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Identification of citrullinated rheumatoid arthritis-specific epitopes in natural filaggrin relevant for antifilaggrin autoantibody detection by line immunoassay.

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ARTHRITIS & RHEUMATISM
Vol. 46, No. 5, May 2002, pp 1185–1195
DOI 10.1002/art.10229
© 2002, American College of Rheumatology
Identification of Citrullinated Rheumatoid Arthritis–Specific
Epitopes in Natural Filaggrin Relevant for
Antifilaggrin Autoantibody Detection by Line Immunoassay
Ann Union,1 Lydie Meheus,1 René Louis Humbel,2 Karsten Conrad,3 Guenter Steiner,4
Henri Moereels,1 Hans Pottel,1 Guy Serre,5 and Filip De Keyser6
Objective. To identify immunodominant epitopes
in natural filaggrin that are reactive with antifilaggrin
autoantibodies (AFA) in the sera of patients with rheumatoid arthritis (RA) and to explore their use in a
diagnostic assay format.
Methods. Based on the results of epitope mapping
of human natural filaggrin as well as molecular modeling and computational chemistry, synthetic peptides
together with recombinant citrullinated filaggrin were
evaluated by a line immunoassay (LIA) for AFA detection. Diagnostic performance was assessed using 336
RA and 253 disease control sera and was compared with
that of reference methods.
Results. Several immunoreactive epitopes were
identified in natural filaggrin, all of which contained at
least 1 citrulline residue. Three antigenic substrates,
including 2 synthetic peptides and recombinant citrullinated filaggrin showing maximal reactivity on LIA,
were finally selected. Using the 3-antigen LIA3, overall
sensitivity, specificity, and positive predictive value for
RA were 65.2%, 98.0%, and 89.1%, respectively, compared with 61.9%, 98.8%, and 92.8% using the 2-antigen
LIA2 (without recombinant protein). Thirty-seven per-
cent of the rheumatoid factor (RF)–negative RA samples (30 of 81) were AFA-positive by LIA2, and 52 of 54
RF-positive control samples had no AFA detected on
LIA2. Higher specificity and sensitivity were obtained
by LIA2 versus anti-RA33 immunoblot, whereas good
agreement was observed with antikeratin antibody
testing. LIA performed significantly better than AFA
immunoblotting using natural filaggrin, at a specificity
level of 99% (P ⴝ 0.0047).
Conclusion. Citrullinated residues are present in
immunoreactive epitopes of natural human filaggrin.
AFA can be readily detected by citrullinated peptides in
an LIA-based test, resulting in high specificity and
positive predictive value for RA. The LIA could serve as
a user-friendly alternative to existing immunofluorescence tests and AFA immunoblot techniques. Given its
complementarity to RF, this test can be a valuable tool
in the differential diagnosis of arthritis.
Rheumatoid arthritis (RA) is a major crippling
systemic joint disease of unknown etiology that affects
⬃1% of the population. It has many features of an
autoimmune disease, including the presence of a variety
of autoantibodies in patients’ sera and, in animal models, the capacity to induce illness by transfer of pathogenic T cells (1,2). Inflammation of the joints—the
hallmark of RA—is, however, not restricted to this
disease but also occurs in osteoarthritis (OA), reactive
arthritis, and gout. Because early diagnosis and prompt
treatment of RA markedly improve outcome, using
reliable diagnostic tools to rule out overlapping disorders is of major importance. However, clinical features
characteristic of established RA are often lacking in the
early stages of disease. Serologic support for the diagnosis, on the other hand, is limited and is based mainly
on the presence of rheumatoid factor (RF). The rather
Supported by grant 960267 from the Vlaams Instituut voor de
Bevordering van het Wetenschappelijk-Technologisch Onderzoek in
de Industrie.
1
Ann Union, PhD, Lydie Meheus, PhD, Henri Moereels,
Hans Pottel, PhD: Innogenetics NV, Ghent, Belgium; 2René Louis
Humbel: Centre Hospitalier de Luxembourg, Luxembourg; 3Karsten
Conrad, MD: Institute of Immunology, Technical University Dresden,
Dresden, Germany; 4Guenter Steiner, PhD: University of Vienna,
Vienna, Austria; 5Guy Serre, MD, PhD: University of Toulouse,
Toulouse, France; 6Filip De Keyser, MD, PhD: University Hospital
Ghent, Ghent, Belgium.
Address correspondence and reprint requests to Ann Union,
PhD, Immune Diseases Group, Innogenetics NV, Industriepark Zwijnaarde 7/4, 9052 Ghent, Belgium. E-mail: annuni@innogenetics.be.
Submitted for publication July 31, 2001; accepted in revised
form December 19, 2001.
1185
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UNION ET AL
Table 1. Overview of serum panels*
Serum
panel
No. of RA
samples
A
B
C
107
127
336
D
50
No. of disease controls
No. of
healthy controls
61 SLE, 62 OA
84 SLE/SpA/OA
90 SLE/MCTD, 53 SSc, 50 SpA,
30 OA, 15 Sjögren’s syndrome,
15 myositis
25 non-RA inflammatory joint pain
51
71
Origin
Application
Ghent
Ghent
Luxembourg, Dresden,
Vienna
Epitope mapping of natural filaggrin
Optimization of INNO-LIA RA
Validation of INNO-LIA RA
Ghent
Comparison of AFA blot with LIA
* RA ⫽ rheumatoid arthritis; SLE ⫽ systemic lupus erythematosus; OA ⫽ osteoarthritis; SpA ⫽ spondylarthropathy; INNO-LIA ⫽ Innogenetics
line immunoassay; MCTD ⫽ mixed connective tissue disease; SSc ⫽ systemic scleroderma; AFA ⫽ antifilaggrin autoantibodies.
low specificity of RF and the substantial number of
RF-negative patients in whom chronic destructive disease subsequently develops (3–5) point to a clear need
for RA-specific markers.
During recent years, efforts to identify novel
RA-specific autoantigens have led to the characterization of a group of antifilaggrin autoantibodies (AFA)
that recognize human epidermal filaggrin, rat esophagus
epithelial deiminated proteins, as well as buccal mucosa
cell–derived profilaggrin (6–10). The high specificity of
these antibodies for RA makes them valuable diagnostic
markers. In this respect, the usefulness of natural filaggrin in Western blotting has been demonstrated (9), and
its use in an enzyme-linked immunosorbent assay
(ELISA) also yielded positive results (11).
Because isolation of antigen from natural sources
is inconvenient, alternative antigen forms are required.
Substitution with the modified arginine residue citrulline
(Cit) has recently been shown to be an indispensable
peptide modification and one that is necessary for
proper immune recognition of filaggrin by the autoantibodies present in human RA sera (10). In vitro deimination of recombinant human filaggrin by the peptidylarginine deiminase (PAD) enzyme generates a protein
that is reactive with human RA sera during immunoblotting, while the noncitrullinated protein remains unreactive. Furthermore, 2 synthetic peptides sharing a central
tripeptide (Ser-Cit-His) showed reactivity to a small
series of RA sera in an ELISA (10). Several other
citrullinated peptides have also been evaluated by
ELISA and showed different patterns of reactivity with
RA sera (12,13). In order to compare these non-native
antigenic substrates with naturally occurring forms, we
investigated the presence of citrulline-containing
epitopes in natural filaggrin and evaluated the diagnostic
usefulness of naturally derived and synthetic citrullinated peptides in the line immunoassay (LIA) system.
MATERIALS AND METHODS
Serum samples. Different panels of human sera were
used throughout this study (Table 1) and were collected in
accordance with local ethics legislation. The epitope mapping
studies used 107 serum samples from patients who fulfilled the
American College of Rheumatology (ACR; formerly, the
American Rheumatism Association) criteria for RA (14) and
174 control sera, derived from 51 healthy persons, 61 with
systemic lupus erythematosus (SLE), and 62 with OA.
Optimization of the INNO-LIA RA (Innogenetics,
Ghent, Belgium) was used to test 127 sera from patients with
established RA and 134 sera derived from 84 disease controls
(patients with SLE, spondylarthropathy [SpA], or OA) and 71
healthy controls. In addition, 26 samples from patients with
Crohn’s disease and 24 from patients with gout were tested.
At a later stage, validation of the INNO-LIA RA was
performed using 336 sera from patients with RA fulfilling the
ACR criteria and 253 sera from disease controls (90 from
patients with SLE or mixed connective tissue disease, 53 from
scleroderma patients, 50 from SpA patients, 30 from OA
patients, 15 from patients with Sjögren’s syndrome, and 15
from patients with myositis), all of which were obtained from
the Centre Hopitalier de Luxembourg, the Division of Rheumatology in Vienna, Austria, and the University Hospital Carl
Gustav Carus in Dresden, Germany.
In addition, a group of 75 sera were tested for AFA in
a blinded manner, using the standard immunoblotting reference method, at the Hopital Purpan, Toulouse, France. These
sera included 25 samples from patients with RA fulfilling the
ACR criteria in whom disease duration was ⬍1 year, 25
samples from RA patients with disease duration ⬎4 years, and
25 from patients who were examined for inflammatory joint
pain but in whom RA could be excluded.
Preparation of human filaggrin. Using the protocol
described by Simon et al (6), natural filaggrin was purified
from human skin that was obtained anonymously after abdominoplasty, in accordance with local ethics regulations. The
epidermis was separated from the dermis by incubating the
skin fragments at 56°C in phosphate buffered saline (PBS)
containing 5 mM EDTA. The material was stored dry at ⫺20°C
until used. The epidermis was cut into small pieces that were
homogenized in 40 mM Tris HCl (pH 7.4), 150 mM NaCl, 5
mM EDTA, 0.5% Nonidet P40, 0.1% sodium azide, and 0.1
CITRULLINATED PEPTIDES FOR DETECTION OF AFA ON LIA
mM phenylmethylsulfonyl fluoride (0.2 ml/cm2) and stirred
overnight at 4°C. The homogenate was centrifuged at 15,000g
for 15 minutes, and the extracted proteins in the supernatant
were precipitated overnight at ⫺20°C by adding 5 volumes of
absolute ethanol. After centrifugation for 15 minutes at
10,000g, the protein pellet was vacuum dried and subsequently
resuspended in water. The amount of protein was determined
by the Bradford protein assay as modified by Peterson (15),
using bovine serum albumin (BSA) standard curves.
For AFA detection, the crude filaggrin preparation
was subjected to 10% tricine–sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) using the
Bio-Rad (Philadelphia, PA) mini-gel apparatus and then electrophoresed and blotted under standard conditions (8). Blot
strips were probed with human sera overnight at a 1:100
dilution in PBS, 0.05% Tween 20, and 0.1% gelatin. As
secondary antibody, the anti-human IgG–alkaline phosphatase
conjugate (Sigma, St. Louis, MO) was added at a 1:1,000
dilution; visualization occurred upon addition of nitroblue
tetrazolium (NBT)/BCIP chromogenic substrate.
Two-dimensional (2-D) gel electrophoresis of filaggrin. The semipurified filaggrin material was separated by 2-D
gel electrophoresis using Immobiline DryStrips (pH 3–10;
Amersham Pharmacia Biotech, Uppsala, Sweden) in the first
dimension and 10% tricine–SDS-PAGE in the second dimension according to standard procedures. Preparative gels were
loaded with 300 ␮g of protein and stained with Coomassie
Brilliant Blue R-250 (Bio-Rad). The large, comma-shaped
filaggrin spot was identified by immunoreaction with the
antifilaggrin monoclonal antibody (Biomedical Technologies,
Stoughton, MA). The pI of this heterogeneous protein ranged
from 6.7 to 8.5, and molecular-weight forms of 35–68 kd were
detected, with the more acidic isoforms representing the
highest masses.
Electroelution of filaggrin. The large filaggrin spot was
cut out of the gel and separated into 3 parts: 1) acidic fraction
(pI 6.7–7.5), 2) neutral fraction (pI 7.5–8.1), and 3) basic
fraction (pI 8.1–8.5). Each fraction was electroeluted according to the method described by Hunkapiller et al (16), using 50
mM (NH4)HCO3, 0.1% SDS as elution buffer. Coomassie
stain and SDS were removed from the eluted proteins by
ion-pair extraction as described by Königsberg and Henderson
(17). Vacuum-dried protein pellets were redissolved in the
appropriate buffer for further analysis.
Peptide mapping. Purified electroeluted filaggrin fractions of ⬃100 ␮g were dissolved in 40 ␮l of 100 mM
(NH4)HCO3 (pH 8.0), 10% acetonitrile and digested with
trypsin (enzyme:substrate ratio 1:40). After overnight incubation at 37°C, the digest was stored at ⫺20°C until used.
Peptides were separated by reverse-phase high-performance
liquid chromatography (RP-HPLC) on a 2.1 ⫻ 250–mm C4
Vydac column (Hesperia, CA) using a 140B solvent delivery
system (Applied Biosystems, Foster City, CA) and eluted with
linear gradient (8–70%) of 70% acetonitrile in 0.1% trifluoroacetic acid (TFA). Detection using a 1,000S diode array
detector (Applied Biosystems) occurred at 214 nm, and peptides were manually recovered.
Dot-spot analysis and microsequencing. Ninety percent of each peak fraction was vacuum dried and subsequently
resuspended in a small volume of 10% acetonitrile, 50 mM
NaCO3 buffer (pH 9.5). The peptides were dot-spotted onto
1187
Immunodyne ABC membranes (Pall Europe, Portsmouth,
UK), which were probed with human sera in order to assign
the immunoreactive epitopes. First, membranes were blocked
with PBS, 0.5% caseine, and incubated overnight with sera
diluted 1:50 in PBS, 0.5% caseine, 0.1% Triton X-705 (Sigma),
10 mM MgCl2.6H2O. After washing with PBS, 0.05% Tween
20, anti-human IgG was added, and membranes were developed in 100 mM NaCl, 100 mM Tris HCl (pH 9.8), 50 mM
MgCl2.6H2O substrate buffer containing the chromogenic
substrate NBT/BCIP. Reaction was stopped by addition of
0.2N H2SO4. The remaining 10% of the immunoreactive
fractions was used for microsequencing. For this, fractions
were directly analyzed on a model 477A pulsed liquid sequencer equipped with an on-line 120 phenylthiohydantoin
analyzer (Applied Biosystems).
Cloning, expression, purification, and citrullination of
human filaggrin. Four candidate human filaggrin sequences
were cloned by polymerase chain reaction (PCR) using
genomic DNA isolated from human lymphocytes as template.
The PCR sense primer was chosen to overlap the filaggrin
linker sequence and was designed to introduce a functional
translation initiation codon upstream from the linker sequence
FLYQVST. The antisense primer was located just upstream
from the next filaggrin linker sequence introducing the translation stop codon TAG. The amplified filaggrin sequence
therefore consisted of the filaggrin linker followed by an
integral filaggrin repeat and 3 additional amino acids resulting
from the cloning strategy. The PCR-amplified fragments were
cloned in a pBLSK vector (Stratagene, Amsterdam, The
Netherlands) as an Eco RI–Xba I fragment, allowing sequencing of the amplified complementary DNA (cDNA). The cDNA
inserts were recloned as Eco RV–Ecl 136II fragments of 1,030
bp in the Escherichia coli expression vector pI GRHISA
(Innogenetics). The filaggrin proteins were expressed as recombinant filaggrin–His-6 fusion proteins in 3 different E coli
strains, resulting in high Coomassie-stainable expression levels,
and were further purified by metal-affinity chromatography
under denaturing conditions. Enrichment of the full-size 50-kd
protein was obtained by RP-HPLC. Enzymatic citrullination
was performed as described by Senshu et al (18), using PAD
enzyme (Sigma) at a 1:60–1:120 enzyme:substrate ratio, for 15
minutes to 6 hours at 37°C. The reaction was stopped by
addition of 0.1% TFA. The degree of citrullination was
verified by analyzing mobility shifts on SDS-PAGE or by
isoelectrofocusing and amino acid analysis.
Peptide synthesis, molecular modeling, and computational chemistry. Biotinylated peptides were synthesized according to standard 9-fluorenylmethoxycarbonyl chemistry as
described previously (19) and purified by RP-HPLC using a
linear gradient (8–70%) of 70% acetonitrile in 0.1% TFA.
Molecular modeling and computational chemistry were performed using Insight II (version 97.2; Accelrys, San Diego,
CA) and Discover software (Molecular Simulations, Orsay,
France).
LIA for antifilaggrin detection. Peptides and recombinant antigens were applied directly on a nylon membrane with
a plastic backing, and strips were developed, with minor
modifications, as previously described (20). In a final layout of
the INNO-LIA RA, recombinant citrullinated filaggrin and 2
citrulline-containing peptides were retained. During each test
run, a reference cutoff control strip was included, providing a
1188
UNION ET AL
Figure 1. Epitope mapping of human natural filaggrin. A, Two-dimensional gel electrophoretic protein pattern of human epidermal extracts
enriched for filaggrin. Separation was performed using Immobiline DryStrips (pH 3–10) and 10% Tricine–sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE). Preparative gels were loaded with 300 ␮g of protein and stained with Coomassie Brilliant
Blue R-250. The heterogeneous filaggrin protein (arrow) was divided into 3 fractions: acidic (ac), neutral (n), and basic filaggrin (ba). B–E,
Tryptic digests of acidic and neutral filaggrin were separated by reverse-phase high-performance liquid chromatography (B and C), and
peptides were dot-spotted on membranes (D and E) and probed with rheumatoid arthritis or control sera. ⴱ indicates positive controls (1 ␮g
natural filaggrin from 2 preparations). IEF ⫽ isoelectric focusing; AU ⫽ absorbance units; OD ⫽ optical density; nr ⫽ number.
cutoff intensity for each antigen line. Air-dried strips were read
visually or scanned using a Scanjet 5P scanner (HewlettPackard, McMinnville, OR) and an in-house LIA interpretation software program. A serum sample was considered to be
positive when at least 1 of the 3 antigen lines scored positive.
The performance of the INNO-LIA RA in terms of
sensitivity and specificity was calculated using clinical diagnosis
as the gold standard. Receiver operating characteristic (ROC)
curves were determined for each antigen using an independent
sample population of 127 sera from RA patients, 84 from
disease controls, and 71 from healthy subjects, which enabled
us to define optimal cutoff values and to generate a corre-
sponding cutoff serum. Subsequently, validation of the INNOLIA RA was performed in a multicenter analysis, using 336
RA and 253 disease control samples, as indicated above.
Reference serum tests. For serum panel D, AFA were
identified by standard immunoblotting using human epidermal
extracts enriched for the neutral-acidic variant of filaggrin (8).
The intensity of color development was estimated according to
a semiquantitative scale. The cutoff values for this assay (95%
or 99% specificity) were based on a larger independent data
set consisting of 190 RA and 480 non-RA control sera and
were fixed at 0.25 and 2.25, respectively.
For the multicenter validation, each center used either
CITRULLINATED PEPTIDES FOR DETECTION OF AFA ON LIA
1189
Table 2. Identification of immunoreactive peptide fractions derived from epitope mapping of acidic and neutral human natural filaggrin*
Synthetic peptide
Immunoreactive peptide fraction†
Acidic filaggrin
R2HSASQDGQDTIRGHPG
R2HSGIGHGQASSAVR
R2DSGHRGYSGSQASDNEGH
R2HSTSQEGQDTIHGHRGS
R2QGSRHQQAR
R2AGHGHSADSSR
R2HGSHHQQSADSSR
Neutral filaggrin
R2HSGIGHGQASSAVR
R2HSASQDGQDTIRGHPG
R2HSTSQEGQDTIHGHRGS
R2DSGHRGYSGSQASDNEGH
R2GYSGSQASDNEGHSE
R2NDEQSGDGSR
Citrulline-containing
IGP1155:
IGP1156:
IGP1157:
IGP1158:
IGP1249:
HSASQDGQDTIRGHPGSS
HSGIGHGQASSAVRDSGHRGYS
DSGHRGYSGSQASDNEGH
HSTSQEGQDTIHGHRGS
GGQGSRHQQAR
Nonmodified
IGP1179:
IGP1180:
IGP1181:
IGP1182:
HSASQDGQDTIRGHPGSS
HSGIGHGQASSAVRDSGHRGYS
DSGHRGYSGSQASDNEGH
HSTSQEGQDTIHGHRGS
IGP1250: GGAGHGHSADSSR
IGP1251: GGHGSHHQQSADSSR
IGP1156
IGP1155
IGP1158
IGP1157
IGP1252: NDEQSGDGSR
* R ⫽ citrulline; R2 indicates tryptic cleavage site.
† Amino acid sequences were retrieved by microsequencing on a 477A sequencer equipped with an online 120 phenylthiohydantoin analyzer.
nephelometric methods, an Imtec ELISA (Zepernick, Germany), or an in-house ELISA prepared at the laboratory of R. L.
Humbel, Luxembourg) for RF detection. The presence of
anti-RA33 antibodies was determined by immunoblotting (20)
or ELISA (Imtec), and testing for antikeratin antibody (AKA)
was carried out by indirect immunofluorescence on rat esophagus sections (9). Evaluation for antiperinuclear factor (APF)
was performed using human buccal mucosa cells as previously
described (9).
Statistical methods. Results of the comparisons of the
INNO-LIA RA with AFA immunoblotting and with testing for
anti-RA33 or AKA were validated using McNemar’s test
statistics and Cohen’s kappa. Discriminant and principal component analyses were performed to determine the individual
peptide contribution to maximal variation and sensitivity.
RESULTS
Epitope mapping of human natural filaggrin.
Human filaggrin isolated from epidermal sheets was
electroeluted from preparative 2-D gels (Figure 1A).
Because the protein consisted of a heterogeneous group
of polypeptides, 3 different fractions were isolated according to their pI. Tryptic digests of the acidic, neutral,
and basic filaggrin were separated by HPLC, and peptide fractions were subsequently analyzed in a dot-spot
assay system (Figures 1B–E). Because of the low amount
of material in the basic fraction, no further analysis was
performed. Four APF-positive RA sera that were also
reactive with human filaggrin on Western blot and 1
APF-negative control serum sample were used to determine immunoreactivity.
For the acidic filaggrin, peptide fractions T32–41
showed a clear positive reaction (Figure 1D), in contrast
to the APF-negative serum. A similar procedure was
performed for neutral filaggrin, resulting in reactive
fractions T28, T65, T81, and the series T29–39 (Figure
1E). Amino acid sequencing revealed that most of these
fractions contained ⬎1 filaggrin-derived peptide, as
could be expected based on the very heterogeneous
nature of the protein (Table 2). During sequencing,
citrulline was found in a number of peptides, as shown
by 1) its specific retention time, which was different from
that of arginine, 2) the lack of cleavage (which is to be
expected if arginine were present), and 3) the absence of
arginine residues during sequencing. Use of a citrulline
standard confirmed these findings.
Use of synthetic peptides for AFA detection on
LIA. Based on the epitope mapping results, 12 peptides
(IGP1155–1158, IGP1179–1182, and IGP1249–1252)
were synthesized and evaluated in the LIA system
(Table 2). For 4 citrullinated peptides, the nonmodified
counterparts containing arginine instead of citrulline
residues were synthesized in order to compare their
respective reactivities. Using serum panel A, reactivity
was noted with citrullinated peptides IGP1155, 1156,
1157, 1158, and 1249, with a combined sensitivity of 48%
(Table 3). Upon taking into account only those sera that
reacted with human natural filaggrin on Western blot,
markedly higher sensitivities were reached for each
synthetic peptide compared with the filaggrin-negative
RA sera. Consequently, the combined sensitivity of
77.0% (95% CI confidence interval [95% CI] 63–87%)
1190
UNION ET AL
Table 3. Immunoreactivity of 5 synthetic citrullinated peptides identified by epitope mapping of human natural filaggrin and of recombinant
citrullinated filaggrin (Rec fg) on line immunoassay*
Serum group
No. of sera
IGP1155
IGP1156
IGP1157
IGP1158
IGP1249
Combination
of peptides
Rec fg
Peptides ⫹ Rec fg
RA
Filaggrin blot positive
Filaggrin blot negative
Control
Healthy
SLE
Osteoarthritis
107
52
55
22 (20.6)
17 (32.7)
5 (9.1)
31 (29.0)
24 (46.2)
7 (12.7)
4 (3.7)
3 (5.8)
1 (1.8)
26 (24.3)
21 (40.4)
5 (9.1)
16 (15.0)
13 (25.0)
3 (5.5)
51 (47.7)
40 (77.0)
11 (20.0)
52 (48.6)
37 (71.2)
15 (27.2)
64 (59.8)
45 (86.5)
19 (34.5)
51
61
62
1 (2.0)
1 (1.6)
1 (1.6)
3 (5.9)
–
1 (1.6)
1 (2.0)
1 (1.6)
1 (1.6)
1 (2.0)
–
–
1 (2.0)
2 (3.3)
1 (1.6)
3 (5.9)
4 (6.6)
2 (3.2)
1 (2.0)
7 (11.5)
3 (4.8)
4 (7.8)
9 (14.8)
5 (8.0)
* Combined reactivity was defined by positivity toward at least 1 antigen. Values are the number (%) of reactive sera. RA ⫽ rheumatoid arthritis;
Filaggrin blot ⫽ Western blotting using human natural filaggrin as antigenic substrate; SLE ⫽ systemic lupus erythematosus.
in the filaggrin-positive group was higher than that of
20.0% (95% CI 10–33%) in the filaggrin-negative group.
Control sera showed only minor reactivities with the
peptides, resulting in a specificity of ⬎96%. In all
groups, IGP1156 and 1158 reacted most strongly; immunoreaction toward IGP1157 was always very faint and
did not increase diagnostic sensitivity. Interestingly, the
positive LIA signals observed with these citrullinecontaining peptides were not seen with the nonmodified
counterparts IGP1179, 1180, 1181, or 1182. The group of
peptides (derived from the epitope mapping) that did
not contain citrulline (IGP1250, 1251, and 1252) showed
no substantial immunoreactivity.
Diagnostic value of citrullinated recombinant
filaggrin. Two of 4 available human filaggrin clones that
differed in their amino acid sequence by 5% were
expressed in E coli. Upon SDS gel analysis, in addition to
the full-size protein, a 25-kd filaggrin band was present
(Figure 2). Kinetic experiments were performed for the
enzymatic deimination of filaggrin using PAD at a
1:60–1:120 enzyme:substrate ratio. A clear mobility shift
on SDS gel was observed with respect to time of
deimination: from 15 minutes onward, the protein had
already migrated to a higher molecular-weight position,
reaching a plateau after 2 hours of citrullination (Figure
2). The loss of positive charges, resulting from the
modification of arginines to citrullines, was demonstrated by isoelectric focusing electrophoresis, which
confirmed the change in pI associated with longer
incubation times (results not shown). Amino acid analysis also showed an increased degree of citrullination
over time: within 15 minutes, half the arginines were
converted into citrullines, reaching near completion
within 6 hours (Table 4).
Immunoreactivity of recombinant proteins both
with and without citrulline residues was further assessed
on LIA. Reactivity with RA sera was noted within 15
minutes of citrullination and thereafter, while the nonmodified protein was mainly unreactive. The diagnostic
value of the recombinant citrullinated filaggrin was
compared with the reactivity of the 5 synthetic filaggrin
peptides sprayed as individual lines, using the same
battery of sera (Table 3). About half of the RA sera
showed a positive reaction with the recombinant citrullinated filaggrin, which was similar to the combined
reactivity with the peptides. Because of the partial
complementary reactivity observed between recombinant protein and peptides, the sensitivity of the test for
RA could be increased to nearly 60%, with a corresponding specificity of 95%.
Figure 2. Citrullination of recombinant filaggrin. Escherichia coli–
derived filaggrin was enzymatically deiminated using peptidyl-arginine
deiminase at a 1:60 enzyme:substrate ratio, and the degree of citrullination at different time points was analyzed by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). In addition
to a major 50-kd full-size protein, a 25-kd breakdown protein was
observed. A clear mobility shift was visible with respect to time of
deimination, indicating the conversion of arginine residues to citrullines.
CITRULLINATED PEPTIDES FOR DETECTION OF AFA ON LIA
1191
Table 4. Amino acid composition of in vitro citrullinated recombinant human filaggrin*
L-citrulline ⫹ L-ornithine
Arginine
Time 0
Time 1
Time 2
Time 3
Time 4
Time 5
0.6 (0.3)
25.5 (12.9)
10.6 (6.3)
10.4 (6.1)
13.3 (8.7)
5.4 (3.5)
15.4 (10.7)
2.9 (2.0)
15.0 (11.5)
1.5 (1.1)
15.9 (11.3)
1.0 (0.7)
* Amino acid composition at different time points was determined by hydrolysis at 156°C in 6N HCl and analysis on a 477 amino acid analyzer.
Ornithine is a well-known degradation product of citrulline formed during hydrolysis, and is taken into account when calculating the degree of
citrullination. Time 1 ⫽ 15 minutes, Time 2 ⫽ 30 minutes, Time 3 ⫽ 1 hour, Time 4 ⫽ 2 hours, Time 5 ⫽ 6 hours. Values are the quantity in
nanograms (%).
Design of variant peptides by molecular modeling and computational chemistry. In order to further
improve LIA sensitivity and shorten serum incubation
times, novel peptides were designed so that citrulline
residues would be spatially positioned for maximal accessibility. Following this modeling approach, numerous
peptide conformations were considered, and peptides
were subsequently synthesized and analyzed for immunoreactivity on LIA to investigate the influence of
individual residue changes. Statistical processing of the
results revealed that a limited number of peptides
contributed to maximal variation and the highest combined sensitivity. ROC curve analysis was performed for
each peptide in order to determine sensitivities at each
specificity level.
In contrast to the signals from the peptides
derived from the epitope mapping studies, much stronger signals were obtained, and serum incubation time
was reduced from overnight to just 1 hour. Finally, 2
peptide variants (peptides A and B) mapping in the
filaggrin region of amino acid 51–100 (SWISS-PROT
accession number P20930), together with the recombinant citrullinated filaggrin, were selected for further
evaluation with serum panel B. A theoretical cutoff
value was defined by ROC curve analysis, corresponding
to a specificity of 99% for each antigen line (Figure 3).
Figure 3. Receiver operating characteristic curves. Serum panel B
(127 rheumatoid arthritis sera, 84 sera from disease controls, and 71
sera from healthy controls) was used to establish the curves.
On the basis of these values, a cutoff control serum was
prepared for use on a reference strip, thereby providing
a cutoff intensity for each antigen line (Figure 4). Using
this cutoff control, an overall specificity of 98.1% with a
corresponding sensitivity of 67% was obtained (serum
panel B). Evaluation of an additional 26 sera from
patients with Crohn’s disease and 24 from patients with
gout revealed no aspecific reactivity of these control
samples.
Assay validation. In a multicenter retrospective
clinical study, performance of the INNO-LIA RA was
assessed and compared with that of individual reference
methods, using serum panel C. As seen in Table 5, a
distinction was made between LIA3, containing the
recombinant filaggrin and 2 citrullinated peptides, and
LIA2, which took only the 2 peptides into consideration.
Sensitivity for RA using LIA3 was slightly higher than
that using LIA2 (65.2% and 61.9%, respectively), but
specificity was lower (98.0% and 98.8%, respectively).
The number of false positives with LIA2 was 3 of 253
and included 2 SSc samples and 1 Sjögren’s syndrome
Figure 4. Immunoreactivity of recombinant filaggrin (Rec fg) and
synthetic citrullinated peptides (pepA, pepB) on line immunoassay.
Ten antifilaggrin autoantibody–positive rheumatoid arthritis (RA)
sera were compared with a control (cut-off) strip that provided cutoff
intensities for each corresponding antigen line.
1192
UNION ET AL
Table 5. Performance of the INNO-LIA RA in a multicenter, retrospective study*
Center
LIA3
LIA2
No. of RA
samples
No. of control
samples
Sensitivity, %
Specificity, %
Sensitivity, %
Specificity, %
125
112
99
336
25
60
168
253
80.0
53.5
59.6
65.2
100
98.4
97.6
98.0
78.4
49.1
55.6
61.9
100
98.4
98.8
98.8
1
2
3
Total
* Strips were read against a cutoff control serum applied on a separate strip. LIA3 ⫽ recombinant citrullinated filaggrin ⫹ peptide A ⫹ peptide B;
LIA2 ⫽ peptide A ⫹ peptide B (see Table 1 for other definitions).
sample. Two of these sera were tested for RF and were
also found to be positive. Because the prevalence of RA
patients in normal rheumatologic practice is ⬃20%, and
assuming that the disease controls are representative,
the positive predictive values of LIA3 and LIA2 for RA
were 89.1% and 92.8%, respectively. Therefore, only
LIA2 was used for further comparison with other serologic tests.
Because of the sample bias with respect to RF
(use of RF-positive controls and the fact that presence of
RF is one of the ACR criteria for RA), we could not
statistically compare results of RF determination and
LIA, but complementarity between these methods could
be investigated (Table 6). A total of 30 (37%) of 81
RF-negative RA samples proved to be AFA-positive on
LIA2. Furthermore, all RF-negative control samples
were also AFA-negative, while 52 of 54 RF-positive
control sera had no AFA detected on LIA2. In addition,
LIA showed a higher sensitivity and specificity compared with the anti-RA33 assay (P ⬍ 0.0001), whereas
good agreement was observed with AKA testing (kappa
value 0.725; 95% CI 0.633–0.817).
Comparison between LIA and AFA immunoblot.
In order to compare the results of LIA with detection of
AFA by the standard immunoblotting procedure using
natural filaggrin, a limited serum panel (panel D) that
was blinded to observers was analyzed on blot and
scored semiquantitatively. Cutoff values for the LIA
were adapted to obtain an overall specificity of either
95% or 99%, equal to the blot specificities. As shown in
Table 7, LIA performed similarly (46% sensitivity) at
both preset specificities (95% and 99%). The AFA blot
resulted in higher sensitivity (56% at the 95% specificity
level) but detected only 30% of the RA sera at the 99%
specificity level. The 3 false-positive AFA blot control
sera that were also APF-positive were diagnosed as OA,
scapulohumeral periarthritis, and lumbar arthrosis.
Comparison between the total number of correct or
incorrect results showed that the 2 tests performed
similarly at the 95% specificity level, while LIA proved
statistically better than the AFA blot at the 99% level
(P ⫽ 0.0047 by McNemar’s test). Among the 8 samples
that were correctly identified by LIA only, 4 were from
patients with early RA, and 4 were from patients with
longstanding RA. Furthermore, 2 of 17 RF-negative RA
samples were detected on LIA but not on Western blot.
When this sample collection was evaluated using the
prototype LIA2, LIA performance at the 99% specificity
level was significantly better than that of the AFA blot
(P ⫽ 0.007; Table 7).
DISCUSSION
In recent years, the presence of AFA in the sera
of RA patients has been well-documented. However, the
precise epitopes in filaggrin that are responsible for the
Table 6. Comparison between antifilaggrin detection on LIA2 and reference methods for detection of RF, anti-RA33, and AKA*
RF
Anti-RA33
Reference test/LIA2
RA
(n ⫽ 334)
Disease control
(n ⫽ 188)
RA
(n ⫽ 211)
Disease control
(n ⫽ 154)
AKA: RA
(n ⫽ 232)
–/–
–/⫹
⫹/–
⫹/⫹
51
30
76
177
134
0
52
2
77
83
24
27
138
2
14
0
72
20
10
130
* Disease controls were from serum panel C (see Table 1). Values are the number of positive (⫹) or negative (⫺) sera. LIA2 ⫽ line immunoassay
with peptide A ⫹ peptide B; RF ⫽ rheumatoid factor; RA ⫽ rheumatoid arthritis; AKA ⫽ antikeratin antibodies.
CITRULLINATED PEPTIDES FOR DETECTION OF AFA ON LIA
1193
Table 7. Sensitivity and specificity comparison of AFA detection by AFA blot using natural filaggrin versus LIA*
Preset specificity 95%†
RA samples (n ⫽ 50)
Disease controls (n ⫽ 25)
Preset specificity 99%‡
Cutoff control
INNO-LIA RA§
AFA blot
INNO-LIA RA
AFA blot
INNO-LIA RA
LIA3
LIA2
28 (56)
3
23 (46)
1
15 (30)
1
23 (46)
1
24 (48)
2
24 (48)
1
* AFA ⫽ antifilaggrin autoantibodies; INNO-LIA ⫽ Innogenetics line immunoassay; RA ⫽ rheumatoid arthritis; LIA3 ⫽ recombinant citrullinated
filaggrin ⫹ peptide A ⫹ peptide B; LIA2 ⫽ peptide A ⫹ peptide B. Values are the number (%) of reactive sera.
† P ⫽ 0.37 by McNemar’s test.
‡ P ⫽ 0.0047 by McNemar’s test.
§ P ⫽ 0.007 by McNemar’s test.
observed immunoreaction have not been determined
until now, and their identification might help to improve
diagnostic performance of assays relying on non-native
antigenic substrates.
The epitope mapping described in the present
study indicates that the immunoreactive epitopes in
natural filaggrin contain citrulline residues. Although
the presence of citrulline in natural human filaggrin has
been described previously (22,23), our results confirm
the specific recognition by AFA of a citrullinated linear
epitope in the natural antigen. These results are in
accordance with previous findings that the presence of
citrulline is necessary in order to obtain proper immunoreactivity of patient sera toward recombinant filaggrin
or synthetic peptides (10,12). Based on the recognition
of this essential secondary modification, it is now apparent why previous attempts to produce reactive recombinant antigen intended for AFA detection were not
successful (Union A, et al: unpublished observations).
Upon citrullination of recombinant E coli filaggrin by
the enzyme PAD, however, a reactive antigen was
generated (10). Other studies using citrullinated peptides have also proven useful for the development of a
diagnostic ELISA (12,13).
The peptides selected in this study partially overlapped those described by Schellekens et al (12) but did
not contain the cfc1 sequence (13). This is most probably
attributable to our use of trypsin for enzymatic cleavage
of the protein, which completely destroys the RGRSRGRSGRSGS cfc1 sequence, hence generating fragments
that are too small to be detected in the epitope mapping
dot-spot analysis. In addition to our previous findings
indicating that the citrulline residue can be situated in
the particular protein context Ser-Cit-His (10), the current results also show that several other environmental
amino acid residues are found in the natural protein
(Table 2). Synthetic peptides from the earlier study,
however, showed only minor reactivity in the LIA system
(10). According to our calculations of molecular modeling and computational chemistry, a His residue appears
to form a hydrogen bond with the adjacent citrulline, by
which the accessibility of the citrulline is reduced. In
general, an inverse correlation was found between the
availability of citrulline for AFA and the size of the
neighboring amino acids.
In order to design novel peptides with higher
immunoreactivity than those derived from the epitope
mapping, the directed approach of molecular modeling
was used instead of random synthesis or combinatorial
chemistry. Evaluation by LIA revealed 2 selected peptides that were highly useful for AFA detection, with
62% sensitivity in established RA samples at a specificity
of 98.4%. The additional use of recombinant deiminated
filaggrin slightly increased sensitivity of the test but at
the expense of specificity. We observed a partial complementarity with RF determination, because on LIA, 37%
of the RF-negative RA samples were positive for the
peptides. This percentage can vary according to the
particular study and detection method but seems largely
dependent on the study population: 30% or 42% in
patients with established RA who fulfill the ACR criteria
(9,13) compared with 14–18% in early arthritis cohorts
(11,24,25). This difference may be attributable to the
fact that RF levels fluctuate during the course of the
disease, while AFA titers are more stable over time
(26,27). Nevertheless, both autoantibodies are markers
of the underlying immunopathologic process in RA,
because specific plasma cells can be identified from
rheumatoid synovium (28,29).
There is a pressing need for new serologic RA
tests that provide diagnostic information that is more
specific than that provided by the currently available RF
determination. The LIA results of the present study
(98.8% specificity) as well as those obtained with the
anti–citrullinated cyclic peptide ELISA (98% specificity)
(13) meet this challenge. Furthermore, it has been
1194
shown that appearance of AFA occurs early in the
course of RA and may precede telltale clinical signs
(30–32), while there is also evidence that RF-negative
RA patients who are AFA-positive have a severe disease
course and poor prognosis (26,33). Recently, it was
demonstrated that positivity for both RF and AFA is
associated with an even worse prognosis (24). In view of
current, expensive biologic treatments, detection of both
AFA and RF could help to define which patients are
eligible for such treatment.
In this study, it was noteworthy that RG sequences in filaggrin were the preferential citrullination
positions and were also observed to be the favorite
dimethylation site in the SmD protein acting as a specific
autoantigen in SLE (19). The glycine residue, being the
smallest, probably favors the amount of available space
around the arginine, which may facilitate arginine modification. Furthermore, citrullinated myelin basic protein
also occurs in patients with multiple sclerosis (34).
Apparently, a number of posttranslational modification
phenomena play a key role in the breakdown of immune
tolerance and triggering of autoimmune reactivities. In
addition to citrullinated and dimethylated autoantigens,
epitopes generated by caspase cleavage and phosphorylation are all targeted by autoantibodies (35; for review
see ref. 36). The biologic significance of deimination and
its involvement in autoimmune responses remain to be
elucidated.
Intracellular and highly specific anticitrulline
staining was recently observed in synovial immunohistochemistry studies (37). Filaggrin itself was not detectable
(37,38), but biochemical characterization of deiminated
synovial tissue proteins led to the identification of ␣ and
␤ chains of fibrin, which could correspond to the genuine target of AFA (38). A defect in the apoptotic process
may be responsible for the uncontrollable proliferation
of synovial fibroblasts. Although extensive DNA fragmentation has been observed in rheumatoid synovium,
only a few cells actually complete the apoptotic process
(39). Because citrullination of vimentin has been linked
to apoptosis (40), such deiminated proteins could be
released at the inflammatory site, hence creating additional neoepitopes for both B cell and T cell immune
recognition.
In conclusion, our results indicate that citrulline
is present in the immunoreactive epitopes of natural
filaggrin, and that AFA can be readily detected on a
membrane solid phase using citrullinated peptides. Due
to its partial complementarity to RF and its high prognostic value, this LIA provides a valuable tool for the
diagnosis of RA and may be of use in the choice of
UNION ET AL
particular therapies. The specificity of AFA undoubtedly makes them intruiging candidates for further study.
ACKNOWLEDGMENTS
We are very thankful to An Vos, Liesbet Amerijckx,
Martine Dauwe, and Patrick Schmit for their skillful technical
assistance; to Stéphanie Dincq, Katrien De Bosschere, and
Frank Hulstaert (Medical Affairs), as well as to the Department of Protein Expression, Purification/Peptide Synthesis of
Innogenetics for valuable contributions; and to Fred Shapiro
for editorial support. We also wish to thank Dr. P. Verlende
(Plastische Heelkunde, Volkskliniek, Ghent) for providing
human skin from abdominoplasty, Prof. Dr. G. Verbruggen
and Dr. I. Peene (Rheumatology, UZ Ghent) for collection of
osteoarthritis samples, and Dr. K. Lüthke (Rheumatology,
Dresden) for serum sample collection and clinical data. We are
especially indebted to Jos Raymackers (Innogenetics) who so
unexpectedly and regrettably died during the course of this
study.
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