genetic) factors necessary to break tolerance against
carbamylated proteins such a fibrinogen remains to be eluci-
dated. Although the molecular structures of homocitrulline
and citrulline are very alike, ACPA and anti-CarP are distinct
autoantibody classes, with anti-CarP being present in both
ACPA-positive and ACPA-negative patients [
2
]. Moreover,
anti-CarP is associated with radiographic progression in pa-
tients negative for RF and ACPA. Diagnostic classification of
RA patients did not improve by adding anti-CarP testing, as
RF and ACPA are already good predictors for disease [
40
].
Assays to test for the presence of anti-CarP most often use
fetal calf serum (FCS), containing a mixture of carbamylated
proteins. The exact autoantigens that anti-CarP bind in vivo
remains unclear.
Anti-acetylated protein antibodies
The latest addition to AMPAs in RA patients is anti-acetylated
protein antibodies which have been described in approximate-
ly 40% of RA patients, mainly in the ACPA-positive group.
Detection rates in seronegative RA patients were comparable
to patients with resolving arthritis, rendering it unlikely that
these antibodies will be a new biomarker helpful for diagnos-
ing RA [
3
]. However, anti-acetylated protein antibodies might
provide useful new insights in pathophysiology, especially in
the era in which the microbiome seems to become increasing-
ly important. Acetylation is an enzymatic process, which can
be affected by bacteria, although the underlying mechanism is
unclear. Therefore, anti-acetylated antibodies could provide a
possible new link between microbiome dysbiosis and the de-
velopment of autoimmunity in RA [
3
,
41
].
Development of the autoantibody response over time
The presence of the different autoantibodies in serum of
(future) RA patients can be detected years before actual dis-
ease onset [
42
–
44
]. Most research on the details of the auto-
antibodies response prior to clinical disease manifestation is
done on ACPA. From all RA patients, 50% presents with
ACPA. About 4 years before disease onset, 50% of the pa-
tients that will be ACPA positive will harbor ACPA. At that
time point, the ACPA levels start to increase. The profile of
citrullinated antigens recognized expands extensively and
isotype switching occurs. These events predict RA develop-
ment in patients with undifferentiated arthritis and correlate
with a rise in inflammatory cytokine levels [
45
–
49
]. Also,
the Fc glycosylation pattern changes before disease onset.
Galactosylation decreases while fucosylation increases, lead-
ing towards a more pro-inflammatory phenotype of the anti-
bodies [
50
].
There is one feature of ACPA, which strikingly differs from
the normal development of antibody responses. In general,
during B cell maturation, class switching, somatic
hypermutation, and affinity maturation are physiological pro-
cesses occurring in germinal centers. B cells producing anti-
bodies with sufficient avidity will proliferate, improving the
440
Semin Immunopathol (2017) 39:437
–
446
efficacy of the immune response. In contrast to an immune
