Recently, we showed the unexpectedly high abundance of N-linked glycans on the Fab-domain of Anti-Citrullinated Protein Antibodies (ACPA). As N-linked glycans can mediate a variety of biological ...functions, we now aimed at investigating the structural composition of the Fab-glycans of ACPA-IgG to better understand their mediated biological effects. ACPA-IgG and noncitrulline specific (control) IgG from plasma and/or synovial fluid of nine ACPA positive rheumatoid arthritis patients were affinity purified. The N-linked glycosylation of total, Fc and F(ab′)2 fragments, as well as heavy and light chains of ACPA-IgG and control IgG were analyzed by UHPLC and MALDI-TOF mass spectrometry. The Fc-glycosylation of ACPA-IgG and IgG was analyzed at the glycopeptide level using LC-MS. The structural analyses revealed that ACPA-IgG molecules contain highly sialylated glycans in their Fab-domain. Importantly, Fab-glycans were estimated to be present on over 90% of ACPA-IgG, which is five times higher than in control IgG isolated from the same patients. This feature was more prominent on ACPA isolated from synovial fluid compared with peripheral blood. These observations provide the first evidence pointing to the ability of ACPA-IgG to mediate novel immunological activities, for example through binding specific lectins via hyper-sialylated Fab-glycans.
In the mantle underneath the Tethyan suture zone, large volumes of positive velocity anomalies have been imaged by seismic tomography and interpreted as the present‐day signature of subducted Tethyan ...lithosphere. We investigate the Mesozoic‐Cenozoic subduction history of the region by integrating independent information from mantle tomography and tectonic reconstructions. Three different subduction scenarios for the Tethyan oceanic lithosphere, representative for the available tectonic reconstructions, are used to predict the present thermally anomalous volumes associated with the lithospheric surface subducted since the late Mesozoic. Next, these predicted thermal volumes and their expected positions are compared to the relevant anomalous volumes derived from seismic tomographic images. In this analysis we include, among others, the possible effects of ridge subduction and slab detachment after the Cenozoic continental collisions, absolute plate motion, and slab thickening in the mantle. Our preferred subduction model comprises the opening of large back‐arc oceanic basins within the Eurasian margin. The model points to slab thickening by a factor of 3 in the mantle, in which case the estimated volumes allow for active oceanic spreading (∼1–2.5 cm/yr) in the Tethyan lithosphere during convergence. Our results further indicate the occurrence of early Oligocene slab detachment underneath the northern Zagros suture zone, followed by both westward and eastward propagation of the slab tear and diachronous Eocene to Miocene slab detachment below the eastern to western Himalayas. Free sinking rates of the detached material of ∼2 cm/yr in the lower mantle provide the best fit between the tomographic mantle structure and our Tethyan subduction model.
Objective
Anti–citrullinated protein antibodies (ACPAs) are disease‐specific biomarkers in rheumatoid arthritis (RA). More than 90% of IgG ACPAs harbor N‐linked glycans in the antibody variable (V) ...domain. The corresponding N‐glycosylation sites in ACPA V‐region sequences result from somatic hypermutation, a T cell–dependent process. As ample evidence indicates that T cells drive the maturation of the ACPA response prior to arthritis onset, we undertook this study to investigate whether the presence of glycans in IgG ACPA V domains predicts the transition from predisease autoimmunity to overt RA.
Methods
We analyzed 2 independent sets of serum samples obtained from 126 ACPA‐positive first‐degree relatives (FDRs) of RA patients. Both sets originated from an Indigenous North American population and comprised cross‐sectional and longitudinal samples of individuals who did or did not develop inflammatory arthritis. Serum IgG ACPAs were affinity‐purified and subjected to ultra high‐performance liquid chromatography–based glycan analysis.
Results
In both data sets, FDR‐derived IgG ACPA displayed markedly lower levels of V domain glycans (<50%) compared to IgG ACPA from RA patients. Notably, FDRs who later developed RA showed extensive V‐domain glycosylation before the onset of arthritis. Moreover, IgG ACPA V‐domain glycosylation was strongly associated with future development of RA (hazard ratio 6.07 95% confidence interval 1.46–25.2; P = 0.013).
Conclusion
Extensive glycosylation of the IgG ACPA V domain is present in a subset of predisposed FDRs of Indigenous North American RA patients. The presence of this feature substantially increases the risk of RA development. Based on these findings, we propose that glycosylation of the IgG ACPA V domain represents a predictive marker for RA development in ACPA‐positive individuals and may serve to better target prevention measures.
Anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) patients display a unique feature defined by the abundant presence of
-linked glycans within the variable domains ...(V-domains). Recently, we showed that
-glycosylation sites, which are required for the incorporation of V-domain glycans, are introduced following somatic hypermutation. However, it is currently unclear when V-domain glycosylation occurs. Further, it is unknown which factors might trigger the generation of V-domain glycans and whether such glycans are relevant for the transition towards RA. Here, we determined the presence of ACPA-IgG V-domain glycans in paired samples of pre-symptomatic individuals and RA patients.
ACPA-IgG V-domain glycosylation was analysed using ultra-high performance liquid chromatography (UHPLC) in paired samples of pre-symptomatic individuals (median interquartile range (IQR) pre-dating time: 5.8 (5.9) years; n=201; 139 ACPA-positive and 62 ACPA-negative) and RA patients (n=99; 94 ACPA-positive and 5 ACPA-negative).
V-domain glycans on ACPA-IgG were already present up to 15 years before disease in pre-symptomatic individuals and their abundance increased closer to symptom onset. Noteworthy, human leucocyte antigen class II shared epitope (HLA-SE) alleles associated with the presence of V-domain glycans on ACPA-IgG.
Our observations indicate that somatic hypermutation of ACPA, which results in the incorporation of
-linked glycosylation sites and consequently V-domain glycans, occurs already years before symptom onset in individuals that will develop RA later in life. Moreover, our findings provide first evidence that HLA-SE alleles associate with ACPA-IgG V-domain glycosylation in the pre-disease phase and thereby further refine the connection between HLA-SE and the development of ACPA-positive RA.
A comparison exercise on thermal-optical elemental carbon/organic carbon (ECOC) analysers was carried out among 17 European laboratories. Contrary to previous comparison exercises, the 17 ...participants made use of an identical instrument set-up, after correcting for temperature offsets with the application of a recently developed temperature calibration kit (Sunset Laboratory Inc, OR, US). Temperature offsets reported by participants ranged from −93 to +100 °C per temperature step. Five filter samples and two sucrose solutions were analysed with both the EUSAAR2 and NIOSH870 thermal protocols. z scores were calculated for total carbon (TC); nine outliers and three stragglers were identified. Three outliers and eight stragglers were found for EC. Overall, the participants provided results between the warning levels with the exception of two laboratories that showed poor performance, the causes of which were identified and corrected through the course of the comparison exercise. The TC repeatability and reproducibility (expressed as relative standard deviations) were 11 and 15% for EUSAAR2 and 9.2 and 12% for NIOSH870; the standard deviations for EC were 15 and 20% for EUSAAR2 and 20 and 26% for NIOSH870. TC was in good agreement between the two protocols, TCNIOSH870 = 0.98 × TCEUSAAR2 (R2 = 1.00, robust means). Transmittance (TOT) calculated EC for NIOSH870 was found to be 20% lower than for EUSAAR2, ECNIOSH870 = 0.80 × ECEUSAAR2 (R2 = 0.96, robust means). The thermograms and laser signal values were compared and similar peak patterns were observed per sample and protocol for most participants. Notable deviations from the typical patterns indicated either the absence or inaccurate application of the temperature calibration procedure and/or pre-oxidation during the inert phase of the analysis. Low or zero pyrolytic organic carbon (POC), as reported by a few participants, is suggested as an indicator of an instrument-specific pre-oxidation. A sample-specific pre-oxidation effect was observed for filter G, for all participants and both thermal protocols, indicating the presence of oxygen donors on the suspended particulate matter. POC (TOT) levels were lower for NIOSH870 than for EUSAAR2, which is related to the heating profile differences of the two thermal protocols.
Autoantibodies against post-translationally modified proteins (anti-modified protein antibodies or AMPAs) are a hallmark of rheumatoid arthritis (RA). A variety of classes of AMPAs against different ...modifications on proteins, such as citrullination, carbamylation and acetylation, have now been described in RA. At present, there is no conceptual framework explaining the concurrent presence or mutual relationship of different AMPA responses in RA. Here, we aimed to gain understanding of the co-occurrence of AMPA by postulating that the AMPA response shares a common 'background' that can evolve into different classes of AMPAs.
Mice were immunised with modified antigens and analysed for AMPA responses. In addition, reactivity of AMPA purified from patients with RA towards differently modified antigens was determined.
Immunisation with carbamylated proteins induced AMPAs recognising carbamylated proteins and also acetylated proteins. Similarly, acetylated proteins generated (autoreactive) AMPAs against other modifications as well. Analysis of anti-citrullinated protein antibodies from patients with RA revealed that these also display reactivity to acetylated and carbamylated antigens. Similarly, anti-carbamylated protein antibodies showed cross-reactivity against all three post-translational modifications.
Different AMPA responses can emerge from exposure to only a single type of modified protein. These findings indicate that different AMPA responses can originate from a common B-cell response that diversifies into multiple distinct AMPA responses and explain the presence of multiple AMPAs in RA, one of the hallmarks of the disease.
The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation ...sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease.
Objective
The autoimmune response in rheumatoid arthritis (RA) is marked by the presence of anti–citrullinated protein antibodies (ACPAs). A notable feature of IgG ACPA is the abundant expression of ...N‐linked glycans in the variable domain. However, the presence of ACPA variable domain glycosylation (VDG) across disease stages, and its response to therapy, are poorly described. To understand its dynamics, we investigated the abundance of IgG ACPA VDG in 1,498 samples from individuals in different clinical stages.
Methods
Using liquid chromatography, we analyzed IgG ACPA VDG profiles in 7 different cohorts from Japan, Canada, The Netherlands, and Sweden. We assessed 106 healthy individuals, 228 individuals with presymptomatic RA, 277 individuals with arthralgia, 307 patients with new‐onset/early RA, and 117 RA patients after prespecified treatment regimens. Additionally, we measured VDG in 234 samples from patients with RA who did or did not achieve long‐term drug‐free remission (DFR) during up to 16 years follow‐up.
Results
IgG ACPA VDG significantly increased (P < 0.0001) toward disease onset and was associated with ACPA levels and epitope spreading prior to diagnosis. A slight increase in VDG was observed in patients with established RA, with a moderate influence of treatment (P = 0.007). In patients in whom DFR was later achieved, IgG ACPA VDG was already reduced at the time of RA onset.
Conclusion
The abundance of IgG ACPA VDG increases toward RA onset and correlates with maturation of the ACPA response. While IgG ACPA VDG levels are fairly stable in established disease, a lower degree of VDG at RA onset correlates with DFR. Although the underlying biologic mechanisms remain elusive, our data support the concept that VDG relates to an expansion of the ACPA response in the pre‐disease phase and contributes to disease development.
Changes in colorectal cancer (CRC) care planning because of the coronavirus disease 2019 (COVID-19) pandemic and associated health-related quality of life (HRQoL) and well-being of patients with CRC ...are unknown. We report changes in CRC care and patient-reported outcomes including HRQoL, distress, and loneliness during the first wave of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
In April 2020, 4984 patients included in the nationwide Prospective Dutch Colorectal Cancer cohort were invited to complete a COVID-19-specific questionnaire, together with the validated European Organisation for Research and Treatment of Cancer Core Quality of Life Questionnaire (QLQ-C30), De Jong Gierveld, and Hospital Anxiety and Depression Scale. Clinical data were obtained from the Netherlands Cancer Registry. Scores were compared with the year prior to COVID-19 and with an age- and sex-matched control population during COVID-19.
In total, 3247 (65.1%) patients responded between April and June 2020. Of the patients, 17% had canceled, postponed, or changed hospital visits to a telephone or video consult, and 5.3% had adjusted, postponed, or canceled treatment. Compared with controls, patients reported worse HRQoL but comparable distress and less social loneliness (patients = 21.2%; controls = 32.9%). Compared with pre-COVID-19, clinically meaningful deterioration of HRQoL was more prevalent in patients with changes in cancer care planning than in patients without changes. Prior to undergoing or currently undergoing treatment and infection worries were associated with lower HRQoL.
CRC patients reported equal anxiety and depression but worse HRQoL than the control population. Changes in care planning were associated with deterioration of HRQoL and increased anxiety. In case of 1 or more risk factors, health-care specialists should discuss (mental) health status and possible support during future SARS-CoV-2 infection waves or comparable pandemics.
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