Background: The interaction of allergens and allergen-specific IgE initiates the allergic cascade after crosslinking of receptors on effector cells. Antibodies of other isotypes may modulate such a reaction. Receptor crosslinking requires binding of antibodies to multiple epitopes on the allergen. Limited information is available on the complexity of the epitope structure of most allergens. Objectives: We sought to allow description of the complexity of IgE, IgG4, and IgG epitope recognition at a global, allergome-wide level during allergen-specific immunotherapy (AIT). Methods: We generated an allergome-wide microarray comprising 731 allergens in the form of more than 172,000 overlapping 16-mer peptides. Allergen recognition by IgE, IgG4, and IgG was examined in serum samples collected from subjects undergoing AIT against pollen allergy. Results: Extensive induction of linear peptide-specific Phl p 1– and Bet v 1–specific humoral immunity was demonstrated in subjects undergoing a 3-year-long AIT against grass and birch pollen allergy, respectively. Epitope profiles differed between subjects but were largely established already after 1 year of AIT, suggesting that dominant allergen-specific antibody clones remained as important contributors to humoral immunity following their initial establishment during the early phase of AIT. Complex, subject-specific patterns of allergen isoform and group cross-reactivities in the repertoires were observed, patterns that may indicate different levels of protection against different allergen sources. Conclusions: The study highlights the complexity and subject-specific nature of allergen epitopes recognized following AIT. We envisage that epitope deconvolution will be an important aspect of future efforts to describe and analyze the outcomes of AIT in a personalized manner.

In this crowdsourced initiative, independent analysts used the same dataset to test two hypotheses regarding the effects of scientists' gender and professional status on verbosity during group meetings. Not only the analytic approach but also the operationalizations of key variables were left unconstrained and up to individual analysts. For instance, analysts could choose to operationalize status as job title, institutional ranking, citation counts, or some combination. To maximize transparency regarding the process by which analytic choices are made, the analysts used a platform we developed called DataExplained to justify both preferred and rejected analytic paths in real time. Analyses lacking sufficient detail, reproducible code, or with statistical errors were excluded, resulting in 29 analyses in the final sample. Researchers reported radically different analyses and dispersed empirical outcomes, in a number of cases obtaining significant effects in opposite directions for the same research question. A Boba multiverse analysis demonstrates that decisions about how to operationalize variables explain variability in outcomes above and beyond statistical choices (e.g., covariates). Subjective researcher decisions play a critical role in driving the reported empirical results, underscoring the need for open data, systematic robustness checks, and transparency regarding both analytic paths taken and not taken. Implications for orga-nizations and leaders, whose decision making relies in part on scientific findings, consulting reports, and internal analyses by data scientists, are discussed.

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann-Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (p(adjusted) = 3 x 10(-9), 3 x 10(-6), and 5 x 10(-6) respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials.

BackgroundPrevious studies and own clinical observations of patients with systemic lupus erythematosus (SLE) suggest that SLE harbors distinct immunophenotypes. This heterogeneity might result in differences in response to treatment in different subgroups and obstruct clinical trials. Our aim was to understand how SLE subgroups may differ regarding underlying pathophysiology and characteristic biomarkers.MethodsIn a cross-sectional study, including 378 well-characterized SLE patients and 316 individually matched population controls, we defined subgroups based on the patients' autoantibody profile at inclusion. We selected a core of an antiphospholipid syndrome-like SLE (aPL+ group; positive in the lupus anticoagulant (LA) test and negative for all three of SSA (Ro52 and Ro60) and SSB antibodies) and a Sjogren's syndrome-like SLE (SSA/SSB+ group; positive for all three of SSA (Ro52 and Ro60) and SSB antibodies but negative in the LA test). We applied affinity-based proteomics, targeting 281 proteins, together with well-established clinical biomarkers and complementary immunoassays to explore the difference between the two predefined SLE subgroups.ResultsThe aPL+ group comprised 66 and the SSA/SSB+ group 63 patients. The protein with the highest prediction power (receiver operating characteristic (ROC) area under the curve=0.89) for separating the aPL+ and SSA/SSB+ SLE subgroups was integrin beta-1 (ITGB1), with higher levels present in the SSA/SSB+ subgroup. Proteins with the lowest p values comparing the two SLE subgroups were ITGB1, SLC13A3, and CERS5. These three proteins, rheumatoid factor, and immunoglobulin G (IgG) were all increased in the SSA/SSB+ subgroup. This subgroup was also characterized by a possible activation of the interferon system as measured by high KRT7, TYK2, and ETV7 in plasma. In the aPL+ subgroup, complement activation was more pronounced together with several biomarkers associated with systemic inflammation (fibrinogen, -1 antitrypsin, neutrophils, and triglycerides).ConclusionsOur observations indicate underlying pathogenic differences between the SSA/SSB+ and the aPL+ SLE subgroups, suggesting that the SSA/SSB+ subgroup may benefit from IFN-blocking therapies while the aPL+ subgroup is more likely to have an effect from drugs targeting the complement system. Stratifying SLE patients based on an autoantibody profile could be a way forward to understand underlying pathophysiology and to improve selection of patients for clinical trials of targeted treatments.

Naturally acquired antibodies to proteins expressed on the Plasmodium falciparum parasitized red blood cell (pRBC) surface steer the course of a malaria infection by reducing sequestration and stimulating phagocytosis of pRBC. Here we have studied a selection of proteins representing three different parasite gene families employing a well-characterized parasite with a severe malaria phenotype (FCR3S1.2). The presence of naturally acquired antibodies, impact on rosetting rate, surface reactivity and opsonization for phagocytosis in relation to different blood groups of the ABO system were assessed in a set of sera from children with mild or complicated malaria from an endemic area. We show that the naturally acquired immune responses, developed during malaria natural infection, have limited access to the pRBCs inside a blood group A rosette. The data also indicate that SURFIN4.2 may have a function at the pRBC surface, particularly during rosette formation, this role however needs to be further validated. Our results also indicate epitopes differentially recognized by rosette-disrupting antibodies on a peptide array. Antibodies towards parasite-derived proteins such as PfEMP1, RIFIN and SURFIN in combination with host factors, essentially the ABO blood group of a malaria patient, are suggested to determine the outcome of a malaria infection.

Plasmodium falciparum invasion into red blood cells (RBCs) is a complex process engaging proteins on the merozoite surface and those contained and sequentially released from the apical organelles (micronemes and rhoptries). Fundamental to invasion is the formation of a moving junction (MJ), a region of close apposition of the merozoite and the RBC plasma membranes, through which the merozoite draws itself before settling into a newly formed parasitophorous vacuole (PV). SURFIN4.2 was identified at the surface of the parasitized RBCs (pRBCs) but was also found apically associated with the merozoite. Using antibodies against the N-terminus of the protein we show the presence of SURFIN4.2 in the neck of the rhoptries, its secretion into the PV and shedding into the culture supernatant upon schizont rupture. Using immunoprecipitation followed by mass spectrometry we describe here a novel protein complex we have named SURGE where SURFIN4.2 forms interacts with the rhoptry neck protein 4 (RON4) and the Glutamate Rich Protein (GLURP). The N-terminal cysteine-rich domain (CRD) of SURFIN4.2 mediates binding to the RBC membrane and its interaction with RON4 suggests its involvement in the contact between the merozoite apex and the RBC at the MJ. Supporting this suggestion, we also found that polyclonal antibodies to the extracellular domain (including the CRD) of SURFIN4.2 partially inhibit merozoite invasion. We propose that the formation of the SURGE complex participates in the establishment of parasite infection within the PV and the RBCs.

Objective The objective of this study was to investigate the association of clinical and renal disease activity with circulating sphingolipids in patients with systemic lupus erythematosus. Methods We used liquid chromatography tandem mass spectrometry to measure the levels of 27 sphingolipids in plasma from 107 female systemic lupus erythematosus patients and 23 controls selected using a design of experiment approach. We investigated the associations between sphingolipids and two disease activity indices, the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index. Damage was scored according to the Systemic Lupus International Collaborating Clinics damage index. Renal activity was evaluated with the British Island Lupus Activity Group index. The effects of immunosuppressive treatment on sphingolipid levels were evaluated before and after treatment in 22 female systemic lupus erythematosus patients with active disease. Results Circulating sphingolipids from the ceramide and hexosylceramide families were increased, and sphingoid bases were decreased, in systemic lupus erythematosus patients compared to controls. The ratio of C-16:0-ceramide to sphingosine-1-phosphate was the best discriminator between patients and controls, with an area under the receiver-operating curve of 0.77. The C-16:0-ceramide to sphingosine-1-phosphate ratio was associated with ongoing disease activity according to the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index, but not with accumulated damage according to the Systemic Lupus International Collaborating Clinics Damage Index. Levels of C-16:0- and C-24:1-hexosylceramides were able to discriminate patients with current versus inactive/no renal involvement. All dysregulated sphingolipids were normalized after immunosuppressive treatment. Conclusion We provide evidence that sphingolipids are dysregulated in systemic lupus erythematosus and associated with disease activity. This study demonstrates the utility of simultaneously targeting multiple components of a pathway to establish disease associations.

Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing.