The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and... Show moreThe type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies. Show less
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease... Show moreFetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available. Show less
Coillie, J. van; Pongracz, T.; Sustic, T.; Wang, W.J.; Nouta, J.; Gars, M. le; ... ; COUGH1 Study Grp 2023
IgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linke... Show moreIgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linked glycan located in the IgG Fc domain. Here, we compared the anti-Spike (S) IgG1 Fc glycosylation profiles in response to mRNA, adenoviral, and protein-based COVID-19 vaccines by mass spectrometry (MS). All vaccines induced a transient increase of antigen-specific IgG1 Fc galactosylation and sialylation. An initial, transient increase of afucosylated IgG was induced by membrane-encoding S protein formulations. A fucose-sensitive ELISA for antigen-specific IgG (FEASI) exploiting FcγRIIIa affinity for afucosylated IgG was used as an orthogonal method to confirm the LC-MS-based afucosylation readout. Our data suggest that vaccine-induced anti-S IgG glycosylation is dynamic, and although variation is seen between different vaccine platforms and individuals, the evolution of glycosylation patterns display marked overlaps. Show less
ObjectiveApplication of a face mask may provoke the trigeminocardiac reflex, leading to apnoea and bradycardia. This study investigates whether re-application of a face mask in preterm infants at... Show moreObjectiveApplication of a face mask may provoke the trigeminocardiac reflex, leading to apnoea and bradycardia. This study investigates whether re-application of a face mask in preterm infants at birth alters the risk of apnoea compared with the initial application, and identify factors that influence this risk. MethodsResuscitation videos and respiratory function monitor data collected from preterm infants ResultsIn total, 111 infants were included with 404 mask applications (102 initial and 302 subsequent mask applications). In 254/404 (63%) applications, infants were breathing prior to mask application, followed by apnoea after 67/254 (26%) mask applications. Apnoea and bradycardia occurred significantly more often after the initial mask application compared with subsequent applications (apnoea initial: 32/67 (48%) and subsequent: 44/187 (24%), p<0.001; bradycardia initial: 61% and subsequent 21%, p<0.001). Apnoea was followed by bradycardia in 73% and 71% of the initial and subsequent mask applications, respectively (p=0.607).In a logistic regression model, a lower breathing rate (OR 0.908 (95% CI 0.847 to 0.974), p=0.007) and heart rate (OR 0.935 (95% CI 0.901 to 0.970), p<0.001) prior to mask application were associated with an increased likelihood of becoming apnoeic following subsequent mask applications. ConclusionIn preterm infants at birth, apnoea and bradycardia occurs more often after an initial mask application than subsequent applications, with lower heart and breathing rates increasing the risk of apnoea in subsequent applications. Show less
Coillie, J. van; Pongracz, T.; Rahmoller, J.; Chen, H.J.; Geyer, C.E.; Vught, L.A. van; ... ; UMC COVID-19 S HCW Study Grp 2022
Background: Afucosylated IgG1 responses have only been found against membrane-embedded epitopes, including anti-S in SARS-CoV-2 infections. These responses, intrinsically protective through... Show moreBackground: Afucosylated IgG1 responses have only been found against membrane-embedded epitopes, including anti-S in SARS-CoV-2 infections. These responses, intrinsically protective through enhanced Fc gamma RIIIa binding, can also trigger exacerbated pro-inflammatory responses in severe COVID-19. We investigated if the BNT162b2 SARS-CoV-2 mRNA also induced afucosylated IgG responses. Methods: Blood from vaccinees during the first vaccination wave was collected. Liquid chromatography-Mass spectrometry (LC-MS) was used to study anti-S IgG1 Fc glycoprofiles. Responsiveness of alveolar-like macrophages to produce proinflammatory cytokines in presence of sera and antigen was tested. Antigen-specific B cells were characterized and glycosyltransferase levels were investigated by Fluorescence-Activated Cell Sorting (FACS). Findings: Initial transient afucosylated anti-S IgG1 responses were found in naive vaccinees, but not in antigen -experienced ones. All vaccinees had increased galactosylated and sialylated anti-S IgG1. Both naive and antigen -experienced vaccinees showed relatively low macrophage activation potential, as expected, due to the low antibody levels for naive individuals with afucosylated IgG1, and low afucosylation levels for antigen-experienced individuals with high levels of anti-S. Afucosylation levels correlated with FUT8 expression in antigen-specific plasma cells in naive individuals. Interestingly, low fucosylation of anti-S IgG1 upon seroconversion correlated with high anti-S IgG levels after the second dose. Interpretation: Here, we show that BNT162b2 mRNA vaccination induces transient afucosylated anti-S IgG1 responses in naive individuals. This observation warrants further studies to elucidate the clinical context in which potent afucosylated responses would be preferred. Copyright (c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Show less
Background: Cyclooxygenase inhibitors are commonly used in infants with patent ductus arteriosus (PDA), but the benefit of these drugs is uncertain. Methods: In this multicenter, noninferiority... Show moreBackground: Cyclooxygenase inhibitors are commonly used in infants with patent ductus arteriosus (PDA), but the benefit of these drugs is uncertain. Methods: In this multicenter, noninferiority trial, we randomly assigned infants with echocardiographically confirmed PDA (diameter, > 1.5 mm, with left-to-right shunting) who were extremely preterm (< 28 weeks' gestational age) to receive either expectant management or early ibuprofen treatment. The composite primary outcome included necrotizing enterocolitis (Bell's stage IIa or higher), moderate to severe bronchopulmonary dysplasia, or death at 36 weeks' postmenstrual age. The noninferiority of expectant management as compared with early ibuprofen treatment was defined as an absolute risk difference with an upper boundary of the one-sided 95% confidence interval of less than 10 percentage points. Results: A total of 273 infants underwent randomization. The median gestational age was 26 weeks, and the median birth weight was 845 g. A primary-outcome event occurred in 63 of 136 infants (46.3%) in the expectant-management group and in 87 of 137 (63.5%) in the early-ibuprofen group (absolute risk difference, -17.2 percentage points; upper boundary of the one-sided 95% confidence interval [CI], -7.4; P < 0.001 for noninferiority). Necrotizing enterocolitis occurred in 24 of 136 infants (17.6%) in the expectant-management group and in 21 of 137 (15.3%) in the early-ibuprofen group (absolute risk difference, 2.3 percentage points; two-sided 95% CI, -6.5 to 11.1); bronchopulmonary dysplasia occurred in 39 of 117 infants (33.3%) and in 57 of 112 (50.9%), respectively (absolute risk difference, -17.6 percentage points; two-sided 95% CI, -30.2 to -5.0). Death occurred in 19 of 136 infants (14.0%) and in 25 of 137 (18.2%), respectively (absolute risk difference, -4.3 percentage points; two-sided 95% CI, -13.0 to 4.4). Rates of other adverse outcomes were similar in the two groups. Conclusions: Expectant management for PDA in extremely premature infants was noninferior to early ibuprofen treatment with respect to necrotizing enterocolitis, bronchopulmonary dysplasia, or death at 36 weeks' postmenstrual age. (Funded by the Netherlands Organization for Health Research and Development and the Belgian Health Care Knowledge Center; BeNeDuctus ClinicalTrials.gov number, ; EudraCT number, .) Show less
As the complexity of emergency care increases, current research methods to improve care are often unable to capture all aspects of everyday clinical practice. Video recordings can visualize... Show moreAs the complexity of emergency care increases, current research methods to improve care are often unable to capture all aspects of everyday clinical practice. Video recordings can visualize clinical care in an objective way. They can be used as a tool to assess care and to reflect on care with the caregivers themselves. Although the use of video recordings to reflect on medical interventions (video-reflection) has increased over the years, it is still not used on a regular basis. However, video-reflection proved to be of educational value and can improve teams' management and performance. It has a positive effect on guideline adherence, documentation, clinical care and teamwork. Recordings can also be used for video-reflexivity. Here, caregivers review recordings together to reflect on their everyday practice from new perspectives with regard to context and conduct in general. Although video-reflection in emergency care has proven to be valuable, certain preconditions have to be met and obstacles need to be overcome. These include gaining trust of the caregivers, having a proper consent-procedure, maintaining confidentiality and adequate use of technical equipment. To implement the lessons learned from video-reflection in a sustainable way and to continuously improve care, it should be integrated in regular simulation training or education. This narrative review will describe the development of video recording in emergency care and how video-reflection can improve patient care and safety in new ways. On our own department, the NICU at the LUMC, video-reflection has already been implemented and we want to further expand this. We will describe the use of video-reflection in our own unit. Based on the results of this narrative review we will propose options for future research to increase the value of video-reflection. Show less
Sustic, T.; Coillie, J. van; Larsen, M.D.; Derksen, N.I.L.; Szittner, Z.; Nouta, J.; ... ; Vidarsson, G. 2022
Background: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (Fc gamma R). Absence of fucose on the highly conserved N-linked glycan in the... Show moreBackground: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (Fc gamma R). Absence of fucose on the highly conserved N-linked glycan in the IgG Fc domain strongly enhances IgG binding and activation of myeloid and natural killer (NK) cell Fc gamma Rs. Although afucosylated IgG can provide increased protection (malaria and HIV), it also boosts immunopathologies in alloimmune diseases, COVID-19 and dengue fever. Quantifying IgG fucosylation currently requires sophisticated methods such as liquid chromatography-mass spectrometry (LC-MS) and extensive analytical skills reserved to highly specialized laboratories. Methods: Here, we introduce the Fucose-sensitive Enzyme-linked immunosorbent assay (ELISA) for Antigen-Specific IgG (FEASI), an immunoassay capable of simultaneously quantitating and qualitatively determining IgG responses. FEASI is a two-tier immunoassay; the first assay is used to quantify antigen-specific IgG (IgG ELISA), while the second gives Fc gamma RIIIa binding-dependent readout which is highly sensitive to both the IgG quantity and the IgG Fc fucosylation (Fc gamma R-IgG ELISA). Findings: IgG Fc fucosylation levels, independently determined by LC-MS and FEASI, in COVID-19 responses to the spike (S) antigen, correlated very strongly by simple linear regression (R-2=0.93, p < 0.0001). The FEASI method was then used to quantify IgG levels and fucosylation in COVID-19 convalescent plasma which was independently validated by LC-MS. Interpretation: FEASI can be reliably implemented to measure relative and absolute IgG Fc fucosylation and quantify binding of antigen-specific IgG to Fc gamma R in a high-throughput manner accessible to all diagnostic and research laboratories. Copyright (c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Show less
Wagner, M.; Boer, M.C. den; Jansen, S.; Groepel, P.; Visser, R.; Witlox, R.S.G.M.; ... ; Pas, A.B. te 2021
Objective The aim of this study was to determine the experience with, and the feasibility of, point-of-view video recordings using eye-tracking glasses for training and reviewing neonatal... Show moreObjective The aim of this study was to determine the experience with, and the feasibility of, point-of-view video recordings using eye-tracking glasses for training and reviewing neonatal interventions during the COVID-19 pandemic. Design Observational prospective single-centre study. Setting Neonatal intensive care unit at the Leiden University Medical Center. Participants All local neonatal healthcare providers. Intervention There were two groups of participants: proceduralists, who wore eye-tracking glasses during procedures, and observers who later watched the procedures as part of a video-based reflection. Main outcome measures The primary outcome was the feasibility of, and the proceduralists and observers' experience with, the point-of-view eye-tracking videos as an additional tool for bedside teaching and video-based reflection. Results We conducted 12 point-of-view recordings on 10 different patients (median gestational age of 30.9 +/- 3.5 weeks and weight of 1764 g) undergoing neonatal intubation (n=5), minimally invasive surfactant therapy (n=5) and umbilical line insertion (n=2). We conducted nine video-based observations with a total of 88 observers. The use of point-of-view recordings was perceived as feasible. Observers further reported the point-of-view recordings to be an educational benefit for them and a potentially instructional tool during COVID-19. Conclusion We proved the practicability of eye-tracking glasses for point-of-view recordings of neonatal procedures and videos for observation, educational sessions and logistics considerations, especially with the COVID-19 pandemic distancing measures reducing bedside teaching opportunities.Single centre evaluation of the use of eye-tracking glasses during neonatal procedures as an additional tool for outside teaching and video-based reflection. The feasibility and potential benefit during a pandemic is reported. Show less
Braster, R.; Bogels, M.; Benonisson, H.; Wuhrer, M.; Plomp, R.; Bentlage, A.E.H.; ... ; Vidarsson, G. 2021
Simple SummaryCancer treatments are increasingly based on therapeutic antibodies to clear tumors. While in vivo mouse models are useful to predict effectiveness of human antibodies it is not... Show moreSimple SummaryCancer treatments are increasingly based on therapeutic antibodies to clear tumors. While in vivo mouse models are useful to predict effectiveness of human antibodies it is not completely clear how useful these models are to test antibodies engineered with enhanced effector functions designed for humans. One of the changes considered for many new antibody-based drugs is the removal of fucose (resulting in afucosylated IgG) which enhances IgG-Fc receptor (Fc gamma R) mediated effector functions in humans through Fc gamma RIIIa. Here we show that afucosylated human IgG1 also have enhanced effector functions against peritoneal metastasis of melanoma cells in mice through the evolutionary related mouse Fc gamma RIV. This shows that afucosylated human IgG is functionally recognized across species and shows that mouse tumor models can be used to assess the therapeutic potential of afucosylated IgG1.Promising strategies for maximizing IgG effector functions rely on the introduction of natural and non-immunogenic modifications. The Fc domain of IgG antibodies contains an N-linked oligosaccharide at position 297. Human IgG antibodies lacking the core fucose in this glycan have enhanced binding to human (Fc gamma R) IIIa/b, resulting in enhanced antibody dependent cell cytotoxicity and phagocytosis through these receptors. However, it is not yet clear if glycan-enhancing modifications of human IgG translate into more effective treatment in mouse models. We generated humanized hIgG1-TA99 antibodies with and without core-fucose. C57Bl/6 mice that were injected intraperitoneally with B16F10-gp75 mouse melanoma developed significantly less metastasis outgrowth after treatment with afucosylated hIgG1-TA99 compared to mice treated with wildtype hhIgG1-TA99. Afucosylated human IgG1 showed stronger interaction with the murine Fc gamma RIV, the mouse orthologue of human Fc gamma RIIIa, indicating that this glycan change is functionally conserved between the species. In agreement with this, no significant differences were observed in tumor outgrowth in Fc gamma RIV-/- mice treated with human hIgG1-TA99 with or without the core fucose. These results confirm the potential of using afucosylated therapeutic IgG to increase their efficacy. Moreover, we show that afucosylated human IgG1 antibodies act across species, supporting that mouse models can be suitable to test afucosylated antibodies. Show less
Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows... Show moreImmunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (Fc gamma RIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger Fc gamma RIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19. Show less
Background Umbilical venous catheters (UVCs) are associated with thrombus formation. Most studies on thrombosis in infants with UVCs focus on only one part of the route, and none assessed a control... Show moreBackground Umbilical venous catheters (UVCs) are associated with thrombus formation. Most studies on thrombosis in infants with UVCs focus on only one part of the route, and none assessed a control group of infants without UVCs.Objective To determine the incidence and location of thrombi in infants after umbilical catheterisation and compare this with a control group of infants without umbilical catheters.Design Prospective observational study with serial ultrasonography of the UVC route from the umbilico-portal confluence to the heart. Ultrasonography was performed until day 14 after catheterisation in cases and day 14 after birth in controls.Results Thrombi in the UVC route were detected in 75% (30/40) of infants with UVCs in the study group, whereas no thrombi were detected in the control group of infants without UVCs (0/20) (p<0.001). Six thrombi (20%) were located in the right atrium. Most of these were also partly present in the ductus venosus. Six thrombi (20%) were located in the ductus venosus only, and in 12 infants (40%), the thrombus was at least partly located in the umbilico-portal confluence. Thrombi persisted after UVC removal in 25/30 cases. Two infants with thrombotic events were treated with low-molecular-weight heparin and resolution was found. In the other 23 infants managed expectantly, 2 died due to necrotising enterocolitis, 1 was lost to follow-up and in 20 spontaneous regression was seen.Conclusions Thrombotic events occur frequently in infants after umbilical catheterisation. Most thrombi were asymptomatic and regressed spontaneously with expectant management. Routine screening for thrombi in UVCs is therefore not advised. Show less
Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical... Show moreImmunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti-CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, Fc gamma R, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti-PD-L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti-PD-L1 showed similar therapeutic efficacy when compared to each other in either wild-type mice or in mice deficient for all Fc gamma R. In contrast, in the CT26 tumor model, anti-PD-L1 mIgG2a, the IgG subclass with the highest affinity for activating Fc gamma R, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD-L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 x BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that Fc gamma R are not crucial for anti-PD-L1 antibody therapy but might play a role in some tumor models. Show less
