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Inflammation Research Unit, School of Pathology, University of New South Wales, Sydney, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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but not IL-4,
indicating a bias toward a type 1 immune response. Chronic low grade
stimulation of autoreactive ß2GP1-specific,
IFN--producing Th1 CD4+ T cells may contribute to the
high risk of thromboses and pregnancy failure in patients with
APS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The physiological function of ß2GP1 remains uncertain, but in vitro studies indicate potential natural anticoagulant properties. ß2GP1 inhibits the contact phase of blood coagulation, impairs ADP-dependent platelet aggregation, and causes a dose-dependent inhibition of the prothrombinase activity of platelets (14, 15, 16). Research into the role of autoimmunity to ß2GP1 in APS has primarily involved characterization of the autoantibodies, with limited focus on the cellular aspects of the immune response. Bone marrow cells from mice with experimental APS transfer the disease to naive mice, and recipient mice display Abs to ß2GP1 within 4 mo of cell transfer (17). T cell-depleted bone marrow cells do not induce aCL Ab production in naive recipients, indicating a dependence upon T lymphocytes.
Taken together, these results suggest that patients with aPL Abs
possess T lymphocytes with specificities to ß2GP1. In
this study, we show that circulating ß2GP1-specific
CD4+ T cells can be demonstrated in approximately one-half
of patients with APS. These cells are not present in individuals with
aPL Abs who do not exhibit the clinical features of APS, indicating
that a cellular immune response may be a more specific marker for the
syndrome than the production of autoantibodies. Stimulation of
ß2GP1-specific cells in vitro leads to the production of
high levels of IFN-, a cytokine that may contribute to the
thrombotic diathesis and pregnancy failure observed in these patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 24 patients with aPL Abs were studied. Of these patients, 22 had aCL Abs of either IgG or IgM isotype (measured using a standardized kit from Medical Innovations, Sydney, Australia), and 2 had LA without aCL Abs. Seven patients with various autoimmune diseases (systemic lupus erythematosus (SLE), psoriatic arthritis, rheumatoid arthritis, or giant cell arthritis) and 15 healthy controls (both groups were aCL Ab-negative) were also studied. The median ages for the three groups were 42, 53, and 33, respectively. Blood was collected in citrate dextrose, diluted 1/3 with PBS, layered over Lymphoprep (Nycomed Pharma, Oslo, Norway), and centrifuged at 1800 rpm. PBMCs were collected, washed twice in PBS, resuspended at 1 x 107/ml in RPMI 1640 (Life Technologies, Gaithersburg, MD) plus 20% FCS (Life Technologies), frozen in 7.5% DMSO, and stored in liquid nitrogen until further use. Serum from patients and controls was also collected.
Anti-ß2GP1 Ab ELISA
Purified ß2GP1 was coated at 5 µg/ml on high-binding polystyrene microtiter plates (Costar 3590, Corning Costar, Cambridge, MA) in 100 µl/well Tris (pH 8.4) and covered overnight at 4°C. Plates were washed four times with PBS (250 µl/well) and blocked with 1% skim milk powder in PBS (200 µl/well) for 1 h at 37°C. Sera from four normal controls were used as negative controls; rabbit anti-human ß2GP1 IgG used as a positive control. For quantitation, a standard curve was established using standards from the Medical Innovations IgG aCL Ab ELISA kit diluted 1/20 in 0.3% gelatin/PBS and added at 100 µl/well. Patient samples were diluted 1/100 in 0.3% gelatin/PBS and added at 100 µl/well in duplicate. Plates were incubated at room temperature for 2 h and subsequently washed as described above. Goat anti-human IgG HRP-conjugated (Dako, Glostrup, Denmark) was diluted 1/500 in 0.3% gelatin/PBS and added at 100 µl/well. Plates were incubated at room temperature for 1 h and washed; next, substrate (trimethylbenzene) was added at 100 µl/well. Plates were covered, and 100 µl of 1 M H2SO4 was added to each well after 15 min. Plates were read on a spectrophotometer at a wavelength of 405 nm using a Titertek Multiskan Plus MKII plate reader (Lierbyen, Norway); absorbance was converted to arbitrary units from the standard curve.
ß2GP1 purification
ß2GP1 was purified from pooled normal human plasma using sequential chromatographic steps. Endotoxin minimization conditions were used during all procedures. A total of 10 mM sodium-EDTA, 1 mM benzamidine, and 2.2 mM PMSF (Sigma, St. Louis, MO) were added to 1.5 liters of plasma and left to mix overnight at 4°C. Plasma was precipitated with 70% ammonium sulfate, resuspended in 10 mM Tris-HCl (pH 7.5), dialyzed twice against 10 mM Tris-HCl (pH 7.5), and loaded onto a heparin Sepharose column (Sigma). The ß2GP1-containing fractions were eluted in 1 M NaCl/10 mM Tris-HCl (pH 7.5), detected by ELISA, pooled, concentrated using an amicon (10,000 m.w. cutoff) membrane, and loaded onto a Sephacryl S-200 column (Sigma). ß2GP1-containing fractions were eluted in 0.5 M NaCl; the fraction pool was concentrated and dialyzed against sodium acetate A (0.05 M acetate and 0.05 M NaCl, pH 4.8) buffer and loaded onto an S-Sepharose fast flow column (Sigma). ß2GP1-containing fractions were eluted using a gradient (0–100%) of acetate A and acetate B (0.05 M acetate and 0.65 M NaCl, pH 5.2) buffer. Fractions containing ß2GP1 were confirmed by ELISA and 10% SDS-PAGE, pooled, and concentrated. The ß2GP1 was filtered sequentially through four Zetapore syringe filters (Cuno, Meriden, CT) and tested for endotoxin in the Limulus amebocyte lysate assay (Pyrotell, Woods Hole, MA) to ensure that endotoxin levels were <0.125 endotoxin units, before being aliquoted and stored at -20°C.
ß2GP1 ELISA
ß2GP1 (10 µg/ml) in sodium carbonate buffer (pH 9.6) was coated on flat-bottom, 96-well plates (Maxisorp, Nunc, Roskilde, Denmark) overnight at 4°C. Plates were washed four times with PBS/0.05% Tween 20 and blocked (200 µl/well) with 1% skim milk powder in PBS for 1 h at 37°C. ß2GP1 standards and fractions (diluted in 0.3% gelatin/PBS, 50 µl/well) were added plus rabbit anti-human ß2GP1 serum (diluted 1/20,000 in 0.3% gelatin/PBS, 50 µl/well). The rabbit anti-human serum was generously supplied by Prof. C. Chesterman (School of Pathology, University of New South Wales, Sydney, Australia). Plates were incubated on a shaker for 40 min at room temperature and subsequently washed as described above. Anti-rabbit IgG HRP (Dako) was added for 40 min at room temperature. Next, the wells were washed and developed with 100 µl of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (Sigma); absorbance was measured on a Titertek Multiskan Plus MKII plate reader at 405 nm.
Lymphocyte proliferation assay
PBMCs from patients and controls were resuspended to 1.5 x
106/ml in AIM V serum-free medium (Life Technologies) and
subsequently incubated in triplicate wells with purified
ß2GP1 (final concentration 25 µg/ml) with or without
1% normal human AB serum or, in some cases, 1% autologous serum to
measure proliferation in response to ß2GP1. PBMCs were
also incubated with PHA (Murex Biotech, Dartford, U.K.) (40 µg/ml) or
tetanus toxoid (TT) (CSL, Melbourne, Australia) (2.5 LF units/ml) as
mitogenic and Ag-specific positive controls, respectively. PBMCs
(150,000 in 100 µl) were added to the wells of 96-well, round-bottom
plates (Nunc) and incubated with 100 µl of each stimulus at 37°C in
the presence of 5% CO2 in air for 5 or 7 days. Next,
cultures were pulsed with [3H]thymidine (25 µl/well)
(Amersham, Buckinghamshire, U.K.) and incubated for 18 h before
harvesting the cells onto filter papers (ICN Biomedicals, Costa Mesa,
CA). Thymidine incorporation was measured using a liquid scintillation
counter (Packard, Canberra, Australia). Stimulation indices (SI) were
calculated as the mean total cpm of Ag-stimulated cells divided by the
mean total cpm of cells cultured in media alone for each individual
patient or control. 
cpm was calculated as the mean total cpm of
Ag-stimulated cells minus the mean total cpm of unstimulated cells for
each individual patient or control. Alternatively, supernatants from
stimulated cultures of PBMCs were collected for the measurement of IL-4
and IFN- using duoset ELISA kits (Genzyme, Cambridge, MA) according
to the manufacturer’s instructions. In some experiments, PBMCs were
incubated with ß2GP1 in the presence of anti-HLA
class II Ab (DP, DQ, DR) (IgG) (Serotec, Raleigh, CA). Different
concentrations of the Ab were tested, and the optimal concentration for
Ab neutralization was found to be 84 µg/ml. An irrelevant mouse IgG
Ab (Dako) was used at the same concentration as a control.
Depletion of ß2GP1 from normal serum (NS)
IgG from rabbit anti-human ß2GP1 serum (Behring Diagnostics, Marburg, Germany) was purified on protein A-Sepharose (Sigma) (18) and subsequently immobilized onto Affi-gel Hz (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. NS (2 ml) was added dropwise to the anti-ß2GP1 immunoaffinity column, and the depleted eluate was collected. To control for nonspecific depletion, a sham immunoaffinity column was prepared as described above using normal rabbit IgG (Dako). NS was passed through this column, and the sham-depleted serum eluate was collected. The ß2GP1-depleted and sham-depleted sera were used as stimuli in PBMC proliferation assays.
Flow cytometric analysis
PBMCs or stimulated cell cultures were double labeled with FITC- or PE-conjugated mAbs in combination (CD14 plus CD45, CD3 plus CD8, CD3 plus CD4, and CD3 plus CD19) (Becton Dickinson, San Jose, CA) (19). A Becton Dickinson FACScan flow cytometer with CellQuest acquisition and analysis software was used to acquire 5000 positive events. Gating for CD14+ CD45+, CD3+ CD8+, CD3+ CD4+, and CD19+ cells was established and used to determine the percentage of CD14+ monocytes, CD8+ T cells, CD4+ T cells, and CD19+ B cells. Percentages of CD8+ and CD4+ T cells were calculated with reference to the percentage of CD45+ lymphocytes using standard methods (20).
Depletion of CD4+, CD8+, CD19+, and CD14+ cells from PBMC populations
PBMCs were washed twice in PBS plus 2% FCS and then resuspended in 100 µl with a predetermined amount of anti-CD4+, anti-CD8+, anti-CD19+, or anti-CD14+ Dynabeads (Dynal AS, Oslo, Norway) to give a ratio of beads to cells of 3:1. Beads were washed twice in PBS plus 2% FCS, isolated with a Dynal magnet, resuspended to the original volume, and incubated with PBMCs for 30 min (CD4+, CD8+, and CD19+) or 1 h (CD14+) at 4°C on a roller apparatus. Unbound cells were removed by using the Dynal magnet and by washing cell rosettes with PBS plus 2% FCS. This procedure was repeated three times; depletion was confirmed by flow cytometric analysis using FITC- or PE-conjugated mAbs in combination: CD14/CD45, CD3 plus CD8, CD3 plus CD4, and CD3 plus CD19.
Statistical calculations
Differences between patient and control groups were determined
using the Student t test and 
2 test.
Correlations between data were examined using Pearson’s test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because ß2GP1 is present at relatively high concentrations in NS, a serum-free system for measuring lymphocyte proliferation was established, with precautions taken to ensure low endotoxin levels (<0.125 endotoxin units/ml) to minimize nonspecific stimulation. Negligible lymphocyte proliferation occurred when PBMCs from patient and control groups were cultured for 7 days in media alone, with a mean [3H]thymidine uptake of 921 ± 398 cpm. In contrast, a significant proliferation of cells from patient and control groups was observed after culture with the nonspecific mitogen, PHA (mean 17,283 ± 4,303 cpm), or the specific Ag, TT (mean 6674 ± 5309 cpm). The percentages of aPL-positive patients, controls, and autoimmune patients responsive to TT were 67%, 53%, and 43%, respectively.
Initial experiments indicated that PBMCs from a number of patients with
aPL Abs proliferated in response to purified ß2GP1. A
dose-response analysis of PBMCs from four of these patients (patients
1, 2, 4, and 5) showed that maximal proliferation consistently occurred
at a final Ag concentration of 25 µg/ml (Table I
); this dose was used for the remainder
of the study. The proliferative responses of PBMCs from the 24 patients
with aPL Abs to 25 µg/ml ß2GP1 are shown in Table II. Of the 24 patients, 8 (patients 1–8)
exhibited unequivocal proliferation to ß2GP1, which
represented SI values that were >4 SD above the mean of the control
group. These eight patients were termed responders, and their mean SI
value was 4.3 ± 0.73 SEM (mean cpm 3963 ± 634). PBMCs from
three additional patients (patients 9–11) exhibited weaker
proliferation, with a mean SI value of 1.85 ± 0.11 SEM (mean cpm
1844 ± 87), which represented SI values between 3 and 4 SD above
the mean of the control group. However, culturing PBMCs from these
patients with higher concentrations of ß2GP1 did not
result in greater stimulation (Table I). There was no significant
proliferation to ß2GP1 by PBMCs from the remaining 13
patients (patients 12–24), with a mean SI value of 1.05 ± 0.14
SEM that was not significantly different from the control group
(p = 0.426). These patients as well as patients
9–11 were termed nonresponders. The mean SI values to
ß2GP1 for the responders were significantly different
from both nonresponder and control populations
(p = 0.002) (Fig. 1).
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, Table II).
Moreover, NS synergized with ß2GP1. PBMCs from the eight
responders exhibited various levels of enhanced proliferation to a
combination of ß2GP1 plus serum compared with
ß2GP1 alone. There were significant differences in mean
SI values between responders and nonresponders to ß2GP1
plus 1% NS (p = 0.017). In addition, PBMCs
from patients 9–11 that showed weak proliferation to
ß2GP1 alone exhibited much stronger proliferation to the
ß2GP1-serum combination (Table II). The proliferative
effect of serum was similar using either 1% NS or 1% autologous
serum, with mean cpm levels of 17,464 ± 2,639 and 18,939 ±
3,140, respectively, for PBMCs from three of three responders tested
(patients 2, 4, and 7).
Because ß2GP1 is a component of human serum, it was
possible that the proliferative response to serum was due to
ß2GP1. To investigate this possibility, NS was depleted
of ß2GP1 by immunoaffinity to levels of <25 µg/ml. The
control was serum adsorbed with normal rabbit IgG, which contained 210
µg/ml ß2GP1. Culturing PBMCs from two responders
(patients 4 and 7) with 1% ß2GP1-depleted serum
abrogated the proliferative response (mean SI 0.56 ± 0.33 and cpm
469 ± 187), whereas proliferation to 1% sham-depleted serum
persisted (mean SI 10.77 ± 1.32 and cpm 6812 ± 2582). The
addition of purified ß2GP1 to
ß2GP1-depleted serum restored PBMC proliferation (mean SI
3.77 ± 0.66 and cpm 5083 ± 1402) (Fig. 2). There were significant differences in
proliferation between responder PBMCs stimulated with
ß2GP1 vs depleted serum (p =
0.031), NS vs depleted serum (p = 0.0007), and
ß2GP1 plus depleted serum vs depleted serum
(p = 0.043). These results suggest that
ß2GP1 contributes to the proliferative responses to serum
in some patients with aPL Abs.
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Of the 24 patients with aPL Abs recruited, 18 were diagnosed with
APS, which manifests as a history of thrombosis and/or recurrent
miscarriages and aCL Ab or LA detected on at least two occasions. Six
of these patients had SLE or a related autoimmune disorder, whereas the
other 12 had primary APS. The six patients with no history of APS had a
variety of conditions, the most common of which was some type of
autoimmune connective tissue disorder. Table II shows that all eight
responders (patients 1–8) had histories of thrombosis or recurrent
miscarriages, whereas the nonresponder group included all six
individuals who had aPL Abs without APS. A significant association
between positive proliferation to ß2GP1 and a history of
APS was apparent (2 test, p = 0.045).
Alternatively, there were no correlations between IgG
anti-ß2GP1 Ab levels and SI values to
ß2GP1 (r = 0.13, p >
0.05), to 1% NS (r = -0.03, p >
0.05), or to ß2GP1 plus 1% NS (r = 0.20,
p > 0.05) for patients with APS.
Proliferation to ß2GP1 is Ag-specific and requires CD4+ T lymphocytes and APCs
Neutralizing anti-HLA class II Ab, but not the control Ab,
abrogated the ß2GP1-induced proliferation of PBMCs from a
ß2GP1 responder (patient 4) (Fig. 3), indicating a requirement for Ag
presentation. The TT-induced proliferation of PBMCs from the same
patient and a normal control was also abrogated by anti-HLA class
II, whereas the responses to the Ag-independent mitogen PHA were
unaffected (Fig. 3).
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). There was no significant change
in either lymphocyte subset when PBMCs from nonresponder patients or
normal or autoimmune controls were cultured with ß2GP1
(data not shown). These results infer a selective expansion of
CD4+ T cells in the PBMCs that proliferate to
ß2GP1.
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but not
IL-4
PBMCs from five ß2GP1 responders (patients 1, 3, 4,
7, and 8) stimulated with ß2GP1 secreted high levels of
IFN- (mean 667 ± 307 pg/ml) compared with
ß2GP1-stimulated PBMCs from four controls (mean 32
± 46 pg/ml) when measured after 7 days of stimulation (Fig. 5). Similar levels of IFN- were
obtained when cells from both groups were cultured with PHA. PBMCs from
ß2GP1 responders cultured with ß2GP1
secreted very low levels of IL-4, whereas significant amounts of IL-4
were generated in the PHA-stimulated culture supernatants of PBMCs from
ß2GP1 responders (mean 345 ± 25 pg/ml) and controls
(mean 145 ± 51 pg/ml). Depletion of CD4+ T
lymphocytes, CD14+ monocytes, or CD19+ B cells
abrogated IFN- production from the PBMCs of two responders (patients
4 and 7) cultured with ß2GP1 (Fig. 6), whereas levels were only minimally
reduced by depletion of CD8+ T cells. IL-4 production
remained at very low levels in depleted PBMC populations and was
unchanged compared with nondepleted PBMC populations (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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but not IL-4. Third, a cellular immune response to
ß2GP1 was strongly correlated with the clinical
manifestations of APS. These results indicate that the measurement of
cellular immunity to ß2GP1 may have predictive
implications and provide potential mechanisms for thrombosis and
recurrent miscarriage in patients with aPL Abs. Whereas the term "antiphospholipid" continues to be used to refer to the Igs associated with the clinical syndrome characterized by recurrent thrombosis and fetal loss, there is little doubt that the immunological specificities of these Abs are not directed to phospholipids, but to epitopes on phospholipid-binding proteins, such as ß2GP1 (2, 3, 4, 5, 6, 7, 8, 9, 10) and prothrombin (21). Furthermore, although much effort has focused on examining the functional effects of these Abs on components of both pro- and anticoagulant pathways (Refs. 16, 22, and 23; reviewed in Refs. 5 and 9), there has been little investigation of potential cell-mediated immunological events in the pathogenesis of the clinical features of APS. This study indicates that specific cell-mediated immunity to ß2GP1 can be demonstrated in at least 8 (44%) and possibly up to 11 (61%) of 18 patients with APS. The finding that such immunity is not observed in individuals with aPL Abs who do not have APS gives additional support to the specificity and potential pathogenic importance of these results.
The cellular immune response to ß2GP1 resides with
circulating CD4+ T cells with a type 1 cytokine profile, as
evidenced by the ß2GP1-induced selective expansion of
CD4+ but not CD8+ T cells (Fig. 4), the
production of IFN- but not IL-4 (Fig. 5), and the abrogation of the
response by the depletion of CD4+ T cells or APCs, but not
CD8+ T cells (Table III and Fig. 6). Both B cells and
monocytes appear to be important in the proliferative response, because
the depletion of either inhibited proliferation and cytokine
production. The ß2GP1 present in serum produced a more
potent proliferative response than purified ß2GP1 (Table II, Fig. 1), possibly because the uptake of ß2GP1 by APCs
including B cells bearing surface anti-ß2GP1 Abs may
be more efficient when the molecule is present in a serum environment
compared with a purified preparation.
T cell involvement in APS is suggested by previous studies
demonstrating the transfer of experimental APS to naive mice with
intact but not T cell-depleted bone marrow cells (17). A cellular
immune response to various well-characterized autoantigens in both
human and animal studies of insulin-dependent diabetes mellitus and SLE
implicate a role for CD4+ T cells in mediating disease
activity (24, 25, 26, 27, 28, 29). There is some evidence that a dichotomy may exist
between cellular and humoral responses to autoantigens in patients with
diabetes mellitus, with a reciprocal relationship observed between
autoantibody levels and cellular immunity (30). However, there was
neither a positive nor reciprocal relationship found between the
IgG-anti-ß2GP1 levels and cellular proliferative
responses to ß2GP1 observed in this study (Table II).
Cellular immunity to ß2GP1 was observed only in patients
with histories of thromboses or fetal loss, indicating that it may be a
more specific, although less sensitive marker for the clinical syndrome
than are aPL or anti-ß2GP1 Abs. Circulating
ß2GP1-specific CD4+ T cells that secrete
IFN- but not IL-4 are likely to be chronically stimulated by
continued exposure to ß2GP1 in serum, and perhaps on cell
surfaces (31, 32). The production of IFN- by these T cells may be a
factor involved in the high occurrence of recurrent miscarriages in APS
patients. Normal pregnancy has been characterized by a dominant humoral
immune response and a concomitantly reduced maternal cell-mediated
anti-fetal immune response. Thus, it has long been considered a
type 2 phenomenon immunologically, supported by the documented
production of IL-4, IL-5, and IL-10 in mouse fetoplacental tissues
(33). Type 1 cytokines such as IFN- have deleterious effects on
embryonic and fetal development and inhibit the proliferation of human
trophoblast cell lines in vitro (34, 35, 36, 37). Our observations indicate
that a switch from a Th2 to a Th1 response could be associated with the
occurrence of unsuccessful pregnancies in patients with APS.
Similarly, a procoagulant diathesis in patients with aPL Abs may be due
to the up-regulation of monocyte procoagulant activity due to products
of activated CD4+ cells (38), including IFN- (39, 40).
There is evidence that monocyte tissue factor (TF) induction occurs in
APS (41, 42, 43, 44); plasma from APS patients up-regulates surface TF
expression on normal human monocytes (45). Moreover, monocytes from
patients with primary APS have increased TF Ag and TF-related
procoagulant activity that correlates with thrombotic episodes (46). It
is plausible that the stimulation of PBMCs with ß2GP1 has
a similar effect on TF expression, because the cytokines produced by
Th1 and Th2 clones have been reported to promote or inhibit monocyte
procoagulant activity, respectively (40). In that study,
anti-IFN- neutralizing Ab inhibited TF production, whereas
neutralizing Abs to other Th1 cytokines failed to do so. Furthermore,
IL-4 and IL-10 inhibited Th1 lymphocyte-induced monocyte TF induction.
These results support the interactive roles of Th1 and Th2 cytokines in
the regulation of T cell-induced monocyte TF, and implicate the Th1
cytokines produced by ß2GP1-specific T cells in TF
up-regulation, thereby providing a mechanism for the thrombotic
episodes typically seen in some APS patients.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. H. Patrick McNeil, Inflammation Research Unit, School of Pathology, University of New South Wales, Sydney, NSW 2052, Australia. E-mail address:
3 Abbreviations used in this paper: aPL, antiphospholipid; APS, aPL syndrome; aCL, anticardiolipin; LA, lupus anticoagulant; ß2GP1, ß2-glycoprotein-1; SLE, systemic lupus erythematosus; SI, stimulation index; TT, tetanus toxoid; NS, normal serum; TF, tissue factor; PAPS, primary APS.
Received for publication December 28, 1998. Accepted for publication March 17, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is cytotoxic to normal, untransformed mouse and rat embryo fibroblast-like cells. Anticancer Res. 9:167.[Medline]
and -interferon against primary human placental trophoblasts. Placenta 15:819.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 1% NS (
), and
ß2GP1 plus 1% NS (
). There was a significant
difference between responders and nonresponders in response to
ß2GP1 (p = 0.002), NS alone
(p = 0.045), and ß2GP1 plus NS
(p = 0.017). Significant differences were also
found between responders and controls in response to
ß2GP1 (p = 0.002), NS alone
(p = 0.036), and ß2GP1 plus NS
(p = 0.010). PBMCs were tested two or more times.
Data represent the mean and SEM for each group.
), 1% sham-depleted serum
(), or ß2GP1 (25 µg/ml) plus 1% DS
(ß2GP1+DS) (
). Data represent the mean and SEM of four
experiments.
