPure (protein A) Purification Kit (Pierce, Rockford, IL), according to the manufacturer’s instructions.
Affinity-purified Abs and normal human IgG were free of endotoxin, as determined by the E-toxate (Limmulus amebocyte lysate) assay (Sigma). Affinity-purified Abs did not have rheumatoid factor, as assessed by standard nephelometry.
Culture of human macrophages. Human macrophages were obtained from peripheral blood of normal volunteers, as described (19). In brief, PBMC were isolated by Ficoll-Hypaque gradient and plated for 2 h in DMEM, in 75-cm2 culture flasks. Nonadherent cells were washed out with HBSS, and adherent macrophages were cultured for 4 days in DMEM supplemented with 10% FCS. Subsequently, macrophages were treated with 0.02% EDTA in PBS and transferred to 24-well culture plates at a density of 2 x 105 cells/well.
Opsonization of apoptotic cells. Cultured human fetal
cardiocytes, 6 h after induction of apoptosis, were thoroughly
washed with HBSS to remove any remaining staurosporine and gently
scraped off the culture flasks, resuspended in DMEM (absent FCS)
containing affinity-purified Abs or normal human IgG (1.5 µg/ml), and
rotated end over end for 30 min at 4°C. After washing three times in
DMEM without serum, cells were resuspended in DMEM with 10% FCS and
added to macrophage cultures, at a density of 6 x
105 apoptotic cells/well. Culture supernatants
were collected 17 h after adding the apoptotic cells, and TNF-
concentration was determined using the high sensitivity Quantikine
Immunoassay by R&D Systems (Minneapolis, MN), according to the
manufacturer’s instructions.
Phagocytosis of apoptotic cells was verified by light microscopy (data not shown).
Cardiocytes cultured under identical conditions absent the staurosporine served as the nonapoptotic control cells. However, because of their adherence, trypsinization was required. After washing once with DMEM containing 10% FCS to neutralize the trypsin and twice with DMEM (absent FCS), the cells were incubated with serum (from a mother whose child had CHB) containing Abs to all three components of the SSA/Ro-SSB/La complex diluted 1/50 in DMEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The initial approach to address the accessibility of autoantigens
to circulating maternal Abs utilized immunoprecipitation of surface
biotinylated proteins in nonapoptotic and apoptotic cardiocytes.
Immunoprecipitation of a biotinylated 48-kDa band was consistently
observed in apoptotic cardiocytes incubated with antiserum Met, which
recognizes 48-kDa SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro (Fig. 1
). This band was not detected in
cardiocytes that had not undergone apoptosis. No reactivities were seen
following immunoprecipitation of either apoptotic or nonapoptotic cells
with normal human serum. The intensity of biotin signal in
nonimmunoprecipitated cell extracts was similar in control and
apoptotic cells, indicating that the efficiency of biotinylation was
equivalent in both conditions.
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). As expected, nonapoptotic and
apoptotic cell lysates were immunoprecipitated by a mAb, W6/32, which
recognizes the 45-kDa heavy chain of HLA class I molecules. W6/32
served both as a positive control for immunoprecipitation of a known
surface protein and facilitated assignment of m.w. No reactivities were
seen following immunoprecipitation of either apoptotic or nonapoptotic
cells with MOPC-21 (an isotype control mAb).
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Scanning electron microscopy
Since the interpretation of surface expression, as assessed by
biotinylation of immunoprecipitated proteins, might be confounded by
minor intracellular uptake of label, scanning electron microscopy was
performed utilizing a large number of well-characterized reagents. To
restrict labeling to the cardiocyte surface, fixation was performed
only after treatment with antisera. Nonpermeabilized apoptotic and
nonapoptotic cardiocytes were incubated with experimental human sera or
murine mAb, followed by second-stage Abs labeled with gold and observed
under a scanning electron microscope. As seen in the backscatter images
of Fig. 3, a serum containing 48-kDa
SSB/La, and 52- and 60-kDa SSA/Ro (Die) did not bind the nonapoptotic
cardiocytes (A). In contrast, gold-labeled particles were
readily observed on early apoptotic cardiocytes in which there was
surface blebbing but size and morphology were relatively conserved
(B). Similarly, there were abundant gold-labeled particles
on cells in the later stages of apoptosis, as defined by increased
numbers of blebs, decreased size, and rounded shape (C). No
gold-labeled particles were seen after incubation with normal human
serum (Hai (D) or Mol (data not shown)).
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). To unambiguously identify the surface
Ags, murine mAb were also used. mAb recognizing 48-kDa SSB/La and
60-kDa SSA/Ro both bound the apoptotic cardiac surfaces (Fig. 5). In all cases, the distribution of
gold particles was diffuse with no differences in pattern between the
Ags. Cells did not label with the secondary Abs when preincubated with
murine isotype control Ab or when primary Ab was omitted (data not
shown). For each condition, the number of gold particles was counted in
at least seven fields on different cells. The mean ± SEM are
summarized in Table I.
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Cardiocytes were coincubated with human macrophages to assess the
functional consequences of Ab binding to the surface of apoptotic
cardiocytes. The Th1 cytokine, TNF-, was chosen as a readout of
inflammation.
Basal production of TNF- by the macrophages was 9.7 ± 0.9 SEM
pg/ml and decreased to 3.3 ± 0.3 SEM pg/ml after coincubation
with apoptotic cells (Fig. 6A). This decrease in basal
production was not observed in initial experiments using cardiocytes
rendered necrotic after hypotonic lysis (Fig. 6B). Apoptotic
cardiocytes preincubated with normal human IgG acted functionally as
nontreated apoptotic cells; TNF- production by the macrophages was
5.7 ± 0.9 SEM pg/ml (Fig. 6A). In contrast, when
macrophages were cocultured with apoptotic cardiocytes incubated with
affinity-purified Abs to each of the components of the SSA/Ro-SSB/La
complex, TNF- production was increased by 3–5-fold over basal
levels and 10–14-fold over that secreted after culture with apoptotic
cells alone (specifically, after coculture with cardiocytes opsonized
with anti-SSB/La, TNF- was 35 ± 2.9 SEM pg/ml; with
anti-52Ro it was 61.7 ± 10.1 SEM; and with anti-60Ro it
was 48.3 ± 9.2 SEM) (Fig. 6A). Nonapoptotic
cardiocytes incubated with medium alone or serum containing Abs
reactive with 48-kDa SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro did not
modify the basal production of TNF- by the macrophages (Fig. 6C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis is an ideal mechanism to account for the problem of accessibility in the fetal heart and absence of injury in the maternal heart, since it is a selective process of physiological cell deletion during embryogenesis and morphogenesis that occurs in isolated cells rather than in extensive areas within an organ. Therefore, any disturbance in the noninflammatory mechanism of removal of apoptotic bodies would result in damage to surrounding healthy tissue. Rosen et al. (9) first demonstrated that SSA/Ro-SSB/La ribonucleoproteins translocate to the surface blebs of apoptotic keratinocytes, a seminal observation subsequently corroborated in human fetal cardiocytes (10). Indirect immunofluorescence surface staining of nonpermeabilized cardiocytes with antisera containing SSA/Ro-SSB/La Abs suggested that these Ags not only localize to the surface blebs, but might be displayed in such a way as to be recognized by maternal extracellular Abs. Translocation of other intracellular proteins, calreticulin (23), the 60-kDa heat-shock chaperonin protein (24), and neutrophil myeloperoxidase (25), to the plasma membrane has been reported.
In the study presented herein, two independent techniques provide evidence that the surface of apoptotic cultured human fetal cardiocytes is indeed bound by Abs to each of the components of the SSA/Ro-SSB/La complex: 48-kDa SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro. Demonstration of surface expression by immunoprecipitation of biotinylated proteins was successful for 48-kDa SSB/La only. It has been reported that biotinylation of proteins can abrogate recognition by immunoprecipitating Abs in certain systems (26), and our results may be explained, in part, by hindrance of conformationally dependent epitopes by the biotin compound. Moreover, immunoprecipitation of 52-kDa SSA/Ro is not consistently detected by most laboratories when using cell extracts (27). Surface binding of Abs to each of the components of the SSA/Ro-SSB/La complex was clearly demonstrated by scanning electron microscopy of the apoptotic cardiocytes. Surface binding was noted in earlier stages of apoptosis, as defined by cell morphology under scanning electron microscopy, and persisted through later stages.
The demonstration of unequivocal Ag-Ab binding at the cell surface
supports the hypothesis that maternal Abs to components of the
SSA/Ro-SSB/La complex are not simply markers of disease, but play an
active part in the pathogenesis of CHB. A molecular explanation for
subsequent damage to the specialized cells of the conduction system and
working myocardium remains speculative. In this context, a mechanism is
envisioned whereby unexpected circumstances convert the physiologic
process of apoptosis into one in which an inflammatory component is
evoked. Perhaps the unexpected event is opsonization. Apoptotic cells
have been regarded as immunosuppressive since internalization of
apoptotic cells by phagocytes inhibits the release of proinflammatory
cytokines (28, 29). In contrast, phagocytosis of opsonized
apoptotic cells has been reported to be proinflammatory (29, 30), an example of which is the observation that ingestion of
apoptotic cells bound by anticardiolipin Abs results in the release of
TNF- from cocultured macrophages (30). Of relevance,
two distinct pathways of phagocytosis, each controlled by different
intracellular signaling cascades involving Rho GTPases, have been
identified (31). Type I follows binding to macrophage
Fc receptors and is considered to be proinflammatory, while type II,
mediated by the complement receptor CR3, is not accompanied by
inflammation. Our results support that nonopsonized apoptotic
cardiocytes are ingested through type II phagocytosis. In contrast,
release of the proinflammatory cytokine, TNF-, by macrophages that
have ingested apoptotic cardiocytes bound by anti-SSA/Ro-SSB/La Abs
is consistent with type I phagocytosis. These findings suggest that
circulating maternal autoantibodies opsonize cells undergoing
physiological apoptosis, which then changes otherwise innocent
degradation products into proinflammatory stimuli. This cascade could
result in damage and ultimately permanent scarring in those tissues
with low regenerative capacity.
In summary, the intracellular protein targets, SSA/Ro and SSB/La, are accessible to transplacentally acquired maternal autoantibodies during the process of apoptosis. The results reported in this study support the hypothesis that opsonization of apoptotic cardiocytes by circulating maternal autoantibodies thwarts the normal scavenging function of tissue macrophages during fetal development. Interference with physiologic remodeling might then lead to inflammation. Furthermore, it would appear that the specialized conducting tissue of the human heart is incapable of healing without fibrotic sequelae.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jill P. Buyon, Department of Rheumatology, Room 1608, Hospital for Joint Diseases, 301 East 17th Street, New York, NY 10003.
3 Abbreviation used in this paper: CHB, congenital heart block.
Received for publication May 23, 2000. Accepted for publication August 8, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-binding proteins in the maternofetal transfer of IgG. APMIS 114:(Suppl. 64):5.
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