Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface.

Biological activities of immunoglobulin G such as effector functions via Fc receptor interactions are influenced by Fc-linked N-glycans. Here we describe a fast, robust and sensitive nano-LC-ESI-MS method for detailed subclass specific analysis of IgG Fc N-glycosylation. A sheath-flow ESI sprayer was used as a sensitive zero dead volume plug-and-play interface for online MS coupling, generating a very constant spray and ionization over the entire LC gradient. The propionic acid containing sheath-liquid effectively suppressed TFA gas-phase ion-pairing, enabling the use of TFA containing mobile phases. The fixed position of the sheath-flow ESI sprayer, far away from the glass capillary inlet, reduced MS contamination as compared to conventional nano-ESI. The method was found to be suitable for fast and detailed subclass specific IgG Fc N-glycosylation profiling in human plasma. The obtained subclass specific IgG Fc N-glycosylation profiles were processed automatically using in house developed software tools. For each of the IgG subclasses the 8 major glycoforms showed an interday analytical variation below 5%. The method was used to profile the IgG Fc N-glycosylation of 26 women at several time points during pregnancy and after delivery, revealing pregnancy-associated changes in IgG galactosylation, sialylation and incidence of bisecting N-acetylglucosamine.

Mannose-binding lectin does not explain the course and outcome of pregnancy in rheumatoid arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) improves during pregnancy and flares after delivery. It has been hypothesized that high levels of the complement factor mannose-binding lectin (MBL) are associated with a favourable disease course of RA by facilitating the clearance of pathogenic immunoglobulin G (IgG) lacking galactose sugar moieties. During pregnancy, increased galactosylation of IgG and simultaneously increased MBL levels can be observed, with the latter being strictly related to maternal MBL genotypes. Therefore, increased MBL levels in concert with increased IgG galactosylation may be associated with pregnancy-induced improvement of RA. The objective of this study was to investigate whether MBL genotypes are associated with changes in RA disease activity and with changes in IgG galactosylation during pregnancy and in the postpartum period. We also studied the association between MBL genotypes and pregnancy outcomes in RA. METHODS: Serum from 216 patients with RA and 31 healthy controls participating in the Pregnancy-induced Amelioration of Rheumatoid Arthritis (PARA) Study was collected before, during and after pregnancy. IgG galactosylation was determined by performing matrix-assisted laser desorption/ionization time of flight mass spectrometry. Disease activity was determined using the internationally recognized Disease Activity Score 28 (DAS28). MBL genotypes were determined. The pregnancy outcome measures studied were gestational age, birth weight, miscarriage and hypertensive disorders. RESULTS: No association was found between the MBL genotype groups and changes in RA disease activity (P = 0.89) or changes in IgG galactosylation (patients, P = 0.75, and controls, P = 0.54) during pregnancy and in the postpartum period. Furthermore, MBL genotype groups were not related to the studied pregnancy outcome measures. CONCLUSIONS: This study does not provide evidence for a role for MBL in the improvement of RA during pregnancy or for a role for MBL in pregnancy outcome.

Effects of mannose-binding lectin polymorphisms on irinotecan-induced febrile neutropenia.

OBJECTIVE: Mannose-binding lectin (MBL) is important in the innate immune response. MBL2 gene polymorphisms affect MBL expression, and genotypes yielding low MBL levels have been associated with an elevated risk for infections in hematological cancer patients undergoing chemotherapy. However, these reported associations are inconsistent, and data on patients with solid tumors are lacking. Here, we investigated the effects of MBL2 genotypes on irinotecan-induced febrile neutropenia in patients with solid tumors. PATIENTS AND METHODS: Irinotecan-treated patients were genotyped for the MBL2 gene. Two promoter (-550 H/L and -221 X/Y) and three exon polymorphisms (52 A/D, 54 A/B, and 57 A/C) were determined, together with known risk factors for irinotecan-induced toxicity. Neutropenia and febrile neutropenia were recorded during the first course. RESULTS: Of the 133 patients, 28% experienced severe neutropenia and 10% experienced febrile neutropenia. No associations were found between exon polymorphisms and febrile neutropenia. However, patients with the H/H promoter genotype, associated with high MBL levels, experienced significantly more febrile neutropenia than patients with the H/L and L/L genotypes (20% versus 13% versus 5%). Moreover, patients with the HYA haplotype encountered significantly more febrile neutropenia than patients without this high MBL-producing haplotype (16% versus 4%). In the subgroup with wild-type exon polymorphisms (A/A), patients with the high MBL promoter phenotype had the highest incidence of febrile neutropenia, regardless of known risk factors. CONCLUSION: Patients with high MBL2 promoter genotypes and haplotypes seem more at risk for developing febrile neutropenia. If confirmed, these preliminary findings may contribute to more individualized approaches of irinotecan treatment.

Immunoglobulin G galactosylation and sialylation are associated with pregnancy-induced improvement of rheumatoid arthritis and the postpartum flare: results from a large prospective cohort study.

INTRODUCTION: Improvement of rheumatoid arthritis (RA) during pregnancy has been causatively associated with increased galactosylation of immunoglobulin G (IgG) N-glycans. Since previous studies were small, did not include the postpartum flare and did not study sialylation, these issues were addressed in the present study. METHODS: Serum from 148 RA cases and 32 healthy controls was collected at several time points before, during and after pregnancy. Improvement during pregnancy and postpartum flare were determined according to the European League Against Rheumatism (EULAR) response criteria. Galactosylation and sialylation of Immunoglobulin G (IgG) and the presence of bisecting N-acetylglucosamine (GlcNAc) were analyzed by matrix-assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS). RESULTS: IgG1 and IgG2 galactosylation of the cases and controls increased during pregnancy with a maximum in the third trimester. Galactosylation decreased directly postpartum. IgG galactosylation of controls was at a higher level than cases (P < 0.001 at all time points) and a similar pattern was observed for sialylation. Moreover, there was a good association between galactosylation and sialylation. The increase in galactosylation was significantly more pronounced for cases with improvement than cases without improvement during pregnancy. The reverse was true for deteriorators and non-deteriorators postpartum. The presence of bisecting GlcNAc was not significantly influenced by pregnancy or postpartum for cases and controls. CONCLUSIONS: This large cohort study demonstrates the association of changes in galactosylation with both pregnancy-induced improvement and postpartum flare in RA-patients, suggesting a role for changes in glycosylation in the pregnancy-induced improvement of RA.

Disease activity of rheumatoid arthritis during pregnancy: results from a nationwide prospective study.

OBJECTIVE: According to common knowledge and retrospective studies, approximately 75-90% of patients with rheumatoid arthritis (RA) will improve during pregnancy. Prospective data on disease activity during pregnancy are limited. Therefore, this study aimed to prospectively determine the disease activity during pregnancy in RA patients treated in an era of new treatment options. METHODS: For 84 RA patients (American College of Rheumatology criteria), a Disease Activity Score in 28 joints (DAS28) and medication use were obtained, before conception if possible, at each trimester of pregnancy and at 6, 12, and 26 weeks postpartum. Improvement and deterioration were determined by assessing changes in DAS28 and by applying the DAS28-derived European League Against Rheumatism (EULAR) response criteria. RESULTS: Disease activity decreased with statistical significance (P = 0.035) during pregnancy and increased postpartum. In patients with at least moderate disease activity in the first trimester (n = 52), at least 48% had a moderate response during pregnancy according to EULAR-defined response criteria. In patients with low disease activity in the first trimester (n = 32), disease activity was stable during pregnancy. Thirty-nine percent of patients had at least a moderate flare postpartum according to reversed EULAR response criteria. Less medication was used during pregnancy compared with before conception and compared with postpartum. CONCLUSION: This study demonstrates that patients achieve remission during pregnancy and deteriorate postpartum, although less frequently than previously described.

Mannose-binding lectin genotypes and pre-eclampsia: a case-control study.

Both immunological and placental factors are involved in the pathogenesis of pre-eclampsia. The complement factor mannose-binding lectin (MBL) is associated with adverse pregnancy outcomes and has been suggested to play a role in abnormal placentation. We investigated whether MBL genotypes are associated with the systemic maternal syndrome pre-eclampsia. MBL2 gene polymorphisms were determined in a case-control study including 157 women with pre-eclampsia (case subjects) and 157 women with uncomplicated pregnancies (control subjects). Considering MBL polymorphisms, case and control subjects were categorized in groups of high (A), intermediate (B), and low (C) MBL production. No association was found between MBL genotypes and pre-eclampsia; adjusted odds ratios and 95% confidence intervals (95% CI) for group B were 0.97 (95% CI = 0.46-2.07) and for group C were 1.44 (95% CI = 0.52-3.94). A trend was found between MBL genotype groups B and C and severe pre-eclampsia or eclampsia. MBL genotypes were not found to be associated with pre-eclampsia; however low-MBL production genotypes might be considered as disease modifier. This suggests that MBL may play a role in modulating placental inflammation by facilitating clearance of apoptotic cells and cell debris.

Glycosylation profiling of immunoglobulin G (IgG) subclasses from human serum.

All four subclasses of human serum IgG contain a single N-glycosylation site in the constant region of their heavy chain, which is occupied by biantennary, largely core-fucosylated and partially truncated oligosaccharides, that may carry a bisecting N-acetylglucosamine and sialic acid residues. IgG glycosylation has been shown to be altered under various physiological and pathological circumstances. IgG N-glycan profiles vary with age, and galactosylation for example is enhanced during pregnancy. Several diseases including rheumatoid arthritis are associated with a reduction in galactosylation of the IgG N-glycans. Here, we describe a robust method for the isolation of IgG subclasses using protein A (binds IgG1, IgG2, and IgG4) and protein G (binds additionally IgG3) at the 96-well plate level, which is suitable for automation. Isolated IgGs were digested with trypsin, and obtained glycopeptides were analyzed by nano-LC-MS. Glycopeptides were characterized by CID as well as electron transfer dissociation (ETD). The method provided glycosylation profiles for IgG1, IgG2, IgG3, and IgG4 and revealed distinct differences in N-glycosylation between the four IgG subclasses. The changes in galactosylation associated with rheumatoid arthritis could readily be monitored. This method is suitable for the subclass-specific analysis of IgG glycosylation from clinical samples.

Measuring disease activity and functionality during pregnancy in patients with rheumatoid arthritis.

OBJECTIVE: Pregnancy has a favorable effect on the course of rheumatoid arthritis (RA), although the magnitude of this effect is equivocal because RA assessment tools have never been validated in pregnancy. The goal of this study was to assess how pregnancy influences the scoring of the Disease Activity Score in 28 joints (DAS28) and the Health Assessment Questionnaire (HAQ), and how both scores perform in pregnant patients with RA. METHODS: Thirty-two healthy women and 30 pregnant patients with RA were prospectively studied during pregnancy and at postpartum. At each trimester and postpartum the components of the DAS28 (global health [GH], erythrocyte sedimentation rate [ESR], and C-reactive protein level [CRP]) and HAQ scores were determined. Maximal influences of healthy pregnancy on each component of the DAS28 were calculated. The performances of different DAS28 scores and the HAQ were also determined in RA patients. Furthermore, variants of the HAQ were developed within the HAQ scoring rules. RESULTS: The components of the DAS28 were influenced by healthy pregnancy, with average increases in DAS28 score of 0.22 (GH), 1.1 (ESR), and 0.25 (CRP). The DAS28 calculated with CRP (DAS28-CRP) and without GH performed the best in pregnant RA patients. In healthy pregnancy, the median HAQ increased to 0.50 in the third trimester and was reduced by the HAQ variants to 0.25. CONCLUSION: Pregnancy considerably influences the scoring of the DAS28 and HAQ. RA disease activity in pregnant patients should preferably be calculated with DAS28-CRP without GH. Even with HAQ variants, influences of pregnancy on the assessment of functionality cannot be precluded.